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 stabilize 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:

The data are of high quality and the description is clear.

Reviewer #1 (Public Review):

Summary:

The current manuscript provides strong evidence that the molecular function of SLC35G1, an orphan human SLC transporter, is citrate export at the basolateral membrane of intestinal epithelial cells. Multiple lines of evidence, including radioactive transport experiments, immunohistochemical staining, gene expression analysis, and siRNA knockdown are combined to deduce a model of the physiological role of this transporter.

Strengths:

The experimental approaches are comprehensive, and together establish a strong model for the role of SLC35G1 in citrate uptake. The observation that chloride inhibits uptake suggests an interesting mechanism that exploits the difference in chloride concentration across the basolateral membrane.

Weaknesses:

Some aspects of the results would benefit from a more thorough discussion of the conclusions and/or model.

For example, the authors find that SLC35G1 prefers the dianionic (singly protonated) form of citrate, and rationalize this finding by comparison with the substrate selectivity of the citrate importer NaDC1. However, this comparison has weaknesses when considering the physiological pH for SLC35G1 and NaDC1. NaDC1 binds citrate at a pH of ~5.4 (the pKa of citrate is 5.4, so there is a lot of dianionic citrate present under physiological circumstances). SLC35G1 binds citrate under pH conditions of ~7.5, where a very small amount of dianionic citrate is present. The data clearly show a pH dependence of transport, and the authors rule out proton coupling, but the discrepancy between the pH dependence and the physiological expectations should be addressed/commented on.

The rationale for the series of compounds tested in Figure 1F, which includes metabolites with carboxylate groups, a selection of drugs including anion channel inhibitors and statins, and bile acids, is not described. Moreover, the lessons drawn from this experiment are vague and should be expanded upon. It is not clear what, if anything, the compounds that reduce citrate uptake have in common.

The transporter is described as a facilitative transporter, but this is not established definitively. For example, another possibility could involve coupling citrate transport to another substrate, possibly even chloride ion.

Reviewer #2 (Public Review):

Summary:

The primary goal of this study was to identify the transport pathway that is responsible for the release of dietary citrate from enterocytes into blood across the basolateral membrane.

Strengths:

The transport pathway responsible for the entry of dietary citrate into enterocytes was already known, but the transporter responsible for the second step remained unidentified. The studies presented in this manuscript identify SLC35G1 as the most likely transporter that mediates the release of absorbed citrate from intestinal cells into the serosal side. This fills an important gap in our current knowledge of the transcellular absorption of dietary citrate. The exclusive localization of the transporter in the basolateral membrane of human intestinal cells and the human intestinal cell line Caco-2 and the inhibition of the transporter function by chloride support this conclusion.

Weaknesses:

(i) The substrate specificity experiments have been done with relatively low concentrations of potential competing substrates, considering the relatively low affinity of the transporter for citrate. Given that NaDC1 brings in not only citrate as a divalent anion but also other divalent anions such as succinate, it is possible that SLC35G1 is responsible for the release of not only citrate but also other dicarboxylates. But the substrate specificity studies show that the dicarboxylates tested did not compete with citrate, meaning that SLc35G1 is selective for the citrate (2-), but this conclusion might be flawed because of the low concentration of the competing substrates used in the experiment.

(ii) The authors have used MDCK cells for assessment of the transcellular transfer of citrate via SLC35G1, but it is not clear whether this cell line expresses NaDC1 in the apical membrane as the enterocytes do. Even though the authors expressed SLC35G1 ectopically in MDCK cells and showed that the transporter localizes to the basolateral membrane, the question as to how citrate actually enters the apical membrane for SLC35G1 in the other membrane to work remains unanswered.

(iii) There is one other transporter that has already been identified for the efflux of citrate in some cell types in the literature (SLC62A1, PLoS Genetics; 10.1371/journal.pgen.1008884), but no mention of this transporter has been made in the current manuscript.

Reviewer #3 (Public Review):

Summary:

Mimura et al describe the discovery of the orphan transporter SLC35G1 as a citrate transporter in the small intestine. Using a combination of cellular transport assays, they show that SLC35G1 can mediate citrate transport in small intestinal cell lines. Furthermore, they investigate its expression and localization in both human tissue and cell lines. Limited evidence exists to date on both SLC35G1 and citrate uptake in the small intestine, therefore this study is an important contribution to both fields. However, the main claims by the authors are only partially supported by experimental evidence.

Strengths:

The authors convincingly show that SLC35G1 mediates uptake of citrate which is dependent on pH and chloride concentration. Putting their initial findings in a physiological context, they present human tissue expression data of SLC35G. Their Transwell assay indicates that SLC35G1 is a citrate exporter at the basolateral membrane.

Weaknesses:

Further confirmation and clarification are required to claim that the SLC indeed exports citrate at the basolateral membrane as concluded by the authors. Most experiments measure citrate uptake, but the authors state that SLC35G1 is an exporter, mostly based on the lack of uptake at physiological conditions faced at the basolateral side. The Transwell assay in Figure 1L is the only evidence that it indeed is an exporter. However, in this experiment, the applied chloride concentration was not according to the proposed model (120mM at the basolateral side). The Transwell assay, or a similar assay measuring export instead of import, should be carried out in knockdown cells to prove that the export indeed occurs through SLC35G1 and not through an indirect effect. Related to the mentioned chloride sensitivity, it is unclear how the proposed model works if the SLC faces high chloride conditions under physiological conditions though it is inhibited by chloride.

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study is solid and clear.

Reviewer #2 (Public Review):

Summary:

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:

The data in this manuscript is robust and the experiments conducted are appropriate.

Weaknesses:

(1) The authors found that SntB played key roles in oxidative stress response of A. flavus by ChIP-seq and RNA sequencing. To confirm the role of SntB in oxidative stress, authors have better to measure the ROS levels in the ΔsntB and WT strains, besides the ΔcatC strain.<br /> (2) Why the authors only studied the function of catC among the 7 genes related to oxidative response listed in Table S14.

Reviewer #1 (Public Review):

By using a series of biochemical methods based on proteomic and metabolomic approaches, this study aims at: (1) validating the specific targeting of a biologically active molecule (MIPS2673) towards a defined (and unique?) protein target within a parasite, and (2) exploring whether it is possible to extrapolate which metabolic pathway has been disrupted.

Strength/Weaknesses

-The chemoproteomic approach, convincingly shows that MIPS2673 more significantly "protects" the putative target (PfA-M1) against thermal degradation or against enzymatic attack (by proteinase K). Proteomic studies are carried using parasite extracts enriched in late trophozoites (30-38 h pi), and are restricted to the soluble proteins fraction.<br /> -The metabolomic approach, documents the ability of MIPS2673 to selectively increase the number of non-hydrolyzed dipeptides in treated versus untreated parasites, further arguing for selective targeting of PfA-M1 and impairment of hemoglobin breakdown by the parasite.<br /> -The revised version now also considers and further studies the additional putative targets identified by one proteomic approach (but not the other one), which is both more critical of the results obtained and more realistic.<br /> The work as a whole is highly interesting, both for the specific topic of PfA-M1's role in parasite biology and for the method, applicable to other malarial drug contexts.

Reviewer #2 (Public Review):

In this manuscript, the authors first developed a new small molecular inhibitor that could target specifically the M1 metalloproteases of both important malaria parasite species Plasmodium falciparum and P. vivax. This was done by a chemical modification of a previously developed molecule that targets PfM1 as well as PfM17 and possibly other Plasmodial metalloproteases. After the successful chemical synthesis, the authors showed that the derived inhibitor, named MIPS2673, has a strong antiparasitic activity with IC50 342 nM and it is highly specific for M1. With this in mind, the authors first carried out two large-scale proteomics to confirm the MIPS2673 interaction with PfM1 in the context of the total P. falciparum protein lysate. This was done first by using thermal shift profiling and subsequently limited proteolysis. While the first demonstrated overall interaction, the latter (limited proteolysis) could map more specifically the site of MIPS2673-PfM1 interaction, presumably the active site. Subsequent metabolomics analysis showed that MIPS2673 cytotoxic inhibitory effect leads to the accumulation of short peptides many of which originate from hemoglobin. Based on that the authors argue that the MIPS2673 mode of action (MOA) involves inhibition of hemoglobin digestion that in turn inhibits the parasite growth and development.

Comments on the revised version:

The authors addressed all my comments from the previous round of reviews.

Reviewer #3 (Public Review):

Summary:

Overall, this is an interesting series of experiments which have identified a putative inhibitor of the Plasmodium M1 alanyl aminopeptidases, PfA-M1 and PvA-M1. The weaknesses include the lack of additional analysis of additional targets identified in the chemoproteomic approaches.

Strengths:

The main strengths include the synthesis of MIPS2673 which is selectively active against the enzyme and in whole cell assay.

Weaknesses:

The authors have addressed the previously identified weaknesses and have now provided additional data and explanations. They have modified their conclusions to indicate the limitations of their work.

Reviewer #1 (Public Review):

Summary:

Khan et. al., investigated the functional redundancy of the non-canonical L-cysteine synthases of M. tuberculosis, CysM and CysK2, focussing on their role in mitigating the effects of host-derived stress. They found that while deletion mutants of the two synthases (Rv∆cysM, Rv∆cysK2) have similar transcriptomes under standard conditions, their transcriptional response to oxidative stress is distinct. The impact of deleting the synthases also differentially affected the pools of L-cysteine-derived metabolites. They show that the mutants (Rv∆cysM, Rv∆cysK2) have impaired survival in peritoneal macrophages and in a mouse model of infection. Importantly, they show that the survival of the mutants increases when the host are defective in producing reactive oxygen and nitrogen species, linking the phenotype to a defect in combating host-derived stress. Finally, they show that compounds inhibiting L-cysteine synthases reduces intracellular survival of M. tuberculosis.

Strengths:

(1) The distinct transcriptome of the Rv∆cysM and Rv∆cysK2 mutants in the presence of oxidative stress provides solid evidence that these mutants are distinct in their response to oxidative stress, and suggests that they are not functionally redundant.<br /> (2) The use of macrophages from phox-/- and INF-/- mice and an iNOS inhibitor for the intracellular survival assays provides solid evidence that the survival defect seen for the Rv∆cysM and Rv∆cysK2 mutants is related to their reduced ability to combat host-derive oxidative and nitrosative stress. This is further supported by the infection studies in phox-/- and INF-/- mice.

Weaknesses:

Inclusion of the complemented strains in the metabolite study would strengthen the data. Furthermore, using an alternate method to quantify the MSH:MSSM ratio would provide insight into the redox homoeostasis in mutants in the presence and absence of CHP to support the statement that "deletion or inhibition of CysM or CysK2 perturbs redox homeostasis of Mtb".

The authors sought to investigate the functional redundancy of the non-canonical L-cysteine synthases CysM and CysK2. While their distinct transcriptional response to oxidative stress suggests distinct physiological roles, the study did not explore these differences, and therefore provides only preliminary insight into the underlying reasons for this observation. In the context of drug development, this work suggests that while L-cysteine synthases inhibitors do not have high potency for killing intracellular M. tuberculosis, they have potential for decreasing the pathogen's survival in the presence of host-derive stress.

Reviewer #3 (Public Review):

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

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

The addition of hydrogen-deuterium exchange mass spectrometry (HDX-MS) complements the results originating from cryo-EM data. Most notable, is the observation of the enhanced exchange (albeit quite subtle) of the GRD domain in the presence of dATP that matches the loss of structural information in this region in the cryo-EM data. The most pronounced and compelling HDX results are seen in the form of dATP-induced protection of peptides immediately adjacent to the b-hairpin at the s-site, where dATP is expected to bind based on cryo-EM. It is clear that the presence of dATP increases the rigidity of this region.

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels (stress) over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most robustly related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, change in intrinsic functional connectivity related to changes in diurnal cortisol release and long-term cortisol exposure. These changes are proposed to result from a combination of factors, which is supported by multivariate analyses showing changes across subfields and training content relate to cortisol changes.

The authors demonstrate that mindfulness training programs are a potential avenue for stress interventions that impact hippocampal structure and cortisol, providing a promising approach to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. The authors note the limitations of their approaches and provide detailed information on the data used and analyses conducted. The results provide a strong basis from which future studies can expand using computational approaches or more fine-grained investigations of the impact of mindfulness training on cortisol levels and the hippocampus.

Reviewer #3 (Public Review):

Summary:

This study profiled the single-cell transcriptome of human spermatogenesis and provided many potential molecular markers for developing testicular puncture-specific marker kits for NOA patients.

Strengths:

Perform single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on testicular tissues from two OA patients and three NOA patients.

Weaknesses:

Most results are analytical and lack specific experiments to support these analytical results and hypotheses.

Reviewer #1 (Public Review):

Summary:

The manuscript is dedicated heavily to cell type mapping and identification of sub-type markers in the human testis but does not present enough results from cross-investigation between NOA cases versus control. Their findings are mostly based on transcriptome and the authors do not make enough use of the scATAC-seq data in their analyses as they put forward in the title. Overall, the authors should do more to include the differential profile of NOA cases at the molecular level - specific gene expression, chromatin accessibility, TF binding, pathway, and signaling that are perturbed in NOA patients that may be associated with azoospermia.

Strengths:

(1) The establishment of single-cell data (both RNA and ATAC) from the human testicular tissues is noteworthy.

(2) The manuscript includes extensive mapping of sub-cell populations with some claimed as novel, and reports marker gene expression.

(3) The authors present inter-cellular cross-talks in human testicular tissues that may be important in adequate sperm cell differentiation.

Weaknesses:

(1) A low sample size (2 OA and 3 NOA cases). There are no control samples from healthy individuals.

(2) Their argument about interactions between germ and Sertoli cells is not based on statistical testing.

(3) Rationale/logic of the study. This study, in its present form, seems to be more about the role of sub-Sertoli population interactions in sperm cell development and does not provide enough insights about NOA.

(4) The authors do not make full use of the scATAC-seq data.

Reviewer #2 (Public Review):

Summary:

Shimin Wang et al. investigated the role of Sertoli cells in mediating spermatogenesis disorders in non-obstructive azoospermia (NOA) through stage-specific communications. The authors utilized scRNA-seq and scATAC-seq to analyze the molecular and epigenetic profiles of germ cells and Sertoli cells at different stages of spermatogenesis.

Strengths:

By understanding the gene expression patterns and chromatin accessibility changes in Sertoli cells, the authors sought to uncover key regulatory mechanisms underlying male infertility and identify potential targets for therapeutic interventions. They emphasized that the absence of the SC3 subtype would be a major factor contributing to NOA.

Weaknesses:

Although the authors used cutting-edge techniques to support their arguments, it is difficult to find conceptual and scientific advances compared to Zeng S et al.'s paper (Zeng S, Chen L, Liu X, Tang H, Wu H, and Liu C (2023) Single-cell multi-omics analysis reveals dysfunctional Wnt signaling of spermatogonia in non-obstructive azoospermia. Front. Endocrinol. 14:1138386.). Overall, the authors need to improve their manuscript to demonstrate the novelty of their findings in a more logical way.

Reviewer #1 (Public Review):

In this manuscript, Leikina et al. investigate the role of redox changes in the ubiquitous protein La in the promotion of osteoclast fusion. In a recently published manuscript, the investigators found that osteoclast multinucleation and resorptive activity are regulated by a de-phosphorylated and proteolytically cleaved form of the La protein that is present on the cell surface of differentiating osteoclasts. In the present work, the authors build upon these findings to determine the physiologic signals that regulate La trafficking to the cell membrane and ultimately, the ability of this protein to promote fusion. Building upon other published studies that show (1) that intracellular redox signaling can elicit changes in the confirmation and localization of La, and (2) that osteoclast formation is dependent on ROS signaling, the authors hypothesize that oxidation of La in response to intracellular ROS underlies the re-localization of La to the cell membrane and that this is necessary for its pro-fusion activity. The authors test this hypothesis in a rigorous manner using antioxidant treatments, recombinant La protein, and modification of cysteine residues predicted to be key sites of oxidation. Osteoclast fusion is then monitored in each condition using fluorescence microscopy. These data strongly support the conclusion that oxidized La is de-phosphorylated, increases in abundance at the cell surface of differentiating osteoclasts, and promotes cell-cell fusion. A strength of this manuscript is the use of multiple complementary approaches to test the hypothesis, especially the use of Cys mutant forms of La to directly tie the observed phenotypes to changes in residues that are key targets for oxidation. The manuscript is also well-written and describes a clearly articulated hypothesis based on a precise summation of the existing literature. The findings of this manuscript will be of interest to researchers in the field of bone biology, but also more generally to cell biologists. The data in this manuscript may also lead to future studies that target La for bone diseases in which there is increased osteoclast activity. The weaknesses of the manuscript are minor and predominantly relate to data presentation choices. These weaknesses do not detract from the overall conclusions of the study.

Reviewer #2 (Public Review):

Summary:

Bone resorption by osteoclasts plays an important role in bone modeling and homeostasis. The multinucleated mature osteoclasts have higher bone-resorbing capacity than their mononuclear precursors. The previous work by authors has identified that increased cell-surface level of La protein promotes the fusion of mononuclear osteoclast precursor cells to form fully active multinucleated osteoclasts. In the present study, the authors further provided convincing data obtained from cellular and biochemical experiments to demonstrate that the nuclear-localized La protein where it regulates RNA metabolism was oxidized by redox signaling during osteoclast differentiation and the modified La protein was translocated to the osteoclast surface where it associated with other proteins and phospholipids to trigger cell-cell fusion process. The work provides novel mechanistic insights into osteoclast biology and provides a potential therapeutic target to suppress excessive bone resorption in metabolic bone diseases such as osteoporosis and arthritis.

Strengths:

Increased intracellular ROS induced by osteoclast differentiation cytokine RANKL has been widely studied in enhancing RANKL signaling during osteoclast differentiation. The work provides novel evidence that redox signaling can post-translationally modify proteins to alter the translocation and functions of critical regulators in the late stage of osteoclastogenesis. The results and conclusions are mostly supported by the convincing cellular and biochemical assays,

Weaknesses:

The lack of in vivo studies in animal models of bone diseases such as postmenopausal osteoporosis, inflammatory arthritis, and osteoarthritis reduces the translational potential of this work.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Beardslee and Schmitz, the authors undertook a screen for potential degrons - short peptide sequences at the C-terminus that would target the toxin VapC for degradation. The authors randomly mutagenized 5 amino acids appended to the C-terminus of VapC and transformed this library into E. coli to look for surviving cells when the VapC gene was expressed. The authors found an enrichment for tags ending Ala-Ala, and found that this enrichment was dependent on the presence of the ClpXP protease, since this sequence was not similarly enriched in a mutant lacking this protease. Moreover, the authors identify the sequence FKLVA as the tag with the highest fold enrichment in the screen and confirm that GFP tagged with this sequence is degraded by ClpXP with similar kinetics to GFP tagged with the ssrA-derived tag.

Strengths:

This study has two major implications for understanding the nature of degrons in E. coli. First, peptides ending Ala-Ala, and especially degrons resembling the ssrA degron are likely the most degradation-promoting sequences in E. coli. Second, these findings suggest that ClpXP is the most central protease, at least for this particular protein with a randomized C-terminus under the particular conditions of this screen. It is also notable that the ribosome quality control protein RqcH tags truncated proteins with an alanine tag in a template-free manner when the large ribosomal subunit is obstructed. Although E. coli doesn't encode RqcH, the utility of alanine-tagging for protein degradation likely extends to other organisms.

Weaknesses:

The authors remark and show that mutations that inactivate the VapC protein are enriched potentially more than the proteolysis tags. This is a limitation of the study and the authors have done well to describe this as it will inform future screens. Perhaps using a protein with more intermediate toxicity in future screens would help to prioritize C-terminal mutations instead of toxin-inactivating mutations.

For clarity, the authors should explain why the NNK structure of the random codons was used. Why is it important that the codon end with a G or T?

Authors state on page 7 that by determining enrichment of individual tags they can rank the relative Km for proteolysis of the individual tags. This statement is not accurate since the tag could variously impact its association with any of the proteases in the cell. Since Km is specific to each particular protease, these can't be ranked in vivo when all proteases are present.

Reviewer #2 (Public Review):

Summary:

The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

Strengths:

The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

Weaknesses:

The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

Reviewer #3 (Public Review):

Summary:

This manuscript by Beardslee and Schmitz reports discoveries made from a genetic screen to identify C-terminal degrons that cause the efficient depletion of a potent toxin, which allowed for a deep assessment of amino acid patterns that promote protein turnover.

Strengths:

The key findings are that SsrA-like C termini are a dominant class of efficient degrons and that ClpP (X/A) mediates the turnover in most cases. Moreover, the data provides insight into the importance of residues situated farther into the degron and reveals aspects of the ClpX engagement and processivity process. The manuscript is clearly written and there is ample supporting data for the conclusions drawn. The figures are also informative.

Weaknesses:

There are only a few minor suggestions on data interpretation.

(1) Page 6: It is stated that "We plated cells on media containing 0 - 1% arabinose inducer, and observed that stronger induction of untagged VapC indeed correlates with smaller colony size; ... We conclude that VapC levels have a titratable effect on growth rate."

In E. coli with intact arabinose import/response systems, sub-saturating levels of arabinose do not generally lower the induction level of the PBAD promoter in each cell; rather, a sub-population of cells becomes induced [PMID: 9223333]. The bulk observation is a reduced expression level, and, in this case, slowed growth, but it seems more likely that the slow growth observed is from the induced cohort dying off as the cultures and colonies develop.

(2) Page 8: "At 6-hours post-induction,..."

Because these experiments were enrichments from initial pools of clones, the number of cell divisions is more informative than the hours of outgrowth or culture densities at harvest. It would be helpful if the authors could indicate, or at least estimate, the number of cell divisions. this could then be included in the results or methods section.

(3) Page 12: "It is possible that these sequences compromise VapC folding and solubility, or mimic inhibitory interactions made by hydrophobic segments of the VapB antitoxin that block VapC activity (43, 59)."

Later in the manuscript, Lon is presented as a minor player in the overall story, but Lon prefers hydrophobic degrons. Could that hydrophobic class be Lon substrates? (Possibly presented as an additional mechanism here or in the discussion of this class of tags.)

(4) Page 13: "Arg in the 2nd position was also associated with proteolysis, yet Arg is virtually absent from proteobacterial ssrA sequences."

The nucleic acid changes required for evolutionary drift from the predominant amino acid codons at this position in proteobacteria to Arg may require moving through several codons that notably impair the performance of the degron. Such a constraint may also be responsible, in part, for the observed conservation.

Reviewer #1 (Public Review):

Summary:

This manuscript addresses the temporal patterns in how cholinergic signaling to the gut affects the lifespan of the worm C. elegans, which should make the manuscript of wide interest to those who study aging, as well as those who study the brain-gut axis in health and disease. The authors show that early acetylcholine (ACh) signaling to the intestine via the ACR-6 receptor shortens worm lifespan, which depends on the DAF-16/FOXO transcription factor. However, later ACh signaling to the intestine via the GAR-3 receptor extends lifespan, which in turn depends on the heat shock factor HSF-1. The authors also show a potential mechanism through which these two temporal patterns of ACh signaling might be coordinated to influence longevity in the worm, and possibly in other animals.

Strengths:

The authors observed that the functional ablation of acr-2-expressing cholinergic neurons in C. elegans (Pacr-2::TeTx) produced a lifespan curve that intersects the lifespan curve of a wild-type population. The first quartile of Pacr-2::TeTx worms shows a longer lifespan than the first quartile of wild-type worms, whereas the last quartile of Pacr-2::TeTx worms exhibits a shorter lifespan than wild-type. These observations raised the hypothesis that cholinergic neurons have two opposing effects on longevity: an early longevity-inhibiting effect and a later longevity-promoting effect. Much of the data supports the authors' conclusions.

Weaknesses:

While the authors have proved their hypothesis by temporally increasing the activity of cholinergic neurons at different life stages through the auxin-inducible degron system, their work raises two major concerns. First, they might want to discuss the conflicting data from Zullo et al (Nature 2019, vol 574, pp 359-364). For example, the authors show that increasing the activity of acr-2-expressing neurons after the 7th day of adulthood increases lifespan. However, Zullo et al (2019) show that the reciprocal experiment, inhibiting cholinergic neuron activity on the 1st day or the 8th day of adulthood, also increases lifespan. Is this because the two studies are using different promoters, that of the acr-2 ACh receptor (this work) versus that of the unc-17 vesicular ACh transporter (Zullo et al., 2019)? The two genes are expressed in different subsets of cells that do not completely overlap. CeNGEN shows that acr-2 is expressed in motor and non-motor neurons, but some of these neurons are also different from those that express unc-17. Is it possible that different cholinergic neurons also have opposite lifespan effects during adulthood? Or is it because both lack of signaling and hypersignaling can lead to a long-life phenotype? Leinwand et al (eLife 2015, vol 4, e10181) previously suggested that disturbing the balance in neurotransmission alone can extend lifespan. A simple discussion of these possibilities in the Discussion section is likely sufficient. Or can the auxin treatment and removal be confounding factors? Loose and Ghazi (Biol Open 2021, vol 10, bio058703) show that auxin IAA alone can affect lifespan and that this effect can depend on the time the animal is exposed to the auxin.

Second, the daf-16-dependence of the early longevity-inhibiting effect of ACh signaling needs clarification and further experimentation. The authors present a model in Figure 6D, where DAF-16 inhibits longevity. This contradicts published literature. Libina et al (Cell 2003, vol 115, pp 489-502) have shown that intestinal DAF-16 increases lifespan. From the authors' data, it is possible that ACh signaling inhibits DAF-16, not promotes it as they have drawn in Figure 6D. In Figure 3F, the authors used Pacr-2::TeTx, which inhibits cholinergic neuron activity, to show an increase in the expression of DAF-16 targets. Why did the authors not use the worms that express the transgene Pacr-2::syntaxin(T254I), which increases cholinergic neuron activity? What happens to the expression of DAF-16 targets in these animals? Do their expression go down? What happens if intestinal daf-16 is knocked down in animals with increased cholinergic neuron activity, instead of reduced cholinergic neuron activity?

Reviewer #2 (Public Review):

Summary:

In the manuscript "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans", Xu and colleagues examine the role of cholinergic signaling by C. elegans motor neurons in modulating lifespan. The authors show that manipulating motor neuronal activity using genetic techniques can be beneficial or detrimental to lifespan, depending on when motor neuron activity is modulated.

Strengths:

A large body of data showing the effects of knockdown of cholinergic receptors and neurotransmitters on lifespan is presented. This would be of value to the community.

Weaknesses:

However, the studies are incomplete. More rigorous approaches would be needed to support the key conclusions, and substantiate the main findings and pathway components.

Reviewer #3 (Public Review):

I very much enjoyed reading Lingxiu Xu et al.'s paper "Temporally controlled nervous system-to-gut signaling bidirectionally regulates longevity in C. elegans," where they investigate the mechanisms by which motor neurons regulate lifespan in C. elegans worms. In this paper, they first discover that interfering with synaptic release in cholinergic motor neurons affects lifespan. Using mutants and gene knockdowns they show that these effects are due to the neurotransmitter acetylcholine. They show that the effects of these motor neurons on lifespan are opposite, depending on timed genetic interventions promoting synaptic release. If these interventions occur during development, the lifespan is shortened, but if they occur starting on day 7 of adulthood, then lifespan is lengthened. They then show that the transcription factor daf-16 is required for the former effect, while the transcription factor hsf-1 is required for the latter one. In addition, these early and late effects, they find, required the acetylcholine receptors acr-6 and gar-3, respectively, and intestinal expression of these genes rescues the respective phenotypes. Interestingly, tagging the endogenous acr-6 and gar-3 genes with mCherry, they find that the ACR-6 and GAR-3 proteins are expressed in the intestine, ACR-6 during development, and GAR-3 during adulthood. Based on these findings they propose a model where acetylcholine from motor neurons regulates lifespan by modulating different receptors expressed at different times. These receptors, in turn, affect lifespan in opposing ways via different transcription factors.

Reviewer #4 (Public Review):

This is a very interesting study, where the authors discovered two neuroendocrine signaling circuits with opposite effects on organismal longevity elicited by motor neurons at different ages.

Interestingly, both systems employ the same neurotransmitter (that is, acetylcholine) and signal the intestine. However, one has effects on early life to shorten lifespan whereas the other system is activated in mid-life to extend lifespan. At the mechanistic level, this bidirectional regulation is possible through the recruitment of two different ACh receptors in the gut: ACR-6 and GAR-3. The authors found that ACR-6 expression in the intestine is restricted to early life, whereas GAR-3 expression in the gut is confined to mid-late life. Interestingly, ACR-6 modulates the transcription factor DAF-16, but GAR-3 regulates HSF-1.

The study combines different approaches, including inducible systems (AID) which are critical for the conclusions of the paper. The conclusions are well supported by the experiments and results. The data provide a potential mechanism for the temporal control of lifespan and shed light on the complex role of the nervous system in organismal aging. These results can have important implications for understanding how organismal aging is regulated in a temporal manner by cell non-autonomous mechanisms. I didn't observe significant weaknesses in the study, but I have several comments that I hope the authors will address.

Reviewer #1 (Public Review):

Summary:

The work provides more evidence of the importance of data quality and representation for ligand-based virtual screening approaches. The authors have applied different machine learning (ML) algorithms and data representation using a new dataset of BRAF ligands. First, the authors evaluate the ML algorithms and demonstrate that independently of the ML algorithm, predictive and robust models can be obtained in this BRAF dataset. Second, the authors investigate how the molecular representations can modify the prediction of the ML algorithm. They found that in this highly curated dataset the different molecule representations are adequate for the ML algorithms since almost all of them obtain high accuracy values, with Estate fingerprints obtaining the worst-performing predictive models and ECFP6 fingerprints producing the best classificatory models. Third, the authors evaluate the performance of the models on subsets of different composition and size of the BRAF dataset. They found that given a finite number of active compounds, increasing the number of inactive compounds worsens the recall and accuracy. Finally, the authors analyze if the use of "less active" molecules affect the model's predictive performance using "less active" molecules taken from ChEMBl Database or using decoys from DUD-E. As results, they found that the accuracy of the model falls as the number of "less active" examples in the training dataset increases while the implementation of decoys in the training set generates results as good as the original models or even better in some cases. However, the use of decoys in the training set worsens the predictive power in the test sets that contain active and inactive molecules.

Strengths:

It is a very interesting topic in medicinal chemistry and drug discovery. This work is very well written and contains up-to-date references. The general structure of the work is adequate, allowing easy reading. The hypotheses are clear and were explored correctly. This work provides new evidence about the importance of inferring models from high-quality data and that, if such a condition is met, it is not necessary to use complex computational methods to obtain predictive models. The generated BRAF dataset is also a valuable benchmark dataset for medicinal chemists working in ligand based virtual screening.

Weaknesses:

Leaving aside the new curated BRAF dataset, the work lacks novelty since it is a topic widely studied in chemoinformatics and medicinal chemistry. Furthermore, the conclusions drawn here correspond to the analysis of only one high-quality dataset where the similarity between the molecules is not quantitatively assessed (maybe active and inactive molecules are very dissimilar and any ML algorithm and fingerprint could obtain good results). To generalize the conclusions, it would be fundamental to repeat the analysis with other high-quality datasets.

Some key tasks are not clearly described, for example, there is no information about the new BRAF dataset (e.g., where the molecules were obtained from or why the inactive molecules provide better results than the "less active" from ChEMBL... what differentiates them?). The defintion of an "inactive" compound is not clear. It is not described if global or balanced accuracy was used in the imbalanced datasets. When using decoys to evaluate the models it is important to consider that decoys were generated to be topologically different from active compounds by the comparison of the ECFP4 fingerprints using the Tanimoto coefficient. Therefore, it is quite obvious that when fingerprints are used to characterize molecules, the models will be able to easily discriminate them. It is important to note that this is not necessarily true for models based on other molecular descriptors, since they are not used in the generation of the decoys. In some cases, the differences between accuracies are very small and there are no statistical analyzes to demonstrate whether they are statistically different or not.

Reviewer #2 (Public Review):

Summary:

The authors explored the importance of data quality and representation for ligand-based virtual screening approaches. I believe the results could be of potential benefit to the drug discovery community, especially to those scientists working in the field of machine learning applied to drug research. The in silico design is comprehensive and adequate for the proposed comparisons.

This manuscript by Chong A. et al describes that it is not necessary to resort to the use of sophisticated deep learning algorithms for virtual screening, since based on their results considering conventional ML may perform exceptionally well if fed by the right data and molecular representations.

The article is interesting and well-written. The overview of the field and the warning about dataset composition are very well thought-out and should be of interest to a broad segment of the AI ​​in drug discovery readership. This article further highlights some of the considerations that need to be taken into consideration for the implementation of data-centric AI for computer-aided drug design methods.

Strengths:

This study contributes significantly to the field of machine learning and data curation in drug discovery. The paper is, in general, well-written and structured. However, in my opinion, there are some suggestions regarding certain aspects of the data analyses.

Weaknesses:

The conclusions drawn in the study are based on the analysis of a single dataset, and I am not sure they can be generalized. Therefore, in my opinion, the conclusions are only partially supported by the data. To generalize the conclusions, it is imperative to conduct a benchmark with diverse datasets, for different molecular targets.<br /> The conclusion cannot be immediately extended to molecular descriptors or features different from the ones used in this study<br /> It is advisable to present statistical analyses to ascertain whether the observed differences in metrics hold statistical significance.

Reviewer #3 (Public Review):

Summary:

The authors presented a data-centric ML approach for virtual ligand screening. They used BRAF as an example to demonstrate the predictive power of their approach.

Strengths:

The performance of predictive models in this study is superior (nearly perfect) with respect to exiting methods.

Weaknesses:

I feel the training and testing datasets may not be rigorously constructed. If that is the case, the results would be significantly affected.

I have 3 major comments:

(1) The authors identified ~4100 BRAF actives, then randomly selected 3600 BRAF actives to be part of the training dataset with the remaining 500 actives becoming a part of the hold-out test set. The problem is that, the authors did not evaluate the chemical similarity between the 3600 actives in the training, and the 500 actives in the testing set. If some of them were similar, the testing results would be very good but partially due to information leakage. The authors should carefully examine the chemical similarity between any pairs of their training and testing datasets, before any conclusion is made.

(2) The authors tried to explore the role of dataset size in the performance, in particular, what would happen when the number of actives are reduced. However the minimal number of actives used is 500 while the number of inactives ranges from 500 to 3600. This is quite different from real applications where the number of expected actives in the screening library would be at most 1-2% of the whole database. The authors should further reduced the number of actives (e.g. 125, 25, 5, 1), and evaluate their model's performance.

(3) The authors chose BRAF as example in this study. BRAF is a well studied drug target with thousands of known actives. In real applications, the target may only have a handful of known actives. The authors should try to apply their approach, to a couple other targets that have less known actives than BRAF, to evaluate their method's transferability.

Reviewer #1 (Public Review):

Using a knock-out mutant strain, the authors tried to decipher the role of the last gene in the mycofactocin operon, mftG. They found that MftG was essential for growth in the presence of ethanol as the sole carbon source, but not for the metabolism of ethanol, evidenced by the equal production of acetaldehyde in the mutant and wild type strains when grown with ethanol (Fig 3). The phenotypic characterization of ΔmftG cells revealed a growth-arrest phenotype in ethanol, reminiscent of starvation conditions (Fig 4). Investigation of cofactor metabolism revealed that MftG was not required to maintain redox balance via NADH/NAD+, but was important for energy production (ATP) in ethanol. Since mycobacteria cannot grow via substrate-level phosphorylation alone, this pointed to a role of MftG in respiration during ethanol metabolism. The accumulation of reduced mycofactocin points to impaired cofactor cycling in the absence of MftG, which would impact the availability of reducing equivalents to feed into the electron transport chain for respiration (Fig 5). This was confirmed when looking at oxygen consumption in membrane preparations from the mutant and would type strains with reduced mycofactocin electron donors (Fig 7). The transcriptional analysis supported the starvation phenotype, as well as perturbations in energy metabolism, and may be beneficial if described prior to respiratory activity data.

While the data and conclusions do support the role of MftG in ethanol metabolism, the title of the publication may be misleading as the mutant was able to grow in the presence of other alcohols (Supp Fig S2). Furthermore, the authors propose that MftG could not be involved in acetate assimilation based on the detection of acetate in the supernatant and the ability to grow in the presence of acetate. The minimal amount of acetate detected in the supernatant but a comparative amount of acetaldehyde could point to disruption of an aldehyde dehydrogenase.

The link between mycofactocin oxidation and respiration is shown, however the mutant has an intact respiratory chain in the presence of ethanol (oxygen consumption with NADH and succinate in Fig 7C) and the NADH/NAD+ ratios are comparable to growth in glucose. Could the lack of growth of the mutant in ethanol be linked to factors other than respiration? To this end, bioinformatic investigation or other evidence to identify the membrane-bound respiratory partner would strengthen the conclusions.

Reviewer #2 (Public Review):

Summary

Patrícia Graça et al., examined the role of the putative oxidoreductase MftG in regeneration of redox cofactors from the mycofactocin family in Mycolicibacerium smegmatis. The authors show that the mftG is often co-encoded with genes from the mycofactocin synthesis pathway in M. smegmatis genomes. Using a mftG deletion mutant, the authors show that mftG is critical for growth when ethanol is the only available carbon source, and this phenotype can be complemented in trans. The authors demonstrate the ethanol associated growth defect is not due to ethanol induced cell death, but is likely a result of carbon starvation, which was supported by multiple lines of evidence (imaging, transcriptomics, ATP/ADP measurement and respirometry using whole cells and cell membranes). The authors next used LC-MS to show that the mftG deletion mutant has much lower oxidised mycofactocin (MFFT-8 vs MMFT-8H2) compared to WT, suggesting an impaired ability to regenerate myofactocin redox cofactors during ethanol metabolism. These striking results were further supported by mycofactocin oxidation assays after over-expression of MftG in the native host, but also with recombinantly produced partially purified MftG from E. coli. The results showed that MftG is able to partially oxidise mycofactocin species, finally respirometry measurements with M. smegmatis membrane preparations from WT and mftG mutant cells show that the activity of MftG is indispensable for coupling of mycofactocin electron transfer to the respiratory chain. Overall, I find this study to be comprehensive and the conclusions of the paper are well supported by multiple complementary lines of evidence that are clearly presented.

Strengths

The major strengths of the paper are that it is clearly written and presented and contains multiple, complementary lines of experimental evidence that support the hypothesis that MftG is involved in the regeneration of mycofactocin cofactors, and assists with coupling of electrons derived from ethanol metabolism to the aerobic respiratory chain. The data appear to support the the authors hypotheses.

