Reviewer #1 (Public review):

Summary:

Knowing that small pupil-size variations accompany brightness variations (even when these are illusory), the authors asked whether pupil constrictions would accompany the synesthetic perception of a brighter color (compared with a darker one), induced by the presentation of a black-white character. This grapheme-colour synesthesia is only experienced by few participants, sixteen of whom were enrolled in this study. The results reliably showed that a relative pupil constriction would "betray" the perception of a brighter color in these participants, while no such effect would be observed in control participants who were asked to report a color in association with each grapheme, even though they did not perceive any.

Strengths:

The main strength of the study lays in its combination of psychophysics (brightness ratings) and pupillometry, which allowed for showing clear-cut results.

Weaknesses:

I only see the following relatively minor weaknesses, namely:

- The pupil traces in Figure3 (main results) are heavily pre-processed (per-participant demeaned), loosing any feature besides the effect of interest. As I argued in my first review, I worry that this format gives unrealistic expectations about the effect (the perception of dark/bright colors do not generate a net dilation/constriction of the pupil; perception-related modulations of pupil size are always relative and generally small compared to the numerous other effects registered in pupil size; these include a pupil dilation that is more prominent in the controls and that gets analyzed later on in the manuscript; I do not think that eliminating one of the effects of interests from a main results figure helps the reader understand the results). In the revised manuscript, the authors addressed this concern by adding a Supplementary Figure 4, where a more complete representation of the results is shown (traces from individual trials are baseline corrected and averaged, resulting in more informative timecourses). I would strongly recommend that Supplementary Figure4 is brought to the main text (Figure3 could be presented in Supplementary).

- Responses to physical brightness modulations were only measured in the synesthethes group, not in controls. The authors point out that pupillary light responses have been thoroughly characterized in previous studies, and conclude that synesthethes' responses were in line with the expectations both in terms of amplitude and latency. However, as we are not dealing with standardized measurements, subtle differences in pupil reactivity across the two populations remain a possibility. I recommend that this possibility is mentioned in the discussion.

Impact:

This work is likely to improve our understanding of synesthesia, providing a new tool to quantify the subjective sensations; an interesting potential extension would be using pupillometry for tracking changes over time of the synesthetic experiences, opening up the possibility to evaluate the importance of learning for this peculiar experience.

Reviewer #1 (Public review):

This work compiles a comprehensive atlas of ncORFs across mammalian tissues and cell types, derived from reanalysis of ~400 public ribosome profiling datasets. The authors then evaluate cross-species conservation and functional signatures, proposing that evolutionarily ancient ncORFs tend to have higher translation potential, stronger expression, and closer relationships with canonical coding sequences.

Strengths:

In general, the study provides a large-scale and timely resource of annotated ncORFs, which could be broadly useful for the community. The authors collected ~400 public ribosome profiling datasets for annotations of ncORFs, which, to my best knowledge, is the largest collection of data for such purpose. The catalog could facilitate future investigations into ncORF biology and broaden understanding of the coding potential of the "non-coding" genome.

Weaknesses:

Based on the ncORF catalog, some of the analyses were not properly done. Some of the results are descriptive.

(1) Bias and representations of data source. Public ribo-seq datasets are unevenly distributed across tissues and cell lines, raising concerns about heterogeneity and underrepresentation of certain contexts. This may limit the generalizability of the catalog.

(2) The discussion on modular domains of ncORFs is unclear, and the claim that they may originate via TE-related mechanisms is not well supported. Stronger evidence or clearer reasoning is needed.

(3) The conservation comparisons are not fully convincing. Figure S7 shows only mild differences between ncORFs and CDS, and statistical significance is not clearly demonstrated. Comparisons with other non-coding RNAs should be added, and overlapping sequences between ncORFs and CDS should be excluded to avoid bias.

(4) Figure 3 indicates that some ncORFs are subject to evolutionary constraints. This is not surprising. The authors should provide further analyses on more detailed features of these "conserved" ncORFs vs. the "non-conserved" ones. Some pretty informative works have been done in drosophila, worms, mouse, and human. Figure 3 suggests some ncORFs are under evolutionary constraint, but this is not unexpected. More granular analyses contrasting "conserved" versus "non-conserved" ncORFs would be informative. In fact, small ORFs, especially uORFs, have been extensively studied, for their functions and corss-species conservations. The authors should explicitly show what is new here in their analyses.

(5) Translation levels are reported using RPF counts. However, translation efficiency (normalized by RNA expression) is a more appropriate measure to account for expression heterogeneity.

(6) The correlation analyses between ncORF translation levels and PhyloCSF are confusing and largely descriptive. These sections need sharper framing and clearer conclusions.

(7) Public ribo-seq datasets, generated by different research labs, are known for their strong batch effects. Representations of tissues and cells are also very unbalanced. Therefore, the co-translation analysis between ncORFs and canonical CDS is not well controlled. This should be done by referring to a recent large-scale ribo-seq meta-analysis (Nat Biotechnol. 2025. doi: 10.1038/s41587-025-02718-5).

Comments on revisions:

The authors have made efforts to address most of the previous concerns, and several points have been clarified or improved in the revision. However, in a number of cases, the responses rely more on acknowledgment and reframing rather than substantive analytical strengthening. Overall, the manuscript is improved, particularly in terms of clarity, transparency, and positioning of claims. I support its publication and look forward to seeing how the field engages with and discusses these claims.

Reviewer #1 (Public review):

Summary:

Meijer et al. sought to investigate the role of cortical layer 6b (L6b) neurons in modulating sleep-wake states and cortical oscillations under baseline and sleep deprived conditions and in response to orexin A and B. Using chronic EEG recordings in mice with silencing of Drd1a+ neurons (via constitutive Cre-dependent knockout of SNAP25), the authors report that while overall baseline sleep-wake architecture and response to sleep deprivation are minimal/unchanged, "L6b silencing leads" to a slowing of theta activity during wakefulness and REM sleep, and a reduction in EEG power during NREM sleep. The manuscript is well written with clarity and transparency. Although Drd1a+ neurons are not exclusive to L6b, the authors describe key future studies to identify a causal role for L6b neurons in brain state regulation. These studies contribute to a growing body of evidence that cortex-in addition to subcortical brain regions-plays a role in brain state regulation.

Strengths:

(1) The text is well written.

(2) The authors are transparent about methodological details and study limitations.

(3) The stated sleep, circadian, and orexin infusion experiments are well designed, executed, and analyzed.

Weaknesses:

(1) Outcomes are attributed to silencing cortical L6b neurons, but the genetic manipulation is not specific to L6b neurons or cortex. The authors acknowledge this as a limitation and offer targets for future studies to identify L6b neuron-specific contributions to stated outcomes that include spatially restricted manipulations.

(2) Experiments use only male mice, which limits generalizability to females.

Comments on revised version:

The authors took great care in addressing my previous comments, and I do not have any additional concerns.

Reviewer #1 (Public review):

Summary:

This manuscript explores the role of the Evening Complex (EC), specifically focusing on ELF3, a disordered protein component of the EC, and its temperature-dependent phase behavior. The study highlights the role of polyQ tracts in modulating temperature-sensitive condensate formation and provides a combination of computational approaches, including REST2 simulations and coarse-grained Martini simulations, to investigate how polyQ tract length and sequence context influence this behavior.

Strengths:

The study addresses a key question in plant biology - how temperature influences circadian clock-mediated growth regulation through protein phase behavior. The manuscript introduces the novel finding that polyQ tract length modulates the temperature-dependent formation of helices and condensates.

Weaknesses:

(1) Coarse-Grained Simulation Results Not Supported by Data:

The results presented in Figure 6A of the manuscript do not seem to show a clear trend in the number of clusters formed as a function of polyQ tract length. This is particularly evident in the comparison between 0Q and 7Q polyQ lengths, which display statistically similar values in terms of the number of clusters. The lack of distinction between these values raises questions about the sensitivity of the coarse-grained simulations to polyQ tract length, which the authors claim as a key modulator of condensate formation. This discrepancy weakens the argument that polyQ length directly impacts the clustering behavior in the simulations.

Suggested Analysis:

a) A more detailed statistical analysis should be performed to assess whether the observed differences between polyQ lengths are significant. This could involve hypothesis testing or the use of error bars in the graphs to better communicate the variability in the data.

b) Additionally, the authors should examine whether there are other features, such as cluster shape or internal structure, that might differentiate between different polyQ lengths, even if the total number of clusters is similar.

(2) Inconsistency in Cluster Size Across Temperatures (Figure 6B):

The results in Figure 6B show a striking difference in the size of the largest cluster between temperatures of 290K and 300K. This abrupt shift in behavior lacks a clear mechanistic explanation. Typically, phase transitions driven by temperature are more gradual, unless there is some underlying structural or chemical shift that the authors have not accounted for. Without a clear explanation, this sudden change in behavior reduces confidence in the simulation results.

Suggested Analysis:

a) The authors should explore possible explanations for the dramatic difference in cluster size between 290K and 300K. For example, they could investigate whether specific interactions (such as the breaking or formation of hydrogen bonds or hydrophobic contacts) might explain the behavior at higher temperatures.

b) It is important to check whether the coarse-grained simulation model has been adequately parameterized and scaled for accurate temperature dependence. Atomistic simulations of monomers and dimers with varying polyQ tract lengths could be used to fine-tune the coarse-grained model, ensuring it accurately reflects molecular behavior. The gross estimate of a 10% scaling factor might be insufficient and could lead to inaccurate representations of cluster formation.

