Reviewer #1 (Public Review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modeling to demonstrate that EPNSst+ neural activity represents movement, choice direction, and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modeling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Figures 1 F-J, Supplementary Figures 1 F, K, etc i.e via the inclusion of faint hash lines connecting individual data points across variables. Additionally, Figure 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, Figure 3C clearly shows that both SXcontra and SXipsi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high vs. low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results is striking. However, this relies on the co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters, etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well-learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitation is transparently discussed by the authors.

Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization, it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduce generalizability and to some extent the impact of their findings.

Warren Zevon at a JP-1 style typewriter in 1977 photo by Joel Bernstein

via https://www.1stdibs.com/art/photography/black-white-photography/joel-bernstein-warren-zevon-1977/id-a_5543612/

Reviewer #1 (Public review):

Using the UK Biobank, this study assessed the value of nuclear magnetic resonance measured metabolites as predictors of progression to diabetes. The authors identified a panel of 9 circulating metabolites that improved the ability in risk prediction of progression from prediabetes to diabetes. In general, this is a well-performed study, and the findings may provide a new approach to identifying those at high risk of developing diabetes.

Comments on the revised version:

Thanks so much for carefully addressing my comments.

Reviewer #1 (Public review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used<br /> (2) This study combines the behavioral tests, MRS, and fMRI.

Major<br /> I have no further comments. The approach and experiment are sound. The only overall drawback is the relatively low sample size.

Weaknesses:<br /> (1) Line 138, "This finding supports the hypothesis that motion perception is associated with neural activity in MT+ area". This sentence is strange because it is a well-established finding in numerous human fMRI papers. I think the authors should be more specific about what this finding implies.

Response: We thank reviewer for pointing this out. We have revised it to:" This finding is in line with prior results, which indicates that motion perception is associated with neural activity in hMT+ area, but not in EVC (primarily in V1)" (lines 156-158)

Reply: This argument should be refined. Numerous studies have shown the key role of V1 in motion perception. V1 contains a vast proportion of direction selective neurons. I am asking how your results here are related to existing literature. This argument is incorrect and too rough. Can you please revise this?

(9) Line 213, as far as I know, the study (Melnick et al., 2013) is a psychophysical study and did not provide evidence that the spatial suppression effect is associated with MT+.

Response: We thank reviewer for pointing this out. It was a mistake to use this reference, and we have revised it accordingly. (line 242)

Reply: Thanks. New citation is good. But that paper is a modeling study. The direct empirical evidence on humans should be as follow:

Tadin, D., Silvanto, J., Pascual-Leone, A. & Battelli, L. (2011) Improved motion perception and impaired spatial suppression following disruption of cortical area MT/V5. Journal of Neuroscience, 31, 1279-1283.

Reviewer #1 (Public Review):

Summary:

In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment. The manuscript has been substantially improved from the initial version by the addition of new experiments and clarification of important figures. Importantly, the overlap of data with previous reports from the same group has been corrected.

Strengths:

(1) Examination of multiple types of calcium and potassium currents, both through electrophysiology and molecular biology.

(2) Focus on arcuate kisspeptin neurons during the surge is relatively conceptually novel as the anteroventral periventricular nucleus (AVPV) kisspeptin neurons have received much more attention as the "surge generator" population.

(3) The modeling studies allow for direct examination of manipulation of single and multiple conductances, whereas the electrophysiology studies necessarily require examination of each current in isolation. Construction of an arcuate kisspeptin neuron model promises to be of value to the reproductive neuroendocrinology field.

Weaknesses:

A remaining weakness in this revised version of the manuscript is that the relevance of the CRISPR experiments is still rather tenuous given that the goal is to understand what happens in the estrogen-treatment condition, and these experiments were performed only in OVX mice. Similar concerns reflect that the computational model examining the effect of E2 infers multiple conductances based on qPCR data and an assumption that the conductances are directionally proportional to the level of gene expression, and then tunes these to the current recordings obtained from OVX mice, without a direct confirmation in OVX+E2 conditions that the model parameters accurately reflect the properties of these currents in the presence of estrogen.

Reviewer #1 (Public Review):

Summary:

An online database called MRAD has been developed to identify the risk or protective factors for AD.

Strengths:

This study is a very intriguing study of great clinical and scientific significance that provided a thorough and comprehensive evaluation with regard to risk or protective factors for AD. It also provided physicians and scientists with a very convenient, free as well as user-friendly tool for further scientific investigation.

Comments on revised version:

The authors have resolved all of my previous comments. It's a decent paper worth to be published in this field.

Reviewer #1 (Public Review):

Summary:

This is a reviewed manuscript submission to better understand mechanisms for why HIV individuals have diastolic dysfunction. Due to a lack of robust animal models, the team developed iPS-CM models to study HFpEF. The revised manuscript has toned down claims regarding diastolic function given the lack of mechanical testing. The team has focused on the altered Ca2+ phenotype, which improves the precision of the claims of the team. There remain questions on the functional relevance of the altered calcium handling given the lack of physiological assays. There also remain some questions about whether SGLT2 protein is expressed in these models without testing it, and whether the effects of SGLT2i could be off-target.

Overall, the revised manuscript is improved. I have no major remaining concerns except that the lack of biomechanical assessments diminishes the significance of the study as altered calcium alone would not be considered sufficient evidence for diastolic dysfunction, which was major task set out to answer by the group.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses: There are no major weaknesses.

Reviewer #2 (Public Review):

Summary:

The authors performed a systematic review and meta-analysis to investigate whether the frequency of emergence of resistance is different if combination antibiotic therapy is used compared to fewer antibiotics. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed.

Strengths:

The strength of the manuscript is that the article addresses a relevant research question which is often debated. The article is well-written and the methodology used is valid. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed. I have several comments and suggestions for the manuscript.

Weaknesses:

Weaknesses of the manuscript are the large clinical and statistical heterogeneity and the lack of clear definitions of acquisition of resistance. Both these weaknesses complicate the interpretation of the study results.

Comments on latest version:

The authors adressed all the comments that were shared in the previous peer review. I still believe that both clinical and statistical heterogeneity remains a problem with the interpretation of the meta-analysis. However, as the authors state, this is in line with the original research question as formulated on Prospero.

Reviewer #1 (Public Review):

Summary:

In this paper the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:

The experiments are comprehensive and clearly described, both in text and in the figures. Statistical analyses are provided throughout.

Weaknesses:

None.

Reviewer #1 (Public Review):

The study investigates Cancer Driving Nucleotides (CDNs) using the TCGA database, finding that these recurring point mutations could greatly enhance our understanding of cancer genomics and improve personalized treatment strategies. Despite identifying 50-150 CDNs per cancer type, the research reveals that a significant number remain undiscovered, limiting current therapeutic applications, and underscoring the need for further larger-scale research.

Strengths:

The study provides a detailed examination of cancer-driving mutations at the nucleotide level, offering a more precise understanding than traditional gene-level analyses. The authors found a significant number of CDNs remain undiscovered, with only 0-2 identified per patient out of an expected 5-8, indicating that many important mutations are still missing. The study indicated that identifying more CDNs could potentially significantly impact the development of personalized cancer therapies, improving patient outcomes.

Weaknesses:

The study is constrained by relatively small sample sizes for each cancer type, which reduces the statistical power and robustness of the findings. ICGC and other large-scale WGS datasets are publicly available but were not included in this study.

To be able to identify rare driver mutations, more samples are needed to improve the statistical power, which is well-known in cancer research.

The challenges in direct functional testing of CDNs due to the complexity of tumor evolution and unknown mutation combinations limit the practical applicability of the findings.

The QC of the TCGA data was not very strict, i.e, "patients with more than 3000 coding region point mutations were filtered out as potential hypermutator phenotypes", it would be better to remove patients beyond +/- 3*S.D from the mean number of mutations for each cancer type. Given some point mutations with >3 hits in the TCGA dataset, they were just false positive mutation callings, particularly in the large repeat regions in the human genome.

The codes for the statistical calculation (i.e., calculation of Ai_e, et al) are not publicly available, which makes the findings hard to be replicated.

Reviewer #1 (Public Review):

Calcium channels are key regulators of synaptic strength and plasticity, yet how these channels are differentially utilized to enable synaptic diversity is not clear. In this manuscript, the authors use new endogenous tagging of the Drosophila CaV2 channel Cac and three auxiliary subunits to investigate distinct calcium channel functions at two motor neuron subtypes at the fly NMJ, Is and Ib. Although it is clear from previous studies that Pr is higher at Is over Ib, it is not clear why. The authors confirm these differences using postsynaptic calcium imaging combined with post-hoc Cac-TdTomato imaging. Then, through a series of confocal and super resolution imaging studies, the authors describe differences in calcium channel and active zone structure between Is and Ib motor neuron terminals, and the role of Brp and homeostatic plasticity in regulating channel abundance. Finally, the authors show that while the CaBeta subunit is present at similar levels at Is and Ib active zones, there is an interesting reduction in Stj at Is active zones. The authors conclude that these differences in active zone structure and architecture contribute to the generation of the observed heterogeneity in synaptic strength.

Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. The key factors generating diversity in synaptic strength beyond simple Ca2+ influx are well articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

Comments on revision:

This is a much improved revised manuscript, where the authors have done an excellent job of responding to my initial concerns. In particular, the key factors generating diversity in synaptic strength beyond simple Ca2+ influx are better articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

Upon reflection, I think my initial review came across as a bit harsh, and I am happy to now update my original evaluation to better reflect the importance and impact of this very nice study. I commend the authors on an outstanding study.

Reviewer #1 (Public Review):

Greter et al. provide an interesting and creative use of lactulose as a "microbial metabolism" inducer, combined with tracking of H2 and other fermentation end products. The topic is timely and will likely be of broad interest to researchers studying nutrition, circadian rhythm, and gut microbiota. However, a couple of moderate to major concerns were noted that may impact the interpretation of the current data:

(1) Much of the data relies on housing gnotobiotic mice in metabolic cages, but I couldn't find any details of methods to assess contamination during multiple days of housing outside of gnotobiotic isolators/cages. Given the complexity of the metabolic cage system used, sterility would likely be incredibly challenging to achieve. More details needed to be included about how potential contamination of the mice was assessed, ideally with 16S rRNA gene sequencing data of the endpoint samples and/or qPCR for total colonization levels relative to the more targeted data shown.

(2) The language could be softened to provide a more nuanced discussion of the results. While lactulose does seem to induce microbial metabolism it also could have direct effects on the host due to its osmotic activity or other off-target effects. Thus, it seems more precise to just refer to lactulose specifically in the figure titles and relevant text. Additionally, the degree to which lactulose "disrupts the diurnal rhythm" isn't clear from the data shown, especially given that the markers of circadian rhythm rapidly recover from the perturbation. It is probably more precise to instead state that lactulose transiently induces fermentation during the light phase or something to that effect. The discussion could also be expanded to address what methods are available or could be developed to build upon the concepts here; for example, the use of genetic inducers of metabolism which may avoid the more complex responses to lactulose.

Despite these concerns, this was still an intriguing and valuable addition to the growing literature on the interface of the microbiome and circadian fields.

Reviewer #1 (Public Review):

Summary:

Recordings were made from the dentate nucleus of two monkeys during a decision-making task. Correlates of stimulus position and stimulus information were found to varying degrees in the neuronal activities.

Strengths:

A difficult decision-making task was examined in two monkeys.

Weaknesses:

One of the monkeys did not fully learn the task. The manuscript lacked a coherent hypothesis to be tested, and no attempt was made to consider the possibility that this part of the brain may have little to do with the task that was being studied.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments.

Strengths:<br /> The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear.

Weaknesses:<br /> I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments. Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.

The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis.

Reviewer #1 (Public Review):

Summary:

This study was to examine the associations of a healthy lifestyle with comprehensive and organ-specific biological ages. It emphasized the importance of lifestyle factors in biological ages, which were defined using common blood biomarkers and body measures.

Strengths:

The data were from a large cohort study and defined comprehensive and six-specified biological ages.

Weaknesses:

(1) Since only 8.5% of participants from the CMEC (China Multi-Ethnic Cohort Study) were included in the study, has any section bias happened?

(2) The authors should specify the efficiency of FFQ. How can FFQ genuinely reflect the actual intake? Moreover, how was the aMED calculated?

(3) HLI (range) and HLI (category) should be clearly defined.

(4) The comprehensive rationale and each specific BA construction should be clearly defined and discussed. For example, can cardiopulmonary BA be reflected only by using cardiopulmonary status? I do not think so.

(5) The lifestyle index is defined based on an equal-weight approach, but this does not reflect reality and cannot fully answer the research questions it raises.

Reviewer #1 (Public Review):

Summary:

The authors set out to understand the role played by a key global metabolic regulator called Crp/cAMP in the formation of persister Escherichia coli that survive antibiotic treatment without acquiring genetic mutations.

In order to achieve this aim, the authors employ an interdisciplinary approach exquisitely integrating standard microbiology assays with cutting-edge genomic, metabolomic, and proteomics screening.

The data presented by the authors convincingly demonstrate that the deletion of two key genes that are part of the Crp/cAMP complex (i.e. crp and cyaA) leads to a significant decrease in the number of persisters, thus pointing towards a key role played by the Crp/cAMP complex in the formation of persisters in E. coli.

The data presented also demonstrate that deletion of the crp gene leads to an overall decrease in energy metabolism and an overall increase in anabolic metabolism at the population level. It is not clear either what the contribution of the cyaA gene is in this respect, or why the deletion of cyaA has an opposite effect on cAMP concentration compared to crp deletion, although the authors present two reasonable untested hypotheses in the discussion. The authors might also want to explicitly acknowledge that these key data are obtained at the whole population level rather than at the level of the persister subpopulation.

Finally, the authors convincingly show that the persisters they investigated are non-growing and have a higher redox activity and that the deletion of key genes involved in energy metabolism leads to a decrease in the number of persisters.

These data will be key for future investigations on the biochemical mechanisms that allow bacteria to adapt to stressors such as nutrient depletion or exposure to antibiotics. As such this work will likely have an impact in a variety of fields such as bacterial biochemistry, antimicrobial resistance research, and environmental microbiology.

Strengths:

Interdisciplinary approach.<br /> Excellent use of replication and ensuring reproducibility.<br /> Excellent understanding and presentation of the biochemical mechanisms underpinning bacterial physiology via an integrated genomic, metabolomic, and proteomic screening.

Weaknesses:

Two genes from the Crp/cAMP complex (crp and cyaA) are hypothesised to be key for persistence but key metabolomics and proteomics data are obtained from only one deletion mutant in the crp gene.

The deletion of crp and cyaA have opposite effects on the concentration of cAMP, a comparison of metabolomics and proteomics data obtained using both mutants might aid in understanding this difference.

Metabolomics, proteomics, and metabolic activity data are obtained at the whole population level rather than at the level of the persister sub-population.

Joint Public Review:

Solitary Fibrous Tumors (SFTs) are a rare malignancy defined by NAB2-STAT6 fusions. Because the molecular understanding of the disease is largely lacking, there are currently no targeted treatment approaches. Using primary tumor and adjacent normal tissue samples and cells inducibly expressing NAB2-STAT6, Hill et al. perform a detailed characterization of the transcriptomic and epigenomic NAB2-STAT6 SFT signatures. They identify enrichment or EGR1/NAB2 (but not STAT6) sites bound by the fusion protein and increased expression of EGR1 targets. Their studies indicate that NAB2-STAT6 fusion may direct the nuclear translocation of NAB2 and EGR1 proteins and potentially NAB1. Transcriptionally, NAB2-STAT6 SFTs most closely resemble neuroendocrine tumors.

This pioneering study provides critical insight into the molecular pathogenesis of SFTs, pivotal for the future development of mechanistically informed treatment approaches. The study is rigorously executed and well-written. This new knowledge is an important addition to the field. Recommendations for minor improvements can be made.

Quotations and Literary Allusions spoken by Willy Wonka in the 1971 film, Willy Wonka and the Chocolate Factory<br /> by Thomas M. Brodhead<br /> https://bmt-systems.com/score/wonka.htm

Archived copy: https://web.archive.org/web/20200111135336/https://bmt-systems.com/score/wonka.htm

Reviewer #1 (Public Review):

Summary:

The overall goal of the manuscript is to delineate pathways that are conditionally essential with the Bam complex and associated chaperones. The Bam complex is made of several proteins, including BamA and BamD, which are essential. The protein complex works to insert proteins in the asymmetric outer membrane. Substrates are translated in the cytoplasm prior to transport across the cell envelope to the Bam complex. Transport includes non-essential periplasmic chaperones, SurA, Skp, and DegP. According to the authors, the pathways were assumed to be redundant. The Bam complex also includes non-essential components, BamBCE. These were thought to be accessory components that interact with BamA and BamD to coordinate optimal activity. While some roles have been assigned to BamE and BamB, a detailed understanding of the role of each accessory Bam protein is lacking. In this study, more specific roles for each non-essential Bam component are proposed.

Strengths:

The overall findings are intriguing and could advance our understanding as to how the Gram-negative cell envelope is assembled. These studies could provide new targets for antimicrobial treatment. In general, the manuscript was well-written.

Weaknesses:

While the overall findings are interesting, I had some concerns with the data analysis, presentation, and conclusions. Not all the conclusions are supported by data. The proposed revisions include experimental and editorial work. The manuscript is generally well-written and could provide impactful data to advance the field if the concerns are addressed.

Major concerns:

Overall Comments:

(1) The cutoffs the authors used to define "conditionally essential" mutants are not reported. The results also lack validation for lethality using a titratable system. It would be ideal to validate several genes in each dataset to determine cutoffs (i.e. 5-fold decrease in insertion mutants) for conditional lethality. It was not done (or described) here.

(2) Also, two mutations that both make the cells sick could provide an additive effect (i.e. dapF and BamB), which doesn't necessarily mean the pathways are linked. The authors should revise their wording. They have not shown genetic linkage in some cases.

(3) Mutations throughout the manuscript are not complemented. It would be ideal to add complementation data to show the gene-phenotype relationship is specific.

(4) Also, I would argue the term "conditionally essential genes" should be replaced with "synthetically lethal". Strains were compared in the same conditions but with different genetic backgrounds.

Reviewer #1 (Public Review):

Summary:

This study explores the roles of dact1 and dact2 in zebrafish embryonic axis formation and craniofacial morphogenesis. The researchers aim to uncover the mechanisms by which dact1/2 modulates Wnt signaling during embryonic development and patterning. They propose distinct spatiotemporal roles for Dact1 and Dact2 proteins in zebrafish embryonic development, particularly their involvement in modulating noncanonical Wnt signaling during convergent extension events. The findings demonstrate that dact1 and dact2 have unique spatiotemporal expression domains during development and that mutations in dact1/2 lead to convergent extension defects. Furthermore, the study attempts to link these defects to craniofacial abnormalities resulting from dact1/2 mutations. Compound mutants were used to investigate the connection between dact1 and dact2, as single mutants did not exhibit craniofacial phenotypes. The research also includes comprehensive transcriptomics and pathway analyses of differentially expressed genes in dact1/2 mutants, revealing the overexpression of calpain 8, a calcium-dependent cysteine protease. The study suggests that the upregulation of calpain 8 is linked to the observed craniofacial dysmorphology in dact1/2 mutants, implying a potential connection between calpain 8 expression and craniofacial abnormalities.

Strengths:

• The study effectively recapitulates previous findings on the role of dact1/2 in modulating convergent extension during zebrafish embryogenesis.<br /> • A combination of multiple approaches, including in vivo time-lapse imaging, is used to elucidate the etiology of the rod-like neurocranial phenotype in dact1/2 double mutants.<br /> • The study utilizes both traditional and newly created mutant lines, analyzing them through single-cell transcriptomics.

Weaknesses:

(1) The authors successfully addressed reviewers' suggestions with revised experiments and explanations. However, the overall narrative struggles to build a more coherent storyline.<br /> (2) The potential activity of truncated and upregulated dact mRNAs (Fig S2) and partially functional dact proteins needs further clarification.<br /> (3) Data-rich figures, specifically Figs 6, 7, and 8D, could be simplified for better clarity.

Reviewer #1 (Public Review):

In this manuscript, Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

Reviewer #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to the identification of a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as, for example, species-specific gamete recognition. Additionally, the use of a TPM >1 threshold for protein-coding transcripts - which, as the authors pointed out, was a necessary compromise due to the high transcriptional noise of the system under study - may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis. Moreover, as mentioned in the Discussion, future variations of the pipeline described in the manuscript may allow us to extend the reach of the present analysis.

Reviewer #2 (Public Review):

Summary:

The authors used state-of-the-art microscopy to analyze the structural changes that occur in sperm tails after the acrosome reaction. They found that midpiece contraction and actin reorganization occurred, which is associated with the cessation of flagellar motility during sperm-egg fusion. The mechanism by which flagellar motility is arrested during sperm-oocyte fusion is unknown, and this study proposes its novel mechanism and provides important insights for cell and reproductive biologists.

In the revised manuscript, the authors addressed most of my concerns.

Strength:

Various microscopy techniques including super-resolution microscopy and scanning electron microscopy were used to analyze structural organization of the midpiece in detail.

Reviewer #1 (Public Review):

Summary

This study was designed to investigate changes in gene expression and associated chromatin accessibility patterns in spermatogonia in mice at different postnatal stages from pups to adults. The objective was to describe dynamic changes in these patterns that potentially correlate with functional changes in spermatogonia as a function of development and reproductive maturation. The potential utility of this information is to serve as a reference against which similar data from animals subjected to various disruptive environmental influences can be compared.

Major Strengths and Weaknesses of the Methods and Results

A strength of the study is that it reviews previously published datasets describing gene expression and chromatin accessibility patterns in mouse spermatogonia. A weakness of the study is that it is not clear what new information is provided by the data provided that was not already known from previously published studies (see below). Specific weaknesses include the following...

- Terminology - In the Abstract and first part of the Introduction the authors use the generic term "spermatogonial cells" in a manner that seems to be referring primarily to spermatogonial stem cells (SSCs) but initially ignores the well-known heterogeneity among spermatogonia - particularly the fact that only a small proportion of developing spermatogonia become SSCs - and ONLY those SSCs and NOT other developing spermatogonia - support steady-state spermatogenesis by retaining the capacity to either self-renew or contribute to the differentiating spermatogenic lineage throughout the male reproductive lifespan. The authors eventually mention other types of developing male germ cells, but their description of prospermatogonial stages that precede spermatogonial stages is deficient in that M-prospermatogonia - which occur after PGCs but before T1-prospermatogonia - are not mentioned. This description also seems to imply that all T2-prospermatogonia give rise to SSCs which is far from the case. It is the case that prospermatogonia give rise to spermatogonia, but only a very small proportion of undifferentiated spermatogonia form the foundational SSCs and ONLY SSCs possess the capacity to either self-renew or give rise to sequential waves of spermatogenesis.

- Introduction - Statements regarding distinguishing transcriptional signatures in spermatogonia at different postnatal stages appear to refer to ALL subtypes of spermatogonia present at each stage collectively, thereby ignoring the well-known fact that there are distinct spermatogonial subtypes present at each postnatal stage and that some of those occur at certain stages but not at others. This brings into question the usefulness of the authors' discussion of what types of genes are expressed and/or what types of changes in chromatin accessibility are detected in spermatogonia at each stage.

