Reviewer #2 (Public review):

Aging poses a significant challenge to the regenerative capacity of oligodendrocyte precursor cells (OPCs) to differentiate and myelinate neuronal axons. Myelin abnormalities accumulate with age, and it is likely that the ability of OPCs to differentiate into myelinating oligodendrocytes becomes progressively impaired during aging, leading to inefficient turnover of damaged myelin and oligodendrocytes, as well as reduced adaptive myelination. Understanding the molecular mechanisms underlying the compromised capacity of aged OPCs is therefore critical for addressing age-related white matter decline.

This study aims to decipher the intrinsic molecular changes that occur in aged OPCs. By profiling differentially expressed transcription factors (TFs) between young and aged OPCs, and by employing a novel bioinformatic tool to identify key TFs that undergo dynamic changes across distinct stages of OPC differentiation, the authors identify Bcl11a as a potential regulator. Bcl11a is highly expressed in young OPCs but markedly reduced in aged cells. Functional experiments further demonstrate that while Bcl11a does not affect OPC proliferation, it significantly promotes the differentiation of aged OPCs. Importantly, this effect is also observed in vivo following demyelinating injury in aged mice.

While the study provides compelling evidence that BCL11A represents a limiting factor for OPC differentiation during ageing, the downstream targets and molecular mechanisms through which BCL11A exerts its effects are not directly addressed. As such, the work should be interpreted primarily as identifying a key regulatory node rather than a fully defined molecular pathway.

Overall, this study offers valuable insights into the age-related loss of regenerative capacity in the central nervous system and introduces a computational framework that may be broadly useful for investigating dynamic gene regulation in other biological contexts.

Major Points:

(1) MACS mouse anti-A2B5 microbeads are not OPC-specific and may also label astrocyte precursor cells or immature astrocytes. How do the authors justify this caveat? Could some of the claimed "OPC-specific" switch genes in fact be enriched in astrocyte lineage cells?

(2) Overall, Figures 1 and 2 are not very informative in terms of biological insight. The authors should provide more detail in the main figures regarding the enriched gene sets associated with each of the Type 1-4 switch categories. For example, summarizing the top Gene Ontology terms for each switch type would greatly enhance interpretability.

(3) A similar issue applies to Figure 3. The authors should explicitly specify the transcription factors in the main figure, particularly the 27 TFs identified through the ENCODE/ReMap2 analysis.

(4) Have the authors validated Bcl11a expression across different CNS cell types and between young and aged conditions using independent methods such as qPCR, immunofluorescence, or western blotting?

(5) Regarding OPC aging, an open question is whether the reduced differentiation capacity of aged OPCs is an intrinsic property of the cells themselves or whether it results from prolonged exposure to an aging environment that induces non-cell-autonomous epigenetic or genetic changes, thereby rendering OPCs less efficient at differentiating. It would be helpful if the authors could expand on this point in the Discussion, with reference to relevant previous studies and experimental evidence.

(6) Do the authors observe a change in the number or density of OPCs between young and aged mice?

(7) The in vivo characterization of Bcl11a overexpression using the AAV-based approach appears incomplete. Do aged mice overexpressing Bcl11a in Sox10⁺ cells exhibit reduced age-related myelin degeneration under baseline conditions? In the LPC model, do the authors observe differences in lesion size and/or remyelination efficiency?

(8) Are the authors presenting gSWITCH for the first time in this manuscript? Given that the gSWITCH framework is novel and central to the study, its conceptual contribution could be emphasized more strongly. A brief comparison with existing trajectory- or pattern-based methods-ideally in the main text around Figure 1-would help readers better appreciate its novelty.

(9) The evolutionary analysis also appears somewhat disconnected from the rest of the study. Could the authors leverage available public datasets to test whether a similar Bcl11a expression trajectory is observed in human oligodendrocyte lineage cells?

Reviewer #2 (Public review):

Summary

This manuscript focuses on the role of social responsibility and guilt in social decision making by integrating neuroimaging and computational modeling methods. Across two studies, participants completed a lottery task in which they made decisions for themselves or for a social partner. By measuring momentary happiness throughout the task, the authors show that being responsible for a partner's bad lottery outcome leads to decreased happiness compared to trials in which the participant was not responsible for their partner's bad outcome. At the neural level, this guilt effect was reflected in increased neural activity in the anterior insula, and altered functional connectivity between the insula and the inferior frontal gyrus. Using computational modeling, the authors show that trial by trial fluctuations in happiness were successfully captured by a model including participant and partner rewards and prediction errors (a 'responsibility' model), and model-based neuroimaging analyses suggested that prediction errors for the partner were tracked by the superior temporal sulcus. Taken together, these findings suggest that responsibility and interpersonal guilt influence social decision making.

Strengths

This manuscript investigates the concept of guilt in social decision making through both statistical and computational modeling. It integrates behavioral and neural data, providing a more comprehensive understanding of the psychological mechanisms. For the behavioral results, data from two different studies is included, and although minor differences are found between the two studies, the main findings remain consistent. The authors share all their code and materials, leading to transparency and reproducibility of their methods.

The manuscript is well-grounded in prior work. The task design is inspired by a large body of previous work on social decision making, and includes the necessary conditions to support their claims (i.e., Solo, Social, and Partner conditions). The computational models used in this study are inspired by previous work, and build on well-established economic theories of decision making. The research question and hypotheses clearly extend previous findings, and the more traditional univariate results align with prior work.

The authors conducted extensive analyses, as supported by the inclusion of different linear models and computational models described in the supplemental materials. Psychological concepts like risk preferences are defined and tested in different ways, and different types of analyses (e.g., univariate and multivariate neuroimaging analyses) are used to try to answer the research questions. The inclusion and comparison of different computational models provides compelling support for the claim that partner prediction errors indeed influence task behavior, as illustrated by the multiple model comparison metrics and the good model recovery.

The authors did a good job acknowledging other factors that could differ between the conditions, including the role of other emotions (like empathy) or agency in the decision making process. These additional analyses and nuances strengthen the manuscript and the interpretability of the findings.

Weaknesses

As the authors already note, they did not directly ask participants to report their feelings of guilt. The authors clearly describe this limitation, and also note that in addition to guilt, other emotions like empathy could also be at play in interpersonal decisions. Despite this limitation, this study provides insights into the neural and behavioral mechanisms of responsibility and guilt in social decision making, and how they influence behavior.

Reviewer #2 (Public review):

In this paper, Rayan et al. report that RNA influences cytotoxic activity of the staphylococcal secreted peptide cytolysin PSMalpha3 versus human cells and E. coli by impacting its aggregation. The authors used sophisticated methods of structural analysis and described the associated liquid-liquid phase separation. They also compare the influence of RNA on the aggregation and activity of LL-37, which shows differences from that on PSMalpha3.

Strengths:

That RNA impacts PSM cytotoxicity when co-incubated in vitro becomes clear.

Weaknesses:

I have two major and fundamental problems with this study:

(1) The premise, as stated in the introduction and elsewhere, that PSMalpha3 amyloids are biologically functional, is highly debatable and has never been conclusively substantiated. The property that matters most for the present study, cytotoxicity, is generally attributed to PSM monomers, not amyloids. The likely erroneous notion that PSM amyloids are the predominant cytotoxic form is derived from an earlier study by the authors that has described a specific amyloid structure of aggregated PSMalpha3. Other authors have later produced evidence that, quite unsurprisingly, indicated that aggregation into amyloids decreases, rather than increases, PSM cytotoxicity. Unfortunately, yet other groups have, in the meantime, published in-vitro studies on "functional amyloids" by PSMs without critically challenging the concept of PSM amyloid "functionality". Of note, the authors' own data in the present study, which show strongly decreased cytotoxicity of PSMalpha3 after prolonged incubation, are in agreement with monomer-associated cytotoxicity as they can be easily explained by the removal of biologically active monomers from the solution.

(2) That RNA may interfere with PSM aggregation and influence activity is not very surprising, given that PSM attachment to nucleic acids - while not studied in as much detail as here - has been described. Importantly, it does not become clear whether this effect has biologically significant consequences beyond influencing, again not surprisingly, cytotoxicity in vitro. The authors do show in nice microscopic analyses that labeled PSMalpha3 attaches to nuclei when incubated with HeLa cells. However, given that the cells are killed rapidly by membrane perturbation by the applied PSM concentrations, it remains unclear and untested whether the attachment to nucleic acids in dying cells makes any contribution to PSM-induced cell death or has any other biological significance.

Overall, the findings can be explained in a much more straightforward way with the common concept of cytotoxicity being due to monomeric PSMs, and the impact of nucleic acids on cytotoxicity being due to lowering of the concentration of that active form by RNA attachment. Further limiting the significance of the findings, whether this interaction has any biological significance on the physiology or infectivity of the PSM producer remains largely unexplored.

Reviewer #2 (Public review):

The authors argue that the Emiliano Aguirre Korongo (EAK) assemblage from the base of Bed II at Olduvai Gorge shows systematic exploitation of elephants by hominins about 1.78 million years ago. They describe it as the earliest clear case of proboscidean butchery at Olduvai and link it to a larger behavioral shift from the Oldowan to the Acheulean.

The manuscript makes a valuable contribution to the Olduvai Gorge record, offering a detailed description of the EAK faunal assemblage. In particular, the paper provides a high-resolution record of a juvenile Elephas recki carcass, associated lithic artifacts, and several green-broken bone specimens. These data are inherently valuable and will be of significant interest to researchers studying Early Pleistocene taphonomy.

Comments on previous round of revisions:

The revised manuscript does a good job of using less definitive language, particularly by adding "possible" qualifiers to several interpretations. This addresses the concern about overstatement.

The main issue raised in the original review, however, remains unresolved. Only two elephant bone specimens at EAK show green-bone breakage interpreted as anthropogenic, and the diagnostic basis for that interpretation is not demonstrated clearly on the EAK material itself. The manuscript discusses a suite of fracture attributes described as diagnostic of dynamic percussive breakage, but these attributes are not explicitly documented on the EAK specimens. Instead, the diagnostic traits are illustrated using material from other Olduvai contexts, and that behavior is then extrapolated to make similar claims at EAK. For a paper making a potentially important behavioral argument, the key diagnostic evidence is not clearly demonstrated at the focal assemblage.

This problem is evident in the presentation of the EAK specimens. In their response, the authors state that one EAK specimen shows "overlapping scars" and constitutes a "long bone flake"; however, these features are not clearly identifiable in the figures or captions as currently presented. The authors state that Figures S21-S23 clearly indicate human agency, including a long bone flake with overlapping scars and a view of the medullary surface, but it is unclear which specimens or surfaces these descriptions refer to. Figure S21 does appear to show green fracture and is described only as an "elephant-sized flat bone fragment with green-bone curvilinear break." Figure S22 shows the same bone and cortical surface in a different orientation, providing no additional information. In Figure S23, I cannot clearly identify a medullary surface or evidence of green-bone fracture from this image. None of these images clearly demonstrates overlapping scars, and the figures would be substantially improved by explicitly identifying the features described in the text. Even if both EAK specimens are accepted as green-broken, they do not demonstrate the co-occurrence of multiple diagnostic fracture traits such as multiple green breaks, large step fractures, hackle marks, and overlapping scars that the authors state is required to attribute dynamic percussive activity to hominins and address equifinality.

I appreciate that the authors are careful to state that spatial association between stone tools and fossils alone does not demonstrate hominin behavior, and that they treat the spatial analyses as supportive rather than decisive. While the association is intriguing, the problem is downstream: spatial association is used to strengthen an interpretation of butchery at EAK that still depends on fracture evidence that is not clearly documented at the assemblage level.

The critique concerning Nyayanga is not addressed in the revision. The manuscript proposes alternative explanations for the Nyayanga material but does not demonstrate why these are more plausible than the interpretation advanced by Plummer et al. (2023). I am not arguing that the Nyayanga material should be accepted as butchery; rather, showing that trampling is possible does not establish it as more probable than cut marks. In contrast, the EAK material is treated as evidence of butchery on the basis of evidence that, in my opinion, is more limited and less clearly demonstrated. Even if this is not the authors' intention, the uneven treatment removes an earlier megafaunal case from the comparison and strengthens the case for interpreting EAK as marking a behavioral shift toward megafaunal butchery by excluding other early cases.

While I remain concerned about how the EAK evidence is documented and interpreted, I think the manuscript is appropriate for publication and will generate useful discussion. Readers can then assess for themselves whether the available evidence supports the strength of the behavioral claims.

[Editors' note: the authors are encouraged to make this version the Version of Record.]

Reviewer #2 (Public review):

Summary:

King et al. present several sets of experiments aimed to address potential impact of UV irradiation on human mitochondrial DNA as well as possible role of mitochondrial TFAM protein in handling UV irradiated mitochondrial genomes. The carefully worded conclusion derived from the results of experiments performed with human HeLa cells, in vitro small plasmid DNA, with PCR-generated human mitochondrial DNA and with UV-irradiated small oligonucleotides is presented in the title of the manuscript: "UV irradiation alters TFAM binding to mitochondrial DNA". Authors also interpret results of somewhat unconnected experimental approaches to speculate that "TFAM as a potential DNA damage sensing protein in that it promotes UVC-dependent conformational changes in the [mitochondrial] nucleoids, making them more compact. They further propose that such a proposed compaction might trigger removal of UV-damaged mitochondrial genomes as well as facilitates replication of undamaged mitochondrial genomes.

Strengths:

(1) Authors presented convincing evidence that a very high dose (1500 J/m2) of UVC applied to oligonucleotides covering the entire mitochondrial DNA genome alleviates sequence specificity of TFAM binding (Figure 3). This high dose was sufficient to cause UV-lesions in a large fraction of individual oligonucleotides. The method has been developed in the lab of one of the corresponding authors (ref. 74) and is technically well refined. This result can be published as is or in combination with other data.

(2) Manuscript also presents AFM evidence (Figure 4) that TFAM, which was long known to facilitate compaction of mitochondrial genome (Alam et al., 2003; PMID 12626705 and follow up citations), causes in vitro compaction of a small pUC19 plasmid and that approximately 3 UVC lesions per plasmid molecule results in slight albeit detectable increase in TFAM compaction of the plasmid.

Both results are discussed in line of a possible extrapolation to in vivo phenomena. The revised version of the discussion includes a clear statement that no in vivo support was provided within the set of experiments presented in the manuscript.

Weaknesses:

The experiments presented on Figures 3 and 4 may support the speculation that TFAM can carry protective role of eliminating mitochondrial genomes with bulky lesions by way of excessive compaction and removal damaged genomes from the in vivo pool, however extensive additional studies that would go well beyond the experiments described in this paper are needed to fill the gap between this set of results and the proposed explanations.

Reviewer #2 (Public review):

Summary:

The manuscript by Chen et al addresses an important aspect of pathogenesis for mycobacterial pathogens, seeking to understand how bacterial effector proteins disrupt the host immune response. To address this question the authors sought to identify bacterial effectors from M. tuberculosis (Mtb) that localize to the host nucleus and disrupt host gene expression as a means of impairing host immune function. Their revised manuscript has strengthened their observations by performing additional experiments with BCG strains expressing tagged MgdE.

Strengths:

The researchers conducted a rigorous bioinformatic analysis to identify secreted effectors containing mammalian nuclear localization signal (NLS) sequences, which formed the basis of quantitative microscopy analysis to identify bacterial proteins that had nuclear targeting within human cells. The study used two complementary methods to detect protein-protein interaction: yeast two-hybrid assays and reciprocal immunoprecipitation (IP). The combined use of these techniques provides strong evidence of interactions between MgdE and SET1 components and suggests the interactions are in fact direct. The authors also carried out rigorous analysis of changes in gene expression in macrophages infected with MgdE mutant BCG. They found strong and consistent effects on key cytokines such as IL6 and CSF1/2, suggesting that nuclear-localized MgdE does in fact alter gene expression during infection of macrophages. The revised manuscript contains additional biochemical analyses of BCG strains expressing tagged MgdE that further supports their microscopy findings.

Reviewer #2 (Public review):

This manuscript examines how disease-associated hyperphosphorylation disrupts tau's role as a cooperative microtubule-binding regulator of intracellular transport. Using in vitro reconstitution assays and live-cell imaging in iPSC-derived neurons, the authors employ phosphomutant tau constructs (E14 to mimic hyperphosphorylation, AP to prevent phosphorylation) at 14 disease-associated residues to isolate phosphorylation effects independent of expression system-dependent PTM heterogeneity. The results show that hyperphosphorylated tau fails to form cooperative envelope-like structures on microtubules, instead binding diffusely and dissociating rapidly. In contrast, wild-type and phospho-resistant tau form cohesive envelopes that regulate motor protein access. At the single-molecule level, hyperphosphorylation reduces KIF5C inhibition while maintaining or enhancing KIF1A inhibition through altered processivity and detachment rates. In live neurons, hyperphosphorylated tau phenocopies tau knockout conditions, weakening tau-mediated inhibition of lysosome transport and increasing processive motility. The authors quantify tau binding using Gaussian mixture model-based image analysis and measure tau kinetics via FRAP, demonstrating that hyperphosphorylation-induced loss of cooperative binding correlates with dysregulated organelle transport. These findings establish a mechanism by which phosphorylation-driven disruption of tau's gatekeeper function on microtubules compromises axonal transport prior to aggregation in tauopathies. The paper provides interesting new knowledge for the field, but there are outstanding concerns that could be further addressed by the authors to strengthen and clarify the current manuscript:

(1) Lack of Phosphatase-Treated Control and Explicit WT Phosphorylation Quantification

Wild-type tau expressed in insect and mammalian cells is known to be phosphorylated by endogenous kinases (eg, GSK3, CDK5, MARK). The manuscript acknowledges this in the Discussion but provides no phosphatase-treated lysate control or quantification of endogenous phosphorylation on WT tau via phospho-specific Western blots. This leaves ambiguity about whether observed differences between WT and E14 reflect purely the introduced mutations or confounding baseline differences in phosphostate content.

(2) Limited Normalization of Motor Effects to Measured Tau Lattice Occupancy

Although kinesin trajectories are classified inside vs. outside tau envelopes (inherently normalizing to local tau density), motor parameters are not systematically reported as functions of tau fluorescence intensity across all constructs. Co-purifying MAPs or microtubule-modifying enzymes in cell lysates is not quantified or excluded, leaving residual uncertainty about tau-specificity of observed motor inhibition. This should be at least acknowledged in the results section.

(3) Insufficient Citation of Prior Neuronal Tau Envelope Evidence

In the Introduction, the authors state, "it was an open question if tau forms envelopes in neurons," but this understates existing evidence. Tan et al. (2019) report tau neuronal staining consistent with envelope formation, while Siahaan et al. (2021) provide more direct evidence in non-neuronal cells. The framing should acknowledge and integrate these prior findings.

(4) Unclear Wording on Expression System-Dependent Phosphorylation

The sentence "The phosphostate of tau is strongly dependent on the expression system" requires rewording. It is ambiguous whether this refers to the final phosphostate achieved after expression or the inherent phosphorylating capacity of each system. Clearer language would strengthen the methodological justification.

(5) Insufficient Quantification of Motor and Lysosome Transport Effect Magnitudes in Results Section

The data on molecular motor motility and lysosome transport are densely described. The magnitude of effects (fold-changes, percentage differences) should be explicitly stated in the Results section when first presenting findings to orient readers to biological significance. For example, effect magnitudes for lysosome run lengths, velocities, and directional bias should be quantified in text, not left to figure inspection.

(6) Incomplete Discussion of Projection Domain Necessity for Envelope Formation

The Discussion states the projection domain is "a critical regulator of both tau-tau and tau-microtubule interactions," but does not engage with prior domain dissection work. Tan et al. (2019) found that the entire projection domain is not necessary for envelope formation in vitro. The authors should discuss which projection domain regions are specifically regulated by phosphorylation vs. required for cooperativity, providing a more nuanced interpretation than implied by their current framing.

Reviewer #2 (Public review):

Summary:

To discover peptides that interact with autophagy-related protein LC3B and profile the key binding determinants, the authors screened a library of ~500,000 36-residue peptides derived from the human proteome using bacterial cell-surface display. Analysis of the screening data revealed exceptions to the reported LIR motif and a strong preference for negatively charged residues adjacent to the LIR.<br /> These results support a refinement of the LIR motif definition and expand the network of candidate LC3B interaction partners.

Strengths:

High-throughput approach.

Weaknesses:

Lack of in vitro data and molecular dynamics simulations.

Reviewer #2 (Public review):

Summary:

In this paper, the authors have characterized Rv2577 as a Fe3+/Zn2+ -dependent metallophosphatase and a nucleomodulin protein. The authors have also identified His348 and Asn359 as critical residues for Fe3+ coordination. The authors show that the proteins encode for two nuclease localization signals. Using C-terminal Flag expression constructs, the authors have shown that MmpE protein is secretory. The authors have prepared genetic deletion strains and show that MmpE is essential for intracellular survival of M. bovis BCG in THP-1 macrophages, RAW264.7 macrophages and mice model of infection. The authors have also performed RNA-seq analysis to compare the transcriptional profiles of macrophages infected with wild type and mmpE mutant strain. The relative levels of ~ 175 transcripts were altered in mmpE mutant infected macrophages and majority of these were associated with various immune and inflammatory signalling pathways. Using these deletion strains, the authors proposed that MmpE inhibits inflammatory gene expression by binding to the promoter region of vitamin D receptor. The authors also showed that MmpE arrests phagosome maturation by regulating the expression of several lysosome associated genes such as TFEB, LAMP1, LAMP2 etc. These findings reveal a sophisticated mechanism by which a bacterial effector protein manipulates gene transcription and promotes intracellular survival.

Strength:

The authors have used a combination of cell biology, microbiology and transcriptomics to elucidate the mechanisms by which Rv2577 contributes to intracellular survival.

Weakness:

The authors should thoroughly check the mice data and show individual replicate values in bar graphs.

Comments on revisions:

Thanks to the authors for addressing the concerns raised during the review of the original manuscript. The data is now presented with clarity, and discrepancies in mouse experiments have also been addressed with additional experiments.

Reviewer #3 (Public review):

Summary:

Metabolic dysfunction associated liver disease (MASLD) describes a spectrum of progressive liver pathologies linked to life style-associated metabolic alterations (such as increased body weight and elevated blood sugar levels), reaching from steatosis over steatohepatitis to fibrosis and finally end stage complications, such as liver failure and hepatocellular carcinoma. Treatment options for MASLD include diet adjustments, weight loss, and the receptor-β (THR-β) agonist resmetirom, but remain limited at this stage, motivating further studies to elucidate molecular disease mechanisms to identify novel therapeutic targets.

In their present study, the authors aim to identify early molecular changes in MASLD linked to obesity. To this end, they study a cohort of 109 obese individuals with no or early-stage MASLD combining measurements from two anatomic sides: 1. bulk RNA-sequencing and metabolomics of liver biopsies, and 2. metabolomics from patient blood. Their major finding is that GTPase-related genes are transcriptionally altered in livers of individuals with steatosis with fibrosis compared to steatosis without fibrosis.

Major comments:

(1) Confounders (such as (pre-)diabetes)

The patient table shows significant differences in non-MASLD vs. MASLD individuals, with the latter suffering more often from diabetes or hypertriglyceridemia. Rather than just stating corrections, subgroup analyses should be performed (accompanied with designated statistical power analyses) to infer the degree to which these conditions contribute to the observations. I.e., major findings stating MASLD-associated changes should hold true in the subgroup of MASLD patients without diabetes/of female sex and so forth (testing for each of the significant differences between groups).

Post-rebuttal update: The authors have performed the requested sub-group analysis and find the gene signatures hold for the non-diabetic sub-cohort, but not the diabetic subgroup. They denote a likely interaction between fibrosis and diabetes, that was not corrected for in the original analysis.

Post-post-rebuttal update: I thank the authors for having added Figure 5-figure supplement 2 to show this analysis.

(2) External validation

Additionally, to back up the major GTPase signature findings, it would be desirable to analyze an external dataset of (pre)diabetes patients (other biased groups) for alternations in these genes. It would be important to know if this signature also shows in non-MASLD diabetic patients vs. healthy patients or is a feature specific to MASLD. Also, could the matched metabolic data be used to validate metabolite alterations that would be expected under GTPase-associated protein dysregulation?

Post-rebuttal update: The authors confirm that with the present data, insulin resistance cannot be fully ruled out as a confounder to the GTP-ase related gene signature. They however plan future mouse model experiments to study whether the GTPase-fibrosis signature differs in diabetic vs. non-diabetic conditions.

(3) 3D liver spheroid MASH model, Fig. 6D/E

This 3D experiment is technically not an external validation of GTPase-related genes being involved in MASLD, since patient-derived cells may only retain changes that have happened in vivo. To demonstrate that the GTPase expression signature is specifically invoked by fibrosis the LX-2 set up is more convincing, however, the up-regulation of the GTPase-related genes upon fibrosis induction with TGF-beta, in concordance with the patient data, needs to be shown first (qPCR or RNA-seq). Additionally, the description of the 3D model is too uncritical. The maintenance of functional PHHs is a major challenge (PMID: 38750036, PMID: 21953633, PMID: 40240606, PMID: 31023926). It cannot be ruled out that their findings are largely attributable to either 1) the (other present) mesenchymal cells (i.e., mesenchyme-derived cells, such as for example hepatic stellate cells, not to be confused with mesenchymal stem cells, MSCs), or 2) related to potential changes in PHHs in culture, and these limitations need to be stated.

Post-rebuttal update: To address the concern of other cells than hepatocytes contributing to the observed effects in culture, the authors performed TGF-beta treatment in independent mono-cultures (Figure R4): LX-2 and hepatocytes, and the spheroid system. Surprisingly, important genes highlighted in Figure 6E for the spheroid system (RAB6A, ARL4A, RAB27B, DIRAS2) are all absent from this qPCR(?) validation experiment. The authors evaluate instead RAC1, RHOU, VAV1, DOCK2, RAB32. ­In spheroids, RHOU and RAB32 are down-regulated with TGF-B. In hepatocytes DOCK2 and RAC seemed up-regulated. They find no difference in these genes in LX-2 cells. Surprisingly, ACTA2 expression values are missing for LX-2 cells. Together, it is hard to judge which individual cell type recapitulates the changes observed in patients in this validation experiment, as the major genes called out in Figure 6E are not analyzed.

Post-post-rebuttal update: I thank the authors for having added Figure 6-figure supplement 5 to show qPCR results for this question.

Unfortunately, the 3D liver spheroid model used (as presente­d in PMID39605182) lacks important functional validation tests of maintained hepatocyte identity in culture (at the very least Albumin expression and secretion plus CYP3A4 assay). This functional data (acquired at the time point in culture when the RNA expression analysis in 6E was performed) is indispensable prior to stating that mature hepatocytes cause the observed effects.

Post-post-rebuttal update: I thank the authors for having added more references, I still think a quick functional validation of the system (at the time point in culture when the RNA expression analysis in 6E was performed) would be beneficial.

(4) Novelty / references

Similar studies that also combined liver and blood lipidomics/metabolomics in obese individuals with and without MASLD (e.g. PMID 39731853, 39653777) should be cited. Additionally, it would benefit the quality of the discussion to state how findings in this study add new insights over previous studies, if their findings/insights differ, and if so, why.

Post-rebuttal update: The authors have included the studies into their discussion.