(3) Scaling of Coarse-Grained Model with Atomistic Simulations:

As mentioned, the coarse-grained model used in the study may not have been properly scaled against atomistic data. A simple scaling factor of 10% may not be appropriate for accurately capturing the behavior of polyQ tracts across different lengths, especially considering their sensitivity to subtle changes in temperature. Without rigorous validation against atomistic simulations, the coarse-grained model's predictions could be skewed.

Suggested Analysis:

a) To address this, the authors should compare the coarse-grained model with atomistic simulations of monomeric and dimeric forms of ELF3 with different polyQ tract lengths. By comparing key structural parameters (e.g., radius of gyration, contact maps, and clustering propensity), the authors could adjust the coarse-grained model to more accurately reflect the atomistic behavior. The authors have wealth of atomistic simulation data that could afford such benchmarking and identification of scaling factor

b) Additionally, the authors should investigate whether the assumed scaling factor of 10% is appropriate for each polyQ length or whether it needs to be refined based on specific properties, such as the number of hydrophobic interactions or secondary structure stability.

(4) Lack of Analysis for Liquid-Like Behavior in Phase Separation:

The simulations presented in the manuscript do not analyze the liquid-like behavior of ELF3 condensates, which is a key characteristic of liquid-liquid phase separation (LLPS). In LLPS systems, condensates are often dynamic, with chains exchanging between clusters, indicating liquid-like rather than solid-like behavior. The authors fail to probe this crucial aspect, which is necessary to support the claim that ELF3 undergoes phase separation.

Suggested Analysis:

a) The authors should conduct additional analyses to probe the liquid-like nature of the clusters formed by ELF3. One approach would be to analyze the dynamics of chain exchange between clusters, measuring how frequently chains leave one cluster and join another over time. This analysis would reveal whether the condensates behave as liquid-like, dynamic structures or more static, solid-like aggregates.

b) Additionally, the temperature dependence of these exchange dynamics should be investigated. In true liquid-liquid phase separation, the rate of chain exchange is often sensitive to temperature. Observing how this rate changes between 290K and 300K, for instance, could help explain the abrupt shift in cluster size seen in Figure 6B.

c) The authors should also analyze whether the internal structures of the condensates are consistent with a liquid-like phase. For example, radial distribution functions and contact lifetimes could be calculated to reveal whether the clusters exhibit liquid-like organization.

(5) Lack of justification of polydispersity of polyQ:

The authors don't provide any rationale for choice of different copies of polyQ used in the manuscript for their chain-growth simulation studies. It will be more apt if it can be motivated via some precedent experimental observations.

(6) Lack of initiative to connect to Experiments:

While the computational models and simulations provide robust theoretical insights, the absence of direct experimental validation weakens the overall impact of the manuscript. For example, experimental data on how specific mutations in the polyQ tract influence ELF3 behavior in vivo would significantly bolster the authors' claims. The manuscript would benefit from either citing existing experimental studies that corroborate these findings or from suggesting future experimental directions.

Comments on revised version:

The authors have now adequately addressed to the key concerns of manuscript. The manuscript in the present form looks significantly improved.

Reviewer #1 (Public review):

Summary:

The authors present a novel approach to subcellular spatial proteomics by combining laser microdissection with expansion microscopy and LC-MS/MS analysis (SPEx). They implement two different workflows for LMD and LC-MS/MS quantification:

(1)The standard approach, where an area of interest is cut out by LMD, subjected to proteomics analysis, and compared to the rest of the cell without the dissected ROI.

(2) The subtraction approach, where ROIs are removed, and the remaining cellular material is compared to samples containing both the surrounding material and the ROI.

The authors assess the technique by applying it to subcellular targets of various sizes, volumes, and protein compositions such as the nucleus, nucleoli, and Golgi. They demonstrate that SPEx can identify proteins enriched or reduced in ROIs.

Strengths:

The broad, relatively easy, and inexpensive applicability of this approach to potentially many cell types and subcellular areas of interest provides an exciting alternative to subcellular fractionation, native immunoprecipitation, or genetically encoded proximity labeling constructs. Moreover, by visually selecting ROIs for subsequent analysis, subcellular context or organelle morphology can be taken into account, as discussed by the authors in the discussion section.

Weaknesses:

While strongly supporting the sharing of this approach, we have a number of comments and questions that will improve the impact of the manuscript:

(1) General:

a) The manuscript would benefit from restructuring and language revision. In its current form, the writing is sometimes dense and verbose (in particular, the Results section). This makes it difficult to follow the authors' arguments.

b) The authors mention the possibility of selecting organelles based on morphology. This is left for the discussion, but it seems like a missed opportunity - the authors could compare individual organelles in different morphological states, e.g., connected vs. fragmented mitochondria.

(2) Technical:

a) Why do the authors strive and optimize for a 10x expansion factor? Is SPEx compatible with a more standard 4x expansion, as e.g., used in the classic U-ExM approach (https://www.nature.com/articles/s41592-018-0238-1)? This could be added to the discussion.

b) The U-ExM approach shows improved ultrastructural preservation when using 3%FA with 0.1% glutaraldehyde fixation (GA). Is SPEx compatible with the use of low amounts of GA for fixation?

c) Related to the above, was the anchoring efficiency reduced only to achieve a 10x expansion factor or does this additionally affect the proteome coverage?

d) Have the authors considered using alternative anchoring approaches, such as GMA (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0291506#pone.0291506.s001), which potentially increase the amount of sample retained in the hydrogel, thus allowing for better proteome coverage? This could be added to the discussion.

e) The limitation of the approach to near-2D samples should be mentioned, and alternative approaches for more 3D samples could be discussed.

f) How are peptides that are directly anchored to the hydrogel dealt with during LC-MS/MS analysis? Are they excluded, or can they be identified during the spectral search? The latter would allow us to get a deeper structural understanding of how proteins are actually anchored into hydrogels, which so far has not been assessed.

An alternative approach to address this question would be to investigate if the peptide coverage of proteins detected by SPEx is enriched for peptides representing the folded core of proteins as opposed to the surface-exposed regions, which likely get more anchored into the hydrogel.

g) Same question regarding peptides with NHS labeling. Can they be identified, or do they just compete for ionization and thus negatively affect coverage and dynamic range of the LC-MS/MS approach?

h) How are the primary and secondary antibodies affecting the proteomics analysis identified as contaminants?

i) Have the authors observed differences in proteomics coverage of only antibody vs NHS-labeling? Depending on the questions above, could pure antibody-based labeling increase proteomic coverage?

Reviewer #1 (Public review):

Summary:

This study investigates a fundamental question in cognitive science: is our ability to reason about the physical world an abstract mental process, or is it "embodied"-directly rooted in our real-time physical interactions with the environment? The authors compared participants' performance in computerized reasoning games with and without Galvanic Vestibular Stimulation (GVS). They suggest that participants failed more often and utilized suboptimal strategies under GVS compared to a sham stimulation condition. Furthermore, they found that this detrimental effect of GVS was reduced when the games were governed by altered gravity (hyper- and hypo-gravity). Consequently, the authors conclude that the physical experience of the body modifies high-level cognitive skills, such as reasoning.

Strengths:

The manuscript is well-written, organized, and easy to follow, making complex concepts accessible. Also, combining a specialized physical reasoning task with real-time vestibular disruption (GVS) is an intriguing approach to testing the boundaries of embodied cognition.

Weaknesses:

(1) Lack of Overall Effects and Inflated Type I Error for Game-Level Effects

The study utilizes a within-subject design. Taking Study 1 as an example, each subject participated in a familiarization session (4 games), a baseline session (12 games without stimulation), a GVS session (14 games), and a sham session (14 games). No game was repeated for any single subject. Performance was quantified using three primary measures (success rate, number of attempts, and time per attempt) and two strategy measures (tool switching and the distance between tool placements).

For Study 1, to identify condition differences at the game level (i.e., Figure 2), the authors effectively conducted 70 independent t-tests (5 measures × 14 games). While 7 significant results were reported, this large number of independent tests invites an inflated Type I error rate, as no multiple-comparison correction appears to have been applied.

A similar inflation is expected in Study 2, where 50 independent t-tests (5 measures × 10 games) yielded 5 significant comparisons (Figure 4). Although the authors might argue the direction of the differences is systematic, implying GVS generally impairs performance, at least one significant comparison shows the opposite effect: tool switching indicates that GVS led to better performance for the 'Table_A' game in Study 2 (Figure 4d), whereas the same variable indicated GVS led to worse performance in Study 1 (Figure 2d). I suspect that none of the significant game-level results would survive a proper statistical correction. If possible, the authors can redo statistical testing with corrections (FDR or Bonferroni) or with LMM using game as a random effect. Before proper statistical analyses, I strongly encourage the authors to refrain from drawing broad conclusions based on these isolated game-level results.

Furthermore, when analyzing data across all games, the study found no significant effect of GVS on overall performance or strategy measures in either Study 1 or Study 2. This lack of an aggregate effect contradicts the authors' conclusion that participants failed more often or utilized suboptimal strategies under GVS.

