Reviewer #2 (Public Review):

Clément Mazeaud et al. identified the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a proviral cellular protein that regulates Zika virus (ZIKV) RNA replication by modulating the biogenesis of virus-induced replication organelles. Based on their findings and previously published data, the authors propose a model outlining the role of IGF2BP2 in the ZIKV infectious cycle. This model details the changes in IGF2BP2 interactions with both cellular and viral proteins and RNAs during viral infection.

Strengths:

This revised manuscript presents an interesting and convincing mechanism by which a cellular RNA-binding protein alters its protein and RNA interactome during viral infection. Using various molecular biology methods, proteomic analysis and a newly described replication-independent vesicle packets induction system, the authors describe the relevance of IGF2BP2 protein during Zika virus infection.

Weaknesses:

In the proposed model, the IGF2BP2 protein specifically binds to the 3' nontranslated region (NTR) of the ZIKV genome, while excluding binding to the 5' NTR. However, the authors cannot rule out the possibility that this host protein associates with other regions of the viral genome, a topic which is discussed in the manuscript.

In this study, the physiological cellular consequences of altering the interaction of IGF2BP2 with its endogenous mRNA ligands due to ZIKV infection remain unexplored. This aspect would be of interest for future studies.

Reviewer #2 (Public Review):

The central theme of the manuscript is to report on the structure of SBPase - an enzyme central to the photosynthetic Calvin-Benson-Bassham cycle. The authors claim that the structure is the first of its kind from a chlorophyte Chlamydomonas reinhardtii, a model unicellular green microalga. The authors use a number of methods like protein expression, purification, enzymatic assays, SAXS, molecular dynamics simulations and x-ray crystallography to resolve a 3.09 A crystal structure of the oxidized and partially reduced state. The results are supported by the claims made in the manuscript. While the structure is the first from a chlorophyte, it is not unique. Several structures of SBPase are available and a comparison has been made between the structure reported here and others that have been previously published.

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-tumourogenic properties of NK cells. This descriptive study characterizes different categories of NK cells in tumor and tumor 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 is 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. The authors have strengthened the study in revision by demonstrating the very strong correlation between Granzyme+ NK cells and the poor prognosis, but the main claims are only partially supported.

Weaknesses:

While this work is interesting, the power of such studies are 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. Further evidence linking mechanistic causality to prognostic outcome would provide significant data for approaches forward.

Reviewer #3 (Public Review):

Summary and Strength:

The manuscript by Amir et al. describes that Sertoli-specific inactivation of the mTORC1 and mTORC2 complex by KO of either Raptor or Rictor, respectively, resulted in progressive changes in blood-testis-barrier (BTB) function, testis weight, and sperm parameters, including counts, morphology, mtDNA content and sperm DNA methylation.

The described studies are based on the hypothesis that a decline of BTB function with increasing chronological age of a male contributes to the DNA methylation changes that are known to occur in sperm DNA of old males when compared to sperm DNA from isogenic young males. In order to demonstrate the relevance of a functioning BTB for the maintenance of sperm methylation patterns, the authors generated mice with genetically disrupted mTORC2 complex or mTORC1 complex in Sertoli cells and determined sperm methylation patterns in comparison to isogenic wild-type males. In line with previously published scientific literature (e.g. Mok et al., 2013; Dong et al, 2015; and others), the manuscript corroborates that a Sertoli-cell specific deletion of mTORC2 caused a loss of BTB function and a progressive spermatogenic defect. The authors further show that sperm DNA is differentially methylated (DMRs) as a consequence of either a mTORC2 disruption (associated with a loss of BTB function) or following a mTORC1 disruption (BTB function either increased or not leaky) when compared to their isogenic age-matched wt controls. Those DMRs overlap partially with changes in sperm DNA methylation that were found when comparing sperm from 8-week males with sperm isolated from 22-week-old male mice.

The authors interpret the observed changes as representative of the sperm DNA methylation changes that occur during normal chronological aging of the male. For an aged control group, the authors use sperm DNA of 22-week-old wild-type mates from the mTORC2 and mTORC2 KO breeding and compare the sperm methylation patterns found in sperm from those 22-week males to 8-week young males, that are intended to represent an old and a young cohort, respectively. DNA methylation analysis indicates that a disruption of mTORC2 (& decrease of BTB function) results in increased DNA methylation of sperm DNA, while a disruption of mTORC1 (and proposed increase of BTB tightness, not shown in the manuscript, though) resulted in increased hypomethylation.

Weaknesses:

While the hypothesis and experimental system are interesting and the data demonstrating the relevance of the mTORC2 complex for BTB function is convincing, several open questions limit the evidence that supports the hypothesis that the sperm DNA methylation changes seen in old males are caused by BTB failure following an imbalance of mTOR signaling complexes. The major critique points are the lack of a chronologically old group and the choice of 8 weeks & 22 weeks age of age:

- Data illustrating the degree of BTB decline and sperm DNA methylation changes from chronologically "old" male mice is missing. 22-week-old mice are not considered old but are of good and mature breeding age, equivalent to humans in their mid-late twenties. (In the manuscript, the 22-week-old wildtype mice show no evidence of BTB breakdown (Figure 3), so why are their sperm used to represent "aged" sperm?

- Adding a group of "old" wild-type mice of 12-14 months of age, which is closer to the end of effective reproduction in mice, more equivalent to 45-59 year-old humans) could be used to illustrate that (a) aging causes a marked decrease in BTB function at this time in mouse life, and that this BTB breakdown chronologically aligns with the age-associated DNA hypermethylation seen in old sperm. Age-matched "old" mTORC1 KO, with a (supposedly) tighter BTB barrier, could then be expected to have a sperm DMA methylation profile closer to that of younger wild-type animals. Such data are currently missing. While the progressive testicular decline observed in the mTORC1 KO (Fig.5) could make it difficult to obtain the appropriately aged mTORC1 KO tissues, it is completely feasible to obtain data from chronologically old wild-type males. (The progressive testicular decline further raises the question of what additional defects the KO causes, and how such additional defects would influence the sperm DNA methylation profile.) The addition of data from an old group to the currently included groups could strengthen the interpretation that the observations in the BTB-defective mTORC2 KO mice are modelling an age-related testicular decline, provided that the DMRs seen in the chronologically old group significantly overlap with the BTB-defective changes.

- In the current form, the described differences in sperm DNA methylation are based on comparisons between pubertal mice (8 weeks) and mature but not old adult males (22 weeks), while a chronologically "old" group is missing from the data sets and comparisons. Thus, it appears that the described sperm methylation changes reflect developmental changes associated with normal maturation and not necessarily declining sperm quality due to aging. (Sperm obtained from 8-week-old mice likely were generated, at least in part, during the 1st wave of spermatogenesis, which is known to differ from the continuously proceeding spermatogenesis during the remained of the mature life. During the 1st wave of spermatogenesis, Sertoli cells are known to undergo gene expression changes which could contribute to varying degrees of BTB function, and thus have effects on the sperm DNA methylation profiles of such 1st wave sperm.)

- It is unclear why the aging-related DMRs between the 8 and 22-week-old wild-type mice vary so dramatically between the two wild-type groups derived from the mTORC1 and the mTORC2 breeding (Fig. S4). If the main difference was due to mTORC1 or mTORC2 activity, both wildtype groups should behave very similarly. Changes seen in a truly "old" mouse (e.g. 20 weeks to 56 weeks), changes in "young mTORC1" and in "old mTORC2" are missing. How do those numbers and profiles compare to the shown samples?

Comments on latest version:

The rebuttal letter and public response indicate the authors' reluctance to consider the limitations of their study, i.e. having chosen chronologically young animals to demonstrate a sperm aging effect and indicate that they are not willing to include adequate controls.

Since there is no evidence that mice at this young age have a deteriorating blood-testis-barrier (indeed, normal intact BTB is clearly visible in the figures included in this study from animals of the relevant age group), the whole central hypothesis that the study is built upon (i.e. that increasing age causes deteriorating BTB integrity which in turn causes age-related changes in sperm DNA methylation), appears irrelevant or invalid.

The authors' claim that age-related DNA methylation changes in sperm occur in linear fashion and that the changes are somewhat proportional with chronological age is in stark contrast of the claim that a decline of the BTB in old animals is causative for age-related sperm epigenetic changes, putting the relevance of the whole study in question.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

We have only few comments to improve an already strong manuscript.

Comments:

(1) Can the authors comment on how they justify K+ ion positions in their models (e.g. the K+ ion bridging G-1 and G+1 nucleotides)? How do they discriminate e.g. in the 'G-1 and G+1' case K+ from water?<br /> (2) The authors comment on Yju2 and Fyv6 assignments in all yeast structures except for the ILS. Can the authors comment on if they have also looked into the assignment of Yju2 in the yeast ILS structure in the same manner? While it is possible that Fyv6 could dissociate and Yju2 reassociate at the P to ILS transition, this would merit a closer look given that in the yeast P complex Yju2 had been misassigned previously.<br /> (3) For accessibility to a general reader, figures 1c, d, e, 2a, b, would benefit from additional headings or labels, to immediately convey what is being displayed. It is also not clear to us if Fig 1e might fit better in the supplement and be instead replaced by Supplementary Figure 1a (wt) , b (delta upf1), and a new c (delta fyv6) and new d (delta upf1, delta fyv6). This may allow the reader to better follow the rationale of the authors' use of the Fyv6/Upf1 double deletion.<br /> (4) The authors carefully interpret the various suppressor mutants, yet to a general reader the authors may wish to focus this section on only the most critical mutants for a better flow of the text.

Reviewer #2 (Public Review):

Summary:

The authors have tested the effects of partial- or whole-chromosome aneuploidy on the m6A RNA modification in Drosophila. The data reveal that overall m6A levels trend up but that the number of sites found by meRIP-seq trend down, which seems to suggest that aneuploidy causes a subset of sites to become hyper-methylated. Subsequent bioinformatic analysis of other published datasets establish correlations between the activity of the H4K16 acetyltransferase dosage compensation complex (DCC) and the expression of m6A components and m6A abundance, suggesting that DCC and m6A can act in a feedback loop on each other. Overall, this paper uses bioinformatic trends to generate a candidate model of feedback between DCC and m6A. It would be improved by functional studies that validate the effect in vivo.

Strengths:

• Thorough bioinformatic analysis of their data.

• Incorporation of other published datasets that enhance scope and rigor.

• Finds trends that suggest that a chromosome counting mechanism can control m6A, as fits with pub data that the Sxl mRNA is m6A modified in XX females and not XY males.

• Suggests this counting mechanism may be due to the effect of chromatin-dependent effects on the expression of m6A components.

Weaknesses:

• The linkage between H4K16 machinery and m6A is indirect and based on bioinformatic trends with little follow-up to test the mechanistic bases of these trends.

• The paper lacks sufficient in vivo validation of the effects of DCC alleles on m6A and vice versa. For example, Is the Ythdc1 genomic locus a direct target of the DCC component Msl-2 ? (see Figure 7).

• Quite a bit of technical detail is omitted from the main text, making it difficult for the reader to interpret outcomes.

(1) Please add the tissues to the labels in Figure 1D.

(2) In the main text, please provide detail on the source tissues used for meRIP; was it whole larvae? adult heads? Most published datasets are from S2 cells or adult heads and comparing m6A across tissues and developmental stages could introduce quite a bit of variability, even in wt samples. This issue seems to be what the authors discuss in lines 197-199.

(3) In the main text, please identify the technique used to measure "total m6A/A" in Fig 2A. I assume it is mass spec.

(4) Line 190-191: the text describes annotating m6A sites by "nearest gene" which is confusing. The sites are mapped in RNAs, so the authors must unambiguously know the identity of the gene/transcript, right?

Reviewer #2 (Public Review):

Summary:<br /> This work aims to characterize the neural signaling cascade underlying the initiation of metamorphosis in Ciona larvae. Combining gene-specific functional analyses, pharmacological experiments, and live imaging approaches, the authors identify the molecular players downstream of GABA to initiate Ciona metamorphosis. The results of this study may serve as a useful framework for future research on animal metamorphosis.

Strengths:<br /> The authors did a great job in connecting their experiments with previous findings on Ciona metamorphosis. Taking advantage of the Ciona model system, they meticulously conducted genetic manipulation and pharmacological experiments to test the epistatic relationships among the signaling players controlling the initiation of Ciona metamorphosis.

Weaknesses:<br /> The causal relationship between cAMP accumulation and the initiation of metamorphosis was not clearly demonstrated by the life-imaging observation with the fluorescent cAMP indicator (Pink Flamindo). It is a pity that this experiment was only conducted using normal larvae to compare those who underwent metamorphosis versus those who failed to initiate metamorphosis. This approach should be applied to some of the genetic manipulation and pharmacological experiments, to strengthen their main thesis on the "cAMP timer" mechanism.<br /> On several occasions, the interpretation of the results seems to be imprecise and may lead to misunderstanding. This should be improved by rewriting the descriptions of those results and carefully comparing the differences in results from various treatments and experiments.

Reviewer #2 (Public Review):

Summary:

The authors provide the first (to my knowledge) detailed characterization of cell wall b-1,6 glucan in the pathogen Candida albicans. The approaches range from biochemistry to genetics to immunology. The study provides fundamental information and will be a resource of exceptional value to the field going forward. Highlights include the construction of a mutant that lacks all b-1,6 glucan and the characterization of its cell wall composition and structure. Figure 5a is a feast for the eyes, showing that b-1,6 glucan is vital for the outer fibrillar layer of the cell wall. Also much appreciated was the summary figure, Figure 7, which presents the main findings in digestible form.

Strengths:

The work is highly significant for the fungal pathogen field especially, and more broadly for anyone studying fungi, antifungal drugs, or antifungal immune responses.

The manuscript is very readable, which is important because most readers will be cell wall nonspecialists.

The authors construct a key quadruple mutant, which is not trivial even with CRISPR methods, and validate it with a complemented strain. This aspect of the study sets the bar high.

The authors develop new and transferable methods for b-1,6 glucan analysis.

Weaknesses:

The one "famous" cell type that would have been interesting to include is the opaque cell. This could be included in a future paper.

Reviewer #2 (Public Review):

Summary: Padder et al. demonstrate that ATG5 mediates lysosomal repair via the recruitment of the retromer components during LLOMe-induced lysosomal damage and that mAtg8-ylation contributes to retromer-dependent cargo sorting of GLUT1. Although previous studies have suggested that during glucose withdrawal, classical autophagy contributes to retromer-dependent GLUT1 surface trafficking via interactions between LC3A and TBC1D5, the experiments here demonstrate that during basal conditions or lysosomal damage, ATGs that are not involved in mATG8ylation, such as FIP200, are not functionally required for retromer-dependent sorting of GLUT1. Overall, these studies suggest a unique role for ATG5 in the control of retromer function, and that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes.

Strengths:

(1) Overall, these studies suggest a unique non-autophagic role for ATG5 in the control of retromer function. They also demonstrate that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes. Overall, these data point to a new role for ATG5 and CASM-dependent mATG8ylation in lysosomal membrane repair and trafficking.

(2) Although the studies are overall supportive of the proposed model that the retromer is controlled by CASM-dependent mATG8-ylaytion, it is noteworthy that previous studies of GLUT1 trafficking during glucose withdrawal (Roy et al. Mol Cell, PMID: 28602638) were predominantly conducted in cells lacking ATG5 or ATG7, which would not be able to discriminate between a CASM-dependent vs. canonical autophagy-dependent pathway in the control of GLUT1 sorting. Is the lack of GLUT1 mis-sorting to lysosomes observed in FIP200 and ATG13KO cells also observed during glucose withdrawal? Notably, deficiencies in glycolysis and glucose-dependent growth have been reported in FIP200 deficient fibroblasts (Wei et al. G&D, PMID: 21764854) so there may be differences in regulation dependent on the stress imposed on a cell.

Weaknesses:

(1) Additional controls are needed to clarify the role of CASM in the control of retromer function. Because the manuscript proposes both CASM-dependent and independent pathways in the ATG5 mediated regulation of the retromer, it is important to provide robust evidence that CASM is required for retromer-dependent GLUT1 sorting to the plasma membrane vs. lysosome. The experiments with monsensin in Fig. 7C-E are consistent with but not unequivocally corroborative of a role for CASM. Based on the results shown with ATG16KO in Fig 4A-D, rescue experiments of these 16KO cells with WT vs. C-terminal WD40 mutant versions of ATG16 will specifically assess the requirement for CASM and potentially provide more rigorous support for the conclusions drawn.

(2) Also, the role of TBC1D5 should be further clarified. In Fig S7, are there any changes in the interactions between TBC1D5 and VPS35 in response to LLOMe or other agents utilized to induce CASM? Does TBC1D5 loss-of-function modulate the numbers of GLUT1 and Gal3 puncta observed in ATG5 deficient cells in response to LLOMe?

(3) Finally, the studies here are motivated by experiments in Fig. S1 (as well as other studies from the Deretic and Stallings labs) suggesting unique autophagy-independent functions for ATG5 in myeloid cells and neutrophils in susceptibility to Mycobacterium tuberculosis infection. However, it is curious that no attempt is made to relate the mechanistic data regarding the retromer or GLUT1 receptor mis-sorting back to the infectious models. Do myeloid cells or neutrophils lacking ATG5 have deficiencies in glucose uptake or GLUT1 cell surface levels?

Reviewer #2 (Public Review):

Chong Wang et al. investigated the role of H3K4me2 during the reprogramming processes in mouse preimplantation embryos. The authors show that H3K4me2 is erased from GV to MII oocytes and re-established in the late 2-cell stage by performing Cut & Run H3K4me2 and immunofluorescence staining. Erasure and re-establishment of H3K4me2 have not been studied well, and profiling of H3K4me2 in germ cells and preimplantation embryos is valuable to understanding the reprogramming process and epigenetic inheritance.

(1) The authors claim that the Cut & Run worked for MII oocytes, zygotes, and the 2-cell embryos. However, it is unclear if H3K4me2 is erased during the stage or if the Cut & Run did not work for these samples. To support the hypothesis of the erasure of H3K4me2, the authors conducted immunofluorescence staining, and H3k4me2 was undetected in the MII oocyte, PN5, and 2-cell stage. However, the published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage ((Ancelin et al., 2016; Shao et al., 2014)). The authors need to cite these papers and discuss the contradictory findings.

The authors used 165 MII oocytes and 190 GV oocytes for the Cut & Run. The amount of DNA in MII oocytes is halved because of the emission of the first polar body. Would it be a reason that H3K4me2 has fewer H3K4me2 peaks in MII oocytes than GV oocytes?

In Figure 3C, 98% (13,183/13,428) of H3K4me2 marked genes in GV oocytes overlap with those in the 4-cell stage. Furthermore, 92% (14,049/15,112) of H3K4me2 marked genes in sperm overlap with those in the 4-cell stage. Therefore, most regions maintain germ line-derived H3K4me2 in the 4-cell stage. The authors need to clarify which regions of germ line-derived H3K4me2 are maintained or erased in preimplantation embryos. Additionally, it would be interesting to investigate which regions show the parental allele-specific H3K4me2 in preimplantation embryos since the authors used hybrid preimplantation embryos (B6 x DBA).

(2) The authors claim that Kdm1a is rarely expressed during mouse embryonic development (Figure 4A). However, the published paper showed that KDM1a is present in the zygote and 2-cell stage using immunostaining and western blotting ((Ancelin et al., 2016)). Additionally, this paper showed that depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage, and therefore, KDM1a is functionally important in early development. The authors should have cited the paper and described the role of KDM1a in early embryos.

(3) The authors used the published RNA data set and interpreted that KDM1B (LSD2) was highly expressed at the MII stage (Figure S3A). However, the heat map shows that KDM1B expression is high in growing oocytes but not at 8w_oocytes and MII oocytes. The authors need to interpret the data accurately.

(4) All embryos in the TCP group were arrested at the four-cell stage. Embryos generated from KDM1b KO females can survive until E10.5 (Ciccone et al., 2009); therefore, TCP-treated embryos show a more severe phenotype than oocyte-derived KDM1b deleted embryos. Depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage ((Ancelin et al., 2016)). The authors need to examine whether TCP treatment affects KDM1a expression. Western blotting would be recommended to quantify the expression of KDM1A and KDM1B in the TCP-treated embryos.

(5) H3K4me2 is increased dramatically in the TCP-treated embryos in Figure 4 (the intensity is 1,000 times more than the control). However, the Cut & Run H3K4me2 shows that the H3K4me2 signal is increased in 251 genes and decreased in 194 genes in the TCP-treated embryos (Fold changes > 2, P < 0.01). The authors need to explain why the gain of H3K4me2 is less evident in the Cut & Run data set than in the immunofluorescence result.

References

Ancelin, K., ne Syx, L., Borensztein, M., mie Ranisavljevic, N., Vassilev, I., Briseñ o-Roa, L., Liu, T., Metzger, E., Servant, N., Barillot, E., Chen, C.-J., Schü le, R., & Heard, E. (2016). Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. https://doi.org/10.7554/eLife.08851.001

Ciccone, D. N., Su, H., Hevi, S., Gay, F., Lei, H., Bajko, J., Xu, G., Li, E., & Chen, T. (2009). KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature, 461(7262), 415-418. https://doi.org/10.1038/nature08315

Shao, G. B., Chen, J. C., Zhang, L. P., Huang, P., Lu, H. Y., Jin, J., Gong, A. H., & Sang, J. R. (2014). Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development. In Vitro Cellular and Developmental Biology - Animal, 50(7), 603-613. https://doi.org/10.1007/s11626-014-9741-6

Reviewer #2 (Public Review):

Summary:<br /> This article, titled "A multi-gene predictive model for the radiation sensitivity of nasopharyngeal carcinoma based on machine learning," utilizes machine learning methods and transcriptomic data from nasopharyngeal carcinoma (NPC) patients to construct a biomarker called NPC-RSS that can predict the radiosensitivity of NPC patients. The authors further explore the biological mechanisms underlying the relationship between NPC-RSS and radiotherapy response in NPC patients. The main objective of this study is to guide the selection of radiotherapy strategies for NPC patients, thereby improving their clinical outcomes and prognosis.

Strengths:<br /> (1) The combination of multiple machine learning algorithms and cross-validation was used to select the best predictive model for radiotherapy sensitivity from 71 differentially expressed genes, enhancing the robustness and reliability of the predictions.<br /> (2) Functional enrichment analysis revealed close associations between NPC-RSS key genes and immune characteristics, expression of radiotherapy sensitivity-related genes, and signaling pathways related to disease progression, providing a biological basis for NPC-RSS in predicting radiotherapy sensitivity.<br /> (3) Grouping NPC samples according to NPC-RSS showed that the radiotherapy-sensitive group exhibited a more enriched and activated state of immune infiltration compared to the radioresistant group. In single-cell samples, NPC-RSS was higher in the radiotherapy-sensitive group, with immune cells playing a dominant role. These results clarify the mechanism of NPC-RSS in predicting radiotherapy sensitivity from an immunological perspective.<br /> (4) The study used public datasets and in-house cohort data for validation, confirming the good predictive performance of NPC-RSS and increasing the credibility of the results.

Limitation:<br /> (1) The study focuses on a specific type of nasopharyngeal carcinoma (NPC) and may not be generalizable to other subtypes or related head and neck cancers. The applicability of NPC-RSS to a broader range of patients and tumor types remains to be determined.<br /> (2) The study does not account for potential differences in radiotherapy protocols, doses, and techniques between the training and validation cohorts, which could influence the performance of the predictive model. Standardization of treatment parameters would be important for future validation studies.<br /> (3) The binary classification of patients into radiotherapy-sensitive and resistant groups may oversimplify the complex spectrum of treatment responses. A more granular stratification system that captures intermediate responses could provide more nuanced predictions and better guide personalized treatment decisions.<br /> (4) The study does not address the potential impact of other relevant factors, such as tumor stage, histological subtype, and concurrent chemotherapy, on the predictive performance of NPC-RSS. Incorporating these clinical variables into the model could enhance its accuracy and clinical utility.

Reviewer #2 (Public Review):

Summary:

This study aims to describe the single-cell transcriptomes of H pylori-associated (Hp) gastric cancers and tumour microenvironment (TME), as a starting point to understand TME diversity stratified by Hp status.

RNAseq was performed for gastric cancers with current Hp+ (from N=9 people), ex-Hp+ (N=6), non-Hp (N=6), and healthy gastric tissue (N=6).

The study expands on previous single-cell transcriptomic studies of gastric cancers and was motivated by previous observations about the effect of H pylori status on therapeutic outcomes. The study includes a brief review of previous work and provides valuable context for this study.

Strengths:

The observations are supported by solid RNAseq study design and analysis. The authors describe correlations between Hp status and inferred molecular characteristics including cell lineages, enrichment for cell subclusters identified as tumour-infiltrating lyphocyte cell types, tumour-infiltrating myeloid cells, and cancer-associated fibroblasts.

The observed correlations between Hp status and enrichment of cell subclusters were broadly corroborated using comparisons to deconvolved bulk RNAseq from publicly available gastric cancer data, providing a convincing starting point for understanding the diversity of tumour microenvironment by Hp-status.

Weaknesses:

The authors acknowledge several limitations of this study.

The correlations with HP-status are based on a small number of participants per Hp category (N=9 with current Hp+; N=6 for ex-HP+ and non-HP), and would benefit from further validation to establish reproducibility in other cohorts.

The ligand-receptor cross-talk analysis and the suggestion that suppressive T cells could interact with the malignant epithelium through TIGIT-NECTIN2/PVR pairs, are preliminary findings based on transcriptomic analysis and immunostaining and will require further validation.

Reviewer #2 (Public Review):

Summary:

This study proposes that the microRNA cluster miR-277/34 controls the generation of sexual dimorphism in Drosophila melanogaster during metamorphosis by acting on specific hormonal and developmental gene pathways.

Strengths:

Using a combination of mRNA and small RNA sequencing together with genome-wide in silico and in vitro analyses the authors identified a microRNA cluster that may be involved in metamorphosis and the generation of sexual dimorphism in Drosophila melanogaster.

Weaknesses:

Biological validation of the identified sexually dimorphic genes and a detailed understanding of how the microRNA cluster miR-277/34 might be involved in the regulation of sesquiterpenoids are needed.