Weaknesses

No major weaknesses were identified, only minor weaknesses mostly surrounding presentation of data in some figures.

Reviewer #1 (Public Review):

Summary:

The authors set out to measure the diffusion of small drug molecules inside live cells. To do this, they selected a range of flourescent drugs, as well as some commonly used dyes, and used FRAP to quantify their diffusion. The authors find that drugs diffuse and localize within the cell in a way that is weakly correalted with their charge, with positively charged molecules displaying dramatically slower diffusion and a high degree of subcellular localization.<br /> The study is important because it points at an important issue related to the way drugs behave inside cells beyond the simple "IC50" metric (a decidedly mesoscopic/systemic value). The authors conclude, and I agree, that their results point to nuanced effects that are governed by drug chemistry that could be optimized to make them more effective.

Strengths:

The work examines an understudied aspect of drug delivery.<br /> The work uses well-established methodologies to measure diffusion in cells<br /> The work provides an extensive dataset, covering a range of chemistries that are common in small molecule drug design<br /> The authors consider several explanations as to the origin of changes in cellular diffusion

Weaknesses:

The results are described qualitatively, despite quantitative data that can be used to infer the strength of the proposed correlations.<br /> The statistical treatment of the data is not rigorous and not visualized according to best practices, making it difficult for readers to assess the significance of the findings.<br /> Some important aspects of drug behavior are not discussed quantitatively, such as the cell-to-cell or subcellular variability in concentration.<br /> It is unclear if the observed behavior of each drug in the cell actually relates to its efficacy - though this is clearly beyond the scope of this specific work.

Reviewer #2 (Public Review):

Summary: Blocking a weak base compound's protonation increased intracellular diffusion and fractional recovery in the cytoplasm, which may improve the intracellular availability and distribution of weakly basic, small molecule drugs and be impactful in future drug development.

Strengths:

1) The intracellular distribution of drugs and the chemical properties that drive their distribution are much needed in the literature. Thus, the idea behind this paper is of relevance.

2) The study used common compounds that were relevant to others.

3) Altering a compound's pKa value and measuring cytosolic diffusion rates certainly is inciteful on how weak base drugs and their relatively high pKa values affect distribution and pharmacokinetics. This particular experiment demonstrated relevance to drug targeting and drug development.

4) The manuscript was fairly well written.

Weaknesses:

1) Small sample sizes. 2 acids and 1 neutral compound vs 6 weak bases (Figure 1).

2) A comparison between the percentage of neutral and weak base drug accumulation in lysosomes would have helped indicate weak base ion trapping. Such a comparison would have strengthened this study.

3) When cytosolic diffusion rates of compounds were measured, were the lysosomes extracted from the image using Imaris to determine a realistic cytosolic value? In real-time, lysosomes move through the cytosol at different rates. Because weak base drugs get trapped, it is likely the movement of a weak base in the lysosome being measured rather than the movement of a weak base itself throughout the cytosol. This was unclear in the methods. Please explain.

4) Because weak base drugs can be protonated in the cytoplasm, the authors need to elaborate on why they thought that inhibiting lysosome accumulation of weak bases would increase cytosolic diffusion rates. Ion trapping is different than "micrometers per second" in the cytosol. Moreover, treating cells with sodium azide de-acidifies lysosomes and acidifies the cytosol; thus, more protons in the cytosol means more protonation of weak base drugs. The diffusion rates were slowed down in the presence of lysosome inhibition (Figure 7), which is more fitting of the story about blocking protonation increases diffusion rates, but in this case, increasing cytosolic protonation via lysosome de-acidification agents decreases diffusion rates. Please elaborate.

A discussion of the likely impact:<br /> The manuscript certainly adds another dimension to the field of intracellular drug distribution, but the manuscript needs to be strengthened in its current form. Additional experiments need to be included, and there are clarifications in the manuscript that need to be addressed. Once these issues are resolved, then the manuscript, if the conclusions are further strengthened, is much needed and would be inciteful to drug development.

Reviewer #2 (Public Review):

In the manuscript by Maio et al, the authors examined the bioenergetic mechanisms involved in the delayed migration of DC's during Mtb infection. The authors performed a series of in vitro infection experiments including bioenergetic experiments using the Agilent Seahorse XF, and glucose uptake and lactate production experiments. Also, data from SCENITH is included in the revised manuscript as well as some clinical data. This is a well written manuscript and addresses an important question in the TB field.

Reviewer #1 (Public Review):

Summary

The authors use an elegant but somewhat artificial heterodimerisation approach to activate the isolated cytoplasmic domains of different receptor kinases (RKs) including the receptor kinase BRI1 and EFR. The developmental RK BRI1 is known to be activated by the co-receptor BAK1. Active BRI1 is then able to phosphorylate downstream substrates. The immune receptor EFR is also an active protein kinase also activated by the co-receptor BAK1. EFR however appears to have little or no kinase activity but seems to use an allosteric mechanism to in turn enable BAK1 to phosphorylate the substrate kinase BIK1. EFR tyrosine phosphorylation by BAK1 appears to trigger a conformational change in EFR, activating the receptor. Likewise, kinase activating mutations can cause similar conformational transitions in EFR and also in BAK1 in vitro and in planta.

Strengths:

I particularly liked The HDX experiments coupled with mutational analysis (Fig. 2) and the design and testing of the kinase activating mutations (Fig. 3), as they provide novel mechanistic insights into the activation mechanisms of EFR and of BAK1. These findings are nicely extended by the large-scale identification of EFR-related RKs from different species with potentially similar activation mechanisms (Fig. 5).

Overall this is an interesting study that aims to advance our understanding of the activation mechanisms of different plant receptor kinases with important functions in plant immunity.

Reviewer #2 (Public Review):

Summary:

Transmembrane signaling in plants is crucial for homeostasis. In this study, the authors set out to understand to what extent catalytic activity in the EFR tyrosine kinase is required in order to transmit a signal. This work was driven by mounting data that suggest many eukaryotic kinases do not rely on catalysis for signal transduction, relying instead on conformational switching to relay information. The crucial findings reported here involve the realisation that a kinase-inactive EFR can still activate (ie lead to downstream phosphorylation) of its partner protein BAK1. Using a convincing set of biochemical, mass spectrometric (HD-exchange) and in vivo assays, the team suggests a model in which EFR is likely phosphorylated in the canonical activation segment (where two Ser residues are present), which is sufficient to generate a conformation that can activate BAK1 through dimersation. A model is put forward involving C-helix positioning in BAK1, and the model is extended to other 'non-RD' kinases in Arabidopsis kinases that likely do not require kinase activity for signaling.

Strengths:

The work uses logical and well-controlled approaches throughout, and is clear and convincing in most areas, linking data from IPs, kinase assays (including clear 32P-based biochemistry), HD-MX data (from non-phosphorylated EFR) structural biology, oxidative burst data and infectivity assays. Repetitions and statistical analysis all appear appropriate.

Overall, the work builds a convincing story and the discussion does a clear job of explaining the potential impact of these findings (and perhaps an explanation of why so many Arabidopsis kinases are 'pseudokinases', including XPS1 and XIIa6, where this is shown explicitly).

Impact:

The work is an important new step in the huge amount of follow-up work needed to examine how kinases and pseudokinases 'talk' to each other in (especially) the plant kingdom, where significant genetic expansions have occurred. The broader impact is that we might understand better how to manipulate signaling for the benefit of plants and mankind; as the authors suggest, their study is a natural progression both of their own work and the kingdom-wide study of the Kannan group.

Reviewer #3 (Public Review):

The study presents strong evidence for allosteric activation of plant receptor kinases, which enhances our understanding of the non-catalytic mechanisms employed by this large family of receptors.

Plant receptor kinases (RKs) play a critical role in transducing extracellular signals. The activation of RKs involves homo- or heterodimerization of the RKs, and it is believed that mutual phosphorylation of their intracellular kinase domains initiates downstream signaling. However, this model faces a challenge in cases where the kinase domain exhibits pseudokinase characteristics. In their recent study, Mühlenbeck et al. reveal the non-catalytic activation mechanisms of the EFR-BAK1 complex in plant receptor kinase signaling. Specifically, they aimed to determine that the EFR kinase domain activates BAK1 not through its kinase activity, but rather by utilizing a "conformational toggle" mechanism to enter an active-like state, enabling allosteric trans-activation of BAK1. The study sought to elucidate the structural elements and mutations of EFR that affect this conformational switch, as well as explore the implications for immune signaling in plants. To investigate the activation mechanisms of the EFR-BAK1 complex, the research team employed a combination of mutational analysis, structural studies, and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. For instance, through HDX-MS analysis, Mühlenbeck et al. discovered that the EFR (Y836F) mutation impairs the accessibility of the active-like conformation. On the other hand, they identified the EFR (F761H) mutation as a potent intragenic suppressor capable of stabilizing the active-like conformation, highlighting the pivotal role of allosteric regulation in BAK1 kinase activation. The data obtained from this methodology strengthens their major conclusion. Moreover, the researchers propose that the allosteric activation mechanism may extend beyond the EFR-BAK1 complex, as it may also be partially conserved in the Arabidopsis LRR-RK XIIa kinases. This suggests a broader role for non-catalytic mechanisms in plant RK signaling.

The allosteric activation mechanism was demonstrated for receptor tyrosine kinases (RTKs) many years ago. A similar mechanism has been suggested for the activation of plant RKs, but experimental evidence for this conclusion is lacking. Data in this study represent a significant advancement in our understanding of non-catalytic mechanisms in plant RK signaling. By shedding light on the allosteric regulation of BAK1, the study provides a new paradigm for future research in this area.

Reviewer #1 (Public Review):

Summary:

The manuscript by Jiayun Li and colleagues aims to provide insight into adipokinetic hormone signaling that mediates the fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Using FISH, qRT-PCR, WB, RNAi, and miRNA-related methods, authors found that knockdown of DcAKH and DcAKHR not only resulted in triacylglycerol accumulation and a decline of glycogen but also significantly decreased fecundity and CLas titer in ovaries. miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes.

Reviewer #2 (Public Review):

Diaphorina citri is the primary vector of Candidatus Liberibacter asiaticus (CLas), but the mechanism of how D. citri maintains a balance between lipid metabolism and increased fecundity after infection with CLas remains unknown. In their study, Li et al. presented convincing methodology and data to demonstrate that CLas exploits AKH/AKHR-miR-34-JH signaling to enhance D. citri lipid metabolism and fecundity, while simultaneously promoting CLas replication. These findings are both novel and valuable, not only have theoretical implications for expanding our understanding of the interaction between insect vectors and pathogenic microorganisms but also provide new targets for controlling D. citri and HLB in practical implications. The conclusions of this paper are well supported by data.

Reviewer #1 (Public Review):

Here, using an organoid system, Wong et al aimed to establish new models of hereditary diffuse leukoencephalopathy with axonal spheroids (HDLS), with which they wanted to understand how CSF1R-mutaions affect the phenotypes of microglia/macrophages. They found metabolic changes in microglia/macrophages with mutations, which were associated with a proinflammatory phenotype. In general, the authors tackle important issues and provide valuable tools to investigate the underlying mechanisms for HDLS.

Strength:

The authors establish two HDLS patient-derived iPS cells with their isogeneic controls and provide possible mechanistic insights into the disease mechanisms.

Weakness:

It is unclear how nicely the organoid system in this study can recapitulate the condition in patients with HDLS (e.g. reduced microglia density, downregulated expression of P2YR12, pathological alterations).

The authors generated two different models with distinct mutations that produce different readouts in CSF1R-mediated cellular responses. It is unclear if the different outcomes between HD1 and HD2 are generated simply through different mutations or due to different differentiation efficiency from iMacs.

Suggestions:

(1) This paper would benefit from additional histological analyses to characterize iMac & iMicro at least histologically, which would be helpful for readers to know how nicely the organoid system recapitulates the condition in patients with HDLS.

(2) In addition, in Fig.5E-J the authors could highlight microglia core genes that would be upregulated if iMacs are successfully differentiated into iMicro.

(3) Since there are no direct evidence to confirm that "microglial dysregulation and IL1b signalling contribute to the degenerative neuro-environment in HDLS", the authors should tone down their argument and rephrase the Abstract.

Reviewer #2 (Public Review):

Summary:

This paper investigates a rare and severe brain disease called Hereditary Diffuse Leukoencephalopathy with Axonal Spheroids (HDLS). The authors aimed to understand how mutations in the gene CSF-1R affect microglia, the resident immune cells in the brain, and which alterations and factors lead to the specific pathophysiology. To model the human brain with the pathophysiology of HDLS, they used the human-specific model system of induced pluripotent stem cell (iPSC)-derived forebrain organoids with integrated iPSC-derived microglia (iMicro) from patients with the HDLS-causing mutation and an isogenic cell line with the corrected genome. They found that iPSC-derived macrophages (iMac) with HDLS mutations showed changes in their response, including increased inflammation and altered metabolism. Additionally, they studied these iMacs in forebrain organoids, where they differentiate into iMicro, and showed transcriptional differences in isolated iMicro when carrying the HDLS mutation. In addition, the authors described the influence of the mutation within iMicro on the transcriptional level of neurons and neural progenitor cells (NPCs) in the organoid. They observed that the one mutation showed implications for impaired development of neurons, possibly contributing to the progression of the disease. Overall, this study provides valuable insights into the mechanisms underlying HDLS and emphasizes the importance of studying diseases like these with a suitable model system. These findings, while promising, represent only an initial step towards understanding HDLS and similar neurodegenerative diseases, and thus, their direct translation into new treatment options remains uncertain.

Strengths:

The strength of the work lies in the successful reprogramming of two HDLS patient-derived induced pluripotent stem cells (iPSCs) with different mutations, which is crucial for the study of HDLS using human forebrain organoid models. The use of corrected isogenic iPSC lines as controls increases the validity of the mutation-specific observations. In addition, the model effectively mimics HDLS, particularly concerning deficits in the frontal lobe, mirroring observations in the human brain. Obtaining iPSCs from patients with different CSF1R mutations is particularly valuable given the limitations of rodent and zebrafish models when studying adult-onset neurodegenerative diseases. The study also highlights significant metabolic changes associated with the CSF1R mutation, particularly in the HD2 mutant line, which is confirmed by the HD1 line. In addition, the work shows transcriptional upregulation of the proinflammatory cytokine IL-1beta in cells carrying the mutation, particularly when they phagocytose apoptotic cells, providing further insight into disease mechanisms.

Weaknesses:

Most of the points have been addressed in the revision, but some points remain (see below) and are well within the scope of the current manuscript in this reviewer's opinion.

(1) The characterization of iMicros is incomplete, with limited protein-level analysis (e.g. validate RNA-seq data via flow cytometry, ELISA etc.).

(2) Additionally, the claim of microglial-like morphology lacks adequate evidence, as the provided image is insufficient for such an assessment (also the newly provided Supp. Fig. 3C is insufficient and looks rather like background). Show single channels for each staining. Show examples for both cell lines.

(3) RNA-seq experiments are still difficult to read. A combination of data from both lines into one big analysis would be advantageous. E.g. showing overlapping GO terms for both lines. What is common, what is different in both lines?

(4) Statistical test information is missing in the legends.

Reviewer #1 (Public Review):

Summary:

Shi and colleagues report the use of modified Cre lines in which the coding region of Cre is disrupted by rox-STOP-rox or lox-STOP-lox sequences to prevent the expression of functional protein in the absence of Dre or Cre activity, respectively. The main purpose of these tools is to enable intersectional or tamoxifen-induced Cre activity with minimal or no leaky activity from the second, Cre-expressing allele. It is a nice study but lacks some functional data required to determine how useful these alleles will be in practice, especially in comparison with the figure line that stimulated their creation.

Strengths:

The new tools can reduce Cre leak in vivo.

Weaknesses:

(1) Activity of R26-loxCre line. As the authors point out, the greatest value of this approach is to accomplish a more complete Cre-mediated gene deletion using CreER transgenes that are combined with low-efficiency floxed alleles using their R26-loxCre line that is similar to the iSure Cre reported by Benedito and colleagues. The data in Figure 5 show strong activity at the Confetti locus, but the design of the newly reported R26-loxCre line lacks a WPRE sequence that was included in the iSure-Cre line to drive very robust protein expression. Thus while the line appears to have minimal leak, as the design would predict, the question of how much of a deletion increase is obtained over simple use of the CreER transgene alone is a key question for use by investigators. This is further addressed in Figure 6 where it is compared with Alb-CreER alone to recombine the Ctnnb1 floxed allele. They demonstrate that recombination frequency is clearly improved, but the western blot in Figure 6E does not look like there was a large amount of remaining b-catenin to remove. These data are certainly promising, but the most valuable experiment for such a new tool would be a head-to-head comparison with iSure (or the latest iSure version from the Benedito lab) using the same CreER and target floxed allele. At the very least a comparision of Cre protein expression between the two lines using identical CreER activators is needed.

(2) In vivo analysis of mCre activities. Why did the authors not use the same driver to compare mCre 1, 4, 7, and 10? The study in Figure 2 uses Alb-roxCre for 1 and 7 and Cdh5-roxCre for 4 and 10, with clearly different levels of activity driven by the two alleles in vivo. Thus whether mCre1 is really better than mCre4 or 10 is not clear.

(3) Technical details are lacking. The authors provide little specific information regarding the precise way that the new alleles were generated, i.e. exactly what nucleotide sites were used and what the sequence of the introduced transgenes is. Such valuable information must be gleaned from schematic diagrams that are insufficient to fully explain the approach.

Reviewer #2 (Public Review):

Summary:

This work presents new genetic tools for enhanced Cre-mediated gene deletion and genetic lineage tracing. The authors optimise and generate mouse models that convert temporally controlled CreER or DreER activity to constitutive Cre expression, coupled with the expression of tdT reporter for the visualizing and tracing of gene-deleted cells. This was achieved by inserting a stop cassette into the coding region of Cre, splitting it into N- and C-terminal segments. Removal of the stop cassette by Cre-lox or Dre-rox recombination results in the generation of modified Cre that is shown to exhibit similar activity to native Cre. The authors further demonstrate efficient gene knockout in cells marked by the reporter using these tools, including intersectional genetic targeting of pericentral hepatocytes.

Strengths:

The new models offer several important advantages. They enable tightly controlled and highly effective genetic deletion of even alleles that are difficult to recombine. By coupling Cre expression to reporter expression, these models reliably report Cre-expressing i.e. gene-targeted cells, and circumvent false positives that can complicate analyses in genetic mutants relying on separate reporter alleles. Moreover, the combinatorial use of Dre/Cre permits intersectional genetic targeting, allowing for more precise fate mapping.

Weaknesses:

The scenario where the lines would demonstrate their full potential compared to existing models has not been tested. Mosaic genetics is increasingly recognized as a key methodology for assessing cell-autonomous gene functions. The challenge lies in performing such experiments, as low doses of tamoxifen needed for inducing mosaic gene deletion may not be sufficient to efficiently recombine multiple alleles in individual cells while at the same time accurately reporting gene deletion. Therefore, a demonstration of the efficient deletion of multiple floxed alleles in a mosaic fashion would be a valuable addition.

In addition, a drawback of this line is the constitutive expression of Cre. When combined with the confetti line, the reporter cassette will continue flipping, potentially leading to misleading lineage tracing results. Constitutive expression of Cre is also associated with toxicity, as discussed by the authors in the introduction. These drawbacks should be acknowledged.

Reviewer #3 (Public Review):

Summary:

The authors report a new version of the iSuRe-Cre approach, which was originally developed by Rui Benedito's group in Spain (https://doi.org/10.1038/s41467-019-10239-4). Shi et al claim that their approach shows reduced leakiness compared to the iSuRe-Cre line. Shi et al elaborate strongly about the leakiness of iSuRe-Cre mice, although leakiness is rather minor according to the original publication and the senior author of the study wrote in a review a few years ago that there is no leakiness (https://doi.org/10.1016/j.jbc.2021.100509). Furthermore, a new R26-roxCre-tdT mouse line was established after extensive testing, which enables efficient expression of the Cre recombinase after activation of the Dre recombinase.

Strengths:

The authors carefully evaluated the efficiency and leakiness of the new strains and demonstrated the applicability by marking peri-central hepatocytes in an intersectional genetics approach, amongst others. I can only find very few weaknesses in the paper, which represents the result of an enormous effort. Carefully conducted technical studies have considerable value. However, I would have preferred to see a study, which uses the wonderful new tools to address a major biological question, rather than a primarily technical report, which describes the ongoing efforts to further improve Cre and Dre recombinase-mediated recombination.

Weaknesses:

Very high levels of Cre expression may cause toxic effects as previously reported for the hearts of Myh6-Cre mice. Thus, it seems sensible to test for unspecific toxic effects, which may be done by bulk RNA-seq analysis, cell viability, and cell proliferation assays. It should also be analyzed whether the combination of R26-roxCre-tdT with the Tnni3-Dre allele causes cardiac dysfunction, although such dysfunctions should be apparent from potential changes in gene expression.

The R26-GFP or R26-tdT reporters, Alb-roxCre1-tdT, Cdh5-roxCre4-tdT, Alb-roxCre7-GFP, and Cdh5-roxCre10-GFP demonstrate no leakiness without Dre-rox recombination (Figure S1-S2). Is there any leakiness when the inducible DreER allele is introduced but no tamoxifen treatment is applied? This should be documented. The same also applies to loxCre mice.

The enhanced efficiency of loxCre and roxCre systems holds promise for reducing the necessary tamoxifen dosage, potentially reducing toxicity and side effects. In Figure 6, the author demonstrates an enhanced recombination efficiency of loxCre mice, which makes it possible to achieve efficient deletion of Ctnnb1 with a single dose of tamoxifen, whereas a conventional driver (Alb-CreER) requires five dosages. It would be very helpful to include a dose-response curve for determining the minimum dosage required in Alb-CreER; R26-loxCre-tdT; Ctnnb1flox/flox mice for efficient recombination.

In the liver panel of Figure 4F, tdT signals do not seem to colocalize with the VE-cad signals, which is odd. Is there any compelling explanation?

The authors claim that "virtually all tdT+ endothelial cells simultaneously expressed YFP/mCFP" (right panel of Figure 5D). Well, it seems that the abundance of tdT is much lower compared to YFP/mCFP. If the recombination of R26-Confetti was mainly triggered by R26-loxCre-tdT, the expression of tdT and YFP/mCFP should be comparable. This should be clarified.

In several cases, the authors seem to have mixed up "R26-roxCre-tdT" with "R26-loxCre-tdT". There are errors in #251 and #256. Furthermore, in the passage from line #278 to #301. In the lines #297 and #300 it should probably read "Alb-CreER; R26-loxCre-tdT;Ctnnb1flox/flox"" rather than "Alb-CreER;R26-tdT2;Ctnnb1flox/flox".

Reviewer #1 (Public Review):

Summary:

This manuscript explores the importance of food type on virus infection dynamics using a nematode virus as a model system. The authors demonstrate that susceptibility to viral infection can change by several orders of magnitude based on the type of bacterial food that potential hosts consume. They go on to show that, for the bacterial food source that reduces susceptibility, the effect is modulated by quorum sensing molecules that the bacteria produce.

Strengths:

This manuscript shows convincingly that nematode susceptibility to viral infection changes by several orders of magnitude (i.e. doses must be increased by several orders of magnitude to infect the same fraction of the population) depending on the bacterial food source on which hosts are reared. The authors then focus on the bacteria that reduce host susceptibility to viral infection and demonstrate that certain bacterial quorum-sensing compounds are required to see this effect of reduced susceptibility. Overall, sample sizes are large, methods are generally rigorous, experiments are repeated, and patterns are clear.

Comments on revised version:

The authors have now addressed all of my previous concerns.

Reviewer #2 (Public Review):

In this study, the authors investigate how diverse bacterial species influence Orsay virus transmission and host susceptibility in C. elegans. They find that Ochrobactrum species increase infection rates, while Pseudomonas species decrease infection rates, and they identify regulators of quorum sensing and the gacA two-component system as genetic factors in the effects of Pseudomonas on infection. These findings provide important insights into the species-specific effects that bacteria can have on viral infection in C. elegans, and they may have relevance for the impact of bacterial species on viral infection in other systems. Overall the manuscript has high rigor. However, a few minor concerns are listed below.

(1) The authors state that the amount of bacteria added to each plate was standardized by seeding plates with equivalent volumes of overnight culture. This approach does not account for differences in bacterial growth rate. A more rigorous approach would be to standardize based on OD600 measurements or CFU's. Alternatively, the authors could include bacterial growth curves to demonstrate that each strain/species has reached a similar growth phase (i.e. late log) at the time of plating, as bacterial physiology and virulence is dependent on the stage of growth. At the least, if it is not possible to perform these experiments, it would be useful to include a statement that potential differences in bacterial growth rate may influence their conclusions.

(2) Line 314-315: The claim "We did not observe any potent effect on host susceptibility to infection by Orsay virus from any supernatant (Supp. Fig. 9)" is not fully supported by the data, as the data in Fig S9 only show pals-5p::GFP levels. To confirm that host susceptibility is not affected, the authors would also measure the viral infection rate and/or viral load. Otherwise, the authors should rephrase the conclusion to increase accuracy. For example, "We did not observe any potent effect on pals-5p::GFP activation upon Orsay virus infection when animals were exposed to bacterial culture supernatant".

(3) The Ct values shown in Fig 3B-F should be normalized to a reference gene (i.e. Ct values for snb-1).

Reviewer #1 (Public Review):

The manuscript addresses a fundamental question about how different types of communication signals differentially affect brain state and neurochemistry. In addition, their manuscript highlights the various processes that modulate brain responses to communication signals, including prior experience, sex, and hormonal status. Overall, the manuscript is well-written and the research is appropriately contextualized.

Reviewer #3 (Public Review):

The work by Ghasemahmad et al. has the potential to significantly advance our understanding of how neuromodulators provide internal-state signals to the basolateral amygdala (BLA) while an animal listens to social vocalizations.

Ghasemahmad et al. made changes to the manuscript that have significantly improved the work. In particular, the transparency in showing the underlying levels of Ach, DA, and 5HIAA is excellent. My previous concerns have been adequately addressed.

Reviewer #1 (Public Review):

Summary:

van der Heijden et al perform an ambitious analysis of single unit activity in the interposed nuclei of multiple mouse models of cerebellar dysfunction. Based on these recordings, they develop a classifier to predict the behavioral phenotype (ataxic, dystonic, or tremor) of each model, suggesting that highly regular spiking is associated with ataxia, irregular spiking is associated with dystonia, and rhythmic spiking is associated with tremor. Interestingly, the "dystonic" and "tremor" patterns appeared to be specific to those disorders, while ataxia could result from at least two different interposed nucleus firing patterns. After developing this classifier, they show that activating Purkinje neurons in different patterns that evoke interposed nuclear activity similar to their "ataxic", "dystonic", and "tremor" firing patterns induce similar behaviors in healthy mice. These results show convincingly that specific patterns of cerebellar output are sufficient to cause specific movement abnormalities. The extent to which cerebellar nuclear firing patterns are solely responsible for phenotypes in human disease remains to be established, however.

Strengths:

Major strengths are the recordings across multiple phenotypic models including genetic and pharmacologic manipulations, and the robust phenotypes elicited by Purkinje neuron stimulation.

Weaknesses:

The number of units recorded was small for each model (on the order of 20), limiting conclusions that can be drawn from the recording/classifier experiments.

Reviewer #2 (Public Review):

Cerebellar diseases can manifest as various behavioral phenotypes, such as ataxia, dystonia, and tremor. In this study, van der Heijden and colleagues aim to understand whether these differing behavioral phenotypes are associated with disease-specific changes in the firing patterns of cerebellar output neurons in the cerebellar nuclei (CN). The authors effectively demonstrate that across different mouse models of cerebellar disease, there are distinct changes in the firing properties of CN neurons. They take a crucial step further by attempting to replicate disease-specific firing patterns in the cerebellar output neurons of healthy (control) mice using optogenetics. When Purkinje cells are stimulated in a manner that results in similar firing properties in CN neurons, the authors observe a variety of atypical behavioral responses, many of which align with the behavioral phenotypes observed in mouse models of the respective diseases.

Overall, the primary results are quite convincing. Specifically, they show that (1) different mouse models of cerebellar disease exhibit different statistics of firing in CN neurons, and (2) driving CN neurons in a time-varying manner that mimics the statistics measured in disease models results in behavioral phenomena reminiscent of the disease states. These findings suggest that aberrant activity in the CN can originate from various sources (e.g., developmental circuit deficits, abnormal plasticity, insult), but ultimately, these changes are funneled through the CN neurons, whose firing rates are affected, and this, in turn, drives some portion of the aberrant behavior. This is a noteworthy observation that underscores the potential of targeting these output neurons in the treatment of cerebellar disease. Moreover, this manuscript provides valuable insights into the firing patterns associated with the most common cerebellar-dependent disease phenotypes.

However, the applicability of the classifier for identifying mice cerebellar behavioral phenotypes directly from the spiking activity of neurons in the cerebellar nuclei remains this paper's weak point. Cross-validated performance of the model on a single mouse model of tremor is, for instance, only 54%. However, a benefit of this classifier is its overall simplicity; only three parameters are required to achieve average classifier performance of 76%. While more sophisticated models might provide improved classifier performance and enhanced generalization, such models would suffer from a lack of interpretability. This paper, therefore, represents a reasonable starting point for understanding the parameter space of cerebellar nuclei firing and its relationship to behavioral phenotypes during disease.

Reviewer #3 (Public Review):

Summary:

This manuscript looks at the single-cell spike signatures taken from in vivo cerebellar nuclear neurons from awake mice suffering from 3 distinct diseases and uses a sophisticated classifier model to predict disease based on a number of different parameters about the spiking patterns, rather than just one or two. Single read-outs of spike firing patterns did not show significant differences between all 4 groups meaning that you need to analyze multiple parameters of the spike trains to get this information. The results are really satisfying and intriguing, with some diseases separating very well, and others having more overlap. It also represents a significant advancement for the rigor and creativity used for analyzing cerebellar output spike patterns. I really like this paper, it's a clever idea and has been done very well.

The authors examine multiple distinct forms of different diseases, including different types of ataxia, dystonia, and tremor. While some of the interpretation of this work remains unclear to this reviewer (in particular Fig. 2, with ataxia models), I applaud the rigor, and sharing complex data that is not always straightforward to understand.

Strengths:

The work is technically impressive and the analysis pushes the envelope of how cerebellar dysfunction is classified, which makes it an important paper for the field.<br /> It's well written. The approach it is taking is clever. The analysis is thorough, and the authors examine a wide array of different disease models, which is time-consuming, costly, and very challenging to do. It's a very strong manuscript.

Reviewer #1 (Public Review):

Summary:

The work by Combrisson and colleagues investigates the degree to which reward and punishment learning signals overlap in the human brain using intracranial EEG recordings. The authors used information theory approaches to show that local field potential signals in the anterior insula and the three sub regions of the prefrontal cortex encode both reward and punishment prediction errors, albeit to different degrees. Specifically, the authors found that all four regions have electrodes that can selectively encode either the reward or the punishment prediction errors. Additionally, the authors analyzed the neural dynamics across pairs of brain regions and found that the anterior insula to dorsolateral prefrontal cortex neural interactions were specific for punishment prediction errors whereas the ventromedial prefrontal cortex to lateral orbitofrontal cortex interactions were specific to reward prediction errors. This work contributes to the ongoing efforts in both systems neuroscience and learning theory by demonstrating how two differing behavioral signals can be differentiated to a greater extent by analyzing neural interactions between regions as opposed to studying neural signals within one region.

Strengths:

The experimental paradigm incorporates both a reward and punishment component that enables investigating both types of learning in the same group of subjects allowing direct comparisons.

The use of intracranial EEG signals provides much needed insight into the timing of when reward and punishment prediction errors signals emerge in the studied brain regions.

Information theory methods provide important insight into the interregional dynamics associated with reward and punishment learning and allows the authors to assess that reward versus punishment learning can be better dissociated based on interregional dynamics over local activity alone.

Weaknesses:

The analysis presented in the manuscript focuses on gamma band activity. Studying slow oscillations could provide additional insights into the interregional dynamics.

Reviewer #2 (Public Review):

Reward and punishment learning have long been seen as emerging from separate networks of frontal and subcortical areas, often studied separately. Nevertheless, both systems are complimentary and distributed representations of reward and punishments have been repeatedly observed within multiple areas. This raised the unsolved question of the possible mechanisms by which both systems might interact, which this manuscript went after. The authors skillfully leveraged intracranial recordings in epileptic patients performing a probabilistic learning task combined with model-based information theoretical analyses of gamma activities to reveal that information about reward and punishment was not only distributed across multiple prefrontal and insular regions, but that each system showed specific redundant interactions. The reward subsystem was characterized by redundant interactions between orbitofrontal and ventromedial prefrontal cortex, while the punishment subsystem relied on insular and dorsolateral redundant interactions. Finally, the authors revealed a way by which the two systems might interact, through synergistic interaction between ventromedial and dorsolateral prefrontal cortex.

Here, the authors performed an excellent reanalysis of a unique dataset using innovative approaches, pushing our understanding on the interaction at play between prefrontal and insular cortex regions during learning. Importantly, the description of the methods and results is truly made accessible, making it an excellent resource to the community. The authors also carefully report individual subjects' data, which brings confidence in the reproducibility of their observations.

This manuscript goes beyond what is classically performed using intracranial EEG dataset, by not only reporting where a given information, like reward and punishment prediction errors, is represented but also by characterizing the functional interactions that might underlie such representations. The authors highlight the distributed nature of frontal cortex representations and proposed new ways by which the information specifically flows between nodes. This work is well placed to unify our understanding of the complementarity and specificity of the reward and punishment learning systems.

Reviewer #3 (Public Review):

Summary:

The authors investigated that learning processes relied on distinct reward or punishment outcomes in probabilistic instrumental learning tasks were involved in functional interactions of two different cortico-cortical gamma-band modulations, suggesting that learning signals like reward or punishment prediction errors can be processed by two dominated interactions, such as areas lOFC-vmPFC and areas aINS-dlPFC, and later on integrated together in support of switching conditions between reward and punishment learning. By performing the well-known analyses of mutual information, interaction information, and transfer entropy, the conclusion was accomplished by identifying directional task information flow between redundancy-dominated and synergy-dominated interactions. Also, this integral concept provided a unifying view to explain how functional distributed reward and/or punishment information were segregated and integrated across cortical areas.

Strengths:

The dataset used in this manuscript may come from previously published works (Gueguen et al., 2021) or from the same grant project due to the methods. Previous works have shown strong evidence about why gamma-band activities and those 4 areas are important. For further analyses, the current manuscript moved the ideas forward to examine how reward/punishment information transfer between recorded areas corresponding to the task conditions. The standard measurements such mutual information, interaction information, and transfer entropy showed time-series activities in the millisecond level and allowed us to learn the directional information flow during a certain window. In addition, the diagram in Figure 6 summarized the results and proposed an integral concept with functional heterogeneities in cortical areas. These findings in this manuscript will support the ideas from human fMRI studies and add a new insight to electrophysiological studies with the non-human primates.

Comments on revised version:

Thank you authors for all efforts to answer questions from previous comments. I appreciated that authors clarified the terminology and added a paragraph to discuss the current limitations of functional connectivity and anatomical connections. This provided clear and fair explanations to readers who are not familiar with methods in systems neuroscience.

Reviewer #1 (Public Review):

Summary:

The study used the sci-Plex system to perform in vitro screen of chemicals and found that 2 compounds improved the reprogramming efficiency in Ascl1-overexpressed MG (Muller glia), and in addition, administration of the identified compounds in the previously established in vivo model (Ascl1, NMDA, TSA) showed that DBZ and metformin increased Otx2+ cells for improved neurogenesis.

Strengths:

The overall study was straightforward and well-designed. The method in the study could be potentially useful for large-scale in vitro screens for compounds to further improve reprogramming efficiency. The data and results of the study are of good quality.

Weaknesses:

Future studies may help provide more in-depth mechanistic examinations of the reprogramming process such as whether the compound treatment indeed affects the corresponding signaling pathways.

Reviewer #2 (Public Review):

Summary:

In the current manuscript, Tresenrider et al., present their recent study focusing on screening of small molecules to enhance the conversion from Müller cells (MG) to retina neurons induced by ectopic Ascl1 expression.

Strengths:

To analyze results from multiple treatment conditions in a single experiment, the authors employed a method called sci-Plex to perform scRNA-seq on mixed samples to investigate the effects of different durations of Ascl1 expression and screen for potential small molecules to promote reprogramming. Ultimately, they identified two compounds with intended activities on mouse retina. The findings may aid in future development of a cell replacement strategy for treating retinal degeneration.

Weaknesses:

The mechanistic insights are limited. Certain claims are confusing or superficial at this point.

Reviewer #1 (Public Review):

Summary:

Using concurrent in vivo whole-cell patch clamp and dendritic calcium imaging, the authors characterized how functional synaptic inputs across dendritic arborizations of mouse primary visual cortex layer 2/3 neurons emerge during the second postnatal week. They were able to identify spatially and functionally separated domains of clustered synapses in these neurons even before eye-opening and characterize how the clustering changes from P8 to P13.

Strengths:

The work is technically challenging and the findings are novel. The results support previous EM and immunostaining studies but really provide in vivo evidence on the time course and the trajectory of how functional synaptic input develop.

Weaknesses:

The authors have provided additional details about the analyses and have adequately addressed all my concerns.

Reviewer #2 (Public Review):

In this study, Leighton et al performed remarkable experiments by combining in-vivo patch-clamp recording with two-photon dendritic Ca2+ imaging. The voltage-clamp mode is a major improvement over the pioneer versions of this combinatorial experiment that had led to major breakthroughs in the neuroscience field for visualizing and understanding synaptic input activities in single cells in-vivo (sharp electrodes: Svoboda et al, Nature 1997, Helmchen et al, Nature Neurosci 1999; whole-cell current-clamp: Jia et al, Nature 2010, Chen et al, Nature 2011. I suggest that these papers would be cited). This is because in voltage-clamp mode, despite a full control of membrane voltage in-vivo is not realistic, is nevertheless most effective in preventing back-propagation action potentials, which would severely confound the measurement of individual synaptically-induced Ca2+ influx events. Furthermore, clamping the cell body at a strongly depolarized potential (here the authors did -30mV) also facilitates the detection of synaptically-induced Ca2+ influx. As a result, the authors successfully recorded high-quality Ca2+ imaging data that can be used for precise analysis. To date, even in view of the rapid progress of voltage-sensitive indicators and relevant imaging technologies in the recent years, this very old 'art' of combining single-cell electrophysiology and two-photon imaging (ordinary, raster-scanned, video-rate imaging) of Ca2+ signals still enable measurements of the best-level precision.