(2) Missing Rationale for Classification Analysis

It is puzzling why the authors pursued two exploratory analyses on tool placement after revealing that the two related primary measures (tool positioning and switching) did not generate significant condition differences in Study 1. These additional analyses-the Dirichlet Process Gaussian Mixture Model and leave-one-out classification-were not pre-registered. In the absence of overall condition differences, the authors appear to be "doubling down" by applying sophisticated classification tools to the raw data without a clear prior rationale.

(3) Insufficient Evidence for the Reduced Effect of GVS Under Altered Gravity

To compare Study 1 and Study 2, the authors devised a "gravity-weighted index," but its definition is not sufficiently justified. The index assigns weights of 1, 2, and 3 to low-, medium-, and high-gravity-dependent games, respectively. The choice of these specific weights appears arbitrary, making the quantitative results difficult to interpret. More importantly, there is no citation or explanation regarding how these three levels of "gravity impact" were defined in the first place (Line 468). This index was also not pre-registered.

The authors state that for the success rate index, a value close to -1 indicates a large negative difference for GVS, 0 indicates no difference, and 1 indicates a large positive difference. These are theoretical bounds; the actual distribution of each index should be examined to validate such claims. However, the paper lacks descriptive statistics for this composite index.

Notably, the "reduction" of the GVS effect in altered gravity was only demonstrated in one of the five available indices (success rate, p = 0.046). In fact, the success rate in Study 2 was 66.7(sham) vs 67.3 (GVS) in Table 2. It is highly debatable whether this marginal result justifies the conclusion that GVS effects "were reduced when the games included reasoning about altered gravity".

(4) Questionable Assumptions Regarding Strategy

The authors assume that "big changes in tool positioning and frequent tool switching indicate poor evaluation of the failed outcome". This assumption is questionable. In solving this cognitive task, participants must explore and exploit solutions based on feedback. Large shifts in positioning or frequent tool switching might reflect active, adaptive exploration based on failed outcomes rather than a failure to evaluate them.

(5) Confounding Factors in GVS Interpretation

The central theoretical question is whether physical reasoning is grounded in physical experience. GVS is used here to manipulate that experience. However, GVS does not selectively target the vestibular nerve; it also activates distributed fronto-parietal attention networks and hippocampal circuits essential for any reasoning task. Additionally, the vestibular system is linked to the limbic system and the cerebellum, which regulate emotional reactivity and arousal. Because attention and emotion are likely affected by GVS, the authors should be much more cautious in attributing their behavioral findings solely to changes in the "physical experience of the body."

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors study two residues in the GHKL ATPase active site of Aq MutL and GyrB, and argue that the catalytic base function is shared between two conserved acidic residues that are 3 residues apart.

They generated mutant versions in MutL and GyrB (both ala and the appropriate Asn/Gln version) and performed ATPase analysis. They also generated high-resolution crystal structures of the GyrB NTD with AMPPnP for WT and mutants of the two acidic residues. The data show that mutation in either of these residues does not fully kill activity (with the exception of the Alanine mutation of the first of the two, which interferes with ATP (or AMPPnP) binding). When the acidic residues are mutated to Asn/Gln, the catalytic water can still be positioned, and hence these mutants are more active than the Ala mutants. In both cases, the double mutation is catalytically dead.

The authors then perform phylogenetic analysis and ancestral gene reconstruction, and based on this, they argue that HSP90 forms a different class of GHKL ATPases, and lost rather than gained this separate status.

Strengths:

The biochemical analysis seems solid.

Weaknesses:

(1) A major question that remains is why the mutations have so much more detrimental effect in MutL (100-fold lower kcat/KM) than they do in GyrB (3-fold lower). Can the authors explain this? Doesn't this argue against the proposed catalytic conservation?

(2) The structure figures all have omit maps for just the AMPPnP and the water, whereas the density for the acidic residues and their mutants is not shown.

Reviewer #1 (Public review):

Summary:

By using an established NAFLD model, choline-deficient high-fat diet, Barros et al show that LPS challenge causes excessive IFN-γ production by hepatic NK cells which further induces recruitment and polarization of a PD-L1 positive neutrophil subset leading to massive TNFα production and increased host mortality. Genetic inhibition of IFN-γ or pharmacological blockade of PD-L1 decreases recruitment of these neutrophils and TNFα release, consequently preventing liver damage and decreasing host death.

Since NAFLD is often accompanied by chronic, low-grade inflammation, it can lead to an overactive but dysfunctional immune response and increase the body's overall susceptibility to infections, therefore this is very important research question.

Strengths:

The biggest strength of the manuscript is vast number of mouse strains used.

Weaknesses:

After the review, there are still some open questions from my side:

(1) I would like the authors to defend their choice of diet type since this has not been done in the review/response to authors. In case they cannot, we need additional proof (HFD or WD model).

(2) Since the authors used same control groups (chow and HFCD), as required by the animal ethics committee, they must have power analysis test to show that the number of controls (but also in other groups) they used is enough to see the effect. Please provide it.

Reviewer #1 (Public review):

Summary:

This study asks whether synapses formed by the same broad neuronal class (excitatory pyramidal neurons, PN) adapt their presynaptic organization in a cortex-specific manner, comparing the prefrontal cortex (PFC) with the primary somatosensory cortex (S1). The authors combine sophisticated electrophysiology (paired recordings and extracellular minimal stimulation), pharmacological perturbations of presynaptic Ca²⁺-secretion coupling, bouton Ca²⁺ imaging, and mechanistic modeling. Across two prominent excitatory connections (Layer 5 (L5) PN-L5PN and L2/3-L5PN), they provide convergent evidence that mature PFC synapses operate with looser Ca²⁺ channel-release sensor coupling than their S1 counterparts.

Overall, the study provides an appealing mechanistic link between synaptic nano/micro-architecture and cortical-area specialization. The idea that PFC synapses retain a more "plasticity-favoring" presynaptic state, while the primary sensory cortex emphasizes reliability and timing precision, is potentially impactful for how we think about circuit computation and plasticity across cortical hierarchies.

Strengths:

A major strength is the multi-pronged experimental strategy. The paper first establishes robust, area-dependent differences in synaptic efficacy, reliability, timing, and short-term plasticity (facilitation prevailing in PFC versus depression in S1), using both paired recordings and minimal extracellular stimulation paradigms. The coupling interpretation is then directly supported by differential sensitivity to EGTA (and appropriate positive-control effects of fast chelators). Finally, volume-averaged calcium signals are reported to be similar across areas, arguing against trivial explanations based on gross differences in calcium influx, and the modeling provides a quantitative framework for interpreting the observed chelator effects.

Weaknesses:

Limitations are minor and concern interpretation/clarity rather than core results. Some key inferences rely on indirect readouts (chelator sensitivity, fluctuation analysis-derived parameters, bouton-averaged calcium signals), each of which carries assumptions and potential confounds that should be discussed more explicitly. In particular, the repatching paradigm for the paired-recording EGTA experiment, though very impressive, and the limited number of extracellular calcium conditions used for fluctuation analysis (three concentrations), can influence quantitative estimates and the confidence intervals around them.

Reviewer #1 (Public review):

Very nice and coherent body of work with appropriate in vitro to in vivo transition in methods.

Lovely and easy to follow figures that can be understood even without the manuscript.

My recommendation is that a sentence or two be added clearly stating the authors think nafamostat is off the table and suggest other approaches/drugs that might be considered instead of just making a general statement. I think all this can be done in a few sentences.

Gabexate was administered to a snakebite victim in this case report from about 20 years ago and also a good example of the now better recognized threat to pregnancy.

Nasu K, Ueda T, Miyakawa I. Intrauterine fetal death caused by pit viper venom poisoning in early pregnancy. Gynecol Obstet Invest. 2004;57(2):114-6. doi: 10.1159/000075676. Epub 2003 Dec 19. PMID: 14691344

Reviewer #1 (Public review):

Summary:

This study presents a new Bayesian approach to estimate importation probabilities of malaria combining epidemiological data, travel history, and genetic data through pairwise IBD estimates. Importation is an important factor challenging malaria elimination, especially in low transmission settings. This paper focus on Magude and Matutuine, two districts in south Mozambique with very low malaria transmission. The results show isolation-by-distance in Mozambique, with genetic relatedness decreasing with distances larger than 100 km, and no spatial correlation for distances between 10 and 100 km. But again strong spatial correlation in distances smaller than 10 km. They report high genetic relatedness between Matutuine and Inhambane, higher than between Matutuine and Magude. Inhambane is the main source of importation in Matutuine, accounting for 63.5% of imported cases. Magude, on the other hand, shows smaller importation and travel rates than Matutuine, as it is a rural area with less mobility. Additionally, they report higher levels of importation and travel in the dry season, when transmission is lower. Also, no association with importation was found for occupation, sex and other factors. These data have practical implications for public health strategies aiming malaria elimination, for example, testing and treating travelers from Matutuine in the dry season.

Strengths:

The strength of this study relies in the combination of different sources of data - epidemiological, travel and genetic data - to estimate importation probabilities, the statistical analyses.

Weaknesses:

The authors recognize the limitations related to sample size and the biases of travel reports.

Reviewer #1 (Public review):

Summary:

Sidarta-Oliveira et al. present TopOMetry, a novel dimensionality reduction method based on the eigendecomposition of approximated Laplace-Beltrami Operator. Shortly, TopOMetry is an iterative version of the existing spectral methods (e.g., Laplacian Eigenmap or Diffusion map). It approximates the Laplacian operators twice, once in a "phenotypic space" and then once again in the eigenbases space. By doing this the approximated operator will contain more information of the manifold, which allows for more robust and accurate downstream analyses.