Overall post-post-rebuttal update: I thank the authors for having added more data, important discussion points, and references, and have no further requests.

Reviewer #2 (Public review):

Summary:

This paper uses an optogenetic approach to either activate or inhibit separate neural pathways projecting to the ventral CA1 hippocampal subregion, from either CA3 or the entorhinal cortex. The authors report that manipulation of the vCA3→vCA1 pathway affected behavioural performance on a number of tasks: elevated plus maze, open field, Vogel conflict test and freezing behaviour to both context and a trace CS cue. In contrast, optogenetic manipulation of neural activity in the EC→vCA1 pathway only affected behaviour on the trace CS/context fear memory test but had no effect on the elevated plus maze, open field or Vogel conflict test. The authors suggest different roles for these two ventral hippocampal pathways in fear versus anxiety.

Strengths:

This is an interesting study addressing an important question in a highly topical subject area. The experiments are well conducted and have generated interesting and important data.

Weaknesses:

While I am broadly sympathetic to the overall narrative of the paper, I have some questions/comments around the specific interpretation of the results presented. In my view, the authors' claims may not be completely supported by their data, but the data are interesting nonetheless.

In terms of the framework presented by the authors for interpreting their data, many would argue that freezing (or at least reduced activity/behavioural inhibition) to the context provides a readout of conditioned anxiety rather than fear. In this sense, the context is a signal of potential threat (i.e. the context becomes associated with both shock and with the absence of shock) and thus generates anxiety rather than fear. Likewise, the trace CS cue could be considered as an ambiguous predictor of shock in that the shock doesn't occur straight away. In contrast, a punctate CS cue which co-terminates with shock would be a reliable signal of imminent threat and thus generates a fear response. Thus, it might be argued that all of the assays adopted by the authors are readouts of anxiety (albeit comprising tests of both conditioned and unconditioned anxiety). For example, from the authors' perspective, it is not clear a priori why the Vogel conflict test is considered anxiety, but contextual freezing is considered fear? Indeed, in the Discussion, the authors mention another study in which the data from the Vogel conflict test align with fear assays rather than anxiety tests. Can the authors elaborate on their distinction? I appreciate that, in practice, it might be difficult to distinguish between fear and anxiety at the behavioural level in rodents (although opposing effects of fear and anxiety on pain responses might be one option). At the very least, this issue merits further discussion.

Another question is whether rather than representing a qualitative difference between the contributions of the vCA3→vCA1 and EC→vCA1 pathways to different aspects of fear/anxiety behaviours, the different results reflect a quantitative difference between the magnitude of effects in vCA1 that are generated from optogenetic manipulation of the two pathways, coupled with the possibility that behaviour on the trace CS/context fear memory task is more sensitive to manipulation than the "anxiety tests". The possibility that vCA3→vCA1 stimulation is more effective is potentially supported by the c-fos measurements in vCA1. vCA3→vCA1 stimulation produced a much bigger vCA1 c-fos response (approx. 350% c-fos cell activation; see Figure 1E) compared to activation of the EC→vCA1 pathway (approx. 170% c-fos cell activation; see Figure 4E).

Furthermore, in some studies, there seem to be quite large differences between the laser OFF conditions for the different groups (which presumably one would not expect to be different). For example, compare laser OFF for the Inhibition group for time in open arms of EPM in Figure 5C (> 40%) versus laser OFF for the Inhibition group for time in open arms of EPM in Fig. 2C (< 20%). This could potentially result in ceiling effects, such that it is very hard to see a further increase in time in the open arms from a level already above 40% when the laser is then switched on. This could complicate the interpretation of the laser ON condition.

Likewise, there is a big difference between the behavioral performance of the two SHAM groups in Figure 3 (compare SHAM in 3 B, C and SHAM in 3 D, E). How is this explained? Could this generate a ceiling effect? This may also merit some discussion. More details on the SHAM procedure(s) in the main manuscript may also be helpful.

According to Figure 3A, the test of freezing response to the trace Tone CS is conducted in a different context from the conditioning context. The data presented in Figure 3 for tone fear are the levels of freezing during the presentation of this cue in the different contexts. It would be important to present both pre-CS and CS freezing levels here to determine how much of the freezing is actually driven by the punctate tone CS. The pre-CS freezing levels in this different context would also provide a nice control for the contextual fear conditioning.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors identify Leiomodin-1 (LMOD1) as a key regulator of early myogenic differentiation, demonstrating its interaction with SIRT1 to influence SIRT1's cellular localization and gene expression. The authors propose that LMOD1 translocates SIRT1 from the nucleus to the cytoplasm to permit the expression of myogenic differentiating genes such as MYOD or Myogenin.

Strengths:

A major strength of this work lies in the robust temporal resolution achieved through a time-course mass spectrometry analysis of in vitro muscle differentiation. This provides novel insights into the dynamic process of myogenic differentiation, often under explored in terms of temporal progression. The authors provide a strong mechanistic case for how LMOD1 exerts its role on muscle differentiation which opens avenues to modulate.

Weaknesses:

In the revised manuscript, the authors begin to translate their in vitro findings to an in vivo context by examining SIRT1 expression across a regeneration time course (Fig. 4I). They observe an increase in SIRT1 expression concomitant with LMOD1, supporting a potential role for SIRT1 in myogenic differentiation. Future studies will be required to provide deeper mechanistic insight into SIRT1 function in vivo.

Discussion:

Overall, the study emphasizes the importance of understanding the temporal dynamics of molecular players during myogenic differentiation and provides valuable proteomic data that will benefit the field. Future studies should explore whether LMOD1 modulates the nuclear-cytoplasmic shuttling of other transcription factors during muscle development and how these processes are mechanistically achieved. Investigating whether LMOD1 can be therapeutically targeted to enhance muscle regeneration in contexts such as exercise, aging, and disease will be critical for translational applications. Additionally, elucidating the interplay among LMOD1, LMOD2, and LMOD3 could uncover broader implications for actin cytoskeletal regulation in muscle biology. The authors have nicely updated their discussion.

Reviewer #2 (Public review):

This manuscript by Carmona, Zagotta, and Gordon is generally well-written. It presents a crude and incomplete structural analysis of the voltage-gated proton channel based on measured FRET distances. The primary experimental approach is Förster Resonance Energy Transfer (FRET), using a fluorescent probe attached to a noncanonical amino acid. This strategy is advantageous because the noncanonical amino acid likely occupies less space than conventional labels, allowing more effective incorporation into the channel structure.

Fourteen individual positions within the channel were mutated for site-specific labeling, twelve of which yielded functional protein expression. These twelve labeling sites span discrete regions of the channel, including P1, P2, S0, S1, S2, S3, S4, and the dimer-connecting coiled-coil domain. FRET measurements are achieved using acridon-2-ylalanine (Acd) as the acceptor, with four tryptophan or four tyrosine residues per monomer serving as donors. In addition to estimating distances from FRET efficiency, the authors analyze full FRET spectra and investigate fluorescence lifetimes on the nanosecond timescale.

Despite these strengths, the manuscript does not provide a clear explanation of how channel structure changes during gating. While a discrepancy between AlphaFold structural predictions and the experimental measurements is noted, it remains unclear whether this mismatch arises from limitations of the model or from the experimental approach. No further structural analysis is presented to resolve this issue or to clarify the conformational states of the protein.

The manuscript successfully demonstrates that Acd can be incorporated at specific positions without abolishing channel function, and it is noteworthy that the reconstituted proteins function as voltage-activated proton channels in liposomes. The authors also report reversible zinc inhibition of the channel, suggesting that zinc induces structural changes in certain channel regions that can be reversed by EDTA chelation. However, this observation is not explored in sufficient depth to yield meaningful mechanistic insight.

Overall, while the study introduces an interesting labeling strategy and provides valuable methodological observations, the analysis appears incomplete. Additional structural interpretation and mechanistic insight are needed.

Major Points

(1) Tryptophan and tyrosine exhibit similar quantum yields, but their extinction coefficients differ substantially. Is this difference accounted for in your FRET analysis? Please clarify whether this would result in a stronger weighting of tryptophan compared to tyrosine.

(2) Is the fluorescence of acridon-2-ylalanine (Acd) pH-dependent? If so, could local pH variations within the channel environment influence the probe's photophysical properties and affect the measurements?

(3) Several constructs (e.g., K125Tag, Y134Tag, I217Tag, and Q233Tag) display two bands on SDS-PAGE rather than a single band. Could this indicate incomplete translation or premature termination at the introduced tag site? Please clarify.

(4) In Figure 5F, the comparison between predicted FRET values and experimentally determined ratio values appears largely uninformative. The discussion on page 9 suggests either an inaccurate structural model or insufficient quantification of protein dynamics. If the underlying cause cannot be distinguished, how do the authors propose to improve the structural model of hHV1 or better describe its conformational dynamics?

(5) Cu²⁺, Ru²⁺, and Ni²⁺ are presented as suitable FRET acceptors for Acd. Would Zn²⁺ also be expected to function as an acceptor in this context? If so, could structural information be derived from zinc binding independently of Trp/Tyr?

(6) The investigated structure is most likely dimeric. Previous studies report that zinc stabilizes interactions between hHV1 monomers more strongly than in the native dimeric state. Could this provide an explanation for the observed zinc-dependent effects? Additionally, do the detergent micelles used in this study predominantly contain monomers or dimers?

(7) hHV1 normally inserts into a phospholipid bilayer, as used in the reconstitution experiments. In contrast, detergent micelles may form monolayers rather than bilayers. Could the authors clarify the nature of the micelles used and discuss whether the protein is expected to adopt the same fold in a monolayer environment as in a bilayer?

Reviewer #2 (Public review):

Summary:

The paper by Stephens and co-workers provides important mechanistic insight into how hyaluronan synthase (HAS) coordinates alternating GlcNAc and GlcA incorporation using a single Type-I catalytic centre. Through cryo-EM structures capturing both "proofreading" and fully "inserted" binding poses of UDP-GlcA, combined with detailed biochemical analysis, the authors show how the enzyme selectively recognizes the GlcA carboxylate, stabilizes substrates through conformational gating, and requires a priming GlcNAc for productive turnover.

These findings clarify how one active site can manage two chemically distinct donor sugars while simultaneously coupling catalysis to polymer translocation.

The work also reports a DDM-bound, detergent-inhibited conformation that possibly illuminates features of the acceptor pocket, although this appears to be a purification artefact (it is indeed inhibitory) rather than a relevant biological state.

Overall, the study convincingly establishes a unified catalytic mechanism for Type-I HAS enzymes and represents a significant advance in understanding HA biosynthesis at the molecular level.

Strengths:

There are many strengths.

This is a multi-disciplinary study with very high-quality cryo-EM and enzyme kinetics (backed up with orthogonal methods of product analysis) to justify the conclusions discussed above.

Comments on revisions:

The suggestions made in the initial comments have all been responded to very well.

Reviewer #3 (Public review):

This manuscript presents a macroevolutionary approach to identification of novel high-level antibiotic resistance determinants that takes advantage of the natural genetic diversity within a genus (mycobacteria, in this case) by comparing antibiotic resistance profiles across related bacterial species and then using computational, molecular, and cellular approaches to identify and characterize the distinguishing mechanisms of resistance. The approach is contrasted with "microevolutionary" approaches based on comparing resistant and susceptible strains of the same species and approaches based on ecological sampling that may not include clinically relevant pathogens or related species. The potential for new discoveries with the macroevolution-inspired approach is evident in the diversity of drug susceptibility profiles revealed amongst the selected mycobacterial species and the identification and characterization of a new group of rifamycin-modifying ADP-ribosyltransferase (Arr) orthologs of previously described mycobacterial Arr enzymes. Additional findings that intra-bacterial antibiotic accumulation does not always predict potency within this genus, that M. marinum is a better proxy for M. tuberculosis drug susceptibility than the commonly used saprophyte M. smegmatis, and that susceptibility to semi-synthetic antibiotic classes is generally less variable than susceptibility to antibiotics more directly derived from natural products strengthen the claim that the macroevolutionary lens is valuable for elucidating general principles of susceptibility within a genus.

There are some limitations to the work. The argument for the novelty of the approach could be better articulated. While the opportunities for new discoveries presented by identification of discrepant susceptibility results between related species is evident, it is less clear how the macroevolutionary approach is further leveraged for the discovery of truly novel resistance mechanisms. The example of the discovery of Arr-X enzymes presented here relied upon foundational knowledge of previously characterized Arr orthologs. There is less clarity about what the pipeline would look like for discovery of previously unknown determinants when one is agnostic to putative mechanisms. From the point at which interspecies differences in susceptibility are noted, does the framework still remain distinct from other discovery frameworks and approaches?

While the experimentation and analyses performed are generally well designed and rigorous, there are a few instances in which broad claims are based on inferences from sample sets or data sets that are, at present, too limited to provide robust support. For example, the claim that rifampicin modification, and precisely ADP-ribosylation, is the dominant mechanism of resistance to rifampicin in mycobacteria is still a bit premature or at least an over-generalization, as other enzymatic modification mechanisms and other mechanisms such as helR-mediated dissociation of rifampicin-stalled RNA polymerases, efflux, etc were not examined. CRISPR interference was used in a demonstrative example to support this assertion, but would need to be applied more systematically to be more conclusive. The general claim that intra-bacterial antibiotic accumulation does not predict potency in mycobacteria may be another over-generalization based on the limited set of drugs and species studied.

Comments on revisions:

Discussion, lines 321-323: "We found that resistance to these antibiotics in mycobacteria do not correlate with by uptake/efflux mechanisms in the species tested..." is an over-generalization and conflicts with the following statement on lines 199-201: "for BDQ we could observe some correlation between antibiotic potency and [BDQ]IB which could be indicative of efflux playing a role in antibiotic efficacy. Given that the current statement in the Discussion only applies to 2 of 3 drugs tested, a more specific or nuanced interpretation seems warranted.

Reviewer #2 (Public review):

Summary:

In this manuscript, submitted to Review Commons (journal agnostic), Coward and colleagues report on the role of insulin/IGF axis in podocyte gene transcription. They knocked out both the insulin and IGFR1 mice. Dual KO mice manifested a severe phenotype, with albuminuria, glomerulosclerosis, renal failure and death at 4-24 weeks.

Long read RNA sequencing was used to assess splicing events. Podocyte transcripts manifesting intron retention were identified. Dual knock-out podocytes manifested more transcripts with intron retention (18%) compared wild-type controls (18%), with an overlap between experiments of ~30%.

Transcript productivity was also assessed using FLAIR-mark-intron-retention software. Intron retention w seen in 18% of ciDKO podocyte transcripts compared to 14% of wild-type podocyte transcripts (P=0.004), with an overlap between experiments of ~30% (indicating the variability of results with this method). Interestingly, ciDKO podocytes showed downregulation of proteins involved in spliceosome function and RNA processing, as suggested by LC/MS and confirmed by Western blot.

Pladienolide (a spliceosome inhibitor) was cytotoxic to HeLa cells and to mouse podocytes but no toxicity was seen in murine glomerular endothelial cells.

The manuscript is generally clear and well-written. Mouse work was approved in advance. The four figures are generally well-designed, bars/superimposed dot-plots.

Methods are generally well described.

Comments on revised version:

Coward and colleagues have done an excellent job of responding to all the reviewer comments.

Reviewer #3 (Public review):

Summary:

The authors revisit the role of DR6 in axon degeneration following physical injury (Wallerian degeneration), examining both its effects on axons and its role in regulating the Schwann cell response to injury. Surprisingly, and in contrast to previous studies, they find that DR6 deletion does not delay the rate of axon degeneration after injury, suggesting that DR6 is not a mediator of this process.

Overall, this is a valuable study. As the authors note, the current literature on DR6 is inconsistent, and these results provide useful new data and clarification. This work will help other researchers interpret their own data and re-evaluate studies related to DR6 and axon degeneration.

Strengths:

(1) The use of two independent DR6 knockout mouse models strengthens the conclusions, particularly when reporting the absence of a phenotype.

(2) The focus on early time points after injury addresses a key limitation of previous studies. This approach reduces the risk of missing subtle protective phenotypes and avoids confounding results with regenerating axons at later time points after axotomy.

Comments on revisions:

I thank the authors for their thorough responses to my previous comments. The revisions have addressed the points raised and have improved the clarity and overall quality of the manuscript. I appreciate the effort taken to strengthen the presentation of the work.

Reviewer #2 (Public review):

Summary:

In this study, the authors set out to determine how a contact-dependent bacterial antagonistic system contributes to the ability of specific bacterial strains to persist within a complex, native gut community derived from wild animals. Rather than focusing on simplified or artificial models, the authors aimed to examine this system in a biologically realistic setting that captures the ecological complexity of the gut environment. To achieve this, they combined controlled laboratory experiments with animal colonization studies and sequencing-based tracking approaches that allow individual strains and mobile genetic elements to be followed over time.

Strengths:

A major strength of the work is the integration of multiple complementary approaches to address the same biological question. The use of defined but complex communities, together with in vivo experiments, provides a strong ecological context for interpreting the results. The data consistently show that the antagonistic system is not required for initial establishment but plays a critical role in long-term strain persistence. This insight that moves beyond traditional invasion-based views of microbial competition. The observation that transferable genetic elements can confer only temporary advantages, and may impose longer-term costs depending on community context, adds important nuance to current understanding of microbial fitness.

Weaknesses:

Overall, there is not a lack of evidence, but a deliberate trade-off between ecological realism and mechanistic resolution, which leaves some causal pathways open to interpretation.

Reviewer #2 (Public review):

Summary:

The manuscript by Aranaz-Novaliches describes a study of Tent5a knockout (KO) mice. The authors demonstrate a severe enamel phenotype in these mice, characterized by hypoplastic enamel with markedly disturbed organization of enamel rods. Additionally, they report that Amelx expression is reduced in the mutant compared to wild type (WT) at both mRNA and protein levels. The authors also examine the distribution and co-localization of Amelx and Ambn in ameloblasts and the enamel matrix. These findings are novel and provide important insights into the role of polyadenylation in regulating enamel matrix protein translation and its downstream effects on protein trafficking, secretion, and enamel formation. However, I have multiple concerns regarding the data and its analysis that need to be addressed.

Specific comments:

(1) Introduction

The structure of the introduction is unconventional. The first sentence of the third paragraph states that the goal of this study is to investigate the role of TENT5A in enamel formation, but the rest of the paragraph focuses on enamel in general. The following paragraph claims that the authors discovered the effects of Tent5a deficiency on enamel formation for the first time, yet most of the paragraph discusses enamel proteins and amelogenesis. The choice of references is problematic. The authors cite Sire et al. (2007), which focuses on the origin and evolution of enamel mineralisation genes, a poor fit for this context. A more appropriate source would be a recent review, e.g., Lacruz R et al., Physiol Rev. 2017;97(3):939-993. Ambn constitutes ~5% of the enamel matrix, not 10%. Reference 16 (Martin) is not ideal for murine enamel; more detailed studies exist, e.g., Smith CE et al., J Anat. 2019;234(2):274-290. References on protein-protein interactions (17-19) are also off: Wald et al. studied Ambn-Ambn and Amelx-Amelx interactions separately; Fang et al. focused on Amelx self-assembly only; Kawasaki and Weiss addressed gene evolution. The authors should cite work from Moradian-Oldak's lab, which clearly demonstrates Amelx-Ambn interactions. The last paragraph contains confusing statements, e.g., "TENT5a localized in rER promotes the expression of AmelX and other secreted protein transcripts." Also, the manuscript does not convincingly show disruption of self-assembly beyond overall enamel disorganization.

(2) Results

(a) microCT

Quantitative microCT analyses of WT and KO enamel are needed. At a minimum, enamel thickness and density should be measured from at least three biological replicates per genotype. Severe malocclusion in KO mice is not discussed. The mandibular incisor appears abraded, while the maxillary incisor is overgrown. Is maxillary enamel as affected as mandibular? The age of the mice is not specified. High-resolution scans of isolated mandibular incisors described in Materials and Methods should be included.

(b) SEM

The term "disorganized crystal structure" is incorrect - SEM cannot reveal crystal structure. This requires electron/X-ray diffraction or vibrational spectroscopy. Likely, the authors meant disorganized rods and interrod enamel. The phrase "weak HAP composition" is unclear. Can the increase in interprismatic matrix volume and reduction in rod diameter be quantified? Since rods are secreted by distal Tomes' processes and interrod by proximal Tomes' processes, an imbalance may indicate alterations in the ameloblast secretory apparatus. TEM studies of demineralized incisors are recommended to assess ameloblast ultrastructure.

(c) EMP expression

There is a discrepancy between WB images and data in Figure S2a. In Figure 2b, Amelx band is stronger than Ambn (expected, as Amelx is ~20× more abundant), but in Figure S2a, Ambn appears higher. How was protein intensity in Fig. S2a calculated? Optical density? Was normalization applied? Co-localization in Figure 2d was performed on LS8 cells, which lack a true ameloblast phenotype. Amelx expression in LS8 cells is ~2% of actin (Sarkar et al., 2014), whereas in murine incisors, it is ~600× higher than actin (Bui et al., 2023). Ambn signal is weaker than Amelx, which may affect co-localization results.

(d) Splicing products in Figure 2e

All isoforms except one contain exon 4. The major functional splice product of Amelx lacks exon 4 (Haruyama et al. J Oral Biosci. 2011;53(3):257-266), and there are some indications that the presence of exon 4 can lead to enamel defects. Can it be that the observed phenotype is due to the presence of exon 4?

(e) Co-localization studies

The presented co-localization studies do not demonstrate self-assembly defects; they reflect enamel microstructural defects observed by SEM. Self-assembly occurs at the nanoscale and cannot be assessed by light microscopy except with advanced optical methods. Conclusions based on single images are weak. The authors should perform experiments at least on three biological replicates per genotype, quantify results (e.g., total gray values per ROI of equal pixel size), and use co-localization metrics such as Mander's coefficient. Claims about alternative secretory pathways require much stronger evidence.

The authors should avoid implying that mRNA is inside the ER lumen. It is likely associated with the outer rER surface, which is expected. The resolution of the methods used is insufficient to confirm ER lumen localization.

DOI: 10.1016/j.celrep.2026.116971

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:ZFIN_ZDB-ALT-040601-2

What is this?

DOI: 10.1016/j.celrep.2026.116971

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:ZFIN_ZDB-ALT-120828-2

What is this?

Reviewer #2 (Public review):

Summary:

The authors studied cognitive control and attention in response to mnemonic prediction errors (MPEs): situations in which the external reality violates internal memory-based predictions. The behavioral task first established strong versus weak predictions, and then either confirmed or violated these predictions. The authors examined markers of cognitive control (frontal theta) and attention (posterior alpha suppression, pupil response) while strong and weak predictions were confirmed or violated. They found increased cognitive control (frontal theta) for strong MPEs, which correlated with subsequent memory. Markers of attention (alpha suppression, pupil response) also accompanied strong MPEs but did not correlate with subsequent memory. Pupil response was investigated using an interesting approach that decomposes the response into different components, finding that different components respond earlier or later and show different correlations with MPEs and their strength. The authors also investigated how EEG, reaction time, and pupil responses correlated with one another, providing further insight into the mechanism underlying the response to MPEs. Together, the study points toward multiple control and attention mechanisms involved in MPE response and memory.

Strengths:

The study has a clear behavioral paradigm with multiple measures - behavioral, EEG, and pupillometry that offer an investigation into different aspects of MPE response and memory.

The study is also very comprehensive in looking at multiple phases in processing MPEs: the prediction phase (prior to the violation), the response to MPEs, and subsequent memory of MPEs, all within one study. Specifically, the link between neural mechanisms and subsequent memory is a major advancement, as most prior studies did not include this component. Mechanisms underlying subsequent memory of MPEs are theoretically important, as a primary function of MPEs is to promote learning and memory. As the authors mention, the different neural and pupillary signals are not robustly correlated, suggesting multiple mechanisms underlying MPE detections, which is interesting, offers avenues for future research, and can facilitate a better theory of how MPEs are processed in the brain. Finally, the decomposition of pupil response into different components and their correlation with behavior (RT during match/MPE detection) is interesting.

Weaknesses:

The methods are rigorous, and the claims are mostly supported by the data, but there are a few weaknesses or places that could be improved:

(1) The authors conduct PCA analysis to identify different components of the pupillary response to MPE and relate them to behavior. Specifically, the authors identify components PC3 and PC4, which they interpret as related to MPE. However, some parts of the interpretation could be clearer or better justified:

(a) The authors refer to PC4 as "post-decision cognitive processing". But, given that RT was between .5-.7s, and PC3 peaked after more than 1s, wouldn't it be cautious to interpret PC3 as post-decision as well?

(b) MPEs overall elicit longer RTs in this study, suggesting that long RT is a behavioral marker of MPE. Nonetheless, the authors argue on p. 12: "Altogether, these findings indicate that when stronger mnemonic predictions (as indexed by shorter RTs) were violated." And, PC3 is correlated with shorter RTs for mismatches, meaning that behaviorally, these trials were more similar to matches. Thus, how do the authors interpret shorter versus longer RTs for MPEs, and what processes do these RT reflect?

(2) The brain to pupil relationship (p. 13-14): If I understand correctly, this was done on a trial-by-trial basis, but the high temporal resolution allows doing the analysis in a time-resolved manner - does brain activity at a certain time point preceding/following the pupil response correlate with the pupil response? It might be that cognitive control influences attention mechanisms or vice versa (because there is some overlap in the response). Although not testing causality, this temporally resolved correlation would be an interesting way to start probing how signals might influence each other.

(3) The relationships the authors find between brain measures and pupil components were largely not specific to mismatches/matches. However, are they specific to this task? I think it would benefit the paper to show that these relationships are potentially specific to making match/mismatch memory decisions, versus, e.g., any stimulus processing. For example, the authors could run the same analyses locked to stimuli in the study phase, anticipating a different pattern, if indeed these findings are specific to the associative memory task.

(4) During memory retrieval (i.e., before the probe), the authors find that frontal theta, a marker of cognitive control, was associated on a trial-by-trial basis with more posterior alpha (i.e., less alpha suppression, potentially reflecting less attention), and that this association was stronger for weaker predictions. The authors interpreted this as weaker predictions necessitating more cognitive control, and that more cognitive control was recruited specifically in trials where retrieval included less content (memory reinstatement) to attend to. Generally, cognitive control is recruited to facilitate memory retrieval. If so, one possible interpretation is that this correlation reflects cognitive control effort that has failed to produce enough memory reinstatement. The other possibility is that this correlation reflects more specific retrieval of the correct probe, without retrieval of interfering items (i.e., overall less content). I believe that the former explanation predicts that this correlation would be associated with longer RTs (more difficult decisions), while the latter predicts shorter RTs (easier decisions due to successful retrieval), at least for matches.

(5) In section 3, the authors found a positive relationship between alpha during memory retrieval and PC3 during MPE. If I understood correctly, this means that less attention during retrieval (less suppression) is correlated with a stronger PC3 response. How do the authors interpret this? Maybe along the same lines as in (5), specifically retrieving the correct information (i.e., less retrieved content to attend to) means a stronger prediction, leading to a stronger MPE, and a stronger MPE response, as reflected by PC3?