- Methodology - The authors based recovery (enrichment) of spermatogonia from male pups on FACS sorting for THY1 and RMV-1. While sorting total testis cells for THY1+ cells does enrich for spermaogonia, this approach is now known to not be highly specific for spermatogonia (somatic cells are also recovered) and definitely not for SSCs. There are more effective means for isolating SSCs from total testis cells that have been validated by transplantation experiments (e.g. use of the Id4/eGFP transgene marker).

The authors then used "deconvolution" of bulk RNA-seq data in an attempt to discern spermatogonial subtype-specific transcriptomes. It is not clear why this is necessary or how it is beneficial given the availability of multiple single-cell RNA-seq datasets already published that accomplish this objective quite nicely - as the authors essentially acknowledge. Beyond this concern, a potential flaw with the deconvolution of bulk RNA-seq data is that this is a derivative approach that requires assumptions/computational manipulations of apparent mRNA abundance estimates that may confound interpretation of the relative abundance of different cellular subtypes within the hetergeneous cell population from which the bulk RNA-seq data is derived. Bottom line, it is not clear that this approach affords any experimental advantage over use of the publicly available scRNA-seq datasets and it is possible that attempts to employ this approach may be flawed yielding misleading data.

- Results & Discussion - In general, much of the information reported in this study is not novel. The authors' discussion of the makeup of various spermatogonial subtypes in the testis at various ages does not really add anything to what has been known for many years on the basis of classic morphological studies. Further, as noted above, the gene expression data provided by the authors on the basis of their deconvolution of bulk RNA-seq data does not add any novel information to what has been shown in recent years by multiple elegant scRNA-seq studies - and, in fact, as also noted above - represents an approach fraught with potential for misleading results. The potential value of the authors' report of "other cell types" not corresponding to major somatic cell types identified in earlier published studies seems quite limited given that they provide no follow-up data that might indicate the nature of these alternative cell types. Beyond this, much of the gene expression and chromatin accessibility data reported by the authors - by their own admission given the references they cite - is largely confirmatory of previously published results. Similarly, results of the authors' analyses of putative factor binding sites within regions of differentially accessible chromatin also appear to confirm previously reported results. Ultimately, it is not at all novel to note that changes in gene expression patterns are accompanied by changes in patterns of chromatin accessibility in either related promoters or enhancers. The discussion of these observations provided by the authors takes on more of a review nature than that of any sort of truly novel results. As a result, it is difficult to discern how the data reported in this manuscript advance the field in any sort of novel or useful way beyond providing a review of previously published studies on these topics.

Likely impact - The likely impact of this work is relatively low because, other than the value it provides as a review of previously published datasets, the new datasets provided are not novel and so do not advance the field in any significant manner.

Reviewer #1 (Public Review):

Summary:<br /> The manuscript provides a novel method for the automated detection of scent marks from urine and feces in rodents. Given the importance of scent communication in these animals and their role as model organisms, this is a welcome tool.

Strengths:<br /> The method uses a single video stream (thermal video) to allow for the distinction between urine and feces. It is automated.

Weaknesses:<br /> The accuracy level shown is lower than may be practically useful for many studies. The accuracy of urine is 80%. This is understandable given the variability of urine in its deposition, but makes it challenging to know if the data is accurate. If the same kinds of mistakes are maintained across many conditions it may be reasonable to use the software (i.e., if everyone is under/over counted to the same extent). Differences in deposition on the scale of 20% would be challenging to be confident in with the current method, though differences of the magnitude may be of biological interest. Understanding how well the data maintain the same relative ranking of individuals across various timing and spatial deposition metrics may help provide further evidence for the utility of the method.

Reviewer #1 (Public Review):

Summary:

De Waele et al. reported a dual-branch neural network model for predicting antibiotic resistance profiles using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry data. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF mass spectrometry data and their associated antibiotic susceptibility profiles. The authors used dual branch neural network to simultaneously represent information about mass spectra and antibiotics for a wide range of species and antibiotic combinations. The authors showed consistent performance of their strategy to predict antibiotic susceptibility for different spectrum and antibiotic representations (i.e., embedders). Remarkably, the authors showed how small datasets collected at one location can improve the performance of a model trained with limited data collected at a second location. The authors also showed that species-specific models (trained in multiple antibiotic resistance profiles) outperformed both the single recommender model and the individual species-antibiotic combination models. Despite the promising results, the authors should explain in more detail some of the analyses reported in the manuscript (see weaknesses).

Strengths:

• A single AMR recommender system could potentially facilitate the adoption of MALDI-TOF based antibiotic susceptibility profiling into clinical practices by reducing the number of models to be considered, and the efforts that may be required to periodically update them.<br /> • Authors tested multiple combinations of embedders for the mass spectra and antibiotics while using different metrics to evaluate the performance of the resulting models. Models trained using different spectrum embedder-antibiotic embedder combinations had remarkably good performance for all tested metrics. The average ROC AUC scores for global and species-specific evaluations were above 0.8.<br /> • Authors developed species-specific recommenders as an intermediate layer between the single recommender system and single species-antibiotic models. This intermediate approach achieved maximum performance (with one type of the species-specific recommender achieving a 0.9 ROC AUC), outlining the potential of this type of recommenders for frequent pathogens.<br /> • Authors showed that data collected in one location can be leveraged to improve the performance of models generated using a smaller number of samples collected at a different location. This result may encourage researchers to optimize data integration to reduce the burden of data generation for institutions interested in testing this method.

Weaknesses:

• Section 4.3 ("expert baseline model"): the authors need to explain how the probabilities defined as baselines were exactly used to predict individual patient susceptible profiles.<br /> • Authors do not offer information about the model features associated with resistance. Although I understand the difficulty of mapping mass spectra to specific pathways or metabolites, mechanistic insights are much more important in the context of AMR than in the context of bacterial identification. For example, this information may offer additional antimicrobial targets. Thus, authors should at least identify mass spectra peaks highly associated with resistance profiles. Are those peaks consistent across species? This would be a key step towards a proteomic survey of mechanisms of AMR. See previous work on this topic: PMIDs: 35586072 and 23297261.

Joint Public Review:

The study offers a compelling molecular model for the organization of rootlets, a critical organelle that links cilia to the basal body. Striations have been observed in rootlets, but their assembly, composition, and function remain unknown. While previous research has explored rootlet structure and organization, this study delivers an unprecedented level of resolution, valuable to the centrosome and cilia field. The authors isolated rootlets from mice's eyes. They apply EM to partially purified rootlets (first negative stain, then cryoET). From these micrographs, they observed striations along the membranes along the rootlet but no regular spacing was observed.

The thickness of the sample and membranes prevented good contrast in the tomograms. Thus they further purified the rootlets using detergent, which allowed them to obtain cryoET micrographs of the rootlets with greater details. The tomograms were segmented and further processed to improve the features of the rootlet structures. They proposed that a number of proteins, including rootletin, form parallel coiled coils that run along the rootlet longitudinally. They described how the cross-striations form 3 types of periodic structures -D1/D2/A bands- connected perpendicularly to filaments along the length of the rootlets and to membranes. Overall their data provide a detailed model for the molecular organization of the rootlet.

The major strength is that this high-quality study uses state-of-the-art cryo-electron tomography, sub-tomogram averaging, and image analysis to provide a model of the molecular organization of rootlets. The micrographs are exceptional, with excellent contrast and details, which also implies the sample preparation was well optimized to provide excellent samples for cryo-ET. The manuscript is also clear and accessible.

This research marks a significant step forward in our understanding of rootlets' molecular organization.

Reviewer #1 (Public review):

Summary:

Ctnnb1 encodes β-catenin, an essential component of the canonical Wnt signaling pathway. In this study, the authors identify an upstream enhancer of Ctnnb1 responsible for the specific expression level of β-catenin in the gastrointestinal track. Deletion of this promoter in mice and analyses of its association with human colorectal tumors support that it controls the dosage of Wnt signaling critical to the homeostasis in intestinal epithelia and colorectal cancers.

Strengths:

This study has provided convincing evidence to demonstrate the functions of a gastrointestinal enhancer of Ctnnb1 using combined approaches of bioinformatics, genomics, in vitro cell culture models, mouse genetics, and human genetics. The results support the idea that the dosage of Wnt/β-catenin signaling plays an important role in pathophysiological functions of intestinal epithelia. The experimental designs are solid and the data presented are of high quality. This study significantly contributes to the research fields of Wnt signaling, tissue-specific enhancers, and intestinal homeostasis.

Weaknesses:

Insufficient discussion on some findings was a major weakness in the previous submission, which has been addressed in the revised submission.

Reviewer #1 (Public Review):

The authors developed a rigorous methodology for identifying all Cancer Driving Nucleotides (CDNs) by leveraging the concept of massively repeated evolution in cancer. By focusing on mutations that recur frequently in pan-cancer, they aimed to differentiate between true driver mutations and neutral mutations, ultimately enhancing the understanding of the mutational landscape that drives tumorigenesis. Their goal was to call a comprehensive catalogue of CDNs to inform more effective targeted therapies and address issues such as drug resistance.

Strengths

(1) The authors introduced a concept of using massively repeated evolution to identify CDNs. This approach recognizes that advantageous mutations recur frequently (at least 3 times) across cancer patients, providing a lens to identify true cancer drivers.

(2) The theory showed the feasibility of identifying almost all CDNs if the number of sequenced patients increases to 100,000 for each cancer type.

Weaknesses

(1) The methodology remains theoretical and no novel true driver mutations were identified in this study.

(2) Different cancer types have unique mutational landscapes. The methodology, while robust, might face challenges in uniformly identifying CDNs across various cancers with distinct genetic and epigenetic contexts.

(3) L223, the statement "In other words, the sequences surrounding the high-recurrence sites appear rather random.". Since it was a pan-cancer analysis, the unique patterns of each cancer type could be strongly diluted in the pan-cancer data.

(4) To solidify the findings, the results need to be replicated in an independent dataset.

(5) The key scripts and the list of key results (i.e., CDN sites with i{greater than or equal to}3) need to be shared to enable replication, validation, and further research. So far, only CDN sites with i{greater than or equal to}20 have been shared.

(6) The versions of data used in this study are not clearly detailed, such as the specific version of gnomAD and the version and date of TCGA data downloaded from the GDC Data Portal.

Reviewer #1 (Public Review):

Summary:

Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

Strengths:

The CAT-tailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. The conclusions of this paper are mostly well-supported by data, but some aspects of image acquisition and data analysis need to be clarified and extended.

Reviewer #1 (Public Review):

Summary:

This work contributes several important and interesting observations regarding the heterotolerance of non-growing Escherichia coli and Pseudomonas aeruginosa to the antimicrobial peptide tachyplesin. The primary mechanism of action of tachyplesin is thought to be disruption of the bacterial cell envelope, leading to leakage of cellular contents after a threshold level of accumulation. Although the MIC for tachyplesin in exponentially growing E. coli is just 1 ug/ml, the authors observe that a substantial fraction of a stationary phase population of bacteria survive much higher concentrations, up to 64 ug/ml. By using a fluorescently-labelled analogue of tachyplesin, the authors show that the amount of per-cell intracellular accumulation of tachyplesin displays a bimodal distribution and that the fraction of "low accumulators" correlates with the fraction of survivors.

Using a microfluidic device, they show that low accumulators exclude propidium iodide, suggesting that their cell envelopes remain largely intact, while high accumulators of tachyplesin also stain with propidium iodide. They show that this phenomenon holds for several clinical isolates of E. coli with different genetic determinants of antibiotic resistance, and for a strain of Pseudomonas aeruginosa. However, the bimodal distribution does not occur in these organisms for several other antimicrobial peptides, or for tachyplesin in Klebsiella pneumoniae or Staphylococcus aureus, indicating some degree of specificity in the interaction between AMP and bacterial cell envelope. They next explore the dynamics of the fluorescent tachyplesin accumulation and show interestingly that a high degree of accumulation is initially seen in all cells, but that the "low accumulator" subpopulation manages to decrease the amount of intracellular fluorescence over time, while the "high accumulator" subpopulation continues to increase its intracellular fluorescence. Focusing on increased efflux as a hypothesised mechanism for the "low accumulator" phenotype, based on transcriptomic analysis of the two subpopulations, the authors screen putative efflux inhibitors to see if they can block the formation of the low accumulator subpopulation. They find that both the protonophore CCCP and the SSRI sertraline can block the formation of this subpopulation and that a combination of sertraline plus tachyplesin kills a greater fraction of the stationary phase cells than either agent alone, similar to the killing observed when growing cells are treated with tachyplesin.

Strengths:

This study provides new insight into the heterogeneous behaviours of non-growing bacteria when exposed to an antimicrobial peptide, and into the dynamics of their response. The single-cell analysis by FACS and microscopy is compelling. The results provide a much-needed single-cell perspective on the phenomenon of tolerance to AMPs and a good starting point for further exploration.

Weaknesses:

My main concerns surround the conclusions drawn about the physiological underpinnings of these behaviours, based in part on transcriptomic analysis and also on the observation of the dynamics. I think deeper consideration of the relative contributions of influx and efflux to the observed accumulation dynamics, and the slow/non-growing context of the observations would be helpful. In particular, these issues seem important:

(1) The initial high accumulation by all cells followed by the emergence of a sub-population that has reduced its intracellular levels of tachyplesin is a key observation and I agree with the authors' conclusion that this suggests an induced response to the AMP is important in facilitating the bimodal distribution. However, I think the conclusion that upregulated efflux is driving the reduction in signal in the "low accumulator" subpopulation is not fully supported. Steady-state amounts of intracellular fluorescent AMP are determined by the relative rates of influx and efflux and a decrease could be caused by decreasing influx (while efflux remained unchanged), increasing efflux (while influx remained unchanged), or both decreasing influx and increasing efflux. Given the transcriptomic data suggest possible changes in the expression of enzymes that could affect outer membrane permeability and outer membrane vesicle formation as well as efflux, it seems very possible that changes to both influx and efflux are important. The "efflux inhibitors" shown to block the formation of the low accumulator subpopulation have highly pleiotropic or incompletely characterised mechanisms of action so they also do not exclusively support a hypothesis of increased efflux.

(2) A conclusion of the transcriptomic analysis is that the lower accumulating subpopulation was exhibiting "a less translationally and metabolically active state" based on less upregulation of a cluster of genes including those involved in transcription and translation. This conclusion seems to borrow from well-described relationships referred to as bacterial growth laws in which the expression of genes involved in ribosome production and translation is directly related to the bacterial growth (and metabolic) rate. However, the assumptions that allow the formulation of the bacterial growth laws (balanced, steady state, exponential growth) do not hold in growth arrest. A non-growing cell could express no genes at all or could express ribosomal genes at a very low level, or efflux pumps at a high level. The distribution of transcripts among the functional classes of genes does not reveal anything about metabolic rates within the context of growth arrest - it only allows insight into metabolic rates when the constraint of exponential growth can be assumed. Efflux pumps can be highly metabolically costly; for example, Tn-Seq experiments have repeatedly shown that mutants for efflux pump gene transcriptional repressors have strong fitness disadvantages in energy-limited conditions. There are no data presented here to disprove a hypothesis that the low accumulators have high metabolic rates but allocate all of their metabolic resources to fortifying their outer membranes and upregulating efflux. This could be an important distinction for understanding the vulnerabilities of this subpopulation. Metabolic rates can be more directly estimated for single cells using respiratory dyes or pulsed metabolic labelling, for example, and these data could allow deeper insight into the metabolic rates of the two subpopulations.

The observation that adding nutrients to the stationary phase cultures pushes most of the cells to the "high accumulator" state is presented as support of the hypothesis that the high accumulator state is a higher metabolism/higher translational activity state. However, it is important to note that adding nutrients will cause most or all of the cells in the population to start to grow, thus re-entering the familiar regime in which bacterial growth laws apply. This is evident in the slightly larger cell sizes seen in the nutrient-amended condition. In contrast to stationary phase cells, growing cells largely do not exhibit the bimodal distribution, and they are much more sensitive to tachyplesin, as demonstrated clearly in the supplement. Growing cells are not necessarily the same as the high-accumulating subpopulation of non-growing cells.

It might also be worth adding some additional context around the potential to employ efflux inhibitors as therapeutics. It is very clear that obtaining sufficient antimicrobial drug accumulation within Gram-negative bacteria is a substantial barrier to effective treatments, and large concerted efforts to find and develop therapeutic efflux pump inhibitors have been undertaken repeatedly over the last 25 years. Sufficiently selective inhibitors of bacterial efflux pumps with appropriate drug-like properties have been challenging to find and none have entered clinical trials. Multiple psychoactive drugs have been shown to impact efflux in bacteria but usually using concentrations in the 10-100 uM range (as here). Meanwhile, the Ki values for their human targets are usually in the sub- to low-nanomolar range. The authors rightly note that the concentration of sertraline they have used is higher than that achieved in patients, but this is by many orders of magnitude, and it might be worth expanding a bit on the substantial challenge of finding efflux inhibitors that would be specific and non-toxic enough to be used therapeutically. Many advances in structural biology, molecular dynamics, and medicinal chemistry may make the quest for therapeutic efflux inhibitors more fruitful than it has been in the past but it is likely to remain a substantial challenge.

Reviewer #1 (Public Review):

Summary:

The study addresses the growing threat of multi-drug-resistant (MDR) pathogens, focusing on the efficacy of colistin (COL), a last-resort antibiotic, and its enhanced activity when combined with artesunate (AS) and ethylenediaminetetraacetic acid (EDTA) against colistin-resistant Salmonella strains. The researchers aim to explore whether these combinations can restore the effectiveness of colistin and understand the underlying mechanisms. The study used a combination of microbiological and molecular techniques to evaluate the antibacterial activity and mechanisms of action of COL, AS, and EDTA.

Key methods include:

(1) Antimicrobial Susceptibility Testing: Determining minimum inhibitory concentrations (MICs) of COL, AS, and EDTA, both alone and in combination, against various Salmonella strains;

(2) Time-Kill Assays: Measuring bacterial growth inhibition over time with different drug combinations;

(3) Fluorescent Probe-Permeability Assays: Assessing cell membrane integrity using fluorescent dyes;

(4) Proton Motive Force Assay: Evaluating the impact on the electrochemical proton gradient (PMF);

(5) Reactive Oxygen Species (ROS) Measurement: Quantifying intracellular ROS levels; (vi) Scanning Electron Microscopy (SEM): Observing morphological changes in bacterial cells; and

(6) Omics Analysis: Transcriptome and metabolome profiling to identify differentially expressed genes (DEGs) and significant differential metabolites (SDMs).

The combination of COL, AS, and EDTA (AEC) showed significant antibacterial activity against colistin-resistant Salmonella strains, reducing the MICs and enhancing bacterial killing compared to individual treatments. The AEC treatment caused extensive damage to both the outer and inner bacterial membranes, as evidenced by increased fluorescence of membrane-impermeant dyes and SEM images showing deformed cell membranes. AEC treatment selectively collapsed the Δψ component of PMF, indicating disruption of vital cellular processes. The combination therapy increased intracellular ROS levels, contributing to bacterial killing. Transcriptome data revealed changes in genes related to two-component systems, flagellar assembly, and ABC transporters. Metabolome analysis highlighted disruptions in pathways such as arachidonic acid metabolism. The findings suggest that AS and EDTA can potentiate the antibacterial effects of colistin by disrupting bacterial membranes, collapsing PMF, and increasing ROS levels. This combination therapy could serve as a promising approach to combat colistin-resistant Salmonella infections.

Strengths:

(1) The study employs a wide range of techniques to thoroughly investigate the antibacterial mechanisms and efficacy of the drug combinations.

(2) The results are consistent across multiple assays and supported by both in vitro and in vivo data.

(3) Combining AS and EDTA with COL represents a novel strategy to tackle antibiotic resistance.

Weaknesses:

(1) The study focuses on a limited number of Salmonella strains, and broader testing on various MDR pathogens would strengthen the findings.

(2) While the study elucidates several mechanisms, further molecular details could provide deeper insights into the interactions between these drugs and bacterial targets.

(3) The time-kill experiment was conducted over 12 hours instead of the recommended 24 hours. To demonstrate a synergistic effect among the drugs, a reduction of at least 2 log10 in colony count should be shown in a 24-hour experiment. Additionally, clarifying the criteria for selecting drug concentrations is important to improve the interpretation of the results.

(4) While the combination of EDTA, artesunate, and colistin shows promising in vitro results against Salmonella strains, the clinical application of this combination warrants careful consideration due to potential toxicity issues associated with these compounds.

Reviewer #1 (Public Review):

Summary:

In this study, the authors investigate the effect of mitochondrial transplantation on post-cardiac arrest myocardial dysfunction (PAMD), which is associated with mitochondrial dysfunction. The authors demonstrate that mitochondrial transplantation enhances cardiac function and increases survival rates after the return of spontaneous circulation (ROSC). Mechanistically, they found that myocardial tissues with transplanted mitochondria exhibit increased mitochondrial complex activity, higher ATP levels, reduced cardiomyocyte apoptosis, and lower myocardial oxidative stress post-ROSC.

Strengths:

Previous studies have reported that mitochondrial transplantation can improve myocardial recovery after regional ischemia, but its potential for treating myocardial injury following cardiac arrest has not been tested yet. Therefore, the findings are somewhat novel. Remarkably, the increased survival in mitochondria treated group post-ROSC is very promising and highlights its translational potential.

Weaknesses:

The organization of the paper, along with the analysis and interpretation of the results, requires significant revision.

Reviewer #1 (Public Review):

Drp1 supports mitochondrial fission (doi: 10.1038/s41586-019-1296-y). Viral sensing triggers mitochondrial fusion, leading to MAVS aggregation and improved type-1 IFN response. It was suggested that impairment of Drp1 upon phosphorylation by Tbk1 enhances mitochondrial fusion in virus-infected cells (doi.org/10.1016/j.molcel.2020.10.018). In this manuscript, Fang et al. describe an unexpected role of caspases activated upon Rift Valley fever virus (RVFV) infection in inactivating Drp1. They show that Drp1 is targeted by multiple caspases, including caspase-3, -6, -7 and -8. Indeed, cleavage of Drp1 leads to mitochondrial elongation, boosting the type-1 IFN response of infected cells. Finally, the authors establish the generalisability of the proposed mechanism in the context of cellular infections with H1N1, SeV, and HSV-1. Caspase-dependent and independent cell death processes provide important host defence mechanisms against obligatorily intracellular viral pathogens. This work suggests that caspases reinforce antiviral response involving also the mitochondria-type 1 IFN axis. As such, the manuscript is well written, and the proposal pertaining to caspase-mediated targeting of Drp1 may have implications beyond host-virus interaction studies. However, several loose ends remain, and these concerns need to be addressed to substantiate the mechanistic model.

Reviewer #1 (Public Review):

Summary:

The current manuscript uses electron spin resonance spectroscopy to understand how the dynamic behavior and conformational heterogeneity of the LPS transport system change during substrate transport and in response to the membrane, bound nucleotide (or transition state analog) and accessory subunits. The study builds on prior structural studies to expand our molecular understanding of this highly significant bacterial transport system.

Strengths

This series of well-designed and well-executed experiments provide new mechanistic insights into the dynamic behavior of the LPS transport system. Notable new insights provided by this study include its indication of the spatial organization of the LptC domain, which was poorly resolved in structures, and how the LptC domain modulates the dynamic behavior of the gate through which lipids access the binding site. In addition, a mass spectrometry approach designed to examine LPS binding at different stages in the nucleotide-dependent conformational cycle provides insight into the order of operations of LPS binding and transport.