Major suggestions:

(1) If AstC-R1 and Kr-h1 are targets of the miR-277/34 cluster and cause their downregulation, it is not clear why there would also be a decrease in the levels of these genes in the miR-277/34 mutants. This would suggest that the mechanism is not straightforward and that further epistatic experiments should be carried out in order to clarify this issue.

(2) The changes in the expression levels of AstC-R1 in pupae of miR-277-KO and mir-34-KO flies must be accompanied by photos of the respective larvae and pupae, as well as an analysis of the larvae-pupa transition on the mutants by gender.

(3) Biological validation of the identified sexually dimorphic genes in vivo will be necessary for the support of this work.

Reviewer #2 (Public Review):

Summary:

This manuscript addresses the intriguing topic of the potential roles of germline-specific proteins in early development. While this issue is quite interesting and generally under-explored, the work falls short of making truly tangible inroads.

Strengths:

The strength of the study is in new proteomic datasets.

Weaknesses:

The manuscript makes some strong statements, beginning with the title "STAG3 (1) promotes exit from pluripotency (2) through post-transcriptional mRNA regulation in the cytoplasm".

Upon reviewing the data it appears that neither (1) or (2) here have strong foundations based on experiments presented. While intriguing, the experimental evidence is still rather inconclusive.

The potential involvement of STAG3 in PGC specification is the most intriguing aspect of this study. Unfortunately, it is not going far enough to derive a fully meaningful biological conclusion. In fact, DPPPA3-GFP, PRDM-GFP, and PGC marker expression results are contradictory and do not form a coherent picture of the biological effect of STAG3 depletion. No effect of the knock-down in PGC specification when PRDM is scored (line 167) is particularly worrisome. As for finding a cytoplasmic role of STAG3, the data also remain inconclusive.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, Lin and colleagues aim to understand the role of different salts on the phase behavior of a model protein of significant biological interest, Caprin1, and its phosphorylated variant, pY-Caprin1. To achieve this, the authors employed a variety of methods to complement experimental studies and obtain a molecular-level understanding of ion partitioning inside biomolecular condensates. A simple theory based on rG-RPA is shown to capture the different salt dependencies of Caprin1 and pY-Caprin1 phase separation, demonstrating excellent agreement with experimental results. The application of this theory to multivalent ions reveals many interesting features with the help of multicomponent phase diagrams. Additionally, the use of CG model-based MD simulations and FTS provides further clarity on how counterions can stabilize condensed phases.

Strengths:<br /> The greatest strength of this study lies in the integration of various methods to obtain complementary information on thermodynamic phase diagrams and the molecular details of the phase separation process. The authors have also extended their previously proposed theoretical approaches, which should be of significant interest to other researchers. Some of the findings reported in this paper, such as bridging interactions, are likely to inspire new studies using higher-resolution atomistic MD simulations.

Weaknesses:<br /> The paper does not have any major issues.

Reviewer #2 (Public Review):

Summary:

During vertebrate gastrulation, the mesoderm and endoderm arise from a common population of precursor cells and are specified by similar signaling events, raising questions as to how these two germ layers are distinguished. Here, Cheng and colleagues use zebrafish gastrulation as a model for mesoderm and endoderm segregation. By reanalyzing published single-cell sequencing data, they identify a common progenitor population for the anterior endoderm and the mesodermal prechordal plate (PP). They find that expression levels of PP genes Gsc and ripply are among the earliest differences between these populations and that their increased expression suppresses the expression of endoderm markers. Further analysis of chromatin accessibility and Ripply cut-and-tag is consistent with direct repression of endoderm by this PP marker. This study demonstrates the roles of Gsc and Ripply in suppressing anterior endoderm fate, but this role for Gsc was already known and the effect of Ripply is limited to a small population of anterior endoderm. The manuscript also focuses extensively on the function of Nodal in specifying and patterning the mesoderm and endoderm, a role that is already well known and to which the current analysis adds little new insight.

Strengths:

Integrated single-cell ATAC- and RNA-seq convincingly demonstrate changes in chromatin accessibility that may underlie the segregation of mesoderm and endoderm lineages, including Gsc and ripply. Identification of Ripply-occupied genomic regions augments this analysis. The genetic mutants for both genes provide strong evidence for their function in anterior mesendoderm development, although these phenotypes are subtle.

Weaknesses:

The use of zebrafish embryonic explants for cell fate trajectory analysis (rather than intact embryos) is not justified. In both transcriptomic comparisons between the two fate trajectories of interest and Ripply cut-and-tag analysis, the authors rely too heavily on gene ontology which adds little to our functional understanding. Much of the work is focused on the role of Nodal in the mesoderm/endoderm fate decision, but the results largely confirm previous studies and again provide few new insights. Some experiments were designed to test the relationship between the mesoderm and endoderm lineages and the role of epigenetic regulators therein, but these experiments were not properly controlled and therefore difficult to interpret.

Reviewer #2 (Public Review):

Summary, strengths, and weaknesses:

This article by Rice et al focuses on the study of limbal epithelial stem cells (LESCs). To obtain high resolution of stem/progenitor cell populations by single-cell RNA sequencing (scRNA-seq), the authors enriched the stem/progenitors by capturing GFP+ epithelial cells undergoing mitosis using the Cyclin-B-GFP transgene. The key novelty in the paper is that they identified Sox9 as a new LSC gene that is important for the health of the cornea. They show that Sox9 is expressed by LSCs at the mRNA and protein level, and that the Sox9-GFP transgene is useful to identify LSCs in live animals. They performed lineage tracing of Sox9-Cre mice that clearly demonstrated that Sox9+ cells are true LSCs. Further, Sox9-knockouts display severe opacification of the cornea, accompanied by the transformation of the central cornea into hyperplastic epidermis - a phenotype similar to previously described Notch1 conditional knockout (cKO). By studying the lifetimes of Notch dominant-negative transgenic individual basal corneal epithelial cells, they suggest that the thickening of the central zone in the Notch model is due to the loss of asymmetric division, and from that, they infer the phenotype of the Sox2-KO. In other words, it is suggested that the increased rate and type of division in the central Sox9-null cornea produce this dramatic epithelial thickening phenotype. This claim makes sense but suggests a role for the Notch, not Sox9, and this role is relevant for the central corneal epithelial cells, and not in LESCs. So I suggest considering revising the conclusions (title of the paper) on this aspect. This would not affect the impact of the paper.

In general, I believe that this is a very interesting and novel study that will be of interest to a broad readership. The methodology and study design are robust and elegant.

However, in some cases, typos, text modifications, additional controls, and new experiments are suggested.

Reviewer #2 (Public Review):

Summary:

The aim was to identify the mechanisms that underlie a form of long-term potentiation (LTP) that requires the activation of dopamine (DA).

Strengths:

The authors have provided multiple lines of evidence that support their conclusions; namely that this pathway involves the activation of a cAMP / PKA pathway that leads to the insertion of calcium-permeable AMPA receptors.

Weaknesses:

Some of the experiments could have been conducted in a more convincing manner.

Reviewer #2 (Public Review):

This study compares neural responses to speech in normal-hearing and hearing-impaired listeners, investigating how different levels of the linguistic hierarchy are impacted across the two cohorts, both in a single-talker and multi-talker listening scenario. It finds that, while normal-hearing listeners have a comparable cortical encoding of speech-in-quiet and attended speech from a multi-talker mixture, participants with hearing impairment instead show a reduced cortical encoding of speech when it is presented in a competing listening scenario. When looking across the different levels of the speech processing hierarchy in the multi-talker condition, normal-hearing participants show a greater cortical encoding of the attended compared to the unattended stream in all speech processing layers - from acoustics to sentence-level information. Hearing-impaired listeners, on the other hand, only have increased cortical responses to the attended stream for the word and phrase levels, while all other levels do not differ between attended and unattended streams.<br /> The methods for modelling the hierarchy of speech features (HM-LSTM) and the relationship between brain responses and specific speech features (ridge-regression) are appropriate for the research question, with some caveats on the experimental procedure. This work offers an interesting insight into the neural encoding of multi-talker speech in listeners with hearing impairment, and it represents a useful contribution towards understanding speech perception in cocktail-party scenarios across different hearing abilities. While the conclusions are overall supported by the data, there are limitations and certain aspects that require further clarification.<br /> (1) In the multi-talker section of the experiment, participants were instructed to selectively attend to the male or the female talker, and to rate the intelligibility, but they did not have to perform any behavioural task (e.g., comprehension questions, word detection or repetition), which could have demonstrated at least an attempt to comply with the task instructions. As such, it is difficult to determine whether the lack of increased cortical encoding of Attended vs. Unattended speech across many speech features in hearing-impaired listeners is due to a different attentional strategy, which might be more oriented at "getting the gist" of the story (as the increased tracking of only word and phrase levels might suggest), or instead it is due to hearing-impaired listeners completely disengaging from the task and tuning back in for selected key-words or word combinations. Especially the lack of Attended vs. Unattended cortical benefit at the level of acoustics is puzzling and might indicate difficulties in performing the task. I think this caveat is important and should be highlighted in the Discussion section.<br /> (2) In the EEG recording and preprocessing section, you state that the EEG was filtered between 0.1Hz and 45Hz. Why did you choose this very broadband frequency range? In the literature, speech responses are robustly identified between 0.5Hz/1Hz and 8Hz. Would these results emerge using a narrower and lower frequency band? Considering the goal of your study, it might also be interesting to run your analysis pipeline on conventional frequency bands, such as Delta and Theta, since you are looking into the processing of information at different temporal scales.<br /> (3) A paragraph with more information on the HM-LSTM would be useful to understand the model used without relying on the Chung et al. (2017) paper. In particular, I think the updating mechanism of the model should be clarified. It would also be interesting to modify the updating factor of the model, along the lines of Schmitt et al. (2021), to assess whether a HM-LSTM with faster or slower updates can better describe the neural activity of hearing-impaired listeners. That is, perhaps the difference between hearing-impaired and normal-hearing participants lies in the temporal dynamics, and not necessarily in a completely different attentional strategy (or disengagement from the stimuli, as I mentioned above).<br /> (4) When explaining how you extracted phoneme information, you mention that "the inputs to the model were the vector representations of the phonemes". It is not clear to me whether you extracted specific phonetic features (e.g., "p" sound vs. "b" sound), or simply the phoneme onsets. Could you clarify this point in the text, please?

Reviewer #3 (Public Review):

In this manuscript, Picard et al. propose a Facial Expression Pain Signature (FEPS) as a distinctive marker of pain processing in the brain. Specifically, they attempt to use functional magnetic resonance imaging (fMRI) data to predict facial expressions associated with painful heat stimulation.

The main strengths of the manuscript are that it is built on an extensive foundation of work from the research group, and that experience can be observed in the analysis of fMRI data and the development of the machine learning model. Additionally, it provides a comparative account of the similarities of the FEPS with other proposed pain signatures. The main weaknesses of the manuscript are the absence of a proper control condition to assess the specificity of the facial pain expressions, as well as several limitations in the experimental setup.

I believe that the authors partially succeed in their aims, as described in the introduction, which are to assess the association between pain facial expression and existing pain-relevant brain signatures, and to develop a predictive brain activation model of the facial responses to painful thermal stimulation. However, they list several limitations in the study that should be addressed in future research in order to establish whether FEPS truly conveys distinctive information about the brain response to nociceptive stimuli.

Reviewer #2 (Public Review):

Summary:

The authors set out to establish the role of the rice LEC1 homolog OsNF-YB7 in embryo development, especially as it pertains to the development of photosynthetic capacity, with chlorophyll production as a primary focus.

Strengths:

The results are well-supported and each approach used complements each other. There are no major questions left unanswered and the central hypothesis is addressed in every figure.

Weaknesses:

There are a handful of sections which could use clarifying for readers, but overall this is a solidly composed manuscript.

The authors clearly achieved their aims; the results compellingly establish a disparity between how this system operates in rice and Arabidopsis. Conclusions are thoroughly supported by the provided data and interpretations. This work will force a reconsideration of the value of Arabidopsis as a model organism for embryo chlorophyll biosynthesis and possibly photosynthesis during embryo maturation more broadly, as rice is a major crop organism and it very clearly does not follow the Arabidopsis model. It will thus be useful to carry out similar tests in other organisms rather than relying on Arabidopsis and attempt to more fully establish the regulatory mechanism in rice.

Reviewer #2 (Public Review):

Summary:

In this manuscript by Cannarsa et. al., the authors describe the engineering of a light-entrainable synthetic biological oscillator in bacteria. It is based on an upgraded version of one of the first synthetic circuits to be constructed, the repressilator. The authors sought to make this oscillator entrainable by an external forcing signal, analogous to the way natural biological oscillators (like the circadian clock) are synchronized. They reasoned that an optogenetic system would provide a convenient and flexible means of manipulation. To this end, the authors exploited the CcaS-CcaA light-switchable system, which allows activation and deactivation of transcription by green and red light, respectively. They used this system to make the expression of one of the repressilator's transcription factors (lacI) light-controlled, from a construct separated from the main repressilator plasmid. This way, under red light the oscillator runs freely, but exposure to green light causes overexpression of the lacI, pushing the system into a specific state. Consequently, returning to red light will restore the oscillations from the same phase in all cells, effectively synchronizing the cell population.

After demonstrating the functionality of the basic concept, the authors combined modeling and experiments to show how periodic exposure to green light enables efficient entrainment, and how the frequency of the forcing signal affects the oscillatory behavior (detuning).

This work provides an important demonstration of engineering tunability into a foundational genetic circuit, expands the synthetic biology toolbox, and provides a platform to address critical questions about synchronization in biological oscillators. Due to the flexibility of the experimental system, it is also expected to provide a fertile ground for future testing of theoretical predictions regarding non-linear oscillators.

Strengths:

* The study provides a simple and elegant mechanism for the entertainment of a synthetic oscillator. The design relies on optogenetic proteins, which enable efficient experimentation compared to alternative approaches (like using chemical inducers). This way, a static culture (without microfluidics or change of growth media) can be easily exposed to flexible temporal sequences of the zeitgeber, and continuously measured through time.

* The study makes use of both plate-reader-based population-level readout and mother-machine single-cell measurements. Synchronization through entrainment is a single cell level phenomenon, but with a clear population-level manifestation. Thus, this experimental approach combination provides a strong validation to their system. At the same time, differences between the readout from the two systems have emerged, and provided a further opportunity for model refinement and testing.

* The authors correctly identified the main optimization goal, namely the effective leakiness of their construct even under red light. Then, they successfully overcame this issue using synthetic biology approaches.

* The work is supported by a simplified model of the repressilator, which provides a convenient analytical and numerical means to draw testable predictions. The model predictions are well aligned with the experimental evidence.

Weaknesses:

* Even after optimizing the expression level of the light-sensitive gene, the system is very sensitive, i.e., a very short exposure is sufficient to elicit the strongest entertainment. This limited dynamic range might hamper some model testing and future usage.

* As a result of the previous point, the system is entrained by transiently "breaking" the oscillator: each pulse of green light represents a Hopf bifurcation into a single attractor. it means that the system cannot oscillate in constant green light. In comparison, this is generally not the case for natural zeitgebers like light and temperature for the circadian rhythms. Extreme values might prevent oscillations (not necessarily due to breaking the core oscillator), but usually, free running is possible in a wide range of constant conditions. In some cases, the free-running period length will vary as a function of the constant value.

While the approach presented in this manuscript is valid, a comprehensive analysis of more subtle modes of repressilator entrainment could also be of value.

* The entire work makes use of a single intensity and single duration of the green pulse to force entrainment. While the model has clear predictions for how different modalities should affect entrainment, more experiments are needed to validate those predictions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Bradley and his colleagues represented the cryo-EM structure of the nuclear cap-binding complex (CBC) in complex with an mRNA export factor, ALYREF, providing a structural basis for understanding CBC regulating gene expression.

Strengths:

The authors successfully modeled the N-terminal region and the RRM domain of ALYREF (residues 1-183) within the CBC-ALYREF structure, which revealed that both the NCBP1 and NCBP2 subunits of the CBC interact with the RBM domain of ALYREF. Further mutagenesis and pull-down studies provided additional evidence to the observed CBC-ALYREF interface. Additionally, the authors engaged in a comprehensive discussion regarding other cellular complexes containing CBC and/or ALYREF components. They proposed potential models that elucidated coordinated events during mRNA maturation. This study provided structural evidence to show how CBC effectively recruits mRNA export factor machinery, enhancing our understanding of CBC regulating gene expression during mRNA transcription, splicing, and export.

Weaknesses:

Absence of functional data to support the proposed models in this study.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public Review):

Summary:

An analysis of images in the biology literature that are problematic for people with a color-vision deficiency (CVD) is presented, along with a machine learning-based model trained on an eLife dataset to identify such images and a web application that uses the model to flag problematic images. Their analysis reveals that about 13% of the images could be problematic for people with CVD and that the frequency of such images decreased over time. Their best model (convolutional neural network, CNN) yields 0.89 AUROC score and 0.77 AUPRC on held-out eLife articles, but lower scores (0.78 and 0.39, respectively). It is proposed that their approach could help making biology literature accessible to diverse audiences.

Strengths:

The manuscript focuses on an important yet mostly overlooked problem and makes contributions both in expanding our understanding of the extent of the problem and in developing solutions to mitigate the problem. The paper is generally well-written and clearly organized. Their CVD simulation combines five different metrics. The dataset has been assessed by two researchers and is likely to be of high-quality. Machine learning algorithm used (CNN) is an appropriate choice for the problem. The evaluation of various hyperparameters for the CNN model is extensive.

Weaknesses:

While the study has significant strengths, it also has some limitations. Specifically, the focus on one type of CVD (deuteranopia) and selecting images from a single journal (eLife) for training limit the generalizability of the models. This is, to some extent, shown by applying the model to PMC articles, which yields lower performance. "Probably problematic" and "probably okay" classes are excluded from the analysis.

Reviewer #2 (Public Review):

Summary:

The authors provided a novel antigen delivery system which showed remarkable efficacy in transporting antigens to develop cancer therapeutic vaccine.

Strengths:

This manuscript was innovative, meaningful, and had a rich amount of data.

Comments on the revised version:

All my concerns have been addressed by the authors

Reviewer #2 (Public Review):

Summary:

This work investigates the enzymatic properties of lysostaphin (LSS) and LytM, two enzymes produced by Staphylococcus aureus and previously described as glycyl-glycyl endopeptidases. The authors use synthetic peptide substrates mimicking peptidoglycan fragments to determine the substrate specificity of both enzymes and identify the bonds they cleave.

Strengths:

- This work is addressing a real gap in our knowledge since very little information is available about the substrate specificity of peptidoglycan hydrolases.<br /> - The experimental strategy and its implementation are robust and provide a thorough analysis of LSS and LytM enzymatic activities. The results are very convincing and demonstrate that the enzymatic properties of the model enzymes studied need to be revisited.<br /> - I think the experimental work is extremely well designed and way beyond what people usually do in the field. I believe that this sets a precedent that is worth acknowledging.

Reviewer #2 (Public Review):

This is an important and very interesting report on a change in newborns' neural abilities to distinguish auditory signals as a function of the gestational age (GA) of the infant at birth (from 35 weeks GA to 40 weeks GA). The authors tested neural discrimination of sounds that were labeled 'happy' vs 'neutral' by listeners that represent two categories of sound, either human voices or auditory signals that mimic only certain properties of the human vocal signals. The finding is that a change occurs in neural discrimination of the happy and neutral auditory signals for infants born at or after 37 weeks of gestation, and not prior (at 35 or 36 weeks of gestation), and only for discrimination of the human vocal signals; no change occurs in discrimination of the nonhuman signals over the 35- to 40-week gestational ages tested. The neural evidence of discrimination of the vocal happy-neutral distinction and the absence of the discrimination of the control signals is convincing. The authors interpret this as a 'landmark' in infants' ability to detect changes in emotional vocal signals, and remark on the potential value of the test as a marker of the infants' interest in emotional signals, underscoring the fact that children at risk for autism spectrum disorder may not show the discrimination.

[Editors' note: The authors addressed the reviewers' main concern by clearly stating the limits of the study's implications.]

Reviewer #2 (Public Review):

This study identified multiple locally evolving lineages transmitted between cattle and humans persistently associated with E. coli O157:H7 illnesses for up to 13 years. Furthermore, this study mentions a dramatic shift in the local persistent lineages toward strains with the more virulent stx2a-only profile. The authors hypothesized that this phenomenon is the large proportion of disease associated with local transmission systems is a principal cause of Alberta's high E. coli O157:H7 incidence. These opinions more effectively explain the role of the cattle reservoir in the dynamics of E. coli O157:H7 human infections.

(1) The authors acknowledge the possibility of intermediate hosts or environmental reservoirs playing a role in transmission. Further discussion on the potential roles of other animal species commonly found in Alberta (e.g., sheep, goats, swine) could enhance the understanding of the transmission dynamics. Were isolates from these species available for analysis? If not, the authors should clearly state this limitation.

(2) The focus on E. coli O157:H7 is understandable given its prominence in Alberta and the availability of historical data. However, a brief discussion on the potential applicability of the findings to non-O157 STEC serogroups, and the limitations therein, would be beneficial. Are there reasons to believe the transmission dynamics would be similar or different for other serogroups?

(3) The authors briefly mention the need for elucidating local transmission systems to inform management strategies. A more detailed discussion on specific public health interventions that could be targeted at the identified LPLs and their potential reservoirs would strengthen the paper's impact.

(4) "Understanding the relationship between specific risk factors and E. coli O157:H7 infections is essential for developing effective prevention strategies. Have case-control or cohort studies been conducted to assess the correlation between identified risk factors and the incidence of E. coli O157:H7 infections? What methodologies were employed to control for potential confounders in these studies?"

(5) The study's findings are noteworthy, particularly in the context of E. coli O157:H7 epidemiology. However, the extent to which these results can be replicated across different temporal and geographical settings remains an open question. It would be constructive for the authors to provide additional data that demonstrate the replication of their sampling and sequencing experiments under varied conditions. This would address concerns regarding the specificity of the observed patterns to the initial study's parameters.

Reviewer #2 (Public Review):

Summary:<br /> This work by Zhao et al. demonstrates the role of the Edwardsiella tarda type 3 secretion system translocon in activating human macrophage inflammation and pyroptosis. The authors show the requirement of both the bacterial translocon proteins and particular host inflammasome components for E. tarda-induced pyroptosis. In addition, the authors show that the C-terminal region of the translocon protein, EseB, is both necessary and sufficient to induce pyroptosis when present in the cytoplasm. The most terminal region of EseB was determined to be highly conserved among other T3SS-encoding pathogenic bacteria and a subset of these exhibited functionally similar effects on inflammasome activation. Overall, the data support the conclusions and interpretations and provide interesting insights into interactions between bacterial T3SS components and the host immune system.

Strengths:<br /> The authors use established and reliable molecular biology and bacterial genetics strategies to characterize the roles of the bacterial T3SS translocon and host inflammasome pathways to E. tarda-induced pyroptosis in human macrophages. These observations are naturally expanded upon by demonstrating the specific regions of EseB that are required for inflammasome activation and the conservation of this sequence among other pathogenic bacteria.

Weaknesses:<br /> The functional assessment of EseB homologues is limited to inflammasome activation at the protein level but does not include the effects on cell viability as shown for E. tarda EseB. Confirmation that EseB homologues have similar effects on cell death would strengthen this portion of the manuscript.

Reviewer #2 (Public Review):

Summary:

Mitochondria import hundreds of precursor proteins from the cytosol. The TOM and TIM23 complexes facilitate the import of the matrix-targeting pathway of mitochondria. In yeast, Tim50 is a critical and essential subunit of the TIM23 complex that mediates the transition of precursors from the outer to the inner membrane. The human Tim50 homolog TIMM50 is highly similar in structure and a comparable function of Tim50 and TIMM50 was proven by several biochemical and genetic studies in the past.

In this study, the authors characterize human cells that express lower levels or mutated versions of TIMM50. They found that in these TIMM50-depletion cells, the levels of other TIM23 core subunits are also diminished but many mitochondrial proteins are unaffected. Moreover, they observed alterations in the electrical activity and the levels of potassium channels in neuronal cells of TIMM50-deficient mice. They propose that these changes explain the pathology of patients who often suffer from epilepsy.

Strengths:

The paper is written by experts in the field, and it is very clear. The experiments are of high quality and sufficiently well-controlled. The study is interesting for a broad readership.

Weaknesses:

However, there is a problem that needs to be fixed: The interpretation of the results needs to be downgraded. The authors claim in the abstract, the introduction, and the discussion that TIMM50 and the TIM23 translocase might not be relevant for mitochondrial protein import in mammals. This is misleading and certainly wrong!!! What they do show is that the depletion of TIMM50 to about 20% of the normal levels is still tolerated in cultured cells grown in a glucose-rich medium. This is similar to the situation of cytochrome oxidase deficiency where lower levels are often well tolerated, and only very low levels (<20%) lead to Leigh syndrome. Still, it would be wrong to claim that respiration can occur without the terminal enzyme. Thus, it is essential that the authors change the wording here. In the discussion they offer several explanations: Mitochondria might import more proteins than they need and just degrade the excess. The TIMM50 depletion cells might increase the expression of mitochondrial proteins as a compensatory mechanism. Translocation across the TIM23 complex might not be the rate-limiting step in mitochondrial protein biogenesis. The authors do not have to sort this out on a molecular level, however, they have to be more cautious with the conclusions they make! Except for this issue, this is a very interesting study that will raise the interest of the mitochondrial community.

Reviewer #2 (Public Review):

Sensory experiences during developmental critical periods have long-lasting impacts on neural circuit function and behavior. However, the underlying molecular and cellular mechanisms that drive these enduring changes are not fully understood. In Drosophila, the antennal lobe is composed of synapses between olfactory sensory neurons (OSNs) and projection neurons (PNs), arranged into distinct glomeruli. Many of these glomeruli show structural plasticity in response to early-life odor exposure, reflecting the sensitivity of the olfactory circuitry to early sensory experiences.