Strengths:

- Introduces operator-native fidelity scores and Riemannian diagnostics to single-cell analysis, enabling researchers to evaluate and trust embeddings - functionality absent in prior methods.<br /> - The approach was rigorously tested based on synthetic and real single-cell RNA-seq datasets.<br /> - The package is well-made and easily scalable to millions of cells.<br /> - The comprehensive documentation helps the end-users to run desired analyses.

Weaknesses:

- The method is an extension of the current state-of-art methods, not a fundamentally new one.

Comments on revised version:

The revised manuscript partially addresses the concerns raised in the prior review. The jargon weakness has been substantially mitigated by relocating mathematical derivations to the Methods section and simplifying language in the main text; this weakness has been updated accordingly.

The introduction of operator-native fidelity scores and Riemannian diagnostics represents a meaningful addition and has been added to the Strengths. The benchmarking scope has also been notably expanded.

The core weakness - that the method is an extension of existing spectral methods rather than a fundamentally new contribution - remains unchanged, as the authors' rebuttal did not provide a sufficiently precise mathematical argument to overturn it.

Reviewer #1 (Public review):

Summary:

In this manuscript, Ding et al. use genetic mouse models to demonstrate that atrial trabeculation is more dependent on Tie1/Tie2 signaling than ventricular trabeculation. With additional experimentation that would support the current claims, the results may hold significant value, as atrial trabeculation remains an understudied phenomenon in cardiac biology with potential implications for atrial cardiomyopathy and atrial fibrillation.

Strengths:

Detailed characterization of atrial versus ventricular trabeculation across different developmental timepoints, and the use of appropriate animal models to address the scientific question at hand.

Weaknesses:

The authors have consistently treated mice with tamoxifen after ventricular, but not atrial, trabeculation has already started. As such, the observed cardiac phenotypes - where predominantly atrial trabeculation is affected - might be a mere consequence of the precise time window in which Tie1/2 signaling was impaired, rather than a direct measurement of its relative importance for atrial versus ventricular trabeculation. The conclusions of the paper may thus be significantly strengthened by depleting Tie1/2 signaling prior to the onset of ventricular trabeculation, as is done for atrial trabeculation.

Reviewer #1 (Public review):

Summary:

In this review paper, the authors describe the concept of neural correlates of consciousness (NCC) and explain how noninvasive neuroimaging methods fall short of being able to properly characterise an unconfounded NCC. They argue that intracranial research is a means to address this gap and provide a review of many intracranial neuroimaging studies that have sought to answer questions regarding the neural basis of perceptual consciousness.

Strengths:

The authors have provided an in-depth, timely, and scholarly contribution to the study of NCCs. First and foremost, the review surveys a vast array of literature. The authors synthesise findings such that a coherent narrative of what invasive electrophysiology studies have revealed about the neural basis of consciousness can be easily grasped by the reader. The authors also succeed in describing how single-cell recordings can interface with task-design to help mitigate the impact of confounded neural activity when searching for NCCs.

The review is also, to the best of my knowledge, the first review to specifically target intracranial approaches to consciousness and to describe their results in a single article. This is a credit to the authors - as it becomes ever harder to apply strict tests to theories of consciousness using methods such as fMRI and M/EEG, it is important to have informative resources describing the results of human intracranial research so that theorists will have to constrain their theories further in accordance with such data. Additionally, the authors provide a compelling case for single-celled research in consciousness science, despite the dominance of theories situated at the system and circuit level of analysis. As far as the authors were aiming to provide a complete and coherent overview of intracranial approaches to the study of NCCs, I believe they have achieved their aim.

Weaknesses:

Overall, I feel positive about this paper. The authors have addressed my comments from my previous review and I see no significant weaknesses in the current version.

Comment on revised version:

No comments - congratulations to the authors!

Reviewer #1 (Public review):

Summary

In this review paper, the authors describe the concept of neural correlates of consciousness (NCC) and explain how noninvasive neuroimaging methods fall short of being able to properly characterise an unconfounded NCC. They argue that intracranial research is a means to address this gap and provide a review of many intracranial neuroimaging studies that have sought to answer questions regarding the neural basis of perceptual consciousness.

Strengths

The authors have provided an in-depth, timely, and scholarly contribution to the study of NCCs. First and foremost, the review surveys a vast array of literature. The authors synthesise findings such that a coherent narrative of what invasive electrophysiology studies have revealed about the neural basis of consciousness can be easily grasped by the reader. The review is also, to the best of my knowledge, the first review to specifically target intracranial approaches to consciousness and to describe their results in a single article. This is a credit to the authors, as it becomes ever harder to apply strict tests to theories of consciousness using methods such as fMRI and M/EEG it is important to have informative resources describing the results of human intracranial research so that theorists will have to constrain their theories further in accordance with such data. As far as the authors were aiming to provide a complete and coherent overview of intracranial approaches to the study of NCCs, I believe they have achieved their aim.

Weaknesses

Overall, I feel positive about this paper. However, there are a couple of aspects to the manuscript that I think could be improved.

(1) Distinguishing NCCs from their prerequisites or consequences

This section in the introduction was particularly confusing to me. Namely, in this section, the authors' aim is to explain how intracranial recordings can help distinguish 'pure' NCCs from their antecedents and consequences. However, the authors almost exclusively describe different tasks (e.g., no-report tasks) that have been used to help solve this problem, rather than elaborating on how intracranial recordings may resolve this issue. The authors claim that no-report designs rely on null findings, and invasive recordings can be more sensitive to smaller effects, which can help in such cases. However, this motivation pertains to the previous sub-section (limits of noninvasive methods), since it is primarily concerned with the lack of temporal and spatial resolution of fMRI and M/EEG. It is not, in and of itself, a means to distinguish NCCs from their confounds.

As such, in its current formulation, I do not find the argument that intracranial recordings are better suited to identifying pure NCCs (i.e. separating them from pre- or post-processing) convincing. To me, this is a problem solved through novel paradigms and better-developed theories. As it stands, the paper justifies my position by highlighting task developments that help to distinguish NCCs from prerequisites and consequences, rather than giving a novel argument as to why intracranial recordings outperform noninvasive methods beyond the reasons they explained in the previous section. Again, this position is justified when, from lines 505-506, the authors describe how none of the reported single-cell studies were able to dissociate NCCs from post-perceptual processing. As such, it seems as if, even with intracranial recording, NCCs and their confounds cannot be disentangled without appropriate tasks.

The section 'Towards Better Behavioural Paradigms' is a clear attempt to address these issues and, as such, I am sure the authors share the same concerns as I am raising. Still, I remain unconvinced that the distinguishing of NCCs from pre-/post- processing is a fair motivation for using intracranial over noninvasive measures.

(2) Drawing misleading conclusions from certain studies

There are passages of the manuscript where the authors draw conclusions from studies that are not necessarily warranted by the studies they cite. For instance:

Lines 265 - 271: "The results of these two studies revealed a complex pattern: on the one hand, HGA in the lateral occipitotemporal cortex and the ventral visual cortex correlated with stimulus strength. On the other hand, it also correlated with another factor that does not appear to play a role in visibility (repetition suppression), and did not correlate with a non-sensory factor that affects visibility reports (prior exposure). These results suggest that activity in occipitotemporal cortex regions reflecting higher-order visual processing may be a precursor to the NCC but not an NCC proper."

It's possible to imagine a theory that would predict HGA could correlate with stimulus strength and repetition suppression, or that it would not correlate with prior exposure (e.g. prior exposure could impact response bias without affecting subjective visibility itself). The authors describe this exact ambiguity in interpretation later in the article (line 664), but in its current form, at least in line 270 (when the study is most extensively discussed), the manuscript heavily implies that HGA is not an NCC proper. This generates a false impression that intracranial recordings have conclusively determined that occipitotemporal HGA is not a pure NCC, which is certainly a premature conclusion.

Line 243: "Altogether, these early human intracranial studies indicate that early-latency visual processing steps, reflected in broadband and low gamma activity, occur irrespective of whether a stimulus is consciously perceived or not. They also identified a candidate NCC: later (>200 ms) activity in the occipitotemporal region responsible for higher-order visual processing."

The authors claim in this section that later (>200ms) activity in occipitotemporal regions may be a candidate for an NCC. However, the Fisch et al. (2009) study they describe in support of this conclusion found that early (~150ms) activity could dissociate conscious and unconscious processing. This would suggest that it is early processing that lays claim to perceptual consciousness. The authors explicitly describe the Fisch et al results as showing evidence for early markers of consciousness (line 240: '...exhibited an early...response following recognized vs unrecognised stimuli.) Yet only a few lines later they use this to support the conclusion that a candidate NCC is 'later (>200ms) activity in the occipitotemporal region' (line 245). As such, I am not sure what conclusion the authors want me to make from these studies.

This problem is repeated in lines 386-387: "Altogether, studies that investigated the cortical correlates of visual consciousness point to a role of neural responses starting ~250 ms after stimulus onset in the non-primary visual cortex and prefrontal cortex."

This seems to be directly in conflict with the Fisch et al results, which show that correlates of consciousness can begin ~100ms earlier than the authors state in this passage.