Reviewer #1 (Public Review):

In this study, the Authors used a stopped-flow method to investigate the kinetics of substrate translocation through the channel in hexameric ClpB, an ATP-dependent bacterial protein disaggregase. They engineered a series of polypeptides with the N-terminal RepA ClpB-targeting sequence followed by a variable number of folded titin domains. The Authors detected translocation of the substrate polypeptides by observing the enhancement of fluorescence from a probe located at the substrate's C-terminus. The total time of the substrates' translocation correlated with their lengths, which allowed the Authors to determine the number of residues translocated by ClpB per unit time.

Strengths:

This study confirms a previously proposed model of processive translocation of polypeptides through the channel in ClpB. The novelty of this work is in a clever design of a series of kinetic experiments with an engineered substrate that includes stably folded domains. This approach produced a quantitative description of the reaction rates and kinetic step sizes. Another valuable aspect is that the method can be used for other translocases from the AAA+ family to characterize their mechanism of substrate processing.

Weaknesses:

The main limitation of the study is in using a single non-physiological substrate of ClpB, which does not replicate physical properties of the aggregated cellular proteins and includes a non-physiological ClpB-targeting sequence. Another limitation is in the use of ATPgammaS to stimulate the substrate processing. It is not clear how relevant the results are to the ClpB function in living cells with ATP as the source of energy, a multitude of various aggregated substrates without targeting sequences that need ClpB's assistance, and in the presence of the co-chaperones.

Evidence that ATPgammaS without ATP can provide sufficient energy for substrate translocation and unfolding is missing in the paper because the rate of phosphate release from ATPgammaS has not been determined. Thus, it is not clear if the observed translocation is linked to an actual chemical energy input or is a result of a diffusion-driven ratchet mediated by a substrate-trapping ClpB conformation obtained in the presence of ATPgammaS.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors delineate the crucial role of the SIRT2-ACSS2 axis in ACSS2 degradation. They demonstrate that SIRT2 acts as an ACSS2 deacetylase specifically under nutrient stress conditions, notably during amino acid deficiency. The SIRT2-mediated deacetylation of ACSS2 at K271 consequently triggers its proteasomal degradation. Additionally, they illustrate that acetylation of ACSS2 at K271 enhances ACSS2 protein levels, thereby promoting De Novo lipogenesis.

Strengths:

The findings presented in this manuscript are clearly interesting.

Weaknesses:

Further support is required for the model put forward by the authors.

Reviewer #1 (Public Review):

Summary:

The authors want to determine the role of the sperm hook of the house mouse sperm in movement through the uterus. They use transgenic lines with fluorescent labels to sperm proteins, and they cross these males to C57BL/6 females in pathogen-free conditions. They use 2-photon microscopy on ex vivo uteri within 3 hours of mating and the appearance of a copulation plug. There are a total of 10 post-mating uteri that were imaged with 3 different males. They provide 10 supplementary movies that form the basis for some of the quantitative analysis in the main body figures. Their data suggest that the role of the sperm hook is to facilitate movement along the uterine wall.

Strengths:

Ex vivo live imaging of fluorescently labeled sperm with 2-photon microscopy is a powerful tool for studying the behavior of sperm.

Weaknesses:

The paper is descriptive and the data are correlations.

The authors cannot directly test their proposed function of the sperm hook in sliding and preventing backward slipping.

Reviewer #1 (Public Review):

Summary:

This study investigated the co-option of IGF2BP2, an RNA binding protein by ZIKV proteins. Designed experiments evaluated if IFG2BP2 co-localized to sites of viral RNA replication, interacted with ZIKV proteins and how ZIKV infection changed the IGF2BP2 interactome.

Strengths:

The authors have used multiple interdisciplinary techniques to address several questions regarding the interaction of ZIKV proteins and IGF2BP2.

The findings could be exciting if concerns are addressed, specifically regarding how ZIKV infection alters the interactome of IGF2BP2.

Comments on thee revised version:

Following response to reviews, the authors have addressed a majority of the concerns with the exception of the western blots:

As requested in the previous review, the authors did quantify the western blot data for half of the blot in 2A, but did not quantify blots in D and E. Please quantify ALL blots. Also, the first two lanes of 2A. The same goes for 4A only infected is quantified, please quantify Mock as well. In the quantification of 4C, all lanes should be quantified, not only the NS5 from C. Also, unclear which lanes were quantified (H/PF/2013 or MR766)? Also, quantification needs to be generally shown as a graph and not included on top of the western blot.

Combined Public Reviews:

Summary:

This study presents an immunotherapeutic strategy for treating mouse cutaneous squamous cell carcinoma (mCSCC) using a passive immunity-like strategy. The researcher induced tumors in healthy mice skin, then isolated the tumor cells and injected into other healthy mice to produce anti-tumor antibodies, and then administered these antibodies back into tumor-bearing mice. Results showed a reduction in tumor volume and altered expression of several cancer markers (p53, Bcl-xL, NF-κB, Bax). The analysis of results suggests a promising impact of antibody-rich serum in treating mouse cutaneous squamous cell carcinoma (mCSCC).

Strengths:

The approach does seem to have effect on preventing tumor progression, from both the tumor size and the cancer hallmarks expression level.

Weaknesses:

Despite the strength of the study, there are a few drawbacks in the study design and statistical analysis:

(1) Regarding the statistical analysis, the use of a paired t-test might be suboptimal for assessing the trend from weeks 15 to 17. It is recommended to consider alternative methods such as repeated measures ANOVA or linear regression to better capture and interpret the trend over this time period.

(2) To affirm the antibodies' role in the observed immune response, isolating antibodies rather than employing whole serum could provide more conclusive evidence. Comparative analyses with antibody-free serum or serum from healthy, non-immunized mice would clarify antibodies' specific contributions versus other serum components. The control group does not account for the potential immunostimulatory effects of serum injection itself. A better control would be tumor-bearing mice receiving serum from healthy non-mCSCC-exposed mice.

Response to author's rebuttal:

I acknowledge the value of evaluating serum therapy as a whole, considering the complex interactive networks and potential synergies involved. However, to scientifically understand and assess serum therapy, it remains essential to decompose the serum and identify the effective components. This decomposition would allow for a comparison of individual components with the overall effectiveness, thereby elucidating any synergistic effects.<br /> While I agree that identifying specific epitopes and paratopes is indeed challenging and may exceed the scope of academic research, the use of methods such as Protein A purification or other techniques to isolate antibodies and cytokines from the serum is both necessary and feasible. This approach would enable a more detailed analysis of the individual effects of these components. I understand that the authors might not have that much resource, and I acknowledge this limitation. Nonetheless, other than this aspect, I believe the authors have adequately addressed my other concerns.

Reviewer #1 (Public Review):

In this study, Le Moigne and coworkers shed light on the structural details of the Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii. The SBPase is part of the Calvin cycle and catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate (SBP), which is a crucial step in the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate for Rubisco. The authors determine the crystal structure of the CrSBPase in an untreated, oxidized state. Based on this structure, potential active site residues and sites of post-translational modifications are identified. Furthermore, the authors determine the CrSBPase structure in a reduced state revealing the disruption of a disulfide bond in close proximity to the dimer interface. The authors then use molecular dynamics (MD) to gain insights into the redox-controlled dynamics of the CrSBPase and investigate the oligomerization of the protein using small-angle X-ray scattering (SAXS) and size-exclusion chromatography. Despite the difference in oligomerization, disruption of this disulfide bond did not impact the activity of CrSBPase, suggesting additional thiol-dependent regulatory mechanisms modulating the activity of the CrSBPase.

The authors provide interesting new findings on a redox-mechanism that modulates the oligomeric behavior of the SBPase. Comparisons of the Chlamydomonas structure to the previously determined SBPase structure from the moss Physcomitrium patens confirm a high structural similarity between the two proteins suggesting that this mechanism might be evolutionary conserved. Future research will have to address this question experimentally, also considering potential cooperativity between the subunits to confirm the link between oligomerization and SBPase activity.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weakness:

The authors provided insufficient experimental evidence to support their claim that GZMK+ NK cells contribute to worse prognosis for cancer patients or promote cancer progression. While one can visually observe an increased fraction of GZMK+ NK cells compared to KIR2DL4+ NK cells in cancer tissues, no quantification is shown. They did not present any preclinical (animal model) or clinical data suggesting a causal relationship between NK cells and tumour growth. Thus, while a correlation may exist between the presence of GZMK+ NK cells and poorer tumour prognosis, causation cannot be claimed based on the available evidence. Furthermore, the in vitro data provided is limited to a single NK cell line derived from a lymphoma patient, which does not fully represent the diversity and functionality of human NK cells.

Reviewer #1 (Public Review):

In the manuscript "Mechanistic target of rapamycin (mTOR) pathway in Sertoli cells regulates age-dependent changes in sperm DNA methylation", the authors proposed to test if the balance of mTOR complexes in Sertoli cells may play a significant role in age-dependent changes in the sperm epigenome. The paper could be of interest and has a good scientific aim but there are too many drawbacks that hamper the initial enthusiasm. All sections need extensive revision. The paper is mostly descriptive without a mechanistic-orientated explanation for the observed results.

Comments on revised version:

I am not sure that the authors have made an attempt to clearly answer the reviewers comments that aimed to improve the quality of the manuscript. It stands as mostly descriptive and with limited interest as it is.

Reviewer #1 (Public Review):

Summary:

A key challenge at the second chemical step of splicing is the identification of the 3' splice site of an intron. This requires recruitment of factors dedicated to the second chemical step of splicing and exclusion of factors dedicated to the first chemical step of splicing. Through the highest resolution cyroEM structure of the spliceosome to-date, the authors show the binding site for Fyv6, a factor dedicated to the second chemical step of splicing, is mutually exclusive with the binding site for a distinct factor dedicated to the first chemical step of splicing, highlighting that splicing factors bind to the spliceosome at a specific stage not only by recognizing features specific to that stage but also by competing with factors that bind at other stages. The authors further reveal that Fyv6 functions at the second chemical step to promote selection of 3' splice sites distal to a branch point and thereby discriminate against proximal, suboptimal 3' splice site. Lastly, the authors show by cyroEM that Fyv6 physically interacts with the RNA helicase Prp22 and by genetics Fyv6 functionally interacts with this factor, implicating Fyv6 in 3'SS proofreading and mRNA release from the spliceosome. The evidence for this study is robust, with the inclusion of genomics, reporter assays, genetics, and cyroEM. Further, the data overall justify the conclusions, which will be of broad interest.

Strengths:

(1) The resolution of the cryoEM structure of Fyv6-bound spliceosomes at the second chemical step of splicing is exceptional (2.3 Angstroms at the catalytic core; 3.0-3.7 Angstroms at the periphery), providing the best view of this spliceosomal intermediate in particular and the core of the spliceosome in general.<br /> (2) The authors observe by cryoEM three distinct states of this spliceosome, each distinguished from the next by progressive loss of protein factors and/or RNA residues. The authors appropriately refrain from overinterpreting these states as reflecting distinct states in the splicing cycle, as too many cyroEM studies are prone to do, and instead interpret these observations to suggest interdependencies of binding. For example, when Fyv6, Slu7, and Prp18 are not observed, neither are the first and second residues of the intron, which otherwise interact, suggesting an interdependence between 3' splice site docking on the 5' splice site and binding of these second step factors to the spliceosome.<br /> (3) Conclusions are supported from multiple angles.<br /> (4) The interaction between Fyv6 and Syf1, revealed by the cyroEM structure, was shown to account for the temperature-sensitive phenotypes of a fyv6 deletion, through a truncation analysis.<br /> (5) Splicing changes were observed in vivo both by indirect copper reporter assays and directly by RT-PCR.<br /> (6) Changes observed by RNA-seq are validated by RT-PCR.<br /> (7) The authors go beyond simply observing a general shift to proximal 3'SS usage in the fyv6 deletion by RNA-seq by experimentally varying branch point to 3' splice site distance experimentally in a reporter and demonstrating in a controlled system that Fyv6 promotes distal 3' splice sites.<br /> (8) The importance of the Fyv6-Syf1 interaction for 3'SS recognition is demonstrated by truncations of both Fyv6 and of Syf1.<br /> (9) In general, the study was executed thoroughly and presented clearly.

Weaknesses:

(1) Despite the authors restraint in interpreting the three states of the spliceosome observed by cyroEM as sequential intermediates along the splicing pathway, it would be helpful to the general reader to explicitly acknowledge the alternative possibility that the difference states simply reflect decomposition from one intermediate during isolation of the complex (i.e., the loss of protein is an in vitro artifact, if an informative one).<br /> (2) The authors acknowledge that for prp8 suppressors of the fyv6 deletion, suppression may be indirect, as originally proposed by the Query and Konarska labs - that is, that defects in the second step conformation of the spliceosome can be indirectly suppressed by compensating, destabilizing mutations in the first step spliceosome. Whereas some of the other suppressors of the fyv6 deletion can be interpreted as impacting directly the second step spliceosome (e.g., because the gene product is only present in the second step conformation), it seems that many more suppressors beyond prp8 mutants, especially those corresponding to bulky substitutions, which would more likely destabilize than stabilize, could similarly act indirectly by destabilization of first step conformation. The authors should acknowledge this where appropriate (e.g., for factors like Prp8 that are present in both first and second step conformations).

Reviewer #1 (Public Review):

Summary:

This study sought to reveal the potential roles of m6A RNA methylation in gene dosage regulatory mechanisms, particularly in the context of aneuploid genomes in Drosophila. Specifically, this work looked at the relationships between the expression of m6A regulatory factors, RNA methylation status, classical and inverse dosage effects, and dosage compensation. Using RNA sequencing and m6A mapping experiments, an in-depth analysis was performed to reveal changes in m6A status and expression changes across multiple aneuploid Drosophila models. The authors propose that m6A methylation regulates MOF and, in turn, deposition of H4K16Ac, critical regulators of gene dosage in the context of genomic imbalance.

Strengths:

This study seeks to address an interesting question with respect to gene dosage regulation and the possible roles of m6A in that process. Previous work has linked m6A to X-inactivation in humans through the Xist lncRNA, and to the regulation of the Sxl in flies. This study seeks to broaden that understanding beyond these specific contexts to more broadly understand how m6A impacts imbalanced genomes in other contexts.

Weaknesses:

The methods being used particularly for analysis of m6A at both the bulk and transcript-specific level are not sufficiently specific or quantitative to be able to confidently draw the conclusions the authors seek to make. MeRIP m6A mapping experiments can be very valuable, but differential methylation is difficult to assess when changes are small (as they often are, in this study but also m6A studies more broadly). For instance, based on the data presented and the methods described, it is not clear that the statement that "expression levels at m6A sites in aneuploidies are significantly higher than that in wildtype" is supported. MeRIP experiments are not quantitative, and since there are far fewer peaks in aneuploidies, it stands to reason that more antibody binding sites may be available to enrich those fewer peaks to a larger extent. But based on the data as presented (figure 2D) this conclusion was drawn from RPKM in IP samples, which may not fully account for changing transcript abundances in absolute (expression level changes) and relative (proportion of transcripts in input RNA sample) terms.

The bulk-level m6A measurements as performed here also cannot effectively support these conclusions, as they are measured in total RNA. The focus of the work is mRNA m6A regulators, but m6A levels measured from total RNA samples will not reflect mRNA m6A levels as there are other abundance RNAs that contain m6A (including rRNA). As a result, conclusions about mRNA m6A levels from these measurements are not supported.

Reviewer #1 (Public Review):

Summary:<br /> In this manuscript, the authors use gene functional analysis, pharmacology and live imaging to develop a proposed model of diverse G protein family signalling that takes place in the papillae during the ascidian Ciona larval adhesion to regulate the timing of initiation of the morphological changes of metamorphosis. Their experiments provide solid evidence that antagonistic G protein signalling regulates cAMP levels in the papillae, which provides a threshold for triggering metamorphosis that is reflective of a larva keeping a strong and sustained level of contact with a substrate for a minimum period of approximately half an hour. The authors discuss their reasoning and address different specific aspects of their proposed timing mechanism to provide a logical flow to the manuscript. The results are nicely linked to<br /> the ecology of Ciona larval settlement and will be of interest to developmental biologists, neurobiologists, molecular biologists, marine biologists as well as provide information relevant to antifouling and aquaculture sectors.

First, they knock down the G proteins Gaq and Gas to show that these genes are important for Ciona larval metamorphosis. They then provide evidence that the Gaq protein acts through a Ca2+ pathway mediated by phospholipase C and inositol triphosphate by showing that inositol phosphate and phospholipase C gene knockdown also inhibits metamorphosis, while overexpression of Gaq or phospholipase C allows larvae to undergo metamorphosis even in the absence of their mechanosensory cue, which is deprived by removing the posterior half of the tail and culturing the larvae on agar-coated dishes. The authors used calcium imaging which is a genetically encoded fluorescent calcium sensor to show that Gq knockdown larvae lack a Ca2+ spike in their papillae after mechanostimulation, confirming that Gaq acts through a Ca2+ pathway. Similarly the authors show that overexpression of Gas also enables larvae to metamorphose in the absence of mechanostimulation, suggesting a role for both Gaq and Gas in this process.

To confirm that Gas acts through cAMP signalling, the authors use pharmacological treatment or overexpression of a photoactivating adenylate cyclase to increase cAMP, and show that this also enables larvae to metamorphose in the absence of mechanostimulation, but only<br /> when their adhesive papillae are still present. Transcriptome data indicate that both Gs and Gq pathway genes are expressed in the adhesive papillae of the Ciona larva. One missing detail seems to be the need for evidence that cAMP is elevated in the papillae directly as a result of Gs activation. The authors use a fluorescent cAMP indicator, Pink Flamindo, to show that cAMP increases in the papillae upon adhesion to a substrate. Complementary to this, larvae that fail to undergo metamorphosis lack a cAMP increase in papillae. However, it is unclear whether the measured larvae that failed to undergo metamorphosis were wildtype or Gas knockdown larvae. If they were Gas knockdown larvae, this could provide evidence that cAMP does act downstream of the Gas activation.

The authors then provide evidence that GABA signalling within the papillae is acting downstream of the G proteins to induce metamorphosis. Transcriptome data shows that the genes for the GABA-producing enzyme, and for GABAb receptors, are both expressed in papillae. Pharmacological experiments show that GABA induces metamorphosis in the absence of mechanosensory cues, but only in larvae that retain their papillae. To show that GABA signalling within the papillae, rather than from the brain of the larva is important, the authors also demonstrate that anterior segments of larvae lacking the brain can also be stimulated to metamorphose by GABA, and show changes in gene expression caused by GABA.

The authors then use a combination of pharmacology and knockdown experiments in the presence or absence of mechanosensory cues to show that Gq/Ca2+ signalling acts upstream of Gs/cAMP signalling. As the elevation of cAMP by pharmacology or photoactivating adenylate cyclase rescued GABA pathway mutant larvae, the Gq and Gs pathways were concluded to be downstream of GABA signaling. However, GABA treatment could still induce Gaq- and Gas-knockdown larvae to metamorphose, suggesting an alternative pathway to metamorphosis, which the authors deduce to be through a third G protein, Gai. They identify an unusual Gai protein that based on transcriptome data is strongly expressed in the papillae. Gai knockdown larvae fail to metamorphose but are rescued by GABA treatment, which can be explained by a potential additional Gai protein being still present (transcriptome evidence suggests this although it is not further confirmed experimentally, for example by hybridization, immunohistochemistry, fluorescent labelling, or knockdown). The authors then use overexpression and knockdown experiments to show that the Gai protein acts through Gβγi complex to activate phospholipase C. Their experiments also indicate a potential for a complementary or compensatory role for Gai and Gaq signalling through Gβγi. By inhibiting the potassium channel GIRK through knockdown, and the MAPK pathway gene MEK1/2 by pharmacology, the authors also establish a role for these in their proposed model of signalling, allowing GABA and cAMP to compensate or interact with each other.

Strengths:<br /> The strength of this paper is the meticulous and extensive experiments, which are carefully designed to be able to precisely target specific genes in the putative signalling pathway to build step by step a complex model that can demonstrate how metamorphosis of the ascidian larva is timed so as to only undergo metamorphosis when strongly attached to a<br /> suitable substrate. The unique possibility of inhibiting mechanosensory-induced metamorphosis by removing some of the tail and smoothing the attachment substrate allows the authors to investigate potential effects on both activation and inhibition of metamorphosis, and to confirm that specific signalling pathways are clearly downstream of the initial<br /> mechanosensory stimulation. The study is also clear about which aspects of the model still remain unknown, such as which ligands and receptors may be responsible for the binding and activation of Gaq and Gas. Experiments testing metamorphosis of just the anterior region of the larvae nicely demonstrates the need for signalling in the region of the papillae, as do experiments where the papillae are removed, which then block metamorphosis in treatments that would otherwise stimulate it. The final model is a nice end point and makes a clear summary of how the extensive experiments all fit together into a cohesive potential signalling network, which can be built upon in the future.

Weaknesses:<br /> The paper has few weaknesses, however the main difficulty it poses is that due to the sheer number of precise experiments carried out and the complexity of the interwoven signalling pathways, it quickly becomes very difficult to follow exactly what is going on when and why or to keep track of the story as it develops. To improve this, an initial section in the results could be included showing a summary of the known G proteins in Ciona, their types and potential downstream signalling or upstream receptors, where known, and their expression levels in papillae. This could be in the form of a table and/or include the phylogenetic tree from the supplementary data. This would help clarify why the study first focuses on Gaq and Gas, and only later looks at Gai. This could be supplemented by a schematic workflow giving an overview of the experimental process of the study. A second minor weakness (understandable as the focus of the study is metamorphosis induced by mechanosensory stimulation) is that the study does not take into account any potential role for other types of sensory modalities (light, chemicals) that may also feed into the regulation of Ciona larval metamorphosis. This aspect would be interesting to discuss in light of the recent paper suggesting that some sensory cells in the Ciona adhesive papillae are polymodal and detect both chemicals and mechanical stimuli (Hoyer et al. 2024 Current Biology 34(6): 1168 - 1182).

Reviewer #1 (Public Review):

Summary:

The fungal cell wall is a very important structure for the physiology of a fungus but also for the interaction of pathogenic fungi with the host. Although a lot of knowledge on the fungal cell wall has been gained, there is a lack of understanding of the meaning of ß-1,6-glucan in the cell wall. In the current manuscript, the authors studied in particular this carbohydrate in the important human-pathogenic fungus Candida albicans. The authors provide a comprehensive characterization of cell wall constituents under different environmental and physiological conditions, in particular of ß-1,6-glucan. Also, β-1,6-glucan biosynthesis was found to be likely a compensatory reaction when mannan elongation was defective. The absence of β-1,6-glucan resulted in a significantly sick growth phenotype and complete cell wall reorganization. The manuscript contains a detailed analysis of the genetic and biochemical basis of ß-1,6-glucan biosynthesis which is apparently in many aspects similar to yeast. Finally, the authors provide some initial studies on the immune modulatory effects of ß-1,6-glucan.

Strengths:

The findings are very well documented, and the data are clear and obtained by sophisticated biochemical methods. It is impressive that the authors successfully optimized methods for the analyses and quantification of ß-1-6-glucan under different environmental conditions and in different mutant strains.