In their study, the authors explored the role of glia in the development of the antennal lobe in young adult flies, proposing that glial cells might also play a role in experience-dependent plasticity. They identified a critical period during which both structural and functional plasticity of OSN-PN synapses occur within the ethyl butyrate (EB)-responsive VM7 glomerulus. When flies were exposed to EB within the first two days post-eclosion, significant reductions in glomerular volume, presynaptic terminal numbers, and postsynaptic activity were observed. The study further highlights the importance of the highly conserved engulfment receptor Draper in facilitating this critical period plasticity. The authors demonstrated that, in response to EB exposure during this developmental window, ensheathing glia increase Draper expression, infiltrate the VM7 glomerulus, and actively phagocytose OSN presynaptic terminals. This synapse pruning has lasting effects on circuit function, leading to persistent decreases in both OSN-PN synapse numbers and spontaneous PN activity as analyzed by perforated patch-clamp electrophysiology to record spontaneous activity from PNs postsynaptic to Or42a OSNs.

In my view, this is an intriguing and potentially valuable set of data. However, since I am not an expert in critical periods or habituation, I do not feel entirely qualified to assess the full significance or the novelty of their findings, particularly in relation to existing research.

Reviewer #2 (Public Review):

Summary:

The goal is to ask if common species when studied across their range tend to have larger ranges in total. To do this the authors examined a very large citizen science database which gives estimates of numbers, and correlated that with the total range size, available from Birdlife. The average correlation is positive but close to zero, and the distribution around zero is also narrow, leading to the conclusion that, even if applicable in some cases, there is no evidence for consistent trends in one or other direction.

Strengths:

The study raises a dormant question, with a large dataset.

Weaknesses:

This study combines information from across the whole world, with many different habitats, taxa, and observations, which surely leads to a quite heterogeneous collection.

First, scale. Many of the earlier analyses were within smaller areas, and for example, ranges are not obviously bounded by a physical barrier. I assume this study is only looking at breeding ranges; that should be stated, as 40% of all bird species migrate, and winter limitation of populations is important. Also are abundances only breeding abundances or are they measured through the year? Are alien distributions removed?

Second, consider various reasons why abundance and range size may be correlated (sometimes positively and sometimes negatively) at large scales. Combining studies across such a large diversity of ecological situations seems to create many possibilities to miss interesting patterns. For example:

(1) Islands are small and often show density release.

(2) North temperate regions have large ranges (Rapoport's rule) and higher population sizes than the tropics.

(3) Body size correlates with global range size (I am unsure if this has recently been tested but is present in older papers) and with density. For example, cosmopolitan species (barn owl, osprey, peregrine) are relatively large and relatively rare.

(4) In the consideration of alien species, it certainly looks to me as if the law is followed, with pigeon, starling, and sparrow both common and widely distributed. I guess one needs to make some sort of statement about anthropogenic influences, given the dramatic changes in both populations and environments over the past 50 years.

(5) Wing shape correlates with ecological niche and range size (e.g. White, American Naturalist). Aerial foraging species with pointed wings are likely to be easily detected, and several have large ranges reflecting dispersal (e.g. barn swallow).

Third, biases. I am not conversant with ebird methodology, but the number appearing on checklists seems a very poor estimate of local abundance. As noted in the paper, common species may be underestimated in their abundance. Flocking species must generate large numbers, skulking species few. The survey is often likely to be in areas favorable to some species and not others. The alternative approach in the paper comes from an earlier study, based on ebird but then creating densities within grids and surely comes with similar issues.<br /> Biases are present in range as well. Notably, tropical mountain-occupying species have range sizes over-estimated because holes in the range are not generally accounted for (Ocampo-Peñuela et al Nature communications). These species are often quite rare too.

Fourth, random error. Random error in ebird assessments is likely to be large, with differences among observers, seasons, days, and weather (e.g. Callaghan et al. 2021, PNAS). Range sizes also come with many errors, which is why occupancy is usually seen as the more appropriate measure.

If we consider both range and abundance measurements to be subject to random error in any one species list, then the removal of all these errors will surely increase the correlation for that list (the covariance shouldn't change but the variances will decrease). I think (but am not sure) that this will affect the mean correlation because more of the positive correlations appear 'real' given the overall mean is positive. It will definitely affect the variance of the correlations; the low variance is one of the main points in the paper. A high variance would point to the operation of multiple mechanisms, some perhaps producing negative correlations (Blackburn et al. 2006).

On P.80 it is stated: "Specifically, we can quantify how AOR will change in relation to increases in species richness and sampling duration, both of which are predicted to reduce the magnitude of AORs" I haven't checked the references that make this statement, but intuitively the opposite is expected? More species and longer durations should both increase the accuracy of the estimate, so removing them introduces more error? Perhaps dividing by an uncertain estimate introduces more error anyway. At any rate, the authors should explain the quoted statement in this paper.

It would be of considerable interest to look at the extreme negative and extreme positive correlations: do they make any biological sense?

Discussion:

I can see how publication bias can affect meta-analyses (addressed in the Gaston et al. 2006 paper) but less easily see how confirmation bias can. It seems to me that some of the points made above must explain the difference between this study and Blackburn et al. 2006's strong result.

Certainly, AOR really does seem to be present in at least some cases (e.g. British breeding birds) and a discussion of individual cases would be valuable. Previous studies have also noted that there are at least some negative and some non-significant associations, and understanding the underlying causes is of great interest (e.g. Kotiaho et al. Biology Letters).

Reviewer #2 (Public Review):

Summary:<br /> simulation study of magnetotactic bacteria in microfluidic channels containing sediment-mimicking obstacles. The obstacles were produced based on micro-computer tomography reconstructions of bacteria-rich sediment samples. The swimming of bacteria through these channels is found experimentally to display the highest throughput for physiological magnetic fields. Computer simulations of active Brownian particles, parameterized based on experimental trajectories are used to quantify the swimming throughput in detail. Similar behavior as in experiments is obtained, but also considerable variability between different channel geometries. Swimming at strong field is impeded by the trapping of bacteria in corners, while at weak fields the direction of motion is almost random. The trapping effect is confirmed in the experiments, as well as the escape of bacteria with reducing field strength.

Strengths:<br /> This is a very careful and detailed study, which draws its main strength from the fruitful combination of the construction of novel microfluidic devices, their use in motility experiments, and simulations of active Brownian particles adapted to the experiment. Based on their results, the authors hypothesize that magnetotactic bacteria may have evolved to produce magnetic properties that are adapted to the geomagnetic field in order to balance movement and orientation in such crowded environments. They provide strong arguments in favor<br /> of such a hypothesis.

Weaknesses:<br /> Some of the issues touched upon here have been studied also in other articles. It would be good to extend the list of references accordingly and discuss the relation briefly in the text.

Reviewer #2 (Public Review):

This manuscript develops well-controlled microfluidic experiments and mathematical modelling to resolve how the temporal development of P. aeruginosa biofilms is shaped by ambient flow. The experiment considers a simple rectangular channel on which a constant flow rate is applied and UV LEDs are used to confine the biofilm to a relatively small length of device. While there is often considerable geometrical complexity in confined environments and feedback between biofilm/flow (e.g. in porous media), these simplified conditions are much more amenable to analysis. A non-dimensional mathematical model that considers nutrient transport, biofilm growth and detachment is developed and used to interpret experimental data. Regimes with both gradual detachment and catastrophic sloughing are considered. The concentration of nutrients in the media is altered to resolve the effect of nutrient limitation. In addition, the role of a couple of major polysaccharide EPS components are explored with mutants, which leads results in line with previous studies.

There has been a vast amount of experimental and modelling work done on biofilms, but relatively rarely are the two linked together so tightly as in this paper. Predictions on influence of the non-dimensional Damkohler number on the longitudinal distribution of biofilm and functional dependence of flow on the maximum amount of biofilm (phi_max) are demonstrated. The study reconfirms a number of previous works that showed the gradual detachment rate of biofilms scales with the square root of the shear stress. More challenging are the rapid biofilm detachment events where a large amount of biofilm is detached at once. These events occur are identified experimentally using an automated analysis pipeline and are fitted with probability distributions. The time between detachment events was fitted with a Gamma distribution and the amplitude of the detachment events was fitted with a log-normal distribution, however, it is not clear how good these fits are. Experimental data was then used as an input for a stochastic differential equation, but the output of this model is compared only qualitatively to that of the experiments. Overall, this paper does an admirable job of developing a well-constrained experiments and a tightly integrated mathematical framework through which to interpret them. However, the new insights this provides the underlying physical/biological mechanisms are relatively limited.

Reviewer #2 (Public Review):

This manuscript by Wu, Liao et al. reports that simultaneous knockdown of P27Kip1 with overexpression of Cyclin D can stimulate Muller glia to re-enter the cell cycle in the mouse retina. There is intense interest in reprogramming mammalian muller glia into a source for neurogenic progenitors, in the hopes that these cells could be a source for neuronal replacement in neurodegenerative diseases. Previous work in the field has shown ways in which mouse Muller glia can be neurogenically reprogrammed and these studies have shown cell cycle re-entry prior to neurogenesis. In other works, typically, the extent of glial proliferation is limited, and the authors of this study highlight the importance of stimulating large numbers of Muller glia to re-enter the cell cycle with the hopes they will differentiate into neurons. While the evidence for stimulating proliferation in this study is convincing, the evidence for neurogenesis in this study is not convincing or robust, suggesting that stimulating cell cycle-reentry may not be associated with increasing regeneration without another proneural stimulus.

Below are concerns and suggestions.

Intro:

(1) The authors cite past studies showing "direct conversion" of MG into neurons. However, these studies (PMID: 34686336; 36417510) show EdU+ MG-derived neurons suggesting cell cycle re-entry does occur in these strategies of proneural TF overexpression.

(2) Multiple citations are incorrectly listed, using the authors first name only (i.e. Yumi, et al; Levi, et al;). Studies are also incompletely referenced in the references.

Figure 1:<br /> (3) When are these experiments ending? On Figure 1B it says "analysis" on the end of the paradigm without an actual day associated with this. This is the case for many later figures too. The authors should update the paradigms to accurately reflect experimental end points.

(4) Are there better representative pictures between P27kd and CyclinD OE, the EdU+ counts say there is a 3 fold increase between Figure 1D&E, however the pictures do not reflect this. In fact, most of the Edu+ cells in Figure 1E don't seem to be Sox9+ MG but rather horizontally oriented nuclei in the OPL that are likely microglia.

(5) Is the infection efficacy of these viruses different between different combinations (i.e. CyclinD OE vs. P27kd vs. control vs. CCA combo)? As the counts are shown in Figure 1G only Sox9+/Edu+ cells are shown not divided by virus efficacy. If these are absolute counts blind to where the virus is and how many cells the virus hits, if the virus efficacy varies in efficiency this could drive absolute differences that aren't actually biological.

(6) According to the Jax laboratories, mice aren't considered aged until they are over 18months old. While it is interesting that CCA treatment does not seem to lose efficacy over maturation I would rephrase the findings as the experiment does not test this virus in aged retinas.

(7) Supplemental Figure 2c-d. These viruses do not hit 100% of MG, however 100% of the P27Kip staining is gone in the P27sh1 treatment, even the P27+ cell in the GCL that is likely an astrocyte has no staining in the shRNA 1 picture. Why is this?

Figure 2<br /> (8) Would you expect cells to go through two rounds of cell cycle in such a short time? The treatment of giving Edu then BrdU 24 hours later would have to catch a cell going through two rounds of division in a very short amount of time. Again the end point should be added graphically to this figure.

Figure 3<br /> (9) I am confused by the mixing of ratios of viruses to indicate infection success. I know mixtures of viruses containing CCA or control GFP or a control LacZ was injected. Was the idea to probe for GFP or LacZ in the single cell data to see which cells were infected but not treated? This is not shown anywhere?

(10) The majority of glia sorted from TdTomato are probably not infected with virus. Can you subset cells that were infected only for analysis? Otherwise it makes it very hard to make population judgements like Figure 3E-H if a large portion are basically WT glia.

(11) Figure 3C you can see Rho is expressed everywhere which is common in studies like this because the ambient RNA is so high. This makes it very hard to talk about "Rod-like" MG as this is probably an artifact from the technique. Most all scRNA-seq studies from MG-reprogramming have shown clusters of "rods" with MG hybrid gene expression and these had in the past just been considered an artifact.

(12) It is mentioned the "glial" signature is downregulated in response to CCA treatment. Where is this shown convincingly? Figure H has a feature plot of Glul , which is not clear it is changed between treatments. Otherwise MG genes are shown as a function of cluster not treatment.

Figure 4<br /> (13) The authors should be commended for being very careful in their interpretations. They employ the proper controls (Er-Cre lineage tracing/EdU-pulse chasing/scRNA-seq omics) and were very careful to attempt to see MG-derived rods. This makes the conclusion from the FISH perplexing. The few puncta dots of Rho and GNAT in MG are not convincing to this reviewer, Rho and GNAT dots are dense everywhere throughout the ONL and if you drew any random circle in the ONL it would be full of dots. The rigor of these counts also comes into question because some dots are picked up in MG in the INL even in the control case. This is confusing because baseline healthy MG do not express RNA-transcripts of these Rod genes so what is this picking up? Taken together, the conclusion that there are Rod-like MG are based off scRNA-seq data (which is likely ambient contamination) and these FISH images. I don't think this data warrants the conclusion that MG upregulate Rod genes in response to CCA.

Figure 5<br /> (14) Similar point to above but this Glul probe seems odd, why is it throughout the ONL but completely dark through the IPL, this should also be in astrocytes can you see it in the GCL? These retinas look cropped at the INL where below is completely black. The whole retinal section should be shown. Antibodies exist to GS that work in mouse along with many other MG genes, IHC or western blots could be done to better serve this point.

Figure 6<br /> (15) Figure 6D is not a co-labeled OTX2+/ TdTomato+ cell, Otx2 will fill out the whole nucleus as can be seen with examples from other MG-reprogramming papers in the field (Hoang, et al. 2020; Todd, et al. 2020; Palazzo, et al. 2022). You can clearly see in the example in Figure 6D the nucleus extending way beyond Otx2 expression as it is probably overlapping in space. Other examples should be shown, however, considering less than 1% of cells were putatively Otx2+, the safer interpretation is that these cells are not differentiating into neurons. At least 99.5% are not.

(16) Same as above Figure 6I is not convincingly co-labeled HuC/D is an RNA-binding protein and unfortunately is not always the clearest stain but this looks like background haze in the INL overlapping. Other amacrine markers could be tested, but again due to the very low numbers, I think no neurogenesis is occurring.

(17) In the text the authors are accidently referring to Figure 6 as Figure 7.

Figure 7<br /> (18) I like this figure and the concept that you can have additional MG proliferating without destroying the retina or compromising vision. This is reminiscent of the chick MG reprogramming studies in which MG proliferate in large numbers and often do not differentiate into neurons yet still persist de-laminated for long time points.

General:<br /> (19) The title should be changed, as I don't believe there is any convincing evidence of regeneration of neurons. Understanding the barriers to MG cell-cycle re-entry are important and I believe the authors did a good job in that respect, however it is an oversell to report regeneration of neurons from this data.

(20) This paper uses multiple mouse lines and it is often confusing when the text and figures switch between models. I think it would be helpful to readers if the mouse strain was added to graphical paradigms in each figure when a different mouse line is employed.

Reviewer #2 (Public Review):

Traditional thinking has been that cortical oligodendrocyte progenitor cells (OPCs) arise in the development of the brain from the medial ganglionic eminence (MGE), lateral/caudal ganglionic eminence (LGE/CGE), and cortical radial glial cells (RGCs). Indeed a landmark study demonstrated some time ago that cortical OPCs are generated in three waves, starting with a ventral wave derived from the medial ganglionic eminence (MGE) or the anterior entopeduncular area (AEP) at embryonic day E12.5 (Nkx2.1+ lineage), followed by a second wave of cortical OLs derived from the lateral/caudal ganglionic eminences (LGE/CGE) at E15.5 (Gsx2+/Nkx2.1- lineage), and then a final wave occurring at P0, when OPCs originate from cortical glial progenitor cells (Emx1+ lineage). However, the authors challenge the idea in this paper that cortical progenitors are produced from the LGE. They have found previously that cortical glial progenitor cells were also found to express Gsx2, suggesting this may not have been the best marker for LGE-derived OPCs. They have used fate mapping experiments and lineage analyses to suggest that cortical OPCs do not derive from the LGE.

Strengths:

(1) The data is high quality and very well presented, and experiments are thoughtful and elegant to address the questions being raised.

(2) The authors use two elegant approaches to lineage trace LGE derived cells, namely fate mapping of LGE-derived OPCs by combining IUE (intrauterine electroporation) with a Cre recombinase-dependent IS reporter, and Lineage tracing of LGE-derived OPCs by combining IUE with the PiggyBac transposon system. Both approaches show convincingly that labelled LGE-derived cells that enter the cortex do not express OPC markers, but that those co-labelling with oligodendrocyte markers remain in the striatum.

(3) The authors then use further approaches to confirm their findings. Firstly they lineage trace Emx1-Cre; Nkx2.1-Cre; H2B-GFP mice. Emx1-Cre is expressed in cortical RGCs and Nkx2.1-Cre is specifically expressed in MGE/AEP RGCs. They find that close to 98% of OPCs in the cortex co-label with GFP at later times, suggesting the contribution of OPCs from LGE is minimal.

(4) They use one further approach to strengthen the findings yet further. They cross Nkx2.1-Cre mice with Olig2 F/+ mice to eliminate Olig2 expression in the SVZ/VZ of the MGE/AEP (Figures 4A-B). The generation of MGE/AEP-derived OPCs is inhibited in these Olig2-NCKO conditional mice. They find that the number of cortical progenitors at E16.5 is reduced 10-fold in these mice, suggesting that LGE contribution to cortical OPCs is minimal.

Impact of Study:

The authors show elegantly and convincingly that the contribution of the LGE to the pool of cortical OPCs is minimal. The title should perhaps be that the LGE contribution is minimal rather than no contribution at all, as they are not able to rule out some small contribution from the LGE. These findings challenge the traditional belief that the LGE contributes to the pool of cortical OPCs. The authors do show that the LGE does produce OPCs, but that they tend to remain in the striatum rather than migrate into the cortex. It is interesting to wonder why their migration patterns may be different from the MGE-derived OPCs which migrate to the cortex. The functional significance of these different sources of OPCs for adult cortex in homeostatic or disease states remains unclear though.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shore et al. investigate the consequent changes in excitability and synaptic efficacy of diverse neuronal populations in an animal model of juvenile epilepsy. Using electrophysiological patch-clamp recordings from dissociated neuronal cultures, the authors find diverging changes in two major populations of inhibitory cell types, namely somatostatin (SST)- and parvalbumin (PV)-positive interneurons, in mice expressing a variant of the KCNT1 potassium channel. They further suggest that the differential effects are due to a compensatory increase in the persistent sodium current in PV interneurons in pharmacological and in silico experiments. It remains unclear why this current is selectively enhanced in PV-interneurons.

Strengths:

(1) Heterozygous KCNT1 gain of function variant was used which more accurately models the human disorder.

(2) The manuscript is clearly written, and the flow is easy to follow. The authors explicitly state the similarities and differences between the current findings and the previously published results in the homozygous KCNT1 gain of function variant.

(3) This study uses a variety of approaches including patch clamp recording, in silico modeling and pharmacology that together make the claims stronger.

(4) Pharmacological experiments are fraught with off-target effects and thus it bolsters the authors' claims when multiple channel blockers (TTX and VU170) are used to reconstruct the sodium-activated potassium current.

Reviewer #2 (Public Review):

Summary:

Photoreceptor neurons are crucial for vision, and discovering pathways necessary for photoreceptor health and survival can open new avenues for therapeutics. Studies have shown that metabolic dysfunction can cause photoreceptor degeneration and vision loss, but the metabolic pathways maintaining photoreceptor health are not well understood. This is a fundamental study that shows that glutamine catabolism is critical for photoreceptor cell health using in vivo model systems.

Strengths:

The data are compelling, and the consideration of potential confounding factors (such as glutaminase 2 expression) and additional experiments to examine the synaptic connectivity and inner retina added strength to this work. The authors were also careful not to overstate their claims, but to provide solid conclusions that fit the results and data provided in their study. The findings linking asparagine supplementation and the inhibition of the integrated stress response to glutamine catabolism within the rod photoreceptor cell are intriguing and innovative. Overall, the authors provide convincing data to highlight that photoreceptors utilize various fuel sources to meet their metabolic needs, and that glutamine is critical to these cells for their biomass, redox balance, function, and survival.

Weaknesses:

Recent studies have explored the metabolic "crosstalk" that exists within the mammalian retina, where metabolites are transferred between the various retinal cells and the retinal pigment epithelium. It would be of interest to test whether the conditional knockout mice have changes in metabolism (via qPCR such as shown in Figure 4 - Supplemental Figure 1) within the retinal pigment epithelium that may be contributing to the authors' findings in the neural retina. Additionally, the authors have very compelling data to show that inhibition of eIF2a or supplementation with asparagine can delay photoreceptor death via OCT measurements in their conditional knockout mouse model (Figure 6G, H). However, does inhibition of eIF2a or asparagine adversely impact the WT retina? It would also be impactful to know whether this has a prolonged effect, or if it is short-term, as this would provide strength to potential therapeutic targeting of these pathways to maintain photoreceptor health.

Reviewer #3 (Public Review):

The manuscript from Amen et al reports the isolation and characterization of human antibodies that recognize proteins expressed at different sexual stages of Plasmodium falciparum. The isolation approach was antigen agnostic and based on the sorting, activation, and screening of memory B cells from a donor whose serum displays high transmission-reducing activity. From this effort, 14 antibodies were produced and further characterized. The antibodies displayed a range of transmission-reducing activities and recognized different Pf sexual stage proteins. However, none of these antibodies had substantially higher TRA than previously described antibodies.

The authors then performed further characterization of antibody B1E11K, which was unique in that it recognized multiple proteins expressed during sexual and asexual stages. Using protein microarrays, B1E11K was shown to recognize glutamate-rich repeats, following an EE-XX-EE pattern. An impressive set of biophysical experiments were performed to extensively characterize the interactions of B1E11K with various repeat motifs and lengths. Ultimately, the authors succeeded in determining a 2.6 A resolution crystal structure of B1E11K bound to a 16AA repeat-containing peptide. Excitingly, the structure revealed that two Fabs bound simultaneously to the peptide and made homotypic antibody-antibody contacts. This had only previously been observed before with antibodies directed against CSP repeats.

Overall I found the manuscript to be very well written. Strengths of the manuscript include the target-agnostic screening approach and the thorough characterization of antibodies. The demonstration that B1E11K is cross-reactive to multiple proteins containing glutamate-rich repeats, and that the antibody recognizes the repeats via homotypic interactions, similar to what has been observed for CSP repeat-directed antibodies, should be of interest to many in the field.

Reviewer #2 (Public Review):

Summary:

Breyton and colleagues analysed the emergent dynamics from a neural mass model, characterised the resultant complexity of the dynamics, and then related these signatures of complexity to datasets in which individuals had been anaesthetised with different pharmacological agents. The results provide a coherent explanation for observations associated with different time series metrics, and further help to reinforce the importance of modelling when integrating across scientific studies.

Strengths:

* The modelling approach was clear, well-reasoned, and explicit, allowing for direct comparison to other work and potential elaboration in future studies through the augmentation with richer neurobiological detail.

* The results serve to provide a potential mechanistic basis for the observation that the Perturbational Complexity Index changes as a function of the consciousness state.

Weaknesses:

* Coactivation cascades were visually identified, rather than observed through an algorithmic lens. Given that there are numerous tools for quantifying the presence/absence of cascades from neuroimaging data, the authors may benefit from formalising this notion.

* It was difficult to tell, graphically, where the model's operating regime lay. Visual clarity here will greatly benefit the reader.

Reviewer #2 (Public Review):

Summary:

Disruption of the excitatory/inhibitory (E/I) balance have been reported in Autism Spectrum disorders (ASD) to which PTEN mutations have been associated. Giunti et al choose to explore the impact of PTEN mutations on the balance between E/I signaling using as a platform the C. elegans neuromuscular system where both cholinergic (E) and GABAergic (I) motor neurons regulate muscle contraction and relaxation. Mutations in daf-18/PTEN specifically affect morphologically and functionally the GABAergic (I) system, while leaving the cholinergic (E) system unaffected. The study further reveals that the observed defects in the GABAergic system in daf-18/PTEN mutants are attributed to reduced activity of DAF-16/FOXO during development.<br /> Moreover, ketogenic diets (KGDs), known for their effectiveness in disorders associated with E/I imbalances such as epilepsy and ASD, are found to induce DAF-16/FOXO during early development. Supplementation with β-hydroxybutyrate in the nematode at early developmental stages proves to be both necessary and sufficient to correct the effects on GABAergic signaling in daf-18/PTEN mutants.

Strengths:

The authors combined pharmacological, behavior and optogenetic experiments to show the GABAergic signaling impairment at the C. elegans neuromuscular junction in DAF-18/PTEN and DAF-16/FOXO mutants. Moreover, by studying the neuron morphology, they point towards neurodevelopmental defects in the GABAergic motoneurons involved in locomotion. Using the same set of experiments, they demonstrate that a ketogenic diet can rescue the inhibitory defect in the daf-18/PTEN mutant at an early stage.

Weaknesses:

The morphological experiments hint towards a pre-synaptic defect to explain the GABAergic signaling impairment, but it would have also been interesting to check the post-synaptic part of the inhibitory neuromuscular junctions such as the GABA receptor clusters to assess if the impairment is only presynaptic or both post and presynaptic. Moreover, analysing post-synaptic functionality in-depth using electrophysiology would be beneficial too.<br /> Nevertheless, this question alone could be entirely the subject of another paper and is not essential to the primary message of the paper.

Conclusion:

Giunti et al provide fundamental insights into the connection between PTEN mutations and neurodevelopmental defects through DAF-16/FOXO and shed light on the mechanisms through which ketogenic diets positively impact neuronal disorders characterized by E/I imbalances.

Reviewer #2 (Public Review):

Summary:

Canonically cerebellar neurons are derived from 2 primary germinal zones within the anterior hindbrain (dorsal rhombomere 1). This manuscript identifies an important, previously underappreciated origin for a subset of early cerebellar nuclei neurons - likely the mesencephalon. This is an exciting finding.

Strengths:

The authors have identified a novel early population of cerebellar neurons with likely novel origin in the midbrain. They have used multiple assays to support their conclusions, including immunohistochemistry and in situ analyses of a number of markers of this population which appear to stream from the midbrain into the dorsal anterior cerebellar anlage.