(3) Justifying single-neuron cortical correlates of consciousness

The purpose of the present manuscript is to highlight why and how intracortical measures of neural activity can help reveal the neural correlates of perceptual consciousness. As such, in the section 'Single-neuron cortical correlates of perceptual consciousness', I think the paper is lacking an argument as to why single-neuron research is useful when searching for the NCC. Most theories of consciousness are based around circuit or system-level analyses (e.g., global ignition, recurrent feedback, prefrontal indexing, etc.) and usually do not make predictions about single cells. Without any elaboration or argument as to why single-cell research is necessary for a science of consciousness, the research described in this section, although excellent and valuable in its own right, seems out of place in the broader discussion of NCCs. A particularly strong interpretation here could be that intracranial recordings mislead researchers into studying single cells simply because it is the finest level of analysis, rather than because it offers helpful insight into the NCCs.

(4) No mention of combined fMRI-EEG research

A minor point, but I was surprised that the authors did not mention any combined fMRI-EEG research when they were discussing the limits of noninvasive recordings. Intracortical recordings are one way to surpass the spatial and temporal resolution limits of M/EEG and fMRI respectively, but studies that combine fMRI and EEG are also an alternative means to solve this problem: by combining the spatial resolution of fMRI with the temporal resolution of EEG, researchers can - in theory - compare when and where certain activity patterns (be they univariate ERPs or multivariate patterns) arise. The authors do cite one paper (Dellert et al., 2021 JNeuro) that used this kind of setup, but they discuss it only with respect to the task and ignore the recording method. The argument for using intracranial recordings is weaker for not mentioning a viable, noninvasive alternative that resolves the same issues.

Reviewer #1 (Public review):

Summary:

LRRK2 protein is familially linked to Parkinson's disease by the presence of several gene variants that confer a gain-of-function effect on LRRK2 kinase activity.

The authors examine the effects of BDNF stimulation in immortalized neuron-like cells, cultured mouse primary neurons, hIPSC-derived neurons, and brain tissue from genetically modified mice. They examine a LRRK2 regulatory phosphorylation residue, LRRK2 binding relationships, other kinase phosphorylation status, and measures of synaptic structure and function.

Strengths:

The study addresses an important research question: how does a PD-linked protein interact with other proteins, and contribute to responses to a well-characterized neuronal signalling pathway involved in the regulation of synaptic function and cell health.

They employ a range of models and techniques to convincingly demonstrate that BDNF stimulation alters LRRK2 phosphorylation at pS935 and binding to many proteins. Several independent data sets lead to some exciting conclusions.<br /> In this re-revised manuscript, some aspects are very convincing and well validated e.g., drebrin binding to LRRK2, increased by BDNF, and reduced LRRK2 protein levels in young (but not mature) drebrin KO mice. A phosphoproteomic analysis of PD mutant Knock-in mouse brain is included. Overall, the links between LRRK2, LRRK2 activity, and the changes to synaptic molecules, structures, and activity are intriguing.

Weaknesses:

Enthusiasm for the title claim that "LRRK2 regulates synaptic function through BDNF signalling" is tempered by disconnected results across different model systems and inconsistent alterations upon kinase phosphorylation in SHSY5Y cell line and primary neurons. Exciting conclusions are sometimes not consistently supported by the data and/or only conducted in one of the models.

BDNF increasing pS935 LRRK2 is quite well supported in cell lines, as is BDNF regulation of derbrin-LRRK2 binding. However, there is a lack of connection between this result and subsequent alterations to LRRK2 substrates e.g., phosphorylation of Rab GTPases, especially in neurons. Interesting omic data sets are provided, but with very little or no validation. For example, only drebrin protein was assessed in BDNF treatment omic, and the phosphoproteomic analysis of PD mutant Knock-in mouse is stand alone with no validation and G2019S is not explored elsewhere in the study.

The major disconnect this reviewer struggles with is the conclusion that the quite clear data in SHSY5Y cells is the same as that from neurons regarding BDNF / LRRK2 and ERK / Akt. It seems they are not.

ERK and Akt phosphorylation by BDNF is absent in CRISPR KO SHSY5Y cells.<br /> This conclusion is at odds with interpretation of neuronal data. To explain; in div14 neurons, BDNF's transient increase in pLRRK2 is seen and strongly prevented by MLi2. BDNF also increased pAkt & pERK1&2 in WT... but also in LRRK2 KO. Furthermore, this happened in the presence of MLi2 in WT despite no pLRRK2 increase. While the 5min BDNF induced increase to pAkt appears reduced in LKO, the same time BDNF in LKO with MLi2 is as high as WT (in these unquantified examples) and ERK is almost identical. This is described as "significantly reduced" but I see no replicates or quantification, and face value assessment of the blot argues against this.<br /> Thus, there is little or no evidence supporting that LRRK2 activity is involved in BDNF-stimulated increases in pAkt or pERK, upstream, in neurons as neither Mli2 nor KO prevented this.

Synapse markers increased in WT neuron with BDNF treatment which did not happen in LKO neurons. So this process requires pLRRK2, but is unrelated to pAkt or pERK (which do still go up with BDNF in KO)? Similarly, an increase in synaptic activity in WT hiPSC neurons in response to BDNF seems lost in LRRK2 KO hiPSC neurons, although their activity is already increased and depending on the age of the cells the effects were different. Both of these experiments lack supporting evidence by other measures e.g., LRRK2 inhibition effects on BDNF-induced increases in WT and parallel biochemistry of p'd LRRK2, Akt, ERK in WT & KO.

LRRK2 activating Akt1 has been published before (e.g., Ohta 2011 - not cited), but Ohta also conclude that LRRK2 gain of function mutations (more LRRK2 kinase activity) were associated with a reduced ability of LRRK2 to bind AND phosphorylate Akt at the same residue, in contradiction to the mechanism proposed here? This should be discussed. Here the authors also conclude Akt is Upstream of LRRK2. However, it appears from the data here in neurons that pLRRK2 increases in response to BDNF are separate from BDNF signalling to Akt.

Of note, in comparison to bTubulin control, LKO total Akt levels appear consistently higher in this single example blot; a large increase in Akt would skew the ratio down, while absolute levels of pAkt (probably the most important matter for an active enzyme - what is the ratio against total protein stain) are similar or increased. These are major problems for the conclusions as presented.

BDNF increased mEPSC frequency in hIPSC neurons; which didn't happen in LKO, which already had high frequency. Earlier in the manuscript BDNF is shown to alter synapse number in WT but not LKO mouse neurons, but no increase in synapse number was seen following BDNF treatment in any WT or LKO hiPSC neurons +/- BDFN.

If we are to assume that the WT neurons have LRRK2 (not demonstrated), and that LRRK2 KO neurons have similar drebrin (not demonstrated) it is unclear how to interpret this result in the model of BDNF-LRRK2 being upstream of pERK/Akt. There is no evidence that the BDNF increase in WT is blocked by LRRK2 inhibition, nor has it been associated with changes (or not) to pAkt or ERK1, which would be expected in both WT and KO based on Figure 4C.

There are many reports of acute and longer term BDNF application increasing event frequency in brain slices & primary neurons. Overexpression of BDNF in NPCs has also been shown to increase synapse function in hiPSC neurons derived from them. Here, BDNF has an effect on frequency in only one 6 comparisons (3 timepoints, two lines). Is it not concerning that expected BDNF effects occur at only one time point in WT, and that generally a lack of effect is more common both in WT and LKO... is this due to slow appearance of TrkB receptors and degeneration at 90 days?

There are no other data provided to show that BDNF was having a consistent expected effect in human neurons (pAkt, pLRRK2 etc etc), and there is little to link between this data and that in previous figures of the study.

The discussion of some of the weaknesses is mostly fair, asides the disparities noted above which are not.

Reviewer #2 (Public review):

Summary:

The authors investigated whether early-life malaria exposure has long-term effects on immune responses to unrelated antigens. They leveraged a natural experiment in coastal Kenya where two adjacent communities (Junju and Ngerenya) experienced divergent malaria transmission patterns after 2004. Using 15 years of longitudinal data from 123 children with weekly malaria surveillance and annual serological sampling, they measured antibody responses to multiple pathogens using a protein microarray technology and ELISA.

Strengths:

(1) Extensive longitudinal data collection with weekly malaria surveillance, enabling precise exposure classification.

(2) Use of a natural experiment design that allows for causal inference about malaria's immunological effects.

(3) Broad panel of antigens tested, demonstrating generalized rather than antigen-specific effects.

(4) Within-cohort analysis in Ngerenya controls for geographic and environmental factors.

(5) Validation of key findings using both serologic microarray and ELISA.

(6) Important public health implications for vaccine strategies in malaria-endemic regions.

Weaknesses:

(1) Due to its nature, the study lacks the ability to determine the direction of the associations found between malaria exposure and other IgG levels to unrelated pathogens.

(2) No evaluation of the clinical Implications of the reduced IgG levels observed in the area with high malaria exposure.

Assessment of Claims:

The data appear to support the authors' primary claims. The strength of the evidence is limited by the observational nature of the study and the results should be interpreted in that light. Together with the currently available evidence of P. falciparum's impact on the host's immune function, this natural experiment design provides further evidence for a relationship between early malaria exposure and reduced antibody responses to other pathogens and vaccine-derived antigens. The within-Ngerenya analysis controls for geographic factors and thus enhances the quality of the evidence; there is limited physical, nutritional, and socio-economic information on factors that may have driven the observed changes.