On the other hand, the interpretation of data in this study is a bit narrow-minded and lacks a comprehensive picture. Some suggestions to improve the manuscript are as follows:

(1) The authors made a segregation of 'spine synapse' and 'shaft synapse' based solely on the two-photon images in-vivo. However, caution shall be taken here, because the optical resolution under in-vivo imaging conditions like this cannot reliably tell apart whether a bright spot within or partially overlapping a segment of dendrite is a spine on top (or below) of it. Therefore, what the authors consider as a 'shaft synapse' (by detecting Ca2+ hotspots) has an unknown probability to be just a spine on top or below the dendrite. If there is other imaging data of higher axial resolution to validate or calibrate, the authors shall take some further considerations or analysis to check the consistency of their data, as the authors do need such a segregation between spine and shaft synapses to show how they evolve over the brain development stages.<br /> (2) The use of terminology 'bursts of spontaneous inputs' for describing voltage-clamp data seems improper. Conventionally, 'burst' refers to suprathreshold spike firing events, but here, the authors use 'burst' to refer to inward synaptic currents collected at the cell body. It is obvious that not every excitatory synaptic input (or ensemble of inputs) activation will lead to spike firing under naturalistic conditions, therefore, these two concepts are not equivalent. It is recommended to use 'barrage of inputs' instead of 'burst of inputs'. Imagine a full picture of the entire dendritic tree, the fact that the authors could always capture spontaneous Ca2+ events here and there within a few pieces of dendrites within an arbitrary field-of-view suggest that, the whole dendritic tree must have many more such events going on as a barrage while the author's patch electrode picks up the summed current flow from the whole dendritic tree.<br /> (3) Following the above issue, an analysis of the temporal correlation between synaptic (not segregating 'spine' or 'shaft') Ca2+ events and EPSCs is absent. Again, the authors drew arbitrary time windows to clump the events for statistical analysis. However, the demonstrated example data already show that the onset times of individual synaptic Ca2+ events do not necessarily align with the beginning of a 'barrage' inward current event.<br /> (4) The authors claim that "these observations indicate that the activity patterns investigated here are not or only slightly affected by low-level anesthesia". It would be nice to show some of the recordings in this work without any anesthesia to support this claim.<br /> (5) I suggest the authors should provide the number of cells and mice recorded in the figure legends.<br /> (6) Instead of showing only cartoon illustrations of dendrites in Figure 3-6, I suggest showing the two-photon images as well together with the cartoon.

The authors have addressed most of my issues, but I miss the responses to my points 5 and 6. I have no additional comments.

Reviewer #3 (Public Review):

Summary:

There is a growing body of literature on the clustering of co-active synapses in adult mice, which has important implications for understanding dendritic integration and sensory processing more broadly. However, it has been unclear when this spatial organization of co-active synapses arises during development. In this manuscript, Leighton et al. investigate the emergence of spatially organized, co-active synapses on pyramidal dendrites in the mouse visual cortex before eye opening. They find that some dendrite segments contain highly active synapses that are co-active with their neighbors as early as postnatal day (P) 8-10, and that these domains of co-active synapses increase their coverage of the dendritic arbor by P12-13. Interestingly, Leighton et al. demonstrate that synapses co-active with their neighbors are more likely to increase their activity across a single recording session, compared to synapses that are not co-active with their neighbors, suggesting local plasticity driven by coincident activity before eye opening.

The current manuscript includes some replication of earlier results from the same research group (Winnubst et al., 2015), including the presence of clustered, co-active synapses in the visual cortex of mouse pups, and the finding that synapses co-active with their neighbors show an increase in transmission frequency during a recording session. The main novelty in the current study compared to Winnubst et al. (2015) is the inclusion of younger animals (P8-13 in the current study compared to P10-15 in Winnubst et al., 2015). The current manuscript is the first demonstration that active synapses are clustered on specific dendrite segments as early as P8-10 in the mouse visual cortex, and the first to show the progression in active synapse distribution along the dendrite during the 2nd postnatal week. These results from visual cortex may help inform our understanding of sensory development more broadly.

Strengths:

The authors ask a novel question about the emergence of synaptic spatial organization, and they use well-chosen techniques that directly address their questions despite the challenging nature of these techniques. To capture both structural and functional information from dendrites simultaneously, the authors performed whole-cell voltage clamp to record synaptic currents arriving at the soma while imaging calcium influx at individual synaptic sites on dendrites. The simultaneous voltage clamp and calcium imaging allowed the authors to isolate individual synaptic inputs without their occlusion by widespread calcium influx from back-propagating action potentials. Achieving in vivo dendrite imaging in live mice that are as young as P8 is challenging, and the resulting data provides a unique view of synaptic activity along individual dendrites in the visual cortex at an early stage in development that is otherwise difficult to assess.<br /> The authors provide convincing evidence that synapses are more likely to be co-active with their neighbors compared to synapses located farther away (Fig. 6F-H), and that synapses co-active with their neighbors increase their transmission frequency during a recording session (Fig. 7C). These findings are particularly interesting given that the recordings occur before eye opening, suggesting a relationship between co-activity and local synaptic plasticity even before the onset of detailed visual input. These results replicate previously published findings from P10-15 pups (Winnubst et al., 2015), increasing confidence in the reproducibility of the data.<br /> The authors also provide novel data documenting for the first time spatially organized, co-active synapses in pups as young as P8. Comparing the younger (P8-10) and older (P12-13) pups, provides insight into how clusters of co-active synapses might emerge during development.

Weaknesses:

The P8-10 vs P12-13 age comparisons are the primary novel finding in this manuscript, and it is therefore critical to avoid systematic age differences in the methods and analysis whenever possible. In their rebuttal and revised manuscript the authors have acceptably addressed prior concerns regarding this important point, as well as most of the other methodological issues raised.<br /> One point addressed in the rebuttal, but not corrected in the manuscript relates to the reliable localization of cells to visual cortex.

Reviewer #1 (Public Review):

This is an interesting and well-written paper reporting on a novel approach to studying cerebellar function based on the idea of selective recruitment using fMRI. The study is well-designed and executed. Analyses are sound and results are properly discussed. The paper makes a significant contribution to broadening our understanding of the role of cerebellum in human behavior.

In the revision, the authors did an excellent job in addressing my concerns.

Reviewer #2 (Public Review):

Summary:

Shahshahani and colleagues used a combination of statistical modelling and whole-brain fMRI data in an attempt to separate the contributions of cortical and cerebellar regions in different cognitive contexts.

Strengths:

* The manuscript uses a sophisticated integration of statistical methods, cognitive neuroscience and systems neurobiology.<br /> * The authors use multiple statistical approaches to ensure robustness in their conclusions.<br /> * The consideration of the cerebellum as not a purely 'motor' structure is excellent and important.

Weaknesses:

* The assumption that cortical BOLD responses in cognitive tasks should be matched irrespective of cerebellar involvement does not cohere directly with the notion of 'forcing functions' introduced by Houk and Wise, suggesting the need for future work.

Reviewer #1 (Public Review):

Summary:

This paper introduces a new approach to modeling human behavioral responses using image-computable models. They create a model (VAM) that is a combination of a standard CNN coupled with a standard evidence accumulation model (EAM). The combined model is then trained directly on image-level data using human behavioral responses. This approach is original and can have wide applicability. However, many of the specific findings reported are less compelling.

Strengths:

(1) The manuscript presents an original approach to fitting an image-computable model to human behavioral data. This type of approach is sorely needed in the field.<br /> (2) The analyses are very technically sophisticated.<br /> (3) The behavioral data are large both in terms of sample size (N=75) and in terms of trials per subject.

Weaknesses:

Major

(1) The manuscript appears to suggest that it is the first to combine CNNs with evidence accumulation models (EAMs). However, this was done in a 2022 preprint (https://www.biorxiv.org/content/10.1101/2022.08.23.505015v1) that introduced a network called RTNet. This preprint is cited here, but never really discussed. Further, the two unique features of the current approach discussed in lines 55-60 are both present to some extent in RTNet. Given the strong conceptual similarity in approach, it seems that a detailed discussion of similarities and differences (of which there are many) should feature in the Introduction.

(2) In the approach here, a given stimulus is always processed in the same way through the core CNN to produce activations v_k. These v_k's are then corrupted by Gaussian noise to produce drift rates d_k, which can differ from trial to trial even for the same stimulus. In other words, the assumption built into VAM appears to be that the drift rate variability stems entirely from post-sensory (decisional) noise. In contrast, the typical interpretation of EAMs is that the variability in drift rates is sensory. This is also the assumption built into RTNet where the core CNN produces noisy evidence. Can the authors comment on the plausibility of VAM's assumption that the noise is post-sensory?

(3) Figure 2 plots how well VAM explains different behavioral features. It would be very useful if the authors could also fit simple EAMs to the data to clarify which of these features are explainable by EAMs only and which are not.

(4) VAM is tested in two different ways behaviorally. First, it is tested to what extent it captures individual differences (Figure 2B-E). Second, it is tested to what extent it captures average subject data (Figure 2F-J). It wasn't clear to me why for some metrics only individual differences are examined and for other metrics only average human data is examined. I think that it will be much more informative if separate figures examine average human data and individual difference data. I think that it's especially important to clarify whether VAM can capture individual differences for the quantities plotted in Figures 2F-J.

(5) The authors look inside VAM and perform many exploratory analyses. I found many of these difficult to follow since there was little guidance about why each analysis was conducted. This also made it difficult to assess the likelihood that any given result is robust and replicable. More importantly, it was unclear which results are hypothesized to depend on the VAM architecture and training, and which results would be expected in performance-optimized CNNs. The authors train and examine performance-optimized CNNs later, but it would be useful to compare those results to the VAM results immediately when each VAM result is first introduced.

(6) The authors don't examine how the task-optimized models would produce RTs. They say in lines 371-2 that they "could not examine the RT congruency effect since the task-optimized models do not generate RTs." CNNs alone don't generate RTs, but RTs can easily be generated from them using the same EAM add-on that is part of VAM. Given that the CNNs are already trained, I can't see a reason why the authors can't train EAMs on top of the already trained CNNs and generate RTs, so these can provide a better comparison to VAM.

(7) The Discussion felt very long and mostly a summary of the Results. I also couldn't shake the feeling that it had many just-so stories related to the variety of findings reported. I think that the section should be condensed and the authors should be clearer about which explanations are speculations and which are air-tight arguments based on the data.

(8) In one of the control analyses, the authors train different VAMs on each RT quantile. I don't understand how it can be claimed that this approach can serve as a model of an individual's sensory processing. Which of the 5 sets of weights (5 VAMs) captures a given subject's visual processing? Are the authors saying that the visual system of a given subject changes based on the expected RT for a stimulus? I feel like I'm missing something about how the authors think about these results.

Reviewer #2 (Public Review):

In an image-computable model of speeded decision-making, the authors introduce and fit a combined CCN-EAM (a 'VAM') to flanker-task-like data. They show that the VAM can fit mean RTs and accuracies as well as the congruency effect that is present in the data, and subsequently analyze the VAM in terms of where in the network congruency effects arise.

Overall, combining DNNs and EAMs appears to be a promising avenue to seriously model the visual system in decision-making tasks compared to the current practice in EAMs. Some variants have been proposed or used before (e.g., doi.org/10.1016/j.neuroimage.2017.12.078 , doi.org/10.1007/s42113-019-00042-1), but always in the context of using task-trained models, rather than models trained on behavioral data. However, I was surprised to read that the authors developed their model in the context of a conflict task, rather than a simpler perceptual decision-making task. Conflict effects in human behavior are particularly complex, and thereby, the authors set a high goal for themselves in terms of the to-be-explained human behavior. Unfortunately, the proposed VAM does not appear to provide a great account of conflict effects that are considered fundamental features of human behavior, like the shape of response time distributions, and specifically, delta plots (doi.org/10.1037/0096-1523.20.4.731). The authors argue that it is beyond the scope of the presented paper to analyze delta plots, but as these are central to studies of human conflict behavior, models that aim to explain conflict behavior will need to be able to fit and explain delta plots.

Theories on conflict often suggest that negative/positive-trending delta plots arise through the relative timing of response activation related to relevant and irrelevant information. Accumulation for relevant and irrelevant information would, as a result, either start at different points in time or the rates vary over time. The current VAM, as a feedforward neural network model, does not appear to be able to capture such effects, and perhaps fundamentally not so: accumulation for each choice option is forced to start at the same time, and rates are a static output of the CNN.

The proposed solution of fitting five separate VAMs (one for each of five RT quantiles) is not satisfactory: it does not explain how delta plots result from the model, for the same reason that fitting five evidence accumulation models (one per RT quantile) does not explain how response time distributions arise. If, for example, one would want to make a prediction about someone's response time and choice based on a given stimulus, one would first have to decide which of the five VAMs to use, which is circular. But more importantly, this way of fitting multiple models does not explain the latent mechanism that underlies the shape of the delta plots.

As such, the extensive analyses on the VAM layers and the resulting conclusions that conflict effects arise due to changing representations across layers (e.g., "the selection of task-relevant information occurs through the orthogonalization of relevant and irrelevant representations") - while inspiring, they remain hard to weigh, as they are contingent on the assumption that the VAM can capture human behavior in the conflict task, which it struggles with. That said, the promise of combining CNNs and EAMs is clearly there. A way forward could be to either adjust the proposed model so that it can explain delta plots, which would potentially require temporal dynamics and time-varying evidence accumulation rates, or perhaps to start simpler and combine CCNs-EAMs that are able to fit more standard perceptual decision-making tasks without conflict effects.

Reviewer #3 (Public Review):

Summary:

In this article, the authors combine a well-established choice-response time (RT) model (the Linear Ballistic Accumulator) with a CNN model of visual processing to model image-based decisions (referred to as the Visual Accumulator Model - VAM). While this is not the first effort to combine these modeling frameworks, it uses this combination of approaches uniquely. Specifically, the authors attempt to better understand the structure of human information representations by fitting this model to behavioral (choice-RT) data from a classic flanker task. This objective is made possible by using a very large (by psychological modeling standards) industry data set to jointly fit both components of this VAM model to individual-level data. Using this approach, they illustrate (among other results) (1) how the interaction between target and flanker representations influence the presence and strength of congruency effects, (2) how the structure of representations changes (distributed versus more localized) with depth in the CNN model component, and (3) how different model training paradigms change the nature of information representations. This work contributes to the ML literature by demonstrating the value of training models with richer behavioral data. It also contributes to cognitive science by demonstrating how ML approaches can be integrated into cognitive modeling. Finally, it contributes to the literature on conflict modeling by illustrating how information representations may lead to some of the classic effects observed in this area of research.

Strengths:

(1) The data set used for this analysis is unique and is made publicly available as part of this article. Specifically, they have access to data for 75 participants with >25,000 trials per participant. This scale of data/individual is unusual and is the foundation on which this research rests.

(2) This is the first time, to my knowledge, that a model combining a CNN with a choice-RT model has been jointly fit to choice-RT data at the level of individual people. This type of model combination has been used before but in a more restricted context. This joint fitting, and in particular, learning a CNN through the choice-RT modeling framework, allows the authors to probe the structure of human information representations learned directly from behavioral data.

(3) The analysis approaches used in this article are state-of-the-art. The training of these models is straightforward given the data available. The interesting part of this article (opinion of course) is the way in which they probe what CNN has learned once trained. I find their analysis of how distractor and target information interfere with each other particularly compelling as well as their demonstration that training on behavioral data changes the structure of information representations when compared to training models on standard task-optimized data.

Weaknesses:

(1) Just as the data in this article is a major strength, it is also a weakness. This type of modeling would be difficult, if not impossible to do with standard laboratory data. I don't know what the data floor would be, but collecting tens of thousands of decisions for a single person is impractical in most contexts. Thus this type of work may live in the realm of industry. I do want to re-iterate that the data for this study was made publicly available though!

2) While this article uses choice-RT data it doesn't fully leverage the richness of the RT data itself. As the authors point out, this modeling framework, the LBA component in particular, does not account for some of the more nuanced but well-established RT effects in this data. This is not a big concern given the already nice contributions of this article and it leads to an opportunity for ongoing investigation.

Reviewer #3 (Public Review):

Summary:

Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme in glycolysis and its translocation to the nucleus in astrocytes in various nervous system pathologies has been associated with a metabolic switch to glycolysis which is a sign of reactive astrogliosis. The authors investigated whether this occurs in experimental autoimmune encephalomyelitis (EAA), an animal model of multiple sclerosis (MS). They show that in EAA, PKM2 is ubiquitinated by TRIM21 and transferred to the nucleus in astrocytes. Inhibition of TRIM21-PKM2 axis efficiently blocks reactive gliosis and partially alleviates symptoms of EAA. Authors conclude that this axis can be a potential new therapeutic target in the treatment of MS.

Strengths:

The study is well-designed, controls are appropriate and a comprehensive battery of experiments has been successfully performed. Results of in vitro assays, single-cell RNA sequencing, immunoprecipitation, RNA interference, molecular docking, and in vivo modeling etc. complement and support each other.

Weaknesses:

Though EAA is a valid model of MS, a proposed new therapeutic strategy based on this study needs to have support from human studies.

Reviewer #1 (Public Review):

Summary:

Yang, Hu et al. examined the molecular mechanisms underlying astrocyte activation and its implications for multiple sclerosis. This study shows that the glycolytic enzyme PKM2 relocates to astrocyte nuclei upon activation in EAE mice. Inhibiting PKM2's nuclear import reduces astrocyte activation, as evidenced by decreased proliferation, glycolysis, and inflammatory cytokine release. Crucially, the study identifies TRIM21 as pivotal in regulating PKM2 nuclear import via ubiquitination. TRIM21 interacts with PKM2, promoting its nuclear translocation and enhancing its activity, affecting multiple signaling pathways. Confirmatory analyses using single-cell RNA sequencing and immunofluorescence demonstrate TRIM21 upregulation in EAE astrocytes. Modulating TRIM21 expression in primary astrocytes impacts PKM2-dependent glycolysis and proliferation. In vivo experiments targeting this mechanism effectively mitigate disease severity, CNS inflammation, and demyelination in EAE.

The authors supported their claims with various experimental approaches, however, some results should be supported with higher-quality images clearly depicting the conclusions and additional quantitative analyses of Western blots.

Strength:

This study presents a comprehensive investigation into the function and molecular mechanism of metabolic reprogramming in the activation of astrocytes, a critical aspect of various neurological diseases, especially multiple sclerosis. The study uses the EAE mouse model, which closely resembles MS. This makes the results relevant and potentially translational. The research clarifies how TRIM21 regulates the nuclear import of PKM2 through ubiquitination by integrating advanced techniques. Targeting this axis may have therapeutic benefits since lentiviral vector-mediated knockdown of TRIM21 in vivo significantly reduces disease severity, CNS inflammation, and demyelination in EAE animals.

Weaknesses:

The authors reported that PKM2 levels are elevated in the nucleus of astrocytes at different EAE phases compared to cytoplasmic localization. However, Figure 1 also shows elevated cytoplasmic expression of PKM2. The authors should clarify the nuclear localization of PKM2 by providing zoomed-in images. An explanation for the increased cytoplasmic PKM2 expression should provided. Similarly, while PKM2 translocation is inhibited by DASA-58, in addition to its nuclear localization, a decrease in the cytoplasmic localization of PKM2 is also observed. This situation brings to mind the possibility of a degradation mechanism being involved when its nuclear translocation of PKM2 is inhibited.

In Figure 3D, the authors claim that PKM2 expression causes nuclear retention of STAT3, p65, and p50, and inhibiting PKM2 localization with DASA-58 suppresses this retention. The western blot results for the MOG-stimulated group show high levels of STAT3, p50, and p65 in nuclear localization. However, in the MOG and DASA-58 treated group, one would expect high levels of p50, p65, and STAT3 proteins in the cytoplasm, while their levels decrease in the nucleus. These western blot results could be expanded. Additionally, intensity quantification for these results would be beneficial to see the statistical difference in their expressions, especially to observe the nuclear localization of PKM2.

The discrepancy between Figure 7A and its explaining text is confusing. The expectation from the knocking down of TRIM21 is the amelioration of activated astrocytes, leading to a decrease in inflammation and the disease state. The presented results support these expectations, while the images showing demyelination in EAE animals are not highly supportive. Clearly labeling demyelinated areas would enhance readers' understanding of the important impact of TRIM21 knockdown on reducing the disease severity.

Reviewer #2 (Public Review):

This study significantly advances our understanding of the metabolic reprogramming underlying astrocyte activation in neurological diseases such as multiple sclerosis. By employing an experimental autoimmune encephalomyelitis (EAE) mouse model, the authors discovered a notable nuclear translocation of PKM2, a key enzyme in glycolysis, within astrocytes.

Preventing this nuclear import via DASA 58 substantially attenuated primary astrocyte activation, characterized by reduced proliferation, glycolysis, and inflammatory cytokine secretion.<br /> Moreover, the authors uncovered a novel regulatory mechanism involving the ubiquitin ligase TRIM21, which mediates PKM2 nuclear import. TRIM21 interaction with PKM2 facilitated its nuclear translocation, enhancing its activity in phosphorylating STAT3, NFκB, and c-myc. Single-cell RNA sequencing and immunofluorescence staining further supported the upregulation of TRIM21 expression in astrocytes during EAE.

Manipulating this pathway, either through TRIM21 overexpression in primary astrocytes or knockdown of TRIM21 in vivo, had profound effects on disease severity, CNS inflammation, and demyelination in EAE mice. This comprehensive study provides invaluable insights into the pathological role of nuclear PKM2 and the ubiquitination-mediated regulatory mechanism driving astrocyte activation.

The author's use of diverse techniques, including single-cell RNA sequencing, immunofluorescence staining, and lentiviral vector knockdown, underscores the robustness of their findings and interpretations. Ultimately, targeting this PKM2-TRIM21 axis emerges as a promising therapeutic strategy for neurological diseases involving astrocyte dysfunction.

While the strengths of this piece of work are undeniable, some concerns could be addressed to refine its impact and clarity further; as outlined in the recommendations for the authors.

Reviewer #4 (Public Review):

Summary:

The authors report the role of the Pyruvate Kinase M2 (PKM2) enzyme nuclear translocation as fundamental in the activation of astrocytes in a model of autoimmune encephalitis (EAE). They show that astrocytes, activated through culturing in EAE splenocytes medium, increase their nuclear PKM2 with consequent activation of NFkB and STAT3 pathways. Prevention of PKM2 nuclear translocation decreases astrocyte counteracts this activation. The authors found that the E3 ubiquitin ligase TRIM21 interacts with PKM2 and promotes its nuclear translocation. In vivo, either silencing of TRIM21 or inhibition of PKM2 nuclear translocation ameliorates the severity of the disease in the EAE model.

Strengths:

This work contributes to the knowledge of the complex action of the PKM2 enzyme in the context of an autoimmune-neurological disease, highlighting its nuclear role and a novel partner, TRIM21, and thus adding a novel rationale for therapeutic targeting.

Weaknesses:

Despite the relevance of the work and its goals, some of the conclusions drawn would require more thorough proof:

I believe that the major weakness is the fact that TRIM21 is known to have per se many roles in autoimmune and immune pathways and some of the effects observed might be due to a PKM2-independent action. Some of the experiments to link the two proteins, besides their interaction, do not completely clarify the issue. On top of that, the in vivo experiments address the role of TRIM21 and the nuclear localisation of PKM2 independently, thus leaving the matter unsolved.

Some experimental settings are not described to a level that is necessary to fully understand the data, especially for a non-expert audience: e.g. the EAE model and MOG treatment; action and reference of the different nuclear import inhibitors; use of splenocyte culture medium and the possible effect of non-EAE splenocytes.

The statement that PKM2 is a substrate of TRIM21 ubiquitin ligase activity is an overinterpretation. There is no evidence that this interaction results in ubiquitin modification of PKM2; the ubiquitination experiment is minimal and is not performed in conditions that would allow us to see ubiquitination of PKM2 (e.g. denaturing conditions, reciprocal pull-down, catalytically inactive TRIM21, etc.).

Reviewer #1 (Public Review):

Summary:

Wang, Y. et al. used a silicone wire embolus to definitively and acutely clot the pterygopalatine ophthalmic artery in addition to carotid artery ligation to completely block the blood supply to the mouse inner retina, which mimics clinical acute retinal artery occlusion. A detailed characterization of this mouse model determined the time course of inner retina degeneration and associated functional deficits, which closely mimic human patients. Whole retina transcriptome profiling and comparison revealed distinct features associated with ischemia, reperfusion, and different model mechanisms. Interestingly and importantly, this team found a sequential event including reperfusion-induced leukocyte infiltration from blood vessels, residual microglial activation, and neuroinflammation that may lead to neuronal cell death.

Strengths:

Clear demonstration of the surgery procedure with informative illustrations, images, and superb surgical videos.

Two-time points of ischemia and reperfusion were studied with convincing histological and in vivo data to demonstrate the time course of various changes in retinal neuronal cell survivals, ERG functions, and inner/outer retina thickness.

The transcriptome comparison among different retinal artery occlusion models provides informative evidence to differentiate these models.

The potential applications of the in vivo retinal ischemia-reperfusion model and relevant readouts demonstrated by this study will certainly inspire further investigation of the dynamic morphological and functional changes of retinal neurons and glial cell responses during disease progression and before and after treatments.

Weaknesses:

It would be beneficial to the manuscript and the readers if the authors could improve the English of this manuscript by correcting obvious grammar errors, eliminating many of the acronyms that are not commonly used by the field, and providing a reason why this complicated but clever surgery procedure was designed and a summary table with the time course of all the morphological, functional, cellular, and transcriptome changes associated with this model.

Reviewer #2 (Public Review):

Summary:

The authors of this manuscript aim to develop a novel animal model to accurately simulate the retinal ischemic process in retinal artery occlusion (RAO). A unilateral pterygopalatine ophthalmic artery occlusion (UPOAO) mouse model was established using silicone wire embolization combined with carotid artery ligation. This manuscript provided data to show the changes in major classes of retinal neural cells and visual dysfunction following various durations of ischemia (30 minutes and 60 minutes) and reperfusion (3 days and 7 days) after UPOAO. Additionally, transcriptomics was utilized to investigate the transcriptional changes and elucidate changes in the pathophysiological process in the UPOAO model post-ischemia and reperfusion. Furthermore, the authors compared transcriptomic differences between the UPOAO model and other retinal ischemic-reperfusion models, including HIOP and UCCAO, and revealed unique pathological processes.

Strengths:

The UPOAO model represents a novel approach to studying retinal artery occlusion. The study is very comprehensive.

Weaknesses:

Some statements are incorrect and confusing. It would be helpful to review and clarify these to ensure accuracy and improve readability.

Reviewer #1 (Public Review):

Summary:

This manuscript by Aybar-Torres et al investigated the effect of common human STING1 variants on STING-mediated T cell phenotypes in mice. The authors previously made knock-in mice expressing human STING1 alleles HAQ or AQ, and here they established a new knock-in line Q293. The authors stimulated cells isolated from these mice with STING agonists and found that all three human mutant alleles resist cell death, leading to the conclusion that R293 residue is essential for STING-mediated cell death (there are several caveats with this conclusion, more below). The authors also bred HAQ and AQ alleles to the mouse Sting1-N153S SAVI mouse and observed varying levels of rescue of disease phenotypes with the AQ allele showing more complete rescue than the HAQ allele. The Q293 allele was not tested in the SAVI model. They conclude that the human common variants such as HAQ and AQ have a dominant negative effect over the gain-of-function SAVI mutants.

Strengths:

The authors and Dr. Jin's group previously made important observations of common human STING1 variants, and these knock-in mouse models are essential for understanding the physiological function of these alleles.

Weaknesses:

However, although some of the observations reported here are interesting, the data collectively does not support a unified model. The authors seem to be drawing two sets of conclusions from in vitro and in vivo experiments, and neither mechanism is clear. Several experiments need better controls, and these knock-in mice need more comprehensive functional characterization.

(1) In Figure 1, the authors are trying to show that STING agonist-induced splenocytes cell death is blocked by HAQ, AQ and Q alleles. The conclusion at line 134 should be splenocytes, not lymphocytes. Most experiments in this figure were done with mixed population that may involve cell-to-cell communication. Although TBK1-dependence is likely, a single inhibitor treatment of a mixed population is not sufficient to reach this conclusion.<br /> (2) Q293 knock-in mouse needs to be characterized and compared to HAQ and AQ. Is this mutant expressed in tissues? Does this mutant still produce IFN and other STING activities? Does the protein expression level altered on Western blot? Is the mutant protein trafficking affected? In the authors' previous publications and some of the Western blot here, expression levels of each of these human STING1 protein in mice are drastically different. HAQ and AQ also have different effects on metabolism (pmid: 36261171), which could complicate interoperation of the T cell phenotypes.<br /> (3) HAQ/WT and AQ/WT splenocytes are protected from STING agonist-induced cell death equally well (Figure 1G). HAQ/SAVI shows less rescue compared to AQ/SAVI. These are interesting observations, but mechanism is unclear and not clearly discussed. E.g., how does AQ protect disease pathology better than HAQ (that contains AQ)? Does Q293 allele also fully rescue SAVI?<br /> (4) Figure 2 feels out of place. First of all, why are the authors using human explant lung tissues? PBMCs should be a better source for lymphocytes. In untreated conditions, both CD4 and B cells show ~30% dying cells, but CD8 cells show 0% dying cells. This calls for technical concerns on the CD8 T cell property or gating strategy because in the mouse experiment (Figure 1A) all primary lymphocytes show ~30% cell death at steady-state. Second, Figure 2C, these type of partial effect needs multiple human donors to confirm. Three, the reconstitution of THP1 cells seems out of place. STING-mediated cell death mechanism in myeloid and lymphoid cells are likely different. If the authors want to demonstrate cell death in myeloid cells using THP1, then these reconstituted cell lines need to be better validated. Expression, IFN signaling, etc. The parental THP1 cells is HAQ/HAQ, how does that compare to the reconstitutions? There are published studies showing THP1-STING-KO cells reconstituted with human variants do not respond to STING agonists as expected. The authors need to be scientifically rigorous on validation and caution on their interpretations.<br /> (5) Figure 2G, H, I are confusing. AQ is more active in producing IFN signaling than HAQ and Q is the least active. How to explain this?<br /> (6) The overall model is unclear. If HAQ, AQ and Q are loss-of-function alleles and Q is the key residue for STING-mediated cell death, then why AQ is the most active in producing IFN signaling and AQ/SAVI rescues disease most completely? If these human variants act as dominant negatives, which would be consistent with the WT/het data, then how do you explain AQ is more dominant negative than HAQ?<br /> (7) As a general note, SAVI disease phenotypes involve multiple cell types. Lymphocyte cell death is only one of them. The authors' characterization of SAVI pathology is limited and did not analyze immunopathology of the lung.<br /> (8) Line 281, the discussion on HIV T cell death mechanism is not relevant and over-stretching. This study did not evaluate viral infection in T cells at all. The original finding of HAQ/HAQ enrichment in HIV/AIDS was 2/11 in LTNP vs 0/11 in control, arguably not the strongest statistics.

Reviewer #2 (Public Review):

Aybar-Torres and colleagues utilize common human STING alleles to dissect the mechanism of SAVI inflammatory disease. The authors demonstrate that these common alleles alleviate SAVI pathology in mice, and perhaps more importantly use the differing functionality of these alleles to provide insight into requirements of SAVI disease induction. Their findings suggest that it is residue A230 and/or Q293 that are required for SAVI induction, while the ability to induce an interferon-dependent inflammatory response is not. This is nicely exemplified by the AQ/SAVI mice that have an intact inflammatory response to STING activation, yet minimal disease progression. As both mutants seem to be resistant STING-dependent cell death, this manuscript also alludes to the importance of STING-dependent cell death, rather than STING-dependent inflammation, in the progression of SAVI pathology. While I have some concerns, I believe this manuscript makes some important connections between STING pathology mouse models and human genetics that would contribute to the field.

Some points to consider:

(1) While the CD4+ T cell counts from HAQ/SAVI and AQ/SAVI mice suggest that these T cells are protected from STING-dependent cell death, an assay that explores this more directly would strengthen the manuscript. This is also supported by Fig 2C, but I believe a strength of this manuscript is the comparison between the two alleles. Therefore, if possible, I would recommend the isolation of T cells from these mice and direct stimulation with diABZI or other STING agonist with a cell death readout.<br /> (2) Related to the above point - further exemplifying that the Q293 locus is essential to disease, even in human cells, would also strengthen the paper. It seems that CD4 T cell loss is a major component of human SAVI. While not completely necessary, repeating the THP1 cell death experiments from Fig 2 with a human T cell line would round out the study nicely.<br /> (3) While I found the myeloid cell counts and BMDM data interesting, I think some more context is needed to fully loop this data into the story. Is myeloid cell expansion exemplified by SAVI patients? Do we know if myeloid cells are the major contributors to the inflammation these patients experience? Why should the SAVI community care about the Q293 locus in myeloid cells?<br /> (4) The functional assays in Figure 4 are exciting and really connect the alleles to disease progression. To strengthen the manuscript and connect all the data, I would recommend additional readouts from these mice that address the inflammatory phenotype shown in vitro in Figure 5. For example, measuring cytokines from these mice via ELISA or perhaps even Western blots looking for NFkB or STING activation would be supportive of the story. This would also allow for some tissue specificity. I believe looking for evidence of inflammation and STING activation in the lungs of these mice, for example, would further connect the data to human SAVI pathology.

Reviewer #1 (Public Review):

Bacterial effectors that interfere with the inner molecular workings of eukaryotic host cells are of great biological significance across disciplines. On the one hand they help us to understand the molecular strategies that bacteria use to manipulate host cells. On the other hand they can be used as research tools to reveal molecular details of the intricate workings of the host machinery that is relevant for the interaction/defence/symbiosis with bacteria. The authors investigate the function and biological impact of a rhizobial effector that interacts with and modifies, and curiously is modified by, legume receptors essential for symbiosis. The molecular analysis revealed a bacterial effector that cleaves a plant symbiosis signaling receptor to inhibit signaling and the host counterplay by phosphorylation via a receptor kinase. These findings have potential implications beyond bacterial interactions with plants.

Bao and colleagues investigated how rhizobial effector proteins can regulate the legume root nodule symbiosis. A rhizobial effector is described to directly modify symbiosis-related signaling proteins, altering the outcome of the symbiosis. Overall, the paper presents findings that will have a wide appeal beyond its primary field.

Out of 15 identified effectors from Sinorhizobium fredii, they focus on the effector NopT, which exhibits proteolytic activity and may therefore cleave specific target proteins of the host plant. They focus on two Nod factor receptors of the legume Lotus japonicus, NFR1 and NFR5, both of which were previously found to be essential for the perception of rhizobial nod factor, and the induction of symbiotic responses such as bacterial infection thread formation in root hairs and root nodule development (Madsen et al., 2003, Nature; Tirichine et al., 2003; Nature). The authors present evidence for an interaction of NopT with NFR1 and NFR5. The paper aims to characterize the biochemical and functional consequences of these interactions and the phenotype that arises when the effector is mutated.

Evidence is presented that in vitro NopT can cleave NFR5 at its juxtamembrane region. NFR5 appears also to be cleaved in vivo. and NFR1 appears to inhibit the proteolytic activity of NopT by phosphorylating NopT. When NFR5 and NFR1 are ectopically over-expressed in leaves of the non-legume Nicotiana benthamiana, they induce cell death (Madsen et al., 2011, Plant Journal). Bao et al., found that this cell death response is inhibited by the coexpression of nopT. Mutation of nopT alters the outcome of rhizobial infection in L. japonicus. These conclusions are well supported by the data.

The authors present evidence supporting the interaction of NopT with NFR1 and NFR5. In particular, there is solid support for cleavage of NFR5 by NopT (Figure 3) and the identification of NopT phosphorylation sites that inhibit its proteolytic activity (Figure 4C). Cleavage of NFR5 upon expression in N. benthamiana (Figure 3A) requires appropriate controls (inactive mutant versions) that have been provided, since Agrobacterium as a closely rhizobia-related bacterium, might increase defense related proteolytic activity in the plant host cells.

Key results from N. benthamiana appear consistent with data from recombinant protein expression in bacteria. For the analysis in the host legume L. japonicus transgenic hairy roots were included. To demonstrate that the cleavage of NFR5 occurs during the interaction in plant cells the authors build largely on western blots. Regardless of whether Nicotiana leaf cells or Lotus root cells are used as the test platform, the Western blots indicate that only a small proportion of NFR5 is cleaved when co-expressed with nopT, and most of the NFR5 persists in its full-length form (Figures 3A-D). It is not quite clear how the authors explain the loss of NFR5 function (loss of cell death, impact on symbiosis), as a vast excess of the tested target remains intact. It is also not clear why a large proportion of NFR5 is unaffected by the proteolytic activity of NopT. This is particularly interesting in Nicotiana in the absence of Nod factor that could trigger NFR1 kinase activity.

It is also difficult to evaluate how the ratios of cleaved and full-length protein change when different versions of NopT are present without a quantification of band strengths normalized to loading controls (Figure 3C, 3D, 3F). The same is true for the blots supporting NFR1 phosphorylation of NopT (Figure 4A).

It is clear that mutation of nopT results in a quantitative infection phenotype. Nodule primordia and infection threads are still formed when L. japonicus plants are inoculated with ∆nopT mutant bacteria, but it is not clear if these primordia are infected or develop into fully functional nodules (Figure 5). A quantification of the ratio of infected and non-infected nodules and primordia would reveal whether NopT is only active at the transition from infection focus to thread or perhaps also later in the bacterial infection process of the developing root nodule.

Reviewer #2 (Public Review):

Summary:

This manuscript presents data demonstrating NopT's interaction with Nod Factor Receptors NFR1 and NFR5 and its impact on cell death inhibition and rhizobial infection. The identification of a truncated NopT variant in certain Sinorhizobium species adds an interesting dimension to the study. These data try to bridge the gaps between classical Nod-factor-dependent nodulation and T3SS NopT effector-dependent nodulation in legume-rhizobium symbiosis. Overall, the research provides interesting insights into the molecular mechanisms underlying symbiotic interactions between rhizobia and legumes.

Strengths:

The manuscript nicely demonstrates NopT's proteolytic cleavage of NFR5, regulated by NFR1 phosphorylation, promoting rhizobial infection in L. japonicus. Intriguingly, authors also identify a truncated NopT variant in certain Sinorhizobium species, maintaining NFR5 cleavage but lacking NFR1 interaction. These findings bridge the T3SS effector with the classical Nod-factor-dependent nodulation pathway, offering novel insights into symbiotic interactions.