(6) The results with subsequent memory are important and address a major gap in the field that largely did not relate neural effects of MPE to subsequent memory. However, one major limitation of the study is that the authors did not test memory for matches. I understand the logic of avoiding testing matches. Because matches were repeated more times in the study, it's not a fair comparison, and could change participants' overall criterion for old/new decisions. However, one possibility would have been to test only the weak prediction; this could have given some specificity to the neural subsequent memory findings.

(7) The authors nicely characterized the different PC of pupillary MPE response. But, with respect to subsequent memory, they only present pupil size. Unless there is some methodological reason that prevents testing subsequent memory on the PC, I think this will be very informative about the potential mechanisms underlying memory of MPE.

(8) This paper includes many interesting findings, and I am not sure how they all come together into a cohesive mechanistic understanding of MPE response and subsequent memory. I think the paper would benefit from either a conceptual mechanism figure or, in the Discussion, have a summary of a proposed mechanism integrating the findings together.

(9) Relatedly, the section "Immediate, strength-sensitive neurocognitive impacts of MPEs" does not link the arguments to specific data points, so it's hard to follow which data specifically the authors are interpreting.

(10) If I understand correctly, the authors did not find improved memory for strong compared to weak MPE. First, I think this behavioral result should be incorporated in the main paper and in the interpretation of the results. Second, given that the neural effects the authors tested either correlated with memory for strong MPE or did not show a relationship with memory, what neural/pupil response could explain memory for weak MPE?

Reviewer #2 (Public review):

Summary:

The manuscript proposes interesting synaptic plasticity rules grounded in experimental data. Its main features are:

(1) plasticity depends on local calcium concentration driven by presynaptic activity and is independent of somatic action potentials,

(2) the rules incorporate metaplasticity, and (3) they demonstrate how a single neuron could address the feature-binding problem at the dendritic level.

The work extends a previous study (https://doi.org/10.7554/eLife.97274.2), to which the author also contributed.

The author models two calcium thresholds (LTP/LTD) from two different calcium sources (NMDA/VGCC), and these thresholds are flexible (metaplasticity rule, similar to BCM), which is claimed to be necessary for successful learning of both FBP and NFBP (linear and nonlinear feature binding problem with 1 or 2 patterns). The role of each threshold seems to be opposite and complementary. One extra condition has been added: an upper threshold for LTP. This threshold serves to stop synaptic strengthening once synapses are strong enough to evoke a plateau. With that, synapses are not strengthened to the maximal value, avoiding strong supralinear integration for irrelevant patterns.

Strengths:

The current model implements not only local synaptic plasticity but also metaplasticity and solves the FBP at the dendrite level. Another strong aspect of the model is that metaplasticity in the LTD threshold protects strengthened synapses from weakening. In this way, as the author mentioned, metaplasticity is able to protect learned patterns from being forgotten or weakened and prevent irrelevant patterns from being stored. This is a nice modelling example of metaplasticity being helpful in preventing the catastrophic interference or forgetting (as has been explicitly discussed in a recent article https://doi.org/10.1016/j.tins.2022.06.002 ). The author might want to briefly mention or emphasize this aspect of the model, which might be interesting also for the AI community.

Weaknesses:

(1) What is novel in the current paper as compared to Khodadadi et al. eLife 2025? That is not completely clear and should be made clearer. Is it only a minor difference related to the fact that the new learning rule has metaplasticity in both calcium thresholds and is simpler? This seems to be just an incremental increase in knowledge/methods. Can the author defend his paper against this point from the „devil's advocate"? How is the conclusion of the author in the abstract that „metaplasticity in both thresholds is necessary" reconcilable with his previous publication (Khodadadi et al. eLife 2025), in which only metaplasticity in one threshold was successful in solving the nonlinear feature binding problem?

(2) As far as I can judge without testing the model, metaplasticity causes thresholds to monotonically increase during systematic pattern presentation, which stabilizes weights and allows pattern separation. Due to the closed-loop nature of the current implementation, where metaplasticity only happens if plasticity happens, this also effectively locks patterns in place. However, flexible learning is an essential mechanism for survival. Imagine a mutation event takes place and bananas suddenly become red and/or strawberries turn yellow. It seems that the current model would be unable to adapt to these new patterns even if rewards were to be shifted. While out of the scope of the study, due to its importance, I feel that pattern shifting/relearning should at least be briefly discussed. How could the model be improved to allow relearning?

Reviewer #2 (Public review):

Summary:

This manuscript reports the application of a combined targeted therapeutic approach to gastric cancer treatment. The RTK, FGFR2 and the phosphatase, SHP2 are targeted with existing drugs; AZD457 and SHP099, respectively. Having shown increased mRNA levels of FGFR2 and SHP2 in a patient population and highlighted the issue of resistance to single therapies the combination of inhibitors is shown to reduce cancer-related signalling in two gastric cell lines. The efficacy of the dual therapy is further demonstrated in a single patient case study and mouse xenograft models. Finally, the rationale for SHP2 inhibition is shown to be linked to immune response.

Strengths:

The data is generally well presented, and the study invokes a novel patient data set which could have wider value. The study provides additional evidence to support the combined therapeutic approach of RTK and phosphatase inhibition.

Weaknesses:

Combined therapy approaches targeting RTKs and SHP2 have been widely reported. Indeed, SHP099 in combination with FGFR inhibitors has been shown to overcome adaptive resistance in FGFR-driven cancers. Furthermore, the inhibition of SHP2 has been documented to have important implications in both targeting proliferative signalling as well as immune response. Thus, it is difficult to see novelty or a significant scientific advance in this manuscript. Although the data is generally well presented, there is inconsistency in the interpretation of the experimental outcomes from ex vivo, patient and mouse systems investigated. In addition, the study provides only minor or circumstantial understanding of the dual mechanism.

Using data from a 161 patient cohort FGFR2 was identified as displaying amplification of FGFR2 in ~6% with concomitant elevation of mRNA of patients which correlated with PTPN11 (SHP2) mRNA expression. The broader context of this data is of value and could add a different patient demographic to other data on gastric cancer. However, there is no detail on patient stratification or prior therapeutic intervention.

Comments on revisions: This has been attended to in the revised version

In SNU16 and KATOIII cells the combined therapy is shown to be effective and appears to be correlated with increase apoptotic effects (i.e. not immune response).

Fig 2E suggests that the combined therapy in SNU16 cells is little better than FGFR2-directed AZD457 inhibitor alone, particularly at the higher dose.

The individual patient case study described via Fig 3 suggests efficacy of the combined therapy (at very high dosage), however the cell biopsies only show reduced phosphorylation of ERK, but not AKT. This is at odds with the ex vivo cell-based assays. Thus, it is not clear how relevant this study is.

The mouse xenograft study shows a convincing reduction in tumor mass/volume and a clear reduction in pAKT, whilst pERK remains largely unaffected by the combined therapeutic approach. This is in conflict with the previous data which seems to show the opposite effect.

Comments on revisions: The authors have clarified this point

In all, the impact of the dual therapy is unclear with respect to the two pathways mediated by ERK and AKT.

Finally, the authors demonstrate the impact of SHP2 on PD-1 expression and propose that the SHP099/AZD4547 combination therapy significantly induces the production of IFN-γ in CD8+ T cells. This part of the study is unconvincing and would benefit from an investigation of the tumor micro-environment to assess T cell infiltration.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors combined coarse-grained structure-based model simulation, optical tweezer experiments, and AI-based analysis to assess the knotting behavior of the TrmD-Tm1570 protein. Interestingly, they found that while the structure-based model can fold the single knot from TrmD and Tm1570, the double-knot protein TrmD-Tm1570 cannot form a knot itself, suggesting the need for chaperone proteins to facilitate this knotting process. This study has strong potential to understand the molecular mechanism of knotted proteins, supported by many experimental and simulation evidence. However, there are a few places that appear to lack sufficient details, and more clarification in the presentation is needed.

Strengths:

A combination of both experimental and computational studies. The authors have addressed my questions in their revised manuscript. I appreciate their efforts.

Reviewer #2 (Public review):

Summary:

This is a mechanistic study that provides new insights into the inhibition of SARS-CoV-2 Mpro.

Strengths:

The identification of dimer interface stabilization/destabilization as distinct inhibitory mechanisms and the discovery of C300 as a potential allosteric site for ebselen are important contributions to the field. The experimental approach is modern, multi-faceted, and generally well-executed.

Weaknesses:

The primary weaknesses relate to linking the biophysical observations more directly to functional enzymatic outcomes and providing more quantitative rigor in some analyses. While the study is overall strong, addressing its weaknesses and limitations would elevate the impact and translational relevance of the current manuscript.

(1) Correlation with Functional Activity:

The most significant gap is the lack of direct enzymatic activity assays under the exact conditions used for MS and HDX. While EC50 values are listed from literature, demonstrating how the observed dimer stabilization (by peptidomimetics) or dimer disruption (by ebselen) directly correlates with inhibition of proteolytic activity in the same experimental setup would solidify the functional relevance of the biophysical observations. For instance, does the fraction of monomer measured by native MS quantitatively predict the loss of activity? Also, the single inhibitor concentration used in each MS experiment needs to be specified in the main text and legends. A discussion on whether the inhibitor concentrations required to observe these dimerization effects (in native MS) or structural dynamics (in HDX-MS) align with EC50 values would be helpful for contextualizing the findings.

(2) For the two Cys residues found to be targeted by ebselen, what are their respective modification stoichiometry related to the ebselen concentration? Especially for the covalent binding site C300, which is proposed in this study to represent a novel allosteric inhibition mechanism of ebselen, more direct experimental evidence is needed to support this major hypothesis. Does mutation or modification of C300 affect the Mpro dimerization/monomer equilibrium and alter the enzymatic activity? If ebselen acts as a covalent inhibitor linked to multiple Cys, why is its activity only in the uM range?

(3) For the allosteric inhibitor pelitinib with low-uM activity, no significant differences in deuterium uptake of Mpro were observed. In terms of the binding affinity, what is the difference between pelitinib and ebselen? Some explanations could be provided about the different HDX-MS results between the two non-peptidomimetic inhibitors with similar activities.

(4) Native MS Quantification:

The analysis of monomer-dimer ratios from native MS spectra appears qualitative or semi-quantitative. A more rigorous and quantified analysis of the percentage of dimer/monomer species under each condition, with statistical replicates, would strengthen the equilibrium shift claims. For native MS analysis of each inhibitor, the representative spectrum can be shown in the main figure together with quantified dimer/monomer fractions from replicates to show significance by statistical tests.

(5) Changes of HDX rates in certain regions seem very subtle. For example, as it states 'residues 296-304 in the C-terminal region of M pro were more flexible upon ebselen binding (Figure 4c)', the difference is barely observable. The percentage of HDX rate changes between two conditions (with p values) can be specified in the text for each fragment discussed, and any change below 5% or 10% is negligible.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.

[Editors' note: The authors have substantially addressed all the reviewers' concerns and comments.]

Reviewer #2 (Public review):

This is an interesting study that shows that mRNA acetylation at synapses is dynamically regulated at synapses by spatial memory in the mouse hippocampus. The dynamic changes of ac4C-mRNAs regulated by memory were validated by methods including ac4C dot-blot and liquid 13 chromatography-tandem mass spectrometry (LC-MS/MS).

Reviewer #2 (Public review):

Summary:

The authors performed a genetic screen using deficiency lines and identified Uev1a as a factor that protects nurse cells from RasG12V-induced cell death. According to a previous study from the same lab, this cell death is caused by aberrant mitotic stress due to CycA upregulation (Zhang et al.). This paper further reveals that Uev1a forms a complex with APC/C to promote proteasome-mediated degradation of CycA.

In addition to polyploid nurse cells, the authors also examined the effect of RasG12V-overexpression in diploid germline cells, where RasG12V-overexpression triggers active proliferation not cell death. Uev1a was found to suppress its overgrowth as well.

Finally, the authors show that the overexpression of the human homolog, UBE2V1 and UBE2V2, suppresses tumor growth in human colorectal cancer xenografts and cell lines. Notably, these genes' expression correlates with the survival of colorectal cancer patients carrying Ras mutation.

Strength:

This paper presents a significant finding that UBE2V1/2 may serve as a potential therapy for cancers harboring Ras mutations. The authors propose a fascinating mechanism in which Uev1a forms a complex with APC/C to inhibit aberrant cell cycle progression.

Comments on revisions:

The authors have addressed several of the major concerns, including the addition of new data and improved figure presentation. However, some issues remain insufficiently resolved, particularly regarding control reuse (Major Comment 3) and experimental interpretation (Major Comments 5 and 8).

Regarding Major Comment 5, the authors state that UAS copy number affects the frequency of egg chamber degradation in Fig. 2D, and thus explains the reduced phenotype in RasG12V + GFP-RNAi compared to RasG12V alone. However, this explanation is not consistent with other data in the manuscript. UAS-RasG12V combined with UAS-lacZ in Fig. 2G shows a phenotype comparable to UAS-RasV12 alone, despite also increasing the UAS copy number. This suggests that the effect is not simply due to copy number.

I understand that the authors used UAS-RasG12V + GFP-RNAi as a control for the RNAi experiments and UAS-RasG12V + lacZ for the overexpression experiments. I suggest examining the phenotype frequency of UAS-RasG12V + UAS-GFP, to figure the reason out. Overall, these results indicate that there is a spectrum of phenotype frequencies, and therefore appropriate controls should be included for each experiment rather than reusing the same dataset across different experiments, as also noted in Major Comment 3.

Reviewer #2 (Public review):

Summary:

The authors performed bioinformatic analyses to trace the genomic history of the clinically relevant pT181 plasmid. Specifically, they:

(1) tracked the presence of pT181 across different S. aureus strain backgrounds through time. It was first found in one, later multiple strains, though this may reflect changes in sampling over time.

(2) estimated the mutation rate of the chromosome and plasmid.

(3) estimated the plasmid copy number of pT181, and found that it decreased over time. The latter was supported by two sets of statistical analyses, first showing that the number of single-copy isolates increased over time, and second, that the multicopy isolates demonstrated a lower PCN over time.

(4) reported the different integration sites at which pT181 integrated into the genome.

As a caveat, they mentioned that identical plasmid sequences have variable plasmid copy numbers across different genomes in their dataset.

Strengths:

This is a very solid, well-considered bioinformatic study on publicly available data. I greatly appreciate the thoughtful approach the authors have taken to their subject matter, neither over- nor underselling their results. It is a strength that the authors focussed on a single plasmid in a single bacterial species, as it allowed them to take into account unique knowledge about the biology of this system and really dive deep into the evolution of this specific plasmid. It makes for a compelling case study. At the same time, I think the introduction and discussion can be strengthened to demonstrate what lessons might be drawn from this case study for other plasmids.

Weaknesses:

The finding that the pT181 copy number declined over time is the most interesting claim of the paper to me, and not something that I have seen done before. While the authors have looked at some confounders in this analysis, I think this could be strengthened further in a revision.

For the flow of the storyline, I also think the estimation of mutation rates (starting L181) and integration into the chromosome (starting L255) could be moved to the supplement or a later position in the main text.

Clearly, the use of publicly available data prevents the authors from controlling the growth and sequencing conditions of the isolates. It is striking that they observe a clear signal in spite of this, but I would have loved to see more discussion of the metadata that came with the publicly available sequences and even more use of that metadata to control for confounding.

Reviewer #2 (Public review):

Summary:

This manuscript studies the impacts of knocking out a protein known to be involved in synapse maturation in mice, measuring their ability to hunt prey items (and to discriminate simple visual patterns) under binocular and monocular viewing conditions. The main results are that the mice with this protein knocked out are impaired when performing visual tasks with binocular viewing, but are actually better when they perform monocularly. The interpretation is that the knocked-out protein has affected binocular visual integration.

Strengths:

Overall, the attempt to connect a protein to behavior/perception, via known mechanistic effects on synapse development and visual critical periods, is admirable.

The use of multiple visual conditions and behavioral paradigms (binocular/monocular, cricket hunting/orientation discrimination, light/dark) strengthens and enriches the results.

Weaknesses:

The primary interpretation - that binocular integration is affected in the PSD-95 knockouts- is not supported by the behavioral evidence. Using behavior to isolate a particular stage in visual processing (and further, to distinguish it from elements of generating the behavioral response and/or acquiring the visual information in the first place) is notoriously difficult. Such attempts are, of course, the domain of psychophysics. In fact, the most classical and loveliest success is in the domain of binocular integration- Bela Julesz's "psychoanatomy" that used random dot stereograms to isolate stereoscopic computations.

I mention this example because it is, in fact, directly relevant to my primary concern about the evidence used as support for the favored interpretation here. Julesz's stimuli were extremely clever in isolating binocular mechanisms (i.e., binocular mechanisms MUST be used to perform the task), and any perceptual/behavioral reports are very straightforward to interpret (i.e., a stereoscopically-defined shape can be identified, or not).

Now compare this to the work described in this manuscript. KO (knockout) mice are worse than wild types at chasing prey items or at moving towards a rewarded orientation, but they get better when performing this task monocularly. No argument that that is an interesting bit of scientific phenomenology to characterize. However, the behaviors do not require binocular integration, the freely-moving paradigms involve a variety of gaze and body-movement strategies, and the metrics used to quantify performance are similarly high-dimensional. Bottom line, it is not possible to glean whether the KO's intriguing binocular-vs-monocular differences are due to binocular integration per se, or something better thought of as fundamentally sensorimotor in origin. The tasks do not isolate visual from sensorimotor processing, and the behaviors and associated metrics cannot definitely adjudicate between a multitude of possible specific interpretations.

More specifically, the KO mice may have abnormal patterns of binocular coordination. Eye movements were not tracked in these studies, despite the availability of such instrumentation and their successful application in many preceding studies of mouse prey capture. If the KO mice do not coordinate their eye movements (in task-specific/task-relevant ways), they might receive binocular input that is abnormal. Under monocular conditions, that mismatched or inappropriately coordinated binocular input is absent, which would relieve them of the confusing visual information. That is rather different than having an impairment of binocular integration, as it is basically a question of whether the visual system is impaired, or whether the inputs to the visual system are abnormal due to differences in binocular coordination.

It is also possible that the binocular deficit, as measured in behavior,r occurs in a distinct part of the sensorimotor loop. Even if the binocular eye movements are normal, and binocular visual integration is normal, PSD-95 KO mice may be confused or distracted by the larger visual field that comes from binocular viewing (quite profound in species with mostly lateralized eyes). Such a "post-sensory" interpretation related to target selection (from what could be a totally normal visual representation) is difficult to rule out as well.

In summary, this reviewer appreciates the value of trying to connect this molecular mechanism to sensory processing and behavior. The use of naturalistic tasks and freely-moving paradigms is also something to commend. However, the sorts of visual stimuli and behavioral paradigms used here are not well-suited to supporting the rather specific interpretation that has been put forth in this manuscript.

Reviewer #2 (Public review):

Summary:

The basic helix-loop-helix transcription factor TCF4 (also known, as ITF2, SEF2, or E2-2) is a protein involved in the development and functioning of many different cell types. TCF4 plays important roles in the nervous system, both in health and disease. Its importance in the nervous system is underlined by its association with common and rare cognitive disorders. Specifically, variants of the TCF4 gene are implicated in increased susceptibility to schizophrenia, and mutations in the TCF4 gene cause Pitt-Hopkins syndrome (PTHS) or mild to moderate non-syndromic intellectual disability.

In this manuscript, the authors have studied whether reinstating TCF4 later in postnatal development in juvenile PTHS model mice could reverse behavioral phenotypes, thereby simulating gene therapy. Previous research by the same group has demonstrated that restoring TCF4 during embryonic or neonatal stages, corresponding to prenatal or neonatal periods in humans, improved phenotypes in a PTHS mouse model. In the current study, a conditional TCF4 reinstatement mouse model, Tcf4-lox-stop-lox (Tcf4-LSL), previously developed and characterized by their lab, where Cre-mediated recombination removes a floxed transcriptional stop cassette downstream of exon 17, leading to reinstatement of all TCF4 isoforms at appropriate levels in neurons, was used. The study showed that this later intervention failed to correct most phenotypes, suggesting that perinatal reinstatement of TCF4 holds the greatest potential to treat behavioral symptoms of PTHS. However, the study also suggests that some cognitive behaviors may still be responsive to TCF4 reinstatement later in life.

Strengths:

This is a very important study aimed at developing gene therapy for PTHS. The study is technically very well performed and written.

Weaknesses:

The only weakness is that a human disease is modelled in a mouse, which is evolutionarily not the closest mammal to humans. Hopefully, in the future, similar studies will also be performed in a nonhuman primate model, for example rhesus macaque.

Reviewer #2 (Public review):

Summary:

The authors derived a time-specific signature of reactogenicity from mouse muscle following exposure to vaccines /TLRs for capturing the reactogenicity patterns. They tested this reactogenicity signature in mouse blood, and then they applied the reactogenicity signature to human blood from subjects having received different vaccines. They identified biomarkers in mouse muscle which are also observed in mouse and human blood and could be used as a reactogenicity signature in mice, instead of CRP.

Strengths:

(1) The authors used transcriptomic response following vaccination and used common genes to human and mice for defining a reactogenic signature.

(2) As the authors used different formulations in mice, the model was trained across a broad reactogenicity spectrum, which has the advantage of being used for evaluating new vaccines/vaccine platforms.

Weaknesses:

(1) The muscle gene signature reflects local reactogenicity. Systemic reactogenicity is not specifically addressed, except where overlapping gene signatures are observed for both local and systemic reactogenicity.

(2) In the same logic, could we find additional genes in the blood which are not captured in the muscle?

(3) The peak of the reactogenicity is usually 24h; it is not certain that additional TPs have helped the findings. If they have, the authors should explain.

Reviewer #2 (Public review):

Summary

This manuscript proposes an original and conceptually interesting model in which anti-apoptotic BCL-2 family proteins, particularly BCL-XL and MCL-1, not only sequester BIM but also act as adaptor "co-receptors" that recruit BIM to the CUL5-WSB2 ubiquitin ligase complex for degradation. The authors present a mechanistic framework supported by structure-guided mutagenesis, BH3 mimetic perturbations and co-immunoprecipitation assays performed in RPE1 cells. In parallel, the study shows that neuroblastoma cell lines are highly dependent on WSB2 for survival. These observations give the work both conceptual and translational relevance.

Strengths

The principal strength of the study lies in its conceptual novelty. Reframing BCL-XL and MCL-1 not only as sequestration factors but also as adaptors that facilitate substrate engagement by an E3 ligase substantially extends current models of apoptotic regulation. The mechanistic narrative developed in RPE1 cells is clear and internally consistent: the combination of AlphaFold-guided motif identification with complementary mutagenesis provides a persuasive framework for how WSB2 associates with anti-apoptotic BCL-2 family members and promotes BIM turnover. The definition of a BCL-XL/MCL-1 co-receptor mechanism for WSB2-mediated BIM degradation is therefore both intuitive and mechanistically appealing. In parallel, the authors present a distinct experimental series showing that neuroblastoma cells exhibit pronounced sensitivity to WSB2 loss, undergo apoptosis upon its depletion and display reduced competitiveness in mixed-culture assays. Although the mechanistic connection between these observations requires further clarification, the convergence of a well-defined biochemical model with a clear cancer-relevant phenotype enhances the potential biological significance of WSB2 and raises the possibility that its regulation may hold therapeutic relevance.

Weaknesses

There are several limitations that readers should consider when interpreting the study. The most fundamental issue is the disconnect between the mechanistic model established in RPE1 cells and the apoptotic phenotype observed in neuroblastoma. Although the manuscript convincingly demonstrates the WSB2-BCL-XL/MCL-1-BIM axis in RPE1 cells and independently shows that WSB2 loss compromises neuroblastoma viability, it does not examine whether BIM levels are elevated upon WSB2 depletion in neuroblastoma, nor whether apoptosis in these cells requires BIM. Without demonstrating WSB2-BCL-2-BIM complex formation or BIM dependence in the disease-relevant context, it remains unclear whether the co-receptor mechanism characterised in RPE1 cells explains the phenotype. This gap is compounded by the observation that PUMA, another potent pro-apoptotic factor, also increases following WSB2 loss, raising the possibility that multiple death pathways contribute to the outcome. The absence of a genetic rescue experiment, such as re-expression of an shRNA-resistant WSB2 restoring viability and suppressing apoptosis, further limits causal inference regarding WSB2's role in neuroblastoma.

Many central claims rely on single Western blots and pulldown assays without quantification or assessment of reproducibility. This complicates the interpretation of CHX chase experiments (where initial steady-state levels differ between samples) and limits confidence in BH3 mimetic experiments, which use a single concentration and short exposure time. Without dose-response curves, time-course analyses, caspase inhibition, or orthogonal genetic perturbation of BCL-XL or MCL-1, indirect or off-target drug effects cannot be excluded. Reduced co-IP signals in these assays could therefore reflect early apoptotic events or compound instability rather than specific disruption of protein-protein interactions.

A further limitation concerns the inference of a direct WSB2-BCL-XL interaction. The mutagenesis analyses are performed in lysates that contain endogenous or overexpressed BIM, and BH3 mimetics disrupt the WSB2 interaction only when the BCL-XL-BIM heterodimer is dismantled. The study thus cannot distinguish whether the mapped WSB2 motifs mediate direct contact with BCL-XL or whether they influence the architecture or stability of the BCL-XL-BIM complex. Because no purified protein reconstitution or biophysical binding assays are presented, the evidence for direct binding remains suggestive rather than conclusive.

The ubiquitination data also remain incomplete. Although the WSB2 mutation reduces the ubiquitin smear on BIM, the assay does not demonstrate dependence on CUL5, RBX2 or ARIH2, leaving open which ligase components are directly responsible. MLN4924 implicates CRLs more broadly, but the ubiquitination assay itself does not assign activity to the CUL5-WSB2 module.

Finally, several methodological details are insufficiently described, including the generation and validation of the doxycycline-inducible WSB2 and HA-WSB2 lines and the suitability of the WSB2-overexpressing control line used in immunoprecipitations.

Collectively, these issues do not undermine the conceptual interest of the proposed co-receptor model, but they do limit the strength of the mechanistic claims and weaken the connection between the defined mechanism and the neuroblastoma phenotype.

Reviewer #2 (Public review):

Summary:

In this elegant study, the authors employ live iGluSnFR-based imaging of glutamate release from cortical boutons to dissect the distinct roles of the Ca²⁺ sensor synaptotagmin-7 (Syt7) in synaptic transmission. Although multiple functions have been attributed to Syt7 over the years, the field remains conflicted. The authors argue that one major obstacle for resolving some of these discrepancies lies in a fundamental limitation of electrophysiological recordings, which aggregate signals across all synapses to yield averaged readouts, dominated by strong, high-release-probability synapses. By using a live glutamate imaging approach combined with sensitive detection of action potential-evoked activity across different stimulation regimes, and a dedicated analysis pipeline, the authors confirm a role for Syt7 in facilitating synchronous release and in regulating the magnitude of asynchronous release. In contrast, they find no evidence that Syt7 contributes to the facilitation of asynchronous release, do not find evidence for a role for Syt7 in synaptic vesicle replenishment during AP trains, and provide evidence suggesting that the maintenance of facilitation by Syt7 may occur independently of vesicle depletion.