Weaknesses:

However, although already very interesting, at this stage there are some loose ends that need to be combined to strengthen the manuscript. For example, the immunological studies are rather preliminary and need at least some substantiation. Also, at this stage, the manuscript in some places remains a bit too descriptive and needs the elucidation of potential causalities.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Masroor Ahmad Paddar and his/her colleagues explore the noncanonical roles of ATG5 and membrane atg8ylation in regulating retromer assembly and function. They begin by examining the interactomes of ATG5 and expand the scope of these effects to include homeostatic responses to membrane stress and damage.

Strengths:<br /> This study provides novel insights into the noncanonical function of ATG8ylation in endosomal cargo sorting process.

Weaknesses:<br /> The direct mechanism by which ATG8ylation regulates the retromer remains unsolved.

Reviewer #1 (Public Review):

By mapping H3K4me2 in mouse oocytes and pre-implantation embryos, the authors aim to elucidate how this histone modification is erased and re-established during the parental-to-zygotic transition, as well as how the reprogramming of H3K4me2 regulates gene expression and facilitates zygotic genome activation.

Employing an improved CUT&RUN approach, the authors successfully generated H3K4me2 profiling data from a limited number of embryos. While the profiling experiments are very well executed, several weaknesses, particularly in data analysis, are apparent:

(1) The study emphasizes H3K4me2, which often serves as a precursor to H3K4me3, a well-studied modification during early development. Analyzing the new H3K4me2 dataset alongside published H3K4me3 data is crucial for comprehensively understanding epigenetic reprogramming post-fertilization and the interplay between histone modifications. However, the current analysis is preliminary and lacks depth.

(2) Tranylcypromine (TCP) is known as an irreversible inhibitor of monoamine oxidase and LSD1. While the authors suggest TCP inhibits the expression of LSD2, this assertion is questionable. Given TCP's potential non-specific effects in cells, conclusions related to the experiments using TCP should be made with caution.

(3) Some batches of H3K4me2 antibody are known to cross-react with H3K4me3. Has the H3K4me2 antibody used in CUT&RUN been tested for such cross-reactivity? Heatmaps in the figures indeed show similar distribution for H3K4me2 and H3K4me3, further raising concerns about antibody specificity.

(4) Certain statements lack supporting references or figures (examples on page 9 can be found on line 245, line 254, and line 258).

(5) Extensive language editing is recommended to clarify ambiguous sentences. Additionally, caution should be taken to avoid overstatement - most analyses in this study only suggest correlation rather than causality.

Reviewer #1 (Public Review):

Summary:<br /> In this study, the authors developed a novel radiotherapy sensitivity score (NPC-RSS) for nasopharyngeal carcinoma patients using machine learning algorithms. They identified 18 key genes associated with radiosensitivity and demonstrated that NPC-RSS could effectively predict radiotherapy response in both public and in-house datasets. Furthermore, they found that the key genes of NPC-RSS were closely related to immune characteristics, the expression of radiosensitivity-related genes, and signaling pathways involved in disease progression. The authors validated the consistency of expression of two key genes, SMARCA2 and CD9, with NPC-RSS in their own cell lines. They also showed that the radiosensitive group, classified by NPC-RSS, exhibited a more enriched and activated state of immune infiltration compared to the radioresistant group.

Strengths:<br /> (1) The study employed a comprehensive approach by integrating multiple machine learning algorithms to develop a robust predictive model for radiotherapy sensitivity in nasopharyngeal carcinoma patients.<br /> (2) The predictive performance of NPC-RSS was validated using both public and in-house datasets, demonstrating its potential clinical applicability.<br /> (3) The authors conducted extensive analyses to investigate the biological mechanisms underlying the association between NPC-RSS and radiotherapy response, including immune characteristics, radiosensitivity-related gene expression, and relevant signaling pathways.<br /> (4) The consistency of key gene expression with NPC-RSS was validated in the authors' own cell lines, providing additional experimental evidence.

Weaknesses:<br /> (1) The sample size of the in-house dataset used for training the model was relatively small (34 patients), which might limit the generalizability of the findings.<br /> (2) The authors did not perform functional experiments to directly validate the roles of the identified key genes in radiotherapy sensitivity, relying instead on associations with immune features and signaling pathways.<br /> (3) The study did not discuss the potential limitations of using machine learning algorithms, such as the risk of overfitting and the need for larger, diverse datasets for more robust model development and validation.

Reviewer #1 (Public Review):

In this study, the authors conducted a single-cell RNA sequencing analysis of the cellular and transcriptional landscape of the gastric cancer tumor microenvironment, stratifying patients according to their H. pylori status into currently infected, previously infected, and non-infected patients. The authors comprehensively dissect various cellular compartments, including epithelial, stromal, and immune cells, and describe specific cell types and signatures to be associated with H. pylori infection, including i) inflammatory and EMT signatures in malignant epithelial cells, ii) inflammatory CAFs in stromal cells, iii) Angio-TAMs, TREM2+ TAMs, exhausted and suppressive T cells in immune cells. Looking at ligand-receptor interactions as well as correlations between cell type abundances, they suggest that iCAFs interact with immunosuppressive T cells via a NECTIN2-TIGIT axis, as well as Angio-TAMs through a VEGFA/B-VEGFR1 axis and thereby promote immune escape, tumor angiogenesis and resistance to immunotherapy.

The authors conduct a comprehensive and thorough analysis of the complex tumor microenvironment of gastric cancer, both single-cell RNA sequencing data as well as the analysis seem of high quality and according to best practices. The authors validate their findings using external datasets, and include some prognostic value of the identified signatures and cell types. However, most of their conclusions throughout the manuscript are based on the comparison between HPGC and healthy controls, which is not a valid comparison to determine which of the phenotypes are specifically driven by HP infection, e.g. Tregs are high in all GC types, independent of HP status. The same holds true for TREM+ TAMs and iCAFs, which are higher in GC in general. This makes it very difficult to assess the actual HP-driven signatures and cell types. Also, when looking at the correlation/transcriptional differences across different cell types and cellular interactions, the authors do not explicitly define if they are looking at the whole dataset (including healthy controls?) or only at certain patients (HPGC?), which again makes it difficult to interpret the results.

The authors aim to confirm some of their findings via immunofluorescence, which in principle is a great approach to validate their results. However, to be able to conclude that e.g. suppressive TIGIT+ T cells are located close to NECTIN2+ malignant epithelium and that this might facilitate immune escape in HPGC (Figure 4K), the authors should include stains that show that this is not the case in the other groups (nonHPGC, exHPGC and HC). The same holds true for Figure 5G.

In summary, this study provides a valuable resource on the cellular and transcriptional heterogeneity of the tumor microenvironment in gastric cancers, distinguishing between positive, negative, and previously positive HP-infected gastric cancer patients. Given that HP is the main risk factor for gastric cancer development, the study provides valuable insights into HP-driven transcriptional signatures and how these might contribute to this increased risk, however, the study would highly benefit from a clearer and more stringent comparison between HPGC and nonHPGC.

Reviewer #1 (Public Review):

Summary:

In this paper, Li and colleagues have found mircoRNAs that affect levels of metamorphosis-regulating genes that can also affect levels of sesquiterpenoids (juvenile hormone and related compounds) and ecdysteriods, which regulate the timing and stages of insects, respectively. They first compared the transcriptomes of Drosophila at the third larval instar and at the white pre-pupa stage. They found thousands of differences in gene transcript levels between males and females, and between the two different stages. Among those genes that were differentially regulated they saw that genes involved in insect hormone biosynthesis were disproportionately represented. Many of the differentially regulated genes were involved in the insect hormone biosynthesis pathway and ascorbate and alderete metabolism. MicroRNAs were also differentially expressed during metamorphosis and were separately identified. The authors then considered genes and whether the differentially expressed microRNAs might regulate transcripts known to be involved in sesquiterpenoid production. In silico analysis of microRNAs predicted a list of 17 microRNAs that can regulate transcripts of sesquiterpenoid biosynthesis genes. The authors then used an in vitro luciferase assay to validate the binding and downregulation of 10 of the microRNAs to genes involved with sesquiterpenoid production in S2 cells.

Li and colleagues then focus on two genes they found were bound by microRNAs that have established roles in metamorphosis. The microRNAs miR-34 and miR-277 bind transcripts of two protein-coding genes that regulate metamorphosis Kr-h1, which encodes a transcription factor that is a JH-inducible transcription factor, and Allatostatin C Receptor 1, (AstC-R1), a G-protein coupled receptor that regulates the corpora allatum, the gland that produces sesquiterpenoids. Using a LAMP assay, one of the microRNAs, miR-277 was shown to bind to both AstC-R1 and Kr-h1 in in vivo whole-animal extracts. There is no mention of binding between either protein-coding transcript and the miR-34 microRNA. Temporal expression of all four transcripts shows that their abundance is anti-correlated; stages of high miR-34 or miR-277 expression correlate with low AstC-R1 or Kr-h1 expression. Homozygous deletions of both mircroRNAs result in 23% lethality, five days after adult eclosion. The authors also generated specific mutants in miR-34 or miR-277 and find differences in the expression of AstC-R1 and Kr-h1 and sex-specific differences in both sesquiterpenoids and ecdysteroids in the knock-out lines. If there were phenotypes associated with the specific knock-outs, those were not mentioned. Next, the authors examined the transcriptomes of the miR-3277 and miR-34 mutants and found several other GO-terms enriched among the differentially expressed genes. However, the sesquiterpenoid pathway and ascorbate and alderete metabolism are not listed.

Strengths:

This is an interesting manuscript that could make an important contribution to our understanding of the roles of micro RNAs at metamorphosis, and potentially of how sex-specific differences arise during metamorphosis. Strengths of the paper include the functional validation of microRNA binding, in vitro and in vivo-, as well as the characterization of sesquiterpenoid and ecdysteroid titers. The authors have also used CRISPR to generate specific knock-outs of miR-34 and miR-277. The transcriptomes will be a resource for future work to mine for differences in gene expression during metamorphosis.

Weaknesses:

(1) Spatial Expression of miR-34 and miR-277. If miR-34 and miR-277 regulate AstC-R1 and Kr-h1, then they must be expressed in the same cells. Although the authors show that the microRNAs do bind to the transcripts of AstC-R1 and Kr-h1 in S2 cells, and miR-277 binds AstC-R1 and Kr-h1 in vivo whole-animal homogenates, we do not know if the microRNAs are ever in the cells where AstC-R1 or Kr-h1 are expressed. AstC-R1 is only expressed in a few cells in the brain, so it is not at all certain that it is co-expressed with either microRNA. The creation of enhancer lines or in situ hybridization in Drosophila is straightforward and would sort this out.

(2) Phenotypes. Although a double deletion was used and specific knock-outs of both miR-34 and miR-277 were generated, the analysis of the mutants is very superficial. For the homozygous deletion of both microRNAs miR-34 and miR-277, only a decrease in survivorship was observed a full six days after adult eclosion - after the end of metamorphosis. No phenotype for either miR-34KO or miR-277-KO was given. The authors cite the work of others who have found specific phenotypes after manipulation of sesquiterpenoids or ecdysteroids, like Riddiford and Ashburner, but do not use any of these many studies to help them characterize the phenotype. If the loss of miR-34 and miR-277 affects so many pathways (including MAPK signaling, TGF-beta signaling, FoxO signaling, and Wnt signaling), as well as global titers of metamorphic hormones, then there shouldn't there be something different in the development to discuss?

(3) I think the reliance on GO term enrichment is getting in the way of biology. For instance, I would not describe Kr-h1 as a sesquiterpenoid biosynthesis pathway gene. Yet the authors say they were motivated to examine microRNA regulation of Kr-h1 because they saw differences in levels of the sesquiterpenoid biosynthesis pathway between WL3 and WPP, a period which also saw differences in expression of some microRNAs. I understand that Kr-h1 expression is regulated by JH, a sesquiterpenoid, but it is not directly involved with JH production, so relying on GO term enrichment has made the decision to focus on Kr-h1 feel arbitrary.

(4) The transcriptomes of miR-34 and miR-277 should have revealed genes encoding members of the sesquiterpenoid biosynthesis pathway as well as AstC-R1 and Kr-h1, but neither was mentioned. The functional tests of miR-34 and miR-277 were performed because they were shown to affect the levels of expression of genes in the sesquiterpenoid biosynthesis pathway. Figure 2 shows a significant decrease in AstC-R1 and Kr-h1 transcripts after the loss of miR-34 and miR-277. However, the results do not mention either (Lines 250-264). Instead, there is a list of 10 different GO terms (like arginine and proline metabolism or fatty acid degradation) that were enriched in miR-34 and miR-277 transcriptomes. If any of those ten types have any relationship to Kr-h1, AstC-R1, or metamorphosis, that has not been explained.

(5) Not enough care was taken in describing the stages. The methods describe wandering larvae (WL3) and white pre-pupa (WPP) for the transcriptomes, but in the text, different terms are used, like "larva", "pupa" and "L3 larvae instars" "early pupae" "late L3". Also, it seems like the small RNA libraries for sequencing were taken from "L3 larvae", but the stage of the L3 larvae was not mentioned. Staging is important, especially during metamorphosis, since differences in expression are expected to exist between different stages of L3, between early vs late wandering, and between WPP and early pupal stages.

Reviewer #1 (Public Review):

The paper titled "STAG3 promotes exit from pluripotency through post-transcriptional mRNA regulation in the cytoplasm" suggests a new and unexpected role for STAG3, a protein traditionally associated with the cohesin complex during meiosis, in regulating the exit from pluripotency in mouse embryonic stem cells (mESCs). While STAG3 is traditionally studied for its role in meiosis, this paper reveals that STAG3 is expressed in mouse embryonic stem cells (mESCs) and primordial germ cell-like cells (PGCLCs) and may be necessary for PGCLC-like specification and exit from pluripotency. In ESCs, the study reports that STAG3 is found in the cytoplasm, where it interacts with various RNA-binding proteins (RBPs) and localizes to centrosomes. Knockdown of STAG3 disrupts centrosome stability and RNA-induced silencing complex (RISC) components, leading to the misregulation of mRNAs such as DPPA3, Nanog, and TNRC6C. In summary, this study expands the known functions of STAG3 beyond cohesin, highlighting a potential role in cytoplasmic post-transcriptional regulation.

The authors perform a comprehensive characterization of RNA and protein changes in ESCs and differentiated cells upon loss of STAG3, providing preliminary and intriguing insights. However, there are several aspects that require further exploration:

(1) A rescue experiment for the STAG3 RNAi is missing, making it unclear whether the observed effects are indeed due to the knockdown of STAG3.

(2) While the paper identifies several interactions and effects of STAG3, it lacks detailed mechanistic insights into how STAG3 regulates specific mRNAs and proteins. Specifically, it is unclear which proteins directly interact with STAG3 or recruit STAG3 to RNP complexes. AlphaFold may help in this analysis.

(3) It is unclear whether this is an alternative STAG3 isoform or if STAG3 is modified. What dictates its interaction with cohesin versus RNPs?

(5) Are there unique features or sequence barcodes present on the misregulated RNAs?

(6) Does STAG3 associate with a single type of RNP or is it present in all types?

Reviewer #1 (Public Review):

Summary:<br /> The authors used multiple approaches to study salt effects in liquid-liquid phase separation (LLPS). Results on both wild-type Caprin1 and mutants and on different types of salts contribute to a comprehensive understanding.

Strengths:<br /> The main strength of this work is the thoroughness of investigation. This aspect is highlighted by the multiple approaches used in the study, and reinforced by the multiple protein variants and different salts studied.

Weaknesses:<br /> (1) The multiple computational approaches are a strength, but they're cruder than explicit-solvent all-atom molecular dynamics (MD) simulations and may miss subtle effects of salts. In particular, all-atom MD simulations demonstrate that high salt strengthens pi-types of interactions (ref. 42 and MacAinsh et al, https://www.biorxiv.org/content/10.1101/2024.05.26.596000v3).

(2) The paper can be improved by distilling the various results into a simple set of conclusions. By example, based on salt effects revealed by all-atom MD simulations, MacAinsh et al. presented a sequence-based predictor for classes of salt dependence. Wild-type Caprin1 fits right into the "high net charge" class, with a high net charge and a high aromatic content, showing no LLPS at 0 NaCl and an increasing tendency of LLPS with increasing NaCl. In contrast, pY-Caprin1 belongs to the "screening" class, with a high level of charged residues and showing a decreasing tendency of LLPS.

(3) Mechanistic interpretations can be further simplified or clarified. (i) Reentrant salt effects (e.g., Fig. 4a) are reported but no simple explanation seems to have been provided. Fig. 4a,b look very similar to what has been reported as strong-attraction promotor and weak-attraction suppressor, respectively (ref. 50; see also PMC5928213 Fig. 2d,b). According to the latter two studies, the "reentrant" behavior of a strong-attraction promotor, CL- in the present case, is due to Cl-mediated attraction at low to medium [NaCl] and repulsion between Cl- ions at high salt. Do the authors agree with this explanation? If not, could they provide another simple physical explanation? (ii) The authors attributed the promotional effect of Cl- to counterion-bridged interchain contacts, based on a single instance. There is another simple explanation, i.e., neutralization of the net charge on Caprin1. The authors should analyze their simulation results to distinguish net charge neutralization and interchain bridging; see MacAinsh et al.

(4) The authors presented ATP-Mg both as a single ion and as two separate ions; there is no explanation of which of the two versions reflects reality. When presenting ATP-Mg as a single ion, it's as though it forms a salt with Na+. I assume NaCl, ATP, and MgCl2 were used in the experiment. Why is Cl- not considered? Related to this point, it looks like ATP is just another salt ion studied and much of the Results section is on NaCl, so the emphasis of ATP ("Diverse Roles of ATP" in the title is somewhat misleading.

Reviewer #1 (Public Review):

Summary:

During vertebrate gastrulation, mesendoderm cells are initially specified by morphogens (e.g. Nodal) and segregate into endoderm and mesoderm in part based on Nodal concentrations. Using zebrafish genetics, live imaging, and single-cell multi-omics, the manuscript by Cheng et al presents evidence to support a claim that anterior endoderm progenitors derive primarily from prechordal plate progenitors, with transcriptional regulators goosecoid (Gsc) and ripply1 playing key roles in this cell fate determination. Such a finding would represent a significant advance in our understanding of how anterior endoderm is specified in vertebrate embryos.

Strengths:

Live imaging-based tracking of PP and endo reporters (Figure 2) is well executed and convincing, though a larger number of individual cell tracks will be needed. Currently, only a single cell track (n=1) is provided.

Weaknesses:

(1) The central claim of the paper - that the anterior endoderm progenitors arise directly from prechordal plate progenitors - is not adequately supported by the evidence presented. This is a claim about cell lineage, which the authors are attempting to support with data from single-cell profiling and genetic manipulations in embryos and explants. The construction of gene expression (pseudo-time) trajectories, while a modern and powerful approach for hypothesis generation, should not be used as a substitute for bona fide lineage tracing methods. If the authors' central hypothesis is correct, a CRE-based lineage tracing experiment (e.g. driving CRE using a PP marker such as Gsc) should be able to label PP progenitor cells that ultimately contribute to anterior endoderm-derived tissues. Such an experiment would also allow the authors to quantify the relative contribution of PP (vs non-PP) cells to the anterior endoderm, which is not possible to estimate from the indirect data currently provided. Note: while the present version of the manuscript does describe a sox17:CRE lineage tracing experiment, this actually goes in the opposite direction that would be informative (sox:17:CRE-marked descendants will be a mixture of PP-derived and non-PP derived cells, and the Gsc-based reporter does not allow for long-term tracking the fates of these cells).

(2) The authors' descriptions of gene expression patterns in the single-cell trajectory analyses do not always match the data. For example, it is stated that goosecoid expression marks progenitor cells that exist prior to a PP vs endo fate bifurcation (e.g. lines 124-130). Yet, in Figure 1C it appears that in fact goosecoid expression largely does not precede (but actually follows) the split and is predominantly expressed in cells that have already been specified into the PP branch. Likewise, most of the cells in the endo branch (or prior) appear to never express Gsc. While these trends do indeed appear to be more muddled in the explant data (Figure 1H), it still seems quite far-fetched to claim that Gsc expression is a hallmark of endoderm-PP progenitors.

(3) The study seems to refer to "endoderm" and "anterior endoderm" somewhat interchangeably, and this is potentially problematic. Most single-cell-based analyses appearing in the study rely on global endoderm markers (sox17, sox32) which are expressed in endodermal precursors along the entire ventrolateral margin. Some of these cells are adjacent to the prechordal plate on the dorsal side of the gastrula, but many (most in fact) are quite some distance away. The microscopy-based evidence presented in Figure 2 and elsewhere, however, focuses on a small number of sox17-expressing cells that are directly adjacent to, or intermingled with, the prechordal plate. It, therefore, seems problematic for the authors to generalize potential overlaps with the PP lineage to the entire endoderm, which includes cells in ventral locations. It would be helpful if the authors could search for additional markers that might stratify and/or mark the anterior endoderm and perform their trajectory analysis specifically on these cells.

(4) It is not clear that the use of the nodal explant system is allowing for rigorous assessment of endoderm specification. Why are the numbers of endoderm cells so vanishingly few in the nodal explant experiments (Figure 1H, 3H), especially when compared to the embryo itself (e.g. Figures 1C-D)? It seems difficult to perform a rigorous analysis of endoderm specification using this particular model which seems inherently more biased towards PP vs. endoderm than the embryo itself. Why not simply perform nodal pathway manipulations in embryos?

(5) The authors should not claim that proximity in UMAP space is an indication of transcriptional similarity (lines 207-208), especially for well-separated clusters. This is a serious misrepresentation of the proper usage of the UMAP algorithm. The authors make a similar claim later on (lines 272-274).

Reviewer #1 (Public Review):

Summary:

The authors wanted to identify genes that are critical for regulating the asymmetric fates of limbal stem cells and their transit amplified progeny in the central cornea. To this end, they utilized an in vivo cell cycle reporter to isolate proliferating basal cells from the anterior ocular surface epithelium and performed single-cell RNA-seq. This strategy revealed distinct basal cell identities with unique expression profiles of structural genes and transcription factors. The authors then focused on the Sox9 transcription factor implicated in stem cell regulation. It was differentially expressed between limbal stem cells and their progeny in the central cornea. Lineage tracing analysis confirmed that Sox9 marks long-lived limbal stem cells. Conditional deletion of Sox9 led to abnormal differentiation and squamous metaplasia in the central cornea. The authors suggest that Sox9 is required for the switch to asymmetric fate and commitment toward differentiation, as transit cells exit the limbal niche. By inhibiting the terminal differentiation of corneal progenitors and forcing them into continuous symmetric divisions, the Sox9 loss-of-function phenotype was replicated.

Strengths:

Thus, the paper shows the important role of Sox9 in the spatial regulation of asymmetric fate in the corneal epithelium and its proliferation and cell differentiation. The work is elegantly done using several models that converge on the main conclusions. It is very novel and delineates a new player in determining corneal epithelial cell fate. The experiments are well done, and the data are credible.