Weaknesses:

(1) In the previous study, when transiently expressed NopT alone in Nicotiana tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. However, this phenotype was not observed when expressing the same NopT in Nicotiana benthamiana (Figure 1A). Conversely, cell death and a hypersensitive reaction were observed in Figure S8. This raises questions about the suitability of the exogenous expression system for studying NopT proteolysis specificity.

(2)NFR5 Loss-of-function mutants do not produce nodules in the presence of rhizobia in lotus roots, and overexpression of NFR1 and NFR5 produces spontaneous nodules. In this regard, if the direct proteolysis target of NopT is NFR5, one could expect the NGR234's infection will not be very successful because of the Native NopT's specific proteolysis function of NFR5 and NFR1. Conversely, in Figure 5, authors observed the different results.

(3) In Figure 6E, the model illustrates how NopT digests NFR5 to regulate rhizobia infection. However, it raises the question of whether it is reasonable for NGR234 to produce an effector that restricts its own colonization in host plants.

(4) The failure to generate stable transgenic plants expressing NopT in Lotus japonicus is surprising, considering the manuscript's claim that NopT specifically proteolyzes NFR5, a major player in the response to nodule symbiosis, without being essential for plant development.

Reviewer #1 (Public Review):

Summary:

The authors study the effects of myelin alterations in working memory via the complementary use of two computational approaches: one based on the de- and re-myelination in multicompartmental models of pyramidal neurons, and one based on synaptic changes in a spiking bump attractor model for spatial working memory. The first model provides the most precise angle (biophysically speaking) of the different effects (loss of myelin lamella or segments, remyelination with thinner and shorter nodes, etc), while the second model allows to infer the consequences of myelin alterations in working memory performance, including memory stability, duration, and bump diffusion, while also exploring the case of myeling alterations in a novel silent working memory model. The results indicate (i) a slowing down and failure of propagation of spikes with demyelination and partial recovery with remyelination, with detailed predictions on the role of nodes and myelina lamella, and (ii) a decrease in memory duration and an increase in memory drift as a function of the demyelination, in agreement with multiple experimental studies.

Strengths:

Overall, the work offers a very interesting approach of a topic which is hard to accomplish experimentally --therefore the computational take is entirely justified and extremely useful. The authors carefully designed the computational experiments to shed light into the demyelination effects on working memory from multiple levels of description, increasing the reliability of their conclusions. I think this work provides now convincing evidence and has the potential to be influential in future studies of myelin alterations (and related disorders such as multiple sclerosis).

Weaknesses:

In its current form, the authors have improved the clarity of the results and the model details, and have provided a new set of simulations to complement and reinforce the original ones (including the development of a new spatial working memory model based on silent working memory principles). I do not appreciate any significant weaknesses at this point.

Reviewer #2 (Public Review):

This paper analyzes the effect of axon de-myelination and re-myelination on action potential speed, and propagation failure. Next, the findings are then incorporated in a standard spiking ring attractor model of working memory.

I think the results are not very surprising or solid and there are issues with method and presentation.<br /> The authors did many simulations with random parameters, then averaged the result, and found for instance that the Conduction Velocity drops in demyelination. It gives the reader little insight into what is really going on. My personal preference is for a well understood simple model rather than a poorly understood complex model. The link between the model outcome of WM and data remains qualitative and is further weakened by the existence of known other age-related effects in PFC circuits.

Comments on revised version:

The paper has improved in the revision, although I still think a reduced model would have been nice.

Reviewer #1 (Public Review):

Carignano et al propose an extension of the self-returning random walk (SRRW) model for chromatin to include excluded volume aspects and use it to investigate generic local and global properties of the chromosome 3D organization inside eukaryotic nuclei. In particular, they focus on chromatin volumic density, contact probability, and domain size and suggest that their framework can recapitulate several experimental observations and predict the effect of some perturbations.

Strengths:

• The developed methodology is convincing and may offer an alternative - less computationally demanding - framework to investigate the single-cell and population structural properties of 3D genome organization at multiple scales.

• Compared to the previous SRRW model, it allows for investigation of the role of excluded volume locally.

• They perform some experiments to compare with model predictions and show consistency between the two.

Weaknesses:

• The model is a homopolymer model and currently cannot fully account for specific mechanisms that may shape the heterogeneous, complex organization of chromosomes (TAD at specific positions, A/B compartmentalization, promoter-enhancer loops, etc.).

• By construction of their framework, the effect of excluded volume is only local and larger-scale properties for which excluded volume could be a main actor (formation of chromosome territories [Rosa & Everaers, PLoS CB 2009], bottle-brush effects due to loop extrusion [Polovnikov et al, PRX 2023], etc.) cannot be captured.

• Apart from being a computationally interesting approach to generating realistic 3D chromosome organization, the method offers fewer possibilities than standard polymer models (eg, MD simulations) of chromatin (no dynamics, no specific mechanisms, etc.) with likely the same predictive power under the same hypotheses. In particular, authors often claim the superiority of their approach to describing the local chromatin compaction compared to previous polymer models without showing it or citing any relevant references that would show it.

• Comparisons with experiments are solid but are not quantified.

Impact:

Building on the presented framework in the future to incorporate TAD and compartments may offer an interesting model to study the single-cell heterogeneity of chromatin organization. But currently, in this reviewer's opinion, standard polymer modeling frameworks may offer more possibilities.

Reviewer #2 (Public Review):

Summary:

The authors introduce a simple Self Returning Excluded Volume (SR-EV) model to investigate the 3D organization of chromatin. This is a random walk with a probability to self-return accounting for the excluded volume effects. The authors use this method to study the statistical properties of chromatin organization in 3D. They compute contact probabilities, 3D distances, and packing properties of chromatin and compare them with a set of experimental data.

Strengths:

(1) Typically, to generate a polymer with excluded volume interactions, one needs to run long simulations with computationally expensive repulsive potentials like the Weeks-Chanlder-Anderson potential. However, here, instead of performing long simulations, the authors have devised a method where they can grow polymer, enabling quick generation of configurations.

(2) Authors show that the chromatin configurations generated from their models do satisfy many of the experimentally known statistical properties of chromatin. Contact probability scalings and packing properties are comparable with Chromatin Scanning Transmission Electron Microscopy (ChromSTEM)  experimental data from some of the cell types.

Weaknesses:

This can only generate broad statistical distributions. This method cannot generate sequence-dependent effects, specific TAD structures, or compartments without a prior model for the folding parameter alpha. It cannot generate a 3D distance between specific sets of genes. This is an interesting soft-matter physics study. However, the output is only as good as the alpha value one provides as input.

Reviewer #1 (Public Review):

The manuscript describes a GAN-based approach that generates parameters for HH-like channels for multiple C. Elengans neurons. The network is trained on generated data to produce parameter sets that, on the one hand, reproduce voltage responses and IV curves, and on the other hand, are indistinguishable from the ground truth parameters, as tested by the discriminator. It is then shown that these generated parameter sets lead to reasonable reproductions of the recorded responses (but see the section "weaknesses" below for some reservations).

Strengths:

In itself, I find the methodology of high interest, particularly in that it can generate parameter sets to construct models of new recordings at a very low computational cost.

Weaknesses:

Nevertheless, I believe there are some weaknesses in the evaluation of the models that should be addressed before the quality of the methodology can be fully assessed. Firstly, at the methodological level, the authors should provide more clarity on the inverse gradient operation they use, as opposed to just simulating the models, as such an inversion depends not only on the parameters but also on the state of the model. How the state is obtained remains unclear here. Secondly, in the evaluation of their models, the authors could provided more information such as IV curves, as whether these would be accurate is difficult to visually infer from their figures. Thirdly, the authors do not address the question of whether all obtained parameter sets are stable when simulated over longer times, while their figures do include hints that this might not be the case for at least some of their models (e.g. voltage traces that do not converge back to the equilibrium after the stimulus, but rather seem to diverge).

Reviewer #2 (Public Review):

Summary:

Generating biophysically detailed computational models that capture the characteristic physiological properties of biological neurons for diverse cell types is an important and difficult problem in computational neuroscience. One major challenge lies in determining the large number of parameters of such models, which are notoriously difficult to fit into experimental data. Thereby, the computational and energy costs can be significant. The study 'ElectroPhysiomeGAN: Generation of Biophysical Neuron Model Parameters from Recorded Electrophysiological Responses' by Kim et al. describes a computationally efficient approach for predicting model parameters of Hodgkin-Huxley neuron models using Generative Adversarial Networks (GANs) trained on simulation data. The method is applied to generate models for 9 non-spiking neurons in C. elegans based on electrophysiological recordings. While the generated models capture the responses of these neurons to some degree, they generally show significant deviations from the empirically observed responses in important features. While interesting, in its current form, the method has not been demonstrated to generate models that faithfully capture empirically observed responses.

Strengths:

The authors work on an important and difficult problem. A noteworthy strength of their approach is that once trained, the GANs can generate models from new empirical data with very little computational effort. The generated models reproduce the average voltage during current injections reasonably well.

Weaknesses:

Major 1: While the models generated with EP-GAN reproduce the average voltage during current injections reasonably well, the dynamics of the response are not well captured. For example, for the neuron labeled RIM (Figure 2), the most depolarized voltage traces show an initial 'overshoot' of depolarization, i.e. they depolarize strongly within the first few hundred milliseconds but then fall back to a less depolarized membrane potential. In contrast, the empirical recording shows no such overshoot. Similarly, for the neuron labeled AFD, all empirically recorded traces slowly ramp up over time. In contrast, the simulated traces are mostly flat. Furthermore, all empirical traces return to the pre-stimulus membrane potential, but many of the simulated voltage traces remain significantly depolarized, far outside of the ranges of empirically observed membrane potentials. While these deviations may appear small in the Root mean Square Error (RMSE), the only metric used in the study to assess the quality of the models, they likely indicate a large mismatch between the model and the electrophysiological properties of the biological neuron.

Major 2: Other metrics than the RMSE should be incorporated to validate simulated responses against electrophysiological data. A common approach is to extract multiple biologically meaningful features from the voltage traces before, during and after the stimulus, and compare the simulated responses to the experimentally observed distribution of these features. Typically, a model is only accepted if all features fall within the empirically observed ranges (see e.g. https://doi.org/10.1371/journal.pcbi.1002107). However, based on the deviations in resting membrane potential and the return to the resting membrane potential alone, most if not all the models shown in this study would not be accepted.

Major 3: Abstract and introduction imply that the 'ElectroPhysiome' refers to models that incorporate both the connectome and individual neuron physiology. However, the work presented in this study does not make use of any connectomics data. To make the claim that ElectroPhysiomeGAN can jointly capture both 'network interaction and cellular dynamics', the generated models would need to be evaluated for network inputs, for example by exposing them to naturalistic stimuli of synaptic inputs. It seems likely that dynamics that are currently poorly captured, like slow ramps, or the ability of the neuron to return to its resting membrane potential, will critically affect network computations.

Reviewer #1 (Public Review):

Summary:

This valuable study by Wu and Zhou combined neurophysiological recordings and computational modelling to investigate the neural mechanisms that underpin the interaction between sensory evaluation and action selection. The neurophysiological results suggest non-linear modulation of decision-related LIP activity by action selection, but some further analysis would be helpful in order to understand whether these results can be generalised to LIP circuitry or might be dependent on specific spatial task configurations. The authors present solid computational evidence that this might be due to projections from choice target representations. These results are of interest for neuroscientists investigating decision-making.

Strengths:

Wu and Zhou combine awake behaving neurophysiology for a sophisticated, flexible visual-motion discrimination task and a recurrent network model to disentangle the contribution of sensory evaluation and action selection to LIP firing patterns. The correct saccade response direction for preferred motion direction choices is randomly interleaved between contralateral and ipsilateral response targets, which allows the dissociation of perceptual choice from saccade direction.<br /> The neurophysiological recordings from area LIP indicate non-linear interaction between motion categorisation decisions and saccade choice direction.

The careful investigation of a recurrent network model suggests that feedback from choice target representations to an earlier sensory evaluation stage might be the source for this non-linear modulation and that it is an important circuit component for behavioural performance.

The paper presents a possible solution to a central controversy about the role of LIP in perceptual decision-making, but see below.

Weaknesses:

The paper presents a possible solution to a central controversy about the role of LIP in perceptual decision-making. However, the authors could be more clear and upfront about their interpretational framework and potential alternative interpretations.<br /> Centrally, the authors' model and experimental data appears to test only that LIP carries out sensory evaluation in its RFs. The model explicitly parks the representation of choice targets outside the "LIP" module receiving sensory input. The feedback from this separate target representation provides then the non-linear modulation that matches the neurophysiology. However, they ignore the neurophysiological results that LIP neurons can also represent motor planning to a saccade target.<br /> The neurophysiological results with a modulation of the direction tuning by choice direction (contralateral vs ipsilateral) are intriguing. However, the evaluation of the neurophysiological results are difficult, because some of the necessary information is missing to exclude alternative explanations. It would be good to see the actual distributions and sizes of the RF, which were determined based on visual responses not with a delayed saccade task. There might be for example a simple spatial configuration, for example, RF and preferred choice target in the same (contralateral) hemifield, for which there is an increase in firing. It is a shame that we do not see what these neurons would do if only a choice target would be put in the RF, as has been done in so many previous LIP experiments. The authors exclude also some spatial task configurations (vertical direction decisions), which makes it difficult to judge whether these data and models can be generalised. The whole section is difficult to follow, partly also because it appears to mix reporting results with interpretation (e.g. "feedback").

The model and its investigation is very interesting and thorough, but given the neurophysiological literature on LIP, it is not clear that the target module would need to be in a separate brain area, but could be local circuitry within LIP between different neuron types.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors recorded activity in the posterior parietal cortex (PPC) of monkeys performing a perceptual decision-making task. The monkeys were first shown two choice dots of two different colors. Then, they saw a random dot motion stimulus. They had to learn to categorize the direction of motion as referring to either the right or left dot. However, the rule was based on the color of the dot and not its location. So, the red dot could either be to the right or left, but the rule itself remained the same. It is known from past work that PPC neurons would code the learned categorization. Here, the authors showed that the categorization signal depended on whether the executed saccade was in the same hemifield as the recorded PPC neuron or in the opposite one. That is, if a neuron categorized the two motion directions such that it responded stronger for one than the other, then this differential motion direction coding effect was amplified if the subsequent choice saccade was in the same hemifield. The authors then built a computational RNN to replicate the results and make further tests by simulated "lesions".

Strengths:

Linking the results to RNN simulations and simulated lesions.

Weaknesses:

Potential interpretational issues due to a lack of evidence on what happens at the time of the saccades.

Reviewer #1 (Public Review):

Summary:

Two important factors in visual performance are the resolving power of the lens and the signal-to-noise ratio of the photoreceptors. These both compete for space: a larger lens has improved resolving power over a smaller one, and longer photoreceptors capture more photons and hence generate responses with lower noise. The current paper explores the tradeoff of these two factors, asking how space should be allocated to maximize eye performance (measured as encoded information).

Strengths:

The topic of the paper is interesting and not well studied. The approach is clearly described and seems appropriate (with a few exceptions - see weaknesses below). In most cases, the parameter space of the models are well explored and tradeoffs are clear.

Weaknesses:

- Light level<br /> The calculations in the paper assume high light levels (which reduces the number of parameters that need to be considered). The impact of this assumption is not clear. A concern is that the optimization may be quite different at lower light levels. Such a dependence on light level could explain why the model predictions and experiment are not in particularly good agreement. The paper would benefit from exploring this issue.

- Discontinuities<br /> The discontinuities and non-monotonicity of the optimal parameters plotted in Figure 4 are concerning. Are these a numerical artifact? Some discussion of their origin would be quite helpful.

- Discrepancies between predictions and experiment<br /> As the authors clearly describe, experimental measurements of eye parameters differ systematically from those predicted. This makes it difficult to know what to take away from the paper. The qualitative arguments about how resources should be allocated are pretty general, and the full model seems a complex way to arrive at those arguments. Could this reflect a failure of one of the assumptions that the model rests on - e.g. high light levels, or that the cost of space for photoreceptors and optics is similar? Given these discrepancies between model and experiment, it is also hard to evaluate conclusions about the competition between optics and photoreceptors (e.g. at the end of the abstract) and about the importance for evolution (end of introduction).

Reviewer #2 (Public Review):

Summary:

In short, the paper presents a theoretical framework that predicts how resources should be optimally distributed between receptors and optics in eyes.

Strengths:

The authors build on the principle of resource allocation within an organism and develop a formal theory for optimal distribution of resources within an eye between the receptor array and the optics. Because the two parts of eyes, receptor arrays and optics, share the same role of providing visual information to the animal it is possible to isolate these from resource allocation in the rest of the animal. This allows for a novel and powerful way of exploring the principles that govern eye design. By clever and thoughtful assumptions/constraints, the authors have built a formal theory of resource allocation between the receptor array and the optics for two major types of compound eye as well as for camera-type eyes. The theory is formalized with variables that are well characterized in a number of different animal eyes, resulting in testable predictions.

The authors use the theory to explain a number of design features that depend on different optimal distribution of resources between the receptor array and the optics in different types of eyes. As an example, they successfully explain why eye regions with different spatial resolution should be built in different ways. They also explain differences between different types of eyes, such as long photoreceptors in apposition compound eyes and much shorter receptors in camera type eyes. The predictive power in the theory is impressive.

To keep the number of parameters at a minimum, the theory was developed for two types of compound eye (neural superposition, and apposition) and for camera-type eyes. It is possible to extend the theory to other types of eyes, although it would likely require more variables and assumptions/constraints to the theory. It is thus good to introduce the conceptual ideas without overdoing the applications of the theory.

The paper extends a previous theory, developed by the senior author, that develops performance surfaces for optimal cost/benefit design of eyes. By combining this with resource allocation between receptors and optics, the theoretical understanding of eye design takes a major leap and provides entirely new sets of predictions and explanations for why eyes are built the way they are.

The paper is well written and even though the theory development in the Results may be difficult to take in for many biologists, the Discussion very nicely lists all the major predictions under separate headings, and here the text is more tuned for readers that are not entirely comfortable with the formalism of the Results section. I must point out though that the Results section is kept exemplary concise. The figures are excellent and help explain concepts that otherwise may go above the head of many biologists.

Reviewer #3 (Public Review):

Summary:

This is a proposal for a new theory for the geometry of insect eyes. The novel cost-benefit function combines the cost of the optical portion with the photoreceptor portion of the eye. These quantities are put on the same footing using a specific (normalized) volume measure, plus an energy factor for the photoreceptor compartment. An optimal information transmission rate then specifies each parameter and resource allocation ratio for a variable total cost. The elegant treatment allows for comparison across a wide range of species and eye types. Simple eyes are found to be several times more efficient across a range of eye parameters than neural superposition eyes. Some trends in eye parameters can be explained by optimal allocation of resources between the optics and photoreceptors compartments of the eye.

Strengths:

Data from a variety of species roughly align with rough trends in the cost analysis, e.g. as a function of expanding the length of the photoreceptor compartment.

New data could be added to the framework once collected, and many species can be compared.

Eyes of different shapes are compared.

Weaknesses:

Detailed quantitative conclusions are not possible given the approximations and simplifying assumptions in the models and poor accounting for trends in the data across eye types.

Reviewer #1 (Public Review):

Summary:

This study uses an online cognitive task to assess how reward and effort are integrated in a motivated decision-making task. In particular the authors were looking to explore how neuropsychiatric symptoms, in particular apathy and anhedonia, and circadian rhythms affect behavior in this task. Amongst many results, they found that choice bias (the degree to which integrated reward and effort affects decisions) is reduced in individuals with greater neuropsychiatric symptoms, and late chronotypes (being an 'evening person').

Strengths:

The authors recruited participants to perform the cognitive task both in and out of sync with their chronotypes, allowing for the important insight that individuals with late chronotypes show a more reduced choice bias when tested in the morning.<br /> Overall, this is a well-designed and controlled online experimental study. The modelling approach is robust, with care being taken to both perform and explain to the readers the various tests used to ensure the models allow the authors to sufficiently test their hypotheses.

Weaknesses:

This study was not designed to test the interactions of neuropsychiatric symptoms and chronotypes on decision making, and thus can only make preliminary suggestions regarding how symptoms, chronotypes and time-of-assessment interact.

Reviewer #2 (Public Review):

Summary:

The study combines computational modeling of choice behavior with an economic, effort-based decision-making task to assess how willingness to exert physical effort for a reward varies as a function of individual differences in apathy and anhedonia, or depression, as well as chronotype. They find an overall reduction in effort selection that scales with apathy and anhedonia and depression. They also find that later chronotypes are less likely to choose effort than earlier chronotypes and, interestingly, an interaction whereby later chronotypes are especially unwilling to exert effort in the morning versus the evening.

Strengths:

This study uses state-of-the-art tools for model fitting and validation and regression methods which rule out multicollinearity among symptom measures and Bayesian methods which estimate effects and uncertainty about those estimates. The replication of results across two different kinds of samples is another strength. Finally, the study provides new information about the effects not only of chronotype but also chronotype by timepoint interactions which are previously unknown in the subfield of effort-based decision-making.

Weaknesses:

The study has few weaknesses. One potential concern is that the range of models which were tested was narrow, and other models might have been considered. For example, the Authors might have also tried to fit models with an overall inverse temperature parameter to capture decision noise. One reason for doing so is that some variance in the bias parameter might be attributed to noise, which was not modeled here. Another concern is that the manuscripts discuss effort-based choice as a transdiagnostic feature - and there is evidence in other studies that effort deficits are a transdiagnostic feature of multiple disorders. However, because the present study does not investigate multiple diagnostic categories, it doesn't provide evidence for transdiagnosticity, per se.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Mehrhof and Nord study a large dataset of participants collected online (n=958 after exclusions) who performed a simple effort-based choice task. They report that the level of effort and reward influence choices in a way that is expected from prior work. They then relate choice preferences to neuropsychiatric syndromes and, in a smaller sample (n<200), to people's circadian preferences, i.e., whether they are a morning-preferring or evening-preferring chronotype. They find relationships between the choice bias (a model parameter capturing the likelihood to accept effort-reward challenges, like an intercept) and anhedonia and apathy, as well as chronotype. People with higher anhedonia and apathy and an evening chronotype are less likely to accept challenges (more negative choice bias). People with an evening chronotype are also more reward sensitive and more likely to accept challenges in the evening, compared to the morning.

Strengths:

This is an interesting and well-written manuscript which replicates some known results and introduces a new consideration related to potential chronotype relationships which have not been explored before. It uses a large sample size and includes analyses related to transdiagnostic as well as diagnostic criteria. I have some suggestions for improvements.

Weaknesses:

(1) The novel findings in this manuscript are those pertaining to transdiagnostic and circadian phenotypes. The authors report two separate but "overlapping" effects: individuals high on anhedonia/apathy are less willing to accept offers in the task, and similarly, individuals tested off their chronotype are less willing to accept offers in the task. The authors claim that the latter has implications for studying the former. In other words, because individuals high on anhedonia/apathy predominantly have a late chronotype (but might be tested early in the day), they might accept less offers, which could spuriously look like a link between anhedonia/apathy and choices but might in fact be an effect of the interaction between chronotype and time-of-testing. The authors therefore argue that chronotype needs to be accounted for when studying links between depression and effort tasks.<br /> The authors argue that, if X is associated with Y and Z is associated with Y, X and Z might confound each other. That is possible, but not necessarily true. It would need to be tested explicitly by having X (anhedonia/apathy) and Z (chronotype) in the same regression model. Does the effect of anhedonia/apathy on choices disappear when accounting for chronotype (and time-of-testing)? Similarly, when adding the interaction between anhedonia/apathy, chronotype, and time-of-testing, within the subsample of people tested off their chronotype, is there a residual effect of anhedonia/apathy on choices or not?<br /> If the effect of anhedonia/apathy disappeared (or got weaker) while accounting for chronotype, this result would suggest that chronotype mediates the effect of anhedonia/apathy on effort choices. However, I am not sure it renders the direct effect of anhedonia/apathy on choices entirely spurious. Late chronotype might be a feature (induced by other symptoms) of depression (such as fatigue and insomnia), and the association between anhedonia/apathy and effort choices might be a true and meaningful one. For example, if the effect of anhedonia/apathy on effort choices was mediated by altered connectivity of the dorsal ACC, we would not say that ACC connectivity renders the link between depression and effort choices "spurious", but we would speak of a mechanism that explains this effect. The authors should discuss in a more nuanced way what a significant mediation by the chronotype/time-of-testing congruency means for interpreting effects of depression in computational psychiatry.

(2) It seems that all key results relate to the choice bias in the model (as opposed to reward or effort sensitivity). It would therefore be helpful to understand what fundamental process the choice bias is really capturing in this task. This is not discussed, and the direction of effects is not discussed either, but potentially quite important. It seems that the choice bias captures how many effortful reward challenges are accepted overall which maybe captures general motivation or task engagement. Maybe it is then quite expected that this could be linked with questionnaires measuring general motivation/pleasure/task engagement. Formally, the choice bias is the constant term or intercept in the model for p(accept), but the authors never comment on what its sign means. If I'm not mistaken, people with higher anhedonia but also higher apathy are less likely to accept challenges and thus engage in the task (more negative choice bias). I could not find any discussion or even mention of what these results mean. This similarly pertains to the results on chronotype. In general, "choice bias" may not be the most intuitive term and the authors may want to consider renaming it. Also, given the sign of what the choice bias means could be flipped with a simple sign flip in the model equation (i.e., equating to accepting more vs accepting less offers), it would be helpful to show some basic plots to illustrate the identified differences (e.g., plotting the % accepted for people in the upper and lower tertile for the SHAPS score etc).

(3) None of the key effects relate to effort or reward sensitivity which is somewhat surprising given the previous literature and also means that it is hard to know if choice bias results would be equally found in tasks without any effort component. (The only analysis related to effort sensitivity is exploratory and in a subsample of N=56 per group looking at people meeting criteria for MDD vs matched controls.) Were stimuli constructed such that effort and reward sensitivity could be separated (i.e., are uncorrelated/orthogonal)? Maybe it would be worth looking at the % accepted in the largest or two largest effort value bins in an exploratory analysis. It seems the lowest and 2nd lowest effort level generally lead to accepting the challenge pretty much all the time, so including those effort levels might not be sensitive to individual difference analyses?

(4) The abstract and discussion seem overstated (implications for the school system and statements on circadian rhythms which were not measured here). They should be toned down to reflect conclusions supported by the data.

Reviewer #1 (Public Review):

Summary:

This manuscript by Shea and Villeda furnishes the field with a valuable scRNAseq data set detailing microglial aging in the mouse hippocampus. They provide clear evidence that changes in microglial attributes begin in mid-life, well before time points when mice are traditionally considered to be "aging." It also adds to a growing body of data in the field demonstrating that there is substantial heterogeneity in microglial responses to aging. Using in vitro experiments and transgenic manipulations in mice, the authors show that transforming growth factor beta (TGFb1)-based signaling can potently impact microglial state, consistent with previous findings in the field. They also demonstrate that manipulation of microglial TGFb1-based signaling can impact hippocampus-dependent behaviors.

Limitations of the study lie primarily in reaching too far with interpretations of the data. The authors argue that changes in microglial transcriptome during midlife represent a type of "checkpoint," after which microglial aging can progress along distinct trajectories depending on the status of TGFb1 signaling. They also posit that a specific intermediate "stress response" state in midlife is mechanistically linked to a translational burst that drives the subsequent progression of microglia to an "inflammatory state." Unequivocal data to support these causal links is lacking, however. similarly, key additional experiments would be needed to demonstrate that TGFb1 signaling and microglial progression through these identified intermediate states are causally linked to cognitive decline.

Guidance for readers along with study strengths and caveats:

The present manuscript provides valuable strengthening and expansion to a growing body of data showing prominent changes in the microglial state during aging. Microarray(1), bulkRNAseq(2-5), scRNAseq(6,7), snRNAseq(8,9), and spatial transcriptomic(10) approaches have been leveraged to map changes in microglial transcriptome during aging in rodents, non-human primates, and humans. A number of these studies include the hippocampus (1,8,9,11) and have highlighted variation across brain regions in microglial transcriptomic changes during aging (1,11). They have also revealed differences across sex (7) as well as increased cell-to-cell heterogeneity (6-10), consistent with the idea that individual microglia can follow distinct aging trajectories. Several of these studies revealed that changes in microglial attributes begin in middle age (1,7,11), supporting similar observations from studies that did not use omics (12-14). The present manuscript utilizes scRNAseq of hippocampal microglia at adulthood (6mo), middle age (12mo), late middle age (18mo) and aging (24mo) to show that aging-induced changes in microglia begin in middle age and that microglia exhibit ample phenotypic heterogeneity during the progression of aging.

To gain further insight into the dynamics of microglial aging in the hippocampus, the authors used a bioinformatics method known as "pseudotime" or "trajectory inference" to understand how cells may progress through different functional states, as defined by cellular transcriptome (15,16). These bioinformatics approaches can reveal key patterns in scRNAseq / snRNAseq datasets and, in the present study, the authors conclude that a "stress response" module characterized by expression of TGFb1 represents a key "checkpoint" in microglial aging in midlife, after which the cells can move along distinct transcriptional trajectories as aging progresses. This is an intriguing possibility. However, pseudotime analyses need to be validated via additional bioinformatics as well as follow-up experiments. Indeed, Heumos et al, in their Nature Genetics "Expert Guidelines" Review, emphasize that "inferred trajectories might not necessarily have biological meaning." They recommend that "when the expected topology is unknown, trajectories and downstream hypotheses should be confirmed by multiple trajectory inference methods using different underlying assumptions."(15) Numerous algorithms are available for trajectory inference (e.g. Monocle, PAGA, Sligshot, RaceID/StemID, among many others) and their performance and suitability depends on the individual dataset and nature of the trajectories that are to be inferred. It is recommended to use dynGuidelines(16) for the selection of optimal pseudotime analysis methods. In the present manuscript, the authors do not provide any justification for their use of Monocle 3 over other trajectory inference approaches, nor do they employ a secondary trajectory inference method to confirm observations made with Monocle 3. Finally, follow-up validation experiments that the authors carry out have their own limitations and caveats (see below). Hence, while the microglial aging trajectories identified by this study are intriguing, they remain hypothetical trajectories that need to be proven with additional follow-up experiments.

To follow up on the idea that TGFb1 signaling in microglia plays a key role in determining microglial aging trajectories, the authors use RNAscope to show that TGFb1 levels in microglia peak in middle age. They also treat primary LPS-activated microglia with TGFb1 and show that this restores expression of microglial homeostatic gene expression and dampens expression of stress response and, potentially, inflammatory genes. Finally, they utilize transgenic approaches to delete TGFb1 from microglia around 8-10mo of age and scRNAseq to show that homeostatic signatures are lost and inflammatory signatures are gained. Hence, findings in this study support the idea that TGFb1 can strongly regulate microglial phenotype. Loss of TGFb1 signaling to microglia in adulthood has already been shown to cause decreased microglial morphological complexity and upregulation of genes typically associated with microglial responses to CNS insults(17-19). TGFb1 signaling to microglia has also been implicated in microglial responses to disease and manipulations to increase this signaling can improve disease progression in some cases(19). In this light, the findings in the present study are largely confirmatory of previous findings in the literature. They also fall short of unequivocally demonstrating that TGFb1 signaling acts as a "checkpoint" for determining subsequent microglial aging trajectory. To show this clearly, one would need to perturb TGFb1 signaling around 12mo of age and carry out sequencing (bulkRNAseq or scRNAseq) of microglia at 18mo and 24mo. Such experiments could directly demonstrate whether the whole microglial population has been diverted to the TGFb1-low aging trajectory (that progresses through a translational burst state to an inflammation state as proposed). Future development of tools to tag TGFb1 high or low microglia could also enable fate tracing type experiments to directly show whether the TGFb1 state in middle age predicts cell state at later phases of aging.

The present study would also like to draw links between features of microglial aging in the hippocampus and a decline in hippocampal-dependent cognition during aging. To this end, they carry out behavioral testing in 8-10mo old mice that have undergone microglial-specific TGFb1 deletion and find deficits in novel object recognition and contextual fear conditioning. While this provides compelling evidence that TGFb1 signaling in microglia can impact hippocampus-dependent cognition in midlife, it does not demonstrate that this signaling accelerates or modulates cognitive decline (see below). Age-associated cognitive decline refers to cognitive deficits that emerge as a result of the normative brain aging process(20-21). For a cognitive deficit to be considered age-associated cognitive decline, it must be shown that the cognitive operation under study was intact at some point earlier in the adult lifespan. This requires longitudinal study designs that determine whether a manipulation impacts the relationship between brain status and cognition as animals age (22-24). Alternatively, cross-sectional studies with adequate sample sizes can be used to sample the variability in cognitive outcomes at different points of the adult lifespan(22-24) and show that this is altered by a particular manipulation. For this specific study, one would ideally demonstrate that hippocampal-based learning/memory was intact at some point in the lifespan of mice with microglial TGFb1 KO but that this manipulation accelerated or exacerbated the emergence of deficits in hippocampal-dependent learning/memory during aging. In the absence of these types of data, the authors should tone down their claims that they have identified a cellular and molecular mechanism that contributes to cognitive decline.

A final point of clarification for the reader pertains to the mining of previously generated data sets within this study. The language in the results section, methods, and figure legends causes confusion about which experiments were actually carried out in this study versus previous studies. Some of the language makes it sound as though parabiosis experiments and experiments using mouse models of Alzheimer's Disease were carried out in this study. However, parabiosis and AD mouse model experiments were executed in previous studies (25,26), and in the present study, RNAseq datasets were accessed for targeted data mining. It is fantastic to see further mining of datasets that already exist in the field. However, descriptions in the results and methods sections need to make it crystal clear that this is what was done.

References:

(1) Grabert, K. et al. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat. Neurosci. (2016). doi:10.1038/nn.4222<br /> (2) Hickman, S. E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. (2013). doi:10.1038/nn.3554<br /> (3) Deczkowska, A. et al. Mef2C restrains microglial inflammatory response and is lost in brain ageing in an IFN-I-dependent manner. Nat. Commun. (2017). doi:10.1038/s41467-017-00769-0<br /> (4) O'Neil, S. M., Witcher, K. G., McKim, D. B. & Godbout, J. P. Forced turnover of aged microglia induces an intermediate phenotype but does not rebalance CNS environmental cues driving priming to immune challenge. Acta Neuropathol. Commun. (2018). doi:10.1186/s40478-018-0636-8<br /> (5) Olah, M. et al. A transcriptomic atlas of aged human microglia. Nat. Commun. (2018). doi:10.1038/s41467-018-02926-5<br /> (6) Hammond, T. R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50, 253-271 (2019).<br /> (7) Li, X. et al. Transcriptional and epigenetic decoding of the microglial aging process. Nat. aging 3, 1288-1311 (2023).<br /> (8) Zhang, H. et al. Single-nucleus transcriptomic landscape of primate hippocampal aging. Protein Cell 12, 695-716 (2021).<br /> (9) Su, Y. et al. A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan. Cell Stem Cell 29, 1594-1610.e8 (2022).<br /> (10) Allen, W. E., Blosser, T. R., Sullivan, Z. A., Dulac, C. & Zhuang, X. Molecular and spatial signatures of mouse brain aging at single-cell resolution. Cell 186, 194-208.e18 (2023).<br /> (11) Soreq, L. et al. Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging. Cell Rep. 18, 557-570 (2017).<br /> (12) Hefendehl, J. K. et al. Homeostatic and injury-induced microglia behavior in the aging brain. Aging Cell (2014). doi:10.1111/acel.12149<br /> (13) Nikodemova, M. et al. Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week. J. Neuroimmunol. 0, 280-288 (2015).<br /> (14) Moca, E. N. et al. Microglia Drive Pockets of Neuroinflammation in Middle Age. J. Neurosci. 42, 3896-3918 (2022).<br /> (15) Heumos, L. et al. Best practices for single-cell analysis across modalities. Nat. Rev. Genet. 24, 550-572 (2023).<br /> (16) Saelens, W., Cannoodt, R., Todorov, H. & Saeys, Y. A comparison of single-cell trajectory inference methods: towards more accurate and robust tools. (2018). doi:10.1101/276907<br /> (17) Zöller, T. et al. Silencing of TGFβ signalling in microglia results in impaired homeostasis. Nat. Commun. 9, (2018).<br /> (18) Bedolla, A. et al. Microglia-derived TGF-β1 ligand maintains microglia homeostasis via autocrine mechanism and is critical for normal cognitive function in adult mouse brain. bioRxiv Prepr. Serv. Biol. (2023). doi:10.1101/2023.07.05.547814<br /> (19) Spittau, B., Dokalis, N. & Prinz, M. The Role of TGFβ Signaling in Microglia Maturation and Activation. Trends Immunol. 41, 836-848 (2020).<br /> (20) L. Nyberg, M. Lövdén, K. Riklund, U. Lindenberger, L. Bäckman, Memory aging and brain maintenance. Trends Cogn. Sci. 16, 292-305 (2012).<br /> (21) L. Luo, F. I. M. Craik, Aging and memory: A cognitive approach. Can. J. Psychiatry 53, 346-353 (2008).<br /> (22) Y. Stern, M. Albert, C. Barnes, R. Cabeza, A. Pascual-Leone, P. Rapp.<br /> A framework for concepts of reserve and resilience in aging. Neurobiol. Aging, 124 (2022), pp. 100-103, 10.1016/j.neurobiolaging.2022.10.015<br /> (23) Y. Stern, C.A. Barnes, C. Grady, R.N. Jones, N. Raz. Brain reserve, cognitive reserve, compensation, and maintenance: operationalization, validity, and mechanisms of cognitive resilience. Neurobiol. Aging, 83 (2019), pp. 124-129, 10.1016/j.neurobiolaging.2019.03.022<br /> (24) R. Cabeza, M. Albert, S. Belleville, F.I.M. Craik, A. Duarte, C.L. Grady, U. Lindenberger, L. Nyberg, D.C. Park, P.A. Reuter-Lorenz, M.D. Rugg, J. Steffener, M.N. Rajah. Maintenance, reserve and compensation: the cognitive neuroscience of healthy ageing. Nat. Rev. Neurosci., 19 (11) (2018), Article 11, 10.1038/s41583-018-0068-2<br /> (25) Palovics, R. et al molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature 603, 309-314 (2022)<br /> (26) Sala Frigerio, C. et al. The major risk factors for Alzheimer's Disease: age, sex, and genes modulate the microglial response to Abeta plaques. Cell Rep, 27, 1293-1306 (2019)

Reviewer #2 (Public Review):

Summary:

The goal of the paper was to trace the transitions hippocampal microglia undergo along aging. ScRNA-seq analysis allowed the authors to predict a trajectory and hypothesize about possible molecular checkpoints, which keep the pace of microglial aging. E.g. TGF1b was predicted as a molecule slowing down the microglial aging path and indeed, loss of TGF1 in microglia led to premature microglia aging, which was associated with premature loss of cognitive ability. The authors also used the parabiosis model to show how peripheral, blood-derived signals from the old organism can "push" microglia forward on the aging path.