Impact and Utility:

This work has fundamental implications for understanding vaccine effectiveness in malaria-endemic regions and may contribute to inform vaccination strategies. The findings, if confirmed, would suggest that children in areas of high malaria transmission may require modified immunization approaches. The dataset provides a valuable resource for future studies of malaria's immunological legacy.

Context:

This study builds on prior work showing acute immunosuppressive effects of malaria but uniquely attempts to demonstrate the durability of these effects years after exposure. The natural experiment design addresses limitations of previous observational studies by providing a more controlled comparison.

Reviewer #1 (Public review):

Sebag et al. addressed the role of ADH5 in BAT in the development of aging and metabolic disarrangements associated with it. This is a follow-up study after the authors' demonstration of the role of BAT ADH5 in glucose homeostasis, obesity, and cold tolerance. By ablating ADH5 specifically in brown adipocytes or pharmacologically modulating ADH5 through activation of its transcription factor, the authors conclude that preservation of BAT function is crucial for healthy aging and ADH5 is causally involved in this process. The topic is appealing given the rise in the aging population and the unclear role of BAT function in this process. Overall, the study uses several techniques and addresses several physiological and molecular manifestations of aging. Therefore, the findings contribute to the growing body of literature pointing to the biological role of BAT activity in aging.

Comments on revised version:

I have no further comments other than to congratulate the authors on the nice piece of work.

Reviewer #1 (Public review):

Summary:

In their manuscript, Metz Reed and colleagues present an exceptionally thorough analysis of three-dimensional genome reorganization during breast cancer progression using the well-characterized MCF10 model system. The integration of high-resolution Micro-C contact maps with multi-omics profiling provides compelling insights into stage-specific dynamics of chromatin compartments, TAD boundaries, and looping events. The discovery that stable chromatin loops enable epigenetic reprogramming of cancer genes while structural changes selectively drive metastasis-associated pathways represents a significant conceptual advance. This work substantially deepens our understanding of genome topology in malignancy.

Strengths:

This work sets a benchmark for integrative 3D genomics in oncology. Its methodological sophistication and conceptual advances establish a new paradigm for studying nuclear architecture in disease.

Comments on revised version:

The authors made a significant effort to improve the manuscript. My comments were sufficiently addressed.

Reviewer #1 (Public review):

Summary:

Patients with STX11 mutations develop familial hemophagocytic lymphohistiocytosis Type 4, a fatal immune disorder marked by defective T and NK cell cytotoxicity and cytokine storm. The conventional explanation attributes this to impaired cytotoxic granule release, but this has never fully accounted for the broader disease picture. This study proposes an alternative mechanism. The authors show that STX11 is required for store-operated calcium entry through ORAI1 channels, which are essential for both cytotoxic killing and NFAT-driven gene expression in T cells. In STX11-deficient cells, ORAI1 currents drop, NFAT nuclear translocation fails, IL-2 expression is suppressed, and degranulation is impaired. These defects are largely rescued by ionomycin or a constitutively active ORAI1 mutant, placing the primary lesion at calcium signaling rather than the fusion machinery. Mechanistically, STX11 binds the C-terminal tail of ORAI1 via its Habc domain and maintains ORAI1 in a state competent for productive assembly prior to STIM1-dependent gating, a step the authors call "priming."

Strengths:

The paper identifies a novel and disease-relevant role for STX11 in calcium channel regulation and raises the possibility of using channel agonists as a therapeutic strategy in the disease. The biochemical and functional data are of high quality and generally consistent with the interpretation. The proposal that a non-conventional syntaxin directly interacts with ion channels to prime its activation is novel and interesting.

Weaknesses:

For readers to appreciate the value of patient experiments derived from a single individual, the authors should quote prior studies showing that STX11 protein levels are abolished in all known human STX11 mutations. The priming model, while functionally well-supported, rests on indirect structural evidence, and the precise conformational transition involved remains to be defined. These are acknowledged limitations, but alternate mechanisms have not been explored and formally excluded. More direct evidence should be provided to exclude the possibility that STX11 could act as a conventional SNARE and sustain calcium fluxes by promoting the delivery of additional ORAI1 channels from vesicles.

Reviewer #1 (Public review):

Summary:

This paper by Boni and colleagues presents the engineering of a multi-step differentiation program in Escherichia coli based on synthetic gene circuits. The motivation behind the study was to engineer a system capable of undergoing differentiation in a step-wise manner without the presence of external spatial cues and without inducers added during the differentiation process. To achieve this, the authors created several synthetic gene circuits, one being a toggle switch, and the others being quorum-sensing-mediated gene expression modules. The outputs of the differentiation process are fluorescent proteins, which allowed the authors to quantify the behavior of the system using fluorescence intensity measurements. The authors additionally built a multi-component mathematical model which is able to reproduce the experimental data.

The data presented are convincing and support the claims; the work is well executed.

Strengths:

(1) The differentiation process proceeds autonomously after the initial step in liquid culture in the presence of external inducers.

(2) It is indeed a step-wise process.

(3) The mathematical model predicts the outcome (% of green, blue and red FP-expressing cells in the population) when changing the initial ratio of green:blue FP-expressing cells.

Weaknesses:

(1) No spatial pattern emerges. There are some isolated colonies that turn on the downstream FPs, but I do not see a pattern, really. Nonetheless, some colonies do differentiate (i.e. they turn on additional FPs).

(2) The mathematical model appears somewhat superfluous. While it can clearly reproduce the data, it is not used to make interesting predictions, changing parameters (and not initial conditions) that guide further experimental implementations.

Future directions

The utility of this differentiation process (e.g. in metabolic engineering or for the study of biofilm formation and antibiotic resistance) will become clearer once the FPs are substituted with functional proteins that exert an effect on the cells.

Reviewer #1 (Public review):

Summary:

The metabolic profiles of immune cells under steady-state or immune-activated conditions remain poorly characterized. The authors find that embryonically derived hemocytes in Drosophila larvae predominantly utilize mitochondrial respiration to generate energy and exhibit minimal glycolysis rates under unchallenged conditions. Hemocytes developmentally elevate ATP production rates. Mitochondrial respiration drives metabolic activation in larval hemocytes. More specifically, lamellocytes exhibit unique metabolic activities, including enhanced trehalose catabolism and mitochondrial remodeling, required for their encapsulation response.

Strengths:

The study shows the metabolism that is most likely to operate in different immune cells in Drosophila during development and also during infection. This is related to mitochondrial organization and proliferation and/or differentiation state.

Weaknesses:

Even though there is a rigorous analysis of mitochondrial activity using the Sea Horse analyzer, the analysis of diverse mitochondrial activities in the different immune cell types across development and in infection could be carried out using microscopy. ROS, mitochondrial membrane potential, NADH/+ and FADH/+ levels in vivo are likely to give a more specific readout of change in cellular activities. The activities of mitochondrial fusion and fission need to be collectively tested to understand their role in development and also in infection. The relevance of the change in mitochondrial activity for development or immunity remains to be tested.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

This manuscript addresses an important methodological issue-the fragility of meta-analytic findings-by extending fragility concepts beyond trial-level analysis. The proposed EOIMETA framework provides a generalizable and analytically tractable approach that complements existing methods such as the traditional Fragility Index and Atal et al.'s algorithm. The findings are significant in showing that even large meta-analyses can be highly fragile, with results overturned by very small numbers of event recodings or additions. The evidence is clearly presented, supported by applications to vitamin D supplementation trials, and contributes meaningfully to ongoing debates about the robustness of meta-analytic evidence. Overall, the strength of evidence is moderate to strong.

Strengths:

(1) The manuscript tackles a highly relevant methodological question on the robustness of meta-analytic evidence.

(2) EOIMETA represents an innovative extension of fragility concepts from single trials to meta-analyses.

(3) The applications are clearly presented and highlight the potential importance of fragility considerations for evidence synthesis.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have provided new data and text that addresses all of the reviewers' comments on the previous versions in a wholly satisfactory way.]

Summary:

This study presents evidence that addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg+2 ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

Strengths:

This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This represents a significant development in the long-term effort to produce synthetic cells.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

The paper from Hudait and Voth details a number of coarse-grained simulations as well as some experiments focused on the stability of HIV capsids in the presence of the drug lenacapavir. The authors find that LEN hyperstabilizes the capsid, making it fragile and prone to breaking inside the nuclear pore complex.

Comments on previous round of revisions:

I found that the authors addressed my concerns satisfactorily. The other reviewer raised a number of important points regarding the nuances of the model and the interpretation of the simulations, which the authors rebutted. I think the paper in its current form now is a worthwhile addition to the literature.

Reviewer #1 (Public review):

Summary:

Zinn and colleagues investigated the role of proteases 2A and 3C of enterovirus D68 (EVD68), an emerging pathogen associated with outbreaks of acute flaccid myelitis (AFM), a polio-like disease, on the nucleocytoplasmic trafficking in different systems, including human neurons derived from pluripotent cells. They found that 2A specifically cleaved Nup98 and POM121. Using reporter proteins and RNA synthesis and trafficking assays in cells expressing viral proteases, they showed that 2A induces broad loss of the nuclear pore barrier function, but, surprisingly, the RNA export appears to be minimally affected. Since nucleocytoplasmic trafficking defects are known to be associated with neuropatologies, they propose a hypothesis that 2A-dependent cleavage of nucleoporins in motoneurons underlies the development of EVD68-induced AFM. They further show that a 2A-specific inhibitor increases the survival of human neurons differentiated from stem cells upon EVD68 infection.