Weaknesses:

This reviewer has some minor concerns mostly related to data interpretation and the use of the LSC term.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Fuchsberger et al. demonstrate a set of experiments that ultimately identifies the de novo synthesis of GluA1-, but not GluA2-containing Ca2+ permeable AMPA receptors as a key driver of dopamine-dependent LTP (DA-LTP) during conventional post-before-pre spike-timing dependent (t-LTD) induction. The authors further identify adenylate cyclase 1/8, cAMP, and PKA as the crucial mitigators of these actions. While some comments have been identified below, the experiments presented are thorough and address the aims of the manuscript, figures are presented clearly (with minor comments), and experimental sample sizes and statistical analyses are suitable. Suitable controls have been utilized to confirm the role of Ca2+ permeable AMPAR. This work provides a valuable step forward built on convincing data toward understanding the underlying mechanisms of spike-timing-dependent plasticity and dopamine.

Strengths:

Appropriate controls were used.

The flow of data presented is logical and easy to follow.

The quality of the data, except for a few minor issues, is solid.

Weaknesses:

The drug treatment duration of anisomycin is longer than the standard 30-45 minute duration (as is the 500uM vs 40uM concentration) typically used in the field. Given the toxicity of these kinds of drugs long term it's unclear why the authors used such a long and intense drug treatment.

With some of the normalizations (such as those in S1) there are dramatic differences in the baseline "untreated" puromycin intensities - raising some questions about the overall health of slices used in the experiments.

Reviewer #1 (Public Review):

The authors are attempting to use the internal workings of a language hierarchy model, comprising phonemes, syllables, words, phrases, and sentences, as regressors to predict EEG recorded during listening to speech. They also use standard acoustic features as regressors, such as the overall envelope and the envelopes in log-spaced frequency bands. This is valuable and timely research, including the attempt to show differences between normal-hearing and hearing-impaired people in these regards.

I will start with a couple of broader questions/points, and then focus my comments on three aspects of this study: The HM-LSTM language model and its usage, the time windows of relevant EEG analysis, and the usage of ridge regression.

Firstly, as far as I can tell, the OSF repository of code, data, and stimuli is not accessible without requesting access. This needs to be changed so that reviewers and anybody who wants or needs to can access these materials.

What is the quantification of model fit? Does it mean that you generate predicted EEG time series from deconvolved TRFs, and then give the R2 coefficient of determination between the actual EEG and predicted EEG constructed from the convolution of TRFs and regressors? Whether or not this is exactly right, it should be made more explicit.

About the HM-LSTM:

• In the Methods paragraph about the HM-LSTM, a lot more detail is necessary to understand how you are using this model. Firstly, what do you mean that you "extended" it, and what was that procedure? And generally, this is the model that produces most of the "features", or regressors, whichever word we like, for the TRF deconvolution and EEG prediction, correct? A lot more detail is necessary then, about what form these regressors take, and some example plots of the regressors alongside the sentences.<br /> • Generally, it is necessary to know what these regressors look like compared to other similar language-related TRF and EEG/MEG prediction studies. Usually, in the case of e.g. Lalor lab papers or Simon lab papers, these regressors take the form of single-sample event markers, surrounded by zeros elsewhere. For example, a phoneme regressor might have a sample up at the onset of each phoneme, and a word onset regressor might have a sample up at the onset of each word, with zeros elsewhere in the regressor. A phoneme surprisal regressor might have a sample up at each phoneme onset, with the value of that sample corresponding to the rarity of that phoneme in common speech. Etc. Are these regressors like that? Or do they code for these 5 linguistic levels in some other way? Either way, much more description and plotting is necessary in order to compare the results here to others in the literature.<br /> • You say that the 5 regressors that are taken from the trained model's hidden layers do not have much correlation with each other. However, the highest correlations are between syllable and sentence (0.22), and syllable and word (0.17). It is necessary to give some reason and interpretation of these numbers. One would think the highest correlation might be between syllable and phoneme, but this one is almost zero. Why would the syllable and sentence regressors have such a relatively high correlation with each other, and what form do those regressors take such that this is the case?<br /> • If these regressors are something like the time series of zeros along with single sample event markers as described above, with the event marker samples indicating the onset of the relevant thing, then one would think e.g. the syllable regressor would be a subset of the phoneme regressor because the onset of every syllable is a phoneme. And the onset of every word is a syllable, etc.

For the time windows of analysis:

• I am very confused, because sometimes the times are relative to "sentence onset", which would mean the beginning of sentences, and sometimes they are relative to "sentence offset", which would mean the end of sentences. It seems to vary which is mentioned. Did you use sentence onsets, offsets, or both, and what is the motivation?<br /> • If you used onsets, then the results at negative times would not seem to mean anything, because that would be during silence unless the stimulus sentences were all back to back with no gaps, which would also make that difficult to interpret.<br /> • If you used offsets, then the results at positive times would not seem to mean anything, because that would be during silence after the sentence is done. Unless you want to interpret those as important brain activity after the stimuli are done, in which case a detailed discussion of this is warranted.<br /> • For the plots in the figures where the time windows and their regression outcomes are shown, it needs to be explicitly stated every time whether those time windows are relative to sentence onset, offset, or something else.<br /> • Whether the running correlations are relative to sentence onset or offset, the fact that you can have numbers outside of the time of the sentence (negative times for onset, or positive times for offset) is highly confusing. Why would the regressors have values outside of the sentence, meaning before or after the sentence/utterance? In order to get the running correlations, you presumably had the regressor convolved with the TRF/impulse response to get the predicted EEG first. In order to get running correlation values outside the sentence to correlate with the EEG, you would have to have regressor values at those time points, correct? How does this work?<br /> • In general, it seems arbitrary to choose sentence onset or offset, especially if the comparison is the correlation between predicted and actual EEG over the course of a sentence, with each regressor. What is going on with these correlations during the middle of the sentences, for example? In ridge regression TRF techniques for EEG/MEG, the relevant measure is often the overall correlation between the predicted and actual, calculated over a longer period of time, maybe the entire experiment. Here, you have calculated a running comparison between predicted and actual, and thus the time windows you choose to actually analyze can seem highly cherry-picked, because this means that most of the data is not actually analyzed.<br /> • In figures 5 and 6, some of the time window portions that are highlighted as significant between the two lines have the lines intersecting. This looks like, even though you have found that the two lines are significantly different during that period of time, the difference between those lines is not of a constant sign, even during that short period. For instance, in figure 5, for the syllable feature, the period of 0 - 200 ms is significantly different between the two populations, correct? But between 0 and 50, normal-hearing are higher, between 50 and 150, hearing-impaired are higher, and between 150 and 200, normal-hearing are higher again, correct? But somehow they still end up significantly different overall between 0 and 200 ms. More explanation of occurrences like these is needed.

Using ridge regression:

• What software package(s) and procedure(s) were specifically done to accomplish this? If this is ridge regression and not just ordinary least squares, then there was at least one non-zero regularization parameter in the process. What was it, how did it figure in the modeling and analysis, etc.?<br /> • It sounds like the regressors are the hidden layer activations, which you reduced from 2,048 to 150 non-acoustic, or linguistic, regressors, per linguistic level, correct? So you have 150 regressors, for each of 5 linguistic levels. These regressors collectively contribute to the deconvolution and EEG prediction from the resulting TRFs, correct? This sounds like a lot of overfitting. How much correlation is there from one of these 150 regressors to the next? Elsewhere, it sounds like you end up with only one regressor for each of the 5 linguistic levels. So these aspects need to be clarified.<br /> • For these regressors, you are comparing the "regression outcomes" for different conditions; "regression outcomes" are the R2 between predicted and actual EEG, which is the coefficient of determination, correct? If this is R2, how is it that you have some negative numbers in some of the plots? R2 should be only positive, between 0 and 1.

Reviewer #2 (Public Review):

Summary:

Cells cultured in high glucose tend to repress mitochondrial biogenesis and activity, a prevailing phenotype type called Crabtree effect that observed in different cell types and cancer. Many signaling pathways have been put forward to explain this effect. Vengayil et al proposed a new mechanism involved in Ubp3/Ubp10 and phosphate that controls the glucose repression of mitochondria. The central hypothesis is that ∆ubp3 shift the glycolysis to trehalose synthesis, therefore lead to the increase of Pi availability in the cytosol, then mitochondrial received more Pi and therefore the glucose repression is reduced.

Strengths:

The strength is that the authors used an array of different assays to test their hypothesis. Most assays were well-designed and controlled.

Weaknesses:

The author addressed my major concerns.

Reviewer #2 (Public Review):

Summary.

The objective of this study was to further our understanding of the brain mechanisms associated with facial expressions of pain. To achieve this, participants' facial expressions and brain activity were recorded while they received noxious heat stimulation. The authors then used a decoding approach to predict facial expressions from functional magnetic resonance imaging (fMRI) data. They found a distinctive brain signature for pain facial expressions (FEPS). This signature had minimal overlap with brain signatures reflecting other components of pain phenomenology, such as signatures reflecting subjective pain intensity or negative effects.

Strength.

The authors used a rigorous approach involving multivariate brain decoding to predict the occurrence and intensity of pain facial expressions during noxious heat stimulation. The analyses are solid and well-conducted. This is an important study of fundamental and clinical relevance.

Weakness.

Despite those major strengths, the main weakness of the study is that the design and analyses do not allow us to know if the FEPS is really specific to pain expressions. Based on the analysis, it is possible to conclude that this brain signature is present when a participant is in a state of pain and displays a facial expression. However, it is possible that it would also be present when a participant experiences (another) negative state and displays (another) facial expression. It will be important, in future work, to investigate the specificity of this brain signature.

Reviewer #1 (Public Review):

Summary:

This manuscript investigates the regulation of chlorophyll biosynthesis in rice embryos, focusing on the role of OsNF-YB7. The rigorous experimental approach, combining genetic, biochemical, and molecular analyses, provides a robust foundation for these findings. The research achieves its objectives, offering new insights into chlorophyll biosynthesis regulation, with the results convincingly supporting the authors' conclusions.

Strengths:

The major strengths include the detailed experimental design and the findings regarding OsNF-YB7's inhibitory role.

Weaknesses:

However, the manuscript's discussion on the practical implications for agriculture and the evolutionary analysis of regulatory mechanisms could be expanded.

Reviewer #3 (Public Review):

Summary:

Kundu et al. investigated the effects of pre-exposure to a non-pathogenic Leptospira strain in prevention of severe disease following subsequent infection by a pathogenic strain. They utilized a single or double exposure method to the non-pathogen prior to challenge with a pathogenic strain. They found that prior exposure to a non-pathogen prevented many of the disease manifestations of the pathogen. Bacteria, however, were able to disseminate, colonize the kidneys, and be shed in the urine. This is important foundational work to describe a novel method of vaccination against leptospirosis. Numerous studies have attempted to use recombinant proteins to vaccinate against leptospirosis, with limited success. The authors provide a new approach that takes advantage of the homology between a non-pathogen and a pathogen to provide heterologous protection. This will provide a new direction in which we can approach creating vaccines against this re-emerging disease.

Strengths:

The major strength of this paper is that it is one of the first studies utilizing a live non-pathogenic strain of Leptospira to immunize against severe disease associated with leptospirosis. They utilize two independent experiments (a single and double vaccination) to define this strategy. This represents a very interesting and novel approach to vaccine development. This is of clear importance to the field.

The authors use a variety of experiments to show the protection imparted by pre-exposure to the non-pathogen. They look at disease manifestations such as death and weight loss. They define the ability of Leptospira to disseminate and colonize the kidney. They show the effects infection has on kidney architecture and a marker of fibrosis. And they begin to define the immune response in both of these exposure methods. This provides evidence of the numerous advantages this vaccination strategy may have. Thus, this study provides an important foundation for future studies utilizing this method to protect against leptospirosis.

Weaknesses:

A direct comparison between single and double exposure to the non-pathogen is not possible with the data presented. The ages of mice infected were different between the single (8 weeks) and double (10 weeks) exposure methods, thus the phenotypes associated with LIC infection are different at these two ages. The authors state that this is expected, but do not provide a reasoning for this drastic difference in phenotypes. It cannot be determined if double-vaccination would provide an additional benefit, which is of importance to future work developing any vaccine treatment. An experiment directly comparing the two exposure methods while infecting mice at the same age would be of great relevance to and strengthen this work.

Reviewer #1 (Public Review):

Summary:

The "optorepressilator", an optically controllable genetic oscillator based on the famous E. coli 3-repressor (LacI, TetR, CI) oscillator "repressilator", was developed. An individual repressilator shows a stable oscillation of the protein levels with a relatively long period that extends a few doubling times of E. coli, but when many cells oscillate, their phases tend to desynchronize. The authors introduced an additional optically controllable promoter through a conformal change of CcaS protein and let it control how much additional CI is produced. By tightly controlling the leak from the added promoter, the authors successfully kept the original repressilator oscillation when the added promoter was not activated. In contrast, the oscillation was stopped by expressing the additional CI. Using this system, the authors showed that it is possible to synchronise the phase of the oscillation, especially when the activation happens as a short pulse at the right phase of the repressilator oscillation. The authors further show that, by changing the frequency of the short pulses, the repressilator was entrained to various ratios to the pulse period, and the author could reconstruct the so-called "Arnold tongues", the signature of entrainment of the nonlinear oscillator to externally added periodic perturbation. The behaviour is consistent with the simplified mathematical model that simulates the protein concentration using ordinary differential equations.

Strengths:

Optical control of the oscillation of the protein clock is a powerful and clean tool for studying the synthetic oscillator's response to perturbation in a well-controlled and tunable manner. The article utilizes the plate reader setup for population average measurements and the mother machine setup for single-cell measurements, and they complement nicely to acquire necessary information.

Reviewer #1 (Public Review):

Summary:

The authors use a combination of biochemistry and cryo-EM studies to explore a complex between the cap binding complex and an RNA binding protein, ALYREF, that coordinates mRNA processing and export.

Strengths:

The biochemistry and structural biology are supported by mutagenesis that tests the model in vitro. The structure provides new insight into how key events in RNA processing and export are likely to be coordinated.

Weaknesses:

The authors provide biochemical studies to confirm the interactions that they identify; however, they do not perform any studies to test these models in cells or explore the consequences for mRNA export from the nucleus. In fact, several of the amino acids that they identified in ALYREF that are critical for the interaction, as determined by their own biochemical studies are conserved in budding yeast Yra1 (residues E124/E128 are E/Q in budding yeast and residues Y135/V138/P139 are F/S/P), where the impact on poly(A) RNA export from the nucleus could be readily evaluated. The authors mention the potential for future studies in the manuscript, but they do not perform any analysis in this study that would explore the contributions of these new interactions.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

Novel findings of how OGT promotes tumor progression.

Reviewer #1 (Public Review):

The authors of this study developed a software application, which aims to identify images as either "friendly" or "unfriendly" for readers with deuteranopia, the most common color-vision deficiency. Using previously published algorithms that recolor images to approximate how they would appear to a deuteranope (someone with deuteranopia), authors first manually assessed a set of images from biology-oriented research articles published in eLife between 2012 and 2022, as well as an additional hold-out set of 2000 articles selected randomly from the PubMed Central Open Access Subset. The researchers identified 636 out of 4964 images as difficult to interpret ("unfriendly") for deuteranopes in the eLife dataset. In the PubMed Central dataset 104 out of 1191 non-grayscale images were identified as unfriendly. The results for the eLife dataset show a decrease in "unfriendly" images over time and a higher probability for articles from cell-oriented research fields to contain "unfriendly" images.

The researchers used the manually classified images from eLife to develop, train, and validate an automated screening tool. They also created a user-friendly web application of the tool, where users can upload images and be informed about the status of each image as "friendly" or "unfriendly" for deuteranopes.

Strengths:

The authors have identified an important accessibility issue in the scientific literature: the use of color combinations that make figures difficult to interpret for people with color-vision deficiency. The metrics proposed and evaluated in the study are a valuable theoretical contribution. The automated screening tool they provide is well-documented, open source, and relatively easy to install and use. It has the potential to provide a useful service to the scientists who want to make their figures more accessible. The data are open and freely accessible, well documented, and a valuable resource for further research. The manuscript is well-written, logically structured, and easy to follow.

Weaknesses:

(1) The authors themselves acknowledge the limitations that arise from the way they defined what constitutes an "unfriendly" image. There is a missed chance here to have engaged deuteranopes as stakeholders earlier in the experimental design. This would have allowed to determine to what extent spatial separation and labelling of problematic color combinations responds to their needs and whether setting the bar at a simulated severity of 80% is inclusive enough. A slightly lowered barrier is still a barrier to accessibility.

(2) The use of training images from a single journal limits the generalizability of the empirical findings as well as of the automated screening tool itself. This is evidenced by a decrease in performance of the tool on the holdout dataset from PubMed Central. Machine-learning algorithms are highly configurable but also notorious for their lack of transparency and for being easily biased by the training data set. A quick and unsystematic test of the web application shows that the classifier works well for electron microscopy images but fails at recognizing the classical diagnostic images for color-vision deficiency (Ishihara test images) as "unfriendly". A future iteration of the tool should be trained on a wider variety of images, ideally enriched with diagnostic images found in scientific publications.

Reviewer #1 (Public Review):

Summary:

The authors fabricated a novel cancer vaccine using endogenous virus-like particles with tumor neoantigen. The vaccine ePAC was proven to elicit strong immune stimulation with an increased killing effect against tumor cells in 2 mouse models.

Strengths:

The author achieved high protein loading and transfection efficiency using PEG10 self-assembly while packaging tumor neoantigens inside for cancer immunotherapy. The author also enhanced the targeting effect towards dendritic cells by surface modification using CpG-ODN.

Weaknesses:

There were some minor issues but they have been resolved in the revision process. It would be great if the authors could compare this with commercially available treatments and other vaccines.

Discussion:

Since the ePAC vaccine particle functions as a delivery platform, it can be tailored to different tumors when packed with their specific tumor neoantigens. Thus, the ePAC platform can be potentially employed in a broad range of cancer vaccine therapies. It would be exciting to see this platform being developed for other major cancer types.

Reviewer #1 (Public Review):

Summary:

The manuscript aimed at elucidating the substrate specificity of two M23 endopeptidase Lysostaphin (LSS) and LytM in S. aureus. Endopeptidases are known to cleave the glycine-bridges of staphylococcal cell wall peptidoglycan (PG). To address this question, various glycine-bridge peptides were synthesized as substrates, the catalytic domain of LSS and LytM were recombinantly expressed and purified, and the reactions were analyzed using solution-state NMR. The major finding is that LytM is not only a Gly-Gly endopeptidase, but also cleaves D-Ala-Gly. Technically, the advantage of using real-time NMR was emphasized in the manuscript. The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM. However, the biological significance and relevance of the conclusions remain clear, as the results are mostly from synthetic substrates.

Strengths:

The study explores an interesting aspect of cell wall hydrolases in terms of substrate-level regulation. It potentially identified new enzymatic activity of LytM.

Comments on the revised version:

The authors have addressed most of my concerns. I agree that the physiological functions of LytM are not in the scope of the current study.

Reviewer #1 (Public Review):

Summary:

This manuscript aimed to investigate the emergence of emotional sensitivity and its relationship with gestational age. Using an oddball paradigm and event-related potentials, the authors conducted an experiment in 120 healthy neonates with a gestational age range of 35 to 40 weeks. A significant developmental milestone was identified at 37 weeks gestational age, marking a crucial juncture in neonatal emotional responsiveness.

Strengths:

This study has several strengths, by providing profound insights into the early development of social-emotional functioning and unveiling the role of gestational age in shaping neonatal perceptual abilities. The methodology of this study demonstrates rigor and well-controlled experimental design, particularly involving matched control sounds, which enhances the reliability of the research. Their findings not only contribute to the field of neurodevelopment, but also showcase potential clinical applications, especially in the context of autism screening and early intervention for neurodevelopmental disorders.

Reviewer #1 (Public Review):

Summary:

A nice study trying to identify the relationship between E. coli O157 from cattle and humans in Alberta, Canada.

Strengths:

(1) The combined human and animal sampling is a great foundation for this kind of study.

(2) Phylogenetic analyses seem to have been carried out in a high-quality fashion.

Weaknesses:

I think there may be a problem with the selection of the isolates for the primary analysis. This is what I'm thinking:

(1) Transmission analyses are strongly influenced by the sampling frame.

(2) While the authors have randomly selected from their isolate collections, which is fine, the collections themselves are not random.

(3) The animal isolates are likely to represent a broad swathe of diversity, because of the structured sampling of animal reservoirs undertaken (as I understand it).

(4) The human isolates are all from clinical cases. Clinical cases of the disease are likely to be closely related to other clinical cases, because of outbreaks (either detected, or undetected), and the high ascertainment rate for serious infections.

(5) Therefore, taking an equivalent number of animal and clinical isolates, will underestimate the total diversity in the clinical isolates because the sampling of the clinical isolates is less "independent" (in the statistical sense) than sampling from the animal isolates.

(6) This could lead to over-estimating of transmission from cattle to humans.

(7) "We hypothesize that the large proportion of disease associated with local transmission systems is a principal cause of Alberta's high E. coli O157:H7 incidence" - this seems a bit tautological. There is a lot of O157 because there's a lot of transmission. What part of the fact it is local means that it is a principal cause of high incidence? It seems that they've observed a high rate of local transmission, but the reasons for this are not apparent, and hence the cause of Alberta's incidence is not apparent. Would a better conclusion not be that "X% of STEC in Alberta is the result of transmission of local variants"? And then, this poses a question for future epi studies of what the transmission pathway is.

Reviewer #1 (Public Review):

Summary:<br /> In this study, Zhao and colleagues investigate inflammasome activation by E. tarda infections. They show that E. tarda induces the activation of the NLRC4 inflammasome as well as the non-canonical pathway in human THP1 macrophages. Further dissecting NLRC4 activation, they find that T3SS translocon components eseB, eseC and eseD are necessary for NLRC4 activation and that delivery of purified eseB is sufficient to trigger NAIP-dependent NLRC4 activation. Sequence analysis reveals that eseB shares homology within the C-terminus with T3SS needle and rod proteins, leading the authors to test if this region is necessary for inflammasome activation. They show that the eseB CT is required and that it mediates interaction with NAIP. Finally, they that homologs of eseB in other bacteria also share the same sequence and that they can activate NLRC4 in a HEK293T cell overexpression system.

Strengths:<br /> This is a very nice study that convincingly shows that eseB and its homologs can be recognized by the human NAIP/NLRC4 inflammasome. The experiments are well designed, controlled and described, and the papers is convincing as a whole.

Weaknesses:<br /> The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.

The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?

Reviewer #1 (Public Review):

Mitochondria are essential organelles consisting in mammalian cells of about 1500 different proteins. Most of those are synthesized in the cytosol as precursor proteins, imported into mitochondria, and sorted into one of the four sub-mitochondrial compartments. The TIM23 complex, which is embedded in the mitochondrial inner membrane, facilitates the import of proteins that harbor Mitochondrial Targeting Sequence (MTS) at their N-terminus. Such proteins are sorted mainly to the mitochondrial matrix while some sub-groups are destined also to the inner membrane or the intermembrane space. TIMM50 (Tim50 in yeast) is an essential component of the TIM23 complex and mutations in this protein were reported to cause several diseases.

Summary:

In the current study, the authors analyzed the impact of TIMM50 mutations on the mitochondrial proteome in both patients' cells and mouse neurons. They provide compelling evidence for several surprising and highly interesting observations: (i) TIMM50 mutations affect the steady-state levels of only a portion of the putative TIMM50 substrates, (ii) such mutations result in increased electrical activity in mice neurons and in reduced levels of some potassium ion channels in the plasma membrane. These findings shed new light on mitochondrial biogenesis in mammalian cells and hint at an unexpected link between mitochondria and ion channels at the plasma membrane.