Strengths:

This study offers a fresh perspective on a debated issue, using a new experimental approach that the authors previously explored in the context of Synaptotagmin 1 (Mendonca et al. 2022). The authors record the response to a series of pair-pulse stimulations, followed by an AP train. By carefully quantifying individual events and by sorting events based on their efficacy, the authors extract quantitative information that they assign to different properties of synaptic function. They also devised an interesting approach for monitoring aspects of facilitation, in which they isolate PPR events where the first response did not elicit detectable release (thus regarding the release in response to the second AP as facilitating), and compare them with successful events. Together, the authors provide semi-quantitative descriptions of synchronous and asynchronous release during single, paired, and AP trains, yielding a weighted estimate of Syt7's contribution to distinct features of synaptic vesicle release that are independent of postsynaptic readouts. A major strength of the study is the confirmation of two principal proposed functions of Syt7: facilitation of synchronous release and regulation of the magnitude of asynchronous release.

Weaknesses:

The experimental approach presented here is elegant and well-executed. However, a principal limitation lies in translating electrophysiological terminology to imaging-based measurements. For instance, interpreting signals persisting beyond 10 ms as a proxy for asynchronous release relies on assumptions that would be good to experimentally justify. Could such signals arise from iGluSnFR saturation, or be affected by desensitization?. Moreover, the quantification of asynchronous release is based on very small signals that represent only a fraction of the already small synchronous release component, raising concerns about signal-to-noise limitations. A key issue is that failures to evoke glutamate release may arise from AP failures, such that the second response in a PPR does not necessarily represent facilitation. Given that many of the findings largely confirm existing literature, the study might have benefited from a different framing, for example, as an additional validation of the correspondence between electrophysiological measures and the authors' imaging-based readouts. Another point concerns the analysis of synaptic vesicle replenishment following depletion, which would ideally be addressed using alternative stimulation protocols, such as quantifying the response/success rate to single APs at varying time points after a train. Although the authors are appropriately cautious in their conclusions (e.g., with respect to Figure 5b), this limitation remains. Finally, the use of heterogeneous cortical neuronal cultures is likely to introduce substantial variability, as the authors themselves acknowledge, which may arise from the co-expression of multiple Ca²⁺ sensors across diverse cell types.

In summary, the authors were able to confirm previously-described changes in neurotransmission properties upon the loss of Syt7 using live imaging of glutamate release at the level of single boutons. They also present preliminary evidence for the interdependence of Syt7 function, synaptic vesicle replenishment, and the facilitation of asynchronous release, although these results will need to be substantiated in future studies using alternative stimulation protocols and complementary methodologies. Taken together with the group's prior work on synaptotagmin-1, this study illustrates that live imaging of glutamate release offers an alternative approach that recapitulates some elements detectable via electrophysiological analysis, while possibly revealing new insights into the function of synaptic proteins. As a whole, taking a live imaging approach may be a broadly accessible way forward to analyze synaptic function. The potential of studying synaptic proteins in diverse cell types that are difficult to access with patch-clamp electrophysiology is particularly compelling.

Reviewer #2 (Public review):

Summary:

Abdelmageed et al., demonstrate POLK expression in nervous tissue and focus mainly on neurons. Here, they describe an exciting age-dependent change in POLK subcellular localization, from the nucleus in young tissue to the cytoplasm in old tissue. They argue that the cytosolic POLK associates with stress granules. They also investigate cell-type specific expression of POLK, and quantitate expression changes induced by cell autonomous (activity) and cell nonautonomous (microglia) factors.

Comments on revisions:

Do the authors have any explanation or reason for why they weren't able to achieve a higher knockdown of POLK using siRNA in Figure 1A2? It does not seem statistically different by eye, as all values in the KD overlap with the control. This does not seem like strong evidence that their antibody works.

Reviewer #2 (Public review):

Summary:

This is a very interesting study focusing on a remarkable oligomerization domain, the LisH-CTLH-CRA module. The module is found in a diverse set of proteins across evolution. The present manuscript focuses on the extraordinary elaboration of this domain in GID/CTLH RING E3 ubiquitin ligases, which assemble into a gigantic, highly ordered, oval-shaped megadalton complex with strict subunit specificity. The arrangement of LisH-CTLH-CRA modules from several distinct subunits is required to form the oval on the outside of the assembly, allowing functional entities to recruit and modify substrates in the center. Although previous structures had shown that data revealed that CTLH-CRA dimerization interfaces share a conserved helical architecture, the molecular rules that govern subunit pairing have not been explored. This was a daunting task in protein biochemistry that was achieved in the present study, which defines this "assembly specificity code" at the structural and residue-specific level.

The authors used X-ray crystallography to solve high-resolution structures of mammalian CTLH-CRA domains, including RANBP9, RANBP10, TWA1, MAEA, and the heterodimeric complex between RANBP9 and MKLN. They further examined and characterized assemblies by quantitative methods (ITC and SEC-MALS) and qualitatively using nondenaturing gels. Some of their ITC measurements were particularly clever and involved competitive titrations and titrations of varying partners depending on protein behavior. The experiments allowed the authors to discover that affinities for interactions between partners is exceptionally tight, in the pM-nM range, and to distill the basis for specificity while also inferring that additional interactions beyond the LisH-CTLH-CRA modules likely also contribute to stability. Beyond discovering how the native pairings are achieved, the authors were able to use this new structural knowledge to reengineer interfaces to achieve different preferred partnerings.

Strengths:

Nearly everything about this work is exceptionally strong.

(1) The question is interesting for the native complexes, and even beyond that, has potential implications for the design of novel molecular machines.

(2) The experimental data and analyses are quantitative, rigorous, and thorough.

(3) The paper is a great read - scholarly and really interesting.

(4) The figures are exceptional in every possible way. They present very complex and intricate interactions with exquisite clarity. The authors are to be commended for outstanding use of color and color-coding throughout the study, including in cartoons to help track what was studied in what experiments. And the figures are also outstanding aesthetically.

Weaknesses:

There are no major weaknesses of note, but I can make a few recommendations for editing the text.

Reviewer #2 (Public review):

Summary:

The fascinating topic of the host range of arthropods, including insects, and the detoxification of host secondary metabolites has been elucidated through studies of the host specificity of two closely related species. The discovery that key genes were acquired from fungi through horizontal gene transfer (HGT) is particularly significant.

Strengths:

(1) The discovery that the TkDOG15 enzyme, acquired through HGT from fungi, plays a key role in the detoxification of green tea catechins in the Kanzawa mite, revealing a new mechanism of plant-herbivore interactions, is highly encouraging.

(2) The verification of this finding through various experiments, including behavioral, toxicological, transcriptomic, and proteomic analyses, RNAi-based gene function analysis, and recombinant enzyme activity assays, is also highly commendable.

(3) By proposing a two-step model in which amino acid substitutions and expression regulation of a specific enzyme gene (TkDOG15) enable host adaptive evolution, this study contributes significantly to our understanding of the evolutionary mechanisms of speciation and plant defense overcoming.

Weaknesses:

While transcriptome/proteome analyses reported changes in the expression of other detoxification-related enzymes, including CCEs, UGTs, ABC transporters, DOG1, DOG4, and DOG7, it is regrettable that the contribution of each enzyme, including its interaction with TkDOG15 and the functional analysis of each enzyme within the overall catechin detoxification system, was not investigated.

Reviewer #2 (Public review):

The work by Spokaite et al describes the discovery of a novel Rab5 binding site present in complex II of class III PI3K using a combination of HDX and Cryo EM. Extensive mutational and sequence analysis define this as the primordial Rab5 interface. The data presented are convincing that this is indeed a biologically relevant interface, and is important in defining mechanistically how VPS34 complexes are regulated.

This paper is a very nice expansion of their previous cryo-ET work from 2021, and is an excellent companion piece on high-resolution cryo-EM of the complex I class III complex bound to Rab1 from the Hurley lab in 2025. Overall, this work is of excellent technical quality and answers important unexplained observations on some unexpected mutational analysis from the previous work.

They used their increased affinity VPS34 mutant to determine the 3.2 ang structure of Rab5 bound to VPS34-CII. Clear density was seen for the original Rab5 interface, but an additional site was observed. Based on this structure, they mutated out the VPS34 interface, allowing for a high-resolution structure of the Rab5 bound at the VPS15 interface.

They extensively validated the VPS15 interface in the yeast variant of VPS34, showing that the Vp215-Rab5 (VPS21) interface identified is critical in controlling complex II VPS34 recruitment.

The major strengths of this paper are that the experiments appear to be done carefully and rigorously, and I have very few experimental suggestions.

Here is what I recommend based on some very minor weaknesses I observed

(1) My main concern has to do a little bit with presentation. My main issue is how the authors use mutant description. They clearly indicate the mutant sequence in the human isoform (for example, see Figure 2A, VPS15 described as 579-SHMIT-583>DDMIE); however, when they shift to the yeast version, they shift to saying VPS15 mutant, but don't define the mutant, Figure 2G). I would recommend they just include the same sequence numbering and WT to mutant replacement every time a new mutant (or species) is described. It is always easier to interpret what is being shown when the authors are jumping between species, when the exact mutant is included. This is particularly important in this paper, where we are jumping between different subunits and different species, so a clear description in the figure/figure legends makes it much easier to read for non-specialists.

(2) The HDX data very clearly shows that Rab5 is likely able to bind at both sites, which back ups the cryo EM data nicely. I am slightly confused by some of the HDX statements described in the methods.

(3) The authors state, "Only statistically significant peptides showing a difference greater than 0.25 Da and greater than 5% for at least two timepoints were kept." This seems to be confusing as to why they required multiple timepoints, and before they also describe that they required a p-value of less than 0.05. It might be clearer to state that significant differences required a 0.25 Da, 5%, and p-value of <0.05 (n=3). Also, what do they mean by kept? Does this mean that they only fully processed the peptides with differences?

(4) They show peptide traces for a selection in the supplement, but it would be ideal to include the full set of HDX data as an Excel file, including peptides with no differences, as there is a lot of additional information (deuteration levels for everything) that would be useful to share, as recommended from the Masson et al 2019 recommendations paper. This may be attached, but this reviewer could not see an example of it in the shared data dropbox folder.

Reviewer #2 (Public review):

Summary:

The authors conducted a brain-wide survey of Avp (arginine vasopressin) and its Avpr1a gene expression in the mouse brain using RNAscope, a high-resolution in situ hybridization method. Overall, the findings are useful and important because they identify brain regions that express the Avpr1a transcript. A comprehensive overview of Avpr1a expression in the mouse brain could be highly informative and impactful. The authors used RNAscope (a proprietary in situ hybridization method) to assess transcript abundance of Avp and one of its receptors, Avpr1a. The finding of Avp-expressing cells outside the hypothalamus and the extended amygdala is novel and is nicely demonstrated by new photomicrographs in the revised manuscript. The Avpr1a data suggest expression in numerous brain regions. In the revised manuscript, reworked figures make the data easier to interpret.

Strengths:

A survey of Avpr1a expression in the mouse brain is an important tool for exploring vasopressin function in the mammalian brain and for developing hypotheses about cell- and circuit-level function.

Future considerations:

The work contained in the manuscript is substantial and informative. Some questions remain and would be addressed in the current manuscript. How many cells are impacted? Are transcripts spread across many cells or only present in a few cells? Is density evenly distributed through a brain region or compacted into a subfield?

Reviewer #2 (Public review):

Summary:

The authors have used a knock-in mouse model to explore late-in-life amyloid effects on sleep. This is an excellent model as the mutated genes are regulated by the endogenous promoter system. The sleep study techniques and statistical analyses are also first-rate.

The group finds an age-dependent increase in motor activity in advanced age in the NLGF homozygous knock-in mice (NLGF), with a parallel age-dependent increase in body temperature, both effects predominate in the dark period. Interestingly, the sleep patterns do not quite follow the sleep changes. Wake time is increased in NLGF mice, and there is no progression in increased wake over time. NREMS and REM sleep are both reduced, and there is no progression. Sleep-wake effects, however, show a robust light:dark effect with larger effects in the dark period. These findings support distinct effects of this mutation on activity and temperature and on sleep. This is the first description of the temporal pattern of these effects. NLGF mice show wake stability (longer bout durations in the dark period (their active period) and fewer brief arousals from sleep. Sleep homeostasis across the lights-on period is normal. Wake power spectral density is unaffected in NLGF mice at either age. Only REM power spectra are affected, with NLGF mice showing less theta and more delta. There are interesting sex differences, with females showing no gene difference in wake bout number, while males show a gene effect. Similarly, gene effects on NREM bout number seem larger in males than in females. Although there was no difference in homeostatic response, there was normalization of sleep-wake activity after sleep deprivation.

Strengths:

Approach (model extent of sleep phenotyping), analysis.

Weaknesses:

The weaknesses are summarized below and are viewed as "addressable".

(1) The term insomnia. Insomnia is defined as a subjective dissatisfaction with sleep, which cannot be ascertained in a mouse model. The findings across baseline sleep in NLGF mice support increased wake consolidation in the active period. The predominant sleep period (lights on) is largely unaffected, and the active period (lights off) shows increased activity and increased wake with longer bouts. There is a fantastic clue where NLGF effects are consistent with increased hypocretinergic (orexinergic) neuron activity in the dark period, and/or increased drive to hypocretin neurons from PVH.

(2) Sleep-wake transitions are impaired: This should not be termed an impairment. It could actually be beneficial to have greater state stability, especially wake stability in the dark or active period. There is reduced sleep in the model that can be normalized by short-term sleep loss. It is fascinating that recovery sleep normalized sleep in the NLGF in the immediate lights-on and light-off period. This is a key finding.

Reviewer #2 (Public review):

This study presents an important analysis of how interactions between muscles can serve as biomarkers to quantify therapeutic responses in post-stroke patients. To do so, the authors employ an information-theoretical metric (co-information) to define muscle networks and perform cluster analysis.

I thank the authors for improving the clarity of the Methods section; the newly added Figure 5 is very helpful.

One minor suggestion is that the authors should avoid overloading the notation "m" for both the EEG measurement and the matrix of II values (Eq. 1.1), which I now realise was the source of some of my initial confusion. I suggest that the authors use separate notation for these two quantities.

Reviewer #2 (Public review):

Summary:

The authors developed a cell-type-specific fluorescence-tagging approach using a CRISPR/Cas9 induced spilt-GFP reconstitution system to visualize endogenous Bruchpilot (BRP) clusters at presynaptic active zones (AZ) in specific cell types of the mushroom body (MB) in the adult Drosophila brain. This AZ profiling approach was implemented in a high-throughput quantification process allowing to compare synapse profiles within single cells, cell-types, MB compartments and between different individuals. Aim is to in more detail analyze neuronal connectivity and circuits in this center of associative learning, notoriously difficult to investigate due to the density of cells and structures within the cells. The authors detect and characterize cell-type specific differences in BRP-dependent profiling of presynapses in different compartments of the MB, while intracellular AZ distribution was found to be stereotyped. Next to the descriptive part characterizing various AZ profiles in the MB, the authors apply an associative learning assay and Rab3 knock-down and detected consequent AZ reorganization.

Strengths:

The strength of this study lies in the outstanding resolution of synapse profiling in the extremely dense compartments of the MB. This detailed analysis will serve as an entry point for many future studies of synapse diversity in connection with functional specificity to uncover the molecular mechanisms underlying learning and memory formation and neuronal network logic. Therefore, this approach is of high importance to the scientific community and represents a valuable tool to investigate and correlate AZ architecture and synapse function in the CNS.

Weaknesses:

The results and conclusions presented in this study are conclusively and well supported by the data presented and appropriate controls. As a comment that could possibly aid and strengthen the manuscript (but not required for acceptance of the manuscript): The experiments in the study are based on spilt-GFP lines (BRP:GFP11 and UAS-GFP1-10). The authors clearly validate the new on-locus construct with a genomic GFP insertion (qPCR, confocal and STED imaging of the brain with anti-BRP (Nc82), MB morphology and memory formation). It would be important to comment on the significant overall intensity decrease of anti-BRP (Nc82) in Fig. S1B (R57C10>BRP::rGFP) and possibly a Western Blot with a correlative antibody staining against BRP might help to show that BRP protein level are not affected. Additionally, it would be important to state, at least in the Materials and Methods section, that the flies are not homozygous viable (and to offer an explanation) and to state that all experiments were performed with heterozygous flies.

Reviewer #2 (Public review):

Summary:

In this EEG study, Huang et al. investigated the relative contribution of two accounts to the process of conflict control, namely the stimulus-control association (SC), which refers to the phenomenon that the ratio of congruent vs. incongruent trials affects the overall control demands, and the stimulus-response association (SR), stating that the frequency of stimulus-response pairings can also impact the level of control. The authors extended the Stroop task with novel manipulation of item congruencies across blocks in order to test whether both types of information are encoded and related to behaviour. Using decoding and RSA they showed that the SC and SR representations were concurrently present in voltage signals and they also positively co-varied. In addition, the variability in both of their strengths was predictive of reaction time. In general, the experiment has a sold design and the analyses are appropriate for the research questions.

Strength:

(1) The authors used an interesting task design that extended the classic Stroop paradigm and is effective in teasing apart the relative contribution of the two different accounts regarding item-specific proportion congruency effect.

(2) Linking the strength of RSA scores with behavioural measure is critical to demonstrating the functional significance of the task representations in question.

Weakness:

(1) The distinction between Phase 2 and Phase 1&3 behavioral results, specifically the opposite effect of MC/MI in congruent trials raises some concerns with regard to the effectiveness of the ISPC manipulation. Why do RTs and error rates under MC congruent condition in Phase 2 seem to be worse than MI congruent? Could there be other factors at play here, e.g. order effect? How does this potentially affect the neural analyses where trials from different phases were combined? Also, the manuscript does not mention whether there is counterbalancing for the color groups across participants, so far as I can tell.

Reviewer #2 (Public review):

Summary and overall evaluation:

The authors assessed how visual discrimination of stimuli in the foveola changes before, during, and after small instructed eye movements (in the "micro" range). Consistent with (and advancing) related prior work, their main finding regards a pre-saccadic modulation of visual performance at the saccade target vs. the opposite location. This pre-saccadic modulation in foveal vision peaks ~70 ms prior to the instructed small saccade.

Strengths:

The study uses an impressive, technically advanced set-up and zooms in on peri-saccadic modulations in visual acuity at the micro scale. The findings build on related prior findings from the literature on smaller and larger eye movements and add temporal granularity over prior work from the same lab. The writing is easy to follow, and the figures are clear.

Weaknesses:

At the same time, the findings remain relatively empirical in nature and do not profoundly advance theoretical understanding beyond adding valuable granularity to existing knowledge. Relevant prior literature could be better introduced and acknowledged. In addition, there remain concerns regarding potential cue-driven attentional influences that may confound the reported effects (leaving the possibility that the reported effects may be related to cue-driven attention, rather than saccade planning/execution per se). There are also some issues regarding specific statistical inferences. I detail these points below.

Major Points:

(1) Novelty framing and introduction of relevant prior literature

At times, this study is introduced as if no prior study explored the time course of changes in visual perception surrounding small (micro) saccades. Yet, it appears that a prior study from the same lab, using a very similar task, already showed a time course (Figure 5 in Shelchkova & Poletti, 2020). While this study is discussed in the introduction, it is not mentioned that at least some pre-saccade time course was already reported there, albeit a more crude one than the one in the current article. Moreover, the 2013 study by Hafed also specifically looked at "peri-microsaccade modulation in visual perception" and also already showed a temporal modulation that peaked ~50 ms before microsaccade onset. I appreciate how the current study differs in a number of ways (focusing on visual acuity in the foveola), but I was nevertheless surprised to see the first reference to this relevant prior finding in the discussion (and without any elaboration). Though more recent, the same could be argued for the 2025 study by Bouhnik et al. on pre-microsaccade modulations in visual processing in V1, which, like the Hafed study, is first mentioned only in the discussion. Perhaps these studies could be introduced in the paragraph starting at line 48, or in the next paragraph, to do better justice to the existing literature on this topic when motivating the study. This would likely also help to better point out the major advances provided by the current study.

Relatedly, in Shelchkova & Poletti (PNAS, 2020), an apparently similar congruency effect on performance was reported >200 ms milliseconds before saccade onset, as evident from Fig 5 in that article. How should readers rhyme this with the current findings? Ideally, the authors would not only acknowledge that such a time course was already reported previously, but also discuss the discrepancies between these findings further: why may the performance effects appear much earlier in this prior study compared to in the current study, where the congruency effect emerges only ~100 ms prior to the instructed small saccade?

(2) Saccade- or cue-driven? (assumption that attention is unaltered in failed saccade trials)

Because the authors used a cue to instruct saccade direction, it remains a possibility that the reported modulations in visual performance may be driven directly by the spatial cue (cue-related attentional allocation), rather than the instructed small saccade per se. While the authors are clearly aware of this potential confound, questions remain regarding the convincingness of the presented control analyses. In my view, a more compelling control would require an additional experiment.

The central argument against a cue-locked (purely attentional) modulation is the absence of a performance modulation in so-called "failed" saccade trials. However, a key assumption here is that putative cue-driven attention was unaltered in these trials. This is never verified and, in my opinion, highly unlikely. Rather, trials with failed microsaccades could very well be the result of failing to process the cue in the first place (indeed, if the task is to make a saccade to the cue, failure to make a saccade equates failure to perform the task). In such trials, any putative cue-driven influences over spatial attention would also be expected to be substantially reduced. Accordingly, just because failed saccade trials show little performance modulation does not rule out cue-driven attention effects, because attention may also have "failed" in these failed saccade trials. The control for potential cue-driven attention effects would be more convincing if the authors included a condition with the same cues, where participants are simply not instructed to make any saccades to the cues. Unfortunately, such an experimental condition appears not to have been included here. The author may still consider adding such a control experiment.

Another argument against a cue-driven effect is that the authors found no interaction with time in the cue-locked data, whereas they did find such an interaction in the saccade-locked data. However, the lack of significance in the cue-locked data but significance in the saccade-locked data is not strong evidence against a cue-driven influence. Statistically, there is no direct comparison here, and more importantly, with longer delays, the cue-locked data may also start to show a dip (this could potentially be tested by the authors if they have enough trials available to extend their cue-locked analysis further in time). Indeed, exogenous attention, that may have been automatically evoked by the spatial cue, is known to be transient and to eventually even reverse after a brief initial facilitation (see e.g., Klein TiCS, 2000).

Finally, the authors consistently refer to "endogenous" attention (starting at line 221) when addressing potential cue-driven attention confounds. However, because the cue is not predictive, but is a spatial cue that differs in a bottom-up manner between left and right cues, "exogenous" attention is a more likely confound here in my view. Specifically, the spatial cue may automatically trigger attention in the direction of the target location it points to (and such exogenous effects would be expected even for unpredictive cues).

(3) Benefit and cost, or just cost?

Line 151 states that no statistically significant benefit for the saccade target was found compared to the neutral baseline. Yet, the claim throughout the article is distinct, such as in line 159: "These results show that approximately 100 milliseconds before microsaccade onset, discrimination rapidly improved at the intended target location". I do not question the robustness of the congruency effect, but the authors should be more careful when inferring "improved" perception at the target location because, as far as I could tell (as well as in the authors' own writing in line 151), this is not substantiated statistically when compared to the neutral baseline.

Related to this point, in Figure 3B, it would be informative to also see the average performance in the neutral cue condition (for example, as a straight line as in some other figures). This would help to better appreciate the relative benefits and/or costs compared to the neutral condition, also in the time-resolved data.

(4) Statistical inference for the comparison between failed and non-failed trials

Currently, the lack of modulation in the failed saccade trials hinges on a null effect. It would be stronger to support the claims with a significant difference in the congruency effect between failed and non-failed trials. Indeed, lack of significance in failed saccade trials does by itself not constitute valid evidence that the congruency effect is larger in saccade compared to failed saccade trials. For this, a significant interaction between saccade-trial-type (failed/non-failed) and congruency (congruent/incongruent) should be established (see e.g., Nieuwenhuis et al., Nat Neurosci, 2011).

(5) Time window justification

While the authors nicely depict their data across the full time axis, all statistics are currently performed on data extracted from specific time windows. How exactly were these time windows determined and justified? Likewise, how were the specific times picked for visualizing and statistically quantifying the data in e.g., Figures 3D and E? It would be reassuring to add justification for these specific time windows and/or to verify (using follow-up analyses) that the presented results are robust when different time windows are chosen.

(6) Microsaccade definition

Microsaccades are explicitly defined as being below half a degree. This appears rather arbitrary and rigid. Does the size of saccades not ultimately depend on the task and stimulus (e.g., Otero-Millan et al., PNAS, 2013) rather than being a fixed biological property? Perhaps this could be stated less rigidly, such as by stating how microsaccades are often observed below 0.5 degrees.

(Relatedly, one may wonder whether the type of instructed saccades that the authors studied here involves the same type of eye movements as the type of fixational microsaccades that have been the focus of ample prior studies. However, I recognize that this specific reflection may open a debate that is beyond the scope of this article.

Reviewer #2 (Public review):

Summary:

This study investigates the role of spinal astrocytes in mediating stress-induced pain hypersensitivity, focusing on the LC (locus coeruleus)-to-SDH (spinal dorsal horn) circuit and its mechanisms. The authors aimed to delineate how LC activity contributes to spinal astrocytic activation under stress conditions, explore the role of noradrenaline (NA) signaling in this process, and identify the downstream astrocytic mechanisms that influence pain hypersensitivity.

The authors provide strong evidence that 1-hour restraint stress-induced pain hypersensitivity involves the LC-to-SDH circuit, where NA triggers astrocytic calcium activity via alpha1a adrenoceptors (alpha1aRs). Blockade of alpha1aRs on astrocytes-but not on Vgat-positive SDH neurons-reduced stress-induced pain hypersensitivity. These findings are rigorously supported by well-established behavioral models and advanced genetic techniques, uncovering the critical role of spinal astrocytes in modulating stress-induced pain.

However, the study's third aim-to establish a pathway from astrocyte alpha1aRs to adenosine-mediated inhibition of SDH-Vgat neurons-is less compelling. While pharmacological and behavioral evidence is intriguing, the ex vivo findings are indirect and lack a clear connection to the stress-induced pain model. Despite these limitations, the study advances our understanding of astrocyte-neuron interactions in stress-pain contexts and provides a strong foundation for future research into glial mechanisms in pain hypersensitivity.

Strengths:

The study is built on a robust experimental design using a validated 1-hour restraint stress model, providing a reliable framework to investigate stress-induced pain hypersensitivity. The authors utilized advanced genetic tools, including retrograde AAVs, optogenetics, chemogenetics, and subpopulation-specific knockouts, allowing precise manipulation and interrogation of the LC-SDH circuit and astrocytic roles in pain modulation. Clear evidence demonstrates that NA triggers astrocytic calcium activity via alpha1aRs, and blocking these receptors effectively reduces stress-induced pain hypersensitivity.

Weaknesses:

The study offers mainly indirect evidence for astrocyte-released adenosine acting on SDH-VGAT neurons. The potential contributions of astrocyte-derived D-serine and adenosine to different spinal neuron subtypes, as well as the transient "dip" in astrocytic calcium following LC optostimulation, merit further clarification in future work once appropriate tools become available.

Comments on revisions:

The authors have thoroughly addressed my previous comments, resolving most of the points I raised except those noted in the "Weaknesses" section above. I understand that some of these aspects will require future tool development.