Strengths:

Use of multiple methods to investigate the effect of 2A and 3C expression on nucleoporin cleavage and nucleocytoplasmic trafficking.

Comments on revisions:

The following issues remain unresolved:

First, the authors still do not show representative images confirming specific nucleoporin degradation (Fig.1), which is the main focus of the work.

Second, the conclusion that 2A-mediated degradation of the nucleo-cytoplasmic barrier does not affect export of the RNA from the nucleus is not supported by the presented data. The representative images shown in Fig 3C do not have the signal for GFP (like in Fig. 2), and therefore it is impossible to see if those cells indeed express EVD68 proteases.

Moreover, to show RNA export, not only the decrease of nuclear EU signal should be quantified, but also the increase of the cytoplasmic signal. The diminishing of the nuclear staining may not necessarily reflect RNA export, but may well be explained by nuclease activity, all the more relevant in cells expressing 2A, where the nuclear-cytoplasmic barrier is disrupted and cytoplasmic nucleases may enter the nucleus.

The same applies to images in Fig. 3D. There are no markers of infection; moreover, the experiment description indicates that EU labeling began at 24 h post-infection with an MOI of 5, i.e., essentially all cells should have been infected. This is difficult to believe as the replication cycle of most EVD68 strains in HeLa cells is no longer than 12 h, yet the images do not show any signs of CPE, and demonstrate a strong EU signal, inconsistent with the expected inhibition of nuclear transcription, a known attribute of enterovirus infections.

The claim that nuclear transcription and RNA export remain unaffected in conditions of 2A-mediated disruption of the nucleo-cytoplasmic barrier is very strong and requires equally strong evidence.

Reviewer #1 (Public review):

Summary

Fogel & Ujfalussy report an extension of a visualization tool that was originally designed to enable an understanding of detailed biophysical neuron models. Named "extended currentscape", this new iteration enables visual assessment of individual currents across a neuron's spatially extended dendritic arbor with simultaneous readout of somatic currents and voltage. The overall aim was to permit a visually intuitive understanding for how a model neuron's inputs determine its output. This goal was worthwhile and the authors achieved it. Demonstrating the utility of extended currentscape, the authors leverage their models to generate interesting and detailed biophysical insights into widely studied neurophysiological phenomena with clear behavioral relevance. Overall, this study provides a valuable and well-characterized biophysical modeling resource to the neuroscience community.

Strengths

The authors significantly extended a previously published open-source biophysical modeling tool. Beyond providing important new capabilities, the potential impact of extended currentscape is boosted by its integration with preexisting resources in the field.

In keeping with the authors' goal to provide an approachable platform with intuitive visualizations of how current flows through neurons, the manuscript is approachable to non-computationalists. In particular, a dedicated glossary and elegant illustrations in Figure 2 boost accessibility for biologists.

Extended currentscape produces intriguing and detailed predictions spanning neurophysiological phenomena such as local dendritic spikes, complex spike generation, and feature selectivity (hippocampal place fields). By triggering analysis of modeled synaptic inputs on these events, the authors trace their origins from dendritic integration to synaptic input patterns.

The authors cleverly apply a graph theoretical approach to efficiently model bidirectional current flow throughout a neuron's dendritic arbor. As a result, extended currentscape can run on a standard personal computer.

The code is well-documented and freely available via GitHub.

Weaknesses

While extended currentscape meets its objective of modeling and illustrating the propagation of axial currents throughout a model neuron in great detail, it requires simulation and measurement of synaptic input currents. For this reason, there currently exists a very high technical barrier to conclusively test its intriguing predictions: simultaneous readout of synaptic inputs throughout a neuron's dendritic arbor. Mitigating this weakness, the authors propose a relatively more feasible alternative approach in Discussion: simultaneous voltage imaging of dendrites and their soma while estimating synaptic inputs from the distributions of voltage dynamics along individual dendritic branches.

Reviewer #1 (Public review):

Summary:

In this article by Xiao et al. the authors aimed to identify the precise targets by which magnesium isoglycyrrhizinate (MgIG) functions to improve liver injury in response to ethanol treatment. The authors found through a series of in-vivo and molecular approaches that MgIG treatment attenuates alcohol-induced liver injury through a potential SREBP2-IdI1 axis. The revised manuscript adds to a previous set of literature showing MgIG improves liver function across a variety of etiologies, and also provides mechanistic insight into its mechanism of action. All major weaknesses were addressed in the revised submission.

Strengths:

(1) The authors use a combination of approaches from both in-vivo mouse models to in-vitro approaches with AML12 hepatocytes to support the notion that MgIG does improve liver function in response to ethanol treatment.

(2) The authors use both knockdown and overexpression approaches, in-vivo and in-vitro, to support most of the claims provided.

(3) Identification of HSD11B1 as the protein target of MgIG, as well as confirmation of direct protein-protein interactions between HSD11B1/SREBP2/IDI1 is novel.

Weaknesses:

The authors addressed all my concerns.

Reviewer #2 (Public review):

In this paper, Biswas et al. describe the role of acetylcholine (ACh) signaling in protection against chronic oxidative stress in C. elegans. They showed that disruption of ACh signaling in either unc-17 mutant or gar-3 mutants led to sensitivity to toxicity caused by chronic paraquat (PQ) treatment. Using RNA seq, they found that approximately 70% of the genes induced by chronic PQ exposure in wild type failed to upregulate in these mutants. The overexpression of gar-3 selectively in cholinergic neurons was sufficient to promote protection against chronic PQ exposure in an ACh-dependent manner. The study points to a previously undescribed role for ACh signaling in providing organism-wide protection from chronic oxidative stress likely through the transcriptional regulation of numerous oxidative stress-response genes. The paper is well-written, and the data are robust. While the study identifies the muscarinic ACh receptor gar-3 as an important regulator of the response to PQ, the specific neurons in which gar-3 functions were not unambiguously identified, and the sources of ACh that regulate GAR-3 signaling and the identities of the tissues targeted by gar-3 remain unknown.

Comments on revisions:

No further comments.

Reviewer #1 (Public review):

Summary:

In the manuscript "Conformational Variability of HIV-1 Env Trimer and Viral Vulnerability", the authors study the fully glycosylated HIV-1 Env protein using an all-atom forcefield. It combines long all-atom simulations of Env in a realistic asymmetric bilayer with careful data analysis. This work clarifies how the CT domain modulates the overall conformation of the Env ectodomain and characterizes different MPER-TMD conformations. The authors also carefully analyze the accessibility of different antibodies to the Env protein.

Strengths:

This paper is state-of-the-art given the scale of the system and the sophistication of the methods. The biological question is important, the methodology is rigorous, and the results will interest a broad elife audience. The authors also establish strong connections to previous literature and acknowledge the limitations of the CT-truncated protein construct, which enhances the manuscript's relevance to the community.

Reviewer #1 (Public review):

Summary:

There is evidence that some genes encode mRNAs from which separate processed transcripts may arise, separating the coding sequence (CDS) from the 3'-UTR, and with both mRNA elements remaining stable in the cell. However, the functional consequences of these mRNA fragments have not been firmly established. In the manuscript by Yang et al., the authors probe the mRNA domain architecture of Nanog in the context of embryonic stem cell colonies and blastocysts. The authors detect spatial separation of Nanog CDS-containing mRNA from abundant Nanog 3'-UTR RNAs depending on the cell position in 2D embryonic stem cell colonies or in blastocysts.

Strengths:

The phenotypic analyses of the Nanog mRNA hold promise for revealing distinct roles for the Nanog encoded protein and a separate RNA encompassing the Nanog 3'-UTR.

Weaknesses:

There are a number of questions about the molecular nature of the mRNA species that the authors should address in order for the results to be firmly established, as noted below.

(1) It is not clear how the authors verified that their probes are specific for Nanog CDS or 3'-UTR regions. Especially for the 3'-UTR probe, it is confusing why colonies show green only regions, suggesting only the CDS is present. I would expect the CDS and 3'-UTR probes to colocalize in the interior cells. Is it possible that the 3'-UTR probe is targeting another RNA?

(2) It would help for the authors to include a graphic similar to Figure 3, Figure Supplement 1A, that diagrams the location of the CDS and 3'-UTR probes (this should also be done for Oct4 and Sox2). This graphic could also show all potential polyadenylation signals.

(3) I think, based on the fluorescence patterns, there is evidence that the signal for the Nanog 3'-UTR probe is nuclear (images with DAPI staining), but this is not commented on that I could find. This should be discussed, as nuclear retention has implications for the noncoding function of the 3'-UTR fragment.

(4) Figure 2, Figure Supplement 1A needs a better explanation. It's not clear how the reads map to the different regions of the Nanog mature mRNA. The authors should show examples at different ratios of CDS to 3'-UTR. Do the reads have a sharp boundary at the junction of where the isolated 3'-UTR is thought to occur?