Strengths:

The authors used both cells from patients and neurons from mice to investigate the impact of mutations in TIMM50 on mitochondrial proteome and function.

Weaknesses:

(1) It will be interesting to monitor the levels of another MIM insertase namely, OXA1. This will help to understand whether some of the observed changes in levels of OXPHOS subunits are related to alterations in the amounts of this insertase.

(2) The authors did not provide explanations for several key findings like:<br /> A. Figure 3: How do the authors explain that although TIMM17 and TIMM23 were found to be significantly reduced by Western analysis they were not detected as such by the Mass Spec. method?<br /> B. How do the authors explain the higher levels of some proteins in the TIMM50 mutated cells?<br /> C. Can the authors elaborate on why mutated cells are impaired in their ability to switch their energetic emphasis to glycolysis when needed?

Reviewer #1 (Public Review):

Time periods in which experience regulates early plasticity in sensory circuits are well established, but the mechanisms that control these critical periods are poorly understood. In this manuscript, Leier and Foden and colleagues examine early-life critical periods that regulate the Drosophila antennal lobe, a model sensory circuit for understanding synaptic organization. Using early-life (0-2 days old) exposure to distinct odorants, they show that constant odor exposure markedly reduces the volume, synapse number, and function of the VM7 glomerulus. The authors offer evidence that these changes are mediated by invasion of ensheathing glia into the glomerulus where they phagocytose connections via a mechanism involving the engulfment receptor Draper.

This manuscript is a striking example of a study where the questions are interesting, the authors spent a considerable amount of time to clearly think out the best experiments to ask their questions in the most straightforward way, and expressed the results in a careful, cogent, and well-written fashion. It was a genuine delight to read this paper. I have two experimental suggestions that would really round out existing work to better support the existing conclusions and some instances where additional data or tempered language in describing results would better support their conclusions. Overall, though, this is an incredibly important finding, a careful analysis, and an excellent mechanistic advance in understanding sensory critical period biology.

Reviewer #1 (Public Review):

Summary:

This article presents a meta-analysis that challenges established abundance-occupancy relationships (AORs) by utilizing the largest known bird observation database. The analysis yields contentious outcomes, raising the question of whether these findings could potentially refute AORs.

Strengths:

The study employed an extensive aggregation of datasets to date to scrutinize the abundance-occupancy relationships (AORs).

Weaknesses:

While the dataset employed in this research holds promise, a rigorous justification of the core assumptions underpinning the analytical framework is inadequate. The authors should thoroughly address the correlation between checklist data and global range data, ensuring that the foundational assumptions and potential confounding factors are explicitly examined and articulated within the study's context.

Reviewer #1 (Public Review):

Summary:<br /> The authors present experimental and numerical results on the motility Magnetospirillum gryphiswaldense MSR-1, a magnetotactic bacterium living in sedimentary environments. The authors manufactured microfluidic chips containing three-dimensional obstacles of irregular shape, that match the statistical features of the grains observed in the sediment via micro-computer tomography. The bacteria are furthermore subject to an external magnetic field, whose intensity can be varied. The key quantity measured in the experiments is the throughput ratio, defined as the ratio between the number of bacteria that reach the end of the microfluidic channel and the number of bacteria entering it. The main result is that the throughput ratio is non-monotonic and exhibits a maximum at magnetic field strength comparable with Earth's magnetic field. The authors rationalize the throughput suppression at large magnetic fields by quantifying the number of bacteria trapped in corners between grains.

Strengths:<br /> While magnetotactic bacteria's general motility in bulk has been characterized, we know much less about their dynamics in a realistic setting, such as a disordered porous material. The micro-computer tomography of sediments and their artificial reconstruction in a microfluidic channel is a powerful method that establishes the rigorous methodology of this work. This technique can give access to further characterization of microbial motility. The coupling of experiments and computer simulations lends considerable strength to the claims of the authors, because the model parameters (with one exception) are directly measured in the experiments.

Weaknesses:<br /> The main weakness of the manuscript pertains to the discussion of the statistical significance of the experimental throughput ratio. Especially when comparing results at zero and 50 micro Tesla. The simulations seem to predict a stronger effect than seen in the experiments. The authors do not address this discrepancy.

Reviewer #1 (Public Review):

Summary:<br /> The paper investigates the interplay between fluid flow and biofilm development using Pseudomonas aeruginosa PAO1 in microfluidic channels. By combining experimental observations with mathematical modeling, the study identifies the significant impact of nutrient limitation and hydrodynamic forces on biofilm growth and detachment. The authors demonstrate that nutrient limitation drives the longitudinal distribution of biomass, while flow-induced detachment influences the maximum clogging and temporal dynamics. The study highlights that pressure buildup plays a critical role in biofilm detachment, leading to cyclic episodes of sloughing and regrowth. A stochastic model is used to describe the detachment process, capturing the apparent randomness of sloughing events. The findings offer insights into biofilm behavior during clogging and fouling, potentially relevant to infections, environmental processes, and engineering applications.

Strengths:<br /> This paper demonstrates a strong integration of experimental work and mathematical modeling, providing a comprehensive understanding of biofilm dynamics in straight microfluidic channel. The simplicity of the microchannel geometry allows for accurate modeling, and the findings have the potential to be applied to more complex geometries. The detailed analysis of nutrient limitation and its impact on biofilm growth offers valuable insights into the conditions that drive biofilm formation. The model effectively describes biofilm development across different stages, capturing both initial growth and cyclic detachment processes. While cyclic pressure buildup has been studied previously, the incorporation of a stochastic model to describe detachment events is a novel and significant contribution, capturing the complexity and randomness of biofilm behavior. Finally, the investigation of pressure buildup and its role in cyclic detachment and regrowth enhances our understanding of the mechanical forces at play, making the findings applicable to a wide range of technological and clinical contexts.

Weaknesses:<br /> The study achieves its primary goal of integrating experiments and modeling to understand the coupling between flow and biofilm growth and detachment in a microfluidic channel, but it should have highlighted the weaknesses of the methods. I list the ones that, in my opinion, are the main ones:

• The study does not consider biofilm porosity, which could significantly affect the flow and forces exerted on the biofilm. Porosity could impact the boundary conditions, such as the no-slip condition, which should be validated experimentally.<br /> • The research suggests EPS development as a stage in biofilm growth but does not probe it using lectin staining. This makes it impossible to accurately assess the role of EPS in biofilm development and detachment processes.<br /> • While the force and flow are three-dimensional, the images are taken in two dimensions. The paper does not clearly explain how the 2D images are extrapolated to make 3D assessments, which could lead to inaccuracies.<br /> • Although the findings are tested using polysaccharide-deficient mutants, the results could have been analyzed in greater detail. A more thorough analysis would help to better understand the role of matrix composition on the stochastic model of detachment.

DOI: 10.1016/j.cub.2023.07.021

Resource: (ZFIN Cat# ZDB-GENO-060207-1,RRID:ZFIN_ZDB-GENO-060207-1)

Curator: @vtello

SciCrunch record: RRID:ZFIN_ZDB-GENO-060207-1

What is this?

Reviewer #1 (Public Review):

This manuscript builds upon the authors' previous work on the cross-talk between transcription initiation and post-transcriptional events in yeast gene expression. These prior studies identified an mRNA 'imprinting' phenomenon linked to genes activated by the Rap1 transcription factor (TF), a surprising role for the Sfp1 TF in promoting RNA polymerase II (RNAPII) backtracking, and a role for the non-essential RNAPII subunits Rpb4/7 in the regulation of mRNA decay and translation. Here the authors aimed to extend these observations to provide a more coherent picture of the role of Sfp1 in transcription initiation and subsequent steps in gene expression. They provide evidence for (1) a physical interaction between Sfp1 and Rpb4, (2) Sfp1 binding and stabilization of mRNAs derived from genes whose promoters are bound by both Rap1 and Sfp1 and (3) an effect of Sfp1 on Rpb4 binding or conformation during transcription elongation.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Wu et al. introduce a novel approach to reactivate the Muller glia cell cycle in the mouse retina by simultaneously reducing p27Kip1 and increasing cyclin D1 using a single AAV vector. The approach effectively promotes Muller glia proliferation and reprograming without disrupting retinal structure or function. Interestingly, reactivation of the Muller glia cell cycle downregulates IFN pathway, which may contribute to the induced retinal regeneration. The results presented in this manuscript may offer a promising approach for developing Müller glia cell-mediated regenerative therapies for retinal diseases.

Strengths:

The data are convincing and supported by appropriate, validated methodology. These results are both technically and scientifically exciting and are likely to appeal to retinal specialists and neuroscientists in general.

Weaknesses:

There are some data gaps that need to be addressed.

(1) Please label the time points of AAV injection, EdU labeling, and harvest in Figure 1B.

(2) What fraction of Müller cells were transduced by AAV under the experimental conditions?

(3) It seems unusually rapid for MG proliferation to begin as early as the third day after CCA injection. Can the authors provide evidence for cyclin D1 overexpression and p27 Kip1 knockdown three days after CCA injection?

(4) The authors reported that MG proliferation largely ceased two weeks after CCA treatment. While this is an interesting finding, the explanation that it might be due to the dilution of AAV episomal genome copies in the dividing cells seems far-fetched.

Reviewer #1 (Public Review):

Summary:

In this manuscript the authors re-examine the developmental origin of cortical oligodendrocyte (OL) lineage cells using a combination of strategies, focussing on the question of whether the LGE generates cortical OL cells. The paper is interesting to myelin biologists, the methods used are appropriate and, in general, the study is well-executed, thorough, and persuasive, but not 100% convincing.

Strengths, weaknesses, and recommendations:

The first evidence presented that the LGE does not generate OLs for the cortex is that there are no OL precursors 'streaming' from the LGE during embryogenesis, unlike the MGE (Figure 1A). This in itself is not strong evidence, as they might be more dispersed. In fact, in the images shown, there is no obvious 'streaming' from the MGE either. Note that in Figure 1 there is no reference to the star that is shown in the figure.

The authors then electroporate a reporter into the LGE at E13.5 and examine the fate of the electroporated cells (Figures 1C-E). They find that electroporated cells became neurons in the striatum and in the cortex but no OLs for the cortex. There are two issues with this: first, there is no quantification, which means there might indeed be a small contribution from the LGE that is not immediately obvious from snapshot images. Second, it is unexpected to find labelled neurons in the cortex at all since the LGE does not normally generate neurons for the cortex! Electroporations are quite crude experiments as targeting is imprecise and variable and not always discernible at later stages. For example, in Figure 1D, one can see tdTOM+ cells near the AEP, as well as the striatum. Hence, IUE cannot on its own be taken as proof that there is no contribution of the LGE to the cortical OL population.

The authors then use an alternative fate-mapping approach, again with E13.5 electroporations (Figure 2). They find only a few GFP+ cells in the cortex at E18 (Figures 2C-D) and P10 (Figure 2E) and these are mainly neurons, not OL lineage cells. Again, there is no quantification.

Figure 3 is more convincing, but the experiments are incomplete. Here the authors generate triple-transgenic mice expressing Cre in the cortex (Emx1-Cre) and the MGE (Nkx2.1-Cre) as well as a strong nuclear reporter (H2B-GFP). They find that at P0 and P10, 97-98% of OL-lineage cells (SOX10+ or PDGFRA+) in the cortex are labelled with GFP (Figure 3). This is a more convincing argument that the LGE/CGE might not contribute significant numbers of OL lineage cells to the cortex, in contrast to the Kessaris et at. (2006) paper, which showed that Gsh2-Cre mice label ~50% of SOX10+ve cells in the motor cortex at P10. The authors of the present paper suggest that the discrepancy between their study and that of Kessaris et al. (2006) is based on the authors' previous observation (Zhang et al 2020) (https://doi.org/10.1016/j.celrep.2020.03.027) that GSH2 is expressed in intermediate precursors of the cortex from E18 onwards. If correct, then Kessaris et al. might have mistakenly attributed Gsh2-Cre+ lineages to the LGE/CGE when they were in fact intrinsic to the cortex. However, the evidence from Zhang et al 2020 that GSH2 is expressed by cortical intermediate precursors seems to rest solely on their location within the developing cortex; a more convincing demonstration would be to show that the GSH2+ putative cortical precursors co-label for EMX1 (by immunohistochemistry or in situ hybridization), or that they co-label with a reporter in Emx1-driven reporter mice. This demonstration should be simple for the authors as they have all the necessary reagents to hand. Without these additional data, the assertion that GSX2+ve cells in the cortex are derived from the cortical VZ relies partly on an act of faith on the part of the reader.

Note that Tripathi et al. (2011, "Dorsally- and ventrally-derived oligodendrocytes have similar electrical properties but myelinate preferred tracts." J. Neurosci. 31, 6809-6819) found that the Gsh-Cre+ OL lineage contributed only ~20% of OLs to the mature cortex, not ~50% as reported by Kessaris et al. (2006). If it is correct that these Gsh2-derived OLs are from the cortical anlagen as the current paper claims, then it would raise the possibility that the ventricular precursors of GSH2+ intermediate progenitors are not uniformly distributed through the cortical VZ but are perhaps localized to some part of it. Then the contribution of Gsh2-derived OLs to the cortical population could depend on precisely where one looks relative to that localized source. It would be a nice addition to the current manuscript if the authors could explore the distribution of their GSH2+ intermediate precursors throughout the developing cortex. In any case, Tripathi et al. (2011) should be cited.

Finally, the authors deleted Olig2 in the MGE and found a dramatic reduction of PDGFRA+ and SOX10+ cells in the cortex at E14 and E16 (Figure 4A-F). This further supports their conclusion that, at least at E16, there is no significant contribution of OLs from ventral sources other than the MGE/AEP. This does not exclude the possibility that the LGE/CGE generates OLs for the cortex at later stages. Hence, on its own, this is not completely convincing evidence that the LGE generates no OL lineage cells for the cortex.

Comments on the latest version:

The revised manuscript has addressed the issues we raised previously. The addition of the new Figure 3 supplement 1A-C demonstrating that Gsx2+ve cells in the cortex are generated from Emx1-Cre precursors is convincing, although there is nothing to prove that the GFP+, Gsh2+ double-labelled nuclei are oligodendrocyte lineage and not, for example, astrocytes. It would be helpful to include a Gsh2, Olig2 (or Gsh2, Sox10) double-label image to prove this point. Also, to make the figure more clear, the authors should also show a small area at high magnification, splitting the green and red channels so that the reader can see more clearly that all the red cells are also green.

Reviewer #1 (Public Review):

Summary:

This manuscript reports the effects of a heterozygous mutation in the KCNT1 potassium channels on the properties of ion currents and firing behavior of excitatory and inhibitory neurons in the cortex of mice expressing KCNT1-Y777H. In humans, this mutation as well as multiple other heterozygotic mutations produce very severe early-onset seizures and produce a major disruption of all intellectual function. In contrast, in mice, this heterozygous mutation appears to have no behavioral phenotype or any increased propensity to seizures. A relevant phenotype is, however, evident in mice with the homozygous mutation, and the authors have previously published the results of similar experiments with the homozygotes. As perhaps expected, the neuronal effects of the heterozygous mutation presented in this manuscript are generally similar but markedly smaller than the previously published findings on homozygotes. There are, however, some interesting differences, particularly on PV+ interneurons, which appear to be more excitable than wild type in the heterozygotes but more excitable in the heterozygotes. This raises the interesting question, which has been explicitly discussed by the authors in the revised manuscript, as to whether the reported changes represent homeostatic events that suppress the seizure phenotype in the mouse heterozygotes or simply changes in excitability that do not reach the threshold for behavioral outcomes.

Reviewer #1 (Public Review):

Summary:

The authors show for the first time that deleting GLS from rod photoreceptors results in the rapid death of these cells. The death of photoreceptor cells could result from loss of synaptic activity because of a decrease in glutamate, as has been shown in neurons, changes in redox balance, or nutrient deprivation.

Strengths:

The strength of this manuscript is that the author shows a similar phenotype in the mice when Gls was knocked out early in rod development or the adult rod. They showed that rapid cell death is through apoptosis, and there is an increase in the expression of genes responsive to oxidative stress.

Weaknesses:

In this manuscript, the authors show a "metabolic dependency of photoreceptors on glutamine catabolism in vivo". However, there is a potential bias in their thinking that glutamine metabolism in rods is similar to cancer cells where it feeds into the TCA cycle. They should consider that as in neurons, GLS1 activity provides glutamate for synaptic transmission. The modest rescue shown by providing α-ketoglutarate in the drinking water suggests that glutamine isn't a key metabolic substrate for rods when glucose is plentiful. The ERG studies performed on the iCre-Glsflox/flox mice showed a large decrease in the scotopic b wave at saturating flashes which could indicate a decrease in glutamate at the rod synapse as stated by the authors. While EM micrographs of wt and iCre-Glsflox/flox mice were shown for the outer retina at p14, the synapse of the rods needs to be examined by EM.

The authors note that the outer segments are shorter but they do not address whether there is a decrease in the number of cones.

Rod-specific Gls ko mice with an inducible promoter were generated by crossing the Pde6g-CreERT2 and homozygous for either the WT or floxed Gls allele (IND-cKO). In Figure 3 the authors document that by western blots and antibody labeling the GLS1 expression is lost in the IND-cKO 10 days post tamoxifen. OCT images show a decrease in the thickness of the outer nuclear layer between 17 and 38 days post-TAM. Ergs should be performed on the animals at 10 and 30 days post TAM, before and after major structural changes in rod photoreceptor cells, to determine if changes in light-stimulated responses are observed. These studies could help to parse out the cause of photoreceptor cell death.

The studies in Figure 4 were all performed on iCre-Glsflox/flox and control mice at p14, why weren't the IND-cKO mice used for these studies since the findings would not be confounded by development?

In all rescue studies, the endpoint was an ONL thickness, which only addressed rod cell death. The authors should also determine whether there are small improvements in the ERG, which would distinguish the role of GLS in preventing oxidative stress.

Reviewer #1 (Public Review):

The research by Lin Chao, Chun Kuen Chen, Chao Shi, and Camilla U. Rang addresses the asymmetric distribution of ribosomes in single E. coli cells during aging by time-lapse microscopy, as well as its correlation to protein misfolding. The presented research is an important contribution to the field of protein biosynthesis pathways and their link to aging, especially in regard to the thorough analysis of variation in cells elongation rate in old and new daughter cells derived from old and new mother cells.

Comments on current version:

I thank the authors for their thoughtful responses. Yet the centrality of protein aggregate distribution analysis to this manuscript requires further evidence to support the link to ribosome asymmetrical distribution and aging.

The authors suggest this is beyond the scope of this study. This then requires a major revising of the study, as in its current form, it is one of its main claims.

Reviewer #1 (Public Review):

Summary:

This study presents careful biochemical experiments to understand the relationship between LRRK2 GTP hydrolysis parameters and LRRK2 kinase activity. The authors report that incubation of LRRK2 with ATP increases the KM for GTP and decreases the kcat. From this they suppose an autophosphorylation process is responsible for enzyme inhibition. LRRK2 T1343A showed no change, consistent with it needing to be phosphorylated to explain the changes in G-domain properties. The authors propose that phosphorylation of T1343 inhibits kinase activity and influences monomer-dimer transitions.

Strengths:

The strengths of the work are the very careful biochemical analyses and interesting results for wild type LRRK2.

Weaknesses:

The conclusions related to the involvement of a monomer-dimer transition are to this reviewer, premature and an independent method needs to be utilized to bolster this aspect of the story.

Reviewer #1 (Public Review):

Summary:

In this paper the researchers aimed to address whether bees causally understand string-pulling through a series of experiments. I first briefly summarize what they did:

- In experiment 1, the researchers trained bees without string and then presented them with flowers in the test phase that either had connected or disconnected strings, to determine what their preference was without any training. Bees did not show any preference.

- In experiment 2, bees were trained to have experience with string and then tested on their choice between connected vs. disconnected string.

- Experiment 3 was similar except that instead of having one option which was an attached string broken in the middle, the string was completely disconnected from the flower.

- In experiment 4, bees were trained on green strings and tested on white strings to determine if they generalize across color.

- In experiment 5, bees were trained on blue strings and tested on white strings.

- In experiment 6, bees were trained where black tape covered the area between the string and the flower (i.e. so they would not be able to see/ learn whether it was connected or disconnected).

- In experiments 2-6, bees chose the connected string in the test phase.

- In experiment 7, bees were trained as in expt 3 and then tested where string was either disconnected or coiled i.e. still being 'functional' but appearing different.

- In experiment 8, bees were trained as before and then tested on string that was in a different coiled orientation, either connected or disconnected.

- In experiments 7 and 8 the bees showed no preference.

Strengths:

I appreciate the amount of work that has gone into these experiments and think they are a nice, thorough set of experiments. I enjoyed reading the paper and felt that it was overall well-written and clear. I think experiment 1 shows that bees do not have an untrained understanding of the function of the string in this context. The rest of the experiments indicate that with training, bees have a preference for unbroken over broken string and likely use visual cues learned during training to make this choice. They also show that as in other contexts, bees readily generalize across different colors.

The 'weaknesses' that I previously listed were dealt with by the authors in the revised version of the manuscript. I think the only point that we disagreed on was relating to the ecological relevance of the task to the bees.

Here is my previous comment:

I think the paper would be made stronger by considering the natural context in which the bee performs this behavior. Bees manipulate flowers in all kinds of contexts, and scrabble with their legs to achieve nectar rewards. Rather than thinking that it is pulling a string, my guess would be that the bee learns that a particular motor pattern within their usual foraging repertoire (scrabbling with legs), leads to a reward. I don't think this makes the behavior any less interesting - in fact, I think considering the behavior through an ecological lens can help make better sense of it.

The authors disagreed, writing the following:

"Here we respectfully disagree. The solving of Rubik s cube by humans could be said to be version of finger movements naturally required to open nuts or remove ticks from fur, but this is somewhat beside the point: it s not the motor<br /> sequences that are of interest, but the cognition involved. A general approach in work on animal intelligence and cognition is to deliberately choose paradigms that are outside the animals daily routines this is what we have done here, in asking whether there is means end comprehension in bee problem solving. Like comparable studies on this question in other animals, the experiments are designed to probe this question, not one of ecological validity."

I think the difference would be that humans know that they are doing a rubik's cube whereas I do not think that the bee knows that it is pulling string- I think the bee thinks that it is foraging on a flower. Therefore, I stand by my statement that I think it's worth considering what the bee is experiencing in this task and how it relates to what it would be doing while foraging. I think that as animal cognition researchers we can design tasks that are distinct from what the animal would naturally encounter to ask specific questions about what they are thinking- but that we can never remove the ecological context since the animal will always be viewing the task through that lens. However, I think this may be a philosophical difference in opinion and I am happy with the manuscript as it stands.