The inclusion of Otx2-GFP short term lineage analyses and analysis of Atoh1 -/- animals also provide considerable support for the midbrain origin of these neurons as streams of cells seem to emanate from the midbrain. However, without live imaging there remains the possibility that these streams of cells are not actually migrating and rather, gene expression is changing in static cells. Hence the authors have conducted midbrain diI labelling experiments of short term and long term cultured embryos showing di-labelled cells in the developing cerebellum. These studies confirm migration of cells from the midbrain into the early cerebellum.

The authors have appropriately responded to review issues, replacing panels in figures and updating legends and text. They have also appropriately noted the limitations of their work.

Reviewer #2 (Public Review):

Summary:

Complexin (Cplx) is expressed at nearly all chemical synapses. Mammalian Cplx comes in four different paralogs which are differentially expressed in different neurons or secretory cell types, either selectively or in combination with one or two other Cplx isoforms. Cplx binds with high affinity to assembled SNARE complexes and promotes evoked synchronous release. Cplx is assumed to preclude premature SV fusion by preventing full SNARE assembly, thereby arresting subsequent SNARE-driven fusion ("fusion-clamp" theory). The protein has multiple domains, the functions of which are controversially discussed. Cplx's function has been studied in a variety of model organisms including mouse, fly, worm, and fish with seemingly conflicting results which led to partly contradicting conclusions.<br /> Makee et al. study the function of mammalian Cplx2 in chromaffin cells by making use of Cplx2 ko mice to overexpress and functionally characterize mutant Cplx2 forms in cultured chromaffin cells. The main conclusion of the present study are:

The hydrophobic character of the amphipathic helix in Cplx's C-terminal domain is essential for inhibiting premature vesicle fusion at a [Ca2+]i of several hundreds of nM (pre-flash [Ca2+]i). The Cplx-mediated inhibition of fusion under these conditions does not rely on expression of either Syt1 or Syt7.

Slow-down of exocytosis by N-terminally truncated Cplx mutants in response to a [Ca2+]i of several µM (peak flash [Ca2+]i) occurs regardless of the presence or absence of Syt7 demonstrating that Cplx2 does not act as a switch favoring preferential assembly of the release machinery with Syt1,2 rather than the "slow" sensor Syt7.

Cplx's N-terminal domain is required for the Cplx2-mediated increase in the speed of exocytosis and faster onset of exocytosis which likely reflect an increased apparent Ca2+ sensitivity and faster Ca2+ binding of the release machinery.

Strengths:

The authors perform systematic truncation/mutational analyses of Cplx2. They analyze the impact of single and combined deficiencies for Cplx2 and Syt1 to establish interactions of both proteins.<br /> State-of-the-art methods are employed: Vesicle exocytosis is assayed directly and with high resolution using capacitance measurements. Intracellular [Ca2+] is controlled by loading via the patch-pipette and by UV-light induced flash-photolysis of caged [Ca2+]. The achieved [Ca2+ ] is measured with Ca2+ -sensitive dyes.<br /> The data is of high quality and the results are compelling.

Weaknesses:

With the exception of mammalian retinal ribbon synapses (and some earlier RNAi knock down studies which had off-target effects), there is little experimental evidence for a "fusion-clamp"-like function of Cplxs at mammalian synapses. At conventional mammalian synapses, genetic loss of Cplx (i.e. KO) consistently decreases AP-evoked release, and generally either also decreases spontaneous release rates or does not affect spontaneous release, which is inconsistent with a "fusion-clamp" theory. This is in stark contrast to invertebrate (D. m. and C. e.) synapses where genetic Cplx loss is generally associated with a strong upregulation of spontaneous release.

There are alternative scenarios explaining how Cplx may phenomenological "clamp" vesicle fusion rates without mechanistically assigning a "clamping" function to Cplx (Neher 2010, Neuron). In fact, changes in asynchronous release kinetics following conditioning AP trains observed at Cplx1 ko calyx of Held synapses do not favor a "fusion clamp" model (Chang et al., 2015, J.Neurosci.), while an alternative model, assigning Cplx the role of a "checkpoint" protein in SNARE assembly, quantitatively reproduces all experimental observations (Lopez et al., 2024, PNAS). It might be helpful for a reader to mention such alternative scenarios.

Reviewer #2 (Public Review):

Summary:

In this revised manuscript, Glaser et al. have responded to the reviewer comments by removing some of the overstated claims from the prior manuscript and editing portions of the manuscript text to enhance the clarity. Although the manuscript would be stronger if the authors had been able to provide data that justified the original high-impact claims from the initial publication (e.g. that the images could be used for robust and automated neuronal tracing across large volumes), the amended manuscript text now more closely matches the supporting data. As with the initial submission, I believe that the microscope design and characterization is a useful contribution to the field and the data are quite stunning. However, I still feel like there are some overstated claims in this revision that should be addressed so as not to mislead readers.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Reviewer #2 (Public Review):

Summary:<br /> In their manuscript the authors address the question of whether the inversion polymorphism in D. melanogaster can be explained by sexually antagonistic selection. They designed a new simulation tool to perform computer simulations, which confirmed their hypothesis. They also show a tradeoff between male reproduction and survival. Furthermore, some inversions display sex-specific survival.

Strengths:<br /> It is an interesting idea on how chromosomal inversions may be maintained

Weaknesses:<br /> General points:<br /> The manuscript lacks clarity of writing. It is impossible to fully grasp what the authors did in this study and how they reached their conclusions. Therefore, I cannot guarantee that I spotted all the problems in the study and in my review I will only highlight some cases that I found problematic.<br /> Although this is an interesting idea, but it clearly cannot explain the apparent influence of seasonal and clinal variation on inversion frequencies.

Comments on the latest version:

I would like to give an example of the confusing terminology of the authors:

"Additionally, fitness conveyed by an allele favoring display quality is also frequency-dependent: since mating success depends on the display qualities of other males, the relative advantage of a display trait will be diminished as more males carry it..."

I do not understand the difference to an advantageous allele, as it increases in frequency the frequency increase of this allele decreases, but this has nothing to do with frequency dependent selection. In my opinion, the authors re-define frequency dependent selection, as for frequency dependent selection needs to change with frequency, but from their verbal description this is not clear.

One example of how challenging the style of the manuscript is comes from their description of the DNA extraction procedure. In principle a straightforward method, but even here the authors provide a convoluted uninformative description of the procedure.

It is not apparent to the reviewer why the authors have not invested more effort to make their manuscript digestible.

Reviewer #2 (Public Review):

Summary:

Blackwell et al. investigated the structure, localization, and physiological function of Plasmodium falciparum (Pf) heme oxygenase (HO). Pf and other malaria parasites scavenge and digest large amounts of hemoglobin from red cells for sustenance. To counter the potentially cytotoxic effects of heme, it is biomineralized into hemozoin and stored in the food vacuole. Another mechanism to counteract heme toxicity is through its enzymatic degradation via heme oxygenases. However, it was previously found by the authors that PfHO lacks the ability to catalyze heme degradation, raising the intriguing question of what the physiological function of PfHO is. In the current contribution, the authors determine that PfHO localizes to the apicoplast, determine its targeting sequence, establish the essentiality of PfHO for parasite viability, and determine that PfHO is required for proper maintenance of apicoplasts and apicoplast gene expression. In sum, the authors establish an essential physiological function for PfHO, thereby providing new insights into the role of PfHO in plasmodium metabolism.

Strengths:

The studies are rigorously conducted and the results of the experiments unambiguously support a role for PfHO as being an apicoplast-targeted protein required for parasite viability and maintenance of apicoplasts.

Weaknesses:

While the studies conducted are rigorous and support the primary conclusions, the lack of experiments probing the molecular function of PfHO limits the impact of the work. Nevertheless, the knowledge that PfHO is required for parasite viability and plays a role in the maintenance of apicoplasts is still an important advance.

Reviewer #2 (Public Review):

Summary:

Sleep has not only been shown to support the strengthening of memory traces but also their transformation. A special form of such transformation is the abstraction of general rules from the presentation of individual exemplars. The current work used large online experiments with hundreds of participants to shed further light on this question. In the training phase, participants saw composite items (scenes) that were made up of pairs of spatially coupled (i.e., they were next to each other) abstract shapes. In the initial training, they saw scenes made up of six horizontally structured pairs, and in the second training phase, which took place after a retention phase (2 min awake, 12 h incl. sleep, 12 h only wake, 24 h incl. sleep), they saw pairs that were horizontally or vertically coupled. After the second training phase, a two-alternatives-forced-choice (2-AFC) paradigm, where participants had to identify true pairs versus randomly assembled foils, was used to measure the performance of all pairs. Finally, participants were asked five questions to identify, if they had insight into the pair structure, and post-hoc groups were assigned based on this. Mainly the authors find that participants in the 2-minute retention experiment without explicit knowledge of the task structure were at chance level performance for the same structure in the second training phase, but had above chance performance for the vertical structure. The opposite was true for both sleep conditions. In the 12 h wake condition these participants showed no ability to discriminate the pairs from the second training phase at all.

Strengths:

All in all, the study was performed to a high standard and the sample size in the implicit condition was large enough to draw robust conclusions. The authors make several important statistical comparisons and also report an interesting resampling approach. There is also a lot of supplemental data regarding robustness.

Weaknesses:

My main concern regards the small sample size in the explicit group and the lack of experimental control.

Reviewer #2 (Public Review):

The paper by Nelson KA, et al. explored the collective migration, coalescence, and positioning of the posterior signaling center (PSC) cells in Drosophila embryo. With live imaging, the authors observed the dynamic progress of PSC migration. Throughout this process, visceral mesoderm (VM), alary muscles (Ams), and cardioblasts (CBs) are in proximity to PSC. Genetic ablation of these tissues reveals the requirement for VM and CBs, but not AMs in this process. Genetic manipulations further demonstrated that Slit-Robo signaling was critical during PSC migration and positioning. While the genetic mechanisms of positioning the PSC were explored in much detail, including using live imaging, the functional consequence of mispositioning or (partial) absence of PSC cells has not been addressed, but would much increase the relevance of their findings. A few additional issues need to be addressed as well in this otherwise well-done study.

Major points:

(1) The only readout in their experiments is the relative correctness of PSC positioning. Importantly, what is the functional consequence if PSC is not properly positioned? This would be particularly important with robo-sli manipulations, where the PSC is present but some cells are misplaced. What is the consequence? Are the LGs affected, like the specification of their cell types, structure, and function? To address this for at least the robo-slit requirement in the PSC, it may be important to manipulate them directly in the PSC with a split Gal4 system, using Antp and Odd promoters.

(2) The densely, parallel aligned fibers in the part of Figure 1J seemed to be visceral mesoderm, but further up (dorsally) that may be epidermis. It is possible that the PSC migrate together with the epidermis? This should be addressed.

(3) Although the authors described the standards of assessing PSC positioning as "normal" or "abnormal", it is rather subtle at times and variable in the mutant or KD/OE examples. The criteria should be more clearly delineated and analyzed double-blind, also since this is the only readout. Further examples of abnormal positioning in supplementary figures would also help.

(4) The Discussion is very lengthy and should shortened.

Reviewer #2 (Public Review):

Summary:

In their paper "Massively Parallel Polyribosome Profiling Reveals Translation Defects of Human Disease‐Relevant UTR Mutations" the authors use massively parallel polysome profiling to determine the effects of 5' and 3' UTR SNPs (from dbSNP/ClinVar) on translational output. They show that some UTR SNPs cause a change in the polysome profile with respect to the wild-type and that pathogenic SNPs are enriched in the polysome-shifting group. They validate that some changes in polysome profiles are predictive of differences in translational output using transiently expressed luciferase reporters. Additionally, they identify sequence motifs enriched in the polysome-shifting group. They show that 2 enriched 5' UTR motifs increase the translation of a luciferase reporter in a protein-dependent manner, highlighting the use of their method to identify translational control elements.

Strengths:

This is a useful method and approach, as UTR variants have been more difficult to study than coding variants. Additionally, their evidence that pathogenic mutations are more likely to cause changes in polysome association is well supported.

Weaknesses:

The authors acknowledge that they "did not intend to immediately translate the altered polysome profile into an increase or decrease in translation efficiency, as the direction of the shift was not readily evident. Additionally, sedimentation in the sucrose gradient may have been partially affected by heavy particles other than ribosomes." However, shifted polysome distribution is used as a category for many downstream analyses. Without further clarity or subdivision, it is very difficult to interpret the results (for example in Figure 5A, is it surprising that the polysome shifting mutants decrease structure? Are the polysome "shifts" towards the untranslated or heavy fractions?)

Reviewer #2 (Public Review):

Summary:

The authors first conducted whole exome sequencing for infertile male patients and families where they co-segregated the biallelic mutations in the Dynein Axonemal Heavy Chain 12 (DNAH12) gene.<br /> Sperm from patients with biallelic DNAH12 mutations exhibited a wide range of morphological abnormalities in both tails and heads, reminiscing a prevalent cause of male infertility, asthenoteratozoospermia. To deepen the mechanistic understanding of DNAH12 in axonemal assembly, the authors generated two distinct DNAH12 knockout mouse lines via CRISPR/Cas9, both of which showed more severe phenotypes than observed in patients. Ultrastructural observations and biochemical studies revealed the requirement of DNAH12 in recruiting other axonemal proteins and that the lack of DNAH12 leads to the aberrant stretching in the manchette structure as early as stage XI-XII. At last, the authors proposed intracytoplasmic sperm injection as a potential measure to rescue patients with DNAH12 mutations, where the knockout sperm culminated in the blastocyst formation with a comparable ratio to that in WT.

Strengths:

The authors convincingly showed the importance of DNAH12 in assembling cilia and flagella in both human and mouse sperm. This study is not a mere enumeration of the phenotypes, but a strong substantiation of DNAH12's essentiality in spermiogenesis, especially in axonemal assembly.

The analyses conducted include basic sperm characterizations (concentration, motility), detailed morphological observations in both testes and sperm (electron microscopy, immunostaining, histology), and biochemical studies (co-immunoprecipitation, mass-spec, computational prediction). Molecular characterizations employing knockout animals and recombinant proteins beautifully proved the interactions with other axonemal proteins.

Many proteins participate in properly organizing flagella, but the exact understanding of the coordination is still far from conclusive. The present study gives the starting point to untangle the direct relationships and order of manifestation of those players underpinning spermatogenesis. Furthermore, comparing flagella and trachea provides a unique perspective that attracts evolutional perspectives.

Weaknesses:

Seemingly minor, but the discrepancies found in patients and genetically modified animals were not fully explained. For example, both knockout mice vastly reduced the count of sperm in the epididymis and the motility, while phenotypes in patients were rather milder. Addressing the differences in the roles that the orthologs play in spermatogenesis would deepen the comprehensive understanding of axonemal assembly.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors combined imaging approaches with molecular and genetic experiments to

(i) for the first time establish a chromatin regulatory role for SDG7;<br /> (ii) examine its role in H3K36 methylation, along with its homolog SDG8;<br /> (iii) examine its potential role in mediating antagonism to PRC2, thereby mediating transcriptional activation.

Strengths:

The manuscript explores interesting and relevant mechanistic hypotheses about chromatin-mediated gene regulation by combining a range of experimental tools and a genome-wide perspective. The writing is very clear. The study makes good connections to existing data and generates datasets that complement existing datasets, providing a valuable resource to the community.

Weaknesses:

Some of the claims appear to need further supporting evidence to establish their robustness.

Review of the main conclusions and supporting evidence:

(1) SDG7 contributes to H3K36 methylation at several loci along with SDG8:<br /> This conclusion is supported by the genome-wide differences (measured by ChIP-seq) in H3K36me3 and me2 levels observed between WT, the sdg7 and sdg8 single mutants, and the double mutant. The reduction in H3K36 methylation levels observed at a large number of genes in the sdg7 mutant, and the further reduction in H3K36 methylation levels in the double mutant compared to sdg8 (observed at multiple genes) indicates that SDG7 promotes this transcription-associated modification. The significantly larger number of H3K36 hypomethylated genes in the double mutant (3380) compared to either of the single mutants (523 and 605) indicates lower overall H3K36me3 levels in the double mutant.<br /> While direct evidence of SDG7 methyltransferase activity on H3K36 is lacking, the study reports structural comparisons using AlphaFold predictions that suggest the possibility of such a role.

(2) SDG7 influences gene expression together with SDG8:<br /> This conclusion is supported by a range of phenotypic differences observed between sdg8 and the double mutant sdg7 sdg8. The reported genome-wide differential gene expression between the WT and the double mutant as well as expression differences in specific genes detected by imaging are also consistent with SDG7 and SDG8 together influencing gene expression. However, a role specifically for SDG7 could be further supported by a direct differential expression analysis between the single mutant sdg8 and the double mutant sdg7 sdg8. A role for SDG7 in gene expression is also consistent with its observed effect on H3K36 methylation, which is generally associated with productive transcription.

(3) SDG8 is exclusively nuclear, but SDG7 localises to the cytosol and the nucleus in meristematic cells:<br /> This conclusion is supported by imaging of fluorescent-tagged SDG8 and SDG7 in the root tip, which shows nuclear localisation of SDG8-GFP, consistent with previous reports, and a combination of nuclear and cytosolic localisation for SDG7-VENUS. However, the study presents only one replicate - more replicates are needed to establish the robustness of this conclusion.

(4) Distinct binding patterns of SDG7 and SDG8 on chromatin:<br /> This conclusion is supported by the analysis of genome-wide binding patterns of these proteins measured by ChIP-seq (using fluorescent tagged versions). This data indicates that while SDG7 tends to localise to TSS and TES regions of genes, SDG8 tends to be more uniformly spread across gene loci. These patterns suggest that SDG7 and SDG8 may influence H3K36 methylation through distinct mechanisms, but do not indicate what these mechanisms may be.

(5) SDG7/SDG8 antagonism with PRC2:<br /> a. SDG7 overlaps with PRC2 and its recruiters on chromatin and can bind to PREs<br /> This conclusion is supported by statistically significant overlaps genome-wide between cis-elements at SDG7 binding peaks and those previously reported to be Polycomb associated. This is further supported by the observation of SDG7 binding peaks close to PRC2 subunit binding peaks at known PRC2 targets.<br /> b. SDG7/SDG8 antagonism with PRC2<br /> This conclusion is supported by the observation that the sdg7 sdg8 double mutant phenotypes are partially rescued by disrupting CLF, one of the PRC2 methyltransferases. However, this does not necessarily suggest a direct antagonism with PRC2, but could be part of a more general antagonism between productive transcription (in which H3K36 methylation plays an active role), and PRC2-mediated silencing.<br /> c. SDG7 can evict PRC2 from PREs to overcome H3K27me3-mediated silencing<br /> This conclusion - that SDG7 directly antagonises PRC2 interaction with cis-elements that mediate its targeting - is partly supported by the observation that inducing overexpression of SDG7 (through dexamethasone induction) can cause changes in CLF occupancy and H3K27me3 levels at certain designated PREs. To establish the robustness of these conclusions, it will be necessary to examine designated regions to be used as a negative control and include a non-transgenic control for the SDG-7-HA ChIP. A suitable negative control may be a non-PRE region known to have a CLF peak and high H3K27me3 levels, where the levels would not be expected to change in this experiment.<br /> It remains to be established whether this apparent antagonism of PRC2 by SDG7 is only part of a more general antagonism of PRC2 by productive transcriptional activity, or whether SDG7 can evict PRC2 from PREs independent of transcripts and therefore is a precursor to switching to an active transcriptional state.<br /> Another aspect that would be interesting to examine in the light of recent findings is the single-cell-level behaviour at these genes targeted by SDG7-to examine whether SDG7 induction is causing individual copies of these genes to stochastically switch to an active transcriptional state so that the observed changes in H3K27me3 result from a fraction of copies completely losing silencing rather than all copies losing some H3K27me3.

(6) H3K36me3 levels are co-regulated by SDG8 and Paf1c, SDG8 associates with Pol II to deliver transcription-coupled H3K36 methylation<br /> This conclusion is supported by analysis of a subset of loci previously reported to be regulated by Paf1c, which also exhibit changes in the sdg7 sdg8 double mutant as well as a clf mutant. This analysis is based on a qualitative examination of ChIP-seq signal at a small number of loci, and therefore provides indirect support for this conclusion. It is, therefore, difficult to draw mechanistic insights from this analysis that go beyond correlation.

Reviewer #2 (Public Review):

Summary:

The authors generated analogs consisting of modified neurotensin (NT) peptides capable of binding to low-density lipoprotein (LDL) and NT receptors. Their lead analog was further evaluated for additional validation as a novel therapeutic. The putative mechanism of action for NT in its antiseizure activity is hypothermia, and as therapeutic hypothermia has been demonstrated in epilepsy, NT analogs may confer antiseizure activity and avoid the negative effects of induced hypothermia.

Strengths:

The authors demonstrate an innovative approach, i.e. using LDLR as a means of transport into the brain, that may extend to other compounds. They systematically validate their approach and its potential through binding, brain penetration, in vivo antiseizure efficacy, and neuroprotection studies.

Weaknesses:

Tolerability studies are warranted, given the mechanism of action and the potential narrow therapeutic index. In vivo studies were used to assess the efficacy of the peptide conjugate analogs in the mouse KA model. However, it would be beneficial to have shown tolerability in naïve animals to better understand the therapeutic potential of this approach.

Mice may be particularly sensitive to hypothermia. It would be beneficial to show similar effects in a rat model.

Reviewer #2 (Public Review):

Summary:

In this study, Karashchuk et al. develop a hierarchical control system to control the legs of a dynamic model of the fly. They intend to demonstrate that temporal delays in sensorimotor processing can destabilize walking and that the fly's nervous system may be operating with as long of delays as could possibly be corrected for.

Strengths:

Overall, the approach the authors take is impressive. Their model is trained using a huge dataset of animal data, which is a strength. Their model was not trained to reproduce animal responses to perturbations, but it successfully rejects small perturbations and continues to operate stably. Their results are consistent with the literature, that sensorimotor delays destabilize movements.

Weaknesses:

The model is sophisticated and interesting, but the reviewer has great concerns regarding this manuscript's contributions, as laid out in the abstract:

(1) Much simpler models can be used to show that delays in sensorimotor systems destabilize behavior (e.g., Bingham, Choi, and Ting 2011; Ashtiani, Sarvestani, and Badri-Sproewitz 2021), so why create this extremely complex system to test this idea? The complexity of the system obscures the results and leaves the reviewer wondering if the instability is due to the many, many moving parts within the model. The reviewer understands (and appreciates) that the authors tested the impact of the delay in a controlled way, which supports their conclusion. However, the reviewer thinks the authors did not use the most parsimonious model possible, and as such, leave many possible sources for other causes of instability.

(2) In a related way, the reviewer is not sure that the elements the authors introduced reflect the structure or function of the fly's nervous system. For example, optimal control is an active field of research and is behind the success of many-legged robots, but the reviewer is not sure what evidence exists that suggests the fly ventral nerve cord functions as an optimal controller. If this were bolstered with additional references, the reviewer would be less concerned.

(3) "The model generates realistic simulated walking that matches real fly walking kinematics...". The reviewer appreciates the difficulty in conducting this type of work, but the reviewer cannot conclude that the kinematics "match real fly walking kinematics". The range of motion of several joints is 30% too small compared to the animal (Figure 2B) and the reviewer finds the video comparisons unpersuasive. The reviewer would understand if there were additional constraints, e.g., the authors had designed a robot that physically could not complete the prescribed motions. However the reviewer cannot think of a reason why this simulation could not replicate the animal kinematics with arbitrary precision, if that is the goal.

Reviewer #2 (Public Review):

Summary:

Pyrotinib, a pan-HER tyrosine kinase inhibitor, has shown significant survival benefits in patients with HER2-positive breast cancer. However, diarrhea is a frequent and important adverse event during pyrotinib treatment. Severe diarrhea may require interruption of pyrotinib treatment, thus affecting its anti-tumor effect and becoming a clinical safety issue. This study evaluated the incidence of diarrhea in patients with early-stage HER2-positive breast cancer treated with nab-paclitaxel and pyrotinib using 42- and 21-day loperamide primary prevention strategies. No significant differences were observed between the 21- and 42-day loperamide durations in the prevention of grade 3 and above diarrhea. Considering the economic cost and patient compliance, 21-day loperamide prophylaxis might represent a more pragmatic and appropriate approach for clinical application.

Strengths:

This study has a reasonable design, a clear hypothesis and methodology, and clear results, making the conclusion reliable. Most importantly, the findings have practical implications for the clinical management of pyrotinib-induced diarrhea.

Weaknesses:

Although the paper does have strengths in the practical implications for the clinical management of pyrotinib-induced diarrhea, there are still many data presentations that are not clear:

(1) The baseline characteristics mention that at the cut-off date on September 27, 2023, two patients withdrew from the 21-day group due to intolerable diarrhea and received other anti-tumor therapies for liposarcoma on the face. In the 42-day group, one patient was lost to follow-up and two withdrew from the study due to intolerable diarrhea and bone metastases. So, in the study, how many cases are in each group? The numbers of cases indicated in Figures 1 and 2 are inconsistent.

(2 ) Why does Figure 3a only compare 3 months of data, while Figure 3b compares 12 months of data?

(3) It is mentioned in lines 222-229 that a combination treatment of nab-paclitaxel plus pyrotinib is 1-3 months, whereas those of pyrotinib alone is 3-12 months. So, what is the exact duration of the combination treatment and pyrotinib alone treatment?

(4) This study found that no significant differences were observed between 21-day and 42-day loperamide durations in preventing grade 3 and above diarrhea. However, nab-paclitaxel can also cause diarrhea, and the conclusions would be more reliable if a control group that was not given loperamide were added. It is suggested that the authors add relevant data to further confirm the conclusion.

Reviewer #2 (Public Review):

Summary:

Colomb et al. investigated here the heparin-binding activity of the HpARI family proteins from H. polygyrus. HpARIs bind to IL-33, a pleiotropic cytokine, and modulate its activities. HpARI1/2 has suppressive functions, while HpARI3 can enhance the interaction between IL-33 and its receptor. This study builds upon their previous observation that HpARI2 binds DNA via its CCP1 domain. Here, the authors tested the CCP1 domain of HpARIs in binding heparan sulfate, an important component of the extracellular matrix, and found that 1/2 bind heparan, but 3 cannot, which is related to their half-lives in vivo.