Strengths:

A major strength and uniqueness of this work is the in-depth single-cell dataset, which may be a useful resource for the community, as well as the data showing what happens to young microglia in heterochronic parabiosis setting and upon loss of TGFb in their environment.

Weaknesses:

That said, given what we recently learned about microglia isolation for RNA-seq analysis, there is a danger that some of the observations are a result of not age, but cell stress from sample preparation (enzymatic digestion 10min at 37C; e.g. PMID: 35260865). Changes in cell state distribution along aging were made based on scRNA-seq and were not corroborated by any other method, such as imaging of cluster-specific marker expression in microglia at different ages. This analysis would allow confirming the scRNA-seq data and would also give us an idea of where the subsets are present within the hippocampus, and whether there is any interesting distribution of cell states (e.g. some are present closer to stem cells?). Since TGFb is thought to be crucial to microglia biology, it would be valuable to include more analysis of the mice with microglia-specific Tgfb deletion e.g. what was the efficiency of recombination in microglia? Did their numbers change after induction of Tgfb deletion in Cx3cr1-creERT2::Tgfb-flox mice.

Overall:

In general, I think the authors did a good job following the initial observations and devised clever ways to test the emerging hypotheses. The resulting data are an important addition to what we know about microglial aging and can be fruitfully used by other researchers, e.g. those working on microglia in a disease context.

Reviewer #1 (Public Review):

Summary:

This manuscript by Vogt et al examines how the synaptic composition of AMPA and NMDA receptors changes over sleep and wake states. The authors perform whole-cell patch clamp recordings to quantify changes in silent synapse numbers across conditions of spontaneous sleep, sleep deprivation, and recovery sleep after deprivation. They also perform single nucleus RNAseq to identify transcriptional changes related to AMPA/NMDA receptor composition following spontaneous sleep and sleep deprivation. The findings of this study are consistent with a decrease in silent synapse number during wakefulness and an increase during sleep. However, these changes cannot be conclusively linked to sleep/wake states. Measurements were performed in the motor cortex, and sleep deprivation was achieved by forced locomotion, raising the possibility that recent levels of neuronal activity/induction of plasticity, rather than sleep/wake states, are responsible for the observed results.

Strengths:

This study examines an important question. Glutamatergic synaptic transmission has been a focus of studies in the sleep field, but AMPA receptor function has been the primary target of these studies. Silent synapses, which contain NMDA receptors but lack AMPA receptors, have important functional consequences for the brain. Exploring the role of sleep in regulating silent synapse numbers is important to understanding the role of sleep in brain function. The electrophysiological approach of measuring the failure rate ratio, supported by AMPA/NMDA ratio measurements, is a rigorous tool to evaluate silent synapse numbers.

The authors also perform snRNAseq to identify genes differentially expressed in the spontaneous sleep and sleep deprivation groups. This analysis reveals an intriguing pattern of upregulated genes controlled by HDAC4 and Mef2c, along with synaptic shaping component genes and genes associated with autism spectrum disorder, across cell types in the sleep deprivation group. This unbiased approach identifies candidate genes for follow-up studies.

Weaknesses:

A major weakness of this study is the experimental design. Measurements are made from the motor cortex, and sleep deprivation was achieved using forced locomotion on a treadmill. Therefore, the effects observed could be due to recent high levels of activity or plasticity induction in the motor cortex from locomotion, rather than lack of sleep per se. In support of this interpretation, other groups have failed to find a difference in AMPA/NMDA ratio in mice with different spontaneous sleep/wake histories, although sleep deprivation was not performed (Bridi et al., Neuron 2020).

The electrophysiological measurements are problematic in several ways. First, the methods lack crucial details such as inclusion/exclusion criteria for each cell based on input and series resistance, stability of input/series resistance, polysynaptic responses, etc. that make it difficult to interpret the data. The holding potential (-90mV) used for AMPA receptor current recordings is much more hyperpolarized than typically used for these measurements. The statistical analysis of these experiments is also problematic. The number of mice used is low (3/group) and more should be added to account for inter-animal variability. Comparing the raw data with the statistical tests in supplementary table 1 (FR ratio), it appears that a data point has been dropped from the analysis, but it is unclear why. In addition, a false discovery rate (FDR) correction for multiple comparisons is used to evaluate group differences following the ANOVAs. Correcting for the FDR is less stringent and is typically used when a large number of hypotheses are tested and false positives are more acceptable. In this analysis, few comparisons are made, and the standard approach of correcting for the family-wise error rate is more appropriate.

The snRNAseq data are intriguing, but a more thorough discussion of the candidate genes and pathways that are upregulated during sleep deprivation is warranted. Several genes relevant to the AMPA/NMDA ratio are mentioned, but upregulation of most of these genes would not be expected to increase the AMPA/NMDA ratio based on the literature cited. The model presented in Figure 4C is not consistent with the data (e.g. many candidate genes could alter NMDAR function without receptor insertion/removal), and it is unclear how the current study fits into the model presented in 4D.

Reviewer #2 (Public Review):

Summary:

Here Vogt et al., provide new insights into the need for sleep and the molecular and physiological response to sleep loss. The authors expand on their previously published work (Bjorness et al., 2020) and draw from recent advances in the field to propose a neuron-centric molecular model for the accumulation and resolution of sleep need and the basis of restorative sleep function. While speculative, the proposed model successfully links important observations in the field and provides a framework to stimulate further research and advances on the molecular basis of sleep function. In my review, I highlight the important advances of this current work, and the clear merits of the proposed model, and indicate areas of the model that can serve to stimulate further investigation.

Strengths:

Reviewer comment on new data in Vogt et al., 2024<br /> Using classic slice electrophysiology, the authors conclude that wakefulness (sleep deprivation (SD)) drives a potentiation of excitatory glutamate synapses, mediated in large part by "un-silencing" of NMDAR-active synapses to AMPAR-active synapses. Using a modern single nuclear RNAseq approach the authors conclude that SD drives changes in gene expression primarily occurring in glutamatergic neurons. The two experiments combined highlight the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons. This view is entirely consistent with a large body of extant and emerging literature and provides important direction for future research.

Consistent with prior work, wakefulness/SD drives an LTP-type potentiation of excitatory synaptic strength on principle cortical neurons. It has been proposed that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity. This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon by introducing the concept of silent synapses. The new data show that in mice well rested, a substantial number of synapses are "silent", containing an NMDAR component but not AMPARs. Silent synapses provide a type of reservoir for learning in that activity can drive the un-silencing, increasing the number of functional synapses. SD depletes this reservoir of silent synapses to essentially zero, explaining how SD can exhaust learning capacity. Recovery sleep led to restoration of silent synapses, explaining how recovery sleep can renew learning capacity. In their prior work (Bjorness et al., 2020) this group showed that SD drives an increase in mEPSC frequency onto these same cortical neurons, but without a clear change in pre-synaptic release probability, implying a change in the number of functional synapses. This prediction is now born out in this new dataset.

The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies. First, this conclusion is corroborated by an independent, contemporary snRNAseq analysis recently available as a pre-print (Ford et al., 2023 BioRxiv https://doi.org/10.1101/2023.11.28.569011). A recently published analysis on the effects of SD in drosophila imaged synapses in every brain region in a cell-type dependent manner (Weiss et al., PNAS 2024), concluding that SD drives brain wide increases in synaptic strength almost exclusively in excitatory neurons. Further, Kim et al., Nature 2022, heavily cited in this work, show that the newly described SIK3-HDAC4/5 pathway promotes sleep depth via excitatory neurons and not inhibitory neurons.

The new experiments provided in Fig1-3 are expertly conducted and presented. This reviewer has no comments of concern regarding the execution and conclusions of these experiments.

Reviewer comment on the model in Vogt et al., 2024

In the view of this reviewer the new model proposed by Vogt et al., is an important contribution. The model is not definitively supported by new data, and in this regard should be viewed as a perspective, providing mechanistic links between recent molecular advances, while still leaving areas that need to be addressed in future work. New snRNAseq analysis indicates that SD drives the expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function. SD-induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes. As pointed out by the authors, sleep problems are commonly reported in ASD, but the emphasis has been on sleep amount. This new analysis highlights the need to understand the impact on sleep's functional output (synapses) to fully understand the role of sleep problems in ASD.

Importantly, SD-induced gene expression in excitatory neurons overlaps with genes regulated by the transcription factor MEF2C and HDAC4/5 (Figure 4). In their prior work, the authors show loss of MEF2C in excitatory neurons abolished the SD transcriptional response and the functional recovery of synapses from SD by recovery sleep. Recent advances identified HDAC4/5 as major regulators of sleep depth and duration (in excitatory neurons) downstream of the recently identified sleep-promoting kinase SIK3. In Zhou et al., and Kim et al., Nature 2022, both groups propose a model whereby "sleep-need" signals from the synapse activate SIK3, which phosphorylates HDAC4/5, driving cytoplasmic targeting, allowing for the de-repression and transcriptional activation of "sleep genes". Prior work shows that HDAC4/5 are repressors of MEF2C. Therefore, the "sleep genes" derepressed by HDAC4/5 may be the same genes activated in response to SD by MEF2C. The new model thereby extends the signaling of sleep need at synapses (through SIK3-HDAC4/5) to the functional output of synaptic recovery by expression of synaptic/sleep genes by MEF2C. The model thereby links aspects of the expression of sleep need with the resolution of sleep need by mediating sleep function: synapse renormalization.

Weaknesses:

Areas for further investigation

In the discussion section Vogt et al., explore the links between excitatory synapse strength, arguably the major target of "sleep function", and NREM slow-wave activity (SWA), the most established marker of sleep need. SIK3-HDAC4/5 have major effects on the "depth" of sleep by regulating NREM-SWA. The effects of MEF2C loss of function on NREM SWA activity are less obvious, but clearly impact the recovery of glutamatergic synapses from SD. The authors point out how adenosine signaling is well established as a mediator of SWA, but the links between adenosine and glutamatergic strength are far from clear. The mechanistic links between SIK3/HDAC4/5, adenosine signaling, and MEF2C, are far from understood. Therefore, the molecular/mechanistic links between a synaptic basis of sleep need and resolution with NREM-SWA activity require further investigation.

Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity. The authors point out that constitutively nuclear (cn) HDAC4/5 (acting as a repressor) will mimic MEF2C loss of function. This is reasonable, however, there are notable differences in the reported phenotypes of each. Notably, cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020). These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes. Likely HDAC4/5 have functionally important interactions with other transcription factors, and likewise for MEF2C, suggesting areas for future analysis.

One emerging theme may be that the SIK3-HDAC4/5 axis is a major regulator of the sleep state, perhaps stabilizing the NREM state once the transition from wakefulness occurs. MEF2C is less involved in regulating sleep per se, and more involved in executing sleep function, by promoting restorative synaptic modifications to resolve sleep need.

Finally, advances in the roles of the respective SIK3-HDAC4/5 and MEF2C pathways point towards transcription of "sleep genes", as clearly indicated in the model of Figure 4. Clearly, more work is needed to understand how the expression of such genes ultimately leads to the resolution of sleep need by functional changes at synapses. What are these sleep genes and how do they mechanistically resolve sleep need? Thus, the current work provides a mechanistic framework to stimulate further advances in understanding the molecular basis for sleep need and the restorative basis of sleep function.

Reviewer #1 (Public Review):

The study reports that STN neurons recorded while monkeys performed a random-dot motion task show diverse activation timecourses relative to task events and dependencies on coherence, reaction time, and saccade-choice direction. Different neuron types could be grouped into functional subpopulations, e.g., coherence sensitivity emerging early only in choice-coding neurons. Clustering techniques identified three functionally defined neuron clusters whose dynamic activity profiles related to computational predictions of different decision models in the literature. Microstimulation at different STN recording sites affected behavioral performance in varying but well-conceptualized ways that were captured by the parameters of drift-diffusion models and related to the presence of STN functional clusters at recording sites. The authors conclude that their results validate key aspects of decision models and identify novel aspects of decision-related STN activity.

This is an interesting and high-quality paper that will be of interest across computational and decision neuroscience fields. The recordings and data analyses seem carefully conducted. The study has an attractive theoretical starting point of three specific computational signals that are then mapped onto identified neuron clusters. The combination of single-cell recordings, microstimulation, and computational modelling is a distinct strength of the paper. I only have a few questions and suggestions for clarification.

(1) It would be helpful to explain the criteria for choosing a given number of clusters and for accepting the final clustering solution more clearly. The quantitative results (silhouette plots, Rand index) in Supplementary Figure 2 should perhaps be included in the main figure to justify the parameter choices and acceptance of specific clustering solutions.

(2) It would be helpful to show how the activity profiles in Figure 3 would look like for 3 or 5 (or 6) clusters, to give the reader an impression of how activity profiles recovered using different numbers of clusters would differ.

(3) The authors attempt to link the microstimulation effects to the presence of functional neuron clusters at the stimulation site. How can you rule out that there were other, session-specific factors (e.g., related to the animal's motivation) that affected both neuronal activity and behavior? For example, could you incorporate aspects of the monkey's baseline performance (mean reaction time, fixation breaks, error trials) into the analysis?

(4) Line 84: What was the rationale for not including both coherence and reaction time in one multiple regression model?

Reviewer #2 (Public Review):

This study uses single-unit recordings in the monkey STN to examine the evidence for three theoretical models that propose distinct roles for the STN in perceptual decision-making. Importantly, the proposed functional roles are predictive of unique patterns of neural activity. Using k-means clustering with seeds informed by each model's predictions, the current study identified three neural clusters with activity dynamics that resembled those predicted by the described theoretical models. The authors are thorough and transparent in reporting the analyses used to validate the clustering procedure and the stability of the clustering results. To further establish a causal role for the STN in decision-making, the researchers applied microstimulation to the STN and found effects on response times, choice preferences, and latent decision parameters estimated with a drift diffusion model. Overall, the study provides strong evidence for a functionally diverse population of STN neurons that could indeed support multiple roles involved in perceptual decision-making. The manuscript would benefit from stronger evidence linking each neural cluster to specific decision roles in order to strengthen the overall conclusions.

The interpretation of the results, and specifically, the degree to which the identified clusters support each model, is largely dependent on whether the artificial vectors used as model-based clustering seeds adequately capture the expected behavior under each theoretical model. The manuscript would benefit from providing further justification for the specific model predictions summarized in Figure 1B. Further, although each cluster's activity can be described in the context of the discussed models, these same neural dynamics could also reflect other processes not specific to the models. That is, while a model attributing the STN's role to assessing evidence accumulation may predict a ramping up of neural activity, activity ramping is not a selective correlate of evidence accumulation and could be indicative of a number of processes, e.g., uncertainty, the passage of time, etc. This lack of specificity makes it challenging to infer the functional relevance of cluster activity and should be acknowledged in the discussion.

Additionally, although the effects of STN microstimulation on behavior provide important causal evidence linking the STN to decision processes, the stimulation results are highly variable and difficult to interpret. The authors provide a reasonable explanation for the variability, showing that neurons from unique clusters are anatomically intermingled such that stimulation likely affects neurons across several clusters. It is worth noting, however, that a substantial body of literature suggests that neural populations in the STN are topographically organized in a manner that is crucial for its role in action selection, providing "channels" that guide action execution. The authors should comment on how the current results, indicative of little anatomical clustering amongst the functional clusters, relate to other reports showing topographical organization.

Overall, the association between the identified clusters and the function ascribed to the STN by each of the models is largely descriptive and should be interpreted accordingly. For example, Figure 3 is referenced when describing which cluster activity is choice/coherence dependent, yet it is unclear what specific criteria and measures are being used to determine whether activity is choice/coherence "dependent." Visually, coherence activity seems to largely overlap in panel B (top row). Is there a statistically significant distinction between low and high coherence in this plot? The interpretation of these plots and the methods used to determine choice/coherence "dependence" needs further explanation.

In general, the association between cluster activity and each model could be more directly tested. At least two of the models assume coordination with other brain regions. Does the current dataset include recordings from any of these regions (e.g., mPFC or GPe) that could be used to bolster claims about the functional relevance of specific subpopulations? For example, one would expect coordinated activity between neural activity in mPFC and Cluster 2 according to the Ratcliff and Frank model. Additionally, the reported drift-diffusion model (DDM) results are difficult to interpret as microstimulation appears to have broad and varied effects across almost all the DDM model parameters. The DDM framework could, however, be used to more specifically test the relationships between each neural cluster and specific decision functions described in each model. Several studies have successfully shown that neural activity tracks specific latent decision parameters estimated by the DDM by including neural activity as a predictor in the model. Using this approach, the current study could examine whether each cluster's activity is predictive of specific decision parameters (e.g., evidence accumulation, decision thresholds, etc.). For example, according to the Ratcliff and Frank model, activity in cluster 2 might track decision thresholds.

Reviewer #3 (Public Review):

Summary:

The authors provide compelling evidence for the causal role of the subthalamic nucleus (STN) in perceptual decision-making. By recording from a large number of STN neurons and using microstimulation, they demonstrate the STN's involvement in setting decision bounds, scaling evidence accumulation, and modulating non-decision time.

Strengths:

The study tested three hypotheses about the STN's function and identified distinct STN subpopulations whose activity patterns support predictions from previous computational models. The experiments are well-designed, the analyses are rigorous, and the results significantly advance our understanding of the STN's multi-faceted role in decision formation.

Weaknesses:

While the study provides valuable insights into the STN's role in decision-making, there are a few areas that could be improved. First, the interpretation of the neural subpopulations' activity patterns in relation to the computational models should be clarified, as the observed patterns may not directly correspond to the specific signals predicted by the models. Second, the authors could consider using a supervised learning method to more explicitly model the pattern correlations between the three profiles. Third, a neural population model could be employed to better understand how the STN population jointly contributes to decision-making dynamics. Finally, the added value of the microstimulation experiments should be more directly addressed in the Results section, as the changes in firing patterns compared to the original patterns are not clearly evident.

Reviewer #1 (Public Review):

What neurophysiological changes support the learning of new sensorimotor transformations is a key question in neuroscience. Many studies have attempted to answer this question at the neuronal population level - with varying degrees of success - but few, if any, have studied the change in activity of the apical dendrites of layer 5 cortical neurons. Neurons in layer 5 of the sensory cortex appear to play a key role in sensorimotor transformations, showing important decision and reward-related signals, and being the main source of cortical and subcortical projections from the cortex. In particular, pyramidal track (PT) neurons project directly to subcortical regions related to motor activity, such as the striatum and brainstem, and could initiate rapid motor action in response to given sensory inputs. Additionally, layer 5 cortical neurons have large apical dendrites that extend to layer 1 where different neuromodulatory and long-range inputs converge, providing motor and contextual information that could be used to modulate layer 5 neurons output and/or to establish the synaptic plasticity required for learning a new association.

In this study, the authors aimed to test whether the learning of a new sensorimotor transformation could be supported by a change in the evoked response of the apical dendrites of layer 5 neurons in the mouse whisker primary somatosensory cortex. To do this, they performed longitudinal functional calcium imaging of the apical dendrites of layer 5 neurons while mice learned to discriminate between two multi-whisker stimuli. The authors used a simple conditioning task in which one whisker stimulus (upward or backward air puff, CS+) is associated with a reward after a short delay, while the other whisker stimulus (CS-) is not. They found that task learning (measured by the probability of anticipatory licking just after the CS+) was not associated with a significant change in the average population response evoked by the CS+ or the CS-, nor a change in the average population selectivity. However, when considering individual dendritic tufts, they found interesting changes in selectivity, with approximately equal numbers of dendrites becoming more selective for CS+ and dendrites becoming more selective for CS-.

One of the major challenges when assessing changes in neural representation during the learning of such Go/NoGo tasks is that the movements and rewards themselves may elicit strong neural responses that may be a confounding factor, that is, inexperienced mice do not lick in response to the CS+, while trained mice do. In this study, the authors addressed this issue in three ways: first, they carefully monitored the orofacial movements of mice and showed that task learning is not associated with changes in evoked whisker movements. Second, they show that whisking or licking evokes very little activity in the dendritic tufts compared to whisker stimuli (CS+ and CS-). Finally, the authors introduced into the design of their task a post-conditioning session after the last conditioning session during which the CS+ and the CS- are presented but no reward is delivered. During this post-session, the mice gradually stopped licking in response to the CS+. A better design might have been to perform the pre-conditioning and post-conditioning sessions in non-water-restricted, unmotivated mice to completely exclude any lick response, but the fact that the change in selectivity persists after the mice stopped licking in the last blocks of the post-conditioning session (in mice relying only on their whiskers to perform the task) is convincing.

The clever task design and careful data analysis provide compelling evidence that learning this whisker discrimination task does not result in a massive change in sensory representation in the apical dendritic tufts of layer 5 neurons in the primary somatosensory cortex on average. Nevertheless, individual dendritic tufts do increase their selectivity for one or the other sensory stimulus, likely enhancing the ability of S1 neurons to accurately discriminate the two stimuli and trigger the appropriate motor response (to lick or not to lick).

One limitation of the present study is the lack of evidence for the necessity of the primary somatosensory cortex in the learning and execution of the task. As the authors have strongly emphasized in their previous publications, the primary somatosensory cortex may not be necessary for the learning and execution of simple whisker detection tasks, especially when the stimulus is very salient. Although this new task requires the discrimination between two whisker stimuli, the simplicity and salience of the whisker stimuli used could make this task cortex-independent. Especially when considering that some mice seem to not rely entirely on their whiskers to execute the task.

Nevertheless, this is an important result that shows for the first time changes in the selectivity to sensory stimuli at the level of individual apical dendritic tufts in correlation with the learning of a discrimination task. This study sheds new light on the cortical cellular substrates of reward-based learning and opens interesting perspectives for future research in this area. In future studies, it will be important to determine whether the change in selectivity of dendritic calcium spikes is causally involved in the learning of the task or whether it simply correlates with learning, as a consequence of changes in synaptic inputs caused by reward. The dendritic calcium spikes may be involved in the establishment of synaptic plasticity required for learning and impact the output of layer 5 pyramidal neurons to trigger the appropriate motor response. It would be important also to study the changes in selectivity in the apical dendrite of the identified projection neurons.

Reviewer #2 (Public Review):

Summary:

The authors did not find an increased representation of CS+ throughout reinforcement learning in the tuft dendrites of Rbp4-positive neurons from layer 5B of the barrel cortex, as previously reported for soma from layer 2/3 of the visual cortex.

Alternatively, the authors observed an increased selectivity to both stimuli (CS+ and CS-) during reinforcement learning. This feature:

(1) was not present in repeated exposures (without reinforcement),<br /> (2) was not explained by the animal's behaviour (choice, licking, and whisking), and<br /> (3) was long-lasting, being present even when the mice disengaged from the task.

Importantly, increased selectivity was correlated with learning (% correct choices), and neural discriminability between stimuli increased with learning.

In conclusion, the authors show that tuft dendrites from layer 5B of the barrel cortex increase the representation of conditioned (CS+) and unconditioned stimuli (CS-) applied to the whiskers, during reinforcement learning.

Strengths:

The results presented are very consistent throughout the entire study, and therefore very convincing:

(1) The results observed are very similar using two different imaging techniques (2-photon -planar imaging- and SCAPE-volumetric imaging). Figure 3 and Figure 4 respectively.

(2) The results are similar using "different groups" of tuft dendrites for the analysis (e.g. initially unresponsive and responsive pre- and post-learning). Figure 5.

(3) The results are similar from a specific set of trials (with the same sensory input, but different choices). Figure 7.

(4) Additionally, the selectivity of tuft dendrites from layer 5B of the barrel cortex was higher in the mice that exclusively used the whisker to respond to the stimuli (CS+ and CS-).<br /> The results presented are controlled against a group of mice that received the same stimuli presentation, except for the reinforcement (reward).

Additionally, the behaviour outputs, such as choice, whisking, and licking could not account for the results observed.

Although there are no causal experiments, the correlation between selectivity and learning (percentage of correct choices), as well as the increased neural discriminability with learning, but not in repeated exposure, are very convincing.

Weaknesses:

The biggest weakness is the absence of causality experiments. Although inhibiting specifically tuft dendritic activity in layer 1 from layer 5 pyramidal neurons is very challenging, tuft dendritic activity in layer 1 could be silenced through optogenetic experiments as in Abs et al. 2018. By manipulating NDNF-positive neurons the authors could specifically modify tuft dendritic activity in the barrel cortex during CS presentations, and test if silencing tuft dendritic activity in layer 1 would lead to the lack of selectivity and an impairment of reinforcement learning. Additionally, this experiment will test if the selectivity observed during reinforcement learning is due to changes in the local network, namely changes in local synaptic connectivity, or solely due to changes in the long-range inputs.

Reviewer #1 (Public Review):

Summary:

This study focuses on metabolic changes in the paraventricular hypothalamic (PVH) region of the brain during acute periods of cold exposure. The authors point out that in comparison to the extensive literature on the effects of cold exposure in peripheral tissues, we know relatively little about its effects on the brain. They specifically focus on the hypothalamus, and identify the PVH as having changes in Atgl and Hsl gene expression changes during cold exposure. They then go on to show accumulation of lipid droplets, increased Fos expression, and increased lipid peroxidation during cold exposure. Further, they show that neuronal activation is required for the formation of lipid droplets and lipid peroxidation.

Strengths:

A strength of the study is trying to better understand how metabolism in the brain is a dynamic process, much like how it has been viewed in other organs. The authors also use a creative approach to measuring in vivo lipid peroxidation via delivery of a BD-C11 sensor through a cannula to the region in conjunction with fiber photometry to measure fluorescence changes deep in the brain.

Weaknesses:

Although the topic and findings are of interest, there are a few key weaknesses in the study that would improve the work if addressed. One weakness was many of the experiments were done in a manner that could not distinguish between the contributions of neurons and glial cells, limiting the extent of conclusions that could be made. While this is not easily doable for all experiments, it can be done for some. For example, the Fos experiments in Figure 3 would be more conclusive if done with the labeling of neuronal nuclei with NeuN, as glial cells can also express Fos. To similarly show more conclusively that neurons are being activated during cold exposure, the calcium imaging experiments in Figure S3 can be done with cold exposure. Additionally, many experiments are only done with the minimal three animals required for statistics and could be more robust with additional animals included. Another weakness is that the authors do not address whether manipulating lipid droplet accumulation or lipid peroxidation has any effect on PVH function (e.g. does it change neuronal activity in the region?).

Reviewer #2 (Public Review):

Summary:

Cold-induced lipid metabolism is well-established in adipose tissues. The authors set out to determine whether cold could alter brain lipid metabolism. By QPCR analysis of brain punches after acute cold, they found that mRNA expressions of several lipolysis-related genes were upregulated compared to RT controls. By combining fluorescent sensors and in vivo fiberphotometry, they observed cold-induced lipid peroxidation/lipolysis, which could be blocked by pharmacological inhibitors of neuronal activity (muscimol and kynurenic acid). The brain is not traditionally considered an organ with high lipid metabolism (vs carbohydrate); therefore, the observation and hypothesis proposed by the authors are unexpected and can be interesting. However, the experiments and data were rather preliminary and superficial and did not support the authors' conclusions. In addition, the main hypothesis, in relationship to the role of cold/temperature, remains incoherent and needs a major update.

Strengths:

A set of relatively novel and interesting observations.

Creative use of several in vivo sensors and techniques.

Weaknesses:

(1) The physiological relevance of lipolysis and thermogenesis genes in the PVH. The authors need to provide quantitative and substantial characterizations of lipid metabolism in the brain beyond a panel of qPCRs, especially considering these genes are likely expressed at very low levels. mRNA and protein level quantification of genes in Fig 1, in direct comparison to BAT/iWAT, should be provided. Besides bulk mRNA/protein, IHC/ISH-based characterization should be added to confirm to cellular expression of these genes.

(2) The fiberphotometry work they cited (Chen 2022, Andersen 2023, Sun 2018) used well-established, genetically encoded neuropeptide sensors (e.g., GRABs). The authors need to first quantitatively demonstrate that adapting BD-C11 and EnzCheck for in vivo brain FP could effectively and accurately report peroxidation and lipolysis. For example, the sensitivity, dynamic range, and off-time should all be calibrated with mass spectrometry measurements before any conclusions can be made based on plots in Figures 4, 5, and 6. This is particularly important because the main hypothesis heavily relies on this unvalidated technique.

(3) Generally, the histology data need significant improvement. It was not convincing, for example, in Figure 3, how the Fos+ neurons can be quantified based on the poor IF images where most red signals were not in the neurons.

(4) The hypothesis regarding the direct role of brain temperature in cold-induced lipid metabolism is puzzling. From the introduction and discussion, the authors seem to suggest that there are direct brain temperature changes in responses to cold, which could be quite striking. However, this was not supported by any data or experiments. The authors should consolidate their ideas and update a coherent hypothesis based on the actual data presented in the manuscript.

Reviewer #1 (Public Review):

Little is known about the local circuit mechanisms in the preoptic area (POA) that regulate body temperature. This carefully executed study investigates the role of GABAergic interneurons in the POA that express neurotensin (NTS). The principal finding is that GABA-release from these cells inhibits neighboring neurons, including warm-activated PACAP neurons, thereby promoting hyperthermia, whereas NTS released from these cells has the opposite effect, causing a delayed activation and hypothermia. This is shown through an elegant series of experiments that include slice recordings alongside matched in vivo functional manipulations. The roles of the two neurotransmitters are distinguished using a cell-type-specific knockout of Vgat as well as pharmacology to block GABA and NTS receptors. Overall, this is an excellent study that is noteworthy for revealing local circuit mechanisms in the POA that control body temperature and also for highlighting how amino acid neurotransmitters and neuropeptides released from the same cell can have opposing physiologic effects. I have only minor suggestions for revision.

Reviewer #2 (Public Review):

Summary:

The study has demonstrated how two neurotransmitters and neuromodulators from the same neurons can be regulated and utilized in thermoregulation.

The study utilized electrophysiological methods to examine the characteristics and thermoregulation of Neurotensin (Nts)-expressing neurons in the medial preoptic area (MPO). It was discovered that GABA and Nts may be co-released by neurons in MPO when communicating with their target neurons.

Strengths:

The study has leveraged optogenetic, chemogenetic, knockout, and pharmacological inhibitors to investigate the release process of Nts and GABA in controlling body temperature.

The findings are relevant to those interested in the various functions of specific neuron populations and their distinct regulatory mechanisms on neurotransmitter/neuromodulator activities

Weaknesses:

Key points for consideration include:

(1) The co-release of GABA and Nts is primarily inferred rather than directly proven. Providing more direct evidence for the release of GABA and the co-release of GABA and Nts would strengthen the argument. Further in vitro analysis could strengthen the conclusion regarding this co-releasing process.

(2) The differences between optogenetic and chemogenetic methods were not thoroughly investigated. A comparison of in vitro results and direct observation of release patterns could clarify the mechanisms of GABA release alone or in conjunction with Nts under different stimulation techniques.

(3) Neuronal transcripts were mainly identified through PCR, and alternative methods like single-cell sequencing could be explored.

(4) In Figure 6, the impact of GABA released from Nts neurons in MPO on CBT regulation appears to vary with ambient temperatures, requiring a more detailed explanation for better comprehension.

(5) The model should emphasize the key findings of the study.

Reviewer #3 (Public Review):

Summary:

Understanding the central neural circuits regulating body temperature is critical for improving health outcomes in many disease conditions and in combating heat stress in an ever-warming environment. The authors present important and detailed new data that characterizes a specific population of POA neurons with a relationship to thermoregulation. The new insights provided in this manuscript are exactly what is needed to assemble a neural network model of the central thermoregulatory circuitry that will contribute significantly to our understanding of regulating the critical homeostatic variable of body temperature. These experiments were conducted with the expertise of an investigator with career-long experience in intracellular recordings from POA neurons. They were interpreted conservatively in the appropriate context of current literature.

The Introduction begins with "Homeotherms, including mammals, maintain core body temperature (CBT) within a narrow range", but this ignores the frequent hypothermic episodes of torpor that mice undergo triggered by cold exposure. Although the author does mention torpor briefly in the Discussion, since these experiments were carried out exclusively in mice, greater consideration (albeit speculative) of the potential for a role of MPO Nts neurons in torpor initiation or recovery is warranted. This is especially the case since some 'torpor neurons' have been characterized as PACAP-expressing and a population of PACAP neurons represent the target of MPO Nts neurons.

Reviewer #1 (Public Review):

Summary:

In this technical paper, the authors introduce a useful variation on the fully automated multi-electrode patch-clamp recording technique for probing synaptic connections that they term "patch-walking". The patch-walking approach involves coordinated pipette route-planning and automated pipette cleaning procedures for pipette reuse to improve recording throughput efficiency, which the authors argue can theoretically yield almost twice the number of connections to be probed by paired recordings on a multi-patch electrophysiology setup for a given number of cells compared to conventional manual patch-clamping approaches used in brain slices in vitro. The authors show solid results from recordings in mouse in vitro cortical slices, demonstrating the efficient recording of dozens of paired neurons with a two-patch pipette configuration for paired recordings and detection of synaptic connections. This approach will be of interest and valuable to neuroscientists conducting automated multi-patch in vitro electrophysiology experiments and seeking to increase the efficiency of neuron connectivity detection while avoiding the more complex recording configurations (e.g., 8-10 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Strengths:

(1) The authors introduce the theory and methods and show experimental results for a fully automated electrophysiology dual patch-clamp recording approach, which uses coordinated patch-clamp pipette route-planning and automated pipette cleaning procedures to "patch-walk" across an in vitro brain slice.

(2) The patch-walking approach improves throughput efficiency over manual patch clamp recording approaches, especially for investigators looking to utilize paired patch electrode recordings in electrophysiology experiments in vitro.

(3) Experimental results are presented from in vitro mouse cortical slices demonstrating the efficiency of recording dozens of paired neurons with a two-patch pipette configuration for paired recordings and detecting synaptic connections, demonstrating the feasibility and efficiency of the patch-walking approach.

(4) The authors suggest extensions of their technique while keeping the number of recording pipettes employed and recording rig complexity low, which are important practical technical considerations for investigators wanting to avoid the more complex recording configurations (e.g., 8-10 pipette multi-patch recording configurations) used by several laboratories that are not readily implementable by most of the neuroscience community.

Reviewer #2 (Public Review):

Summary:

In this study, the authors aim to combine automated whole-cell patch clamp recording simultaneously from multiple cells. Using a 2-electrode approach, they are able to sample as many cells (and connections) from one slice, as would be achieved with a more technically demanding and materially expensive 8-electrode patch clamp system. They provide data to show that this approach is able to successfully record from 52% of attempted cells, which was able to detect 3 pairs in 71 screened neurons. The authors state that this is a step forward in our ability to record from randomly connected ensembles of neurons.

Strengths:

The conceptual approach of recording multiple partner cells from another in a stepwise manner indeed increases the number of tested connections. An approach that is widely applicable to both automated and manual approaches. Such a method could be adopted for many connectivity studies using dual recording electrodes.

The implementation of automated robotic whole-cell patch-clamp techniques from multiple cells simultaneously is a useful addition to the multiple techniques available to ex vivo slice electrophysiologists.

The approach using 2 electrodes, which are washed between cells is economically favourable, as this reduces equipment costs for recording multiple cells, and limits the wastage of capillary glass that would otherwise be used once.

Weaknesses:

(1) The premise of this article is based upon the fact that even a "skilled" whole-cell electrophysiologist is only capable of recording ~10 cells per day are flawed. Many studies have shown that capable electrophysiologists can record upwards of 50 cells a day, given adequate slice quality and reliable recording conditions with multiple electrodes (e.g. Pastoll et al., 2020 eLife, Booker et al., 2014, JoVE, Peng et al., 2017); often with over 80% success rates for recording. It is not convincing that this approach is a dramatic improvement on such approaches - except when a less skilled researcher is beginning recordings.

Importantly, could the patch walk protocol not be alternatively implemented using manual recording approaches? Yes, the use of a semi-automated robotic system aids recording from many cells by a less experienced colleague, but the inferences about the number of connections tested are common to the approach, not the technique used. This seems like a crucial conceptual point to include.

(2) A key omission of this study is the absence of brain area, cell type, and layer recorded from. It is mentioned in Figure 2 that this is the somatosensory and visual cortices. Which were these, and how were they confirmed?

(3) A comparison of measurements shown in Figure 2 to other methods - e.g. conventional dual patch, 8-electrode patch, single electrode. How do the values obtained for cell quality measurements compare to those expected for the cell population recorded (which is unclear - see point 2)?

(4) What is the reliability of performing outside-out patch configuration to obtain sealed and biocytin-filled cells under these conditions? A key tenet of performing high-throughput paired recordings is the ability to identify the cell types involved in the local microcircuit, and if their axon has been preserved in the slice configuration (which varies between cell types). Not having confirmation of morphological identity and integrity likely leads to a dramatic underestimation of connection probability, given that main axon collaterals could be severed during acute brain slice preparation.

(5) The quality control criteria used in this manuscript require further clarification. An upper limit of 50 MΩ access resistance is extremely high (i.e. 20-30 MΩ is a more typical and stringent cut-off), which is worsened as no real information is given to the degree of resistance change that could be accepted. This is simply listed as "If the seal quality decreased during recording, the cell is excluded from analysis". Indeed, the range of access resistances plotted in Figure 2 is from 10-100 MΩ, which implies that some neurons included in this data did not meet recording criteria. Also, it is widely accepted in the field that a 10-20% change in access during recording is acceptable - within a more defined range. I would consider re-assessing the recorded cells to only include cells with access resistances <30MΩ and those that did not fluctuate by more than 20%.

Appraisal of aims:

The authors certainly established a system that is useful for interrogating synaptic connectivity in an automated manner. However, it remains unclear how widely used this would be in the field, and whether this truly represents an advancement from manual recordings or >4 electrode recordings.