Reviewer #2 (Public review):

Summary:

This manuscript uses single-molecule run-off experiments and TASEP/HMM models to estimate biophysical parameters, i.e., ribosomal initiation and elongation rates. Combining inferred initiation and elongation rates, the authors quantify ribosomal density. TASEP modeling was used to simulate the mechanistic dynamics of ribosomal translation, and the HMM is used to link ribosomal dynamics to microscope intensity measurements. The authors' main conclusions and findings are:

- Ribosomal elongation rates and initiation rates are strongly coordinated.

- Elongation rates were estimated between 1 and 4.5 aa/sec. Initiation rates were estimated between 1 and 2 ribosomes/min. These values agree with previously reported ones.

- Ribosomal density was determined to be below 12% for all constructs and conditions.

- eIF5A-perturbations (GC7 inhibition) resulted in non-significant changes in translational bursting and ribosome density.

- eIF5A perturbations affected both elongation and initiation rates.

Strengths:

This manuscript presents an interesting scientific hypothesis to study ribosome initiation and elongation concurrently. This topic is relevant for the field. The manuscript presents a novel quantitative methodology to estimate ribosomal initiation rates from Harringtonine run-off assays. This is relevant because run-off assays have been used to estimate, exclusively, elongation rates.

Comments on revisions:

The authors have addressed my concerns. Specifically, they have expanded the discussion on unexpected eIF5A perturbation results, calculated CAI values for all constructs, and made code and data publicly available via GitHub and Zenodo. The mathematical notation is now consistent, and all variables are properly defined.

Reviewer #2 (Public review):

Summary:

Neural stem cells produce a wide variety of neurons during development. The regulatory mechanisms of neural diversity are based on the spatial and temporal patterning of neural stem cells. Although the molecular basis of spatial patterning is well-understood, the temporal patterning mechanism remains unclear. In this manuscript, the authors focused on the roles of cell cycle progression and cytokinesis in temporal patterning and found that both are involved in this process.

Strengths:

They conducted RNAi-mediated disruption on cell cycle progression and cytokinesis. As they expected, both disruptions affected temporal patterning in NSCs.

Weaknesses:

Although the authors showed clear results, they needed to provide additional data to support their conclusion sufficiently.

For example, they can examine the effects of cell cycle acceleration on the temporal patterning.

Reviewer #2 (Public review):

Summary:

This work addresses the question whether artificial deep neural network models of the brain could be improved by incorporating top-down feedback, inspired by the architecture of neocortex.

In line with known biological features of cortical top-down feedback, the authors model such feedback connections with both, a typical driving effect and a purely modulatory effect on the activation of units in the network.

To asses the functional impact of these top-down connections, they compare different architectures of feedforward and feedback connections in a model that mimics the ventral visual and auditory pathways in cortex on an audiovisual integration task.

Notably, one architecture is inspired by human anatomical data, where higher visual and auditory layers possess modulatory top-down connections to all lower-level layers of the same modality, and visual areas provide feedforward input to auditory layers, whereas auditory areas provide modulatory feedback to visual areas.

First, the authors find that this brain-like architecture imparts the models with a light visual bias similar to what is seen in human data, which is the opposite in a reversed architecture, where auditory areas provide feedforward drive to the visual areas.

Second, they find that, in their model, modulatory feedback should be complemented by a driving component to enable effective audiovisual integration, similar to what is observed in neural data.

Overall, the study shows some possible functional implications when adding feedback connections in a deep artificial neural network that mimic some functional aspects of visual perception in humans.

Strengths:

The study contains innovative ideas, such as incorporating an anatomically inspired architecture into a deep ANN, and comparing its impact on a relevant task to alternative architectures.

Moreover, the simplicity of the model allows it to draw conclusions on how features of the architecture and functional aspects of the top-down feedback affects performance of the network.

This could be a helpful resource for future studies of the impact of top-down connections in deep artificial neural network models of neocortex.

Weaknesses:

Some claims not yet supported.

The problem is that results are phrased quite generally in the abstract and discussion, while the actual results shown in the paper are very specific to certain implementations of top-down feedback and architectures. This could lead to misunderstanding and requires some revisions of the claims in the abstract and discussion (see below).

"Altogether our findings demonstrate that modulatory top-down feedback is a computationally relevant feature of biological brain..."

This claim is not supported, since no performance increase is demonstrated for modulatory feedback. So far, only the second half of the sentence is supported: "...and that incorporating it into ANNs affects their behavior and constrains the solutions it's likely to discover."

"This bias does not impair performance on the audiovisual tasks."

This is only true for the composite top-down feedback that combines driving and modulatory effects, whereas modulatory feedback alone can impair the performance (e.g., in the visual tasks VS1 and VS2). The fact that modulatory feedback alone is insufficient in ANNs to enable effective cross-modal integration and requires some driving component is actually very interesting, but it is not stressed enough in the abstract. This is hinted at in the following sentence, but should be made more explicitly:

"The results further suggest that different configurations of top-down feedback make otherwise identically connected models functionally distinct from each other, and from traditional feedforward and laterally recurrent models."

"Here we develop a deep neural network model that captures the core functional properties of top-down feedback in the neocortex" -> this is too strong, take out "the", because very likely there are other important properties that are not yet incorporated.

"Altogether, our results demonstrate that the distinction between feedforward and feedback inputs has clear computational implications, and that ANN models of the brain should therefore consider top-down feedback as an important biological feature."

This claim is still not substantiated by evidence provided in the paper. First, the wording is a bit imprecise, because mechanistically, it is not really the feedforward versus feedback (a purely feedforward model is not considered at all in the paper), but modulatory versus driving. Moreover, the second part of the sentence is problematic: The results imply that, computationally/functionally, driving connections are doing the job, while modulatory feedback does not really seem to improve performance (best case, it does not do any harm). It is true that it is a feature that is inspired by biology, but I don't see why the results imply that (modulatory) top-down feedback should be considered in ANN models of the brain. This would require to show that such models either improve performance, or do improve the ability to fit neural data, both which are beyond the scope of the paper.

The same argument holds for the following sentence, which is not supported by the results of the paper:

"More broadly, our work supports the conclusion that both the cellular neurophysiology and structure of feed-back inputs have critical functional implications that need to be considered by computational models of brain function."

Additional supplementary material required

Although the second version checked the influence of processing time, this was not done for the most important figure of the paper, Figure 4. A central claim in the abstract "This bias does not impair performance on the audiovisual tasks" relies on this figure, because only with composite feedback the performance is comparable between the the "drive-only" and "brain-like" models. Thus, the supplementary Figure 3 should also include the composite networks and drive only network to check the robustness of the claim with respect to process time. This robustness analysis should then also be mentioned in the text. For example, it should be mentioned whether results in these networks are robust or not with respect to process time, whether there are differences between network architectures or types of feedback in general etc.

Moreover, the current analysis for networks with modulatory feedback is a bit confusing. Why is the performance so low for the reverse model for a process time of 3 and 10? This is a very strong effect that warrants explanation. More details should be added in the caption as well. For example, are the models separately trained for the output after 3 and 10 processing steps for the comparison, or just evaluated at these times? Not training these networks separately might explain the low performance for some networks, so ideally networks are trained for each choice of processing steps.

Reviewer #2 (Public review):

Summary:

The authors set out to investigate how well the onset of a self-initiated movement could be predicted at different times prior to action onset. To do so, they collected EEG and MEG data across 15 human participants who watched natural landscape images on a screen. These participants performed active self-initiated movements or observed passive actions to have a new image appear. By comparing the neural activity prior to active and time-matched passive actions, the authors found that even though a build-up of neural activity is visible close to 1s prior to action, action onset could only be reliably predicted around 100ms prior to action. These results confirm what was already suggested in previous literature: the commitment to action is only clear from the late stages in the visible neural ramp-up to action onset.

Strengths:

(1) The paper presents a well-thought-out methodology to assess the predictive value of neural activity prior to a self-initiated movement and passively observed action, while keeping all other experimental factors identical. This methodology can be applied outside the specific scope of this paper as well, in efforts to assess the correspondence of a neural signature with an observed behavior.

(2) The results are a strong confirmation of what was suggested less clearly in previous research (Trevena & Miller, 2010, Consciousness & Cognition; Schmidt et al., 2016, Neuroscience & Biobehavioral Reviews; Travers et al., 2020, NeuroImage).

Weaknesses:

(1) Although the authors conducted a solid confirmatory study, the importance of this confirmation is less clear to me. How do the current results change our interpretation of the relation between conscious intention and neural preparation for action? Do these results affect our interpretation of free will? Why does it matter at all whether we see neural preparatory activity prior to the report of a conscious intention to act, or prior to action observation? This study does not clarify the relationship between the observed neural phenomenon, the action or the experienced intention. It does not explain whether this relation is causal, correlational or something else.

(2) Whereas Derchi et al. (2023, Scientific Reports) were able to keep the entire experimental context similar across intended and unintended conditions, Jeay-Bizot et al. have one big difference between their passive and active conditions: the presence of a movement. Therefore, the present results explain the presence or absence of a movement rather than the presence or absence of an intention to act.

Reviewer #2 (Public review):

In this article, Schmidt et al use iPSC-derived human cortical neurons to test the effects the psychedelic psilocin in different models of neuroplasticity.

Using human iPSC-derived cortical neurons, the authors test the expression of 5-HT2A and subcellular distribution, as well as the effect of different times of exposure to psilocin on 5-HT2A expression. The authors evaluated the effect of the 5-HT2 antagonist ketanserin, as well as the inhibition of dynamin-dependent endocytic pathways with dynasore. Gene expression and plasticity (structural and functional) was also evaluated after different times of exposure to psilocin.

In general, results are interesting since they use the iPSC to evaluate the potentially translationally relevant effects of psilocin (the active metabolite of the psychedelic psilocybin).

Comments on revisions:

The authors have addressed all of my previous concerns. A particular strength of the rebuttal is that the authors corroborated the lack of selectivity/specificity of the anti-5-HT2A antibody used in earlier versions of the manuscript.

Reviewer #2 (Public review):

Summary:

The authors used experimental evolution, repeatedly subjecting Saccharomyces cerevisiae populations to rapid liquid-nitrogen freeze-thaw cycles, while tracking survival, cellular biophysics, metabolite levels, and whole-genome sequence changes. Within 25 cycles, viability rose from ~2 % to ~70 % in all independent lines, demonstrating rapid and highly convergent adaptation despite distinct starting genotypes. Evolved cells accumulated about three-fold more intracellular trehalose, adopted a quiescence-like phenotype (smaller, denser, non-budding cells), showed cytoplasmic stiffening and reduced membrane damage, and re-entered growth with shorter lags-traits that together protected them from ice-induced injury. Whole-genome indicated that multiple genetic routes can yield the same mechano-chemical survival strategy. A population model in which trehalose controls quiescence entry, growth rate, lag, and freeze-thaw survival reproduced the empirical dynamics, implicating physiological state transitions rather than specific mutations as the primary adaptive driver. The study therefore concludes that extreme-stress tolerance can evolve quickly through a convergent, trehalose-rich quiescence-like state that reinforces membrane integrity and cytoplasmic structure.

Strengths:

Experimental design, data presentation and interpretation, writing

Weaknesses:

None

Comments on revisions:

The revised manuscript is improved and addresses the reviews concerns adequately.

Reviewer #2 (Public review):

Summary:

The altered metabolism of tumors enables their growth and survival. Classically, tumor metabolism often involves increased activity of a given pathway in intermediary metabolism to provide energy or substrates needed for growth. Papadopoli et al. investigate the converse - the role of mitochondrial electron transfer flavoprotein dehydrogenase (ETFDH) in cancer metabolism and growth. The authors present compelling evidence that ETFDH insufficiency, which is detrimental in non-malignant tissues, paradoxically enhances bioenergetic capacity and accelerates neoplastic growth in cancer cells in spite of the decreased metabolic fuel flexibility that this affords tumor cells. This is achieved through the retrograde activation of the mTORC1/BCL-6/4E-BP1 axis, leading to metabolic and signaling reprogramming that favors tumor progression.

Strengths:

This review focuses primarily on the cancer metabolism aspects of the manuscript.

The study provides robust evidence linking ETFDH insufficiency to enhanced cancer cell bioenergetics and tumor growth.

The use of multiple cancer cell lines and in vivo models strengthens the generalizability of the findings.

The mechanistic insights into the mTORC1/BCL-6/4E-BP1 axis and its role in metabolic reprogramming are of general interest within and outside the immediate field of tumor metabolism.

Conclusion:

This manuscript provides significant insights into the role of ETFDH insufficiency in cancer metabolism and growth. The findings highlight the potential of targeting the mTORC1/BCL-6/4E-BP1 axis in ETFDH-deficient cancers. The compelling data support the conclusions presented in the manuscript, which will be valuable to the cancer metabolism community.

[Editors' note: The authors have addressed each of the two weaknesses previously listed in the public review, providing new experimental data on nucleotides and showing that the catalytic activity is required via the suggested addback experiment.]

Reviewer #2 (Public review):

The substantially revised paper has increased in clarity and is much more accessibe and straightforward than the first version. The analyses are now clearer and support the conclusions better. There are however some remaining methodological weakness, which in my mind still renders the evidence to not be entirely convincing.

(1) The temporal autocorrelation concern is not fully convincingly addressed. The temporal autocorrelation curves supplied in the supplements are really helpful, but linearly regressing out the temporal distance from the neural distance clearly does not work, as one can see from the right panel of supplementary Figure 1. If the method had worked correctly the line should have been flat. The analysis however shows that decision trials with a lag > 2 are basically independent - so a simple way to address this is to restrict the RSA analysis to trials with a decision lag of > 2. This analysis would strengthen the paper a lot.

(2) In the final analysis, the authors use all the trials to make the claim that the hippocampus represents the characters in a shared social space. However, as within-character distances are still included in the analysis, this result could still be driven by the effects of within-character representations that are not shared across characters. A simple way of addressing this concern would be to only include between-character distances in this analysis, making it truly complementary to the previous within-character analysis. It would also be very interesting to compare the the within- and between-character analyses in the hippocampus directly.

(3) Overall, the correction for multiple comparisons in the fMRI and the resulting corrected p-values are not sufficiently explained and documented in the paper. What was exactly permuted in the tests? Was correction applied in a voxel-wise or cluster-wise fashion? If cluster-wise, the cluster-wise p-values need to be reported.

Reviewer #2 (Public review):

Summary:

This study investigates the involvement of first-order thalamic nuclei in language-related tasks using task-based fMRI in a 3 × 2 design contrasting linguistic and non-linguistic versions of reading, speech comprehension, and speech production. By focusing on the LGN, MGN, and VLN and combining activation, connectivity, lateralization, and multivariate pattern analyses, the authors aim to characterize modality-specific and language-related thalamic contributions.

Strength:

A major strength of the work is its hypothesis-driven and multimodal analytical approach, and the modality-specific engagement of first-order thalamic nuclei is robust and consistent with known thalamocortical organization. This is a very sound study overall.

Weaknesses:

However, several conceptual issues complicate the interpretation of the results as evidence for linguistic modulation per se. A central concern relates to the operationalization of the linguistic versus non-linguistic contrast. In the present design, linguistic and non-linguistic stimuli differ along multiple dimensions beyond linguistic content. For example, written words and scrambled images differ in spatial frequency structure, edge composition, contrast regularities, and familiarity, while intelligible speech and acoustically scrambled sounds differ substantially in temporal and spectral statistics. This is particularly relevant given that first-order thalamic nuclei such as the LGN are known to be highly sensitive to low-level sensory properties. As a result, observed differences in thalamic responses may reflect sensitivity to stimulus properties rather than linguistic processing per se, and this limits the specificity of claims regarding linguistic modulation.

Relatedly, although the manuscript frequently refers to effects "depending on the linguistic nature of the stimuli," the statistical evidence for linguistic versus non-linguistic modulation is uneven across analyses. Whole-brain contrasts collapse across stimulus type and primarily test modality effects. Similarly, the primary ROI analyses of activation amplitude are collapsed across linguistic and non-linguistic conditions and convincingly demonstrate modality-specific engagement of thalamic nuclei, but do not in themselves provide evidence for linguistic modulation. Linguistic effects emerge only in later, more targeted analyses focusing on hemispheric lateralization and multivariate pattern classification, and these effects are nucleus-, modality-, and analysis-specific rather than general. Taken together, these results suggest that linguistic modulation constitutes a secondary and selective finding, whereas modality-specific task engagement represents the primary and most robust outcome of the study.

An additional interpretational issue concerns task engagement and attention. The tasks differ substantially in cognitive demands (e.g., passive reading and listening versus overt speech production), and linguistic and non-linguistic blocks may differ systematically in salience or engagement. This is particularly important given prior evidence, cited by the authors, that LGN and MGN activity can be modulated by task demands and attention. In the absence of behavioral measures indexing task engagement or compliance, it is difficult to determine whether differences between linguistic and non-linguistic conditions reflect linguistic processing per se or are mediated by attentional factors.

Finally, while the manuscript emphasizes the novelty of evaluating thalamic involvement in language, thalamic contributions to language have been documented previously in both lesion and functional imaging studies. The contribution of the present work, therefore, lies less in establishing thalamic involvement in language per se, and more in its focus on specific first-order nuclei, its multimodal design, and its combination of univariate, connectivity, and multivariate analyses. Moderating claims of novelty would help place the findings more clearly within the existing literature.

Reviewer #2 (Public review):

Summary:

The manuscript reports a cryo-EM structure of TMAO demethylase from Paracoccus sp. This is an important enzyme in the metabolism of trimethylamine oxide (TMAO) and trimethylamine (TMA) in human gut microbiota, so new information about this enzyme would certainly be of interest.

Strengths:

The cryo-EM structure for this enzyme is new and provides new insights into the function of the different protein domains, and a channel for formaldehyde between the two domains.

Weaknesses:

(1) The proposed catalytic mechanism in this manuscript does not make sense. Previous mechanistic studies on the Methylocella silvestris TMAO demethylase (FEBS Journal 2016, 283, 3979-3993, reference 7) reported that, as well as a Zn2+ cofactor, there was a dependence upon non-heme Fe2+, and proposed a catalytic mechanism involving deoxygenation to form TMA and an iron(IV)-oxo species, followed by oxidative demethylation to form DMA and formaldehyde.

In this work, the authors do not mention the previously proposed mechanism, but instead say that elemental analysis "excluded iron". This is alarming, since the previous work has a key role for non-heme iron in the mechanism. The elemental analysis here gives a Zn content of about 0.5 mol/mol protein (and no Fe), whereas the Methylocella TMAO demethylase was reported to contain 0.97 mol Zn/mol protein, and 0.35-0.38 mol Fe/mol protein. It does, therefore, appear that their enzyme is depleted in Zn, and the absence of Fe impacts the mechanism, as explained below.

The proposed catalytic mechanism in this manuscript, I am sorry to say, does not make sense to me, for several reasons:

(i) Demethylation to form formaldehyde is not a hydrolytic process; it is an oxidative process (normally accomplished by either cytochrome P450 or non-heme iron-dependent oxygenase). The authors propose that a zinc (II) hydroxide attacks the methyl group, which is unprecedented, and even if it were possible, would generate methanol, not formaldehyde.

(ii) The amine oxide is then proposed to deoxygenate, with hydroxide appearing on the Zn - unfortunately, amine oxide deoxygenation is a reductive process, for which a reducing agent is needed, and Zn2+ is not a redox-active metal ion;

(iii) The authors say "forming a tetrahedral intermediate, as described for metalloproteinase", but zinc metalloproteases attack an amide carbonyl to form an oxyanion intermediate, whereas in this mechanism, there is no carbonyl to attack, so this statement is just wrong.

So on several counts, the proposed mechanism cannot be correct. Some redox cofactor is needed in order to carry out amine oxide deoxygenation, and Zn2+ cannot fulfil that role. Fe2+ could do, which is why the previously proposed mechanism involving an iron(IV)-oxo intermediate is feasible. But the authors claim that their enzyme has no Fe. If so, then there must be some other redox cofactor present. Therefore, the authors need to re-analyse their enzyme carefully and look either for Fe or for some other redox-active metal ion, and then provide convincing experimental evidence for a feasible catalytic mechanism. As it stands, the proposed catalytic mechanism is unacceptable.

(2) Given the metal content reported here, it is important to be able to compare the specific activity of the enzyme reported here with earlier preparations. The authors do quote a Vmax of 16.52 µM/min/mg; however, these are incorrect units for Vmax, they should be µmol/min/mg. There is a further inconsistency between the text saying µM/min/mg and the Figure saying µM/min/µg.

(3) The consumption of formaldehyde to form methylene-THF is potentially interesting, but the authors say "HCHO levels decreased in the presence of THF", which could potentially be due to enzyme inhibition by THF. Is there evidence that this is a time-dependent and protein-dependent reaction? Also in Figure 1C, HCHO reduction (%) is not very helpful, because we don't know what concentration of formaldehyde is formed under these conditions; it would be better to quote in units of concentration, rather than %.

(4) Has this particular TMAO demethylase been reported before? It's not clear which Paracoccus strain the enzyme is from; the Experimental Section just says "Paracoccus sp.", which is not very precise. There has been published work on the Paracoccus PS1 enzyme; is that the strain used? Details about the strain are needed, and the accession for the protein sequence.

Reviewer #2 (Public review):

Summary:

This study aimed to explore dynamic changes in the somatosensory representation of both the body and artificial body parts. The study investigated how proprioceptive localisation along the finger changes when participants wear, actively use, and then remove a hand augmentation device - a rigid finger-extension. By mapping perceived target locations along the biological finger and the extension across multiple stages, the authors aim to characterise how the somatosensory system updates our spatial body representation during and after interaction with body augmentation technology.

Strengths:

The manuscript addresses an interesting question of how augmentation devices alter proprioceptive localisation abilities. Conceptually, the work moves beyond classic tool-use paradigms by focusing on a device that is used with the hand to extend the fingers' abilities (versus a tool that is simply used by the hand), and by attempting to map perceived spatial structure across both biological and artificial segments within the same framework.

A major strength is the multi-stage design, which samples localisation abilities at baseline, the beginning of device wear, post-training, and immediately post-removal. This provides a richer characterisation of short-term adaptation compared to a simple pre/post comparison. The dense sampling across stages and target locations generates a rich behavioural dataset that will be valuable to readers interested in somatosensory body representation. The within-subject, counterbalanced control session further strengthens interpretability, providing a useful comparison for interpreting stage-dependent effects, and to probe how functional training shapes changes in the perceptual representations. Finally, the augmentation device itself appears carefully engineered, with thoughtful design decisions regarding wearability, including comfort and customised fit. The manuscript is also communicated clearly, with transparent reporting of analyses and succinct figures that make the pattern changes across stages straightforward to evaluate.

Weaknesses:

There is conceptual ambiguity in how the regression outcomes are interpreted in relation to perceived length and spatial integration. The manuscript treats regression slope as a proxy for "length perception" and discards the intercept as "spatial bias," but in this localisation task translation (intercept) and scaling (slope) are coupled: changes in anchoring at the proximal baseline (intercept) or distal endpoint can generate slope differences without uniform rescaling across the mapped surface. Relatedly, the analyses do not establish whether the reported effects are global across targets or disproportionately driven by the most distal locations. This limits the strength of inferences about "partitioning" or "reallocation" of representational space across biological and artificial segments. Some interpretive statements also appear stronger than the evidence supports (e.g., describing the stage 2 bio-extension map as "geometrically accurate", despite Bayes factors that provide only anecdotal support for no difference from true length). Extensive repeated judgements to a fixed set of locations may additionally stabilise response strategies or anchoring even without feedback, complicating the separation of body-representation change from task-specific calibration.

The manuscript would also benefit from clearer conceptual framing of what the device is and what its training probes are. The device is described variably as an "artificial finger" versus a rigid "finger extension," with different implications for perception and function. In addition, the training tasks appear to emphasise manipulation and dexterity more than scenarios requiring an extended reachable workspace (indeed, participants appear to have performed at least as well, if not better, in the control training), which brings into question whether participants explored the device's intended functionality and possible proprioceptive consequences. The control experiment is thoughtfully designed to test whether functional training contributes to the stage 3 changes, but because localisation is not performed while wearing the short device, the design does not resolve whether the stage 2 change and the post-removal aftereffect are specific to the augmentative extension versus more general consequences of wearing a device on the finger (and the following possible distorted distal cues).

Finally, the immediate post-removal aftereffects are intriguing, but the mechanistic interpretation remains underspecified. As presented within the internal model framework, the magnitude and consistency of the aftereffect following brief exposure are difficult to reconcile with the stability expected from a lifetime biological finger model, and because the aftereffect is assessed only immediately after removal, its time course and functional significance remain unclear.

Reviewer #2 (Public review):

Summary:

The manuscript by the Root laboratory and colleagues describes how the posterolateral cortical amygdala (plCoA) generates valenced behaviors. Using a suite of methods, the authors demonstrate that valence encoding is mediated by several factors, including spatial localization of neurons within the plCoA, glutamatergic markers, and projection. The manuscript shows convincingly that multiple features (spatial, genetic, and projection) contribute to overall population encoding of valence. Overall, the authors conduct many challenging experiments, each of which contains the relevant controls, and the results are interpreted within the framework of their experiments.

Strengths:

- The manuscript is well constructed, containing lots of data sets and clearly presented, in spite of the abundance of experimental results.

- The authors should be commended for their rigorous anatomical characterizations and post-hoc analysis. In the field of circuit neuroscience, this is rarely done so carefully, and when it is, often new insights are gleaned as is the case in the current manuscript.

- The combination of molecular markers, behavioral readouts and projection mapping together substantially strengthens the results.

- The focus on this relatively understudied brain region in the context is valence is well appreciated, exciting and novel.

Weaknesses:

The weaknesses noted in the primary review have all been addressed adequately.

Reviewer #2 (Public review):

Summary:

This manuscript by Rosenthal and Goldberg investigates interactions between artemisinins and its quinoline partner drugs currently used for treating uncomplicated Plasmodium falciparum malaria. The authors show that chloroquine (CQ), piperaquine, and amodiaquine antagonize dihydroartemisinin (DHA) activity, and in CQ-resistant parasites, the interaction is described as "superantagonism," linked to the pfcrt genotype. Mechanistically, application of the heme-reactive probe H-FluNox indicates that quinolines render cytosolic heme chemically inert, thereby reducing peroxide activation. The work is further extended to triple ACTs and ozonide-quinoline combinations, with implications for artemisinin-based combination therapy (ACT) design, including triple ACTs.

Strengths:

The manuscript is clearly written, methodologically careful, and addresses a clinically relevant question. The pulsing assay format more accurately models in vivo artemisinin exposure than conventional 72-hour assays, and the use of H-FluNox and Ac-H-FluNox probes provides mechanistic depth by distinguishing chemically active versus inert heme. These elements represent important refinements beyond prior studies, adding nuance to our understanding of artemisinin-quinoline interactions.

Weaknesses:

Several points warrant consideration. The novelty of the work is somewhat incremental, as antagonism between artemisinins and quinolines is well established. Multiple prior studies using standard fixed-ratio isobologram assays have shown that DHA exhibits indifferent or antagonistic interactions with chloroquine, piperaquine, and amodiaquine (e.g., Davis et al., 2006; Fivelman et al., 2007; Muangnoicharoen et al., 2009), with recent work highlighting the role of parasite genetic background, including pfcrt and pfmdr1, in modulating these interactions (Eastman et al., 2016). High-throughput drug screens likewise identify quinoline-artemisinin combinations as mostly antagonistic. The present manuscript adds refinement by applying pulsed-exposure assays and heme probes rather than establishing antagonism de novo.