Reviewer #2 (Public Review):

This manuscript by Amen, Yoo and Fabra-Garcia et al describes a human monoclonal antibody B1E11K, targeting EENV repeats which are present in parasite antigens such as Pfs230, RESAs and Pf11.1. The authors isolated B1E11K using an initial target agnostic approach for antibodies that would bind gamete/gametocyte lysate which they made 14 mAbs. Following a suite of highly appropriate characterization methods from Western blotting of recombinant proteins to native parasite material, use of knockout lines to validate specificity, ITC, peptide mapping, SEC-MALS, negative stain EM and crystallography, the authors have built a compelling case that B1E11K does indeed bind EENV repeats. In addition, using X-ray crystallography they show that two B1E11K Fabs bind to a 16 aa RESA repeat in a head-to-head conformation using homotypic interactions and provide a separate example from CSP, of affinity-matured homotypic interactions.

The authors have addressed most of our previous comments in their revised manuscript.

One of the main conclusions in the paper is the binding of B1E11K to RESAs which are blood stage antigens that are exported to the infected parasite surface. In the future, it would be interesting to understand if B1E11K mAb binds to the red cell surface of infected blood stage parasites to understand its cellular localization in those stages.

Materials and Methods:<br /> PBMC sampling: While the authors have provided clarification that they obtained informed consent from the PBMC donor, they have not added the ethics approval codes in this section.

Reviewer #1 (Public Review):

Summary:

This manuscript introduced a new behavioral apparatus to regulate the animal's behavioral state naturally. It is a thermal maze where different sectors of the maze can be set to different temperatures; once the rest area of the animal is cooled down, it will start searching for a warmer alternative region to settle down again. They recorded with silicon probes from the hippocampus in the maze and found that the incidence of SWRs was higher at the rest areas and place cells representing a rest area were preferentially active during rest-SWRs as well but not during non-REM sleep.

Strengths:

The maze can have many future applications, e.g., see how the duration of waking immobility can influence learning, future memory recall, or sleep reactivation. It represents an out-of-the-box thinking to study and control less-studies aspects of the animals' behavior.

Weaknesses:

The impact is only within behavioral research and hippocampal electrophysiology.

Joint Public Review

The present study explored the principles that allow cells to maintain complex subcellular proteinaceous structures despite the limited lifetimes of the individual protein components. This is particularly critical in the case of neurons, where the size and protein composition of synapses define synaptic strength and encode memory.

PSD95 is an abundant synapse protein that acts as a scaffold in the recruitment of transmitter receptors and other signaling proteins and is required for memory formation. The authors used super-resolution microscopy to study PSD95 super-complexes isolated from the brains of mice expressing tagged PSD variants (Halo-Tag, mEos, GFP). Their results show compellingly that a large fraction (~25%) of super-complexes contains two PSD95 copies about 13 nm apart, that there is substantial turnover of PSD95 proteins in super-complexes over a period of seven days, and that ~5-20% of the super-complexes contain new and old PSD95 molecules. This percentage is higher in synaptic fractions as compared to total brain lysates, and highest in isocortex samples (~20%). These important findings support the hypothesis put forward by Crick that sequential subunit replacement gives synaptic super-complexes long lifetimes and thus aids in memory maintenance. Overall, this is a very interesting study that provides key insights into how synaptic protein complexes are formed and maintained. On the other hand, the actual role of these PSD95 super-complexes in long-term memory storage remains unknown. Specifically, a direct correlation between PSD95 stability and memory formation remains hypothetical - but the present findings indicate important new directions for studying the mechanisms that control postsynaptic protein organisation and the maintenance of postsynaptic proteinaceous substructures.

Strengths

(1) The study employed an appropriate and validated methodology.<br /> (2) Large numbers of PSD95 super-complexes from three different mouse models were imaged and analyzed, providing adequately powered sample sizes.<br /> (3) State-of-the-art super-resolution imaging techniques (PALM and MINFLUX) were used, providing a robust, high-quality, cross-validated analysis of PSD95 protein complexes that is useful for the community.<br /> (4) The result that PSD95 proteins in dimeric complexes are on average 12.7 nm apart is useful and has implications for studies on the nanoscale organization of PSD95 at synapses.<br /> (5) The finding that postsynaptic protein complexes can continue to exist while individual components are being renewed is important for our understanding of synapse maintenance and stability.<br /> (6) The data on the turnover rate of PSD95 in super-complexes from different brain regions provide a first indication of potentially meaningful differences in the lifetime of super-complexes between brain regions.

Weaknesses

(1) The manuscript emphasizes the hypothesis that stable super-complexes, maintained through sequential replacement of subunits, might underlie the long-term storage of memory. While an interesting idea, this notion requires considerably more research. The presented experimental data are indeed consistent with this notion, but there is no evidence that these complexes are causally related to memory storage.<br /> (2) Much of the presented work is performed on biochemically isolated protein complexes. The biochemical isolation procedures rely on physical disruption and detergents that are known to alter the composition and structure of complexes in certain cases. Thus, it remains unclear how the protein complexes described in this study relate to PSD95 complexes in intact synapses.<br /> (3) Because not all GFP molecules mature and fold correctly in vitro and the PSD95-mEos mice used were heterozygous, the interpretation of the corresponding quantifications is not straightforward.<br /> (4) It was not tested whether different numbers of PSD95 molecules per super-complex might contribute to different retention times of PSD95, e.g. in synaptic vs. total-forebrain super-complexes.<br /> (5) The conclusion that the population of 'mixed' synapses is higher in the isocortex than in other brain regions is not supported by statistical analysis.<br /> (6) The validity of conclusions regarding PSD95 degradation based on relative changes in the occurrence of SiR-Halo-positive puncta is limited.

Public Review (Joint Version of all Reviewers)

Cav1.4 calcium channels control voltage-dependent calcium influx at photoreceptor synapses, and congenital loss of Cav1.4 function causes stationary night blindness CSNB2. Based on a broad portfolio of methodological approaches - genetic mouse models, immunolabeling and microscopic imaging, serial block-face-SEM, ERGs, and electrophysiology - the authors show that cone photoreceptor synapse development is strongly perturbed in the absence of Cav1.4 protein, and that expression of a nonconducting Cav1.4 channel mitigates these perturbations. Further data indicate that Cav3 channels are present, which, according to the authors, may compensate for the loss of Cav1.4 calcium currents and thus maintain cone synaptic transmission. These data, which are in agreement with a similar study by the same authors on rod photoreceptor synapses, help to explain what functional defects exactly cause CSNB2 and why it is accompanied by only mild visual impairment.

The strengths of the present study are its conceptual and experimental soundness, the broad spectrum of cutting-edge methodological approaches pursued, and the convincing differential analysis of mutant phenotypes. Weaknesses mainly concern the fact that the mechanism by which Cav3 channels might partially compensate for the loss of Cav1.4 calcium currents remains unclear.

Annotation 1

Reviewer #1 (Public Review):

Summary:

This paper attempts to measure the complex changes of consciousness in the human brain as a whole. Inspired by the perturbational complexity index (PCI) from classic research, authors introduce simulation PCI (𝑠𝑃𝐶𝐼) of a time series of brain activity as a measure of consciousness. They first use large-scale brain network modeling to explore its relationship with the network coupling and input noise. Then the authors verify the measure with empirical data collected in previous research.

Strengths:

The conceptual idea of the work is novel. The authors measure the complexity of brain activity from the perspective of dynamical systems. They provide a comparison of the proposed measure with four other indexes. The text of this paper is very concise, supported by experimental data and theoretical model analysis.

Weaknesses:

(1) Consciousness is a network phenomenon. The measure defined by the authors is to consider the maximal sPCI across the nodes stimulated. This measure is based on the time series of one node. The measure may be less effective in quantifying the ill relationship between nodes. This may contribute to the less predictive power of anesthesia (Figure 4b).

(2) One of the focuses of the work is the use of a dynamic model of brain networks. The explanation of the model needs to be in more detail.

(3) The equations should be checked. For example, there should be no max on the left side of the first equation on page 13.

(4) The quality of the figures should be improved.

(5) Figure 4 should be discussed and analyzed more in the text.

(6) The usage of the terms PCI and sPCI should be distinguished.

Reviewer #1 (Public Review):

Summary

This interesting study, which has greatly improved in the current revised version, explores the mechanism behind an increased susceptibility of daf-18/PTEN mutant nematodes to paralyzing drugs that exacerbate cholinergic transmission. The authors use state-of-the-art genetics and neurogenetics coupled with locomotor behavior monitoring and neuroanatomical observations using gene expression reporters to show that the susceptibility occurs due to low levels of DAF-18/PTEN in developing inhibitory GABAergic neurons early during larval development (specifically, during the larval L1 stage). DAF-18/PTEN is convincingly shown to act cell-autonomously in these cells upstream of the PI3K-PDK-1-AKT-DAF-16/FOXO pathway, consistent with its well-known role as an antagonist of this conserved signaling pathway. The authors exclude a role for the TOR pathway in this process and present evidence implicating selectivity towards-developing GABAergic neurons of the ventral nerve cord in comparison to excitatory cholinergic neurons. Finally, the authors show that a diet supplemented with a ketogenic body, β-hydroxybutyrate, which also counteracts the PI3K-PDK-1-AKT pathway, promoting DAF-16/FOXO activity, partially rescues the proper development (morphology and function) of GABAergic neurons in daf-18/PTEN mutants, but only if the diet is provided early during larval development. This strongly suggests that the critical function of DAF-18/PTEN in developing inhibitory GABAergic neurons is to prevent excessive PI3K-PDK-1-AKT activity during this critical and particularly sensitive period of their development in juvenile L1 stage worms. Whether or not the sensitivity of GABAergic neurons to DAF-18/PTEN function is a defining and widespread characteristic of this class of neurons in C. elegans and other animals, or rather a particularity of the early developmental stage of the GABAergic neurons investigated remains to be determined.

Strengths:

The study reports interesting and important findings, advancing the knowledge of how daf-18/PTEN and the PI3K-PDK-1-AKT pathway can influence neurodevelopment, and providing a valuable paradigm to study the selectivity of gene activities towards certain neurons. It also defines a solid paradigm to study the potential of dietary interventions (such as ketogenic diets) or other drug treatments to counteract (prevent or revert?) neurodevelopment defects and stimulate DAF-16/FOXO activity.

Weaknesses:

The fact that other non-GABAergic C. elegans neurons (i.e., AIY and HSN neurons) are also sensitive to DAF-18/PTEN activity during development suggests that the particular sensitivity observed in the GABAergic ventral nerve cord neurons in this study could be unrelated to their neurotransmitter class (GABAergic) per se, but rather to some other neuronal property (a critical period of plasticity or activity-based wiring?) that these neurons share with the AIY and HSN neurons, and not with the other surveyed ventral nerve cord neurons (the excitatory cholinergic neurons). The relevance of this possibility within the framework of understanding the role of DAF-18/PTEN in E/I imbalance across clades is not fully clear at this stage.

Reviewer #1 (Public Review):

Summary:

The authors are interested in the developmental origin of the neurons of the cerebellar nuclei. They identify a population of neurons with a specific complement of markers originating in a distinct location from where cerebellar nuclear precursor cells have been thought to originate that show distinct developmental properties. The cerebellar nuclei have been well studied in recent years to understand their development through an evolutionary lens, which supports the importance of this study. The discovery of a new germinal zone giving rise to a new population of CN neurons is an exciting finding, and it enriches our understanding of cerebellar development, which has previously been quite straightforward, where cerebellar inhibitory cells arise from the ventricular zone and the excitatory cells arise from the rhombic lip.

Strengths:

One of the strengths of the manuscript is that the authors use a wide range of technical approaches, including transgenic mice that allow them to disentangle the influence of distinct developmental organizers such at ATOH.<br /> Their finding of a novel germinal zone and a novel population of CN neurons is important for developmental neuroscientists, cerebellar neuroscientists.

Weaknesses:

One important question raised by this work is what do these newly identified cells eventually become in the adult cerebellum. Are they excitatory or inhibitory? Do they correspond to a novel cell type or perhaps one of the cell classes that have been recently identified in the cerebellum (e.g. Fujita et al., eLife, 2020)? Understanding this would significantly bolster the impact of this manuscript.

The major weakness of the manuscript is that it is written for a very specialized reader who has a strong background in cerebellar development, making it hard to read for eLife's general audience. It's challenging to follow the logic of some of the experiments as well as to contextualize these findings in the field of cerebellar development.

Reviewer #1 (Public Review):

Summary:

Using chromaffin cells as a powerful model system for studying secretion, the authors study the regulatory role of complexin in secretion. Complexin is still enigmatic in its regulatory role, as it both provides inhibitory and facilitatory functions in release. The authors perform an extensive structure-function analysis of both the C- and N-terminal regions of complexin. There are several interesting findings that significantly advances our understanding of cpx/SNARe interactions in regulating release. C-terminal amphipathic helix interferes with SNARE complex assembly and thus clamps fusion. There are acidic residues in the C-term that may be seen as putative interaction partners for Synaptotagmin. The N-terminus of Complexin promoting role may be associated with an interaction with Syt1. In particular the putative interaction with Syt1 is of high interest and supported by quite strong functional and biochemical evidence. The experimental approaches are state of the art, and the results are of the highest quality and convincing throughout. They are adequate and intelligently discussed in the rich context of the standing literature. Whilst there are some concerns about whether the facilitatory actions of complexion have to be tightly linked to Syt1 interactions, the proposed model will significantly advance the field by providing new directions in future research.

Reviewer #1 (Public Review):

Summary:

Glaser et al present ExA-SPIM, a light-sheet microscope platform with large volumetric coverage (Field of view 85mm^2, working distance 35mm ), designed to image expanded mouse brains in their entirety. The authors also present an expansion method optimized for whole mouse brains, and an acquisition software suite. The microscope is employed in imaging an expanded mouse brain, the macaque motor cortex and human brain slices of white matter.<br /> This is impressive work, and represents a leap over existing light-sheet microscopes. As an example, it offers a ~ fivefold higher resolution than mesoSPIM (https://mesospim.org/), a popular platform for imaging large cleared samples. Thus while this work is rooted in optical engineering, it manifests a huge step forward and has the potential to become an important tool in the neurosciences.

Strengths:

-ExA-SPIM features an exceptional combination of field of view, working distance, resolution and throughput.

-An expanded mouse brain can be acquired with only 15 tiles, lowering the burden on computational stitching. That the brain does not need to be mechanically sectioned is also seen as an important capability.

-The image data is compelling, and tracing of neurons has been performed. This demonstrates the potential of the microscope platform.

Weaknesses:

-There is a general question about the scaling laws of lenses, and expansion microscopy, which in my opinion remained unanswered: In the context of whole brain imaging, a larger expansion factor requires a microscope system with larger volumetric coverage, which in turn will have lower resolution (Figure 1B). So what is optimal? Could one alternatively image a cleared (non-expanded) brain with a high resolution ASLM system (Chakraborty, Tonmoy, Nature Methods 2019, potentially upgraded with custom objectives) and get similar effective resolution as the authors get with expansion? This is not meant to diminish the achievement, but it was unclear if the gains in resolution from the expansion factor are traded off by the scaling laws of current optical systems.

-It was unclear if 300 nm lateral and 800 nm axial resolution is enough for many questions in neuroscience. Segmenting spines, distinguishing pre- and postsynaptic densities, or tracing densely labeled neurons might be challenging. A discussion about the necessary resolution levels in neuroscience would be appreciated.

-Would it be possible to characterize the aberrations that might be still present after whole brain expansion? One approach could be to image small fluorescent nanospheres behind the expanded brain, and recover the pupil function via phase retrieval. But even full width half maximum (FWHM) measurements of the nanospheres' images would give some idea of the magnitude of the aberrations.

Review of the revised manuscript:

The authors have carefully addressed my concerns and suggestions.

I appreciate the extended discussion on tissue clearing compared to expansion. I would recommend substantiating some of the statements though with references, or in other instances expanding a little further. I would encourage the authors to consider the points below. But there is also another path to actually reduce that specific discussion, if the conclusion is that it opened more questions than answers.

Specifically, here are some points in the paragraph that discusses tissue clearing and expansion that could be improved:<br /> -The statement "Spherical aberration increases with NA" reads nonspecific to me. I think a more precise formulation would be "The effect of spherical aberration (e.g. loss of Strehl ratio) increases with NA. The stated third power law would also benefit from a reference.<br /> -The statement "the index of refraction gradients in tissue decreases with the third power of the expansion factor..." reads a bit odd. "Gradients in refractive index" would be more consistent with the usage of r.i. throughout the manuscript.<br /> For the third power law, it might be important to know what drives the remaining refractive index variation in expansion microscopy. If it is the labels and their linkers, then indeed, they get increasingly diluted as their amount remains constant. However, if the aberrations are caused by the polymer gel, I would assume you would need more monomer material for higher expansion factors? Thus, I was not fully sure about the scaling law in this case. If there is a reference where this was explored in detail, that would resolve this issue.

-The statement that aberrations scale with gradients in refractive index also needs either a reference, or an explanation for the reader. I think figure S4 was supposed to illustrate this, but was not referenced in the discussion (and could be clarified, see comment below).

To me, the discussion focused strongly on tissue clearing vs expansion. What was left out in the discussion was if larger expansion factors would be favorable (i.e. whole brain imaging with 10-20X expansion instead of 4-5X). Some arguments implicitly seemed to stipulate that a larger expansion factor would optically be favorable. But Figure S7 highlights another tradeoff with the decay in sensitivity and Figure 1b provides the technological constraints on lens design. So as a reader, I was not fully sure if the next frontier should be 10-20X expansion brain imaging, or if 4-5X is currently a sweet spot.

Further comments:

Please explain the variables in Figure S4, such as F, WD and d. It was unclear to me what the RI profile should mean in the bottom row. Naively, the figure of merit would be the optical path length that is integrated along the different rays, as this leads to a variation in the wavefront.

Figure S5: I would caution to say the SNR was quantified, but rather say it was estimated (in the shot noise limit). Was the background subtracted for the SNR measurements?<br /> Squaring the SNR estimates, it looks like the photon counts went down ~10-fold from z=2mm to z=25mm. That is a larger reduction in signal than I had expected. If it was based solely on aberrations, a 10-fold drop in Strehl ratio seems significant (potentially smaller if we assume the light-sheet also underwent aberrations). Are there other factors that could explain the signal reduction (maybe from the labeling side)?<br /> Further on Figure S5: Fourier transforms (power spectrum) and single line profiles are in my opinion not the best way to quantify resolution. Could the authors perform image decorrelation analysis on the region of interest (Descloux, A., Kristin Stefanie Grußmayer, and Aleksandra Radenovic. "Parameter-free image resolution estimation based on decorrelation analysis." Nature methods 2019) or Fourier ring correlation? This would give in some sense an average resolving power in that depth, and would remove the bias from picking a line profile.

Reviewer #1 (Public Review):

Abreo et al., performed a detailed multidisciplinary analysis of a pathogenic variant of the KCNQ2 ion channel subunit identified in a child with neonatal-onset epilepsy and neurodevelopmental disorders. These analyses revealed multiple molecular and cellular mechanisms associated with this variant, and providing important insights into what distinguishes distinct pathogenic variants of KCNQ2 associated with self-limited familial neonatal epilepsy versus those leading to developmental and epileptic encephalopathy, and how they may mechanistically differ, to result in different extents of developmental impairment. The authors first provide a detailed clinical description of the patient heterozygous for a novel pathogenic variant encoding KCNQ2 G256W. They then model the structure of the G256W variant based on recent cryo-EM structures of KCNQ2 and other ion channel subunits and find that while the affected position is quite distinct from the channel pore, it participates in a novel, evolutionarily conserved set of amino acids that form a network of hydrogen bonds that stabilize the structure of the pore domain. They then undertake a series of rigorous and quantitative laboratory experiments in which the KCNQ2 G256W variant is coexpressed exogenously with WT KCNQ2 and KCNQ3 subunits in heterologous cells, and endogenously in novel gene edited mice generated for this study. This includes detailed electrophysiological analyses in the transfected heterologous cells revealing the dominant-negative phenotype of KCNQ2 G256W. They find altered firing properties in hippocampal CA1 neurons in brain slices from the heterozygous KCNQ2 G256W mice. They next show that the expression and localization of KCNQ channels is altered in brain neurons from heterozygous KCNQ2 G256W mice, suggesting that this variant impacts KCNQ2 trafficking and stability. Together, these laboratory studies reveal that the molecular and cellular mechanisms shaping KCNQ channel expression, localization and function are impacted at multiple levels by the variant encoding KCNQ2 G256W, likely contributing to the clinical features of the child heterozygous for this variant relative to patients harboring distinct KCNQ2 pathogenic variants.

Reviewer #1 (Public Review):

The hypothesis is based on the idea that inversions capture genetic variants that have antagonistic effects on male sexual success (via some display traits) and survival of females (or both sexes) until reproduction. Furthermore, a sufficiently skewed distribution of male sexual success will tend to generate synergistic epistasis for male fitness even if the individual loci contribute to sexually selected traits in an additive way. This should favor inversions that keep these male-beneficial alleles at different loci together at a cis-LD. A series of simulations are presented and show that the scenario works at least under some conditions. While a polymorphism at a single locus with large antagonistic effects can be maintained for a certain range of parameters, a second such variant with somewhat smaller effects tends to be lost unless closely linked. It becomes much more likely for genomically distant variants that add to the antagonism to spread if they get trapped in an inversion; the model predicts this should drive accumulation of sexually antagonistic variants on the inversion versus standard haplotype, leading to the evolution of haplotypes with very strong cumulative antagonistic pleiotropic effects. This idea has some analogies with one of predominant hypotheses for the evolution of sex chromosomes, and the authors discuss these similarities. The model is quite specific, but the basic idea is intuitive and thus should be robust to the details of the model assumption. It makes perfect sense in the context of the geographic pattern of inversion frequencies.

To provide empirical support for this idea, the authors study the dynamics of inversions in population cages over one generation, tracking their frequencies through amplicon sequencing at three time points: (young adults), embryos and very old adult offspring of either sex (>2 months from adult emergence). Out of four inversions included in the experiment, two show patterns consistent with antagonistic effects on male sexual success (competitive paternity) and the survival of offspring, especially females, until an old age, which the authors interpret as consistent with their theory.

There are several reasons why the support from these data for the proposed theory is not waterproof.

(1) As I have already pointed out in my previous review, survival until 2 months (in fact, it is 10 weeks and so 2.3 months) of age is of little direct relevance to fitness, whether under natural conditions or under typical lab conditions.

The authors argue this objection away with two arguments<br /> First, citing Pool (2015) they claim that the average generation time (i.e. the average age at which flies reproduce) in nature is 24 days. That paper made an estimate of 14.7 generations per year under the North Carolina climate. As also stated in Pool (2015), the conditions in that locality for Drosophila reproduction and development are not suitable during three months of the year. This yields an average generation length of about 19.5 days during the 9 months during which the flies can reproduce. On the highly nutritional food used in the lab and at the optimal temperature of 25 C, Drosophila need about 11-12 days to develop from egg to adult. Even assuming these perfect conditions, the average age (counted from adult eclosion) would be about 8 days. In practice, larval development in nature is likely longer for nutritional and temperature reasons, and thus the genomic data analyzed by Pool imply that the average adult age of reproducing flies in nature would be about 5 days, and not 24 days, and even less 10 weeks. This corresponds neatly to the 2-6 days median life expectancy of Drosophila adults in the field based on capture-recapture (e.g., Rosewell and Shorrocks 1987).<br /> Second, the authors also claim that survival over a period of 2 month is highly relevant because flies have to survive long periods where reproduction is not possible. However, to survive the winter flies enter a reproductive diapause, which involves profound physiological changes that indeed allow them to survive for months, remaining mostly inactive, stress resistant and hidden from predators. Flies in the authors' experiment were not diapausing, given that they were given plentiful food and kept warm. It is still possible that survival to the ripe old age of 10 weeks under these conditions still correlates well with surviving diapause under harsh conditions, but if so, the authors should cite relevant data. Even then, I do not think this allows the authors to conclude that longevity is "the main selective pressure" on Drosophila (l. 936).