Discussion of impact:

This approach, particularly the conceptual approach to paired testing, is of use to the field. However, in practice, many researchers using conventional dual-electrode paired recording likely implement similar approaches - especially when targeting specific cell types (see Booker et al., 2014 JoVE, Qi et al., 2020 Front Synaptic Neurosci.). This may pave the way for greater implementation of dual and multi-electrode recordings using robotic patch-clamp techniques.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Yip and colleagues incorporated the pipette cleaning technique into their existing dual-patch robotic system, "the PatcherBot", to allow sequential patching of more cells for synaptic connection detection in living brain slices. During dual-patching, instead of retracting all two electrodes after each recording attempt, the system cleaned just one of the electrodes and reused it to obtain another recording while maintaining the other. With one new patch clamp recording attempt, new connections can be probed. By placing one pipette in front of the other in this way, one can "walk" across the tissue, termed "patch-walking." This application could allow for probing additional neurons to test the connectivity using the same pipette in the same preparation.

Strengths:

Compared to regular dual-patch recordings, this new approach could allow for probing more possible connections in brain slices with dual-patch recordings, thus having the potential to improve the efficiency of identifying synaptic connections

Weaknesses:

While this new approach offers the potential to increase efficiency, it has several limitations that could curtail its widespread use.

Loss of Morphological Information: Unlike traditional multi-patch recording, this approach likely loses all detailed morphology of each recorded neuron. This loss is significant because morphology can be crucial for cell type verification and understanding connectivity patterns by morphological cell type.

Spatial Restrictions: The robotic system appears primarily suited to probing connections between neurons with greater spatial separation (~100µm ISD). This means it may not reliably detect connections between neurons in close proximity, a potential drawback given that the connectivity is much higher between spatially close neurons. This limitation could help explain the low connectivity rate (5%) reported in the study.

Limited Applicability: While the approach might be valuable in specific research contexts, its overall applicability seems limited. It's important to consider scenarios where the trade-off between efficiency and specific questions that are asked.

Scalability Challenges: Scaling this method beyond a two-pipette setup may be difficult. Additional pipettes would introduce significant technical and logistical complexities.

Reviewer #1 (Public Review):

Cheng, Yu-Ting, et al. demonstrate the capabilities of three-photon excited fluorescence (3PEF) microscopy for in vivo imaging of the mouse spinal cord. It enables imaging up to ~550 µm in depth, overcoming the limitations of two-photon excited fluorescence (2PEF) microscopy. The authors used 3PEF to visualize and quantify blood flow across different vessel types within the spinal cord and observed the cellular responses following venule occlusion. They showed depth-dependent structural changes in neurites and the behavior of microglia with a high contrast. The findings show that 3PEF can provide high-resolution, multicolor imaging of dynamic cellular interactions and vascular architecture, helping studies of spinal cord physiology and pathology.

The experiments are well done and supported by data but some points need to be clarified:

(1) For the two vs three-photon comparison, the authors should provide more information about how they performed the 2PEF: power and pulse duration. This comparison is primarily focused on imaging depth and signal-to-background ratio (SBR), but imaging speed should also be discussed.

(2) A comparison with state-of-the-art 2PEF would have been more convincing. For instance, the use of adaptive optics, or red-shifted fluorophores allowing better 2PEF SBR, or deeper imaging.

(3) The study focuses on structural imaging and does not provide extensive data on real-time dynamic processes, which could be crucial for understanding rapid cellular responses in the spinal cord.<br /> By addressing these weaknesses, future studies could enhance the applicability and reliability of 3PEF microscopy for spinal cord research.

Reviewer #2 (Public Review):

Summary:

In this work, the authors attempt to advance our capacity to image the intact spinal cord in living mice, with the ultimate goal of allowing optical access to all spinal layers, from the dorsal (sensory-related) to the ventral (motor-related) laminae. They demonstrate the potency of 3-photon excited fluorescence imaging (3PEF) to collect fluorescent signals in anesthetized adult mice to depths of up to 450 µm from the dorsal surface.

Strengths:

• 3PEF is convincingly demonstrated as a significant improvement over previously used 2-photon imaging.

• The images show very good spatial resolution and stable signal-to-noise ratio up to 450 µm from the dorsal surface, providing unprecedented access to intermediate ventral laminae.

Weaknesses:

• The paper in its current form lacks a detailed description of the experimental apparatus used, including its invasiveness (removal of vertebrae and muscles) and its impact on animal behavior. One can hope that, in the future, a similar implantation chamber may be used for awake, freely-moving animals.

• In general, non-optic specialists may find it difficult to appreciate some of the findings due to technical writing at times, and minimally described metrics.

• The possibility that the 3-photon illumination may cause tissue damage, notably by heat induction, is not evaluated or discussed.

• At this stage, no attempt has been made to image cellular activity. The reader should keep in mind that motor neurons, as well as most of their upstream circuits, are located between 500 and 900 µm from the dorsal surface. Hence, although the method is a significant advancement, it still does not allow for the evaluation of morphological (or possibly, activity) changes in the whole spinal cord, particularly excluding motor-related laminae."

Reviewer #1 (Public Review):

Summary:

This manuscript by Meissner and colleagues described a novel take on a classic social cognition paradigm developed for marmosets. The classic pull task is a powerful paradigm that has been used for many years across numerous species, but its analog approach has several key limitations. As such, it has not been feasible to adopt the task for neuroscience experiments. Here the authors capture the spirit of the classic task but provide several fundamental innovations that modernize the paradigm - technically and conceptually. By developing the paradigm for marmosets, the authors leverage the many advantages of this primate model for studies of social brain functions and their particular amenability to freely-moving naturalistic approaches.

Strengths:

The current manuscript describes one of the most exciting paradigms in primate social cognition to be developed in many years. By allowing for freely-moving marmosets to engage in high numbers of trials, while precisely quantifying their visual behavior (e.g. gaze) and recording neural activity this paradigm has the potential to usher in a new wave of research on the cognitive and neural mechanisms underlying primate social cognition and decision-making. This paradigm is an elegant illustration of how naturalistic questions can be adapted to more rigorous experimental paradigms. Overall, I thought the manuscript was well written and provided sufficient details for others to adopt this paradigm. I did have a handful of questions and requests about topics and information that could help to further accelerate its adoption across the field.

Weaknesses:

LN 107 - Otters have also been successful at the classic pull task (https://link.springer.com/article/10.1007/s10071-017-1126-2)

LN 151 - Can you provide a more precise quantification of timing accuracy than the 'sub-second level'. This helps determine synchronization with other devices.

Using this paradigm, the marmosets achieved more trials than in the conventional task (146 vs 10). While this is impressive, given that only ~50 are successful Mutual Cooperation trials it does present some challenges for potential neurophysiology experiments and particular cognitive questions. The marmosets are only performing the task for 20 minutes, presumably because they become sated and are no longer motivated. This seems a limitation of the task and is something worth discussing in the manuscript. Did the authors try other food rewards, reduce the amount of reward, food/water restrict the animals for more than the stated 1-3 hours? How might this paradigm be incorporated into in-cage approaches that have been successful in marmosets? Any details on this would help guide others seeking to extend the number of trials performed each day.

Can you provide more details on the DLC/Anipose procedure? How were the cameras synchronized? What percentage of trials needed to be annotated before the model could be generalized? Did each monkey require its own model, or was a single one applied to all animals?

Will the schematics and more instructions on building this system be made publicly available? A number of the components listed in Table 1 are custom-designed. Although it is stated that CAD files will be made available upon request, sharing a link to these files in an accessible folder would significantly add to the potential impact of this paradigm by making it easier for others to adopt.

In the Discussion, it would be helpful to have some discussion of how this paradigm might be used more broadly. The classic pulling paradigm typically allows one to ask a specific question about social cognition, but this task has the potential to be more widely applied to other social decision-making questions. For example, how might this task be adopted to ask some of the game-theory-type approaches common in this literature? Given the authors' expertise in this area, this discussion could serve to provide a roadmap for the broader field to adopt.

Although this paradigm was developed specifically for marmosets, it seems to me that it could readily be adopted in other species with some modifications. Could the authors speak to this and their thoughts on what may need to be changed to be used in other species? This is particularly important because one of the advantages of the classic paradigm is that it has been used in so many species, providing the opportunity to compare how different species approach the same challenge. For example, though both chimps and bonobos are successful, their differences are notably illuminating about the nuances of their respective social cognitive faculties.

Reviewer #2 (Public Review):

Summary:

This important work by Meisner et al., developed an automated apparatus (MarmoAPP) to collect a wide array of behavioral data (lever pulling, gaze direction, vocalizations) in marmoset monkeys, with the goal of modernizing collection of behavioral data to coincide with the investigation of neurological mechanisms governing behavioral decision making in an important primate neuroscience model. The authors show a variety of "proof-of-principle" concepts that this apparatus can collect a wide range of behavioral data, with higher behavioral resolution than traditional methods. For example, the authors highlight that typical behavioral experiments on primate cooperation provide around 10 trials per session, while using their approach the authors were able to collect over 100 trials per 20-minute session with the MarmoAAP.

Overall the authors argue that this approach has a few notable advantages:<br /> (1) it enhances behavioral output which is important for measuring small or nuanced effects/changes in behavior;<br /> (2) allows for more advanced analyses given the higher number of trials per session;<br /> (3) significantly reduces the human labor of manually coding behavioral outcomes and experimenter interventions such as reloading apparatuses for food or position;<br /> (4) allows for more flexibility and experimental rigor in measuring behavior and neural activity simultaneously.

Strengths:

The paper is well-written and the MarmoAPP appears to be highly successful at integrating behavioral data across many important contexts (cooperation, gaze, vocalizations), with the ability to measure significantly many more behavioral contexts (many of which the authors make suggestions for).

The authors provide substantive information about the design of the apparatus, how the apparatus can be obtained via a long list of information Apparatus parts and information, and provide data outcomes from a wide number of behavioral and neurological outcomes. The significance of the findings is important for the field of social neuroscience and the strength of evidence is solid in terms of the ability of the apparatus to perform as described, at least in marmoset monkeys. The advantage of collecting neural and freely-behaving behavioral data concurrently is a significant advantage.

Weaknesses:

While this paper has many significant strengths, there are a few notable weaknesses in that many of the advantages are not explicitly demonstrated within the evidence presented in the paper. There are data reported (as shown in Figures 2 and 3), but in many cases, it is unclear if the data is referenced in other published work, as the data analysis is not described and/or self-contained within the manuscript, which it should be for readers to understand the nature of the data shown in Figures 2 and 3.

(1) There is no data in the paper or reference demonstrating training performance in the marmosets. For example, how many sessions are required to reach a pre-determined criterion of acceptable demonstration of task competence? The authors reference reliably performing the self-reward task, but this was not objectively stated in terms of what level of reliability was used. Moreover, in the Mutual Cooperation paradigm, while there is data reported on performance between self-reward vs mutual cooperation tasks, it is unclear how the authors measured individual understanding of mutual cooperation in this paradigm (cooperation performance in the mutual cooperation paradigm in the presence or absence of a partner; and how, if at all, this performance varied across social context). What positive or negative control is used to discern gained advantages between deliberate cooperation vs two individuals succeeding at self-reward simultaneously?

(2) One of the notable strengths of this approach argued by the authors is the improved ability to utilize trials for data analysis, but this is not presented or supported in the manuscript. For example, the paper would be improved by explicitly showing a significant improvement in the analytical outcome associated with a comparison of cooperation performance in the context of ~150 trials using MarmoAAP vs 10-12 trials using conventional behavioral approaches beyond the general principle of sample size. The authors highlight the dissection of intricacies of behavioral dynamics, but more could be demonstrated to specifically show these intricacies compared to conventional approaches. Given the cost and expertise required to build and operate the MarmoAAP, it is critical to provide an important advantage gained on this front. The addition of data analysis and explicit description(s) of other analytical advantages would likely strengthen this paper and the advantages of MarmoAAP over other behavioral techniques.

Reviewer #3 (Public Review):

Summary:

The authors set out to devise a system for the neural and behavioral study of socially cooperative behaviors in nonhuman primates (common marmosets). They describe instrumentation to allow for a "cooperative pulling" paradigm, the training process, and how both behavioral and neural data can be collected and analyzed. This is a valuable approach to an important topic, as the marmoset stands as a great platform to study primate social cognition. Given that the goals of such a methods paper are to (a) describe the approach and instrumentation, (b) show the feasibility of use, and (c) quantitatively compare to related approaches, the work is easily able to meet those criteria. My specific feedback on both strengths and weaknesses is therefore relatively limited in scope and depth.

Strengths:

The device is well-described, and the authors should be commended for their efforts in both designing this system but also in "writing it up" so that others can benefit from their R&D.

The device appears to generate more repetitions of key behavior than other approaches used in prior work (with other species).

The device allows for quantitative control and adjustment to control behavior.

The approach also supports the integration of markerless behavioral analysis as well as neurophysiological data.

Weaknesses:

A few ambiguities in the descriptions are flagged below in the "Recommendations for authors".

The system is well-suited to marmosets, but it is less clear whether it could be generalized for use in other species (in which similar behaviors have been studied with far less elegant approaches). If the system could impact work in other species, the scope of impact would be significantly increased, and would also allow for more direct cross-species comparisons. Regardless, the future work that this system will allow in the marmoset will itself be novel, unique, and likely to support major insights into primate social cognition.

Reviewer #1 (Public Review):

The manuscript by Yu et al seeks to investigate the role of neuritin (Nrn1), identified as a marker of anergic cells, in the biology of regulatory (Tregs) and conventional (Tconv) T cells. Although the role of Nrn1 expressed by Tregs has already been explored (Gonzalez-Figueroa 2021 cited in the manuscript), this manuscript shows original new data suggesting that this molecule would be important in promoting Treg function and inhibiting Tconv effector function by acting at the level of membrane potential and molecule transport across the plasma membrane. However, it is disappointing that reading this manuscript leaves an impression of incomplete work done too quickly. Multiple models have been used, but none has been studied thoroughly enough to provide really conclusive and unambiguous data. For example, 5 different models were used to study T cells in vivo. It would have been preferable to use fewer, but to go further in the study of mechanisms. In the absence of a more in-depth study, the conclusions drawn by the authors are often open to question. Major points concern the fact that there are enough biological replicates for most experiments, some critical controls and data are lacking, and the authors have used iTregs rather than nTregs for many experiments (see below). This is unfortunate because the role of neuritin in T cell biology studied here is new and interesting.

Major points (in the order in which they appear in the text):

(1) A real weakness of this work is the fact that in most of the results shown, there are few biological replicates with differences that are often small between Ctrl and Nrn1 -/-. The systematic use of student's t-test may lead to thinking that the differences are significant, which is often misleading given the small number of samples, which makes it impossible to know whether the distributions are Gaussian and whether a parametric test can be used. RNAseq bulk data are based on biological duplicates, which is open to criticism.

(2) The authors use Nrn1+/+ and Nrn1+/- cells indiscriminately as control cells on the basis of similar biology between Nrn1+/+ and Nrn1+/- cells at homeostasis. However, it is quite possible that the Nrn1+/- cells have a phenotype in situations of in vitro activation or in vivo inflammation (cancer, EAE). It would be important to discriminate Nrn1+/- and Nrn1+/+ cells in the data or to show that both cell types have the same phenotype in these conditions too.

(3) Figure 1A-D. Since the authors are using the Nrp1 KO mice, it would be important to confirm the specificity of the anti-Nrn1 mAb by FACS. Once verified, it would be important to add FACS results with this mAb in Figures 1A-C to have single-cell and quantitative data as well.

(4) Figure 1E-H. The authors assume that this immunization protocol induces anergic cells, but they provide no experimental evidence for this. It would be useful to show that T cells are indeed anergic in this model, especially those that are OVA-specific. The lack of IL-2 production by Cltr cells could be explained by the presence of fewer OVA-specific cells, rather than by an anergic status.

(5) Figure 2A-C and Figure 3. The use of iTregs to try to understand what is happening in vivo is problematic. iTregs are cells that have probably no equivalent in vivo, and so may have no physiological relevance. In any case, they are different from pTreg cells generated in vivo. Working with pTreg may be challenging, that is why I would suggest generating data with purified nTreg. Moreover, it was shown in the article of Gonzalez-Figueroa 2021 that Nrn1-/- nTreg retained a normal suppressive function, which would not be what is concluded by the authors of this manuscript. Moreover, we do not even know what the % of Foxp3 cells is in the iTreg used (after differentiation and 20h of re-stimulation) and whether this % is the same between Ctlr and Nrn1 KO cells.

(6) Figure 2D-L. The model is designed to study the role of Nrn1 in nTreg. However, the % of Foxp3+ among CD45.2 nTreg cells fell to 5-15% of CD4+ cells (Figure 2F). Since we do not know what is the % of Foxp3 among the injected cells, we do not know whether this very low % is due to very high Treg instability or to preferential expansion of contaminating Tconvs. It is possible that the % of Tconv contaminant is high since Treg was sorted using beads and not FACS in some experiments. As it is very likely that there are Tconv contaminants that would be Nrn1-/- in the group transferred with Nrn1-/- "nTreg", the higher tumor rejection could be due to an overactivation of Nrn1-/- Tconvs (rather than a defect in Nrn1-/- Treg function).

Reviewer #2 (Public Review):

Summary:

This manuscript explores the role of Nrn1 in T cell tolerance. A previous study has demonstrated that Nrn1 is up-regulated in the Tfr fraction of Foxp3+ T regulatory cells. These authors now confirm the expression of Nrn1 in Tregs as well as report here that Nrn1 is also greatly over-expressed in anergic CD4 T cells, and this is the stepping-off point for this investigation.

Most remarkably, experiments show that anergy induction is defective when T cells cannot express Nrn1. Furthermore, differentiation to a Foxp3+ Treg phenotype is inhibited in the absence of Nrn1, and the Tregs that do develop appear functionally defective. With such defects in the anergy induction and Treg differentiation and function, auto-reactive effector T cell activation is unrestrained, and Nrn1-/- mice are more susceptible to severe EAE development.

Strengths:

The characterizations of T cell Nrn1 expression both in vitro and in vivo are comprehensive and convincing. The in vivo functional studies of anergy development, Treg suppression, and EAE development are also well done to strengthen the notion that Nrn1 is an important regulator of CD4 responsiveness.

Weaknesses:

The major weakness of this study stems from a lack of a clear molecular mechanism involving Nrn1. Previous studies of Nrn1 have suggested its role as a soluble molecule involved in intracellular communication, perhaps influencing cellular ion channel function and/or triggering downstream NFAT and mTOR activation. However, a unique receptor for Nrn1 has not been discovered and it remains unclear whether it acts in a cell-intrinsic or cell-extrinsic fashion for any particular cell type.

Data shown here provide evidence of alterations in the electrical and metabolic state of T cells when the Nrn1 gene is deleted. Nrn1-/- Tregs and Teffector cells each express a unique pattern of genes associated with Neurotransmitter receptor, Metal ion transmembrane transport, Amino acid transport, and mTORC1 signaling activities, different than that seen in wild-type mice. Although the biochemical and informatics studies are well-performed, it is my opinion that these results are inconclusive in part due to the absence of key "naive" control groups. This limits my ability to understand the significance of these data.

Specifically, studies of the electrical and metabolic state of Nrn1-/- inducible Treg cells (iTregs) would benefit from similar data collected from wild-type and Nrn1-/- naive CD4 T cells. Even though naive T cells don't express Nrn1, they may be positively influenced by soluble Nrn1. Does deletion of Nrn1 lead to changes in metabolic and electrical state in naive T cells? Is that why Nrn1 deletion in mice blocks naive T cell activation?

Since the loss of Nrn1 inhibits the activation of T cells, are Nrn1-/- iTregs transcriptionally, electrically, and metabolically similar to naive T cells due to their suboptimal activation? Does this account for their persistent functional defects? Or is up-regulation of Nrn1 (and cell-intrinsic Nrn1 signaling) necessary to complete Treg differentiation and to promote T regulatory function (similar to how cell-intrinsic Nrn1 facilitates anergy induction)? The study of naive cells in parallel with iTregs would address these possibilities.

A comparison of Nrn1-/- naive cells to Teffector cells should also be undertaken to reveal how it is that Nrn1-/- Teffector cells regain the capacity to respond effectively to stimulation (e.g. increased mTOR activation) despite their early activation defects.

Joint Public Review:

In this paper Hui and colleagues investigate how the predictive accuracy of a polygenic score (PGS) for body mass index (BMI) changes when individuals are stratified by 62 different covariates. After showing that the PGS has different predictive power across strata for 18 out of 62 covariates, they turn to understanding why these differences and seeing if predictive performance could be improved. First they investigated which types of covariates result in the largest differences in PGS predictive power, finding that covariates with with larger "main effects" on the trait and covariates with larger interaction effects (interacting with the PGS to affect the trait) tend to better stratify individuals by PGS performance. The authors then see if including interactions between the PGS and covariates improves predictive accuracy, finding that linear models only result in modest increases in performance but nonlinear models result in more substantial performance gains.

Overall, the results are interesting and well-supported. The results will be broadly interesting to people using and developing PGS methods, as well as the broader statistical genetics community.

A few of the important points of the paper are:

A major impediment to the clinical use of PGS is the interaction between the PGS and various other routinely measure covariates, and this work provides a very interesting empirical study along these lines. The problem is interesting, and the work presented here is a convincing empirical study of the problem.

The result that PGS accuracy differs across covariates, but in a way that is not well-captured by linear models with interactions is important for PGS method development.

The quantile regression analysis is an interesting approach to explore how and why PGS may differ in accuracy across different strata of individuals.

Reviewer #2 (Public Review):

Summary

The authors developed new tools for isolating PI3K activity and for labeling newly made membrane proteins for monitoring membrane trafficking. They found that PI3K activity alone was able to explain the increased presence of TRPV1 on the membrane independent of other cascades induced by NGF signaling. They also showed an interesting feedback between PI3K and the insulin receptor trafficking to the membrane.

Strengths:

A major strength of the paper is the innovative combination of techniques. The first technique used the optogenetic PhyB/PIF system. They anchored PhyB to the membrane and fused PIF with the interSH2 domain from PI3K. This allowed them to use 650nm light to induce an interaction between the PhyB and PIF resulting in a recruitment of the endogenous PI3K to the membrane through the iSH2 domain without actual activation of an RTK. This allowed them to dissect out one function, just PI3K recruitment/activation from the vast number of RTK downstream cascades.

The second technique was the development of a new non-canonical amino acid that is cell-impermeant. The authors synthesized the sTSO-sulfa-Cy5 compound that will react with the Tet3 ncAA through click chemistry. They showed that the sulfa-Cy5 did not cross the membrane and would be used to track protein production over time, though the reaction rates were slow as noted by the authors. The comparison of the sulfa-Cy5 data with the standard GFP with TIRF showed a clear difference indicating the useful information that is gained with the ncAA.

Another strength comes from the discovery that an isolated PI3K is responsible for increasing TRPV1 and InR trafficking to the plasma membrane.

Weakness:

The discussion does not go into much detail regarding the importance of their discovery of TRPV1 and InR increases trafficking due to PI3K activation. It also jumps to the limitations of in vivo implementation prematurely. These weaknesses are minor however.

The authors achieved their goal of creating the tools needed to separate out one of the many RTK signals and give a strong proof of concept implementation of their tools. Their results support their conclusions and will help understand how TRPV1 is regulated by signals other than the traditional channel activators. The tools developed in the article will be of use to the broader cell biology and biophysics community, not just the channel community. The opto control of the PhyB/PIF system makes it more convenient than other systems since it does not take the typical wavelengths needed for fluorescence. The cell-impermeant ncAA will also be a great tool for those studying membrane proteins, protein trafficking and protein dynamics.

Reviewer #1 (Public Review):

Summary:

This work seeks to isolate the specific effects of phosphoinositide 3-kinase (PI3K) on the trafficking of the ion channel TRPV1, distinct from other receptor tyrosine kinase-activated effectors. It builds on earlier studies by the same group (Stein et al. 2006; Stratiievska et al. 2018), which described the regulatory relationship between PI3K, nerve growth factor (NGF), and TRPV1 trafficking. A central theme of this study is the development of methods that precisely measure the influence of PI3K on TRPV1 trafficking and vice versa. The authors employ a range of innovative methodologies to explore the dynamics between TRPV1 and PI3K trafficking.

Strengths:

A major strength of this study is the application of innovative methods to understand the interaction between PI3K and TRPV1 trafficking. The key techniques presented include:

(1) The optogenetic trafficking system based on phytochrome B, introduced in this research. Its interaction mechanism, dependent on reversible light activation, is comprehensively explained in Figures 1 and 2, with the system's efficacy demonstrated in Figure 3.

(2) An extracellular labeling method using click chemistry, which although not exclusive to this study, introduces specific reagents engineered for membrane impermeability.

The central biological insight presented here is the sufficiency of PI3K activation to guide TRPV1 trafficking to the plasma membrane. An additional notable discovery is the potential regulation of insulin receptors via this mechanism.

The paper's strengths are anchored in its innovative methodologies and the valuable collaboration between groups specializing in distinct areas of research.

Weaknesses:

The paper might benefit from a more streamlined structure and a clearer emphasis on its findings. A possible way to enhance its impact might be to focus more on its methodological aspects. The methodological facets stand out as both innovative and impactful. These experiments are well-executed and align with biological expectations. It's evident how these techniques could be tailored for many protein trafficking studies, a sentiment echoed in the manuscript (lines 287-288). When seen through a purely biological lens, some findings, like those concerning the PI3K-TRPV1 interaction, are very similar to previous work (Stratiievska et al. 2018). A biological focus demands further characterization of this interaction through mutagenesis. Also, the incorporation of insights on the insulin receptor feels somewhat tangential. A cohesive approach could be to reshape the manuscript with a primary focus on methodology, using TRPV1 and InsR as illustrative examples.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Koh, Stratiievska, and their colleagues investigate the mechanism by which TRPV1 channels are delivered to the plasma membrane following the activation of receptor tyrosine kinases, specifically focusing on the NGF receptor. They demonstrate that the activation of the NGF receptor's PI3K pathway alone is sufficient to increase the levels of TRPV1 at the plasma membrane.

Strengths:

The authors employ cutting-edge optogenetic, imaging, and chemical-biology techniques to achieve their research goals. They ingeniously use optogenetics to selectively activate the PI3K pathway without affecting other NGF pathways. Additionally, they develop a novel, membrane-impermeable fluorescent probe for labeling cell-surface proteins through click-chemistry.

Comment on revised version:

We commend the authors on the significant improvements made to the manuscript. They have adequately addressed our comments. Notably, the new control experiments shown in Figure 4E and Figure 5 Fig. Supp 1 convincingly demonstrate the specificity of the NGF and 650 nm light stimuli, respectively. The addition of quantitative analyses strengthens the findings significantly. Furthermore, the manuscript is now presented in a much linear manner, enhancing its clarity and impact.

Reviewer #2 (Public Review):

Summary:

The study tries to connect energy metabolism with immune tolerance during bacterial infection. The mechanism details the role of pyruvate transporter expression via ERRalpha-PGC1 axis, resulting in pro-inflammatory TNF alpha signalling responsible for acquired infection tolerance.

Strengths:

Overall, the study is an excellent addition to the role of energy metabolism during bacterial infection. The mechanism-based approach in dissecting the roles of metabolic coactivator, transcription factor, mitochondrial transporter, and pro-inflammatory cytokine during acquired tolerance towards infections indicates a detailed and well-written study. The in vivo studies in mice nicely corroborate with the cell line-based data, indicating the requirement for further studies in human infections with another bacterial model system.

Weaknesses:

The authors have involved various mechanisms to justify their findings. However, they have missed out on certain aspects which connect the mechanism throughout the paper. For example, they measured ATP and acetyl COA production linked with bacterial re-exposures and added various targets like MCP1, EER alpha, PGC1 alpha and TNF alpha. However, they skipped PGC1 alpha levels, ATP and acetyl COA in various parts of the paper. Including the details would make the work more comprehensive.

The use of public data sets to support their claim on immune tolerance is missing. Including various data sets of similar studies will strengthen the findings independently.

Reviewer #1 (Public Review):

Summary:

Their findings elucidate the mechanisms underlying 2-AA-mediated reduction of pyruvate transport into mitochondria, which impairs the interaction between ERRα and PGC1α, consequently suppressing MPC1 expression and reducing ATP production in tolerized macrophages. While the data presented is intriguing and the paper is well-written, there are several points that warrant consideration. The authors should enhance the clarity, relevance, and impact of their study.

Strengths:

This paper presents a novel discovery regarding the mechanisms through which PA regulates the bioenergetics of tolerized macrophages.

Weaknesses:

The relevance of the in vivo model to support the conclusions is questionable. Further clarification is needed on this point.

Reviewer #1 (Public Review):

Cheng et al explore the utility of analyte ratios instead of relative abundance alone for biological interpretation of tissue in a MALDI MSI workflow. Utilizing the ratio of metabolites and lipids that have complimentary value in metabolic pathways, they show the ratio as a heat map which enhances the understanding of how multiple analytes relate to each other spatially. Normally, this is done by projecting each analyte as a unique color but using a ratio can help clarify visualization and add to biological interpretability. However, existing tools to perform this task are available in open-source repositories, and fundamental limitations inherent to MALDI MSI need to be made clear to the reader. The study lacks rigor and controls, i.e. without quantitative data from a variety of standards (internal isotopic or tissue mimetic models for example), the potential delta in ionization efficiencies of different species subtracts from the utility of pathway analysis using metabolite ratios.

Reviewer #2 (Public Review):

Summary:

In the article, "Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging" the authors describe their software package in R for visualizing metabolite ratio pairs. I think the novelty of this manuscript is overstated and there are several notable issues with the figures that prevent detailed assessment but the work would be of interest to the mass spectrometry community.

Strengths:

The authors describe a software that would be of use to those performing MALDI MSI. This software would certainly add to the understanding of metabolomics data and enhance the identification of critical metabolites.

Weaknesses:

The authors are missing several references and discussion points, particularly about SIMS MSI, where ratio imaging has been previously performed.

There are several misleading sentences about the novelty of the approach and the limitations of metabolite imaging.

Several sentences lack rigor and are not quantitative enough.

The figures are difficult to interpret/ analyze in their current state and lack some critical components, including labels and scale bars.

Reviewer #1 (Public Review):

This important study uses a wide variety of convincing, state-of-the-art neuroimaging analyses to characterize whole-brain networks and relate them to reward-based motor learning. During early learning, the authors found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks. During late learning, they observed the opposite pattern. It remains to be seen whether these changes reflect generic changes in task engagement during learning or are specific to reward-based motor learning. This study is highly relevant for researchers interested in reward-based motor learning and decision-making.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is compelling and goes beyond the current state of the art. The results are of interest to neuroscientists working on motor control and reward-based learning.

Comments on revised version:

The revision has produced a stronger manuscript. Thank you for your thorough responses to the comments and concerns.

Reviewer #3 (Public Review):

Summary:

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field.

Strengths:

This manuscript is written in a clear and comprehensible way. It introduces a rather novel technique of assessing connectivity across lower-dimensional manifold which has hitherto not been applied in this way to the question of reward-based motor learning. Thus, this presents a unique viewpoint on understanding how the brain changes with motor learning. I particularly enjoyed the combination of connectivity-based, followed by further scrutiny of seed-based connectivity analyses, thus providing a more comprehensive viewpoint. Now it also has added a more comprehensive report on the behavioural changes of learning, and the added statistical quantification, which is useful.

Weaknesses:

The main weakness of the manuscript is the lack of direct connection between the reported neural changes and behavioural learning. Namely, most of the results could also be explained by changes in attention allocation during the session, or changes in movement speed (independent of learning). The authors acknowledge some of these potential confounds and argue that factors like attention are important for learning. While this is true, it is nonetheless very limiting if one cannot ascertain whether the observed effects are due to attention (independent of learning) or attention allocated in the pursuit of learning. The only direct analysis linking behavioural changes to neural changes is based on individual differences in learning performance, where the DAN-A shows the opposite trend than group level effects, which they interpret as differences given the used higher-resolution parcellation. However, it could be that these learning effects are indeed much smaller and subtler compared to more dominant group-level attention effects during the task. The lack of a control condition in the task limits the interpretability of results as learning-related.

Reviewer #1 (Public Review):

The authors investigated how global brain activity varied during reward-based motor learning. During early learning, they found increased covariance between the sensorimotor and dorsal attention networks, coupled with reduced covariance between the sensorimotor and default mode networks; during late learning, they found the opposite pattern. Individual learning performance varied only with changes in the dorsal attention network. The authors certainly used a wide variety of valuable, state-of-the-art techniques to interrogate whole-brain networks and extract the key components of learning behavior. However, the findings are incomplete, tempered by potential confounds in the experimental design. As such, the underlying claim regarding how these networks jointly support reward-based motor learning is unclear.

Reviewer #2 (Public Review):

This useful investigation of learning-driven dynamics of cortical and some subcortical structures combines a novel in-scanner learning paradigm with interesting analysis approaches. The new task for reward-based motor learning is highly compelling and goes beyond the current state-of-the-art, but it is incomplete with respect to examining different signatures of learning, clarifying probed learning processes, and investigating changes in all relevant subcortical structures is incomplete and would benefit from more rigorous approaches. With the rationale and data presentation strengthened this paper would be of interest to neuroscientists working on motor control and reward-based learning.

Reviewer #3 (Public Review):

The manuscript of Nick and colleagues addresses the intriguing question of how brain connectivity evolves during reward-based motor learning. The concept of quantifying connectivity through changes in extraction and contraction across lower-dimensional manifolds is both novel and interesting and the presented results are clear and well-presented. Overall, the manuscript is a valuable addition to the field. The evidence supporting the presented findings is strong, though at times lacking rigorous statistical quantification. Nevertheless, there are several issues that require attention and clarification.

Reviewer #1 (Public Review):

Fang Huang et al found that RBM7 deficiency promotes metastasis by coordinating MFGE8 splicing switch and NF-kB pathway in breast cancer by utilizing clinical samples as well as cell and tail vein injection models.

This study uncovers a previously uncharacterized role of MFGE8 splicing alteration in breast cancer metastasis, and provides evidence supporting RBM7 function in splicing regulation. These findings facilitate the mechanistic understanding of how splicing dysregulation contributes to metastasis in cancer, a direction that has increasingly drawn attention recently, and provides a potentially new prognostic and therapeutic target for breast cancer.

Reviewer #2 (Public Review):

In this manuscript, the authors reported the biological role of RBM7 deficiency in promoting metastasis of breast cancer. They further used a combination of genomic and molecular biology approaches to discover a novel role of RBM7 in controlling alternative splicing of many genes in cell migration and invasion, which is responsible for the RBM7 activity in suppressing metastasis. They conducted an in-depth mechanistic study on one of the main targets of RBM7, MFGE8, and established a regulatory pathway between RBM7, MFGE8-L/MFGE8-S splicing switch, and NF-κB signaling cascade. This link between RBM7 and cancer pathology was further supported by analysis of clinical data.

Overall, this is a very comprehensive study with lots of data, and the evidence is consistent and convincing. Their main conclusion was supported by many lines of evidence, and the results in animal models are pretty impressive.

Reviewer #1 (Public Review):

This paper reports the useful discovery of the roles and signaling components of the TOR pathway in vegetative growth, sexual development, stress response, and aflatoxin production in Aspergillus flavus.

While I acknowledge the authors' effort in conducting Southern blot analysis to address my prior concern regarding the presence of dual copies of torA and tapA, I find their current resolution inadequate. Specifically, the simple deletion of the respective result sections for torA and tapA significantly impacts the overall significance of this study. The repeated unsuccessful attempts to generate correct mutants only offer circumstantial evidence, as technical issues may have been a contributing factor. Therefore, instead of merely removing these sections, it is essential for the authors to present more compelling experimental data demonstrating that torA and tapA are indeed vital for the viability of A. flavus. Such data would enhance the overall significance of this study.

Reviewer #2 (Public Review):

In this study, authors identified TOR, HOG and CWI signaling network genes as modulators of the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation. They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, especially that the conserved site K19 of FKBP3 plays a key role in regulating aflatoxin biosynthesis. In general, the study involved a heavy workload and the findings are potentially interesting and important for understanding or controlling the aflatoxin biosynthesis. However, the findings have not been deeply explored and the conclusions mostly are based on parallel phenotypic observations.

Reviewer #1 (Public Review):

Summary:

The study investigates parafoveal processing during natural reading, combining eye-tracking and MEG techniques, building upon the RIFT paradigm previously introduced by Pan et al. (2021).

The manuscript is well-written with a clear structure, and the data analysis and experimental results are presented in a lucid manner.

Comments on revised version:

I am satisfied with the revisions made by the authors. I believe the study introduces a new research paradigm to the field.

Reviewer #1 (Public Review):

Summary:

The authors study the variability of patient response of NSCLC patients on immune checkpoint inhibitors using single-cell RNA sequencing in a cohort of 26 patients and 33 samples (primary and metastatic sites), mainly focusing on 11 patients and 14 samples for association analyses, to understand the variability of patient response based on immune cell fractions and tumor cell expression patterns. The authors find immune cell fraction, clonal expansion differences, and tumor expression differences between responders and non-responders. Integrating immune and tumor sources of signal the authors claim to improve prediction of response markedly, albeit in a small cohort.

Strengths:

- The problem of studying the tumor microenvironment, as well as the interplay between tumor and immune features is important and interesting and needed to explain the heterogeneity of patient response and be able to predict it.

- Extensive analysis of the scRNAseq data with respect to immune and tumor features on different axes of hypothesis relating to immune response and tumor immune evasion using state-of-the-art methods.

- The authors provide an interesting scRNAseq data set linked to outcomes data.

- Integration of TCRseq to confirm subtype of T-cell annotation and clonality analysis.

- Interesting analysis of cell programs/states of the (predicted) tumor cells and characterization thereof.

Weaknesses:

- Generally, a very heterogeneous and small cohort where adjustments for confounding are hard. Additionally, there are many tests for association with outcome, where necessary multiple testing adjustments would negate signal and confirmation bias likely, so biological takeaways have to be questioned.

- RNAseq is heavily influenced by the tissue of origin (both cell type and expression), so the association with the outcome can be confounded. The authors try to argue that lymph node T-cell and NK content are similar, but a quantitative test on that would be helpful.

- The authors claim a very high "accuracy" performance, however, given the small cohort and lack of information on the exact evaluation it is not clear if this just amounts to overfitting the data.

- Especially for tumor cell program/state analysis the specificity to the setting of ICIs is not clear and could be prognostic.

- Due to the small cohort with a lot of variability, more external validation is needed to be convincingly reproducible, especially when talking about AUC/accuracy of a predictor.

Reviewer #2 (Public Review):

Summary:

The authors have utilised deep profiling methods to generate deeper insights into the features of the TME that drive responsiveness to PD-1 therapy in NSCLC.

Strengths:

The main strengths of this work lie in the methodology of integrating single-cell sequencing, genetic data, and TCRseq data to generate hypotheses regarding determinants of IO responsiveness.