Strengths:

The authors use a comprehensive multidisciplinary approach to assess the binding and their effects in vivo, coupled with molecular modeling.

Weaknesses:

(1) Figure 1C should include Western.

(2) Figure 1E: Why does HpARI1 stop binding DNA at 50%?

(3) ITC binding experiment with HpARI1?Also, the ITC results from HpARI2 do not seem to saturate, thus it is difficult to really determine the affinity.

(4) It would be helpful to add docking results from HpARI1.

(5) Some conclusions are speculative and need to remain in the Discussion. e.g.:<br /> a) That HpARI3 may be able to diffuse farther<br /> b) That DNA/HS may trap HpARI1/2 at the infection site.

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.

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.

Reviewer #2 (Public Review):

The paper by Willems aimed to uncover the neural implementations of threat omissions and showed that the VTA/SN activity was stronger following the omissions of more probable and intense shock stimulation, mimicking a reward PE signal.

My main concern remains regarding the interpretation of the task as a learning paradigm (extinction) or simply an expectation violation task (difference between instructed and experienced probability), though I appreciate some of the extra analyses in the responses to the reviewers. Looking at both the behavioral and neural data, a clear difference emerges among different US intensities and non-0% vs. 0% contrasts, however, the difference across probabilities was not clear in the figures, potentially partly due to the false instructions subjects received about the shock probabilities.

The lack of probability related PE demonstration, both in behavior and to a less extent in imaging data, does not fully support the PE axioms (0% and 100% are by themselves interesting categories since the instruction and experience matched well and therefore might need to be interpreted differently from other probabilities).

As the other reviewers pointed out, the application of instruction together with extinction paradigm complicates the interpretation of results. Also, the trial-by-trial analysis suggestion was responded by the probability x run interaction analysis, which still averaged over trials within each run to estimate a beta coefficient. So my evaluation remains that this is a valuable study to test PE axioms in the human reward and salience systems but authors need to be extremely careful with their wordings as to why this task is not a particularly learning paradigm (or the learning component did not affect their results, which was in conflict with the probability related SCR, pleasantness ratings as well as BOLD signals).

Reviewer #3 (Public Review):<br /> Summary:

Federer et al. describe the laminar profiles of GABA+ and of PV+ neurons in marmoset V1. They also report on the selectivity and efficiency of expression of a PV-selective enhancer (S5E2) across laminae. Three further viruses were tested, with a view to characterizing the expression profiles of a GABA-selective enhancer (h56d), but these results are preliminary.

Strengths:

The derivation of cell-type specific enhancers is key for translating the types of circuit analyses that can be performed in mice - which rely on germline modifications for access to cell-type specific manipulation - in higher-order mammals. Federer et al. further validate the utility of S5E2 as a PV-selective enhancer in NHPs.

Additionally, the authors characterize the laminar distribution pattern of GABA+ and PV+ cells in V1. This survey may prove valuable to researchers seeking to understand and manipulate the microcircuitry mediating the excitation-inhibition balance in this region of the marmoset brain.

Weaknesses:

Enhancer/promoter specificity and efficiency cannot be directly compared, because they were packaged in different serotypes of AAV.

The three different serotypes of AAV expressing reporter under the h56D promoter were only tested once each, and all in the same animal. There are many variables that can contribute to the success (or failure) of a viral injection, so observations with an n=1 cannot be considered reliable.

Added after revision:

The revisions satisfy my concerns. In particular, the new language to qualify the strength of evidence relating to the interpretation of the data relating to the 3 different serotypes of virus used to test h56D is appropriate.

Reviewer #2 (Public Review):

Summary:

The authors identified a new chloride-conducting Channelrhodopsin (MsACR1) that can be activated at low light intensities and within the red part of the visible spectrum. Additional engineering of MsACR1 yielded a variant (raACR1) with increased current amplitudes, accelerated kinetics, and a 20nm red-shifted peak excitation wavelength. Stimulation of MsACR1 and raACR1 expressing neurons with 635nm in mice's primary motor cortices inhibited the animals' locomotion.

Strengths:

The in vitro characterization of the newly identified ACRs is very detailed and confirms the biophysical properties as described by the authors. Notably, the ACRs are very light sensitive and allow for efficient in vitro inhibition of neurons in the nano Watt/mm^2 range. These new ACRs give neuroscientists and cell biologists a new tool to control chloride flux over biological membranes with high temporal and spatial precision. The red-shifted excitation peaks of these ACRs could allow for multiplexed application with blue-light excited optogenetic tools such as cation-conducting channelrhodopsins or green-fluorescent calcium indicators such as GCaMP.

Weaknesses:

(1) The in-vivo characterization of MsACR1 and raACR1 lacks critical control experiments and is, therefore, too preliminary. The experimental conditions differ fundamentally between in vitro and in vivo characterizations. For example, chloride gradients differ within neurons which can weaken inhibition or even cause excitation at synapses, as pointed out by the authors. Notably, the patch pipettes for the in vitro characterization in neurons contained low chloride concentrations that might not reflect possible conditions found in vivo preparations, i.e., increasing chloride gradients from dendrites to synapses.<br /> 2nd review: The authors have addressed this comment in their rebuttal.

(2) Interestingly, the authors used soma-targeted (st) MsACR1 and raACR1 for some of their in vitro characterization yielding more efficient inhibition and reduction of co-incidental "on-set" spiking. Still, the authors do not seem to have utilized st-variants in vivo.<br /> 2nd review: The authors offered an explanation in their rebuttal and aim to add these experiments later.

(3) Most importantly, critical in vivo control experiments, such as negative controls like GFP or positive controls like NpHR, are missing. These controls would exclude potential behavioral effects due to experimental artifacts. Moreover, in vivo electrophysiology could have confirmed whether targeted neurons were inhibited under optogenetic stimulations.<br /> Some of these concerns stem from the fact that the pulsed raACR stimulation at 635 nm at 10Hz (Fig. 3E) was far less efficient compared to MsACR1, yet the in vivo comparison yielded very similar results (Fig. 4D).<br /> Also, the cortex is highly heterogeneous and comprises excitatory and inhibitory neurons. Using the synapsin promoter, the viral expression paradigm could target both types and cause differential effects, which has not been investigated further, for example, by immunohistochemistry. An alternative expression system, for example, under VGLUT1 control, could have mitigated some of these concerns.<br /> 2nd review: The authors have not added control experiments in Fig 4 yet but plan to conduct them later. They added a new set of experiments in Fig.5 in which PV neurons were exclusively targeted. However, the authors show in vivo electrophysiology demonstrating the expected inhibition of firing neurons (presumably PV neurons expressing raACR) under 635 nm light and disinhibition (presumably excitatory neurons).

(4) Furthermore, the authors applied different light intensities, wavelengths, and stimulation frequencies during the in vitro characterization, causing varying spike inhibition efficiencies. The in vivo characterization is notably lacking this type of control. Thus, it is unclear why the 635nm, 2s at 20Hz every 5s stimulation protocol, which has no equivalent in the in vitro characterization, was chosen.<br /> 2nd review: The authors offered a satisfactory explanation.

In summary, the in vivo experiments did not confirm whether the observed inhibition of mouse locomotion occurred due to the inhibition of neurons or experimental artifacts.<br /> 2nd review: New experiments were added which demonstrate the expected inhibition of raACR expressing PV neurons in vivo.

In addition, the author's main claim of more efficient neuronal inhibition would require to threshold MsACR1 and raACR1 against alternative methods such as the red-shifted NpHR variant Jaws or other ACRs to give readers meaningful guidance when choosing an inhibitory tool.<br /> The light sensitivity of MsACR1 and raACR1 are impressive and well characterized in vitro. However, the authors only reported the overall light output at the fiber tip for the in vivo experiments: 0.5 mW. Without context, it is difficult to evaluate this value. Calculating the light power density at certain distances from the light fiber or thresholding against alternative tools such as NpHR, Jaws, or other ACRs would allow for a more meaningful evaluation.<br /> 2nd review: The authors added Supl Fig. 8 in which light power density and propagation of photons at 550 nm and 635nm through brain tissue was calculated when emitted from light fibers of varying diameter.

Reviewer #2 (Public Review):

Summary:<br /> The authors aim at challenging the relevance of cell populations with characteristic selectivity for specific aspects of navigation (e.g. place cells, head direction and border cells) in the processing of spatial information. Their claim is that such cells naturally emerge in any system dealing with the estimation of position in an environment, without the need for a special involvement of these cells in the computations. In particular the work shows how when provided with spatial error signals, networks designed for invariant object recognition spontaneously organize the activity in their hidden layers into a mixture of spatially selective cells, some of them passing classification criteria for place, head direction or border cells. Crucially, these cells are not necessary for position decoding, nor are they the most informative when it comes to the performance of the network in reconstructing spatial position from visual scenes. These results lead the authors to claim that focusing on the classification of specific cell types is hindering rather than helping advancement in the understanding of spatial cognition. In fact they claim that the attention should rather be pointed at understanding highly-dimensional population coding, regardless of its direct interpretability or its appeal to human observers.

Strengths:<br /> Methodologically the paper is consistent and convincingly support the author claims regarding the role of cell types in coding for spatial aspects of cognition. It is also interesting how the authors leverage on established machine learning systems to provide a sort of counter-argument to the use of such techniques to establish a parallel between artificial and biological neural representations. In the recent past similar applications of artificial neural networks to spatial navigation have been directed at proving the importance of specific neural substrates (take for example Banino et al. 2018 for grid cells), while in this case the same procedure is used to unveil them as epiphenomena, so general and unspecific to be of very limited use in understanding the actual functioning of the neural system. I am quite confident that this stance regarding the role of place cells and co. could gather large sympathy and support in the greater part of the neuroscience community, or at least among the majority of theoretical neuroscientists with some interest in the hippocampus and higher cognition.

Weaknesses:<br /> My criticism of the paper can be articulated in three main points:<br /> - What about grid cells? Grid cells are notably not showing up in the analyses of the paper. But they surely can be considered as the 'mother' of all tailored spatial cells of the hippocampal formation. Are they falling outside the author's assessment of the importance of this kind of cells? Some discussion of the place grid cells occupy in the vision of the authors would greatly help.<br /> - The network used in the paper is still guided by a spatial error signal, and the network is trained to minimize spatial decoding error. In a sense, although object classfication networks are not designed for spatial navigation, one could say that the authors are in some way hacking this architecture and turning it into a spatial navigation one through learning. I wonder if their case could be strengthened by devising a version of their experiment based on some form of self-supervised or unsupervised learning.<br /> - The last point is more about my perception of the community studying hippocampal functions, rather than being directed at the merits of the paper itself. My question is whether the paper is fighting an already won battle. That is whether the focus on the minute classification of response profiles of cells in the hippocampus is in fact already considered an 'old' approach, very useful for some initial qualitative assessments but of limited power when asked to provide deeper insight into the functioning of hippocampal computations (or computations of any other brain circuit).

Reviewer #2 (Public Review):

Summary:<br /> Members of a conserved family of flavin-containing monooxygenases (FMOs) are necessary and at least partly sufficient for lifespan extension induced by diet restriction and hypoxia. Of 5 FMOs in C. elegans, fmo-2 has received the majority of attention, but this study identifies that fmo-4 is also an important, positive modulator of lifespan. Based on differential requirements of fmo-2 and fmo-4 in stress resistance and lifespan extension paradigms, the authors conclude that fmo-4 acts through mechanisms that are overlapping, but distinct from fmo-2. Ultimately, the authors place fmo-2 genetically within a pathway involving atf-6, calreticulin, the IP3 receptor, and mitochondrial calcium uniporter, which was previously shown to link ER calcium homeostasis to mitochondrial homeostasis and longevity. Because the known enzymatic activity of FMOs involves oxygenating xenobiotic and endogenous metabolites, these findings highlight a potential new link between redox/metabolic homeostasis and ER-mitochondrial calcium signaling, while revealing that different FMO family members regulate stress resistance and lifespan through distinct mechanisms.

Strengths:<br /> The authors have used genetics to discover an interesting and unanticipated new link between conserved FMOs and ER calcium pathways known to regulate lifespan.

The genetic epistasis patterns for lifespan and stress resistance phenotypes are generally clean and compelling.

Weaknesses:<br /> The effects of carbachol and EDTA on intracellular calcium levels are inferred, especially in the tissues where fmo-4 is acting. Validating that these agents and fmo-4 itself have an impact on calcium in relevant subcellular compartments is important to support conclusions on how fmo-4 regulates and responds to calcium.

Experiments are generally reliant on RNAi. While in most cases experiments reveal positive results, indicating RNAi efficacy, key conclusions could be strengthened with the incorporation of mutants.

While FMO-4 is clearly placed in the ER calcium pathway genetically, a putative molecular mechanism by which FMO-4 would alter ER calcium remains unclear. Notably, Tuckowski et al. highlight this gap in the discussion as well.

Reviewer #2 (Public Review):

(1) Summary<br /> Kleinman and Foster's study investigates the role of dopamine signaling in the ventral tegmental area (VTA) on hippocampal replay and sharp-wave ripples (SWR) in rats exposed to changes in reward magnitude and environmental novelty. The authors utilize chemogenetic silencing techniques to modulate dopamine neuron activity in the VTA while conducting simultaneous electrophysiological recordings from the hippocampal CA1 region. Their findings suggest that VTA dopamine signaling is critical for modulating hippocampal replay in response to changes in reward context and novelty, with specific disruptions observed in replay dynamics when VTA is inhibited, particularly in novel environments.

(2) Strengths<br /> The research addresses a significant gap in our understanding of the neurobiological underpinnings of memory and spatial learning, highlighting the importance of dopamine-mediated processes. The methodological approach is robust, combining chemogenetic silencing with precise electrophysiological measurements, which allows for a detailed examination of the neural circuits involved. The study provides important insights into how hippocampal replay and SWR are influenced by reward prediction errors, as well as the role of dopamine in these processes. Specifically, the authors note that VTA silencing unexpectedly did not prevent increases in ripple activities where reward was increased, but induced significant aberrant increases in environments where reward levels were unchanged, highlighting a novel dependency of hippocampal replay on dopamine and a VTA-independent reward prediction error signal in familiar environments. These findings are critical for understanding the consolidation of episodic memory and the neural basis of learning.

(3) Weaknesses<br /> Despite the strengths in methodology and conceptual framework, the study has several weaknesses that could affect the interpretation of the results. There is a need for more rigorous histological validation to confirm the extent and specificity of viral expression and electrode placements, which is crucial for ensuring the accuracy of the findings. Variability in the dosing and timing of chemogenetic interventions could also lead to inconsistencies in the data, suggesting a need for more standardized experimental protocols.

Reviewer #2 (Public Review):

Summary:

In this work, Degutis and colleagues addressed an interesting issue related to the concurrent coding of sensory percepts and visual working memory contents in visual cortices. They used generalization analyses to test whether working memory representations change over time, diverge from sensory percepts, and vary across distraction conditions. Temporal generalization analysis demonstrated that off-diagonal decoding accuracies were lower than on-diagonal decoding accuracies, regardless of the presence of intervening distractions, implying that working memory representations can change over time. They further showed that the coding space for working memory contents showed subtle but statistically significant changes over time, potentially explaining the impaired off-diagonal decoding performance. The neural coding of sensory distractions instead remained largely stable. Generalization analyses between target and distractor codes showed overlaps but were not identical. Cross-condition decodings had lower accuracies compared to within-condition decodings. Finally, within-condition decoding revealed more reliable working memory representations in the condition with intervening random noises compared to cross-condition decoding using a trained classifier on data from the no-distraction condition, indicating a change in the VWM format between the noise distractor and no-distractor trials.

Strengths:

This paper demonstrates a clever use of generalization analysis to show changes in the neural codes of working memory contents across time and distraction conditions. It provides some insights into the differences between representations of working memory and sensory percepts, and how they can potentially coexist in overlapping brain regions.

Weaknesses:

(1) An alternative interpretation of the temporal dynamic pattern is that working memory representations become less reliable over time. As shown by the authors in Figure 1c and Figure 4a, the on-diagonal decoding accuracy generally decreased over time. This implies that the signal-to-noise ratio was decreasing over time. Classifiers trained with data of relatively higher SNR and lower SNR may rely on different features, leading to poor generalization performance. This issue should be addressed in the paper.

(2) The paper tests against a strong version of stable coding, where neural spaces representing WM contents must remain identical over time. In this version, any changes in the neural space will be evidence of dynamic coding. As the paper acknowledges, there is already ample evidence arguing against this possibility. However, the evidence provided here (dynamic coding cluster, angle between coding spaces) is not as strong as what prior studies have shown for meaningful transformations in neural coding. For instance, the principal angle between coding spaces over time was smaller than 8 degrees, and around 7 degrees between sensory distractors and WM contents. This suggests that the coding space for WM was largely overlapping across time and with that for sensory distractors. Therefore, the major conclusion that working memory contents are dynamically coded is not well-supported by the presented results.

(3) Relatedly, the main conclusions, such as "VWM code in several visual regions did not generalize well between different time points" and "VWM and feature-matching sensory distractors are encoded in separable coding spaces" are somewhat subjective given that cross-condition generalization analyses consistently showed above chance-level performance. These results could be interpreted as evidence of stable coding. The authors should use more objective descriptions, such as 'temporal generalization decoding showed reduced decoding accuracy in off-diagonals compared to on-diagonals.

Reviewer #2 (Public Review):

This study provides an in-depth exploration of the impact of X-linked ZDHHC9 gene mutations on cognitive deficits and epilepsy, with a particular focus on the expression and function of ZDHHC9 in myelin-forming oligodendrocytes (OLs). These findings offer crucial insights into understanding ZDHHC9-related X-linked intellectual disability (XLID) and shed light on the regulatory mechanisms of palmitoylation in myelination. The experimental design and analysis of results are convincing, providing a valuable reference for further research in this field. However, upon careful review, I believe the article still needs further improvement and supplementation in the following aspects:

(1) Regarding the subcellular localization experiment of ZDHHC9 mutants in OL, it is currently limited to in vitro cultured OL, lacking validation in vivo OL or myelin sheath. Additionally, it is necessary to investigate whether the abnormal subcellular localization of ZDHHC9 mutants affects their enzyme activity and palmitoylation modification of substrate proteins.

(2) The experimental period (P21+21 days) using genetic labeling to track the development of myelinating cells may not be long enough. It is recommended to extend the observation time and analyze at more time points to more comprehensively reflect the impact of Zdhhc9 KO.

(3) The author speculates that Zdhhc9 may regulate myelination by affecting the membrane localization of specific myelin proteins, but lacks direct experimental evidence to support this. It is suggested to detect the expression and distribution of relevant proteins in the myelin of Zdhhc9 KO mice.

(4) Although the article mentions the association of Zdhhc9 with intellectual disabilities, it does not involve behavioral analysis of Zdhhc9 KO mice. It is recommended to supplement some behavioral experimental data to support the important role of Zdhhc9 in maintaining normal cognitive function, enhancing the clinical relevance of the article.

(5) For the abnormal myelination observed in Zdhhc9 KO mice, including unmyelinated large-diameter axons and excessively myelinated small-diameter axons, the article lacks in-depth research and explanation on the exact mechanism and mode of action of ZDHHC9 in regulating myelination.

(6) The function of ZDHHC9 in OL may be related to the Golgi apparatus, but its exact role in these structures is still unclear. It is suggested to discuss in more detail the role of ZDHHC9 in the Golgi apparatus in the discussion section.

(7) More experimental support and in-depth research are needed on the detailed mechanism of how ZDHHC9 and Golga7 cooperatively regulate MBP palmitoylation, and how this decrease in palmitoylation level leads to myelination defects.

In summary, it is recommended that the authors address the above issues through additional experiments and improved discussions to further strengthen the credibility and clinical relevance of the article.

Reviewer #2 (Public Review):

Summary:

The current work investigates the neural signature of category representation in infancy. Neural responses during steady-state visually-evoked potentials (ssVEPs) were recorded in four age groups of infants between 3 and 15 months. Stimuli (i.e., faces, limbs, corridors, characters, and cars) were presented at 4.286 Hz with category changes occurring at a frequency of 0.857 Hz. The results of the category frequency analyses showed that reliable responses to faces emerge around 4-6 months, whereas responses to libs, corridors, and characters emerge at around 6-8 months. Additionally, the authors trained a classifier for each category to assess how consistent the responses were across participants (leave-one-out approach). Spatiotemporal responses to faces were more consistent than the responses to the remaining categories and increased with increasing age. Faces showed an advantage over other categories in two additional measures (i.e., representation similarity and distinctiveness). Together, these results suggest a different developmental timing of category representation.

Strengths:

The study design is well organized. The authors described and performed analyses on several measures of neural categorization, including innovative approaches to assess the organization of neural responses. Results are in support of one of the two main hypotheses on the development of category representation described in the introduction. Specifically, the results suggest a different timing in the formation of category representations, with earlier and more robust responses emerging for faces over the remaining categories. Graphic representations and figures are very useful when reading the results.

Weaknesses:

The role of the adult dataset in the goal of the current work is unclear. All results are reported in the supplementary materials and minimally discussed in the main text. The unique contribution of the results of the adult samples is unclear and may be superfluous.

It would be useful to report the electrodes included in the analyses and how they have been selected.

Reviewer #2 (Public Review):

Summary:

This study aimed to test experimentally a theoretical framework that aims to explain the perception of tinnitus, i.e., the perception of a phantom sound in the absence of external stimuli, through differences in auditory predictive coding patterns. To this aim, the researchers compared the neural activity preceding and following the perception of a sound using MEG in two different studies. The sounds could be highly predictable or random, depending on the experimental condition. They revealed that individuals with tinnitus and controls had different anticipatory predictions. This finding is a major step in characterizing the top-down mechanisms underlying sound perception in individuals with tinnitus.

Strengths:

This article uses an elegant, well-constructed paradigm to assess the neural dynamics underlying auditory prediction. The findings presented in the first experiment were partially replicated in the second experiment, which included 80 participants. This large number of participants for an MEG study ensures very good statistical power and a strong level of evidence. The authors used advanced analysis techniques - Multivariate Pattern Analysis (MVPA) and classifier weights projection - to determine the neural patterns underlying the anticipation and perception of a sound for individuals with or without tinnitus. The authors evidenced different auditory prediction patterns associated with tinnitus. Overall, the conclusions of this paper are well supported, and the limitations of the study are clearly addressed and discussed.

Weaknesses:

Even though the authors took care of matching the participants in age and sex, the control could be more precise. Tinnitus is associated with various comorbidities, such as hearing loss, anxiety, depression, or sleep disorders. The authors assessed individuals' hearing thresholds with a pure tone audiogram, but they did not take into account the high frequencies (6 kHz to 16 kHz) in the patient/control matching. Moreover, other hearing dysfunctions, such as speech-in-noise deficits or hyperacusis, could have been taken into account to reinforce their claim that the observed predictive pattern was not linked to hearing deficits. Mental health and sleep disorders could also have been considered more precisely, as they were accounted for only indirectly with the score of the 10-item mini-TQ questionnaire evaluating tinnitus distress. Lastly, testing the links between the individuals' scores in auditory prediction and tinnitus characteristics, such as pitch, loudness, duration, and occurrence (how often it is perceived during the day), would have been highly informative.

Reviewer #2 (Public Review):

Summary:

Here the authors show a novel direct neuronal reprogramming model using a very pure culture system of oligodendrocyte progenitor cells and demonstrate hallmarks of corticospinal neurons to be induced when using Neurogenin2, a dominant-negative form of Olig2 in combination with the CSN master regulator Fezf2.

Strengths:

This is a major achievement as the specification of reprogrammed neurons towards adequate neuronal subtypes is crucial for repair and still largely missing. The work is carefully done and the comparison of the neurons induced only by Neurogenin 2 versus the NVOF cocktail is very interesting and convincingly demonstrates a further subtype specification by the cocktail.

Weaknesses:

As carefully as it is done in vitro, the identity of projection neurons can best be assessed in vivo. If this is not possible, it could be interesting to co-culture different brain regions and see if these neurons reprogrammed with the cocktail, indeed preferentially send out axons to innervate a co-cultured spinal cord versus other brain region tissue.

Reviewer #2 (Public Review):

Summary:

Zhang and colleagues use a combination of behavioral, neural, and computational analyses to test an active inference model of exploration in a novel reinforcement learning task.

Strengths:

The paper addresses an important question (validation of active inference models of exploration). The combination of behavior, neuroimaging, and modeling is potentially powerful for answering this question.

I appreciate the addition of details about model fitting, comparison, and recovery, as well as the change in some of the methods.

Weaknesses:

The authors do not cite what is probably the most relevant contextual bandit study, by Collins & Frank (2018, PNAS), which uses EEG.

The authors cite Collins & Molinaro as a form of contextual bandit, but that's not the case (what they call "context" is just the choice set). They should look at the earlier work from Collins, starting with Collins & Frank (2012, EJN).

Placing statistical information in a GitHub repository is not appropriate. This needs to be in the main text of the paper. I don't understand why the authors refer to space limitations; there are none for eLife, as far as I'm aware.

In answer to my question about multiple comparisons, the authors have added the following: "Note that we did not attempt to correct for multiple comparisons; largely, because the correlations observed were sustained over considerable time periods, which would be almost impossible under the null hypothesis of no correlations." I'm sorry, but this does not make sense. Either the authors are doing multiple comparisons, in which case multiple comparison correction is relevant, or they are doing a single test on the extended timeseries, in which case they need to report that. There exist tools for this kind of analysis (e.g., Gershman et al., 2014, NeuroImage). I'm not suggesting that the authors should necessarily do this, only that their statistical approach should be coherent. As a reference point, the authors might look at the aforementioned Collins & Frank (2018) study.

I asked the authors to show more descriptive comparison between the model and the data. Their response was that this is not possible, which I find odd given that they are able to use the model to define a probability distribution on choices. All I'm asking about here is to show predictive checks which build confidence in the model fit. The additional simulations do not address this. The authors refer to figures 3 and 4, but these do not show any direct comparison between human data and the model beyond model comparison metrics.