The dataset focuses on several parasite lines assayed in vitro, so claims about broad clinical implications should be tempered, and the discussion could more clearly address how in vitro antagonism may or may not translate to clinical outcomes. The conclusion that artemisinins are predominantly activated in the cytoplasm is intriguing but relies heavily on Ac-H-FluNox data, which may have limitations in accessing the digestive vacuole and should be acknowledged explicitly. The term "superantagonism" is striking but may appear rhetorical; clarifying its reproducibility across replicates and providing a mechanistic definition would strengthen the framing. Finally, some discussion points, such as questioning the clinical utility of DHA-PPQ, should be moderated to better align conclusions with the presented data while acknowledging the complexity of in vivo pharmacology and clinical outcomes.

Despite these mild reservations, the data are interesting and of high quality and provide important new information for the field.

Editor's Review of the Revision: The authors have provided a well-reasoned rebuttal to the comments of the three reviewers. Most of the changes were incorporated in their revised Discussion. Their data with the active heme probe H-FluNox are novel and the authors reveal interesting interactions between peroxide and 4-aminoquinoline-based antimalarials that open new avenues of research especially when considering antimalarial combinations that combine these chemical scaffolds. This study will be of broad interest to investigators studying and developing antimalarial drugs and combinations and the impact of Plasmodium falciparum resistance mechanisms. A minor recommendation would be that the authors state H-FluNox when referring to their small molecule probe in the abstract, so that it is captured in PubMed searches.

Reviewer #2 (Public review):

Summary:

In this study, the authors identify a previously uncharacterised regulator of mitochondrial function using a genetic screen and propose a role for this protein in supporting mitochondrial protein production. They provide evidence that the protein localises to mitochondria, interacts with components of the mitochondrial translation machinery, and is required for normal heart function in an animal model.

Strengths:

A major strength of the work is the use of multiple independent approaches to assess mitochondrial activity and protein production, which together provide support for the central conclusions. The in vivo data linking loss of this factor to impaired heart function are particularly compelling and elevate the relevance of the study beyond a purely cell-based context.

Weaknesses:

Given prior reports placing this protein outside mitochondria, its mitochondrial localisation would benefit from more rigorous and quantitative validation, and the proposed mechanism of the interaction with the mitochondrial translation machinery remains only partially explored. In addition, the physiological analysis is largely limited to the heart, leaving open questions about how broadly this pathway operates across tissues.

Major comments:

(1) Evidence for mitochondrial localization of EOLA1<br /> EOLA1 has previously been reported as a nuclear and cytosolic protein and is not annotated in MitoCarta 3.0, making rigorous validation of its mitochondrial localization particularly important. Although the authors provide several lines of evidence, interpretation is complicated by the use of different cell lines across localization, interaction, and functional experiments. Greater consistency in the cellular models used would strengthen the conclusions. The immunofluorescence analysis of tagged EOLA1 would also benefit from quantification across more cells and the inclusion of an additional mitochondrial marker (e.g., an outer membrane marker such as TOM20), as HSP60 staining can vary with mitochondrial state.

(2) Normalization of OCR measurements<br /> Clarification of how Seahorse oxygen consumption rate measurements were normalized (e.g., cell number or protein content) would aid interpretation, particularly given potential effects of Eola1 loss on cell growth.

(3) Linking interaction data to functional phenotypes<br /> Loss-of-function analyses are performed in mouse cell lines, whereas localization and interactome studies are conducted in human HEK293T cells. The absence of a human EOLA1 knockout model makes it difficult to directly connect the interaction data to the observed functional phenotypes. Additional validation or discussion of species conservation would improve clarity.

(4) Mechanistic interpretation of the EOLA1-TUFM-12S rRNA interaction<br /> The identification of TUFM and 12S mt-rRNA as EOLA1 interactors is an interesting finding; however, the basis for prioritizing TUFM among the many mitochondrial proteins identified in the interactome is not fully explained. Providing enrichment statistics and functional categorization of mitochondrial interactors would increase transparency. In addition, the proposed role of the ASCH domain in RNA binding would be strengthened by structure-informed or mutational analysis of the conserved RNA-binding motif.

(5) Interpretation of mitochondrial translation and protein abundance data<br /> Several assays supporting impaired mitochondrial translation would benefit from additional controls and quantification. The de novo mitochondrial translation assay (Fig. 3h) is not quantified, making it difficult to assess the magnitude and reproducibility of the effect. In addition, western blots showing reduced levels of mitochondrially encoded OXPHOS subunits (Figure 3g) lack a mitochondrial loading control (e.g., TOM20 or VDAC). Since loss of EOLA1 may affect mitochondrial mass, normalization to a mitochondrial marker is necessary. Relatedly, it would be informative to assess whether steady-state levels of mitoribosomal proteins (e.g., MRPS15, MRPL37) and nuclear-encoded OXPHOS subunits are altered upon Eola1 loss, both in knockout cell lines and in the knockout mouse.

(6) Physiological scope of the in vivo analysis<br /> The cardiac phenotype observed in the whole-body Eola1 knockout mouse is compelling, but the focus on a single tissue limits interpretation of EOLA1's broader physiological role. Examination of additional high-energy-demand tissues would help clarify whether the observed effects are heart-specific or more general. In addition, the presence of residual EOLA1 protein bands in western blots (Figure 4a) and remaining Eola1 transcripts in qRT-PCR analyses (Extended Figure 4e) from knockout tissues should be addressed. The authors should clarify whether these signals reflect incomplete knockout, alternative isoforms, antibody cross-reactivity, or technical background.

(7) Relationship to previously reported MT2A interaction<br /> Given prior reports of EOLA1 interaction with MT2A, a brief comment on whether MT2A was detected in the authors' co-immunoprecipitation experiments and how this relates to the proposed mitochondrial role would be useful.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors present the development and characterization of a pulsed ponderomotive phase plate for transmission electron microscopy (TEM). The primary goal is to overcome the long-standing challenge of generating stable, tunable phase contrast for weakly scattering biological specimens - a capability that has remained elusive despite decades of development. While the commercially available Volta Phase Plate offers phase enhancement, it suffers from a lack of control and stability. More recent efforts have focused on continuous-wave (CW) laser phase plates; however, these systems face significant practical hurdles, including extreme optical power requirements, thermal instability of mirrors, and the necessity for high-finesse optical cavities that act as diffraction gratings for the electron beam. The authors aim to demonstrate that a pulsed, free-space laser interaction can circumvent these limitations, offering a more robust path toward practically usable phase plates

Strengths:

The most significant strength of this work is the elegant use of a free-space pulsed interaction, which fundamentally simplifies the hardware requirements compared to cavity-based designs. By utilizing a high-intensity pulsed laser focus rather than a standing wave inside a resonator, the authors eliminate the need for complex locking feedback loops and avoid the thermal mirror deformation that currently limits CW systems.

Furthermore, this approach provides a critical theoretical advantage regarding image quality. Current CW cavity-based designs must grapple with the Kapitza-Dirac effect, where the standing wave creates a diffraction grating that generates unwanted "ghost images," delocalizing the signal. Recent proposals have had to resort to complex crossed-beam geometries to mitigate these artifacts. In contrast, the traveling-wave nature of the pulsed interaction described here inherently avoids the creation of a standing wave grating, thereby eliminating ghost images entirely without requiring elaborate compensation strategies.

The authors successfully demonstrate a proof-of-concept implementation, reporting a pronounced peak phase shift of approximately 430 radians and a stable angular deflection of the electron beam. The stability data, covering a 10-hour period, suggests that this approach is robust enough for data collection sessions typical in structural biology.

Weaknesses:

However, the strength of the evidence is modestly tempered by limitations in data presentation and analysis. The agreement between the experimental data and the theoretical simulation in Figure 2b is imperfect; the simulation underestimates the depth of the central signal trough. While the authors acknowledge this "muted" prediction, the discrepancy suggests that the theoretical model or the estimation of experimental parameters (such as electron beam size or laser intensity) requires refinement to fully describe the interaction.

While the authors claim stability over many hours, the data in Figure 3c reveal a significant drift in the baseline reference signal. Although attributed to a weakening electron beam, this drift complicates the reader's ability to assess the true stability of the laser-induced phase shift. A drift-corrected analysis would have provided more compelling evidence of the "stable angular kick" described.

Despite these specific weaknesses in data presentation, the work represents a fundamental step forward. The authors have effectively demonstrated that the trade-off between beam current and spatiotemporal resolution (driven by space-charge effects) can be managed to achieve significant phase modulation. By moving the field away from the tight constraints of optical cavities and toward free-space pulsed interactions, this work establishes a potentially more viable route for integrating laser phase plates into routine biological imaging workflows. This study will be of high value to biophysicists and microscopists seeking to push the boundaries of contrast in cryo-EM

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights on preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification.<br /> This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Comments on revisions:

The critiques have been taken care of appropriately.

Reviewer #2 (Public review):

Using chronic intravital two-photon imaging of calcium dynamics in meningeal macrophages in Pf4Cre:TIGRE2.0-GCaMP6 mice, the study identified heterogeneous features of perivascular and non-perivascular meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD). Analyses of calcium dynamics and blood vessels revealed a subpopulation of perivascular meningeal macrophages whose activity is coupled to behaviorally driven diameter fluctuations of their associated vessels. The analyses also investigated synchrony between different macrophage populations and revealed a role for CGRP/RAMP1 signaling in the CSD-induced increase, but not the decrease, in calcium transients.

This is a timely study at both the technical and conceptual levels, examining calcium dynamics of meningeal macrophages in vivo. The conclusions are well supported by the findings and will provide an important foundation for future research on immune cell dynamics within the meninges in vivo. The paper is well written and clearly presented.

I have only minor comments.

(1) Please indicate the formal definition of perivascular versus non-perivascular macrophages in terms of distance from the blood vessel. This information is not provided in the main text or the Methods. In addition, please explain how the meningeal vasculature was imaged in the main text.

(2) Similarly, the method used to induce acute CSD (pin prick) is not described in the main text and is only mentioned in the figure legends and Methods. Additional background on the neurobiology of acute CSD, as well as the resulting brain activity and neuroinflammatory responses, could be helpful.

Reviewer #2 (Public review):

In the current report, Sun and Colleagues sought to determine the liver-specific role that DHHC7, a DHHC palmitoyltransferase protein, plays in regulating whole-body energy balance and hepatic crosstalk with adipose tissues. The authors generated an inducible, liver-specific DHHC7 knockout mouse to determine how altered palmitoylation in hepatocytes alters hepatokine production/secretion, and in turn, systemic metabolism. The ablation of DHHC7 was found to alter the production of proteoglycan 4 (Prg4), a hepatokine previously linked to metabolic regulation. The authors propose that the change in Prg4 production is mediated by the loss of Gαi palmitoylation, due to DHHC7 ablation, thereby augmenting cAMP-PKA-CREB signaling in hepatocytes, which alleviates the 'brake' on Prg4 production. The authors further propose that Prg4 overexpression leads to excessive binding to GPR146 on adipocytes, which in turn suppresses PKA-mediated HSL activation, promoting impairments in lipolysis, leading to obesity. The report is interesting and generally well-written, but it appears to have some clear gaps in additional data that would aid in interpretation. The addition of confirmatory culture studies would be incredibly helpful for testing the hypotheses being explored. My comments, concerns, and/or suggestions are outlined below in no particular order.

(1) Figures: All data should be presented in dot-boxplot format so the reader knows how many samples were analyzed for each assay and group. n=3 for some assays/experiments is incredibly low, particularly when considering the heterogeneity in responsiveness to HFD, food intake, etc....

(2) Figure 1E-F: It is unclear when the food intake measure was performed. Mice can alter their feeding behavior based on a myriad of environmental and biological cues. It would also be interesting to show food intake data normalized to body mass over time. Mice can counterregulate anorexigenic cues by altering neuropeptide production over time. It is not clear if this is occurring in these mice, but the timing of measuring food intake is important. Additionally, the VO2 measure appears to be presented as being normalized to total body mass, when in fact, it would probably be more accurate to normalize this to lean body mass. Normalizing to total body mass provides a denominator effect due to excessive adiposity, but white fat is not as metabolically active as other high-glucose-consuming tissues. If my memory serves me right, several reports have discussed appropriate normalizations in circumstances such as this.

(3) Figure 1J-N: It is not all that surprising that fasting glucose and/or TGs were found to be similar between groups. It is well-established that mice have an incredible ability to become hyperinsulinemic in an effort to maintain euglycemia and lipid metabolism dynamics. A few relatively easy assays can be performed to glean better insights into the metabolic status of the authors' model. First, fasting insulin concentrations will be incredibly helpful. Secondly, if the authors want to tease out which adipose depot is most adversely affected by ablation, they could take an additional set of CON and KO mice, fast them for 5-6 hours, provide a bolus injection of insulin (similar to that provided during an insulin tolerance test), and then quickly harvest the animals ~15 minutes after insulin injections; followed by evaluating AKT phosphorylation. This will really tell them if these issues have impairments in insulin signaling. The gold-standard approach would be to perform a hyperinsulinemic-euglyemic clamp in the CON and KO mice. I now see GTT and ITT data, but the aforementioned assays could help provide insight.

(4) Figure 3A: This looks overexposed to me.

(5) Figures 3-4: It appears that several of these assays could be complemented with culture-based models, which would almost certainly be cleaner. The conditioned media could then be used from hepatocyte cultures to treat differentiated adipocytes.

(6) Figure 4: It is unclear how to interpret the phospho-HSL data because the fasting state can affect this readout. It needs to be made clear how the harvest was done. Moreover, insulin and glucagon were never measured, and these hormones have a significant influence over HSL activity. I suspect the KO mice have established hyperinsulinemia, which would likely affect HSL activity. This provides an example of why performing some of these experiments in a dish would make for cleaner outcomes that are easier to interpret.

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Reviewer #2 (Public review):

Summary:

The authors sought to validate the use of genetic screening pipelines that assess phenotypes in founders (F0, referred to as "crispants") obtained from CRISPR/Cas9 gene editing in 1-cell zygotes. The application of this approach in mice has generally been avoided due to concerns that results would be confounded by genetic mosaicism, but benefits to this approach include reducing animal numbers needed to achieve goals of identifying knockout phenotypes, as well as improved efficiency in the use of time and resources. The authors targeted seven genes associated with visible recessive phenotypes and observed the expected null phenotype in up to 100% of founders for each gene. Although mosaicism was common in the crispants, the various alleles were generally all functional null alleles and, in fact, some in-frame deletions with null phenotypes revealed critical functional motifs within the gene products. The rigorous data presented support using crispants to assess knockout phenotypes when guide RNAs with strong on-target and low off-target scores are used for gene editing in 1-cell mouse embryos.

Strengths:

By targeting multiple genes with existing, well-characterized mutations, the authors established a robust system for validating the analysis of crispants to assess gene function.

Cutting-edge technologies were used to carefully assess the spectrum of mutations generated.

Weaknesses:

There could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).

Reviewer #2 (Public review):

Summary:

In this manuscript, Thompson et al. investigate the impact of prior ATP exposure on later macrophage functions as a mechanism of immune training. They describe that ATP training enhances bactericidal functions which they connect to the P2x7 ATP receptor, Nlrp3 inflammasome activation, and TWIK2 K+ movement at the cell surface and subsequently at phagosomes during bacterial engulfment. This is an incremental addition to existing literature, which has previously explored how ATP alters TWIK2 and K+, and linked it to Nlrp3 activation. The novelty here is in discovering the persistence of TWIK2 change and exploring the impact this biology may have on bacterial clearance. Additional experiments could strengthen their hypothesis that the in vivo protective effect of ATP-training on bacterial clearance is mediated by alveolar macrophages.

Strengths:

The authors demonstrate three novel findings: 1) prolonged persistence of TWIK2 at the macrophage plasma membrane following ATP that can translocate to the phagosome during particle engulfment, 2) a persistent impact of ATP exposure on remodeling chromatin around nlrp3, and 3) administering mice intra-nasal ATP to 'train' lungs protects mice from otherwise fatal bacterial infection.

Weaknesses:

(1) Some methods remain unclear including the timing and method by which lung cellularity was assessed in Figure 2. It is also difficult to understand how many mice were used in experiments 1, 2 and 6 and thus how rigorous the design was. A specific number is only provided for 1D and the number of mice stated in legend and methods do not match.

(2) The study design is not entirely ideal for the authors' in vivo question. Overall, the discussion would benefit from a clear summary of study caveats, which are primarily that that 1) in vitro studies attributing ATP training-mediated bacterial killing to persistent TWIK2 relocation, K+ influx, a glycolytic metabolic shift , and epigenetic nlrp3 reprogramming were performed in BMDM or RAW cells and not primary AMs, 2) data does not eliminate the possibility that non-AM immune or non-immune cells in the lung are "trained" and responsible for ATP-mediated protection in vivo; flow data examined total lung digest which may obscure important changes in alveolar recruitment, and 3) in vivo work shows data on acute bacterial clearance but does not explore potential risks that "training" for a more responsive inflammasome may have for the severity of lung injury during infection.

(3) The is some lack of transparency on data and rigor of methods. Clear data is not provided regarding the RNA-sequencing results. Specific identities of DEGs is not provided, only one high-level pathway enrichment figure. It would also be ideal if controls were included for subcellular fractionating to confirm pure fractions and for dye microscopy to show negative background.

(4) In results describing 5A, the text states that "ATP-induced macrophage training effects, as measured by augmented bactericidal activity, were diminished in macrophages treated with protease inhibitors". However, these data are not identified significant in the figure; protease dependence can be described as a trend that supports the authors' hypothesis but should not be stated as significant data in text.

In summary, this work contains some useful data showing how ATP can train macrophages via TWIK2/Nlrp3. Revisions have significantly improved methods reporting, added some data to strengthen the conclusions, and toned down on overstatements to bring conclusions more in line with data presented. The title still overstates what the authors have actually tested, since no macrophage-specific targeting in vivo (no conditional gene deletion, macrophage depletion etc) was performed in infection studies. However, in vitro data provide clear evidence that macrophages can be trained by ATP, and through caveats remain, it is plausible that macrophage training is a key mechanism for the protection observed here in the lung.

Reviewer #2 (Public review):

Summary:

Kumar et al. aimed to assess the role of the understudied H3K115 acetylation mark, which is located in the nucleosomal core. To this end, the authors performed ChIP-seq experiments of H3K115ac in mouse embryonic stem cells as well as during differentiation into neuronal progenitor cells. Subsequent bioinformatic analyses revealed an association of H3K115ac with fragile nucleosomes at CpG island promoters, as well as with enhancers and CTCF binding sites. This is an interesting study, which provides important novel insights into the potential function of H3K115ac. However, the study is mainly descriptive, and functional experiments are missing.

Strengths:

(1) The authors present the first genome-wide profiling of H3K115ac and link this poorly characterized modification to fragile nucleosomes, CpG island promoters, enhancers, and CTCF binding sites.

(2) The study provides a valuable descriptive resource and raises intriguing hypotheses about the role of H3K115ac in chromatin regulation.

(3) The breadth of the bioinformatic analyses adds to the value of the dataset

Comments on revisions:

The authors sufficiently addressed my concerns.

Reviewer #2 (Public Review):

This an exciting study investigating the role of OXT in central nervous system (CNS) regulation of different behaviors and physiological processes. The study clearly shows the expression level of Oxt and Oxtr in different brain nuclei and regions.

Sex differences in Oxt expression are also well demonstrated.

Extensions of OXT's function in CNS regulation are sufficiently discussed.

Overall, this provides a good direction for further investigate OXT's role in CNS's regulation on different behaviors and physiological processes.

Reviewer #2 (Public Review):

The manuscript from Chang et al. taps on an important issue in olfactory perceptual plasticity, named the generalization of perceptual learning effect by training using odors. They employed a discrimination training/learning task with either binary odor mixture or odor enantiomers, and tested for post-training effect at several time intervals. Their results showed contrasting patterns of specificity (enantiomers) and transfer (odor mixtures), and the learning effect persisted at 2 weeks post-training. They demonstrated that the effect was independent of task difficulty, olfactory adaptation and gender.

Overall this was a well-controlled study and shows novel results. The strength of the study includes the consideration of odor structure and perceptual (dis)similarity and the control training condition. I have two minor issues that hope the authors could address in the next version of the manuscript.

1) The author used a binary odor mixture with a ration 7:9 or 9:11, why is this ratio chosen and used for the experiment?

2) Over the course of training, has the valence of odor (odor mixture) changed, it would be helpful to include these results in the supplements. As the author indicated in the discussion, the potential site underlying the transfer effect is the OFC, which has been found to represent odor valence previously (Anderson, Christoff et al. 2003). It would be nice to see the author replicate the results with odor/odor mixture valence (change) controlled.

Anderson, A. K., K. Christoff, I. Stappen, D. Panitz, D. G. Ghahremani, G. Glover, J. D. Gabrieli and N. Sobel (2003). "Dissociated neural representations of intensity and valence in human olfaction." Nat Neurosci 6(2): 196-202.

Reviewer #2 (Public Review):

Many tropical montane species live only within narrow elevational ranges. Rapid climate change has led to considerable interest in determining whether these narrow elevational ranges are the result of physiological specialization: if so, then warming temperatures will have direct fitness consequences. Thus far, studies of tropical montane ectotherms have often found patterns consistent with physiological specialization, while the few field studies of tropical montane birds (endotherms) have not. However, these few studies measured the thermal physiology of adult birds. The early life stages of birds may show physiological specialization, as eggs and nestlings function as ectotherms.

In this paper, Ocampo and colleagues provide the first test of the hypothesis that bird eggs are physiologically specialized to the climatic conditions of certain elevational zones. They use experiments and observations to measure water vapor conductance rates and eggshell traits in a diverse set of 197 species that live from the lowland Amazon to the high Andes. Ocampo and colleagues present two principal results: (1) High-elevation eggs lose less water over time than do low-elevation eggs, high elevations tend to be less humid than low elevations and (2) Eggshell traits do not show consistent patterns along the elevational gradient. The pattern in water loss is consistent with the hypothesis that high-elevation eggs are physiologically specialized for the slightly drier environments they experience. The finding that eggshell traits did not vary with elevation, however, means that the pattern of water loss is not driven by single eggshell traits (thicker eggshells could reduce water loss rates, as could fewer or smaller eggshell pores).

This paper represents a strong advance for two main reasons. First, it provides evidence that egg physiology varies with elevation as predicted by the hypothesis that eggs are physiologically adapted to certain climatic conditions. This means egg physiological adaptation is a factor that could influence species' elevational ranges. Second, it is a proof-of-concept study that shows it is possible to measure eggshell physiology for a large number of species in the field in order to test hypotheses. As such, it should inspire many further tests that examine adaptation in egg physiology in the context of species' distributions along environmental gradients.

There are two caveats that readers should be aware of. First, measuring these traits is difficult, and there remain questions about the efficacy of different methods. For example, the authors note that quantifying eggshell structures is very difficult, with several unresolved questions about their method of using scanning electron microscopy images to measure eggshell pores. Similarly, the authors mention that temperature variation may partially influence their main result that high-elevation eggs lose water at slower rates than low-elevation eggs (temperatures were colder for experiments at high elevations than for low elevations). Second, I regard the analyses of eggshell traits for specific families as exploratory. There are no a priori expectations for how different families might be expected to differ in their patterns. These analyses are fruitful in that they generate additional hypotheses that future work can test. However, it does mean that the statistical significance of eggshell trait relationships with elevation for specific families should be interpreted with caution.

Reviewer #2 (Public Review):

In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant revision are included below:

• The introduction should be better constructed and organized. It should be written with more connectors to present facts in a stream that flows naturally, from the broad general facts to the experimental details implemented in the manuscript. It should also discuss other similar approaches used in the literature, such as LaFleur et al. 2019, and relate in which ways these presented methods could be better.

• The scheme in Figure 4A should more clearly indicate the timings, doublings, numbers of cells, and other aspects of the experimental design.

• The volcano plot in Figure 4B is poorly informative and almost redundant. What does one make of it?

• The representation (normalized reads) of each sgRNA in the library and across multiple experiments, including their correlation, should be checked and plotted, to visualize how robust these replicates are.

• In Figure 4E, the distribution of the hit sgRNAs should be compared to all other targeting guides (instead of just to non-targeting controls). Linear density distribution plots or scatter plots of all guides are usually the best way, but there are others (for example, see Figure 4 of LaFleur et al. 2019). Ideally, each independent sgRNA for each gene in the library, as well as biological replicates, should be separately shown, with hits clearly highlighted.

• While in vivo differentiation is shown as possible with these cell lines, it is unclear whether CRISPR screenings could be performed in vivo too. Would sgRNA representation suffice for genome-wide? At least some of the new hits could be validated by testing differentiation in vivo (i.e. WASH complex).

• In the methods section, the RNA-seq analysis pipeline details are missing (versions, software for alignment, quantification, differential gene expression, and enrichment). Also, parameters for MAGeCK and MAGeCKFlute should be explicit and detailed.

• The discussion is mostly a summary of the results. It is lacking in detail and thoughtful discussion regarding novelty and impact beyond the validation of the cell line. What about potential applications? What about extending screenings to test bacterial-killing, as suggested in Figure 2? What about limitations compared to other similar methods out there? There is little discussion of such important potential matters. Also, a large part of the discussion is dedicated to discussing details about Cebpe that are all well known in the literature and add little value.

• Figure legends are typically too succinct and hard to interpret, especially for non-experts. The text should enable the figure reader to correctly interpret what is shown in each panel.

Reviewer #5 (Public review):

While the study presents an innovative and potentially impactful mRNA-based approach for addressing monogenic causes of male infertility, several significant weaknesses limit the strength of the authors' central conclusions.

First, the functional evidence for true fertility restoration remains incomplete. Although the authors convincingly demonstrate partial recovery of sperm motility, the downstream reproductive outcomes, particularly for IVF, are weak. Importantly, these concerns are shared by all three reviewers and the former Reviewing Editor, and to my eye they are both thoughtfully articulated and well warranted. The ICSI data show modest improvement, but this rescue is difficult to interpret.

In parallel, significant mechanistic questions persist regarding the unusually prolonged persistence of naked mRNA and reporter protein expression in germ cells, which is not fully reconciled with established mRNA and protein half-life biology and is supported largely by inference rather than by direct decay measurements.

Finally, although the authors have conducted additional cellular analyses, concerns about the extent and specificity of germ-cell targeting versus Sertoli-cell expression remain unresolved. Together, these issues do not negate the technical novelty of the work, but they do constrain the confidence with which the current dataset can support the authors' strongest therapeutic claims.