Some of the findings in this study are not surprising and well precedented eg. association of Treg, STAT3, and NFkB with ICI resistance and CD8+ activation in ICI responders and thus act as an additional dataset to add weight to this prior body of evidence. Whilst the role of Th17 in PD-1 resistance has been previously reported (eg. Cancer Immunol Immunother 2023 Apr;72(4):1047-1058, Cancer Immunol Immunother 2024 Feb 13;73(3):47, Nat Commun. 2021; 12: 2606 ) these studies have used non-clinical models or peripheral blood readouts. Here the authors have supplemented current knowledge by characterization of the TME of the tumor itself.

Weaknesses:

Unfortunately, the study is hampered by the small sample size and heterogeneous population and whilst the authors have attempted to bring in an additional dataset to demonstrate the robustness of their approach, the small sample size has limited their ability to draw statistically supported conclusions. There is also limited validation of signatures/methods in independent cohorts, no functional characterisation of the findings, and the discussion section does not include discussion around the relevance/interpretation of key findings that were highlighted in the abstract (eg. role of Th17, TRM, STAT3, and NFKb). Because of these factors, this work (as it stands) does have value to the field but will likely have a relatively low overall impact.

Related to the absence of discussion around prior TRM findings, the association between TRM involvement in response to IO therapy in this manuscript is counter to what has been previously demonstrated (Cell Rep Med. 2020;1(7):100127, Nat Immunol. 2017;18(8):940-950., J Immunol. 2015;194(7):3475-3486.). However, it should be noted that the authors in this manuscript chose to employ alternative markers of TRM characterisation when defining their clusters and this could indicate a potential rationale for differences in these findings. TRM population is generally characterised through the inclusion of the classical TRM markers CD69 (tissue retention marker) and CD103 (TCR experienced integrin that supports epithelial adhesion), which are both absent from the TRM definition in this study. Additional markers often used are CD44, CXCR6, and CD49a, of which only CXCR6 has been included by the authors. Conversely, the majority of markers used by the authors in the cell type clustering are not specific to TRM (eg. CD6, which is included in the TRM cluster but is expressed at its lowest in cluster 3 which the authors have highlighted as the CD8+ TRM population). Therefore, whilst there is an interesting finding of this particular cell cluster being associated with resistance to ICI, its annotation as a TRM cluster should be interpreted with caution.

Reviewer #2 (Public Review):

Summary:

Singh and colleagues employ a methodic approach to reveal the function of the transcription factors Rela and Stat3 in the regulation of the inflammatory response in the intestine.

Strengths of the manuscript include the focus on the function of these transcription factors in hepatocytes and the discovery of their role in the systemic response to experimental colitis. While the systemic response to induce colitis is appreciated, the cellular and molecular mechanisms that drive such systemic response, especially those involving other organs beyond the intestine are an active area of research. As such, this study contributes to this conceptual advance. Additional strengths are the complementary biochemical and metabolomics approaches to describe the activation of these transcription factors in the liver and their requirement - specifically in hepatocytes - for the production of bile acids in response to colitis.

In this revised version, the authors have addressed previously raised questions.

Reviewer #3 (Public Review):

Summary:

The authors try to elucidate the molecular mechanisms underlying the intra-organ crosstalks that perpetuate intestinal permeability and inflammation.

Strengths:

This study identifies a hepatocyte-specific rela/stat3 network as a potential therapeutic target for intestinal diseases via the gut liver axis using both murine models and human samples.

Weaknesses:

(1) The mechanism by which DSS administration induces the activation of the Rela and Stat3 pathways and subsequent modification of the bile acid pathway remains clear. As the authors state, intestinal bacteria are one candidate, and this needs to be clarified. I recommend the authors investigate whether gut sterilization by administration of antibiotics or germ free condition affects 1. the activation of the Rela and Stat3 pathway in the liver by DSS-treated WT mice and 2. the reduction of colitis in DSS-treated relaΔhepstat3Δhep mice.

(2) It has not been shown whether DSS administration causes an increase in primary bile acids, represented by CDCA, in the colon of WT mice following activation of the Rela and Stat3 pathways, as demonstrated in Figure 6.

(3) The implications of these results for IBD treatment, especially in what ways they may lead to therapeutic intervention, need to be discussed.

The above weakness points have been resolved by the revision and additional experiments.

Reviewer #2 (Public Review):

This manuscript describes new methodology to study low water potential (drought) stress responses in agar plates. They devote considerable effort in comparing transcriptome data among various previously published experimental systems, examining how different approaches of reducing water potential impact the Arabidopsis root and shoot transcriptome. Each method purported to reduce water potential in plate-grown seedlings has a different effect on Arabidopsis root transcriptome responses, which is problematic for the field. In this reviewer's view, differences in transcriptome are not as important, and often not as informative as measurement of physiological parameters, which they do very little of in their study.

The focus on transcriptome data to the almost complete exclusion of other types of data is a symptom of a broader over-emphasis on the transcriptome that is quite prevalent in plant science now. We measure transcriptomes because we can, not because it is inherently the most informative thing to do. The important thing is protein amount, and even more so protein activity/function, which we know has an imperfect, at best, correlation with transcript level. This reviewer acknowledges that using Arabidopsis transcriptomics is a commonly employed method, and as such, the outcomes of this study will hold value for a broad audience, even if largely as a cautionary tale. If transcriptomics is used to identify candidate genes for future investigations, an approach that has had some success, then appropriate cautions should be taken in translating expectations about gene, protein, and phenotypic responses in field conditions.

Reviewer #3 (Public Review):

This work compares transcriptional responses of shoots and roots harvested from four plate-based assays that aim to simulate drought and from plants subjected to water deficit in pots using the model plant Arabidopsis thaliana with the goal to select a plate-based assay that best recapitulates transcriptional changes that are observed during water-deficit in pots. For the plate-based assays polyethylene glycol (PEG), mannitol, and sodium chloride (salt) treatments were used as well as a 'hard agar' assay which was newly developed by the authors. In the 'hard agar' assay, less water was added to the solid components of the media leading to an increase in agar strength and nutrient concentration. Plants in pots were grown on vermiculite with the same nutrient mix as used in the plates and drought was induced by withholding watering for five days.

The authors observed a good directional agreement of differential expressed genes for shoots between the plate assays on the vermiculite drying experiment. However, less directional agreement was observed for differential expressed genes of roots, except for their newly developed 'hard agar' assay which had good directional agreement. Testing whether the increase in agar strength or more concentrated nutrients are attributed to this, they found that both factors contributed to the effect of the 'hard agar'. Arabidopsis ecotypes that showed a stronger reduction in shoot size when grown on the 'hard agar' tended to have a lower fitness according to an external study which may indicate that the 'hard agar' assay simulates physiological relevant conditions.

The work highlights that transcriptional responses for simulated drought on plates and drought caused by water deficit are highly variable and dependent on the tissues that are observed. The authors demonstrate that transcriptomics can be used to select a suitable plate assay that most closely recapitulates drought through water deficit for plants grown in pots. Interestingly their newly developed 'hard agar' assay provides an alternative to traditional plate-based assays with improved directional agreement of differential expressed genes in roots in comparison to plants experiencing water deficit in vermiculite. It is promising that the impact of 'hard agar' on the shoot size of 20 diverse Arabidopsis accessions shows some association with plant fitness under drought in the field. Their methodology could be powerful in identifying a better substitute for plate-based high-throughput drought assays that have an emphasis on gene expression changes.

Reviewer #1 (Public Review):

Summary:

The paper begins with phenotyping the DGRP for post-diapause fecundity, which is used to map genes and variants associated with fecundity. There are overlaps with genes mapped in other studies and also functional enrichment of pathways including most surprisingly neuronal pathways. This somewhat explains the strong overlap with traits such as olfactory behaviors and circadian rhythm. The authors then go on to test genes by knocking them down effectively at 10 degrees. Two genes, Dip-gamma and sbb, are identified as significantly associated with post-diapause fecundity, and they also find the effects to be specific to neurons. They further show that the neurons in the antenna but not the arista are required for the effects of Dip-gamma and sbb. They show that removing the antenna has a diapause-specific lifespan-extending effect, which is quite interesting. Finally, ionotropic receptor neurons are shown to be required for the diapause-associated effects.

Strengths and Weaknesses:

Overall I find the experiments rigorously done and interpretations sound. I have no further suggestions except an ANOVA to estimate the heritability of the post-diapause fecundity trait, which is routinely done in the DGRP and offers a global parameter regarding how reliable phenotyping is. A minor point is I cannot find how many DGRP lines are used.

Reviewer #2 (Public Review):

Summary

In this study, Easwaran and Montell investigated the molecular, cellular, and genetic basis of adult reproductive diapause in Drosophila using the Drosophila Genetic Reference Panel (DGRP). Their GWAS revealed genes associated with variation in post-diapause fecundity across the DGRP and performed RNAi screens on these candidate genes. They also analyzed the functional implications of these genes, highlighting the role of genes involved in neural and germline development. In addition, in conjunction with other GWAS results, they noted the importance of the olfactory system within the nervous system, which was supported by genetic experiments. Overall, their solid research uncovered new aspects of adult diapause regulation and provided a useful reference for future studies in this field.

Strengths:

The authors used whole-genome sequenced DGRP to identify genes and regulatory mechanisms involved in adult diapause. The first Drosophila GWAS of diapause successfully uncovered many QTL underlying post-diapause fecundity variations across DGRP lines. Gene network analysis and comparative GWAS led them to reveal a key role for the olfactory system in diapause lifespan extension and post-diapause fecundity.

Weaknesses:

(1) I suspect that there may be variation in survivorship after long-term exposure to cold conditions (10ºC, 35 days), which could also be quantified and mapped using genome-wide association studies (GWAS). Since blocking Ir21a neuronal transmission prevented flies from exiting diapause, it is possible that natural genetic variation could have a similar effect, influencing the success rate of exiting diapause and post-diapause mortality. If there is variation in this trait, could it affect post-diapause fecundity? I am concerned that this could be a confounding factor in the analysis of post-diapause fecundity. However, I also believe that understanding phenotypic variation in this trait itself could be significant in regulating adult diapause.

(2) On p.10, the authors conclude that "Dip-𝛾 and sbb are required in neurons for successful diapause, consistent with the enrichment of this gene class in the diapause GWAS." While I acknowledge that the results support their neuronal functions, I remain unconvinced that these genes are required for "successful diapause". According to the RNAi scheme (Figure 4I), Dip-γ and sbb are downregulated only during the post-diapause period, but still show a significant effect, comparable to that seen in the nSyb Gal4 RNAi lines (Figure 4K). In addition, two other RNAi lines (SH330386, 80461) that did not show lethality did not affect post-diapause fecundity. Notably, RNAi (27049, KK104056) substantially reduced non-diapause fecundity, suggesting impairment of these genes affects fecundity in general regardless of diapause experience. Therefore, the reduced post-diapause fecundity observed may be a result of this broader effect on fecundity, particularly in a more "sensitized" state during the post-diapause period, rather than a direct regulation of adult diapause by these genes.

(3) The authors characterized 546 genetic variants and 291 genes associated with phenotypic variation across DGRP lines but did not prioritize them by significance. They did prioritize candidate genes with multiple associated variants (p.9 "Genes with multiple SNPs are good candidates for influencing diapause traits."), but this is not a valid argument, likely due to a misunderstanding of LD among variants in the same gene. A gene with one highly significantly associated variant may be more likely to be the causal gene in a QTL than a gene with many weakly associated variants in LD. I recommend taking significance into account in the analysis.

Reviewer #3 (Public Review):

Summary:

Drosophila melanogaster of North America overwinters in a state of reproductive diapause. The authors aimed to measure 'successful' D. melanogaster reproductive diapause and reveal loci that impact this quantitative trait. In practice, the authors quantified the number of eggs produced by a female after she exited 35 days of diapause. The authors claim that genes involved with olfaction in part contribute to some of the variation in this trait.

Strengths:

The work used the power platform of the fly DRGP/GWAS. The work tried to verify some of the candidate loci with targeted gene manipulations.

Weaknesses:

Some context is needed. Previous work from 2001 established that D. melanogaster reproductive diapause in the laboratory suspends adult aging but reduces post-diapause fecundity. The work from 2001 showed the extent fecundity is reduced is proportional to diapause duration. As well, the 2001 data showed short diapause periods used in the current submission reduce fecundity only in the first days following diapause termination; after this time fecundity is greater in the post-diapause females than in the non-diapause controls.

In this context, the submission fails to offer a meaningful concept for what constitutes 'successful diapause'. There is no biological rationale or relationship to the known patterns of post-diapause fecundity. The phenotype is biologically ambiguous.

I have a serious concern about the antenna-removal design. These flies were placed on cool/short days two weeks after surgery. Adults at this time will not enter diapause, which must be induced soon after eclosion. Two-week-old adults will respond to cool temperatures by 'slowing down', but they will continue to age on a time scale of day-degrees. This is why the control group shows age-dependent mortality, which would not be seen in truly diapaused adults. Loss of antennae increases the age-dependent mortality of these cold adults, but this result does not reflect an impact on diapause.

• Appraisal of whether the authors achieved their aims, and whether the results support their conclusions.

The work falls well short of its aim because the concept of 'successful diapause' is not biologically established. The paper studies post-diapause fecundity, and we don't know what that means. The loci identified in this analysis segregate for a minimally constructed phenotype. The results and conclusions are orthogonal.

• The likely impact of the work on the field, and the utility of the methods and data to the community.

The work will have little likely impact. Its phenotype and operational methods are weakly developed. It lacks insight based on the primary literature on post-diapause. The community of insect diapause investigators are not likely to use the data or conclusions to understand beneficial or pest insects, or the impact of a changing climate on how they over-winter.

Reviewer #1 (Public Review):

Summary:

The study presented by Atsumi et al. is about using smartphone-driven, community-sourced data to enhance biodiversity monitoring. The idea is to leverage the widespread use of smartphones to gather data from the community quickly, contributing to a more comprehensive understanding of biodiversity. The authors discuss the importance of ecosystem services linked to biodiversity and the threats posed by human activities. It emphasizes the need for comprehensive biodiversity data to implement the Kunming-Montreal Global Biodiversity Framework. The 'Biome' mobile app, launched in Japan, uses species identification algorithms and gamification to gather over 6 million observations since 2019. While community-sourced data may have biases, incorporating it into Species Distribution Models (SDMs) improves accuracy, especially for endangered species. The app covers urban-natural gradients uniformly, enhancing traditional survey data biased towards natural areas. Combining these sources provides valuable insights into species distributions for conservation, protected area designation, and ecosystem service assessment.

Strengths:

The use of a smartphone app ('Biome') for community-driven species occurrence data collection represents an innovative and inclusive approach to biodiversity monitoring, leveraging the widespread use of smartphones. The app has successfully accumulated a large volume of species occurrence data since its launch in 2019, showcasing its effectiveness in rapidly gathering information from diverse locations. Despite challenges with certain taxa, the study highlights high species identification accuracy, especially for birds, reptiles, mammals, and amphibians, making the 'Biome' app a reliable tool for species observation. The integration of community-sourced data into Species Distribution Models (SDMs) improves the accuracy of predicting species distributions. This has implications for conservation planning, including the designation of protected areas and assessment of ecosystem services. The rapid accumulation of data and advancements in machine learning methods open up opportunities for conducting time-series analyses, contributing to the understanding of ecosystem stability and interaction strength over time. The study emphasizes the collaborative nature of the platform, fostering collaboration among diverse stakeholders, including local communities, private companies, and government agencies. This inclusive approach is essential for effective biodiversity assessment and decision-making. The platform's engagement with various stakeholders, including local communities, supports biodiversity assessment, management planning, and informed decision-making. Additionally, the app's role in fostering nature-positive awareness in society is highlighted as a significant contribution to creating a sustainable society.

Weaknesses:

While the studies make significant contributions to biodiversity monitoring, they also have some weaknesses. Firstly, relying on smartphone-driven, community-sourced data may introduce spatial and taxonomic biases. The 'Biome' app, for example, showed lower accuracy for certain taxa like seed plants, molluscs, and fishes, potentially impacting the reliability of the gathered data. Furthermore, the effectiveness of Species Distribution Models (SDMs) relies on the assumption that biases in community-sourced data can be adequately accounted for. The unique distribution patterns of the 'Biome' data, covering urban-natural gradients uniformly, might not fully represent the diversity of certain ecosystems, potentially leading to inaccuracies in the models. Moreover, the divergence in data distribution patterns along environmental gradients between 'Biome' data and traditional survey data raises concerns. The app data shows a more uniform distribution across natural-urban gradients, while traditional data is biased towards natural areas. This discrepancy may impact the representation of certain ecosystems and influence the accuracy of Species Distribution Models (SDMs). While the integration of 'Biome' data into SDMs improves accuracy, the study notes that controlling the sampling efforts is crucial. Spatially-biased sampling efforts in community-sourced data need careful consideration, and efforts to control biases are essential for reliable predictions.

Reviewer #1 (Public Review):

Summary:

Debeuf et al. introduce a new, fast method for the selection of suitable T cell clones to generate TCR transgenic mice, a method claimed to outperform traditional hybridoma-based approaches. Clone selection is based on the assessment of the expansion and phenotype of cells specific for a known epitope following immune stimulation. The analysis is facilitated by a new software tool for TCR repertoire and function analysis termed DALI. This work also introduces a potentially invaluable TCR transgenic mouse line specific for SARS-CoV-2.

Strengths:

The newly introduced method proved successful in the quick generation of a TCR transgenic mouse line. Clone selection is based on more comprehensive phenotypical information than traditional methods, providing the opportunity for a more rational T cell clone selection.

The study provides a software tool for TCR repertoire analysis and its linkage with function.

The findings entail general practical implications in the preclinical study of a potentially very broad range of infectious diseases or vaccination.

A novel SARS-CoV-2 spike-specific TCR transgenic mouse line was generated.

Weaknesses:

The authors attempt to compare their novel method with a more conventional approach to developing TCR transgenic mice. In this reviewer's opinion, this comparison appears imperfect in several ways:

• Work presenting the "traditional" method was inadequate to justify the selection of a suitable clone. It is therefore not surprising that it yielded negative results. More evidence would have been necessary to select clone 47 for further development of the TCR transgenic line, especially considering the significant time and investment required to create such a line.

• The comparison is somewhat unfair, because the methods start at different points: while the traditional method was attempted using a pool of peptides whose immunogenicity does not appear to have been established, the new method starts by utilising tetramers to select T cells specific for a well-established epitope.

• Given the costs and time involved, only a single clone could be tested for either method, intrinsically making a proper comparison unfeasible. Even for their new method, the authors' ability to demonstrate that the selected clone is ideal is limited unless they made different clones with varying profiles to show that a particular profile was superior to others.

In my view, there was no absolute need to compare this method with existing ones, as the proposed method holds intrinsic value.

While having more data to decide on clone selection is certainly beneficial, given the additional cost, it remains unclear whether knowing the expression profiles of different proteins in Figure 2 aids in selecting a candidate. Is a cell expressing more CD69 preferable to a cell expressing less of this marker? Would either have been effective? Are there any transcriptional differences between clonotype 1 and 2 (red colour in Figure 2G) that justify selecting clone 1, or was the decision to select the latter merely based on their different frequency? If all major clones (i.e. by clonotype count) present similar expression profiles, would it have been necessary to know much more about their expression profiles? Would TCR sequencing and an enumeration of clones have sufficed, and been a more cost-effective approach?

Lastly, it appears that several of the experiments presented were conducted only once. This information should have been explicitly stated in the figure legends.

Reviewer #2 (Public Review):

Summary:

The authors seek to use single-cell sequencing approaches to identify TCRs specific for the SARS CoV2 spike protein, select a candidate TCR for cloning, and use it to construct a TCR transgenic mouse. The argument is that this process is less cumbersome than the classical approach, which involves the identification of antigen-reactive T cells in vitro and the construction of T cell hybridomas prior to TCR cloning. TCRs identified by single-cell sequencing that are already paired to transcriptomic data would more rapidly identify TCRs that are likely to contribute to a functional response. The authors successfully identify TCRs that have expanded in response to SARS CoV2 spike protein immunization, bind to MHC tetramers, and express genes associated with functional response. They then select a TCR for cloning and construction of a transgenic mouse in order to test the response of resulting T cells in vivo following immunization with spike protein of coronavirus infection.

Strengths:

(1) The study provides proof of principle for the identification and characterization of TCRs based on single-cell sequencing data.

(2) The authors employ a recently developed software tool (DALI) that assists in linking transcriptomic data to individual clones.

(3) The authors successfully generate a TCR transgenic animal derived from the most promising T cell clone (CORSET8) using the TCR sequencing approach.

(4) The authors provide initial evidence that CORSET8 T cells undergo activation and proliferation in vivo in response to immunization or infection.

(5) Procedures are well-described and readily reproducible.

Weaknesses:

(1) The purpose of presenting a failed attempt to generate TCR transgenic mice using a traditional TCR hybridoma method is unclear. The reasons for the failure are uncertain, and the inclusion of this data does not really provide information on the likely success rate of the hybridoma vs single cell approach for TCR identification, as only a single example is provided for either.

(2) There is little information provided regarding the functional differentiation of the CORSET8 T cells following challenge in vivo, including expression of molecules associated with effector function, cytokine production, killing activity, and formation of memory. The study would be strengthened by some evidence that CORSET8 T cells are successfully recapitulating the functional features of the endogenous immune response (beyond simply proliferating and expressing CD44). This information is important to evaluate whether the presented sequencing-based identification and selection of TCRs is likely to result in T-cell responses that replicate the criteria for selecting the TCR in the first place.

(3) While I find the argument reasonable that the approach presented here has a lot of likely advantages over traditional approaches for generating TCR transgenic animals, the use of TCR sequencing data to identify TCRs for study in a variety of areas, including cancer immunotherapy and autoimmunity, is in broad use. While much of this work opts for alternative methods of TCR expression in primary T cells (i.e. CRISPR or retroviral approaches), the process of generating a TCR transgenic mouse from a cloned TCR is not in itself novel. It would be helpful if the authors could provide a more extensive discussion explaining the novelty of their approach for TCR identification in comparison to other more modern approaches, rather than only hybridoma generation.

Reviewer #1 (Public Review):

In their manuscript, Gerlevik et al. performed an integrative analysis of clinical, genetic and transcriptomic data to identify MDS subgroups with distinct outcomes. The study was based on the building of an "immunoscore" and then combined with genotype and clinical data to analyze patient outcomes using multi-omics factor analysis.

Strengths: Integrative analysis of RNA-seq, genotyping and clinical data

Weaknesses: Validation of the bioinformatic pipeline is incomplete

Major comments:

(1) This study considered two RNA-seq data sets publicly available and generated in two distinct laboratories. Are they comparable in terms of RNA-seq technique: polyA versus rRNA depletion, paired-end sequencing, fragment length?

(2) Data quality control (figure 1): the authors must show in a graph whether the features (dimensions) of factor 1 were available for each BMMNC and CD34+ samples.

(3) How to validate the importance of "immunoscore"? If GSEA of RNA-seq data was performed in the entire cohort, in the SF3B1-mutated samples or SRSF2-mutated samples (instead of patients having a high versus low level of factor 1 shown in Sup Fig. 4), what would be the ranking of Hallmarks or Reactome inflammatory terms among the others?

(4) To decipher cell-type composition of BMMNC and CD34+ samples, the authors used van Galen's data (2019; supplementary table 3). Cell composition is expressed as the proportion of each cell population among the others. Surprisingly, the authors found that the promonocyte-like score was increased in SF3B1-mutated samples and not in SRSF2-mutated samples, which are frequently co-mutated with TET2 and associated with a CMML-like phenotype. Is there a risk of bias if bone marrow subpopulations such as megakaryocytic-erythroid progenitors or early erythroid precursors are not considered?

(5) Figures 2a and 2b indicated that the nature of retrotransposons identified in BMMNC and CD34+ was different. ERVs were not detected in CD34+ cells. Are ERVs not reactivated in CD34+ cells? Is there a bias in the sequencing or bioinformatic method?

(6) What is the impact of factor 1 on survival? Is it different between BMMNC and CD34+ cells considering the distinct composition of factor 1 in CD34+ and BMMNC?

(7) In Figure 1e, genotype contributed to the variance of in the CD34+ cell analyses more importantly than in the BMMNC. Because the patients are different in the two cohorts, differences in the variance could be explained either by a greater variability of the type of mutations in CD34 or an increased frequency of poor prognosis mutations in CD34+ compared to BMMNC. The genotyping data must be shown.

(8) Fig. 2a-b: Features with high weight are shown for each factor. For factor 9, features seemed to have a low weight (Fig. 1b and 1c). However, factor 9 was predictive of EFS and OS in the BMMNC cohort. What are the features driving the prognostic value of factor 9?

(9) The authors also provided microarray analyses of CD34+ cell. It could be interesting to test more broadly the correlation between features identified by RNA-seq or microarrays.

(10) The authors should discuss the relevance of immunosenescence features in the context of SRSF2 mutation and extend the discussion to the interest of their pipeline for patient diagnosis and follow up under treatments.

Reviewer #2 (Public Review):

The authors performed a Multi-Omics Factor Analysis (MOFA) on analysis of two published MDS patient cohorts-1 from bone marrow mononuclear cells (BMMNCs) and CD34 cells (ref 17) and another from CD34+ cells (ref 15) --with three data modalities (clinical, genotype, and transcriptomics). Seven different views, including immune profile, inflammation/aging, Retrotransposon (RTE) expression, and cell-type composition, were derived from these modalities to attempt to identify the latent factors with significant impact on MDS prognosis.

SF3B1 was found to be the only mutation among 13 mutations in the BMMNC cohort that indicated a significant association with high inflammation. This trend was also observed to a lesser extent in the CD34+ cohort. The MOFA factor representing inflammation showed a good prognosis for MDS patients with high inflammation. In contrast, SRSF2 mutant cases showed a granulocyte-monocyte progenitor (GMP) pattern and high levels of senescence, immunosenescence, and malignant myeloid cells, consistent with their poor prognosis. Also, MOFA identified RTE expression as a risk factor for MDS. They proposed that this work showed the efficacy of their integrative approach to assess MDS prognostic risk that 'goes beyond all the scoring systems described thus far for MDS'.

Several issues need clarification and response:

(1) The authors do not provide adequate known clinical and molecular information which demonstrates prognostic risk of their sample cohorts in order to determine whether their data and approach 'goes 'beyond all the scoring systems described thus far for MDS'. For example, what data have the authors that their features provide prognostic data independent of the prior known factors related to prognosis (eg, marrow blasts, mutational, cytogenetic features, ring sideroblasts, IPSS-R, IPSS-M, MDA-SS)?

(2) A major issue in analyzing this paper relates to the specific patient composition from whom the samples and data were obtained. The cells from the Shiozawa paper (ref 17) is comprised of a substantial number of CMML patients. Thus, what evidence have the authors that much of the data from the BMMNCs from these patients and mutant SRSF2 related predominantly to their monocytic differentiation state?

(3) In addition, as the majority of patients in the Shiozawa paper have ring sideroblasts (n=59), thus potentially skewing the data toward consideration mainly of these patients, for whom better outcomes are well known.

(4) Further, regarding this patient subset, what evidence have the authors that the importance of the SF3B1 mutation was merely related to the preponderance of sideroblastic patients from whom the samples were analyzed?

(5) An Erratum was reported for the Shiozawa paper (Shiozawa Y, Malcovati L, Gallì A, et al. Gene expression and risk of leukemic transformation in myelodysplasia. Blood. 2018 Aug 23;132(8):869-875. doi: 10.1182/blood-2018-07-863134) that resulted from a coding error in the construction of the logistic regression model for subgroup prediction based on the gene expression profiles of BMMNCs. This coding error was identified after the publication of the article. The authors should indicate the effect this error may have had on the data they now report.

(6) What information have the authors as to whether the differing RTE findings were not predominantly related to the differentiation state of the cell population analyzed (ie higher in BM MNCs vs CD34, Fig 1)? What control data have the authors regarding these values from normal (non-malignant) cell populations?

(7) The statement in the Discussion regarding the effects of SRSF2 mutation is speculative and should be avoided. Many other somatic gene mutations have known stronger effects on prognosis for MDS.

Reviewer #1 (Public Review):

Summary:

Mao and colleagues re-analysed published spatial, bulk and single-cell transcriptomic datasets from primary colorectal cancers and colorectal-cancer-derived liver metastases. The analyses of paired cancer and non-cancer tissue samples showed that T cells are enriched in tumour tissue, accompanied by a reduction in the fraction of NK cells in the cancer tissue transcriptional datasets. Furthermore, authors claim that tumour tissue has a higher fraction of GZMK+ (resting) NK cells and suggest a correlation between the presence of these cells and poorer prognosis for cancer patients. In contrast, the increased frequency of KIR2DL4+ (activated) NK cells correlates with improved survival of cancer patients.

Strengths:

The authors performed a comprehensive analysis of published datasets, integrating spatial and single-cell transcriptomic data, which allowed them to discover the enrichment of GZMK+ NK cells in cancer tissues.

Weaknesses:

Despite their thorough analysis, the authors did not provide sufficient experimental evidence to support their claim that GZMK+ NK cells contribute to a worse prognosis for cancer patients or promote cancer progression. The terms resting and activated NK cells are used without properly defining the characteristics of these populations other than the gene expression of a handful of genes. Furthermore, the criteria used to quantify the NK cell population in spatial data is not entirely clear. While one can visually observe an increased fraction of GZMK+ NK cells compared to KIR2DL4+ NK cells in cancer tissues, no quantification is shown. They did not present any preclinical (animal model) or clinical data suggesting a causal relationship between NK cells and tumour growth. Thus, while a correlation may exist between the presence of GZMK+ NK cells and poorer tumour prognosis, causation cannot be claimed based on the available evidence. Furthermore, the in vitro data provided is limited to a single NK cell line derived from a lymphoma patient, which does not fully represent the diversity and functionality of human NK cells. Moreover, the in vitro experiments suffer from a lack of required controls and inadequate methodology.

Reviewer #2 (Public Review):

Summary:

This manuscript investigates the role of the abundant NK cells that are observed in colon cancer liver metastasis using sequencing and spatial approaches in an effort to clarify the pro and anti-tumourigenic properties of NK cells. This descriptive study characterises different categories of NK cells in tumour and tumour-adjacent tissues and some correlations. An attempt has been made using pseudotime trajectory analysis but no models around how these NK cells might be regulated are provided.

Strengths:

This study integrates multiomics data to attempt to resolve correlates of protection that might be useful in understanding NK cell diversity and activation.

Weaknesses:

While this work is interesting, the power of such studies is in taking the discovered information and applying this to other cohorts to determine the strength and predictive power of the genes identified. It is also clear that these 'snapshots' analysed poorly take account of the dynamic temporal changes that occur within a tumour. It would have been good to see a proposed model of NK cell regulation as it might occur in the tumour (accounting for turnover and recruitment) beyond the static data.

Reviewer #1 (Public Review):

The detection sensitivity and accuracy are unclear.

In this manuscript, Zhou et al describe a deaminase and reader protein-assisted RNA m5C sequencing method. The general strategy is similar to DART-seq for m6A sequencing, but the difference is that in DART-seq, m6A sites are always followed by C which can be deaminated by fused APOBEC1 to provide a high resolution of m6A sites, while in the case of m5C, no such obvious conserved motifs for m5C sites exist, therefore, the detection resolution is much lower. In addition, the authors used two known m5C binding proteins ALYREF and YBX1 to guide the fused deaminases, but it is not clear whether these two binding proteins can bind most m5C sites and compete with other m5C binding proteins.

It is well known that two highly modified m5C sites exist in 28S RNA and many m5C sites exist in tRNA, the authors should validate their methods first by detecting these known m5C sites and evaluate the possible false positives in rRNA and tRNA. In mRNA, it is not clear what is the overlap between the technical replicates. In Figures 4A and 4C, they detected more than 10K m5C sites, and most of them did not overlap with sites uncovered by other methods. These numbers are much larger than expected and possibly most of them are false positives. Besides, it is not clear what is the detection sensitivity and accuracy since the method is neither single base resolution nor quantitative. There are no experiments to show that the detected m5C sites are responsive to the writer proteins such as NSUN2 and NSUN6, and the determination of the motifs of these writer proteins.

Reviewer #2 (Public Review):

The fledgling field of epitranscriptomics has encountered various technical roadblocks with implications for the validity of early epitranscriptomics mapping data. As a prime example, the low specificity of (supposedly) modification-specific antibodies for the enrichment of modified RNAs, has been ignored for quite some time and is only now recognized for its dismal reproducibility (between different labs), which necessitates the development of alternative methods for modification detection. Furthermore, early attempts to map individual epitranscriptomes using sequencing-based techniques are largely characterized by the deliberate avoidance of orthogonal approaches aimed at confirming the existence of RNA modifications that have been originally identified.

Improved methodology, the inclusion of various controls, and better mapping algorithms as well as the application of robust statistics for the identification of false-positive RNA modification calls have allowed revisiting original (seminal) publications whose early mapping data allowed making hyperbolic claims about the number, localization and importance of RNA modifications, especially in mRNA. Besides the existence of m6A in mRNA, the detectable incidence of RNA modifications in mRNAs has drastically dropped.

As for m5C, the subject of the manuscript submitted by Zhou et al., its identification in mRNA goes back to Squires et al., 2012 reporting on >10.000 sites in mRNA of a human cancer cell line, followed by intermittent findings reporting on pretty much every number between 0 to > 100.000 m5C sites in different human cell-derived mRNA transcriptomes. The reason for such discrepancy is most likely of a technical nature. Importantly, all studies reporting on actual transcript numbers that were m5C-modified relied on RNA bisulfite sequencing, an NGS-based method, that can discriminate between methylated and non-methylated Cs after chemical deamination of C but not m5C. RNA bisulfite sequencing has a notoriously high background due to deamination artifacts, which occur largely due to incomplete denaturation of double-stranded regions (denaturing-resistant) of RNA molecules. Furthermore, m5C sites in mRNAs have now been mapped to regions that have not only sequence identity but also structural features of tRNAs. Various studies revealed that the highly conserved m5C RNA methyltransferases NSUN2 and NSUN6 do not only accept tRNAs but also other RNAs (including mRNAs) as methylation substrates, which in combination account for most of the RNA bisulfite-mapped m5C sites in human mRNA transcriptomes. Is m5C in mRNA only a result of the Star activity of tRNA or rRNA modification enzymes, or is their low stoichiometry biologically relevant?

In light of the short-comings of existing tools to robustly determine m5C in transcriptomes, other methods - like DRAM-seq, that allow the mapping of m5C independently of ex-situ RNA treatment with chemicals - are needed to arrive at a more solid "ground state", from which it will be possible to state and test various hypotheses as to the biological function of m5C, especially in lowly abundant RNAs such as mRNA.

Importantly, the identification of >10.000 sites containing m5C increases through DRAM-Seq, increases the number of potential m5C marks in human cancer cells from a couple of 100 (after rigorous post-hoc analysis of RNA bisulfite sequencing data) by orders of magnitude. This begs the question of whether or not the application of these editing tools results in editing artefacts overstating the number of actual m5C sites in the human cancer transcriptome.

Comments:

(1) The use of two m5C reader proteins is likely a reason for the high number of edits introduced by the DRAM-Seq method. Both ALYREF and YBX1 are ubiquitous proteins with multiple roles in RNA metabolism including splicing and mRNA export. It is reasonable to assume that both ALYREF and YBX1 bind to many mRNAs that do not contain m5C.

To substantiate the author's claim that ALYREF or YBX1 binds m5C-modified RNAs to an extent that would allow distinguishing its binding to non-modified RNAs from binding to m5C-modified RNAs, it would be recommended to provide data on the affinity of these, supposedly proven, m5C readers to non-modified versus m5C-modified RNAs. To do so, this reviewer suggests performing experiments as described in Slama et al., 2020 (doi: 10.1016/j.ymeth.2018.10.020). However, using dot blots like in so many published studies to show modification of a specific antibody or protein binding, is insufficient as an argument because no antibody, nor protein, encounters nanograms to micrograms of a specific RNA identity in a cell. This issue remains a major caveat in all studies using so-called RNA modification reader proteins as bait for detecting RNA modifications in epitranscriptomics research. It becomes a pertinent problem if used as a platform for base editing similar to the work presented in this manuscript.

(2) Since the authors use a system that results in transient overexpression of base editor fusion proteins, they might introduce advantageous binding of these proteins to RNAs. It is unclear, which promotor is driving construct expression but it stands to reason that part of the data is based on artifacts caused by overexpression. Could the authors attempt testing whether manipulating expression levels of these fusion proteins results in different editing levels at the same RNA substrate?

(3) Using sodium arsenite treatment of cells as a means to change the m5C status of transcripts through the downregulation of the two major m5C writer proteins NSUN2 and NSUN6 is problematic and the conclusions from these experiments are not warranted. Sodium arsenite is a chemical that poisons every protein containing thiol groups. Not only do NSUN proteins contain cysteines but also the base editor fusion proteins. Arsenite will inactivate these proteins, hence the editing frequency will drop, as observed in the experiments shown in Figure 5, which the authors explain with fewer m5C sites to be detected by the fusion proteins.

(4) The authors should move high-confidence editing site data contained in Supplementary Tables 2 and 3 into one of the main Figures to substantiate what is discussed in Figure 4A. However, the data needs to be visualized in another way than an Excel format. Furthermore, Supplementary Table 2 does not contain a description of the columns, while Supplementary Table 3 contains a single row with letters and numbers.

(5) The authors state that "plotting the distribution of DRAM-seq editing sites in mRNA segments (5'UTR, CDS, and 3'UTR) highlighted a significant enrichment near the initiation codon (Figure 3F).", which is not true when this reviewer looks at Figure 3F.

(6) The authors state that "In contrast, cells expressing the deaminase exhibited a distinct distribution pattern of editing sites, characterized by a prevalence throughout the 5'UTR.", which is not true when this reviewer looks at Figure 3F.

(7) The authors claim in the final conclusion: "In summary, we developed a novel deaminase and reader protein assisted RNA m5C methylation approach...", which is not what the method entails. The authors deaminate As or Us close to 5mC sites based on the binding of a deaminase-containing protein.

(8) The authors claim that "The data supporting the findings of this study are available within the article and its Supplementary Information." However, no single accession number for the deposited sequencing data can be found in the text or the supplementary data. Without the primary data, none of the claims can be verified.

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Reviewer #2 (Public Review):

Summary:

The authors showed the applicability and usefulness of a new AlphaFold2 pipeline called PabFold, which can predict linear antibody epitopes (B-cell epitopes) that can be helpful for the selection of reagents to be applied in competitive ELISA assay.