Reviewer #2 (Public Review):

Summary:

In this interesting and original study, Feng and colleagues set out to address the effect of manipulating endothelin signaling on nerve regeneration, focusing on the crosstalk between endothelial cells (ECs) in dorsal root ganglia (DRG), which secrete ET-1 and satellite glial cells (SGCs) expressing ETBR receptor. The main finding is that ETBR signaling is a default brake on axon growth, and inhibiting this pathway promotes axon regeneration after nerve injury and counters the decline in regenerative capacity that occurs during aging. ET-1 and ETBR are mapped in ECs and SGCs, respectively, using scRNA-seq of DRGs from adult or aged mice. Although their expression does not change upon injury, it is modulated during aging, with a reported increase in plasma levels of ET-1 (a potent vasoconstrictive signal). Using in vitro explant assays coupled with pharmacological inhibition in mouse models of nerve injury, the authors demonstrate that ET-1/ETBR curbs axonal growth, and the ETAR/ETBR antagonist Bosentan boosts regrowth during the early phase of repair. In addition, Bosentan restores the ability of aged DRG neurons to regrow after nerve lesions. Despite Bosentan inhibiting both endothelin receptors A and B, comparison with an ETAR-specific antagonist indicates that the effects can be attributed to the ET-1/ETBR pathway. In the DRGs, ETBR is mostly expressed by SGCs (and a subset of Schwann cells) a cell type that previous studies, including work from this group, have implicated in nerve regeneration. SGCs ensheath and couple with DRG neurons through gap junctions formed by Cx43. Based on their own findings and evidence from the literature, the pro-regenerative effects of ETBR inhibition are in part attributed to an increase in Cx43 levels, which are expected to enhance neuron-SGC coupling. Finally, gene expression analysis in adult vs aged DRGs predicts a decrease in fatty acid and cholesterol metabolism, for which previous work by the authors has shown a requirement in SGCs to promote axon regeneration.

Strengths:

The study is well-executed and the main conclusion that "ETBR signaling inhibits axon regeneration after nerve injury and plays a role in age-related decline in regenerative capacity" (line 77) is supported by the data. Given that Bosentan is an FDA-approved drug, the findings may have therapeutic value in clinical settings where peripheral nerve regeneration is suboptimal or largely impaired, as it often happens in aged individuals. In addition, the study highlights the importance of vascular signals in nerve regeneration, a topic that has gained traction in recent years. Importantly, these results further emphasize the contribution of long-neglected SGCs to nerve tissue homeostasis and repair. Although the study does not reach a complete mechanistic understanding, the results are robust and are expected to attract the interest of a broader readership.

Weaknesses:

Despite these positive comments provided above, the following points should be considered:

(1) This study examines the contribution of the ET-1 pathway in the ganglia, and in vitro assays are consistent with the idea that important signaling events take place there. Nevertheless, it remains to be determined whether the accelerated axon regrowth observed in vivo depends also on cellular crosstalk mediated by ET-1 at the lesion site. Are ECs along the nerve secreting ET-1? What cells are present in the nerve stroma that could respond and participate in the repair process? Would these interactions be sensitive to Bosentan? It may be difficult to dissect this contribution, but it should at least be discussed.

(2) It is suggested that the permeability of DRG vessels may facilitate the release of "vascular-derived signals" (lines 82-84). Is it possible that the ET-1/ETBR pathway modulates vascular permeability, and that this, in turn, contributes to the observed effects on regeneration?

(3) Is the affinity of ET-3 for ETBR similar to that of ET-1? Can it be excluded that ET-3 expressed by fibroblasts is relevant for controlling SGC responses upon injury/aging?

(4) ETBR inhibition in dissociated (mixed) cultures uncovers the restraining activity of endothelin signaling on axon growth (Figure 2C). Since neurons do not express ET-1 receptors, based on scRNA-seq analysis, these results are interpreted as an indication that basal ETBR signaling in SGC curbs the axon growth potential of sensory neurons. For this to occur in dissociated cultures, however, one should assume that SGC-neuron association is present, similar to in vivo, or to whole DRG cultures (Figure 2C). Has this been tested? In both in vitro experimental settings (dissociated and whole DRG cultures) how is ETBR stimulated over up to 7 days of culture? In other words, where does endothelin come from in these cultures (which are unlikely to support EC/blood vessel growth)? Is it possible that the relevant ligand here derives from fibroblasts (see point #6)? Or does it suggest that ETBR can be constitutively active (i.e., endothelin-independent signaling)? Is there any chance that endothelin is present in the culture media or Matrigel?

(5) The discovery that ET-1/ETBR signaling in SGC curtails the growth capacity of axons at baseline raises questions about the physiological role of this pathway. What happens when ETBR signaling is prevented over a longer period of time? This could be addressed with pharmacological inhibitors, or better, with cell-specific knock-out mice. The experiments would certainly be of general interest, although not within the scope of this story. Nevertheless, it could be worth discussing the possibilities.

(6) Assessing Cx43 levels by measuring the immunofluorescence signal (Figure 5E-F) is acceptable, particularly when the aim is to restrict the analysis to SGCs. The modulation of Cx43 expression by ET-1/ETBR plays an important part in the proposed model. Therefore, a complementary analysis of Cx43 expression by quantitative RT-PCR on sorted SGCs would be a valuable addition to the immunofluorescence data. Is this attainable?

(7) The conclusions "We thus hypothesize that ETBR inhibition in SGCs contributes to axonal regeneration by increasing Cx43 levels, gap junction coupling or hemichannels and facilitating SGC-neuron communication" (lines 303-305) are consistent with the findings but seem in contrast with the effect of aging on gap junction coupling reported by others and cited in line 210: "the number of gap junctions and the dye coupling between these cells increases (Huang et al., 2006)". I am confused by what distinguishes a potential, and supposedly beneficial, increase in coupling after ETBR inhibition, from what is observed in aging.

(8) I find it difficult to reconcile the results in Figure 5F with the proposed model since (1) injury increases Cx43 levels in both adult and aged mice, (2) the injured aged/vehicle group has a similar level to the uninjured adult group, (3) upon injury, aged+Bosentan is much lower than adult+Bosentan (significance not tested). It seems hard to explain the effect of Bosentan only through the modulation of Cx43 levels. Whether the increase in Cx43 levels following ETBR inhibition actually results in higher SGC-neuron coupling has not been assessed experimentally.

Reviewer #2 (Public Review):

Summary:

The authors demonstrate heightened susceptibility of Terc-KO mice to S. aureus-induced pneumonia, perform gene expression analysis from the infected lungs, find an elevated inflammatory (NLRP3) signature in some Terc-KO but not control mice, and some reduction in T cell signatures. Based on that, They conclude that disregulated inflammation and T-cell dysfunction play a major role in these phenomena.

Strengths:

The strengths of the work include a problem not previously addressed (the role of the Terc component of the telomerase complex) in certain aspects of resistance to bacterial infection and innate (and maybe adaptive) immune function.

Weaknesses:

The weaknesses outweigh the strengths, dominantly because conclusions are plagued by flaws in experimental design, by lack of rigorous controls, and by incomplete and inadequate approaches to testing immune function. These weaknesses are as follows

(1) Terc-KO mice are a genomic knockout model, and therefore the authors need to carefully consider the impact of this KO on a wide range of tissues. This, however, is not the case. There are no attempts to perform cell transfers or use irradiation chimera or crosses that would be informative.

(2) Throughout the manuscript the authors invoke the role of telomere shortening in aging, and according to them, their Terc-KO mice should be one potential model for aging. Yet the authors consistently describe major differences between young Terc-KO and naturally aging old mice, with no discussion of the implications. This further confuses the biological significance of this work as presented.

(3) Related to #2, group design for comparisons lacks a clear rationale. The authors stipulate that Terc-KO will mimic natural aging, but in fact, the only significant differences seen between groups in susceptibility to S. aureus are, contrary to the authors' expectation, between young Terc-KO and naturally old mice (Figures 1A and B, no difference between young Terc-KO and young wt); or there are no significant differences at all between groups (Figures 1, C, D,).<br /> Another example of inadequate group design is when the authors begin dividing their Terc-KO groups by clinical score into animals with or without "systemic infection" (the condition where a bacterium spreads uncontrollably across the many organs and via blood, which should be properly called sepsis), and then compare this sepsis group to other groups (Supplementary Figures 1G; Figure 2; lines 374-376 and 389-391). This gives them significant differences in several figures, but because they did not clearly indicate where they applied this stratification in the figure legends, the data are somewhat confusing. Most importantly, methodologically it is highly inappropriate to compare one mouse with sepsis to another one without. If Terc-KO mice with sepsis are a comparator group, then their controls have to be wild-type mice with sepsis, who are dealing with the same high bacterial load across the body and are presumably forced to deploy the same set of immune defenses.

(4) The authors conclude that disregulated inflammation and T-cell dysfunction play a major role in S. aureus susceptibility. This may or may not be an important observation, because many KO mice are abnormal for a variety of reasons, and until such reasons are mechanistically dissected, the physiological importance of the observation will remain unclear. Two points are important here. First, there is no natural counterpart to a Terc-KO, which is a complete loss of a key non-enzymatic component of the telomerase complex starting in utero. Second, the authors truly did not examine the key basic features of their model, including the features of basic and induced inflammatory and immune responses. This analysis could be done either using model antigens in adjuvants, defined innate immune stimuli (e.g. TLR, RLR, or NLR agonists), or microbial challenge. The only data provided along these lines are the baseline frequencies of total T cells in the spleen of the three groups of mice examined (not statistically significant, Figure 4B). We do not know if the composition of naïve to memory T cell subsets may have been different, and more importantly, we have no data to evaluate whether recruitment of the immune response (including T cells) to the lung upon microbial challenge is similar or different. So, what are the numbers and percentages of T cells and alveolar macrophages in the lung following S. aureus challenge and are they even comparable or are there issues in mobilizing the T cell response to the site of infection? If, for example, Terc-KO mice do not mobilize enough T cells to the lung during infection, that would explain the paucity in many T-cell-associated genes in their transcriptomic set that the authors report. That in turn may not mean dysfunction of T cells but potentially a whole different set of defects in coordinating the response in Terc-KO mice.

(5) Related to that, immunological analysis is also inadequate. First, the authors pull signatures from the total lung tissue, which is both imprecise and potentially skewed by differences, not in gene expression but in types of cells present and/or their abundance, a feature known to be affected by aging and perhaps by Terc deficiency during infection. Second, to draw any conclusions about immune responses, the authors would have to track antigen-specific T cells, which is possible for a wide range of microbial pathogens using peptide-MHC multimers. This would allow highly precise analysis of phenomena the authors are trying to conclude about. Moreover, it would allow them to confirm their gene expression data in populations of physiological interest

Third, the authors co-incubate AM and T cells with S. aureus. There is no information here about the phenotype of T cells used. Were they naïve, and how many S. aureus-specific T cells did they contain? Or were they a mix of different cell types, which we know will change with aging (fewer naïve and many more memory cells of different flavors), and maybe even with a Terc-KO? Naïve T cells do not interact with AM; only effector and memory cells would be able to do so, once they have been primed by contact with dendritic cells bringing antigen into the lymphoid tissues, so it is unclear what the authors are modeling here. Mature primed effector T cells would go to the lung and would interact with AM, but it is almost certain that the authors did not generate these cells for their experiment (or at least nothing like that was described in the methods or the text).

(6) Overall, the authors began to address the role of Terc in bacterial susceptibility, but to what extent that specifically involves inflammation and macrophages, T cell immunity, or aging remains unclear at present.

Reviewer #2 (Public Review):

Chambers et al. (2024) present a systematic and unbiased approach to explore the evolutionary potential of the human antiviral protein kinase R (PKR) to evade inhibition by a poxviral antagonist while maintaining one of its essential functions.

The authors generated a library of 426 single-nucleotide polymorphism (SNP)-accessible non-synonymous variants of PKR kinase domain and used a yeast-based heterologous virus-host system to assess PKR variants' ability to escape antagonism by the vaccinia virus pseudo-substrate inhibitor K3. The study identified determinant sites in the PKR kinase domain that harbor K3-resistant variants, as well as sites where variation leads to PKR loss of function. The authors found that multiple K3-resistant variants are readily available throughout the domain interface and are enriched at sites under positive selection. They further found some evidence of PKR resilience to viral antagonist diversification. These findings highlight the remarkable adaptability of PKR in response to viral antagonism by mimicry.

Significance of the findings:

The findings are important with implications for various fields, including evolutionary biology, virus-host interfaces, genetic conflicts, and antiviral immunity.

Strength of the evidence:

Convincing methodology using state-of-the-art mutational scanning approach in an elegant and simple setup to address important challenges in virus-host molecular conflicts and protein adaptations.

Strengths:

● Systematic and Unbiased Approach:<br /> The study's comprehensive approach to generating and characterizing a large library of PKR variants provides valuable insights into the evolutionary landscape of the PKR kinase domain. By focusing on SNP-accessible variants, the authors ensure the relevance of their findings to naturally occurring mutations.

● Identification of Key Sites:<br /> The identification of specific sites in the PKR kinase domain that confer resistance or susceptibility to a poxvirus pseudosubstrate inhibition is a significant contribution.

● Evolutionary Implications:<br /> The authors performed meticulous comparative analyses throughout the study between the functional variants from their mutagenesis screen ("prospective") and the evolutionarily-relevant past adaptations ("retrospective").

● Experimental Design:<br /> The use of a yeast-based assay to simultaneously assess PKR capacity to induce cell growth arrest and susceptibility/resistance to various VACV K3 alleles is an efficient approach. The combination of this assay with high-throughput sequencing allows for the rapid characterization of a large number of PKR variants.

Areas for Improvement:

● Validation of the screen:<br /> The results would be strengthened by validating results from the screen on a handful of candidate PKR variants, either using a similar yeast heterologous assay, or - even more powerfully - in another experimental system assaying for similar function (cell translation arrest) or protein-protein interaction.

● Evolutionary Data:<br /> Beyond residues under positive selection, the screen would allow the authors to also perform a comparative analysis with PKR residues under purifying selection. Because they are assessing one of the most conserved ancestral functions of PKR (i.e. cell translation arrest), it may also be of interest to discuss these highly conserved sites.

● Mechanistic Insights:<br /> While the study identifies key sites and residues involved in vaccinia K3 resistance, it could benefit from further investigation into the underlying molecular mechanisms. The study's reliance on a single experimental approach, deep mutational scanning, may introduce biases and limit the scope of the findings. The authors may acknowledge these limitations in the Discussion.

● Viral Diversity:<br /> The study focuses on the viral inhibitor K3 from vaccinia. Expanding the analysis to include other viral inhibitors, or exploring the effects of PKR variants on a range of viruses would strengthen and expand the study's conclusions. Would the identified VACV K3-resistant variants also be effective against other viral inhibitors (from pox or other viruses)? or in the context of infection with different viruses? Without such evidence, the authors may check the manuscript is specific about the conclusions.

Overall Assessment:

The systematic approach, identification of key sites, and evolutionary implications are all notable strengths. While there is room for further investigation into the mechanistic details and broader viral diversity, the findings are robust and already provide important advancements. The manuscript is well-written and clear, and the figures are informative. Specific minor comments are further shared below.

Minor revisions addressing the areas for improvement mentioned above are recommended.

Reviewer #2 (Public Review):

Wang et al. investigated the role of dynein axonemal heavy chain 3 (DNAH3) in male infertility. They found that variants of DNAH3 were present in four infertile men, and the deficiency of DNAH3 in sperm affects sperm mobility. Additionally, they showed that Dnah3 knockout male mice are infertile. Furthermore, they demonstrated that DNAH3 influences inner dynein arms by regulating several DNAH proteins. Importantly, they showed that intracytoplasmic sperm injection (ICSI) can rescue the infertility in Dnah3 knockout mice and two patients with DNAH3 variants.

Strengths:

The conclusions of this paper are well-supported by data.

Weaknesses:

The sample/patient size is small; however, the findings are consistent with those of a recent study on DNAH3 in male infertility involving 432 patients.

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, 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. The biggest drawback is that it does not provide evidence for the idea that a match between chronotype and delay matters is especially relevant for people with depression or continuous measures like anhedonia and apathy. It is unclear whether disorders further interact with chronotype and time of day to determine a bias against effort. On the other hand, the study does provide evidence that future studies should consider such interactions when examining questions about effort expenditure in psychiatric disorders.

Reviewer #2 (Public Review):

Summary:

The authors describe a form of synaptic plasticity at synapses from granule cells onto Purkinje cells in the mouse cerebellum, which is specific to synapses from granule cells close to the cell body but not to distal ones. This plasticity is induced by the paired or associative stimulation of the two types of synapses because it is not observed with stimulation of one type of synapse alone. In addition, this form of plasticity is dependent on the order in which the stimuli are presented and is dependent on NMDA receptors, metabotropic glutamate receptors and to some degree on GABAA receptors.

Strengths:

The focus of the authors on the properties of two different synapse-types on cerebellar Purkinje cells is interesting and relevant, given previous results that ascending and parallel fiber synapses might be functionally different and undergo different forms of plasticity (although it hasn't been proven here that the two types of synapses are indeed ascending vs parallel fiber synapses). Nevertheless, the interaction between proximal vs. distal stimulation driven synapse types during plasticity is important for understanding cerebellar function. The demonstration of timing and order-dependent potentiation of only one pathway, and not another, after associative stimulation of both pathways, changes our understanding of potential plasticity mechanisms. In addition, this observation opens up many new questions on underlying intracellular mechanisms as well as on its relevance for cerebellar learning.

Weaknesses:

A concern with this study is that all recordings demonstrate "rundown", a progressive decrease in the amplitude of the EPSC, starting during the baseline period and continuing after the plasticity-induction stimulus. The issues that are causing rundown are not known and may or may not be related to the cellular processes involved in synaptic plasticity. This concern applies in particular to all the experiments where there is a decrease in synaptic strength. However, a key finding of this paper is the associative potentiation of one pathway, which is clearly different from all conditions where there is a decrease in synaptic strength and raises confidence in the authors' conclusions.

In addition, there is some inconsistency with previous results; specifically, that no PF-LTP was induced by PF-alone repeated stimulation.

It remains for future work to identify what these two synapse types, distinguished by the stimulation location, actually are, and where they are on the Purkinje cell dendritic tree. What this specific timing rule is important for is also something that remains to be discovered. Its potential relevance for plasticity and learning will depend on what information these AA vs PF synapses carry, and why their association is meaningful for the circuit and for a behavior. Overall, this study opens up many new questions for the field.

Reviewer #2 (Public Review):

Summary:

The authors used 2-photon Ca2+-imaging to study the activity of ventral tegmental area (VTA) and locus coeruleus (LC) axons in the CA1 region of the dorsal hippocampus in head-fixed male mice moving on linear paths in virtual reality (VR) environments.

The main findings were as follows:<br /> - In a familiar environment, activity of both VTA axons and LC axons increased with the mice's running speed on the Styrofoam wheel, with which they could move along a linear track through a VR environment.<br /> - VTA, but not LC, axons showed marked reward position-related activity, showing a ramping-up of activity when mice approached a learned reward position.<br /> - In contrast, activity of LC axons ramped up before initiation of movement on the Styrofoam wheel.<br /> - In addition, exposure to a novel VR environment increased LC axon activity, but not VTA axon activity.

Overall, the study shows that the activity of catecholaminergic axons from VTA and LC to dorsal hippocampal CA1 can partly reflect distinct environmental, behavioral and cognitive factors. Whereas both VTA and LC activity reflected running speed, VTA, but not LC axon activity reflected approach of a learned reward and LC, but not VTA, axon activity reflected initiation of running and novelty of the VR environment.

I have no specific expertise with respect to 2-photon imaging, so cannot evaluate the validity of the specific methods used to collect and analyse 2-photon calcium imaging data of axonal activity.

Strengths:

(1) Using a state-of-the-art approach to record separately the activity of VTA and LC axons with high temporal resolution in awake mice moving through virtual environments, the authors provide convincing evidence that activity of VTA and LC axons projecting to dorsal CA1 reflect partly distinct environmental, behavioral and cognitive factors.

(2) The study will help a) to interpret previous findings on how hippocampal dopamine and norepinephrine or selective manipulations of hippocampal LC or VTA inputs modulate behavior and b) to generate specific hypotheses on the impact of selective manipulations of hippocampal LC or VTA inputs on behavior.

Comments on revised version:

I thank the authors for including a sample size justification.

The justification is based on previous studies using similar sample sizes to characterize behavioral correlates of LC and VTA activity and on practical reasons. I note that to improve reproducibility, it would be preferable to have predefined target sample sizes based on predefined plans for statistical analysis.

Reviewer #3 (Public Review):

Summary:

Well-illustrated new material is documented for Acanthomeridion, a formerly incompletely known Cambrian arthropod. The formerly known facial sutures are proposed be associated with ventral plates that the authors homologise with the free cheeks of trilobites (although also testing alternative homologies). An update of a published phylogenetic dataset permits reconsideration of whether dorsal ecdysial sutures have a single or multiple origins in trilobites and their relatives.

Strengths:

Documentation of an ontogenetic series makes a sound case that the proposed diagnostic characters of a second species of Acanthomeridion are variation within a single species. New microtomographic data shed light on appendage morphology that was not formerly known. The new data on ventral plates and their association with the ecdysial sutures are valuable in underpinning homologies with trilobites.

I think the revision does a satisfactory job of reconciling the data and analyses with the conclusions drawn from them. Referee 1's valid concerns about whether a synonymy of Acanthomeridion anacanthus is justified have been addressed by the addition of a length/width scatterplot in Figure 6. Referee 2's doubts about homology between the librigenae of trilobites and ventral plates of Acanthomeridion have been taken on board by re-running the phylogenetic analyses with a coding for possible homology between the ventral plates and the doublure of olenelloid trilobites. The authors sensibly added more trilobite terminals to the matrix (including Olenellus) and did analyses with and without constraints for olenelloids being a grade at the base of Trilobita. My concerns about counting how many times dorsal sutures evolved on a consensus tree have been addressed (the authors now play it safe and say "multiple" rather than attempting to count them on a bushy topology). The treespace visualisation (Figure 9) is a really good addition to the revised paper.

Weaknesses:

The question of how many times dorsal ecdysial sutures evolved in Artiopoda was addressed by Hou et al (2017), who first documented the facial sutures of Acanthomeridion and optimised them onto a phylogeny to infer multiple origins, as well as in a paper led by the lead author in Cladistics in 2019. Du et al. (2019) presented a phylogeny based on an earlier version of the current dataset wherein they discussed how many times sutures evolved or were lost based on their presence in Zhiwenia/Protosutura, Acanthomeridion and Trilobita. The answer here is slightly different (because some topologies unite Acanthomeridion and trilobites). This paper is not a game-changer because these questions have been asked several times over the past seven years, but there are solid, worthy advances made here.

I'd like to see some of the most significant figures from the Supplementary Information included in the main paper so they will be maximally accessed. The "stick-like" exopods are not best illustrated in the main paper; their best imagery is in Figure S1. Why not move that figure (or at least its non-redundant panels) as well as the reconstruction (Figure S7) to the main paper? The latter summarises the authors' interpretation that a large axe-shaped hypostome appears to be contiguous with ventral plates. The specimens depict evidence for three pairs of post-antennal cephalic appendages but it's a bit hard to picture how they functioned if there's no room between the hypostome and ventral plates. Also, a comment is required on the reconstruction involving all cephalic appendages originating against/under the hypostome rather the first pair being paroral near the posterior end of the hypostome and the rest being post-hypostomal as in trilobites.

Reviewer #2 (Public Review):

Summary:

The study describes differences in responses to sounds and whisker deflections as well as combinations of these stimuli in different neurochemically defined subsections of the lateral and dorsal cortex of the inferior colliculus in anesthetised and awake mice.

Strengths:

A major achievement of the work lies in obtaining the data in the first place as this required establishing and refining a challenging surgical procedure to insert a prism that enabled the authors to visualise the lateral surface of the inferior colliculus. Using this approach, the authors were then able to provide the first functional comparison of neural responses inside and outside of the GABA-rich modules of the lateral cortex. The strongest and most interesting aspects of the results, in my opinion, concern the interactions of auditory and somatosensory stimulation. For instance, the authors find that a) somatosensory-responses are strongest inside the modules and b) somatosensory-auditory suppression is stronger in the matrix than in the modules. This suggests that, while somatosensory inputs preferentially target the GABA-rich modules, they do not exclusively target GABAergic neurons within the modules (given that the authors record exclusively from excitatory neurons we wouldn't expect to see somatosensory responses if they targeted exclusively GABAergic neurons) and that the GABAergic neurons of the modules (consistent with previous work) preferentially impact neurons outside the modules, i.e. via long-range connections.

Reviewer #3 (Public Review):

Summary:

The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.

Strengths:

The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.

Weaknesses:

After an additional revision, the bioinformatics section of the methods has changed significantly from previous versions and now indicates a third sequencer was used instead: Ion S5 XL. Important parameters required to replicate analysis have still not been provided. Inspection of the SRA data indicates a mix of Illumina MiSeq and Illumina HiSeq 2500. It is now unclear which sequencing technology was used as authors have variably reported 4 different sequencers for these samples. Appropriate metadata was not provided in the SRA, although some groups may be inferred from sample names. These changing descriptions of the methodologies and ambiguity in making the data available create concerns about rigor of study and results.

Reviewer #2 (Public Review):

Summary:

This manuscript provides a detailed analysis of B-cell lymphomagenesis in mice lacking an alternative exon in region encoding the C-terminal (regulatory) domain of the p53 protein and thus enable to assemble the so-called p53AS isoform. This isoform differs from canonical p53 by the replacement of roughly 30 c-terminal residues by about 10 residues encoded by the alternative exon. There is biochemical and biological evidence that p53AS retains strong transcriptional and somewhat enhanced suppressive activities, with mouse models expressing protein constructs similar to p53AS showing signs of increased p53 activity leading to rapid and lethal anemia. However, the precise role of the alternative p53AS variant has not been addressed so far in a mouse model aimed at demonstrating whether the lack of this particular p53 isoform (trp53ΔAS/ΔAS mice) may cause a specific pathological phenotype.