(2) It appears that the "parental" (in fact, paternal) inversion frequency was estimated by sequencing sires that survived until the end of the two-week mating period. No information is provided on male mortality during the mating period, but substantial mortality is likely given constant courtship and mating opportunities. If so, the difference between the parental and embryo inversion frequency could reflect the differential survival of males until the point of sampling rather than / in addition to sexual selection.

(3) Finally, irrespective of the above caveats, the experimental data only address one of the elements of the theoretical hypothesis, namely antagonistic effects of inversions on reproduction and survival, notably that of females. It does not test for two other key elements of the proposed theory: the assumption of frequency-dependence of selection on male sexual success, and the prediction of synergistic epistasis for male fitness among genetic variants in the inversion. To be fair, particularly testing the latter prediction would be exceedingly difficult. Nonetheless, these limitations of the experiment mean that the paper is much stronger theoretical than empirical contribution.

Reviewer #1 (Public Review):

Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to their disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP reporter fused to the first 83 residues of PfHO (PfHO N-term-GFP). To investigate the functional importance of PfHO, the authors generated an anhydrotetracycline (aTC) controlled PfHO knockdown strain. Strikingly, the parasites lacking PfHO failed to grow and lost their apicoplast. Finally, by chromatin immunoprecipitation (ChIP), quantitative PCR/RT-PCR, and growth assays, the authors showed that both the cognate N-terminus and HO-like domain were required for PfHO function as an apicoplast DNA interacting protein.

The authors systemically performed multidisciplinary approaches to address this difficult question: what is the function of this enzymatically dead PfHO? I enjoyed reading this manuscript and its thoughtful discussion. This study is not of clinical importance for antimalarial treatments but also deepens our understanding of protein function evolution. While I understand these experiments are challenging to conduct in malaria parasites, the data quality of some of the experiments could be improved. For example, most of the Western blots and Southern blots are not of high quality.

Reviewer #1 (Public Review):

Summary:

This paper investigates the effects of the explicit recognition of statistical structure and sleep consolidation on the transfer of learned structure to novel stimuli. The results show a striking dissociation in transfer ability between explicit and implicit learning of structure, finding that only explicit learners transfer structure immediately. Implicit learners, on the other hand, show an intriguing immediate structural interference effect (better learning of novel structure) followed by successful transfer only after a period of sleep.

Strengths:

This paper is very well written and motivated, and the data are presented clearly with a logical flow. There are several replications and control experiments and analyses that make the pattern of results very compelling. The results are novel and intriguing, providing important constraints on theories of consolidation. The discussion of relevant literature is thorough. In summary, this work makes an exciting and important contribution to the literature.

Weaknesses:

There have been several recent papers that have identified issues with alternative forced choice (AFC) tests as a method of assessing statistical learning (e.g. Isbilen et al. 2020, Cognitive Science). A key argument is that while statistical learning is typically implicit, AFC involves explicit deliberation and therefore does not match the learning process well. The use of AFC in this study thus leaves open the question of whether the AFC measure benefits the explicit learners in particular, given the congruence between knowledge and testing format, and whether, more generally, the results would have been different had the method of assessing generalization been implicit. Prior work has shown that explicit and implicit measures of statistical learning do not always produce the same results (eg. Kiai & Melloni, 2021, bioRxiv; Liu et al. 2023, Cognition).

Given that the explicit/implicit classification was based on an exit survey, it is unclear when participants who are labeled "explicit" gained that explicit knowledge. This might have occurred during or after either of the sessions, which could impact the interpretation of the effects.

Reviewer #1 (Public Review):

Summary:

The study by Nelson et al. is focused on the formation of the Drosophila Posterior Signaling Center (PSC) which ultimately acts as a niche to support hematopoietic stem cells of the lymph gland (LG). Using a combination of genetics and live imaging, the authors show that PSC cells migrate as a tight collective and associate with multiple tissues during a trajectory that positions them at the posterior of the LG.<br /> This is an important study that identifies Slit-Robo signaling as a regulator of PSC morphogenesis, and highlights the complex relationship of interacting cell types - PSC, visceral mesoderm (VM), and cardioblasts (CBs) - in the coordinated development of these three tissues during organ development. However, one point requiring clarification is the idea that PSC cells exhibit a collective cell migration; it is not clear that the cells are migrating rather than being pushed to a more dorsal position through dorsal closure and/or other similar large-scale embryo movement. This does not detract from the very interesting analysis of PSC morphogenesis as presented.

Strengths:

(1) Using the expression of Hid or Grim to ablate associated tissues, they find evidence that the VM and CB of the dorsal vessel affect PSC migration/morphology whereas the alary muscles do not. Slit is expressed by both VM and CBs, and therefore Slit-Robo signaling was investigated as PSCs express Robo.

(2) Using a combination of approaches, the authors convincingly demonstrate that Slit expression in the CBs and VM acts to support PSC positioning. A strength is the ability to knockdown slit levels in particular tissue types using the Gal4 system and RNAi.

(3) Although in the analysis of robo mutants, the PSC positioning phenotype is weaker in the individual mutants (robo1 and robo2) with only the double mutant (robo1,robo2) exhibiting a phenotype comparable to the slit RNAi. The authors make a reasonable argument that Slit-Robo signaling has an intrinsic effect, likely acting within PSCs because PSCs show a phenotype even when CBs do not (Figure 4G).

(4) New insight into dorsal vessel formation by VM is presented in Figure 4A, B, as loss of the VM can affect dorsal vessel morphogenesis. This result additionally points to the VM as important.

Weaknesses:

(1) The authors are cautioned to temper the result that Slit-Robo signaling is intrinsic to PSC since the loss of robo may affect other cell types (besides CBs and PSCs) to indirectly affect PSC migration/morphogenesis. In fact, in the robo2, robo1 mutant, the VM appears to be incorrectly positioned (Figure 4G).

(2) If possible, the authors should use RNAi to knockdown Robo1 and Robo2 levels specifically in the PSCs if a Gal4 is available; might Antp.Gal4 (Fig 1K) be useful? Even if knockdown is achieved in PSCs+CBs, this would be a better/complementary experiment to support the approach outlined in Figure 4D.

(3) Movies are hard to interpret, as it seems unclear that the PSCs actively migrate rather than being pushed/moved indirectly due to association with VM and CBs/dorsal vessel.

Reviewer #1 (Public Review):

The authors describe a massively parallel reporter assays (MPRA) screen focused on identifying polymorphisms in 5' and 3' UTRs that affect translation efficiency and thus might have a functional impact on cells. The topic is of timely interest, and indeed, several related efforts have recently been published and preprinted (e.g., https://pubmed.ncbi.nlm.nih.gov/37516102/ and https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635273/). This study has several major issues with the results and their presentation.

Major comments:

(1) The main issue is that it appears that the screen has largely failed, yet the reasons for that are unclear, which makes it difficult to interpret. The authors start with a library that includes approximately 6,000 variants, which makes it a medium-sized MPRA. But then, only 483 pairs of WT/mutated UTRs yield high-confidence information, which is already a small number for any downstream statistical analysis, particularly since most don't actually affect translation in the reporter screen setting (which is not unexpected). It is unclear why >90% of the library did not give high-confidence information. The profiles presented as base-case examples in Figure 2B don't look very informative or convincing. All the subsequent analysis is done on a very small set of UTRs that have an effect, and it is unclear to this reviewer how these can yield statistically significant and/or biologically relevant associations.

(2) From the variants that had an effect, the authors go on to carry out some protein-level validations and see some changes, but it is not clear if those changes are in the same direction as observed in the screen.

(3) The authors follow up on specific motifs and specific RBPs predicted to bind them, but it is unclear how many of the hits in the screen actually have these motifs, or how significant motifs can arise from such a small sample size.

(4) It is particularly puzzling how the authors can build a machine learning predictor with >3,000 features when the dataset they use for training the model has just a few dozens of translation-shifting variants.

(5) The lack of meaningful validation experiments altering the SNPs in the endogenous loci by genome editing limits the impact of the results.

Reviewer #1 (Public Review):

Summary:

Even though this is not the first report that the mutation in the DNAH12 gene causes asthenoteratozoospermia, the current study explores the sperm phenotype in-depth. The authors show experimentally that the said mutation disrupts the proper axonemal arrangement and recruitment of DNALI1 and DNAH1 - proteins of inner dynein arms. Based on these results, the authors propose a functional model of DNAH12 in proper axonemal development. Lastly, the authors demonstrate that the male infertility caused by the studies mutation can be rescued by ICSI treatment at least in the mouse. This study furthers our understanding of male infertility caused by a mutation of axonemal protein DNAH12, and how this type of infertility can be overcome using assisted reproductive therapy.

Strengths:<br /> This is an in-depth functional study, employing multiple, complementary methodologies to support the proposed working model.

Weaknesses:

The study strength could be increased by including more controls such as peptide blocking of the inhouse raised mouse and rat DNAH12 antibodies, and mass spectrometry of control IP with beads/IgG only to exclude non-specific binding. Objective quantifications of immunofluorescence images and WB seem to be missing. At least three technical replicates of western blotting of sperm and testis extracts could have been performed to demonstrate that the decrease of the signal intensity between WT and mutant was not caused by a methodological artifact.

Reviewer #1 (Public Review):

Summary:

In this manuscript, the authors reveal a new role for SDG7 in the regulation of H3K36me2 and me3. SDG7 appear to be functionally redundant to SDG8 as the double mutant presents lower levels of H3K36me and stronger phenotypes than either single mutant, however, their mechanisms of action might differ as the proteins displayed different localization on their target genes, with SDG7 localizing preferentially to TSS and TES while SDG8 covers the gene body. SDG7 binds preferentially to PREs, which recruit PRC2 for H3K27me3 deposition. The authors therefore present an interesting model where SDG7 evicts PRC2 from silenced genes, leading to a loss of H3K27me3. This would allow the transcriptional activation of the genes and the deposition of H3K36me3.

Strengths:

Overall, the manuscript is well-written and organized, although some paragraphs need clarifications. The figures are clear and well designed and the proposed model is compelling. While the manuscript is already interesting as it is, I think addressing the following questions would elevate it even more and refine the proposed model:

Weaknesses/potential aspects to address:

(1) It is still unclear whether SDG7 directly catalyzes H3K36me or if it promotes its deposition simply by eviction of PRC2. The AlphaFold and structure analyses show a significant similarity between the catalytic domains which would support the first possibility, but some more experiments would be required to prove this more definitively.

(2) Does SDG7 directly recognize the PRE (as suggested by the model in Figure 5F) or is it recruited by some transcription factors? Is SDG7 known to interact with any of the PRC2 recruiters?

(3) Line154/Figure 2A: The metagene plot for H3K36me3 shows a lower level on the gene body but a higher peak in sdg7sdg8 double mutants compared to the Wild-type, which is a bit surprising, especially considering that the immunostaining in reference 19 showed a near complete loss of H3K36me3 signal in the same double mutant. Can this higher peak be an artifact from the normalization strategy, or due to the existence of different subpopulations of genes?

Indeed, on the genome tracks presented by the authors, the hypomethylated genes show a loss of signal on the entire gene body, and not a higher peak near the TSS. It might be interesting to generate metagene plots for H3K36me3 hypo and hyper-methylated genes, to see if the higher peak at the TSS is solely due to the hyper-methylated genes.

(4) Figure 2C: More than 40% of differentially methylated genes are actually hypermethylated, but the authors do not discuss this at all. What are those genes, are they targeted by SDG7 or 8? Could they be responsible for the higher peak at the TSS observed in the double mutant? (see previous comment).

(5) Figure 2C and D: The method section states that the ChIP-seq was performed on 5-day-old seedlings, while the legend of this figure mentions root and shoot samples but this does not appear in the figure itself. There is also mention of shoot and root samples in Supplementary Tables 1 and 2. The authors should clarify which tissue was used for the data presented in Figure 2 and correct the legends or the methods accordingly.

(6) Line 270/Fig 4K and L: The text mentions looking at the 838 genes "downregulated in clf sdg7 sdg8 relative to sdg7 sdg8" and in the overlap, the authors identified FLC. However, in Figure 5D, FLC is upregulated in clf sdg7-sdg8 compared to sdg7-sdg8, not downregulated as mentioned in line 270. The Venn diagram in Figure 4L mentions "sdg vs clf sdg up", which would fit the pattern seen in Figure 5D, but the number of genes (838) matches the number of downregulated genes in the sdg7sdg8 vs clf s dg7sdg8 volcano plot.

I would actually expect the phenotype rescue to be caused by genes that are up in Wt vs clf, down in Wt vs sdg7-sdg8, and back up in sdg7-sdg8 vs clf-sdg7-sdg8, not "up/down/down" as mentioned in the text: genes would be downregulated in sdg7-sdg8 because of a loss of H3K36me and therefore hypermethylation of H3K27, but in the absence of CLF, this hypermethylation is reversed and the genes are upregulated in the triple mutant compared to the sdg7-sdg8 mutant. This is also what the authors see and describe in their cluster analysis in Figure 4M and line 280, mentioning an upregulation in clf-sdg7-sdg8 vs sdg7-sdg8. Could the authors please clarify these discrepancies between the different subplots and within the text itself? Was there maybe some error plotting the volcano plot and/or Venn diagram?

In general, as this part is quite complicated, maybe it would benefit from a clearer explanation from the authors as to why they look at those particular overlaps, so that the reader can more easily follow their train of thought.

(7) Figure 4N/Line 286: How were these 828 genes identified? Is it stemming from a clf-sdg7-sdg8 vs sdg7-sdg8 comparison? The legend says "genes shown by white color in Fig. 4M", do the authors mean the two clusters previously described?

(8) Line 300: "suggesting that SDG8 primarily mediates target gene expression in conjunction with PAF1C". This statement is based on overlapping genes that are downregulated in sdg7-sdg8 double mutant and paf1c mutants but concludes only on the role of SDG8. I feel that to state that SDG8 regulates expression in conjunction with PAF1C, the authors should rather examine the genes downregulated in the sdg8 mutant, especially considering the reduced overlap between genes downregulated in sdg8 and sdg7-sdg8 (according to Figure 2C, only 30% of the genes downregulated in sdg8 are also downregulated in the double mutant), or this statement should be corrected to also include SDG7.

Maybe it would be easier to read the figure if the authors created a master list of genes downregulated in at least one of the paf1c mutants they examined (as they anyway do not examine in detail the contribution of each individual paf1c mutant), and overlap it with the genes downregulated in sdg7, sdg8 or sdg7-sdg8.

(9) Line 326: "We also discovered that SDG7 and SDG8 overcome PRC2-mediated silencing, leading to a switch from H3K27 methylation to H3K36 methylation during growth and development." While part of this statement is supported by the ChIP data presented in Figure 4E, I think a ChIP for H3K36me2 and/or me3 is necessary to prove the existence of a K27me to K36me switch.

(10) Line 347: The authors state that SDG8 is located at the TSS and 3' end of genes, but on line 187 they state that it occupies the gene body (which is supported by the plot in Figure 3A).

(11) Line 351: The authors suggest a role of RNApolII in the deposition of K36me, but their data are not sufficient to support this hypothesis. The transcriptome data show that both SDGs and PAF1C regulate a similar set of genes, but they do not show data demonstrating that RNApolII is necessary for the deposition of K36me. It might be interesting to examine H3K36me levels in a paf1c mutant to further consolidate their hypothesis.

Reviewer #1 (Public Review):

In this manuscript, Ferhat and colleagues describe their study aimed at developing a blood-brain barrier (BBB) penetrant agent that could induce hypothermia and provide neuroprotection from the sequelae of status epilepticus (SE) in mice. Hypothermia is used clinically in an attempt to reduce neurological sequelae of injury and disease. Hypothermia can be effective, but physical means used to reduce core body temperature are associated with untoward effects. Pharmacological means to induce hypothermia could be as effective with fewer untoward complications. Intracerebroventricularly applied neurotensin can cause hypothermia; however, neurotensin applied peripherally is degraded and does not cross the BBB. Here the authors develop and characterize a neurotensin conjugate that can reach the brain, induce hypothermia, and reduce seizures, cognitive changes, and inflammatory changes associated with status epilepticus.

Strengths:

(1) In general, the study is well-reasoned, well-designed, and seemingly well-executed.

(2) Strong dose-response assessment of multiple neurotensin conjugates in mice.

(3) Solid assessment of binding affinity, in vitro stability in blood, and brain uptake of the conjugate.

(4) Appropriate inclusion of controls for SE and for drug injections. However, perhaps a vehicle control could have been employed.

(5) Multifaceted assessment of neurodegeneration, inflammation, and mossy fiber sprouting in the different groups.

(6) Inclusion of behavioral assessments.

(7) Evaluates NSTR1 receptor distribution in multiple ways; however, does not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs.

(8) Demonstrates that this conjugate can induce hypothermia and have positive effects on the sequelae of SE. Could have a great impact on the application of pharmacologically-induced hypothermia as a neuroprotective measure in patients.

Weaknesses:

(1) The authors make the claim, repeatedly, that the hypothermia caused by the neurotensin conjugate is responsible for the effects they see; however, what they really show is that the conjugate causes hypothermia AND has favorable effects on the sequelae of SE. They need to discuss that they did not administer the conjugate without allowing the pharmacological hypothermia (e.g., by warming the animal, etc.).

(2) In the status epilepticus studies, it is unclear how or whether they monitored animals for the development of spontaneous seizures. Can the authors please describe this?

(3) They do not evaluate changes in receptor distribution or ping wo/w SE and/or various drugs.

(4) It is not clear why several different mouse strains were employed.

Reviewer #1 (Public Review):

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

Strengths:<br /> • The developed methodology is convincing and may offer an alternative - less computationally demanding - framework to investigate the single-cell and population structural properties of 3D genome organization at multiple scales.<br /> • Compared to the previous SRRW model, it allows for investigation of the role of excluded volume locally.<br /> • They perform some experiments to compare with model predictions and show consistency between the two.

Weaknesses:<br /> • The model currently cannot fully account for specific mechanisms that may shape the heterogeneous, complex organization of chromosomes (TAD at specific positions, A/B compartmentalization, promoter-enhancer loops, etc.).<br /> • By construction of their framework, excluded volume only impacts locally the polymer organization and larger-scale properties for which excluded volume could be a main actor (formation of chromosome territories [Rosa & Everaers, PLoS CB 2009], bottle-brush effects due to loop extrusion [Polovnikov et al, PRX 2023], etc.) cannot be captured.<br /> • Comparisons with experiments are solid but are not clearly quantified.

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

Reviewer #1 (Public Review):

Summary:

In this work, the authors present a novel, multi-layer computational model of motor control to produce realistic walking behaviour of a Drosophila model in the presence of external perturbations and under sensory and motor delays. The novelty of their model of motor control is that it is modular, with divisions inspired by the fly nervous system, with one component based on deep learning while the rest are based on control theory. They show that their model can produce realistic walking trajectories. Given the mostly reasonable assumptions of their model, they convincingly show that the sensory and motor delays present in the fly nervous system are the maximum allowable for robustness to unexpected perturbations.

Their fly model outputs torque at each joint in the leg, and their dynamics model translates these into movements, resulting in time-series trajectories of joint angles. Inspired by the anatomy of the fly nervous system, their fly model is a modular architecture that separates motor control at three levels of abstraction:<br /> (1) oscillator-based model of coupling of phase angles between legs,<br /> (2) generation of future joint-angle trajectories based on the current state and inputs for each leg (the trajectory generator), and<br /> (3) closed-loop control of the joint-angles using torques applied at every joint in the model (control and dynamics).

These three levels of abstraction ensure coordination between the legs, future predictions of desired joint angles, and corrections to deviations from desired joint-angle trajectories. The parameters of the model are tuned in the absence of external perturbations using experimental data of joint angles of a tethered fly. A notable disconnect from reality is that the dynamics model used does not model the movement of the body and ground contacts as is the case in natural walking, nor the movement of a ball for a tethered fly, but instead something like legs moving in the air for a tethered fly.

In order to validate the realism of the generated simulated walking trajectories, the authors compare various attributes of simulated to real tethered fly trajectories and show qualitative and quantitative similarities, including using a novel metric coined as Kinematic Similarity (KS). The KS score of a trajectory is a measure of the likelihood that the trajectory belongs to the distribution of real trajectories estimated from the experimental data. While such a metric is a useful tool to validate the quality of simulated data, there is some room for improvement in the actual computation of this score. For instance, the KS score is computed for any given time-window of walking simulation using a fraction of information from the joint-angle trajectories. It is unclear if the remaining information in joint-angle trajectories that are not used in the computation of the KS score can be ignored in the context of validating the realism of simulated walking trajectories.

The authors validate simulated walking trajectories generated by the trained model under a range of sensorimotor delays and external perturbations. The trained model is shown to generate realistic joint-angle trajectories in the presence of external perturbations as long as the sensorimotor delays are constrained within a certain range. This range of sensorimotor delays is shown to be comparable to experimental measurements of sensorimotor delays, leading to the conclusion that the fly nervous system is just fast enough to be robust to perturbations.

Strengths:

This work presents a novel framework to simulate Drosophila walking in the presence of external perturbations and sensorimotor delay. Although the model makes some simplifying assumptions, it has sufficient complexity to generate new, testable hypotheses regarding motor control in Drosophila. The authors provide evidence for realistic simulated walking trajectories by comparing simulated trajectories generated by their trained model with experimental data using a novel metric proposed by the authors. The model proposes a crucial role in future predictions to ensure robust walking trajectories against external perturbations and motor delay. Realistic simulations under a range of prediction intervals, perturbations, and motor delays generating realistic walking trajectories support this claim. The modular architecture of the framework provides opportunities to make testable predictions regarding motor control in Drosophila. The work can be of interest to the Drosophila community interested in digitally simulating realistic models of Drosophila locomotion behaviors, as well as to experimentalists in generating testable hypotheses for novel discoveries regarding neural control of locomotion in Drosophila. Moreover, the work can be of broad interest to neuroethologists, serving as a benchmark in modelling animal locomotion in general.

Weaknesses:

As the authors acknowledge in their work, the control and dynamics model makes some simplifying assumptions about Drosophila physics/physiology in the context of walking. For instance, the model does not incorporate ground contact forces and inertial effects of the fly's body. It is not clear how these simplifying assumptions would affect some of the quantitative results derived by the authors. The range of tolerable values of sensorimotor delays that generate realistic walking trajectories is shown to be comparable with sensorimotor delays inferred from physiological measurements. It is unclear if this comparison is meaningful in the context of the model's simplifying assumptions. The authors propose a novel metric coined as Kinematic Similarity (KS) to distinguish realistic walking trajectories from unrealistic walking trajectories. Defining such an objective metric to evaluate the model's predictions is a useful exercise, and could potentially be applied to benchmark other computational animal models that are proposed in the future. However, the KS score proposed in this work is calculated using only the first two PCA modes that cumulatively account for less than 50% of the variance in the joint angles. It is not obvious that the information in the remaining PCA modes may not change the log-likelihood that occurs in the real walking data.