Strengths:

The authors showed the accuracy of the pipeline to identify correctly the binding epitope for three different antibody-antigen systems (Myc, HA, and Sars-Cov2 nucleocapsid protein). The design of scFvs from Fab of the three antibodies to speed up the analysis time is extremely interesting.

Weaknesses:

The article justifies correctly the findings and no great weaknesses are present. However, it could be useful for a broader audience to show in detail how pLDDT was calculated for both Simple-Max approach (per residue-pLDDT) and Consensus analysis ( average pLDDT for each peptide), with associated equations.

Reviewer #1 (Public Review):

Metabotropic glutamate receptors (mGLuRs) play a key role in regulating neuronal activity and related behaviors. In different brain regions these receptors can be expressed presynaptically and postsynaptically in different classes of neurons. Therefore, it is difficult to predict the effects of systemically applied drugs that act on these receptors. Here, the authors harness the power of photopharmacology, applying modulators that can be activated or inactivated by light with spatial precision, to address this problem. Their stated goal is to determine the role of mGluRs in regulating pain behaviors, and the circuit mechanisms driving this regulation. Their findings suggest that mGluRs acting in medial prefrontal cortex and thalamus drive antinociception in animals with neuropathic pain, whereas these receptors drive pronociception when acting in the amygdala. Their circuit analysis suggests that, in the amygdala, mGluRs act by decreasing feedforward inhibition of the output neurons. These findings have the potential to affect the development of targeted treatment for pain and related disorders. The elegant photopharmacological approaches will likely inform future studies attempting to distinguish the action of neuroactive drugs in different brain regions.

Reducing the impact of these studies are several methodological, analytical, and interpretation issues.

- The authors report that "the effect of optical manipulations of photosensitive mGlu5 NAMs in individual brain regions in pain models has been studied before". It is, therefore, not immediately clear what is novel in the present study.<br /> - The reliance only on reflexive measures of pain, especially in a study that examines the role of "affective and cognitive aspects of pain and pain modulation".<br /> - The inclusion of only males is unfortunate because of known, significant sex differences in neuronal circuits driving pain conditions, in both preclinical models (including form work by the authors) and in clinical populations.<br /> - The elegant slice experiments (especially Fig. 3) were designed to probe circuit mechanisms through which mGluRs act in different brain regions. These experiments also provide a control to assess whether the photopharmacological compounds act as advertised. Surprisingly, the effect size produced by these compounds on neuronal activity are rather small (and, at times, seems driven by outliers). How this small effect affects the interpretation of the behavioral findings is not clear.<br /> - These small effect sizes should also be considered when interpreting the circuit actions studied here.<br /> - Some of the sample sizes are as small as n=3. Without an a priori power analysis, it is difficult to assess the validity of the analyses.<br /> - The authors present intriguing data on changes in InsP levels in some (but not all) animals after injury, but not in sham animals. They also report an increase in the expression of mGLuRs expression in some, but not all brain regions. These findings are not discussed. It is not clear how these selective changes in mGluR expression and activity might affect the interpretation of the photopharmacological results.<br /> - The behavioral data seem to represent discrete, and not continuous variables. The statistical tests applied are likely inappropriate for these analyses.<br /> - The authors assume (and state in the abstract) that they can selectively stimulate BLA afferents to the neocortex. This is technically highly unlikely.<br /> - The results from the experiment on rostroventral medulla (RVM) neurons are less than convincing because only a "trend" towards decreased excitation is reported. As above, without consideration of effect size, it is hard to appreciate the significance of these findings. The absence of a demonstration of a classical ON Cell firing pattern is also unfortunate.

Reviewer #2 (Public Review):

In this study, Notartomaso et al. used optical activation of systemic JF-NP-26, a caged, baseline inactive, negative allosteric modulator (NAM) of mGlu5 receptors, in cingulate, prelimbic and infralimbic cortices, thalamus, and BLA to investigate the roles of these receptors in various brain regions in pain processing. They found that alloswitch-1, an intrinsically active mGlu5 receptor NAM, caused analgesia, but this analgesic effect was reversed by light-induced drug inactivation in the prelimbic and infralimbic cortices, and thalamus. In contrast, these pharmacological effects were reversed in the BLA. They further found that alloswitch-1 increased excitatory synaptic responses in prelimbic pyramidal neurons evoked by stimulation of BLA input, and decreased feedforward inhibition of amygdala output neurons by BLA. They thus concluded that mGlu5 receptors had differential effects in distinct brain regions. mGlu5 receptors are important receptors in pain processing, and their regional specificity has not been studied in detail. Further, this is an interesting study regarding the use of optical activation of pro-drugs, and the findings are timely. The combination of in vivo pharmacology, biochemistry, and slice EP provides complementary results.

Reviewer #3 (Public Review):

In this manuscript, Notartomaso, Antenucci et al. use two different light-sensitive metabotropic glutamate receptor negative allosteric modulators (NAMs) to determine how mGlu5 receptor signaling in distinct brain regions of mice influences mechanical sensitivity in chronic constriction injury (CCI) model of neuropathic pain. This is an extension of their previous work using photocaged mGlu5 negative allosteric modulators and incorporates a systemically active NAM that can be locally photoswitched off and on with violet and green light, respectively. The authors found that NAM signaling in the thalamus and prefrontal cortical regions consistently reduced mechanical hypersensitivity. However, they observed divergent effects on these measures in the basolateral amygdala. The authors attempted to solve the discrepancy in behavioral measurements between mGlu5 signaling in the basolateral amygdala by determining how NAMs modulate synaptic transmission or in vivo firing and found that these effects were projection-dependent.

Strengths:

This study demonstrates the importance of local signaling by mGlu5 receptors across multiple pain-processing circuits in the brain, and the use of optical activation and inactivation strategies is very intriguing.

Weaknesses:

One major limitation is the lack of sham surgery groups and vehicle/light-only controls in behavior and physiology experiments, though the authors did test mechanical sensitivity in the contralateral paw. The reliance on a single behavioral measure in these groups is also problematic. Many of these brain regions are known to influence distinct aspects of somatosensory processing or other behaviors entirely, which may be interpreted as hypersensitivity (e.g. fear or anxiety-like behaviors in the basolateral amygdala). Details on the light intensities used is also absent, and it is important to test whether violet light had any unintended effects on these cells/mice.

While the effort to provide some mechanistic understanding using slice physiology and in vivo recordings is appreciated, they ignore any effects that these NAMs have directly on the excitability of the recorded output neuron. In the models, mGlu5 is proposed to exist on some upstream inhibitory (mPFC) or excitatory (BLA) projection, but no evidence of a direct effect on these synaptic inputs was observed. Given the widespread distribution of mGlu5 in these brain regions, the proposed model seems unlikely. Perhaps CCI induces changes in functional coupling of mGlu5 in different cell types, and this could be revealed with appropriate control experiments.

Overall the broad profiling taken here across multiple brain regions lacks controls and some cohesion, making it challenging to conclude how mGlu5 signaling is acutely impacting these circuits and/or specific cell types.

Reviewer #1 (Public Review):

How does the brain respond to the input of different complexity, and does this ability to respond change with age?

The study by Lalwani et al. tried to address this question by pulling together a number of neuroscientific methodologies (fMRI, MRS, drug challenge, perceptual psychophysics). A major strength of the paper is that it is backed up by robust sample sizes and careful choices in data analysis, translating into a more rigorous understanding of the sensory input as well as the neural metric. The authors apply a novel analysis method developed in human resting-state MRI data on task-based data in the visual cortex, specifically investigating the variability of neural response to stimuli of different levels of visual complexity. A subset of participants took part in a placebo-controlled drug challenge and functional neuroimaging. This experiment showed that increases in GABA have differential effects on participants with different baseline levels of GABA in the visual cortex, possibly modulating the perceptual performance in those with lower baseline GABA. A caveat is that no single cohort has taken part in all study elements, ie visual discrimination with drug challenge and neuroimaging. Hence the causal relationship is limited to the neural variability measure and does not extend to visual performance. Nevertheless, the consistent use of visual stimuli across approaches permits an exceptionally high level of comparability across (computational, behavioural, and fMRI are drawing from the same set of images) modalities. The conclusions that can be made on such a coherent data set are strong.

The community will benefit from the technical advances, esp. the calculation of BOLD variability, in the study when described appropriately, encouraging further linkage between complementary measures of brain activity, neurochemistry, and signal processing.

Reviewer #2 (Public Review):

Lalwani et al. measured BOLD variability during the viewing of houses and faces in groups of young and old healthy adults and measured ventrovisual cortex GABA+ at rest using MR spectroscopy. The influence of the GABA-A agonist lorazepam on BOLD variability during task performance was also assessed, and baseline GABA+ levels were considered as a mediating variable. The relationship of local GABA to changes in variability in BOLD signal, and how both properties change with age, are important and interesting questions. The authors feature the following results: 1) younger adults exhibit greater task-dependent changes in BOLD variability and higher resting visual cortical GABA+ content than older adults, 2) greater BOLD variability scales with GABA+ levels across the combined age groups, 3) administration of a GABA-A agonist increased condition differences in BOLD variability in individuals with lower baseline GABA+ levels but decreased condition differences in BOLD variability in individuals with higher baseline GABA+ levels, and 4) resting GABA+ levels correlated with a measure of visual sensory ability derived from a set of discrimination tasks that incorporated a variety of stimulus categories.

Strengths of the study design include the pharmacological manipulation for gauging a possible causal relationship between GABA activity and task-related adjustments in BOLD variability. The consideration of baseline GABA+ levels for interpreting this relationship is particularly valuable. The assessment of feature-richness across multiple visual stimulus categories provided support for the use of a single visual sensory factor score to examine individual differences in behavioral performance relative to age, GABA, and BOLD measurements. Weaknesses of the study include the absence of an interpretation of the physiological mechanisms that contribute to variability in BOLD signal, particularly for the chosen contrast that compared viewing houses with viewing faces. Whether any of the observed effects can be explained by patterns in mean BOLD signal, independent of variability would be useful to know. The positive correlation between resting GABA+ levels and the task-condition effect on BOLD variability reaches significance at the total group level, when the young and old groups are combined, but not separately within each group. This correlation may be explained by age-related differences since younger adults had higher values than older adults for both types of measurements. This is not to suggest that the relationship is not meaningful or interesting, but that it may be conceptualized differently than presented. Two separate dosages of lorazepam were used across individuals, but the details of why and how this was done are not provided, and the possible effects of the dose are not considered. The observation of greater BOLD variability during the viewing of houses than faces may be specific to these two behavioral conditions, and lingering questions about whether these effects generalize to other types of visual stimuli, or other non-visual behaviors, in old and young adults, limit the generalizability of the immediate findings.

The observed age-related differences in patterns of BOLD activity and ventrovisual cortex GABA+ levels along with the investigation of GABA-agonist effects in the context of baseline GABA+ levels are particularly valuable to the field, and merit follow-up. Assessing background neurochemical levels is generally important for understanding individualized drug effects. Therefore, the data are particularly useful in the fields of aging, neuroimaging, and vision research.

Reviewer #3 (Public Review):

The role of neural variability in various cognitive functions is one of the focal contentions in systems and computational neuroscience. In this study, the authors used a large-scale cohort dataset to investigate the relationship between neural variability measured by fMRI and several factors, including stimulus complexity, GABA levels, aging, and visual performance. Such investigations are valuable because neural variability, as an important topic, is by far mostly studied within animal neurophysiology. There is little evidence in humans. Also, the conclusions are built on a large-scale cohort dataset that includes multi-model data. Such a dataset per se is a big advantage. Pharmacological manipulations and MRS acquisitions are rare in this line of research. Overall, I think this study is well-designed, and the manuscript reads well. I listed my comments below and hope my suggestions can further improve the paper.

Strength:<br /> (1) The study design is astonishingly rich. The authors used task-based fMRI, MRS technique, population contrast (aging vs. control), and psychophysical testing. I appreciate the motivation and efforts for collecting such a rich dataset.<br /> (2) The MRS part is good. I am not an expert in MRS so cannot comment on MRS data acquisition and analyses. But I think linking neural variability to GABA in humans is in general a good idea. There has been a long interest in the cause of neural variability, and inhibition of local neural circuits has been hypothesized as one of the key factors.<br /> (3) The pharmacological manipulation is particularly interesting as it provides at least evidence for the causal effects of GABA and deltaSDBOLD. I think this is quite novel.

Weakness:<br /> (1) I am concerned about the definition of neural variability. In electrophysiological studies, neural variability can be defined as Poisson-like spike count variability. In the fMRI world, however, there is no consensus on what neural variability is. There are at least three definitions. One is the variability (e.g., std) of the voxel response time series as used here and in the resting fMRI world. The second is to regress out the stimulus-evoked activation and only calculate the std of residuals (e.g., background variability). The third is to calculate variability of trial-by-trial variability of beta estimates of general linear modeling. It currently remains unclear the relations between these three types of variability with other factors. It also remains unclear the links between neuronal variability and voxel variability. I don't think the computational principles discovered in neuronal variability also apply to voxel responses. I hope the authors can acknowledge their differences and discuss their differences.<br /> (2) If I understand it correctly, the positive relationship between stimulus complexity and voxel variability has been found in the author's previous work. Thus, the claims in the abstract in lines 14-15, and section 1 in results are exaggerated. The results simply replicate the findings in the previous work. This should be clearly stated.<br /> (3) It is difficult for me to comprehend the U-shaped account of baseline GABA and shift in deltaSDBOLD. If deltaSDBOLD per se is good, as evidenced by the positive relationship between brainscore and visual sensitivity as shown in Fig. 5b and the discussion in lines 432-440, why the brain should decrease deltaSDBOLD ?? or did I miss something? I understand that "average is good, outliers are bad". But a more detailed theory is needed to account for such effects.<br /> (4) Related to the 3rd question, can you should the relationship between the shift of deltaSDBOLD (i.e., the delta of deltaSDBOLD) and visual performance?<br /> (5) Are the dataset openly available ?? I didn't find the data availability statement.

Reviewer #1 (Public Review):

The study is thorough and systematic, and in comparing three well-separated hypotheses about the mechanism leading from grid cells to hexasymmetry it takes a neutral stand above the fray which is to be particularly appreciated. Further, alternative models are considered for the most important additional factor, the type of trajectory taken by the agent whose neural activity is being recorded. Different sets of values, including both "ideal" and "realistic" ones, are considered for the parameters most relevant to each hypothesis. Each of the three hypotheses is found to be viable under some conditions, and less so in others. Having thus given a fair chance to each hypothesis, nevertheless, the study reaches the clear conclusion that the first one, based on conjunctive grid-by-head-direction cells, is much more plausible overall; the hypothesis based on firing rate adaptation has intermediate but rather weak plausibility; and the one based on clustering of cells with similar spatial phases in practice would not really work. I find this conclusion convincing, and the procedure to reach it, a fair comparison, to be the major strength of the study.

What I find less convincing is the implicit a priori discarding of a fourth hypothesis, that is, that the hexasymmetry is unrelated to the presence of grid cells. Full disclosure: we have tried unsuccessfully to detect hexasymmetry in the EEG signal from vowel space and did not find any (Kaya, Soltanipour and Treves, 2020), so I may be ranting off my disappointment, here. I feel, however, that this fourth hypothesis should be at least aired, for a number of reasons. One is that a hexasymmetry signal has been reported also from several other cortical areas, beyond entorhinal cortex (Constantinescu et al, 2016); true, also grid cells in rodents have been reported in other cortical areas as well (Long and Zhang, 2021; Long et al, bioRxiv, 2021), but the exact phenomenology remains to be confirmed. Second, as the authors note, the conjunctive mechanism is based on the tight coupling of a narrow head direction selectivity to one of the grid axes. They compare "ideal" with "Doeller" parameters, but to me the "Doeller" ones appear rather narrower than commonly observed and, crucially, they are applied to all cells in the simulations, whereas in reality only a proportion of cells in mEC are reported to be grid cells, only a proportion of them to be conjunctive, and only some of these to be narrowly conjunctive. Further, Gerlei et al (2020) find that conjunctive grid cells may have each of their fields modulated by different head directions, a truly surprising phenomenon that, if extensive, seems to me to cast doubts on the relation between mass activity hexasymmetry and single grid cells.

Finally, a variant of the fourth hypothesis is that the hexasymmetry might be produced by a clustering of head direction preferences across head direction cells similar to that hypothesized in the first hypothesis, but without such cells having to fire in grid patterns. If head direction selectivity is so clustered, who needs the grids? This would explain why hexasymmetry is ubiquitous, and could easily be explored computationally by, in fact, a simplification of the models considered in this study.

Reviewer #2 (Public Review):

Grid cells - originally discovered in single-cell recordings from the rodent entorhinal cortex, and subsequently identified in single-cell recordings from the human brain - are believed to contribute to a range of cognitive functions including spatial navigation, long-term memory function, and inferential reasoning. Following a landmark study by Doeller et al. (Nature, 2010), a plethora of human neuroimaging studies have hypothesised that grid cell population activity might also be reflected in the six-fold (or 'hexadirectional') modulation of the BOLD signal (following the six-fold rotational symmetry exhibited by individual grid cell firing patterns), or in the amplitude of oscillatory activity recorded using MEG or intracranial EEG. The mechanism by which these network-level dynamics might arise from the firing patterns of individual grid cells remains unclear, however.

In this study, Khalid and colleagues use a combination of computational modelling and mathematical analysis to evaluate three competing hypotheses that describe how the hexadirectional modulation of population firing rates (taken as a simple proxy for the BOLD, MEG, or iEEG signal) might arise from the firing patterns of individual grid cells. They demonstrate that all three mechanisms could account for these network-level dynamics if a specific set of conditions relating to the agent's movement trajectory and the underlying properties of grid cell firing patterns are satisfied.

The computational modelling and mathematic analyses presented here are rigorous, clearly motivated, and intuitively described. In addition, these results are important both for the interpretation of hexadirectional modulation in existing data sets and for the design of future experiments and analyses that aim to probe grid cell population activity. As such, this study is likely to have a significant impact on the field by providing a firmer theoretical basis for the interpretation of neuroimaging data. To my mind, the only weakness is the relatively limited focus on the known properties of grid cells in rodent entorhinal cortex, and the network level activity that these firing patterns might be expected to produce under each hypothesis. Strengthening the link with existing neurobiology would further enhance the importance of these results for those hoping to assay grid cell firing patterns in recordings of ensemble-level neural activity.

Reviewer #3 (Public Review):

This is an interesting and carefully carried out theoretical analysis of potential explanations for hexadirectional modulation of neural population activity that has been reported in the human entorhinal cortex and some other cortical regions. The previously reported hexadirectional modulation is of considerable interest as it has been proposed to be a proxy for the activation of grid cell networks. However, the extent to which this proposal is consistent with the known firing properties of grids hasn't received the attention it perhaps deserves. By comparing the predictions of three different models this study imposes constraints on possible mechanisms and generates predictions that can be tested through future experimentation.

Overall, while the conclusions of the study are convincing, I think the usefulness to the field would be increased if null hypotheses were more carefully considered and if the authors' new metric for hexadirectional modulation (H) could be directly contrasted with previously used metrics. For example, if the effect sizes for hexadirectional modulation in the previous fMRI and EEG data could be more directly compared with those of the models here, then this could help in establishing the extent to which the experimental hexadirectional modulation stands out from path hexasymmetry and how close it comes to the striking modulation observed with the conjunctive models. It could also be helpful to consider scenarios in which hexadirectional modulation is independent of grid firing, for example perhaps with appropriate coordination of head direction cell firing.

Reviewer #1 (Public Review):

In this work, Plaza-Alonso et al. present a collection of volume electron microscopy (EM) reconstructions of human postmortem medial entorhinal cortex (MEC), and they measure properties of MEC cytoarchitecture and synapses as a function of neuroanatomical subdivision. The authors generate a sampling of 9 smaller (≲10 µm/side) EM reconstructions per subdivision to avoid prohibitively large (petabyte) EM volumes, using 3 reconstructions for each of 3 brain donors to control for inter-individual variability. Conducting in-depth analyses for 7 subdivisions (63 reconstructions total), the authors find little significant inter-subdivision variability in structural composition (volume fractions of cell bodies vs. neuropil vs. blood vessels) and multiple synapse properties (spatial distribution, density, area, shape, excitatory/inhibitory type, and postsynaptic cell compartment). They conclude that human MEC connectivity is largely homogeneous, with synapses arranged in a generally random spatial distribution and a large fraction of synapses being asymmetric (putatively excitatory). Their other findings include that asymmetric synapses are larger than symmetric/putatively inhibitory synapses; that asymmetric synapses prefer dendritic spines whereas symmetric synapses prefer dendritic shafts; and that a small fraction of synapses have larger, complex shapes that may suggest increased synaptic efficacy. They note that inhomogeneities may include inter-subdivision variation in asymmetric synapse area and complex-shaped synapse prevalence, and for some reconstructions (12/63), possible substructure in synapse distributions.

Strengths:<br /> The authors have carefully conducted this work, using reasonable methods and comparing their findings with previous volume EM reconstructions where possible. It represents a substantial effort, given the challenges of producing and annotating volume EM data and of collecting human postmortem tissue. They have thus contributed a brain-region-specific characterization of human postmortem tissue with value as both a data resource and an examination of postmortem EM reconstruction quality, given that postmortem tissue is less-studied with volume EM but could be an important source of human brain samples (for example in regions that are surgically inaccessible). Further, some of the authors' measurements may be of added value, as they suggest functional correlates for less-studied synapse structures (such as the differing sizes of complex and simple "macular" synapses formed onto dendritic spines vs. shafts).

Weaknesses:<br /> Despite these strengths, the analysis in this work may be impacted by multiple sources of experimental variability that may have contributed to the observed lack of structural variability, and the potential contributions of these should be addressed in making their claims.

(1) The authors' approach to tissue sampling may have resulted in under-sampling, which may have reduced the detection power of their tests. More specifically, each reconstructed EM volume measured ~10 µm x 7 µm x 6 µm (360 - 502 µm^3) and contained ~300-400 synapses (Lines 211-212, 772-773). Per donor, this amounts to a sampling volume of ~1500 µm^3 for each MEC subdivision or ~1x104 µm^3 total. By contrast, the volume of the adult human MEC is ~1x10^12 µm^3, roughly 1x10^8 times larger [1]. Thus, while these EM reconstructions reflect a substantial effort, it is likely that they represent an under-sampling of MEC structure, especially since multiple excitatory and inhibitory neuron types are likely interspersed throughout (the authors also note this possibility in Lines 640-659).

(2) The authors' measurements are combined across three donors who are biologically diverse (Table S11), including in terms of characteristics that themselves may impact neuronal connectivity. Without controlling for these variables, the possible reduction in stochastic, biological inter-individual variability that could be achieved by combining data across donors may be offset by increases in phenotype-related variability, which could reduce the detectability of true, conserved connectivity variations across MEC subdivisions. Specifically, these donors represent a mix of males and females; a mix of ages (40, 53, and 66 years) that suggest differing degrees of aging-related changes in neuronal connectivity (according to previous work, a majority of people >55 years of age are estimated to have Alzheimer's-associated neurofibrillary tangles, regardless of whether they have dementia symptomatology; see for instance [1]); and one death from metastatic cancer, indicating that for one donor cellular/neuronal abnormalities associated either with cancer itself or related therapies could be present.

These two factors could substantially increase the dispersion of the authors' measurements in each MEC subdivision and lead to a situation with no detectable differences between subdivisions. It would be important to address these impacts when determining whether to interpret a lack of significant differences as true biological homogeneity for human MEC.

One helpful approach would be to explicitly show the variance of each measurement obtained for each EM reconstruction. For example, error bars showing the interquartile range could be added to each data point in Fig. 3C, to show how much synapse areas vary per reconstruction and to allow some comparison across donors and MEC subdivisions.

(3) A third potential source of variability relates to the authors' approach for synapse annotation. They appear to annotate active zones and postsynaptic densities by thresholding synapse images at some user-defined pixel intensity value, taking only pixels darker than that threshold as their annotations (Lines 806 - 812). This technique seems like it could be prone to producing noisy annotations, particularly since in the EM images provided (Figs. S11-16) the pixel intensities of active zones/postsynaptic densities and surrounding neuropil do not appear to be highly distinct.

It would be important for the authors to support their findings by quantifying the variability that may be associated with this technique.

[1] Price, C.C. et al., J. Int. Neuropsychol. Soc., (2010), doi: 10.1017/S135561771000072X.

Reviewer #2 (Public Review):

Plaza-Alanso et al. characterize synaptic properties across human medial entorhinal cortex across layers and, importantly, across multiple individuals. Using an impressive collection of post-mortem autopsy samples, they generate high resolution 3d FIB-SEM volumes across layers and MEC subregions and measure features such as synapse density, spatial distribution, size, shape and target location. The use of volumes permits a richer local context to synaptic reconstructions, and the methods used to count and quantify synapses appear thorough and convincing, although with limited descriptions at times. The core findings suggest few differences in most properties across either layers or individuals, with some modest exceptions in layers 1 and 6. A particular strength of the dataset is the large number of high quality synaptic contact reconstructions.<br /> However, because the volumes have no specific labels and are too small to associate axons or dendrites with individual cells or cell types, it is not clear how to extrapolate these findings to new insights toward the stated goal of a better understanding of the networks and connectivity characteristics of the MEC. Broadly speaking, the paper would benefit from a better explanation of why these specific parameters were chosen and what the authors hoped to gain from them. It might be useful to think of what would need to be the case to see something substantially different. Many of the measures here reflect the properties of dendrites passing through a small volume, which depends on the number of cells of different cell types, the length and thickness of their dendritic arbors, synapse density distributions, local and long range afferents, and more. One interpretation of these results is that these neuropil volumes across layers and individuals are effectively fully packed with dendrites, with a similar ratio of excitatory and inhibitory neurons, dendrites with roughly similar thickness and synaptic input density and local E/I balance. Can the authors disentangle these cellular-scale contributions or constrain their inter-individual variability across individuals? The lack of variability is perhaps the main observation here, and understanding this more clearly could be useful for thinking about larger volumes where fewer replicates are currently possible.

Reviewer #1 (Public Review):

Article strengths:

(1) Detailed data: The authors provided a large amount of clinical data as support, making the analysis results more persuasive and credible.<br /> (2) Scientific method: Appropriate statistical methods were used to analyze the data, which can accurately reflect the internal laws and trends of the data.<br /> (3) Clear conclusions: The conclusions drawn in the article are clear and explicit, easy for readers to understand and accept.<br /> (4) High practicality: The research results have important guiding significance for obstetrics and gynecology clinical practice, helping to improve patient treatment outcomes and quality of life.

Article weaknesses:

Limitations of research methods: Although the authors used statistical methods to analyze the data, they may be limited by factors such as data sources and sample size, leading to some limitations in the research results. It is recommended that the authors further expand the data sources and increase the sample size in subsequent studies to improve the accuracy and reliability of the research.

Reviewer #2 (Public Review):

This prospective study advances our understanding of the predictive value of preoperative serum CA125, CA19-9, CA72-4, CEA, and AFP in endometrial cancer. The evidence supporting the conclusions is convincing with rigorous analysis of the association between prognostic values of several serum markers with the clinical data of endometrial cancer patients. However, there are a few areas in which the article may be improved through further validation of the prognostic value of the risk score in patients with endometrial cancer at different stages. Moreover, the authors should provide a more detailed explanation of the choice of statistical methods in the manuscript. The work will be of broad interest to clinicians, medical researchers and scientists working in endometrial cancer.

(1) The groups of patients with endometrial cancer in the manuscript are classified according to age greater than/less than 60. Please explain why 60 years old is chosen as the boundary value of age.<br /> (2) Among the patients with endometrial cancer selected in the manuscript, AFP outliers accounted for a relatively small proportion. The authors chose the clinical detection outliers of CA-125, CA19-9, AFP and CEA as the dividing line, instead of re-selecting the optimal cut-off value in this population, which should be classified and the prognostic value explored.<br /> (3) In cancer research, stage is an important prognostic factor to guide the treatment of patients in clinical work. Patients with different stages of endometrial cancer have obvious prognostic differences. The authors constructed a new prognostic risk score based on serum level of AFP， CEA and CA125, the prognostic value of the risk score should be validated in patients with endometrial cancer at different stages。

Reviewer #3 (Public Review):

The authors of this study aimed to enhance the prognostic assessment of endometrial cancer (EC) by identifying and validating a set of serum tumor markers (CA125, CEA, and AFP) that could reliably predict progression-free survival (PFS) and overall survival (OS) in patients. By employing a robust methodology that included the use of LASSO Cox regression analysis to construct a predictive model, the study sought to provide a more nuanced tool for clinical decision-making in the management of EC.

Major Strengths:

Methodological Rigor: The study's use of advanced statistical methods to analyze a large dataset of EC patients stands out. The inclusion of a validation cohort enhances the credibility of the prognostic model developed.<br /> Clinical Relevance: The identification of CA125, CEA, and AFP as independent prognostic factors and the creation of a risk score based on these markers offer valuable tools for clinicians. The predictive accuracy of this model could significantly impact patient management and treatment planning.<br /> Weaknesses:

Generalizability: The study is based on a cohort from a single institution, which may limit the applicability of the findings across different populations and healthcare settings.<br /> Loss to Follow-Up: As acknowledged by the authors, the loss to follow-up of some patients introduces a potential source of bias, possibly affecting the study's conclusions.<br /> Achievement of Aims and Support for Conclusions:

The study successfully achieves its aim of developing a prognostic model for EC that integrates serum levels of CA125, CEA, and AFP. The evidence presented supports the authors' conclusions that this model is a robust tool for predicting patient outcomes, evidenced by its performance in both the training and validation cohorts.

Impact and Utility:

This work is poised to make a significant contribution to the field of gynecological oncology, particularly in the management of endometrial cancer. The study's findings provide a practical approach to stratifying patients based on their risk, which could be instrumental in tailoring individualized treatment plans. Moreover, the model's ability to predict PFS and OS with considerable accuracy offers a promising avenue for further research and application in clinical settings.

Additional Context:

Understanding the role of tumor markers in cancer prognosis is a rapidly evolving area of oncology research. This study's focus on combining multiple serum markers into a comprehensive risk score model represents a significant step forward in the quest for more personalized cancer care. Future studies could expand on this work by exploring the integration of such markers with other clinical and molecular data to further refine prognostic models.

Reviewer #1 (Public Review):

The current manuscript investigates the role of microRNA cluster 221/222 (miR221/222) in rheumatoid arthritis synovial fibroblasts (RA SFs) prompted by previous evidence that this cluster is upregulated in these cells. The authors employed multiple genetic mouse models and genomic approaches demonstrating that global overexpression of miR221/222 in huTNFtg polyarthritic mice further expanded SF proliferation and exacerbated RA, whereas global deletion reduced SF proliferation and dampened RA. Mechanistically, the authors provide sufficient evidence that these effects are mediated through the regulation of cell cycle inhibitors (p27 and p57) and the epigenetic regulator Smarca1. In general, these studies offer strong evidence that miR221/222 contributes to the pathogenic mechanisms underlying SF function in RA and provide new critical information to advance the understanding of RA pathology. However, certain important aspects are not addressed. Specifically, limited information related to the immune and inflammatory nature of this mechanism is offered, which is further complicated by limitations of using global overexpression and knockout. For example, it remains unknown to what is the extent of contribution by immune and inflammatory cells as well as what are the SF-derived effectors that propagate tissue damage and erosion

Reviewer #2 (Public Review):

This study focuses on the role of miR221/222 in the pathogenesis of rheumatoid arthritis. Through the use of different murine models and genome-wide techniques, the authors individuate a miR221/222 elicited mechanism leading to synovial fibroblast hyperproliferation. These discoveries may provide a rationale for future targeted therapies for RA treatment.

miR-221 and miR-222 have been linked with arthritis in previous studies from this and other laboratories: miR-221 and miR-222 have been found upregulated in SFs derived from the huTNFtg mouse model and RA patients, where their expression correlates with disease activity. The novelty of the present study resides in the analysis of the role of miR-221/miR-222 in an in vivo system and provides insight into cellular and molecular mechanisms linking miR-221/222 to RA progression.

Reviewer #3 (Public Review):

In this study, Roumelioti et al demonstrate the role of miR-221/222 in synovial fibroblasts (SFs) in inflammatory arthritis, applying a plethora of methods in three transgenic mouse models (huTNFtg, TgColVI-miR-221/222, huTNFtg;TgColVI-miR-221/222). miR-221/222 is upregulated in SFs, upon stimulation with TNF, both in early and established disease, while its gene is activated, as shown by scATAC-seq data. Using RNA sequencing and KEGG pathway analysis, authors showed that overexpression of miR-221 and miR-222 exacerbates arthritis, mainly due to SFs proliferation, driven by cell cycling inhibition and extracellular matrix remodeling. Although the authors suggest the potential utility of miR-221/222 targeting in inflammatory arthritis treatment, this was only examined through miR-221/222 -/- mice generation and not by direct silencing of miR-221/222 by administering a miR-221/222 antagonist.

Reviewer #1 (Public Review):

The authors tested the hypothesis that protein consumption decreases with decreasing mass-specific growth during development. This hypothesis is firmly grounded in the logical premise that as animals progress from periods of reduced activity and rapid growth to phases of increased activity and reduced mass-specific growth during their development, they are likely to adjust their nutrient intake, reducing protein and increasing carbohydrate consumption accordingly. The authors tested their hypothesis using the South American locust Schistocerca cancellata, combining field observations with laboratory experiments. This approach allowed them to discern how variations in activity history and metabolism between field- and laboratory-raised locusts influenced their nutrient requirements.<br /> Their findings, indeed reveal the predicted shift from high protein: carbohydrate consumption to lower protein: carbohydrate intake from the first instar to adult locust - a decline that strongly correlated with a decrease in mass-specific growth rate. Their comparison between field- and laboratory-raised locusts, showed that protein demand was not different, however, carbohydrate consumption rate was >50% higher in the field locusts. These results add depth and significance to the study, shedding light on how environmental factors influence nutrient requirements.<br /> What truly amplifies the strength and novelty of the authors' hypothesis is their anticipation that this observed trend in Schistocerca cancellata could extend to all animals. This anticipation is rooted in the expectation that growth rates scale hypometrically across various body sizes and developmental stages, introducing a universal dimension to their findings that holds great promise for broader ecological and evolutionary understanding.<br /> However, while the study is commendable in its methodology and core findings, there is room for improvement in clarifying the implications of the results. The current lack of clarity is evident in the somewhat shallow questions outlined in lines 358 to 363. For instance, the practice of administering age-specific diets has been commonplace in human and livestock management for ages. Thus, its continued utility may not be the most stimulating question. Instead, a more thought-provoking inquiry might delve into whether variations in global protein availability play a pivotal role in driving niche specialization and the biogeography of animal body sizes and ontogeny, especially considering the potential impacts of climate change. Such inquiries would further elevate the significance of the author's work and its broader implications in the field.

Reviewer #2 (Public Review):

How and why nutritional requirements and intake targets change over development and differ between species are significant questions with wide-ranging implications spanning ecology to health. In this manuscript, Talal et al. set out to address these questions in laboratory and field experiments with grasshoppers and in a comparative analysis of different species.

The authors conclude that the target intake of protein to non-protein energy (in this case carbohydrate) (P:C) falls over developmental stages and that this occurs because of a decline in mass-specific intake of protein whereas mass-specific carbohydrate intake remains more constant. The decrease in mass-specific protein consumption rate is tightly correlated with a decline in specific growth rate. Hence, protein consumption directly reflects requirements for growth, with hypometric scaling of protein intake serving as a useful relationship in nutritional ecology.

The laboratory experiments on the locust, Schistocerca cancellata, provide an elegant dataset in which different instars have been provided with one of two nutritionally complementary food pairings differing in protein to carbohydrate (P: C) content, and their self-selected protein to carbohydrate "intake target" measured.

These lab locust results were then compared with independently collected field data for late instar nymphs of the same locust species, and the conclusion is drawn that field insects ingested similar protein but 50-90% more carbohydrate (with only 23% increased mass-specific resting oxygen consumption rates). Numerous uncontrolled variables between the lab and field studies make meaningful conclusions difficult to draw from this observation.

A graph is then provided showing comparative data across a selection of species, making the case that protein consumption scales similarly both developmentally and across taxa. Questions need to be addressed for this to be convincing, including which criteria were used to select the examples in the graph and how comprehensively do these represent the available literature.

Reviewer #3 (Public Review):

The main goal of this study was to test how and why the intake of two important macronutrients ‒protein and carbon‒ often changes with ontogeny and body size. To do this, authors examined protein and carbon intake in a locusts lab population, across each instar and adult stages. Then, authors examined how the optimal balance of carbon and protein intake in a wild locusts population corresponded to that observed in the laboratory population. Results of these experiments showed that with ontogenic growth, locust decreased protein while increasing carbohydrate intake. Authors concluded that such decrease in the protein: carbohydrate intake may result from reductions in specific growth rates (growth within each instar). The protein: carbohydrate intake in the lab population appeared to be consistent with that observed in a wild locust population. Finally, authors combined their data with that from the literature to examine how protein intake scales with body mass throughout development, within and across different species.

Strengths:<br /> To determine how locusts balance protein: carbohydrate intake, authors applied the Geometric Framework (GF) of nutrition, which is a powerful approach for studying effects of nutrition and understanding the rules of compromise associated with balancing dietary unbalances.

Captivity can change behavior and physiology of most organisms, making it difficult to establish the relevance of laboratory experiments to what happens in the real world. A strength of this paper is that it compares behavior/physiology of lab vs. wild locusts. Finally, this study takes a step further by proposing a new scaling rule based on this study's results and data from the literature on various species.

Weaknesses:<br /> Although the paper has strengths, there seems to be several methodological issues that obscure the interpretation/conclusions presented in the manuscript.<br /> It appears that authors are not actually estimating "Intake Targets", as stated throughout the manuscript. According to the geometric framework, the intake target (IT) is estimated as the point in the nutritional landscape under which performance/fitness is optimized. The geometric framework also predicts that animals can reach their intake targets by feeding selectivity when given a choice of diets that differ in nutrient amounts, which is what authors did here. However, because the relationship between fitness/performance with diet was not established, in the choice experiments authors seem to be assuming (but not testing) that locusts are reaching their intake target.

You estimated a mass-specific protein intake for each instar. It is not clear why mass-specific intake and not just intake of protein was used for analysis. While mass (or size) of an individual may influence food consumption, it seems like authors calculated mass-specific consumption using each instar's final mass, which would make mass a result of protein consumption (and not the opposite). Importantly, the comparison between mass-specific protein consumption and specific growth rate may be problematic, as both variables seem to be estimated using final mass.