Reviewer #2 (Public Review):

The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do not provide rigorous or convincing data that the cells they derive are truly mature neutrophils. To the contrary, the neutrophil-like cells lack Ly6G expression and so the authors fall back on using CD11b as the primary marker for delineating neutrophils; however, CD11b is expressed by both myeloid progenitors and some premature and mature myeloid lineages that are not neutrophils. They acknowledge this shortcoming, but they make an assumption that Ly6G expression is the only way in which the cells they derive are different from primary neutrophils without presenting any evidence indicating such. The authors use only SCF during the maturation of ER-HoxB8 GMPs into leukocytes, rather than including other cytokines such as G-CSF (or use in vivo maturation) that could have better-induced differentiation and maturation into granulocytes/neutrophils. The authors did not use their transcriptional analyses to further establish that the cells they derive from ER-HoxB8 GMPs are similar/different from primary murine neutrophils. Unfortunately, this shortcoming means that all of the analyses of neutrophil-like cells derived from clonal GMPs may or may not represent the transcriptional, epigenetic, etc. profile of a true mature neutrophil. It is also not rigorously addressed whether what they call PMNs derived from clonal GMPs are a transcriptionally uniform population or if they harbor heterogeneity within the bulk population. Overall, while conceptually intriguing and in pursuit of an experimental system that would be impactful for the field, the study as performed has critical flaws.

Reviewer #2 (Public review):

Summary:

The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control. The paper in innovative, but there are issues in the writing that need to be added and corrected.

Comments on revisions:

The author has addressed my questions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single cell RNAseq analysis on bone marrow derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is, in parts, hard to read due to the use of acronyms and there are some questions about data analysis that still need to be addressed.

Comments on revisions:

Dillard et al have made several improvements to their manuscript.

(1) We previously asked the authors to determine whether any cell types were enriched for BMD-related traits since the premise of the paper is that 'many genes impacting BMD do so by influencing osteogenic differentiation or ... adipogenic differentiation'. Given the potential for the cell culture method to skew the cell type distribution non-physiologically, it is important to establish which cell types in their assay are most closely associated with BMD traits. The new CELLECT analysis and Figure 1E address this point nicely. However, it would still be nice to see the correlations between these cell types and BMD traits in the mice as this would provide independent evidence to support their physiological importance more broadly.

(2) Shortening the introduction.

(3) Addressing limitations that arise from not accounting for founder genome SNPs when aligning scRNA-seq data.

(4) The main take-away of this paper is, to us, the development of a single cell approach to studying BMD-related traits. It is encouraging that the cells post-culture appear to be representative of those pre-culture (supplemental figure 3).

However, the authors seem to have neglected several comments made by both reviewers. While we share the authors' enthusiasm for the single cell analytical approach, we do not understand their reluctance to perform further statistical tests. We feel that the following comments have still not been addressed:

(1) The manuscript still contains the following:

"To provide further support that tradeSeq-identified genes are involved in differentiation, we performed a cell type-specific expression quantitative trait locus (eQTL) analysis for each mesenchymal cell type from the 80 DO mice. We identified 563 genes (eGenes) regulated by a significant cis-eQTL in specific cell types of the BMSC-OB scRNA-seq data (Supplementary Table S14). In total, 73 eGenes were also tradeSeq-identified genes in one or more cell type boundaries along their respective trajectories (Supplementary Table S9)."

The purpose of this paragraph is to convince readers that the eGenes approach aligns with the tradeSeq approach (and that their approach can therefore be trusted). It is essential that such claims are supported by statistical reasoning. Given that it would be very simple to perform permutation/enrichment analyses to address this point, and both reviewers requested similar analyses, we do not understand the author's reluctance here. Otherwise, this section should be rewritten so that it does not imply that the identification of these genes provides support for their approach.

(2) Given that a central purpose of this manuscript is to establish a systematic workflow for identifying candidate genes, the manuscript could still benefit from more explanation as to why the authors chose to highlight Tpx2 and Fgfrl1. Tpx2 does already have a role in bone physiology through the IMPC. The authors should comment on why they did not explore Kremen1, for instance, as this gene seems important for the transition to both OB1 and 2.

A final minor comment is that it would be very helpful if the authors could indicate if the DDGs in Table 1 are also eGenes for the relevant cell type. This is much more meaningful than looking through GTEx.

Reviewer #2 (Public review):

Summary:

Xu et al. used fMRI to examine the neural correlates associated with retrieving temporal information from an external compared to internal perspective ('mental time watching' vs. 'mental time travel'). Participants first learned a fictional religious ritual composed of 15 sequential events of varying durations. They were then scanned while they either (1) judged whether a target event happened in the same part of the day as a reference event (external condition); or (2) imagined themselves carrying out the reference event and judged whether the target event occurred in the past or will occur in the future (internal condition). Behavioural data suggested that the perspective manipulation was successful: RT was positively correlated with sequential distance in the external perspective task, while a negative correlation was observed between RT and sequential distance for the internal perspective task. Neurally, the two tasks activated different regions, with the external task associated with greater activity in the supplementary motor area and supramarginal gyrus, and the internal condition with greater activity in default mode network regions. Of particular interest, only a cluster in the posterior parietal cortex demonstrated a significant interaction between perspective and sequential distance, with increased activity in this region for longer sequential distances in the external task but increased activity for shorter sequential distances in the internal task. Only a main effect of sequential distance was observed in the hippocampus head, with activity being positively correlated with sequential distance in both tasks. No regions exhibited a significant interaction between perspective and duration, although there was a main effect of duration in the hippocampus body with greater activity for longer durations, which appeared to be driven by the internal perspective condition. On the basis of these findings, the authors suggest that the hippocampus may represent event sequences allocentrically, whereas the posterior parietal cortex may process event sequences egocentrically.

Strengths:

The topic of egocentric vs. allocentric processing has been relatively under-investigated with respect to time, having traditionally been studied in the domain of space. As such, the current study is timely and has the potential to be important for our understanding of how time is represented in the brain in the service of memory. The study is well thought out and the behavioural paradigm is, in my opinion, a creative approach to tackling the authors' research question. A particular strength is the implementation of an imagination phase for the participants while learning the fictional religious ritual. This moves the paradigm beyond semantic/schema learning and is probably the best approach besides asking the participants to arduously enact and learn the different events with their exact timings in person. Importantly, the behavioural data point towards successful manipulation of internal vs. external perspective in participants, which is critical for the interpretation of the fMRI data. The use of syllable length as a sanity check for RT analyses as well as neuroimaging analyses is also much appreciated.

Suggestions:

The authors have done a commendable job addressing my previous comments. In particular, the additional analyses elucidating the potential contribution of boundary effects to the behavioural data, the impact of incorporating RT into the fMRI GLMs, and the differential contributions of RT and sequential distance to neural activity (i.e., in PPC) are valuable and strengthen the authors' interpretation of their findings.

My one remaining suggestion pertains to the potential contribution of boundary effects. While the new analyses suggest that the RT findings are driven by sequential distance and duration independent of a boundary effect (i.e., Same vs. Different factor), I'm wondering whether the same applies to the neural findings? In other words, have the authors run a GLM in which the Same vs. Different factor is incorporated alongside distance and duration?

Reviewer #2 (Public review):

The paper from Kulej et al. reports a set of tools for proteogenomic analysis of cancer proteomes. Their approach utilizes modern methods in long-read RNA sequencing to assemble a proteome database that is specific to Ewing sarcoma-derived A673 cells. To maximize proteome coverage and therefore increase the odds of detecting cancer-specific alterations at the protein level, the authors use multiple enzymes (trypsin, gluC, etc.) to digest cellular proteins and then perform multidimensional peptide fractionation. Peptide samples are then analyzed by LC-MS/MS using data-dependent and data-independent schemes on a timstof mass spectrometer. Proteogenomics is an important area of investigation for cancer research and does require new informatics tools.

The authors describe an end-to-end workflow where they claim to have optimized four different steps:

(1) Assembly of a sample-specific protein database using long-read transcriptomic data.

(2) Use of 8 different proteolytic enzymes to maximize diversity of peptides.

(3) Multiple stages of peptide fractionation using SCX and high pH rp chromatography.

(4) Utilize acquisition methods on the timstof mass spec to provide MS/MS data from single-charged peptides and multiply-charged peptides.

The authors published two earlier versions of ProteomeGenerator (versions 1 and 2) in the Journal of Proteome Research. In these earlier versions, 'ProteomeGenerator' was the set of software tools designed to integrate DNA and RNA sequencing to create a sample-specific protein database. To test the performance of each ProteomeGenerator version, the authors generated LC-MS/MS data using a combination of trypsin and LysC, then in the other paper, trypsin, LysC, and GluC. In both papers, they performed some levelof peptide fractionation prior to LC-MS/MS. They acquired LC-MS/MS data on a Thermo Q-Exactive in one paper and a Thermo Orbitrap mass spec in the other paper.

In the current paper, the primary innovation is the use of long-read sequencing to potentially improve the quality of the sample specific protein database. The other three components noted above are incremental compared to the authors' previous two papers and generally accepted practices in the field of proteomics. To note one example, the authors previously digested proteins using three enzymes and now use eight. Similarly, they are now using a timstof Bruker mass spec instead of one from Thermo. The detailed descriptions around the use of many enzymes and peptide fractionation, etc., create a very technically oriented paper, similar to or more so than the authors' earlier papers in J. Proteome Research. So, while there is enthusiasm for the use of long-read sequencing across biomedical research, the impact here for proteogenomic applications is somewhat lost with all of the technical description for experimental details that are not particularly innovative. In this respect, the report is not well matched to a broad readership.

Reviewer #2 (Public review):

Summary:

The authors tested an interesting hypothesis that white flies and planthoppers independently evolved salivary proteins to dampen plant immunity by targeting a receptor-like protein. Unlike previously reported receptor-like proteins with large ligand-binding domains, the NtRLP4 here has a malectin LRR domain. Interestingly, it also associates with the adaptor SOBIR1. While the function of this protein remains to be further explored, the authors provide strong evidence showing it's the target of salivary proteins as the insects' survival strategy.

Major points:

The authors mixed the concepts of LRR-RLPs with malectin LRR-RLPs. These are two different type of receptors. While LRR-RLPs are well studied, little is known about malectin LRR-RLPs. The authors should not simply apply the mode of function of LRR-RLPs to RLP4 which is a malectin LRR-RLP. In addition, LRR-RLPs that function as ligand-binding receptors typically possess >20 LRRs, whereas RLP4 in this work has a rather small ectodomain. It remains unclear whether it will function as a PRR.

I can't agree with the author's logic of testing uninfested plants for proving a PRR's function. The function of a pattern recognition receptor depends on perceiving the corresponding ligand. As shown by the data provided, RLP4-OE plants have altered transcriptional profile indicating activated defense, suggesting it's unlikely a PRR. An alternative explanation is needed.

More work on BAK1 will also help to clarify the ideas proposed by the authors.

Reviewer #2 (Public review):

Summary:

The authors use a postnatal mouse model of E. coli bacterial meningitis and a mouse brain endothelioma cell line combined with cell-type-specific gene deletion to study the function of endothelial TLR4, a cell surface receptor that recognizes gram positive bacterial wall components, in the local leptomeningeal (LPM) response with a focus on endothelial barrier breakdown mediated by TLR4. Single-cell transcriptional profiling and imaging studies using whole-mount preps of the LPM support that LPM endothelial, CD206+ local macrophage and LPM fibroblast and arachnoid barrier cell inflammatory response and is abrogated in endothelial-specific KO of TLR4, pointing to a role for endothelial TLR4 in local LPM response. Culture studies using Bend3.1 cells (a mouse brain endothelioma cell line) support a direct role for TLR4 in the bacteria-mediated inflammatory response and in internalization of Cldn5 via the endosomal-lysosomal pathway, resulting in loss of barrier integrity

Strengths:

The local LPM cell response in meningitis and the role of specific LPM cells in inflammation and CNS barrier breakdown have not been extensively studied, despite ample evidence for primary immune response in the meninges in human patients and in animal models. The authors employ a robust, multi-model approach using both in vivo and in vitro models with cell-type-specific knockout to study the function of TLR4 in brain endothelial cell response. The authors nicely combine functional barrier assays with IF for junctional localization in their experimental design, and they delve into potential mechanisms of Cldn5 internalization using markers of endosomal-lysosomal pathway localization. The authors also describe a new type of barrier assay using a streptavidin-coated plate upon which barrier-forming cell cultures can be placted, this could be a very useful alternative or complement to other size-selective barrier assays and presumably could work for other barrier forming cells types, likely epithelial cells.

Weaknesses:

(1) There are no measures of bacterial burden in peripheral organs, blood, in the LPM or brain in the TLR4 endothelial cKO mice. Lack of TLR4 in endothelial cells could prevent bacterial 'access' into the LPM and brain, essentially preventing meningitis and leading to a lack of inflammatory responses in the LPM-located cells simply because there is no bacteria present. Bacteremia may also be reduced, as might inflammatory responses in peripheral organs with TLR4-deficient peripheral endothelium. Bacterial counts and inflammatory measures in peripheral organs and blood are important to better understand the mechanism(s) underlying the reduced inflammatory profile in LPM cells and no LPM endothelial breakdown in the Tlr4 endothelial cKO mice. In other words, does deleting TLR4 in EC protect against the development of meningitis by somehow blocking bacteria access to the LPM (this would be supported by low or no CFU counts in infected Tlr4 endothelial cKO) or is it what the authors appear to propose in Figure 1J that TLF4 in EC is the only cell responding to the bacteria to trigger the immune cascade in the LPM? More data is needed to resolve this, as this is a major claim of the paper.

(2) The authors look at the underlying cortical response (cerebral vasculature for ICAM and immune cells) but do not use markers that could identify microglia (Iba1), the primary resident immune cell (CD206 is not useful, at this stage, in perivascular macrophages that are extremely sparse in the postnatal brain). This would be important to better study the impact on CNS resident immune cell morphological activation.

(3) The authors suggest that Cldn5 junctional localization is selectively disrupted upon bacterial exposure, mediated by TLR4 - they suggest this based on studying PECAM, GLUT-1, ZO-1 and B-catenin (all normally junction or cell surface located in cultured Bend3.1) in relationship to Cldn5 localization (normally high) - it is possibly these are also impact by bacteria exposure (maybe through different mechanisms?) - a better measure would be to use the similar cyto/PM measure they do for Cldn5 in Fig. 4D and to evaluate this or to use intensity measurements.

(4) The discussion could benefit from delving more into the prior literature on E.coli-mediated breakdown of junctions in cultured human microvascular brain endothelial cell model and critical host-pathogen interactions of the bacteria with ECs (PMID: 14593586), and how this might involve TLR4.

(5) It would be important to discuss how their results relate to earlier studies on TLR4-/- and TLR2-/- global knockout mice and protection vs vulnerability to development of meningitis (see PMCID: PMC3524395) - this paper showed that TLR4 global KO mice have increased susceptibility to die from meningitis and have much higher CFU counts in the CNS. In this manuscript and their prior work (Wang et al., 2023), this group shown that both global TLR4-/- mutants and their EC-specific KO have reduced barrier permeability, but we don't have any information about CFU or susceptibility to death from meningitis in their models.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate the mechanistic link between a Mediterranean-mimicking diet (MedDiet)-specifically the synergy between high fiber and fish oil-and its ability to suppress tumor growth. They successfully identify that this dietary combination alters the gut microbiome to favor the expansion of Bacteroides thetaiotaomicron. This bacterium metabolizes dietary tryptophan into indole-3-acetic acid (3-IAA), which then acts systemically to prevent CD8+ T-cell exhaustion.

Strengths:

The study integrates controlled dietary interventions, microbiome perturbation, metabolite profiling, and immune functional analyses into a coherent and well-organized framework, making the overall logic of the work easy to follow. The dietary design is carefully controlled, allowing clear interpretation of which broad dietary features are associated with the observed antitumor effects. The immune dependence of the phenotype is addressed using appropriate experimental approaches, and the results broadly support a role for gut microbiota-derived metabolites in shaping immune cell function. In addition, analyses of human datasets provide important context and enhance the potential relevance and usefulness of the findings for a broader research community.

Weaknesses:

While the manuscript provides strong support for a role of the microbial metabolite indole-3-acetic acid and downstream stress signaling in shaping immune cell function, the upstream mechanism by which this metabolite exerts its effects remains unresolved. In particular, the specific molecular sensor or binding target through which the metabolite acts has not been identified, and this uncertainty limits mechanistic precision. Framing this point more explicitly as an open question would help align the interpretation with the current data.

In addition, at several points, the presentation may imply that a single microbial species is uniquely responsible for the observed effects. However, the experimental evidence more directly demonstrates sufficiency under the tested conditions rather than necessity. A clearer distinction between "sufficient" and "necessary" claims would help readers better assess the generality of the findings and their applicability to more complex microbial communities.

The interpretation of the human data also warrants some caution. The diet-associated score applied to human datasets is derived from gene-expression signatures identified in mouse models and therefore represents an indirect proxy rather than a direct measure of dietary intake. Although the score correlates with clinical outcomes, it does not establish that patient survival is driven by consumption of specific dietary components such as fiber and fish oil.

Reviewer #2 (Public review):

Summary:

This study demonstrates that METTL5-mediated rRNA m⁶A1832 modification regulates tumor neoantigen generation by maintaining translational fidelity. Loss of METTL5 in tumor cells promotes immune cell infiltration into the tumor microenvironment and enhances the therapeutic efficacy of anti-PD-1 treatment, identifying a novel and potentially important target for cancer immunotherapy.

Strengths:

In murine tumor models, the authors found that Mettl5 depletion increases CD8⁺T cell infiltration and T cell receptor (TCR) repertoire diversity, and revealed a novel mechanism by which reduced ribosomal translation fidelity enhances non-canonical translation, thereby promoting the production of tumor neoantigens.

Weaknesses:

(1) While Mettl5 knockout enhances T-cell infiltration into tumors, it remains unclear whether loss of Mettl5 affects the expression of chemokines involved in immune cell recruitment.

(2) Although the authors report a significant reduction in tumor cell growth as well as tumor volume and weight, direct evidence demonstrating T-cell-mediated cytotoxicity is lacking.

Reviewer #2 (Public review):

In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistic insights, and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering (a) the recent publication by the group (Reference 17) on the role of Eomes+ cNK cells during pregnancy, (b) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and (c) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript. To this end, the following aspects should be discussed and, where applicable, experimentally addressed by the authors:

(1) The authors suggest cNK extravasation and local differentiation into iv- trNK.

Can it be estimated how much this process contributes to the trNK pool vs. a potential local proliferation of already existing trNK? How do absolute numbers of CD49a+ Eomes+ trNK change during pregnancies? (In Figure 1A, the cell numbers of CD49a+ Eomes+ trNK seem to go down dramatically between gd 6.5 and 14.5). The plot in 1B could also include absolute numbers of ILC1s and trNKs. Would recruited cNK cells compensate for a potential loss of CD49a+ Eomes+ trNK?

(2) Figure 1C: 2.5

Mio cNK cells have been transferred, but only very few cells can be detected within the uterus (concatenated FACS plot shown). What may represent the limit to generate uterine trNK out of cNK? Is the niche supporting cNK-trNK differentiation limited? Is it only a specific subset of (splenic) cNK capable of differentiating into trNK? Is gd 0.5 the optimal timepoint for the transfer? Is there continuous recruitment of cNK into the uterus and differentiation into trNK, or is it enhanced at specific timepoints of pregnancy? Could there be local proliferation of cNK-derived trNK? This could be studied by proliferation dye dilution of WT cNK cells in this transfer-setup.

(3) The authors should consider inducible Tgfbr2 deletion (e.g. with Tamoxifen-inducible Cre) to enable development of the uterine NK compartment in virgin mice and only ablate trNK differentiation during pregnancy. This could help to estimate the turnover of cNK into trNK, or to understand if constant cNK recruitment is required to form the uterine trNK compartment during pregnancy.

(4) Did the authors consider transfer of Tgfbr2-floxed Ncr1-Cre cNK in the same setup as in Fig. 1C? This experiment could confirm the requirement of Tgfbr-dependent signalling for cNK to trNK conversion during pregnancy versus effects of Tgfb signals on trNK numbers in the uterus at steady state (before pregnancy).

(5) Figures 2D/E

The authors should state that ILC1s are reduced in the virgin uterus of female Tgfbr2-floxed or Tgfb1-floxed Ncr1-Cre mice and cite the relevant work (the Ref #29 discussed in this context did not show that?). It would be helpful to include an analysis of all three uterine ILC subsets in steady state. This could help to answer the question if the cNK cell changes are pregnancy-specific or a general phenomenon in Tgfbr2-floxed Ncr1-Cre mice.

(6) Figure 2E

Please phrase more carefully about the "concomitant increase" of cNKs, since this increase is much less pronounced compared to the very strong reduction (absence) of trNKs in Tgfbr2-floxed Ncr1-Cre mice. Do the authors suggest that cNKs are halted at this stage and cannot differentiate into trNK, based on these data?

(7) Figure 3/4

Can the reduced litter size and the abnormal spiral artery formation be rescued by transfer of WT cNK into Tgfbr2-floxed Ncr1-Cre mice?

Reviewer #2 (Public review):

Summary

This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

Strength

(1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

(2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

(3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

(4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests-were conducted, and the results are clearly reported.

Comments on revisions:

The authors have addressed our comments in their revised manuscript and in their response to the reviewers. We don't have any further suggestions or comments.

Reviewer #2 (Public review):

Summary:

This article presents a neuromusculoskeletal (NMS) model of the Japanese Macaque. This model is added with a neural feedforward controller based on CPG and synergy that allows for reproducing quadrupedal and bipedal gait as well as the transition between quadrupedal and bipedal gait. The model and controller were validated using experimental data. Results were also compared to an inverted pendulum model to show that the transition between quadrupedal and bipedal in macaque is using this kind of representation for transition and stability. Overall, the article is very interesting, but it sometimes lacks clarity.

Strengths:

The results of the model present impressive results for quadrupedal, bipedal, and transition, validated by experimental data. NMS controllers based on feedforward controllers are very difficult to fine-tune.

Weaknesses:

(1) The movement regulator is not clear and should be better explained. At first, it seems that it is just a new CPG/synergy (feedforward) added, but in the methods, it seems to be a feedback controller.

(2) It is also not clear what is meant by discretizing the weight for the trigger limb from 0 to 1 (page 8).

(3) The controller is mainly using a feedforward controller, allowing only anticipatory movement. Animals are also using a reflex-based feedback controller. A controller with feedback/reflex could reduce failed attempts in training and better represent the transition.

(4) There are small typos throughout the article that should be corrected.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors. Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Reviewer #2 (Public review):

Summary:

Laurent et al. perform in vivo electrophysiological recordings in the retrosplenial cortex of rats foraging in multi-compartment environments with either identical or unique visual features. The authors characterize two types of directional signals in the area that they have previously reported: classic head direction cells anchored to the global allocentric reference frame and multi-direction cells (MDCs), which have a rotationally preserved directional field anchored to local compartments. The primary finding of this work is that MDCs seem sensitive to local environmental geometry rather than visual context. They also show that MDC tuning persists in the absence of hippocampal place field repetition, further dissociating the RSC local directional signal from the broader allocentric representation of space. A novel observation is that RSC non-directional spatial signals are anchored to the local environment, which could and should be explored further. While the data is solid and the analyses are mostly appropriate, the primary findings are incremental, and more interesting novel claims are not explored in detail or not explicitly tested.

Strengths:

The environmental manipulations clearly demonstrate that tuning is not modulated by complex visual information.

The finding that RSC two-dimensional spatial responses are stable and anchored to environmental features is novel and can be further explored in future work.

Weaknesses:

The observation that BDCs and MDCs are insensitive to visual context builds upon the author's previous work (and replicates aspects of Zhang et al., 2022) but leaves many open questions that are not addressed with the current set of experiments. Specifically, what exactly are MDCs anchoring to? The primary theory is that they anchor to environmental geometry, but there are no explicit experimental manipulations to test this theory. It is important to note that 2- and 4-compartment environments share many features, including the same cardinal axes, making any differences/similarities in these two conditions difficult to interpret.

The main finding presented with respect to BDC/MDs tuning is that they are not sensitive to visual context as manipulated by distinct visual patterns on the wall and floor in multicompartment environments. One could argue that the individual rooms are, in actuality, quite similar in low-level visual features - each possesses a large white background square visual feature on a single wall with a fixed relationship to the door(s). How can the authors rule out that i) BDC/MDC responses are modulated by these low-level features rather than geometry and/or ii) that the rats are not paying attention to any visual features at all? There is no task requiring them to indicate which room they are in. Furthermore, the doorways themselves are prominent visual features that are present in each context. It would be interesting to see if MDC/BDC tuning persisted in a square room where the number of doorways was manipulated to rule out this possibility.

A strong possibility is that the rotational symmetry of both MDCs and non-directional spatial neurons is related to i) door-related firing, 2) stereotyped movement, and 3) stereotyped directional sampling. In Supplemental Figure 8, the authors begin to address this by comparing a 'population ratemap' to a 'population speed map.' I do not think this is sufficient and is difficult to interpret. Instead, the authors should assess whether MDC and BDCs fire more at doorways and what the overlap is with the speed-modulated cells they report. Moreover, they should assess whether the spatial speed profile itself is rotationally symmetric within each session. It would also be useful to look at the confluence of the variables simultaneously using some form of regression analysis. The authors could generate a directional predictor that captures the main response property of these cells and see if it accounts for greater variability in spiking than speed or x,y position. Finally, rotationally symmetric directional sampling biases could arise from the doors being present on the same two walls in each room. The authors should assess whether MDC tuning is still present if directional sampling is randomly downsampled to match directional observations in each compartment.

Recent work has demonstrated that neurons with egocentric corner or boundary tuning are observed in RSC. The authors do not address whether egocentric tuning contributes to MDC signals. An explicit analysis of the relationship and potential overlap of MDC and egocentric populations is warranted.

Many of the MDCs presented in the main figures are not especially compelling. This includes alterations to MDC tuning in Figure 2, which is a key datapoint. The authors should show significantly more (if not all) examples of MDCs in each environment. It would similarly be useful to see all/more examples of non-directional spatially tuned neurons with rotationally symmetric firing patterns.

"One might hypothesize that specific environmental cues, such as door orientation or landmark positioning, drive these tuning shifts. However, our results argue against this interpretation. In four-room environments, each room had multiple entry points, yet MDCs never exhibited multidirectional activity within a single room."

I do not understand the logic here. Can the authors unpack this? Also, it is clear that some of the example cells have more than one peak in individual compartments. How is this quantified?

Reviewer #2 (Public review):

Summary:

The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.

Strengths:

A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.

Weaknesses:

In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.

Reviewer #2 (Public review):

Summary:

This is a very interesting paper bringing new and important information about the poorly understood rhodopsin 7 photoreceptive molecule. The very ancient origin of the gene is revealed in addition to data supporting a signaling pathway that is different from the one known for the canonical rhodopsins. Precise expression data, particularly in the optic lobe of the fly, as well as clear behavioral phenotypes in responses to light changes, make this study a strong contribution to the understanding of the still-debated function of rhodopsin 7.

Specific comments

(1) Title and abstract: Contribution of Rh7 to circadian clock regulation

(a) It is not that clear to me what rhodopsin does in terms of circadian regulation (even though its function might be circadianly regulated). The clear role in the light/dark distribution of activity might not be circadian per se, but mostly light/dark-driven, and there is no evidence here for a role in the entrainment of the clock.

(b) The authors should cite Lazopulo, which nicely shows that Rh7 has an important role in peripheral neurons to allow flies to escape from blue light (see below).