Results show that lack of AS expression does not noticeably affect p53 the patterns of protein expression and transcriptional activity but reveals a subtle pathogenic phenotype, with trp53ΔAS/ΔAS males, but not females, tending to develop more frequently and earlier B-cell lymphoma than WT. Next, the authors then introduced ΔAS in transgenic Eμ-Myc mice that show accelerated lymphomagenesis. They show that lack of AS caused increased lethality and larger tumor lymph nodes in p53ΔAS Eμ-Myc males compared to their p53WT Eμ-Myc male counterparts, but not in females. Comparative transcriptomics identified a small set of candidate, differentially expressed gene, including Ackr4 (atypical chemokine receptor 4), which was significantly expressed in the spleens of ΔAS compared to WT controls. Ackr4 encodes a dummy receptor acting as an interceptor for multiple chemokines and thus may negatively regulate a chemokine/cytokine signalling axis involved in lymphomagenesis, which is down-regulated by estrogen signalling. Using in vitro cell models, the authors provide evidence that Ackr4 is a transcriptional target for p53 and that its p53-dependent activation is repressed by 17b-oestradiol. Finally, seeking evidence for a relevance for this gene in human lymphomagenesis, the authors analyse Burkitt lymphoma transcriptomic datasets and show that high ACKR4 expression correlated with better survival in males, but not in females

Reviewer #2 (Public Review):

Summary:

The authors tried to identify novel adult functions of the classical Drosophila juvenile-adult transition axis (i.e. ptth-ecdysone). Surprisingly, larval ptth-expressing neurons expressed the sex-specific doublesex gene, thus belonging to the sexual dimorphic circuit. Lack of ptth during late larval development caused enhanced female sexual receptivity, effect rescued by supplying ecdysone in the food. Among many other cellular players, pC1 neurons control receptivity by encoding the mating status of females. Interestingly, during metamorphosis a subtype of pC1 neurons required Ecdysone Receptor A in order to regulate such female receptivity. A transcriptomic analysis using pC1-specific Ecdyone signaling down-regulation gives some hints of possible downstream mechanisms.

Strengths:

The manuscript showed solid genetic evidence that lack of ptth during development caused enhanced copulation rate in female flies, which includes ptth mutant rescue experiments by over-expressing ptth as well as by adding ecdysone-supplemented food. They also present elegant data dissecting the temporal requirements of ptth-expressing neurons by shifting animals from non-permissive to permissive temperatures, in order to inactivate neuronal function (although not exclusively ptth function). They showed that EcR-A is up-regulated in ptth mutant background. By combining different drivers together with EcR-A RNAi and torso RNAi lines authors also identified the Ecdysone receptor and torso requirements of a particular subtype of pC1 neurons during metamorphosis. Convincing live calcium imaging showed no apparent effect of EcR-A in neural activity, although some effect on morphology is uncovered. Finally, bulk RNAseq shows differential gene expression after EcR-A down-regulation.

Weaknesses:

The paper has three main weaknesses. The first one refers to temporal requirements of ptth and ecdysone signaling. Whereas ptth is necessary during larval development, ecdysone effect appears during pupal development. ptth induces ecdysone synthesis during larval development but there is no published evidence about a similar role for ptth during pupal stages. The down-regulation of EcR-A by RNAi requires at least 8 h to be complete, whereas the activation of ptth neurons in larva stages is immediate. Furthermore, larval and pupal ecdysone functions are different (triggering metamorphosis vs tissue remodeling). The second caveat is the fact that ptth and ecdysone/torso loss-of-function experiments render opposite effects (enhancing and decreasing copulation rates, respectively). The most plausible explanation is that both functions are independent of each other, also suggested by differential temporal requirements. Finally, in order to identify the effect in the transcriptional response of down-regulating EcR-A in a very small population of neurons, a scRNAseq study should have been performed instead of bulk RNAseq.

In summary, despite the authors providing convincing evidence that ptth and ecdysone signaling pathways are involved in female receptivity, the main claim that ptth regulates this process through ecdysone is not supported by results. More likely, they'd rather be independent processes.

Reviewer #2 (Public Review):

Summary:<br /> This research offers a comprehensive analysis of the regenerative process in sea cucumbers and builds upon decades of previous research. The approach involves a detailed examination using single-cell sequencing, making it a crucial reference paper while shedding new light on regeneration in this organism.

Strengths:<br /> Detailed analysis of single-cell sequencing data and high-quality RNA localization images provide significant new insights into regeneration in sea cucumbers and, more broadly, in animals.

Weaknesses:<br /> The spatial context of the RNA localization images is not well represented, making it difficult to understand how the schematic model was generated from the data. In addition, multiple strong statements in the conclusion should be better justified and connected to the data provided.

Reviewer #2 (Public Review):

Summary

This study provides a detailed analysis and dissociation between two effects of activation of lateral inhibitory circuits in the olfactory bulb on ongoing single mitral/tufted cell (MTC) spiking activity, namely enhanced synchronization in the gamma frequency range or lateral inhibition of firing rate.

The authors use a clever combination of single-cell recordings, optogenetics with variable spatial stimulation of MTCs and sensory stimulation in vivo, and established mathematical methods to describe changes in autocorrelation/synchronization of a single MTC's spiking activity upon activation of lateral glomerular MTC ensembles. This assay is rounded off by a gain-of-function experiment in which the authors enhance granule cell (GC) excitation to establish a causal relation between GC activation and enhanced synchronization to gamma (they had used this manipulation in their previous paper Dalal & Haddad 2022, but use a smaller illumination spot here for spatially restricted activation).

Strengths

This study is of high interest for olfactory processing - since it shows directly that interactions between only two selected active receptor channels are sufficient to enhance the synchronization of single neurons to gamma in one channel (and thus by inference most likely in both). These interactions are distance-independent over many 100s of µms and thus can allow for non-topographical inhibitory action across the bulb, in contrast to the center-surround lateral inhibition known from other sensory modalities.

In my view, parallels between vision and olfaction might have been overemphasized so far, since the combinatorial encoding of olfactory stimuli across the glomerular map might require different mechanisms of lateral interaction versus vision. This result is indicative of such a major difference.

Such enhanced local synchronization was observed in a subset of activated channel pairs; in addition, the authors report another type of lateral interaction that does involve the reduction of firing rates, drops off with distance and most likely is caused by a different circuit-mediated by PV+ neurons (PVN; the evidence for which is circumstantial).

Weaknesses/Room for improvement

Thus this study is an impressive proof of concept that however does not yet allow for broad generalization. Therefore the framing of results should be slightly more careful in my opinion.

Along this line, the conclusions regarding two different circuits underlying lateral inhibition vs enhanced synchronization are not quite justified by the data, e.g.

(1) The authors mention that their granule cell stimulation results in a local cold spot (l. 527 ff) - how can they then said to be not involved in the inhibition of firing rate (bullet point in Highlights)? Please elaborate further. In l.406 they also state that GCs can inhibit MTCs under certain conditions. The argument, that this stimulation is not physiological, makes sense, but still does not rule out anything. You might want to cite Aghvami et al 2022 on the very small amplitude of GC-mediated IPSPs, also McIntyre and Cleland 2015.

(2) Even from the shown data, it appears that laterally increased synchronization might co-occur with lateral suppression (See also comment on Figures 1d,e and Figure S1c)

(3) There are no manipulations of PVN activity in this study, thus there is no direct evidence for the substrate of the second circuit.

(4) The manipulation of GC activity was performed in a transgenic line with viral transfection, which might result in a lower permeation of the population compared to the line used for optogenetic stimulation of MTCs.

In some instances, the authors tend to cite older literature - which was not yet aware of the prominent contribution of EPL interneurons including PVN to recurrent and lateral inhibition of MT cells - as if roles that then were ascribed to granule cells for lack of better knowledge can still be unequivocally linked to granule cells now. For example, they should discuss Arevian et al (2006), Galan et al 2006, Giridhar et al., Yokoi et al. 1995, etc in the light of PVN action.

Therefore it is also not quite justified to state that their result regarding the role of GCs specifically for synchronization, not suppression, is "in contrast to the field" (e.g. l.70 f.,, l.365, l. 400 ff).

Why did the authors choose to use the term "lateral suppression", often interchangeably with lateral inhibition? If this term is intended to specifically reflect reductions of firing rates, it might be useful to clearly define it at first use (and cite earlier literature on it) and then use it consistently throughout.

A discussion of anesthesia effects is missing - e.g. GC activity is known to be reportedly stronger in awake mice (Kato et al). This is not a contentious point at all since the authors themselves show that additional excitation of GCs enhances synchrony, but it should be mentioned.

Some citations should be added, in particular relevant recent preprints - e.g. Peace et al. BioRxiv 2024, Burton et al. BioRxiv 2024 and the direct evidence for a glutamate-dependent release of GABA from GCs (Lage-Rupprecht et al. 2020).

The introduction on the role of gamma oscillations in sensory systems (in particular vision) could be more elaborated.

Reviewer #2 (Public Review):

Summary:

To evaluate whether somatic mutations in cancer genomes are enriched with mutations in polyadenylation signal regions, the authors analyzed 1000 genomes data and PCAWG data as a control and experimental set, respectively. They observed increased enrichment of somatic mutations that may affect the function of polyA signals and confirmed that these mutations may influence the expression of the gene through a minigene expression experiment.

Strengths:

This study provides a systematic evaluation of polyA signal, which makes it valuable. Overall, the analytic approach and results are solid and supported by experimental validation.

Weaknesses:

(1) This study uses APARENT2 as a tool to evaluate functional alteration in polyA signal sequences. Based on the original paper and the results shown in this paper, the algorithm appears to be of high quality. However, the whole study is dependent on the output of APARENT2. Therefore, it would be nice to<br /> (a) run and show a positive control run, which can show that the algorithm works well, and<br /> (b) describe the rationale for selecting this algorithm in the main text.

(2) Are there recurrent somatic mutation calls (= exactly the same mutation across different tumor samples) in the poly(A) region of certain genes?

(3) The authors nicely showed that the minigene with A>G mutation altered gene expression. Maybe one can reach a similar conclusion by analyzing a cancer dataset that has mutation and gene expression data? That is, genes with or without polyA mutations show different expression levels.

Reviewer #2 (Public Review):

Summary:

Disruption of the nicotinamide adenine dinucleotide (NAD) de novo Synthesis Pathway, by which L-tryptophan is converted to NAD results in multi-organ malformations which collectively has been termed Congenital NAD Deficiency Disorder (CNDD).

While NAD de novo synthesis is primarily active in the liver postnatally, the site of activity prior to and during organogenesis is unknown. However, mouse embryos are susceptible to CNDD between E7.5-E12.5, before the embryo has developed a functional liver. Therefore, NAD de novo synthesis is likely active in another cell or tissue during this time window of susceptibility.

The body of work presented in this paper continues the corresponding author's lab investigation of the cause and effects of NAD Deficiency and the primary goal was to determine the cell or tissue responsible for NAD de novo synthesis during early embryogenesis.

The authors conclude that visceral yolk sac endoderm is the source of NAD de novo synthesis, which is essential for mouse embryonic development, and furthermore that the dynamics of NAD synthesis are conserved in human equivalent cells and tissues, the perturbation of which results in CNDD.

Strengths:

Overall, the primary findings regarding the source of NAD synthesis, the temporal requirement, and conservation between rodent and human species are quite novel and important for our understanding of NAD synthesis and its function and role in CNDD.

The authors used UHPLC-MS/MS to quantify NAD+ and NAD-related metabolites and showed convincingly that the NAD salvage pathway can compensate for the loss of NAD synthesis in Haao-/- embryos, then determined that Haao activity was present in the yolk sac prior to hepatic development identifying this organ as the site of de novo NAD synthesis. Dietary modulation between E7.5-10.5 was sufficient to induce CNDD phenotypes, narrowing the window of susceptibility, and then re-analysis of RNA-seq datasets suggested the endoderm was the cell source of NAD synthesis.

Weaknesses:

Page 4 and Table S4. The descriptors for malformations of organs such as the kidney and vertebrae are quite vague and uninformative. More specific details are required to convey the type and range of anomalies observed as a consequence of NAD deficiency.

Can the authors define whether the role of the NAD pathway in a couple of tissue or organ systems is the same? By this I mean is the molecular or cellular effect of NAD deficiency is the same in the vertebrae and organs such as the kidney. What unifies the effects on these specific tissues and organs and are all tissues and organs affected? If some are not, can the authors explain why they escape the need for the NAD pathway?

Page 5 and Figure 6C. The expectation and conclusion for whether specific genes are expressed in particular cell types in scRNA-seq datasets depend on the number of cells sequenced, the technology (methodology) used, the depth of sequencing, and also the resolution of the analysis. It is therefore essential to perform secondary validation of the analysis of scRNA-seq data. At a minimum, the authors should perform in situ hybridization or immunostaining for Tdo2, Afmid, Kmo, Kynu, Haao, Qprt, and Nadsyn1 or some combination thereof at multiple time points during early mouse embryogenesis to truly understand the spatiotemporal dynamics of expression and NAD synthesis.

Absolute functional proof of the yolk sac endoderm as being essential and required for NAD synthesis in the context of CNDD might require conditional deletion of Haoo in the yolk sac versus embryo using appropriate Cre driver lines or in the absence of a conditional allele, could be performed by tetraploid embryo-ES cell complementation approaches. But temporal dietary intervention can also approximate the same thing by perturbing NAD synthesis Shen the yolk sac is the primary source versus when the liver becomes the primary source in the embryo.

Reviewer #2 (Public Review):

Summary:

Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study the death of zebrafish spinal motor neurons.

Strengths:<br /> Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish.

Weaknesses:<br /> However, they have ignored significant literature describing the death of an identified zebrafish motor neuron, expression of the mnx gene in interneurons that are closely related to motor neurons, the increase in number of zebrafish motor neurons over developmental time, and potential differences between the limb-innervating motor neurons whose death has been characterized in chicks and rodents and the body wall-innervating motor neurons whose death they characterized using their biosensor. Thus, although their new tool is likely to be useful in the future, it does not provide new insights into zebrafish motor neuron programmed cell death.

Reviewer #2 (Public Review):

Summary:

This study investigated the effects of transcutaneous auricular vagus nerve stimulation (taVNS) on cardiovascular dynamics in subarachnoid hemorrhage (SAH) patients. The researchers conducted a randomized clinical trial with 24 SAH patients, comparing taVNS treatment to a Sham treatment group (20 minutes per day twice a day during the ICU stay). They monitored electrocardiogram (ECG) readings and vital signs to assess acute as well as middle-term changes in heart rate, heart rate variability, QT interval, and blood pressure between the two groups. The results showed that repetitive taVNS did not significantly alter heart rate, corrected QT interval, blood pressure, or intracranial pressure. However, it increased overall heart rate variability and parasympathetic activity after 5-10 days of treatment compared to the sham treatment. Acute taVNS led to an increase in heart rate, blood pressure, and peripheral perfusion index without affecting corrected QT interval, intracranial pressure, or heart rate variability. The acute post-treatment elevation in heart rate was more pronounced in patients who showed clinical improvement. In conclusion, the study found that taVNS treatment did not cause adverse cardiovascular effects, suggesting it is a safe immunomodulatory treatment for SAH patients. The mild acute increase in heart rate post-treatment could potentially serve as a biomarker for identifying SAH patients who may benefit more from taVNS therapy.

Strengths:

The paper is overall well written, and the topic is of great interest. The methods are solid and the presented data are convincing.

Weaknesses:

(1) It should be clearly pointed out that the current paper is part of the NAVSaH trial (NCT04557618) and presents one of the secondary outcomes of that study while the declared first outcomes (change in the inflammatory cytokine TNF-α in plasma and cerebrospinal fluid between day 1 and day 13, rate of radiographic vasospasm, and rate of requirement for long-term CSF diversion via a ventricular shunt) are available as a pre-print and currently under review (doi: 10.1101/2024.04.29.24306598.). The authors should better stress this point as well as the potential association of the primary with the secondary outcomes.

(2) The references should be implemented particularly concerning other relevant papers (including reviews and meta-analysis) of taVNS safety, particularly from a cardiovascular standpoint, such as doi: 10.1038/s41598-022-25864-1 and doi: 10.3389/fnins.2023.1227858).

(3) The dose-response issue that affects both VNS and taVNS applications in different settings should be mentioned (doi: 10.1093/eurheartjsupp/suac036.) as well as the need for more dose-finding preclinical as well as clinical studies in different settings (the best stimulation protocol is likely to be disease-specific).

Overall, the present work has the important potential to further promote the usage of taVNS even on critically ill patients and might set the basis for future randomized studies in this setting.

Rothchild (2014) defined gender bias as behavior showing favoritism towards one gender over another.

Reviewer #2 (Public Review):

Gekko et al investigate the impact of perturbing mitochondrial during early embryo development, through modulation of the mitochondrial fission protein Drp1 using Trim-Away technology. They aimed to validate a role for mitochondrial dynamics in modulating chromosomal segregation, mitochondrial inheritance and embryo development and achieve this through the examination of mitochondrial and endoplasmic reticulum distribution, as well as actin filament involvement, using targeted plasmids, molecular probes and TEM in pronuclear stage embryos through the first cleavages divisions. Drp1 deletion perturbed mitochondrial distribution, leading to asymmetric partitioning of mitochondria to the 2-cell stage embryo, prevented appropriate chromosomal segregation and culminated in embryo arrest. Resultant 2-cell embryos displayed altered ATP, mtDNA and calcium levels. Microinjection of Drp1 mRNA partially rescued embryo development. A role for actin filaments in mitochondrial inheritance is described, however the actin-based motor Myo19 does not appear to contribute.

Overall, this study builds upon their previous work and provides further support for the role of mitochondrial dynamics in mediating chromosomal segregation and mitochondrial inheritance. In particular, Drp1 is required for redistribution of mitochondria to support symmetric partitioning and support ongoing development.

Strengths:<br /> The study is well designed, the methods appropriate and the results clearly presented. The findings are nicely summarised in a schematic.

Understanding the role of mitochondria in binucleation and mitochondrial inheritance is of clinical relevance for patients undergoing infertility treatment, particularly those undergoing mitochondrial replacement therapy.

Weaknesses:

The authors first describe the redistribution of mitochondria during normal development, followed by alterations induced by Drp1 depletion. It would be useful to indicate the time post-hCG for imaging of fertilised zygotes (first paragraph of the results/Figure 1) to compare with subsequent Drp1 depletion experiments.

It is noted that Drp1 protein levels were undetectable 5h post-injection, suggesting earlier times were not examined, yet in Figure 3A it would seem that aggregation has occurred within 2 hours (relative to Figure 1).

Mitochondria appear to be slightly more aggregated in Drp1 fl/fl embryos than in control, though comparison with untreated controls does not appear to have been undertaken. There also appears to be some variability in mitochondrial aggregation patterns following Drp1 depletion (Figure 2-suppl 1 B) which are not discussed.

The authors use western blotting to validate the depletion of Drp1, however do not quantify band intensity. It is also unclear whether pooled embryo samples were used for western blot analysis.

Likewise, intracellular ROS levels are examined however quantification is not provided. It is therefore unclear whether 'highly accumulated levels' are of significance or related to Drp1 depletion.

In previous work, Drp1 was found to have a role as a spindle assembly checkpoint (SAC) protein. It is therefore unclear from the experiments performed whether aggregation of mitochondria separating the pronuclei physically (or other aspects of mitochondrial function) prevents appropriate chromosome segregation or whether Drp1 is acting directly on the SAC.

Reviewer #2 (Public Review):

Summary:

This manuscript describes a very eloquent study of disrupted stimulus -secretion coupling in salivary acinar cells in the early stages of an animal model (DMXAA) of Sjogren's syndrome (SS). The study utilizes a range of technically innovative in vivo imaging of Ca signaling, in vivo salivary secretion, patch clamp electrophysiology to assess TMEM16a activity, immunofluorescence and electron microscopy and a range of morphological and functional assays of mitochondrial function. Results show that in mice with DMXAA-induced Sjogren's syndrome, there was a reduced nerve stimulation induced salivary secretion, yet surprisingly the nerve stimulation induced Ca signaling was enhanced. There was also a reduced carbachol (CCh)-induced activation of TMEM16a currents in acinar cells from DMXAA-induced SS mice, whereas the intrinsic Ca-activated TMEM16a currents were unaltered, further supporting that stimulus-secretion coupling was impaired. Consistent with this, high resolution STED microscopy revealed that there was a loss of close physical spatial coupling between IP3Rs and TMEM16a, which may contribute to the impaired stimulus-secretion coupling. Furthermore, the authors show that the mitochondria were both morphologically and functionally impaired, suggesting that bioenergetics may be impaired in salivary acinar cells of DMXAA-induced SS mice.

Strengths:

Overall, this is an outstanding manuscript, that will have a huge impact on the field. The manuscript is beautifully well-written with a very clear narrative. The experiments are technically innovative, very well executed and with a logical design The data are very well presented and appropriately analyzed and interpreted.

Review of Revised Manuscript:

The authors have now addressed all my comments and concerns in the revised manuscript to my satisfaction.

Reviewer #2 (Public Review):

Summary:<br /> 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 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 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:<br /> 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:<br /> 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 #2 (Public Review):

Summary:

The authors observed an aggravated vascular endothelial dysfunction upon overexpressing circHMGCS1 and inhibiting miR-4521. This study discovered that circHMGCS1 promotes arginase 1 expression by sponging miR-4521, which accelerated the impairment of vascular endothelial function.

Strengths:

The study is systematic and establishes the regulatory role of the circHMGCS1-miR-4521 axis in diabetes-induced cardiovascular diseases.

Weaknesses:

(1) The authors show direct evidence of interaction between circHMGCS1 and miR-4521 by pulldown assay. However, the changes in miRNA expression opposite to the levels of target circRNA could be through Target RNA-Directed MicroRNA Degradation. Since the miRNA level is downregulated, the downstream target gene is expected to be upregulated even in the absence of circRNA.

Reviewer #2 (Public Review):

Summary:

The authors set out to demonstrate a mechanistic link between Fcgamma receptor (IIIA) glycosylation and IgG binding affinity and signaling - resulting in antibody-dependent cellular cytotoxicity - ADCC. The work builds off prior findings from this group about the general impact of glycosylation on FcR (Fc receptor)-IgG binding.

Strengths:

The structural data (NMR) is highly compelling and very significant to the field. A demonstration of how IgG interacts with FcgRIIIA in a manner sensitive to glycosylation of both the IgG and the FcR fills a critical knowledge gap. The approach to demonstrate the selective impact of glycosylation at N162 is also excellent and convincing. The manuscript/study is, overall, very strong.

Weaknesses:

There are a number of minor weaknesses that should be addressed.

(1) Since S164A is the only mutant in Figure 1 that seems to improve affinity, even if minimally, it would be a nice reference to highlight that residue in the structural model in panel B.

(2) It is confusing why some of the mutants in the study are not represented in Figure 1 panel A. Those affinities and mutants should be incorporated into panel A so the reader can easily see where they all fall on the scale. T167Y in particular needs to be shown, as it is one of few mutants that fall between what seems to be ADCC+ and ADCC- lines. Also, that mutant seems to have a stronger affinity compared to wt (judged by panel D), yet less ADCC than wt. This would imply that the relationship between affinity and activity is not as clean as stated, though it is clearly important. Comments about this would strengthen the overall manuscript.

(3) This statement feels out of place: "In summary, this result demonstrates that the sensitivity to antibody fucosylation may be eliminated through FcγRIIIa engineering while preserving antibody-binding affinity." In Figure 2, the authors do indeed show that mutations in FcgRIIIa can alter the impact of IgG core fucosylation, but implying that receptor engineering is somehow translatable or as impactful therapeutically as engineering the antibody itself deflates the real basic science/biochemical impact of understanding these interactions in molecular detail. Not everything has to be immediately translatable to be important.

(4) The findings reported in Figure 2, panel C are exciting. Controls for the quality of digestion at each step should be shown (perhaps in supplementary data).

(5) Figure 3 is confusing (mislabeled?) and does not show what is described in the Results. First, there is a F158V variant in the graph but a V158F variant in the text. Please correct this. Second, this variant (V158F/F158V) does not show the 2-fold increase in ADCC with kifunesine as stated. Finally, there are no statistical evaluations between the groups (+/- kif; +/- fucose). The differences stated are not clearly statistically significant given the wide spread of the data. This is true even for the wt variant.

(6) The kifunensine impact is somewhat confusing. They report a major change in ADCC, yet similar large changes with trimming only occur once most of the glycan is nearly gone (Figure 2). Kifunensine will tend to generate high mannose and possibly a few hybrid glycans. It is difficult to understand what glycoforms are truly important outside of stating that multi-branched complex-type N-glycans decrease affinity.

(7) This is outside of the immediate scope, but I feel that the impact would be increased if differences in NK cell (and thus FcgRIIIA) glycosylation are known to occur during disease, inflammation, age, or some other factor - and then to demonstrate those specific changes impact ADCC activity via this mechanism.

Reviewer #2 (Public Review):

Through RNA analysis, Xie et al found LncRNA Snhg3 was one of the most down-regulated Snhgs by high fat diet (HFD) in mouse liver. Consequently, the authors sought to examine the mechanism through which Snhg3 is involved in the progression of metabolic dysfunction-associated fatty liver diseases (MASLD) in HFD-induced obese (DIO) mice. Interestingly, liver-specific Sngh3 knockout reduced, while Sngh3 over-expression potentiated fatty liver in mice on a HFD. Using the RNA pull-down approach, the authors identified SND1 as a potential Sngh3 interacting protein. SND1 is a component of the RNA-induced silencing complex (RISC). The authors found that Sngh3 increased SND1 ubiquitination to enhance SND1 protein stability, which then reduced the level of repressive chromatin H3K27me3 on PPARg promoter. The upregulation of PPARg, a lipogenic transcription factor, thus contributed to hepatic fat accumulation.

The authors propose a signaling cascade that explains how LncRNA sngh3 may promote hepatic steatosis. Multiple molecular approaches have been employed to identify molecular targets of the proposed mechanism, which is a strength of the study. There are, however, several potential issues to consider before jumping to the conclusion.

(1) First of all, it's important to ensure the robustness and rigor of each study. The manuscript was not carefully put together. The image qualities for several figures were poor, making it difficult for the readers to evaluate the results with confidence. The biological replicates and numbers of experimental repeats for cell-based assays were not described. When possible, the entire immunoblot imaging used for quantification should be presented (rather than showing n=1 representative). There were multiple mis-labels in figure panels or figure legends (e.g., Fig. 2I, Fig. 2K and Fig. 3K). The b-actin immunoblot image was reused in Fig. 4J, Fig. 5G and Fig. 7B with different exposure times. These might be from the same cohort of mice. If the immunoblots were run at different times, the loading control should be included on the same blot as well.

(2) The authors can do a better job in explaining the logic for how they came up with the potential function of each component of the signaling cascade. Sngh3 is down-regulated by HFD. However, the evidence presented indicates its involvement in promoting steatosis. In Fig. 1C, one would expect PPARg expression to be up-regulated (when Sngh3 was down-regulated). If so, the physiological observation conflicts with the proposed mechanism. In addition, SND1 is known to regulate RNA/miRNA processing. How do the authors rule out this potential mechanism? How about the hosting snoRNA, Snord17? Does it involve in the progression of NASLD?

(3) The role of PPARg in fatty liver diseases might be a rodent-specific phenomenon. PPARg agonist treatment in humans may actually reduce ectopic fat deposition by increasing fat storage in adipose tissues. The relevance of the finding to human diseases should be discussed.

Reviewer #2 (Public Review):

Summary:

With this work, the authors tried to expand and integrate the concept of realized niche in the context of movement ecology by using fine-scale GPS data of 55 juvenile Golden eagles in the Alps. Authors found that ontogenic changes influence the percentage of area flyable to the eagles as individuals exploit better geographic uplifts that allow them to reduce the cost of transport.

Strengths:

Authors made insightful work linking changes in ontogeny and energy landscapes in large soaring birds that may not only advance the understanding of how changes in the life cycle affect the exploitability of aerial space but also offer valuable tools for the management and conservation of large soaring species in the changing world.

Weaknesses:

Future research may test the applicability of the present work by including more individuals and/or other species from other study areas.

Reviewer #2 (Public Review):

Summary:

This manuscript uses single-molecule fluorescence resonance energy transfer (smFRET) to identify differences in the molecular mechanisms of CXCR4 and ACKR3, two 7-transmembrane receptors that both respond to the chemokine CXCL12 but otherwise have very different signaling profiles. CXCR4 is highly selective for CXCL12 and activates heterotrimeric G proteins. In contrast, ACKR3 is quite promiscuous and does not couple to G proteins, but like most G protein-coupled receptors (GPCRs), it is phosphorylated by GPCR kinases and recruits arrestins. By monitoring FRET between two positions on the intracellular face of the receptor (which highlights the movement of transmembrane helix 6 [TM6], a key hallmark of GPCR activation), the authors show that CXCR4 remains mostly in an inactive-like state until CXCL12 binds and stabilizes a single active-like state. ACKR3 rapidly exchanges among four different conformations even in the absence of ligands, and agonists stabilize multiple activated states.

Strengths:

The core method employed in this paper, smFRET, can reveal dynamic aspects of these receptors (the breadth of conformations explored and the rate of exchange among them) that are not evident from static structures or many other biophysical methods. smFRET has not been broadly employed in studies of GPCRs. Therefore, this manuscript makes important conceptual advances in our understanding of how related GPCRs can vary in their conformational dynamics.

Weaknesses:

(1) The cysteine mutations in ACKR3 required to site-specifically install fluorophores substantially increase its basal and ligand-induced activity. If, as the authors posit, basal activity correlates with conformational heterogeneity, the smFRET data could greatly overestimate the conformational heterogeneity of ACKR3.

(2) The probes used cannot reveal conformational changes in other positions besides TM6. GPCRs are known to exhibit loose allosteric coupling, so the conformational distribution observed at TM6 may not fully reflect the global conformational distribution of receptors. This could mask important differences that determine the ability of intracellular transducers to couple to specific receptor conformations.

(3) While it is clear that CXCR4 and ACKR3 have very different conformational dynamics, the data do not definitively show that this is the main or only mechanism that contributes to their functional differences. There is little discussion of alternative potential mechanisms.

(4) The extent to which conformational heterogeneity is a characteristic feature of ACKRs that contributes to their promiscuity and arrestin bias is unclear. The key residue the authors find promotes ACKR3 conformational heterogeneity is not conserved in most other ACKRs, but alternative mechanisms could generate similar heterogeneity.

(5) There are no data to confirm that the two receptors retain the same functional profiles observed in cell-based systems following in vitro manipulations (purification, labeling, nanodisc reconstitution).

Reviewer #2 (Public Review):

Summary:

The authors build a colossal anatomical model of juvenile rat non-barrel primary somatosensory cortex, including inputs from the thalamus. This enhances past models by incorporating information on the shape of the cortex and estimated densities of various types of excitatory and inhibitory neurons across layers. This is intended to enable an analysis of the micro- and mesoscopic organisation of cortical connectivity and to be a base anatomical model for large-scale simulations of physiology.

Strengths:

• The authors incorporate many diverse data sources on morphology and connectivity.

• This paper takes on the challenging task of linking micro- and mesoscale connectivity.

• By building in the shape of the cortex, the authors were able to link cortical geometry to connectivity. In particular, they make an unexpected prediction that cortical conicality affects the modularity of local connectivity, which should be testable.

• The author's analysis of the model led to the interesting prediction that layer 5 neurons connect local modules, which may be testable in the future, and provide a basis to link from detailed anatomy to functional computations.

• The visualisation of the anatomy in various forms is excellent.

• A subnetwork of the model is openly shared (but see question below).

Weaknesses:

• Why was non-barrel S1 of the juvenile rat cortex selected as the target for this huge modelling effort? This is not explained.

• There is no effort to determine how specific or generalisable the findings here are to other parts of the cortex.

• Although there is a link to physiological modelling in another paper, there is no clear pathway to go from this type of model to understand how the specific function of the modelled areas may emerge here (and not in other cortical areas).

• In a few places the manuscript could be improved by being more specific in the language, for example:<br /> - "our anatomy-based approach has been shown to be powerful", I would prefer instead to read about specific contributions of past papers to the field, and how this builds on them.<br /> - similarly: "ensuring that the total number of synapses in a region-to-region pathway matches biology." Biology here is a loose term and implies too much confidence in the matching to some ground truth. Please instead describe the source of the data, including the type of experiment.

• Some of the decisions seem a little ad-hoc, and the means to assess those decisions are not always available to the reader e.g.<br /> - pg. 10. "Based on these results, we decided that the local connectome sufficed to model connectivity within a region.". What is the basis for this decision? Can it be formalised?<br /> - "In the remaining layers the results of the objective classification were used to validate the class assignments of individual pyramidal cells. We found the objective classification to match the expert classification closely (i.e., for 80-90% of the morphologies). Consequently, we considered the expert classification to be sufficiently accurate to build the model." The description of the validation is a little informal. How many experts were there? What are their initials? Was inter-rater or intra-rater reliability assessed? What are these numbers? The match with Kanari's classification accuracy should be reported exactly. There are clearly experts among the author list, but we are all fallible without good controls in place, and they should be more explicit about those controls here, in my opinion.<br /> - "Morphology selection was then performed as previously (Markram et al., 2015), that is, a morphology was selected randomly from the top 10% scorers for a given position." A lot of the decisions seem a little ad-hoc, without justification other than this group had previously done the same thing. For example, why 10% here? Shouldn't this be based on selecting from all of the reasonable morphologies?

• I would like to know if one of the key results relating to modularity and cortical geometry can be further explored. In particular, there seem to be sharp changes in the data at the end of the modelled cortical regions, which need to be explored or explained further.

• The shape of the juvenile cortex - a key novelty of this work - was based on merely a scalar reduction of the adult cortex. This is very surprising, and surely an oversimplification. Huge efforts have gone into modelling the complex nonlinear development of the cortex, by teams including the developing Human Connectome Project. For such a fundamental aspect of this work, why isn't it possible to reconstruct the shape of this relatively small part of the juvenile rat cortex?

• The same relative laminar depths are used for all subregions. This will have a large impact on the model. However, relative laminar depths can change drastically across the cortex (see e.g. many papers by Palomero-Gallagher, Zilles, and colleagues). The authors should incorporate the real laminar depths, or, failing that, show evidence to show that the laminar depth differences across the subregions included in the model are negligible.

• The authors perform an affine mapping between mouse and rat cortex. This is again surprising. In human imaging, affine mappings are insufficient to map between two individual brains of the same species and nonlinear transformations are instead used. That an affine transformation should be considered sufficient to map between two different species is then very surprising. For some models, this may be fine, but there is a supposed emphasis here on biological precision in terms of anatomical location.

• One of the most interesting conclusions, that the connectivity pattern observed is in part due to cooperative synapse formation, is based on analyses that are unfortunately not shown.

• Open code:<br /> - Why is only a subvolume available to the community?<br /> - Live nature of the model. This is such a colossal model, and effort, that I worry that it may be quite difficult to update in light of new data. For example, how much person and computer time would it take to update the model to account for different layer sizes across subregions? Or to more precisely account for the shape of the juvenile rat cortex?

Reviewer #2 (Public Review):

Summary of the manuscript:

The authors present MGPfactXMBD, a novel model-based manifold-learning framework designed to address the challenges of interpreting complex cellular state spaces from single-cell RNA sequences. To overcome current limitations, MGPfactXMBD factorizes complex development trajectories into independent bifurcation processes of gene sets, enabling trajectory inference based on relevant features. As a result, it is expected that the method provides a deeper understanding of the biological processes underlying cellular trajectories and their potential determinants.

MGPfactXMBD was tested across 239 datasets, and the method demonstrated similar to slightly superior performance in key quality-control metrics to state-of-the-art methods. When applied to case studies, MGPfactXMBD successfully identified critical pathways and cell types in microglia development, validating experimentally identified regulons and markers. Additionally, it uncovered evolutionary trajectories of tumor-associated CD8+ T cells, revealing new subtypes with gene expression signatures that predict responses to immune checkpoint inhibitors in independent cohorts.

Overall, MGPfactXMBD represents a relevant tool in manifold learning for scRNA-seq data, enabling feature selection for specific biological processes and enhancing our understanding of the biological determinants of cell fate.

Summary of the outcome:

The novel method addresses core state-of-the-art questions in biology related to trajectory identification. The design and the case studies are of relevance.

However, in my opinion, the manuscript requires several clarifications and updates.

Also, how the methods compare with existing Deep Learning based approaches such as TIGON is a question mark. If a comparison would be possible, it should be conducted; if not, it should be clarified why.

Strengths:

(1) Relevant methodology for a current field of research.

(2) Relevant case studies with relevant outcomes.

Weaknesses:

(1) In general, the manuscript may be improved by making the text more accessible to the Journal's audience: (i) intuitive explanation of some concepts; (ii) review the flow of some explanations.

(2) Additionally, several parts require more details on how the methods work, especially the case studies.

(3) Finally, there are missing references to published work and possibly some additional comparisons to make.

Reviewer #2 (Public Review):

This paper examines the reproducibility of results reported by the Murphy lab regarding transgenerational inheritance of a learned avoidance behavior in C. elegans. It has been well established by multiple labs that worms can learn to avoid the pathogen pseudomonas aeruginosa (PA14) after a single exposure. The Murphy lab has reported that learned avoidance is transmittable to 4 generations and dependent on a small RNA expressed by PA14 that elicits the transgenerational silencing of a gene in C. elegans. The Hunter lab now reports that although they can reproduce inheritance of the learned behavior by the first generation (F1), they cannot reproduce inheritance in subsequent generations.

This is an important study that will be useful for the community. Although they fail to identify a "smoking gun", the study examines several possible sources for the discrepancy, and their findings will be useful to others interested in using these assays. The preference assay appears to work in their hands in as much as they are able to detect the learned behavior in the P0 and F1 generations, suggesting that the failure to reproduce the transgenerational effect is not due to trivial mistakes in the protocol. An obvious reason, however, to account for the differing results is that the culture conditions used by the authors are not permissive for the expression of the small RNA by PA14 that the MUrphy lab identified as required for transgenerational inheritance. It would seem prudent for the authors to determine whether this small RNA is present in their cultures, or at least acknowledge this possibility. The authors should also note that their protocol was significantly different from the Murphy protocol (see comments below) and therefore it remains possible that protocol differences cumulatively account for the different results.

Reviewer #2 (Public Review):

Summary:<br /> The authors improve the work of Jallais et al. (2022) by including a novel module capable of automatically learning feature selection from different acquisition protocols inside a supervised learning framework. Combining the module above with an estimation framework for estimating the posterior distribution of model parameters, they obtain rich probabilistic information (uncertainty and degeneracy) on the parameters in a reasonable computation time.

The main contributions of the work are:<br /> (1) The whole framework allows the user to avoid manually defining summary statistics, which may be slow and tedious and affect the quality of the results.<br /> (2) The authors tested the proposal by tackling three different biophysical models for brain tissue and using data with characteristics commonly used by the diffusion-MR-microstructure research community.<br /> (3) The authors validated their method well with the state-of-the-art.

The main weakness is:<br /> (1) The methodology was tested only on scenarios with a signal-to-noise ratio (SNR) equal to 50. It is interesting to show results with lower SNR and without noise that the method can detect the model's inherent degenerations and how the degeneration increases when strong noise is present. I suggest expanding the Figure in Appendix 1 to include this information.

The authors showed the utility of their proposal by computing complex parameter descriptors automatically in an achievable time for three different and relevant biophysical models.

Importantly, this proposal promotes tackling, analyzing, and considering the degenerated nature of the most used models in brain microstructure estimation.

Reviewer #2 (Public Review):

The transient receptor potential mucolipin 1 (TRPML1) functions as a lysosomal organelle ion channel whose variants are associated with lysosomal storage disorder mucolipidosis type IV. Understanding sites that allosterically control the TRPML1 channel function may provide new molecular moieties to target with prototypic drugs.

Gan et al provide the first high-resolution cryo-EM structures of the TRPML1 channel (Y404W) in the open state without any activating ligands. This new structure demonstrates how a mutation at a site some distance away from the pore can influence the channel's conducting state. However, the authors do not provide a structural analysis of the Y404W pore which would validate their open-state claims. Nonetheless, Gan et al provide compelling electrophysiology evidence which supports the proposed Y404W gain of function effect. The authors propose an allosteric mechanism with the following molecular details- the Y404 to W sidechain substitution provides extra van der Waals contacts within the pocket surrounded by helices of the VSD-like domain and causes S4 bending which in turn opens to the pore through the S4-S5 linker. Conversely, the author functionally demonstrates that an alanine mutation at this site causes a loss of function. Although the authors do not provide a structure of the Y404A mutation, they propose that the alanine substitution disrupts the sidechain packing and likely destabilizes the open conformation. TRPM1 channels are regulated by PIP2 species, which is related to their cell function. In the membrane of lysosomes, PI(3,5)P2 activates the channel, whereas PI(4,5)P2 found in the plasma membrane has inhibitory effects. To understand its lipid regulation, the authors solved a cryo-EM structure of TRPM1 bound to PI(4,5)P2 in its presumed closed state. Again, while the provided functional evidence suggests that PI(4,5)P2 occupancy inhibits TRPML1 current, the authors do not provide analysis of the pore which would support their closed state assertion. Within this same structure, the authors observe a density that may be attributed to sphingomyelin (or possibly phosphocholine). Using electrophysiology of WT and the Y404W channels, the authors report sphingomyelins antagonist effect on TRPML1 currents under low luminal (external) pH. Taken together, the results described in Gan et al provide compelling evidence for a gating (open, closed) mechanism of the TRPML1 pore which can be allosterically regulated by altered packing and lipid interactions within the VSDL.

Reviewer #2 (Public Review):

Summary:

The study demonstrates that Znhit1 regulates male meiosis, with deletion causing pachytene failure associated with defective expression of pachytene genes and subtle effects on X-Y pairing and DSB repair. The authors attribute this phenotype to the defective incorporation of the Znhit1 target H2A.Z into chromatin.

Strengths:

The paper and the figures are well presented and the narrative is clear. Evidence that the conditional deletion strategy removes Znhit1 is strong, with multiple orthogonal approaches used. Most of the meiotic phenotyping is well performed, and the omics analysis clearly identifies a dramatic effect on the meiotic gene expression program. The link to H2A.Z and A-MYB adds a mechanistic angle to the study.

Weaknesses:

(1) Current literature demonstrates that meiotic mutants arrest at one of two stages: midpachytene (stage IV of the seminiferous cycle) or metaphase I (stage XII of the seminiferous cycle). This study documents that in the Znhit1 KO the midpachytene marker H1t appears normally, but that cells arrest before diplotene. If this is true, then arrest must occur during late pachytene, which based on my knowledge has never been documented for a meiotic KO. To resolve this, the authors should present stronger histological substaging evidence to support their claim.

(2) The authors overlooked the possible effects of Znhit1 deletion on MSCI. Defective MSCI is a well-established cause of pachytene arrest. Actually, the fact that they see X-Y pairing failure should alert them even more strongly to this possibility because MSCI failure is often associated with defective X-Y pairing. This could be easily addressed by examination of their RNAseq data.

(3) The recombination assays need attention.<br /> - In the text the authors state that they studied RPA2 and DMC1, but the figures show RPA2 and RAD51.<br /> - The RPA counts are not quantitated.<br /> - The conclusion that crossover formation fails (based on MLH1 staining) is not justified. This marker does not appear in wt males until late pachytene, so if cells in this mutant are dying before that stage, MLH1 cannot be assessed.<br /> - The authors state that gH2AZ persists in the KO, but I'm not convinced that they are comparing equivalent stages in the wt and KO. In Figure 3C, the pachytene cell is late, whereas in the mutant the pachytene cell is early or mid (when residual gH2AX is expected, even in wt males).<br /> - Previous work (PMID: 23824539) has shown that antibodies reportedly detecting pATM in the sex body are non-specific. I therefore advise caution with the data shown in Figure 3D.

(4) RNAseq data. The authors show convincingly that Znhit1 activates genes that are normally upregulated at the zyg-pachytene transition. They should repeat the analysis for genes normally upregulated at the prelep- lep and lep-zyg transition to show that this effect is really pachytene-gene specific.

(5) I am puzzled that the title and overall gist of the study focuses on H2A.Z, when it is Znhit1 that has been deleted.

Reviewer #2 (Public Review):

Summary:

In this work, the authors attempt to noninvasively image metabolic aspects of the tumor microenvironment in vivo, in 2 mouse models of glioblastoma. The tumor lesion and its surrounding appearance are extensively characterized using histology to validate/support any observations made with the metabolic imaging approach. The metabolic imaging method builds on a previously used approach by the authors and others to measure the kinetics of deuterated glucose metabolism using dynamic 2H magnetic resonance spectroscopic imaging (MRSI), supported by de-noising methods.

Strengths:

Extensive histological evaluation and characterization.

Measurement of the time course of isotope labeling to estimate absolute flux rates of glucose metabolism.

Weaknesses:

The de-noising method appears essential to achieve the high spatial resolution of the in vivo imaging to be compatible with the dimensions of the tumor microenvironment, here defined as the immediately adjacent rim of the mouse brain tumors. There are a few challenges with this approach. Often denoising methods applied to MR spectroscopy data have merely a cosmetic effect but the actual quantification of the peaks in the spectra is not more accurate than when applied directly to original non-denoised data. It is not clear if this concern is applicable to the denoising technique applied here. However, even if this is not an issue, no denoising method can truly increase the original spatial resolution at which data were acquired. A quick calculation estimates that the spatial resolution of the 2H MRSI used here is 30-40 times too low to capture the much smaller tumor rim volume, and therefore there is concern that normal brain tissue and tumor tissue will be the dominant metabolic signal in so-called tumor rim voxels. This means that the conclusions on metabolic features of the (much larger) tumor are much more robust than the observations attributed to the (much smaller) tumor microenvironment/tumor rim.

To achieve their goal of high-level metabolic characterization the authors set out to measure the deuterium labeling kinetics following an intravenous bolus of deuterated glucose, instead of the easier measurement of steady-state after the labeling has leveled off. These dynamic data are then used as input for a mathematical model of glucose metabolism to derive fluxes in absolute units. While this is conceptually a well-accepted approach there are concerns about the validity of the included assumptions in the metabolic model, and some of the model's equations and/or defining of fluxes, that seem different than those used by others.

Reviewer #2 (Public Review):

This study from Dr. Emura and colleagues addresses the relevance of AGS3 mutations in the execution of asymmetric cell divisions promoting the formation of the micromere during sea-searching development. To this aim, the authors use quantitative imaging approaches to evaluate the localisation of AGS3 mutants truncated at the N-terminal region or at the C-terminal region, and correlate these distributions with the formation of micromere and correct development of embryos to the pluteus stage. The authors also analyse the capacity of these mutated proteins to rescue developmental defects observed upon AGS3 depletion by morpholino antisense nucleotides (MO). Collectively these experiments revealed that the C-terminus of AGS3, coding for four GoLoco motifs binding to cortical Gaphai proteins, is the molecular determinant for cortical localisation of AGS3 at the micromeres and correct pluteus development. Further genetic dissections and expression of chimeric AGS3 mutants carrying shuffled copies of the GoLoco motifs or four copies of the same motifs revealed that the position of GoLoco1 is essential for AGS3 functioning. To understand whether the AGS3-GoLoco1 evolved specifically to promote asymmetric cell divisions, the authors analyse chimeric AGS3 variants in which they replaced the sea urchin GoLoco region with orthologs from other echinoids that do not form micromeres, or from Drosophila Pins or human LGN. These analyses corroborate the notion that the GoLoco1 position is crucial for asymmetric AGS3 functions. In the last part of the manuscript, the authors explore whether SpAGS3 interacts with the molecular machinery described to promote asymmetric cell division in eukaryotes, including Insc, NuMA, Par3, and Galphai, and show that all these proteins colocalize at the nascent micromere, together with the fate determinant Vasa. Collectively this evidence highlighted how evolutionarily selected AGS3 modifications are essential to sustain asymmetric divisions and specific developmental programs associated with them.

Reviewer #2 (Public Review):

Summary:

In this work, the authors show that the camelid single-chain antibody sdAb42 selectivity inhibits Trypanosome pyruvate kinase (PYK) but not human PYK. Through the determination of the crystal structure and biophysical experiments, the authors show that the nanobody binds to the inactive T-state of the enzyme, and in silico analysis shows that the binding site coincides with an allosteric hotspot, suggesting that nanobody binding may affect the enzyme active site. Binding to the T-state of the enzyme is further supported by non-linear inhibition kinetics. PYK is an important enzyme in the glycolytic pathway, and inhibition is likely to have an impact on organisms such a trypanosomes, that heavily rely on glycolysis for their energy production. The nanobody was generated against Trypanosoma congolense PYK, but for technical reasons the authors progressed to testing its impact on cell viability in Trypanosoma brucei brucei. First, they show that sdA42 is able to inhibit Tbb PYK, albeit with lower potency. Cell-based experiments next show that expression of sdA42 has a modest, and dose-dependent effect on the growth rate of Tbb. The authors conclude that their data indicates that targeting this allosteric site affects cell growth and is a valuable new option for the development of new chemotherapeutics for trypanosomatid diseases.

Strengths:

The work clearly shows that sdA42A inhibits Trypanosome and Leishmania PYK selectively, with no inhibition of the human orthologue. The crystal structure clearly identifies the binding site of the nanobody, and the accompanying analysis supports that the antibody acts as an allosteric inhibitor of PYK, by locking the enzyme in its apo state (T-state).

Weaknesses:

(1) The most impactful claim of this work is that sdAb42-mediated inhibition of PYK negatively affects parasite growth and that this presents an opportunity to develop novel chemotherapeutics for trypanosomatid diseases. For the following reasons I think this claim is not sufficiently supported:

- The authors do not provide evidence of target-engagement in cells, i.e. they do not show that sdA42A binds to, or inhibits, Tbb PYK in cells and/or do not provide a functional output consistent with PYK inhibition (e.g. effect on ATP production). Measuring the extent of target engagement and inhibition is important to draw conclusions from the modest effect on growth.

- The authors do not explore the selectivity of sdA42A in cells. Potentially sdA42A may cross-react with other proteins in cells, which would confound interpretation of the results.

- sdA42A only affects minor growth inhibition in Tbb. The growth defect is used as the main evidence to support targeting this site with chemotherapeutics, however based on the very modest effect on the parasites, one could reasonably claim that PYK is actually not a good drug target. The strongest effect on growth is seen for the high expressor clone in Figure 4a, however here the uninduced cells show an unusual profile, with a sudden increase in growth rate after 4 days, something that is not seen for any of the other control plots. This unexplained observation accentuates the growth difference between induced and uninduced, and the growth differences seen in all other experiments, including those with the highest expressors (clones 54 and 55) are much more modest. The loss of expression of sdA42A over time is presented as a reason for the limited effect, and used to further support the hypothesis that targeting the allosteric site is a suitable avenue for the development of new drugs. However, strong evidence for this is missing.

- For chemotherapeutic interventions to be possible, a ligandable site is required. There is no analysis provided of the antibody binding site to indicate that small molecule binding is indeed feasible.

(2) The authors comment on the modest growth inhibition, and refer to the need to achieve over 88% reduction in Vmax of PYK to see a strong effect, something that may or may not be achieved in the cell-based model (no target-engagement or functional readout provided). The slow binding model and switch of species are also raised as potential explanations. While these may be plausible explanations, they are not tested which leaves us with limited evidence to support targeting the allosteric site on PYK.

(3) The evidence to support an allosteric mechanism is derived from structural studies, including the in silico allosteric network predictions. Unfortunately, standard enzyme kinetics mode of inhibition studies are missing. Such studies could distinguish uncompetitive from non-competitive behaviour and strengthen the claim that sdAb42 locks the enzyme complex in the apo form.

(4) As general comment, the graphical representation of the data could be improved in line with recent recommendations: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128, https://elifesciences.org/inside-elife/5114d8e9/webinar-report-transforming-data-visualisation-to-improve-transparency-and-reproducibility.

- Bar-charts for potency are ideally presented as dot plots, showing the individual data points, or box plots with datapoints shown.

- Images in Figure 7 show significant heterogeneity of nanobody expression, but the extent of this can not be gleaned from Figure 7B. It would be much better to use box plots or violin plots for each cell line on this figure panel. The same applies to Figure 10.