(2) Figure 2 C

The finding showing that Galphaz but not Galphaq can trigger signaling from light-excited Rh7 is a very intriguing finding to better understand Rh7 function. Since Galphaz is related to Gi/o, it would be interesting to test those, for example, by expressing RNAi with Rh7-gal4 and testing the Light-dark or light-off response behavior.

(3) Figures 3-4

The change in the locomotor activity distribution between light and dark in LD conditions provides a nice assay for Rh7 function. Since Lazopulo et al. (2019) have shown that wild-type but not Rh7 mutants do escape from blue light, it would be important to compare and discuss these LD behavior data with the Lazopulo results. Precisely, is this nighttime preference linked to blue light?

The expression data are really nice and show that Rh7 is mostly a non-retinal photoreceptor. However, the paper would be strongly reinforced by correlating this with the LD behavior. The LD phenotype should be tested in flies with Rh7 expression rescued under Rh7gal4 control (as done for the startle response). This is important to show whether the expression pattern is likely responsible for the described Rh7 function in LD. If L5 and or M11 drivers are available, they should be used to rescue Rh7? Since expression in some clock neurons is shown, the rescue experiment should also be done with a clock neuron driver.

In the same line, can the LD phenotype (or startle response phenotype of Figure 4) be restored by expressing Rh7 under ppk control, as shown for the blue light avoidance phenotype by Lazopulo et al?

Finally, the Rh7 "darkfly" rescued flies should be tested in LD.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

Reviewer #2 (Public review):

Summary:

This manuscript examines the association between atovaquone/proguanil use, zoster vaccination, toxoplasmosis serostatus and Alzheimer's Disease, using 2 databases of claims data. The manuscript is well written and concise. The major concerns about the manuscript center around the indications of atovaquone/proguanil use, which would not typically be active against toxoplasmosis at doses given, and the lack of control for potential confounders in the analysis.

Strengths:

(1) Use of 2 databases of claims data.

(2) Unbiased review of medications associated with AD, which identified zoster vaccination associated with decreased risk of AD, replicating findings from other studies.

Weaknesses:

(1) Given that atovaquone/proguanil is likely to be given to a healthy population who is able to travel, concern that there are unmeasured confounders driving the association.

(2) The dose of atovaquone in atovaquone/proguanil is unlikely to be adequate suppression of toxo (much less for treatment/elimination of toxo), raising questions about the mechanism.

(3) Unmeasured bias in the small number of people who had toxoplasma serology in the TriNetX cohort.

Reviewer #2 (Public review):

Summary:

The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

Strengths:

The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. They use of state of the art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

Weaknesses:

The results now support the conclusions;however, it is still unclear how direct the relationship is between Prmt1 and SFPQ.

Reviewer #3 (Public review):

Summary:

In this manuscript, Wang and colleagues explore factors contributing to the diversification of wtf meiotic drivers. wtf genes are autonomous, single-gene poison-antidote meiotic drivers that encode both a spore-killing poison (short isoform) and an antidote to the poison (long isoform) through alternative transcriptional initiation. There are dozens of wtf drivers present in the genomes of various yeast species, yet the evolutionary forces driving their diversification remain largely unknown. This manuscript is written in a straightforward and effective manner, and the analyses and experiments are easy to follow and interpret. While I find the research question interesting and the experiments persuasive, they do not provide any deeper mechanistic understanding of this gene family.

Revision update:

Having read the response to the reviewers, I believe the major issues have been addressed. However, I would strongly suggest toning down the claim regarding the chimeric WTF element in the abstract, which currently reads

"As proof-of-principle, we generate a novel meiotic driver through artificial recombination between wtf drivers, and its encoded poison cannot be detoxified by the antidotes encoded by their parental wtf genes but can be detoxified by its own antidote."

As the author reports in their response, despite various attempts, it was not possible to show that this chimeric WTF element was indeed capable of meiotic drive in a natural context (not transgenic overexpression experiment). thus the authors should not claim they generated "a novel meiotic driver"

Strengths:

(1) The authors present a comprehensive compendium and analysis of the evolutionary relationships among wtf genes across 21 strains of S. pombe

(2) The authors found that a synthetic chimeric wtf gene, combining exons 1-5 of wtf23 and exon 6 of wtf18, behaves like a meiotic driver that could only be rescued by the chimeric antidote but neither of the parental antidotes. This is a very interesting observation that could account for their inception and diversification.

Weaknesses:

(1) Deletion strains

The authors separately deleted all 25 Wtf genes in the S. pombe ference strain. Next, the authors performed spot assay to evaluate the effect of wtf gene knockout on the yeast growth. They report no difference to the WT and conclude that the wtf genes might be largely neutral to the fitness of their carriers in the asexual life cycle at least in normal growth condition.

The authors could have conducted additional quantitative growth assays in yeast, such as growth curves or competition assays, which would have allowed them to detect subtle fitness effects that cannot be quantified with a spot assay. Furthermore, the authors do not rule out simpler explanations, such as genetic redundancy. This could have been addressed by crossing mutants of closely related paralogs or editing multiple wtf genes in the same genetic background.

Another concern is the lack of detailed information about the 25 knockout strains used in the study. There is no information provided on how these strains were generated or, more importantly, validated. Many of these wtf genes have close paralogs and are flanked by repetitive regions, which could complicate the generation of such deletion strains. As currently presented, these results would be difficult to replicate in other labs due to insufficient methodological details

Revision update:

The authors measured the fitness of the deletion strains using growth curves (Fig. 2C and D) and no significant differences were found, further supporting their claims. The requested information (details on the generation of the deletion strains) is now available in the methods section.

(2) Lack of controls

The authors found that a synthetic chimeric wtf gene, constructed by combining exons 1-5 of wtf23 and exon 6 of wtf18, behaves as a meiotic driver that can be rescued only by its corresponding chimeric antidote, but not by either of the parental antidotes (Figure 4F). In contrast, three other chimeric wtf genes did not display this property (Figure 4C-E). No additional experiments were conducted to explain these differences, and basic control experiments, such as verifying the expression of the chimeric constructs, were not performed to rule out trivial explanations. This should be at the very least discussed. Also, it would have been better to test additional chimeras.

Revision update:

The authors report that the expression of the construct was measured. However, they do not make reference to any specific figure or section of the main text. It would be very useful if the authors explicitly referenced where exactly changes were made (this is true for all changed made)

(3) Statistical analyses

In line 130 the authors state that: "Given complex phylogenetic mixing observed among wtf genes (Figure 1E), we tested whether recombination occurred. We detected signals of recombination in the 25 wtf genes of the S. pombe reference genome (p = 0) and in the wtf genes of the 21 S. pombe strains (p = 0) using pairwise homoplasy index (HPI) test. "<br /> Reporting a p-value of 0 is not appropriate. Please report exact P-values.

Revision update:

This has been addressed.

Reviewer #2 (Public review):

Summary:

Tian et al. explore the developmental origins of cortical reorganization in blindness. Previous work has found that a set of regions in the occipital cortex show different functional responses and patterns of functional correlations in blind vs. sighted adults. Here, Tian et al. explore how this organisation arises over development, asking whether the infant brain looks more like the blind adult pattern, or more like the sighted adult pattern. Their analyses reveal that the answer depends on the particular networks investigated. Some functional connections in infants look more like blind than sighted adults; other functional connections look more like sighted than blind adults; and others fall somewhere in the middle, or show an altogether different pattern in infants compared with both sighted and blind adults.

Strengths:

The paper addresses very important questions about the "starting state" in the developing visual cortex, and how cortical networks are shaped by experience. Another clear strength lies in the unequivocal nature of many results. Many results have very large effect sizes, critical interactions between regions and groups are tested and found, and infant analyses are replicated in split halves of the data.

Weaknesses:

While potential roles of experience (e.g., visual, cross-modal) are discussed in detail, little consideration is given to the role of experience-independent maturation. The infants scanned are extremely young, only 2 weeks old. It is possible that the sighted adult pattern may still emerge later in infancy or childhood, regardless of infant visual experience. If so, the blind adult pattern may depend on blindness-related experience only (which may or may not reflect "visual" experience per se). In short, it is not clear that the age range studied is a clear-cut "starting point" for development, after which all change can be attributed to experience.

Reviewer #2 (Public review):

Summary:

Lesser et al. present an atlas of Drosophila wing sensory neurons. They proofread the axons of all sensory neurons in the wing nerve of an existing electron microscopy dataset, the female adult fly nerve cord (FANC) connectome. These reconstructed sensory axons were linked with light microscopy images of full-scale morphology to identify their origin in the periphery of the wing and encoded sensory modalities. The authors described the morphology and postsynaptic targets of proprioceptive neurons as well as previously unknown sensory neurons.

Strengths:

The authors present a valuable catalogue of wing sensory neurons, including previously undescribed sensory axons in the Drosophila wing. By providing both connectivity information with linked genetic drive lines, this research facilitates future work on the wing motor-sensory network and applications relating to Drosophila flight. The findings were linked to previous research as well as their putative role in the proprioceptive and nerve cord circuitry, providing testable hypotheses for future studies.

Weaknesses:

With future use as an atlas, it should be noted that the evidence is based on sensory neurons on only one side of the nerve cord. Fruit flies have stereotyped left/right hemispheres in the brain and left/right hemisegments in the nerve cord. Comparison of left and right neurons of the nervous system can give a sense of how robust the morphological and connectivity findings are. Unfortunately, this dataset has damage to the right side, making such comparisons unreliable.

Reviewer #3 (Public review):

This paper addresses, through experiment and simulation, the combined effects of bacterial circular swimming near no-slip surfaces and chemotaxis in simple linear gradients. The authors have constructed a microfluidic device in which a gradient of L-aspartate is established, to which bacteria respond while swimming while confined in channels of different widths. There is a clear effect that the chemotactic drift velocity reaches a maximum in channel widths of about 8 microns, similar in size to the circular orbits that would prevail in the absence of side walls. Numerical studies of simplified models confirm this connection.

The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.<br /> The authors have included a useful intuitive explanation of their results via a geometric model of the trajectories. In future work it would be interesting to analyze further the voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, this might help understand how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis.

The authors argue that these findings may have relevance to a number of physiological and ecological contexts. As these would be characterized by significant heterogeneity in pore sizes and geometries, further work will be necessary to translate the present results to those situations.

Reviewer #2 (Public review):

The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. Overall, the authors conclude that microtubule lattice "expanders" promote probe binding, suggesting that the CTT is generally more accessible under these conditions. Consistent with this, detyrosination is enhanced. Mechanistically, molecular dynamics simulations indicate that the CTT may interact with the microtubule lattice at several sites, and that these interactions are affected by the tubulin nucleotide state.

Strengths and weaknesses:

Key strengths of the work include the use of three distinct probes that yield broadly consistent findings, and a wide variety of experimental manipulations (drugs, motors, MAPs) that collectively support the authors' conclusions, alongside a careful quantitative approach.

The challenges of studying the dynamics of a short, intrinsically disordered protein region within the complex environment of the cellular microtubule lattice, amid numerous other binders and regulators, should not be understated. While it is very plausible that the probes report on CTT accessibility as proposed, the possibility of confounding factors (e.g., effects on MAP or motor binding) cannot be ruled out. Sensitivity to the expression level clearly introduces additional complications. Likewise, for each individual "expander" or "compactor" manipulation, one must consider indirect consequences (e.g., masking of binding sites) in addition to direct effects on the lattice; however, this risk is mitigated by the collective observations all pointing in the same direction.

The discussion does a good job of placing the findings in context and acknowledging relevant caveats and limitations. Overall, this study introduces an interesting and provocative concept, well supported by experimental data, and provides a strong foundation for future work. This will be a valuable contribution to the field.

Reviewer #2 (Public review):

Summary:

In this work, the authors applied a range of computational methods to probe the translocation of cholesterol through the Smoothened receptor. They test whether cholesterol is more likely to enter the receptor straight from the outer leaflet of membrane or via a binding pathway in the inner leaflet first. Their data reveal that both pathways are plausible but that the free energy barriers of pathway 1 is lower suggesting this route is preferable. They also probe the pathway of cholesterol transport from the transmembrane region to the cysteine-rich domain (CRD).

Strengths:

A wide range of computational techniques are used, including potential of mean force calculations, adaptative sampling, dimensionality reduction using tICA, and MSM modelling. These are all applied in a rigorous manner and the data are very convincing. The computational work is an exemplar of a well-carried out study.

Their computational predictions are experimentally supported using mutagenesis, with an excellent agreement between their PMF and mRNA fold change data.

The data are described clearly and coherently, with excellent use of figures. They combine their findings into a mechanism for cholesterol transport, which on the whole seems sound.

Their methods are described well, and much of their analysis methods have been made available via GitHub, which is an additional strength.

Reviewer #2 (Public review):

This is an ambitious and technically powerful study, investigating a long-standing question about the functional organization of area V4. The project combined large-scale single-unit electrophysiology in macaque V4 with deep learning-based activation maximization to characterize neuronal tuning in natural image space. The authors built predictive encoding models for V4 neurons and used these models to synthesize most exciting images (MEIs), which are subsequently validated in vivo using a closed-loop experimental paradigm.

Overall, the manuscript advances three main claims:

(1) Individual V4 neurons showed complex and highly structured selectivity for naturalistic visual features, including textures, curvatures, repeating patterns, and apparently eye-like motifs.

(2) Neurons recorded along the same linear probe penetration tended to have more similar MEIs than neurons recorded at different cortical locations (this similarity was supported by human psychophysics and by distances in a learned, contrastive image embedding space).

(3) MEIs clustered into a limited number of functional groups that resembled feature visualizations observed in deep convolutional neural networks.

Strengths:

(1) The study is important in that it is the first to apply activation maximization to neurons sampled at such fine spatial resolution. The authors used 32-channel linear silicon probes, spanning approximately 2 mm of cortical depth, with inter-contact spacing of roughly 60 µm. This enabled fine sampling across most of the cortical thickness of V4, substantially finer resolution than prior Utah-array or surface-biased approaches.

(2) A key strength is the direct in vivo validation of model-derived synthetic images by stimulating the same neurons used to build the models, a critical step often absent in other neural network-based encoding studies.

(3) More broadly, the study highlights the value of probing neuronal selectivity with rich, naturalistic stimulus spaces rather than relying exclusively on oversimplified stimuli such as Gabors.

Weaknesses:

(1) A central claim is that neurons sampled within the same penetration shared MEI tuning properties compared to neurons sampled in different penetrations because of functional organization. I am concerned about technical correlations in activity due to technical or methodology-related approaches (for example, shared reference or grounding) instead of functional organization alone. These recordings were obtained with linear silicon probes, and there have been observations that neuronal activity along this type of probe (including neuropixels probes) may be correlated above what prior work showed, using manually advanced single electrodes. For example, Fujita et al. (1992) showed finer micro-domains and systematic changes in selectivity along a cortical penetration, and it is not clear if that is true or detectable here. I think that the manuscript would be strengthened by a more thorough and explicit characterization of lower-level response correlations (at the neuronal electrophysiology level) prior to starting with fitting models. In particular, the authors could examine noise correlations along the electrode shaft (using the repeated test images, for example), as well as signal correlations in tuning, both within and across sessions. It would also be helpful to clarify whether these correlations depended on penetration day, recording chamber hole (how many were used?), or spatial separation between penetrations, and whether repeated use of the same hole yielded stable or changing correlations. Illustrations of the peristimulus time histogram changes across the shaft and across penetrations would also help. All of this would help us understand if the reports of clustering were technically inevitable due to the technique.

(2) It is difficult to understand a story of visual cortex neurons without more information about their receptive field locations and widths, particularly given that the stimulus was full-screen. I understand that there was a sparse random dot stimulus used to find the population RF, so it should be possible to visualize the individual and population RFs. Also, the investigators inferred the locations of the important patches using a masking algorithm, but where were those masks relative to the retinal image, and how distributed were they as a function of the shaft location? This would help us understand how similar each contact was.

(3) A major claim is that V4 MEIs formed groups that were comparable to those produced by artificial vision systems, "suggesting potential shared encoding strategies." The issue is that the "shared encoding strategy" might be the authors' use of this same class of models in the first place. It would be useful to know if different functional groups arise as a function of other encoding neural network models, beyond the robust-trained ResNet-50. I am unsure to what extent the reported clustering, depth-wise similarity, and correspondence to artificial features depended on architectural and training bias. It would substantially strengthen the manuscript to test whether a similar organizational structure would emerge using alternative encoding models, such as attention-based vision transformers, self-supervised visual representations, or other non-convolutional architectures. Another important point of contrast would be to examine the functional groups encoded by the ResNet architecture before its activations were fit to V4 neuronal activity: put simply, is ResNet just re-stating what it already knows?

(4) Several comparisons to prior work are presented largely at a qualitative level, without quantitative support. For example, the authors state that their MEIs are consistent with known tuning properties of macaque V4, such as selectivity for shape, curvature, and texture. However, this claim is not supported by explicit image analyses or metrics that would substantiate these correspondences beyond appeal to visual inspection. Incorporating quantitative analyses, for instance, measures of curvature, texture statistics, or comparisons to established stimulus sets, would strengthen these links to prior literature and clarify the relationship between the synthesized MEIs and previously characterized V4 tuning properties.

Reviewer #2 (Public review):

In this paper, the authors investigate the role of the cerebellum in song production in the zebra finch. First, they replicate prior studies to show that lesions of the lateral deep cerebellar nuclei (latDCN, primarily lobules IV-VII and IX) result in shorter duration syllables and song motifs than sham controls. The authors then record neural activity from the cerebellum during both passive auditory exposure in anesthetized birds and in freely singing animals. The authors claim that across multiple lobules, the cerebellum receives "non-selective" auditory inputs locked to syllable boundaries (based on acute recordings) and that cerebellar neurons display song-locked responses that are unaffected by auditory feedback perturbations (in chronic recordings). Moreover, the authors emphasized the distinct properties of lobule IV, which they argue is tightly locked to the onset and offset of syllables, and conclude that the cerebellum might contribute to the duration of song elements.

This paper presents novel and useful descriptions of song-related neural activity in the cerebellum. However, there are multiple serious issues. First, there are major issues with the design and presentation of the analysis of the electrophysiological data; based on these, it is unclear whether the authors are justified in some of their conclusions about neural tuning or are entitled to any of their claims about the specific tuning or function of neurons in particular lobules. Second, because the authors' conceptual framework seems to ignore possible non-auditory inputs to the cerebellum, their results on (minimal) effects of auditory manipulation during singing are over-interpreted with respect to providing evidence of a forward model. Third, the paper's central assertion - that the songbird cerebellum may contribute to the duration of vocal events during song - was firmly established by a prior lesion study (Radic et al., 2024). Although the authors do cite this prior study with respect to longer-term postlesion changes after cerebellar lesions, this paper also showed a large change in syllable duration immediately after cerebellar lesion (Figure 5 in Radic et al). The electrophysiological results in the present paper could provide valuable insights into the neural mechanisms underlying this already-described role of the songbird cerebellum; however, given the other concerns above, it is not clear that the authors have done so.

Reviewer #2 (Public review):

Summary:

The manuscript describes a combined computational and experimental approach to investigate the ABHD5 binding to and insertion into membranes.

Strengths:

Mutational experiments support computational findings obtained on ABHD5 membrane insertion with enhanced-sampling atomistic simulations.

Weaknesses:

While the addressed problem is interesting, I have several concerns, which fall into two categories:

(A) I see statements throughout the manuscript, e.g. on PNPLA activation, that are not supported by the results.

(B) The presentation of the computational and experimental results lacks in part clarity and detail.

Comments and questions on (A):

(1) I think the following statements in the abstract, which go beyond ABHD5 membrane binding, are not supported by the presented data:

the addition "to control lipolytic activation" in the 3rd sentence of the abstract.

further below ".... transforming ABHD5 into an active and membrane-localized regulator".

(2) The authors state in the Introduction (page numbers and line numbers are missing to be more specific):

"We hypothesize that binding of ABHD5 alters the nanoscale chemical and biophysical properties of the LD monolayer, which, combined with direct protein-protein interactions, enables PNPLA paralogs to access membrane-restricted substrates. This regulatory mechanism represents a paradigm shift from conventional enzyme-substrate interactions to sophisticated allosteric control systems that operate at membrane interfaces."

This hypothesis and the suggested paradigm shift are not supported by the data. Protein-protein interactions are not considered. What is meant by "sophisticated allosteric control"?

(3) The authors state in the Results section:

"We hypothesize that this TAG nanodomain is critical for ABHD5-activated TAG hydrolysis by PNPLA2." In previous pages, the authors state the location of the nanodomain: "TAG nanodomain under ABHD5".

If the nanodomain is located under ABHD5, how can it be accessible to PNPLA2? To my understanding, ABHD5 then sterically blocks access of PNPLA2 to the TAG nandomain.

(4) Another statement: "Our findings suggest that ABHD5-mediated membrane remodeling regulates lipolysis in part by regulating PNPLA2 access to its TAG substrate."

I don't see how the reported results support this statement (see point 3 above).

Comments and questions on (B):

(1) The authors state that the GaMD simulations started "from varying conformations observed during CGMD".

What is missing is a clear description of the CGMD simulation conformations, and the CG simulations as a whole, prior to the results section on GaMD. The authors use standard secondary and tertiary constraints in the Martini CG simulations. Do the authors observe some (constrained) conformational changes of ABHD5 already in the CG simulations (depending on the strength of the constraints)? Or do the conformational changes occur exclusively in the GaMD simulations? Both are fine, but this needs to be described.

(2) The authors write: "Three replicas of GaMD were performed."

Do these replicas lead to similar, or statistically identical, membrane-bound ABHD5 conformations? Is this information, i.e. a statistical analysis of differences in the replica runs, already included in the manuscript?

(3) The authors state on the hydrogen exchange results:

"HDX-MS provided orthogonal experimental evidence for the dynamics of the lid. In solution, a peptide (residues 200-226) spanning the lid helix displayed a bimodal isotopic distribution (Fig. S4), indicating the coexistence of different conformations. Upon LD binding, this distribution shifted to a single, low-exchange peak, demonstrating stabilization of the membrane-bound conformation with reduced solvent accessibility. These experimental observations corroborate our MD simulations."

I find this far too short to be understandable. Also, there are no computational results of ABHD5 in solution that show a bimodal conformational distribution of the lid helix, which is observed in the hydrogen exchange experiments. Which aspects of the MD simulations are corroborated?

Reviewer #2 (Public review):

Summary:

This manuscript presents the "NoSeMaze", a novel automated platform for studying social behavior and cognitive performance in group-housed male mice. The authors report that mice form robust, transitive dominance hierarchies in this environment and that individual social rank remains largely stable across multiple group compositions. They further demonstrate that social dominance and aggressive behaviors, like chasing, are partially dissociable and that dominance traits are independent of non-social cognitive performance. The study includes a genetic manipulation of oxytocin receptor expression in the anterior olfactory nucleus, which showed only transient effects on social rank.

Strengths:

(1) Innovative Methodology:<br /> The NoSeMaze platform is a technically elegant and conceptually well-integrated system that enables fully automated, long-term monitoring of both social and cognitive behaviors in large groups of group-housed mice. It combines tube-test-like dominance contests, voluntary chase-escape interactions, and an embedded operant olfactory discrimination task within a single, ethologically relevant environment. This modular design allows for high-throughput, minimally invasive behavioral assessment without the need for repeated handling or artificial isolation.

(2) Experimental Scale and Rigor:<br /> The study includes 79 male mice and over 4,000 mouse-days of observation across multiple group reshufflings. The use of RFID-based identification, automated data logging, and longitudinal design enables robust quantification of individual trait stability and group-level social structure.

(3) Multidimensional Behavioral Profiling:<br /> The integration of social (tube dominance, proactive chasing), physical (body weight), and cognitive (olfactory learning task) measures offers a rich, multi-dimensional profile of each individual mouse. The authors' finding that social dominance traits and non-social cognitive performance are largely uncorrelated reinforces emerging models of orthogonal behavioral trait axes or "animal personalities".

(4) Clarity and Data Analysis:<br /> The analytical framework is well-suited to the study's complexity, with appropriate use of dominance metrics, mixed-effects models, and permutation tests. The analyses are clearly explained, statistically rigorous, and supported by transparent supplementary materials.

Weaknesses:

(1) Conceptual Novelty and Prior Work:<br /> While the study is carefully executed and methodologically innovative, several of its core findings reaffirm concepts already established in the literature. The emergence of stable, transitive social hierarchies, the persistence of individual differences in social behavior, and the presence of non-despotic social structures have all been previously reported in mice, including under semi-naturalistic conditions (e.g., Fan et al., 2019; Forkosh et al., 2019). Although this work extends those findings with greater behavioral resolution and scale, the manuscript would benefit from a clearer articulation of what is genuinely novel at the conceptual level, beyond the technological advance.

(2) Role of OXTR Deletion:<br /> The inclusion of the OXTR manipulation feels somewhat disconnected from the manuscript's central aims. The effects were minimal and transient, and the authors defer full interpretation to a separate study.

(3) Scope Limitations (Sex and Age):<br /> The study is limited to male mice, and although this is acknowledged, the title and overall framing imply broader generalizability. This sex-specific focus represents a common but problematic bias. Additionally, results from the older mouse cohort are under-discussed; if age had no effect, this should be explicitly stated.

(4) Ambiguity of Dominance as a Construct:<br /> While the study robustly quantifies social rank and hierarchy structure, the broader functional meaning of "dominance" remains unclear. As in prior work (e.g., Varholick et al., 2019), dominance rank here shows only weak associations with physical attributes (e.g., body weight), cognitive strategy, or neuromodulatory manipulation (OXTR deletion). This recurring pattern, where rank metrics are reliably established yet poorly predictive of other behavioral or biological traits, raises important questions about what such measures actually capture. In particular, it challenges the assumption that outcomes in paradigms like the tube test or chase frequency necessarily reflect dominance per se, rather than other constructs.

Reviewer #2 (Public review):

Summary:

In this study, the authors were testing the hypothesis that hemagglutination inhibition antibody titers, measured later in life, might be higher against influenza viruses that belong to the same hemagglutinin classification group as the influenza virus that a person was likely first exposed to early in life. This is one conceptualization of a phenomenon termed immune imprinting, which may explain previously observed differences in susceptibility to severe influenza infection between cohorts that were likely first exposed to different hemagglutinin groups. The results of the analysis provide some support for this analysis. However, support for the hypothesis is not consistently observed across sensitivity analyses, and a simulation study finds that antibody patterns consistent with immune imprinting may arise due to other factors in the absence of true imprinting effects. Therefore, overall support for the hypothesis is weak. Nonetheless, this study is important in that it provides guidance and has developed an analytic methodology for additional studies in this area of research. These findings and methods may also be useful for other infectious diseases for which patterns consistent with immune imprinting have been observed.

Strengths:

The strengths of this study include the relatively large cohort data source with broad age representation, rigorous statistical methods, and the use of sensitivity and simulation analyses to assess the robustness of the results.

Weaknesses:

The model outcome includes antibody titers measured against many different viruses, and the imprinting parameter was defined at the subtype level. This may obscure specific imprinting effects related to finer structural similarities between first and subsequent virus exposures. This analysis focuses only on one component of the immune response to influenza; immune imprinting may also involve other immune mechanisms. The analysis was carried out in a Chinese cohort, and vaccination status of the cohort is not discussed; the results may not be generalizable to other populations, particularly if vaccination patterns differ.