(5) I looked in the Zenbu browser at human NANOG CAGE mapping in the FANTOM5 dataset. I could not see evidence for substantial capping of a 3'-UTR fragment when filtering for embryonic cell types. Given the strong signal for the 3'-UTR in border cells, I would expect to see evidence for capping if the RNA were indeed capped. This suggests that if it exists, it is likely uncapped and (as noted in point 3) is likely nuclear retained.

(6) Are there predicted polyadenylation signals near the end of the CDS that would generate a short 3'-UTR, and are these signals conserved across mammals?

(7) It would help to see a zoomed-in view of the region targeted by one of the guide RNAs in the 3'-UTR, and where that site is relative to the polyadenylation signal. Is the polyadenylation signal upstream, i.e., CDS proximal?

(8) A final note, the use of green and red together will be challenging for those who are colorblind. Providing a different false color palette would be helpful.

I am refraining from comments on the cell biology and morphological insights, as they are remote from my core expertise.

Reviewer #1 (Public review):

Summary:

Dalben et al. grafted the fusion loop mature (FLM) modification, based on a previously reported D2-FLM, to another serotype DENV4, and adapted them to replicate in Vero cells for live attenuated vaccine (LAV) manufacturing while retaining favorable antigenic profiles, generating two new strains: D2-vFLM and D4-vFLM. Deep sequencing revealed adapted mutations at the junction of envelope domains I and II (EDI and EDII), and both D2-vFLM and D4-vFLM showed no evidence of ADE in the presence of FL-targeting Abs. Sera from D2-vFLM immunized mice displayed strong homotypic and reduced heterotypic neutralization compared to wild-type viruses, with minimal to no ADE potential in vitro. Moreover, D2-vFLM immunization completely protected AG129 mice from lethal challenge with mouse-adapted D220. They demonstrate that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk. The FLM approach provides a promising path toward the development of a safer tetravalent DENV LAV.

Strengths:

The authors carried out a series of experiments to generate and characterize two new strains (D2-vFLM and D4-vFLM) of FLM-modified viruses, and showed their antigenic and immunogenic profiles. The observation that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk is interesting.

Weaknesses:

However, one concern is the total number of mutations (including originally introduced and compensatory mutations) in this FLM vaccine platform, and it is not clear regarding the future directions for the proof-of-concept vaccine in this study.

Reviewer #1 (Public review):

Summary:

The authors present a simplified neural bursting model with explicitly controllable parameterization of oscillator dynamics designed for neural circuit modeling involved in rhythm generation.

Strengths:

(1) The purpose of the model and applied abstractions are well articulated and justified (2D model, independent parameter control).

(2) Explicit control of burst duration, inter-burst interval, amplitude, resetting-behavior/entrainment. This allows modelers to focus on circuit interactions and is especially useful when details of intrinsic currents and bursting mechanisms are unknown. One could even imagine a scenario where this model would help identify predictions on key underlying burst generation mechanisms.

(3) The model is well described and validated with simulations and comparisons to the base model and one alternative model.

(4) Circuit-level validation is convincing, as it reproduces not only trivial examples.

(5) The underlying mechanism in phase space is well reasoned and justified, extends previous work, e.g., by McKean, by improving usability.

Weaknesses:

(1) The paper heavily relies on numerical demonstrations but does not provide a formal analysis of stability, bifurcations, or entrainment. While appropriate for the intended purposes, a more formal footing could strengthen the model.

(2) Lots of nice demonstrations are shown, but it is less clear how model parameterization was chosen, how behavior depends on parameterization, and in what parameter ranges certain behavior can be expected. A more detailed description of parameterization/exploration of parameter space would greatly benefit anyone using this model in the future.

(3) Some claims on reproduction of prior locomotor CPG model and production of "more biologically realistic activity" by the presented model are overstated. The key feature of the locomotor CPG models cited was that they not only reproduced speed-dependent gait expression of intact mice, but also changes of gait expression after silencing/removal of specific commissural and long propriospinal interneurons (e.g., selective loss of trot after deleting of V0V; changes in gait expression and step-to-step variability after silencing of descending long-propriospinal neurons or ascending V3 LPNs). While likely (at least partially) feasible with the model formulation, the correspondence of these silencing/ablation of neuron classes has not been shown by the model. Importantly, though, it appears that authors didn't show how the model in general behaves under the influence of noise, which is key to reproducing LPN silencing.

Reviewer #1 (Public review):

The idea is super interesting, and the subsequent work is potentially significant because it links peripheral inflammation to remodelling of perinodal adipose tissue and draining lymph nodes. This suggests an antigen-independent manner by which local tissue inflammation can communicate with and reshape immune organ structure and tissue metabolism. However, the evidence is suggestive. For instance, many conclusions rely on correlational weight/cellularity relationships, models with confounders (spontaneous wounding; potentially systemic IMQ), and macrophage dependence inferred from a single pharmacologic approach without definitive depletion/lineage or tracer-based causal link.

Major Comments:

(1) "Wounding/fighting" evidence is confounding.

Unless I am mistaken, a large part of the argument for inflammation-driven perinodal fat pad atrophy and LN expansion relies on spontaneous fighting injuries in co-housed CCR2-/- males, including animals "culled...due to excessive wounding." Because wound severity, duration, infection load, stress, and cage dynamics are uncontrolled, isn't it difficult to assign causality to "cutaneous inflammation"?

(2) The "CCR2-independent macrophage" conclusion.

The manuscript interprets persistence/accumulation of macrophages despite reduced inflammatory monocytes as CCR2-independent recruitment or local proliferation. However, CCR2 deficiency can alter immune baselines and long-term tissue remodelling. Perhaps consider bone marrow chimeras (WT to CCR2-/-, CCR2-/- to WT ????) or an inducible CCR2 deletion approach to separate developmental/systemic effects from acute inflammation-driven mechanisms. If "in situ proliferation" is proposed, include a direct readout (e.g., Ki67 in ATMs in the fat pad).

(3) IMQ and systemic effects.

The work relies on topical Aldara/imiquimod as an "inflammation without antigen" driver of distal LN/fat-pad remodelling. But IMQ is well known (and cited by the authors) to enter circulation and drive systemic responses, which could blur whether effects are truly draining-site specific vs systemic metabolic/inflammatory effects. It would be ideal to provide systemic context: plasma cytokines and/or metabolic readouts (e.g., circulating FFAs) to distinguish local vs systemic drivers.

(4) Macrophage dependence is inferred from CSF1R inhibitor treatment.

However, validation of macrophage depletion and specificity is incomplete. The manuscript uses AZD7507 (CSF1R inhibitor) and observes partial rescue of fat pad/LN phenotype while skin severity (PASI) is unaffected. But, to this reviewer, the data shown do not clearly quantify actual macrophage depletion efficiency in the target fat pad, and LN at endpoint, and CSF1R blockade can affect multiple myeloid populations. Therefore, show absolute macrophage counts (and likely other myeloid populations) in fat pad and LN with/without AZD7507 at the analysed timepoints, not only outcome weights. (The methods describe dosing but not endpoint depletion quantification??)

(5) Fat pad atrophy/LN expansion is a correlation.

The paper emphasises negative correlations between fat pad and LN weights/cellularity at baseline and with inflammation. But correlation does not establish whether fat pad lipolysis drives LN expansion, whether LN changes drive fat remodelling, or whether both reflect systemic mediators. Add tissue-level evidence distinguishing true adipocyte loss vs other contributors to "weight change" (e.g., oedema/fibrosis).

(6) Evidence for "fatty acid donation" from fat pad to LN.

The lipid data are described as "exemplary," and the inference that LN fatty acids originate from the fat pad is based on temporal ordering and relative abundance. This does not rule out plasma spillover, LN-intrinsic metabolism, or altered lymph flow.

Joint Public Review:

Summary:

Calle-Schuler et. al. reconstruct all the pre- and post-synaptic neurons to the bristle mechanosensory neurons on the adult fly head to understand if neural circuits support the parallel mechanosensory pathways, which could be instrumental in shaping the sequential motor patterns during fly grooming. They find that most presynaptic neurons, interneurons and excitatory post synaptic neurons are also somatotopically organized, such that each neuron is more connected to bristles mechanosensory neurons that are closer on the head and less connected to bristles mechanosensory neurons that are further away. These include the direct BMN-BMN circuits, excitatory interneurons, as well as the inhibitory networks. They also identify that the one entire hemi-lineage 23b form excitatory postsynaptic circuit with BMNs, highlighting how these circuits and hence their function could be developmentally determined.

Strengths:

This is a complete map of the all the neurons which make 5 or more pre- and post-synaptic connections of the fly head BMNs. Using this, the authors have identified various trends such as ascending neurons provide most of the GABAergic inhibitory input, which could provide the presynaptic inhibition essential for the parallel model for sequential grooming generation. Moreover, they identified that the entire cholinergic hemilineage 23b is postsynaptic to BMNs. Both their excitatory postsynaptic connectivity and inhibitory presynaptic connectivity demonstrate core motifs of the parallel circuits necessary for the hierarchical suppression model of grooming sequence.

Weaknesses:

Somatotropic organization with hierarchical suppression is an elegant mechanism to generate sequential motor sequence during grooming. Yet, anatomical connectivity alone, in absence of functional connectivity, cannot explain the grooming motor sequences. Future work should be aimed at mapping the functional connectivity with behavioral sequence.

Closing statement:

The authors have addressed the major concerns regarding clarity, scope, and interpretation. The manuscript is now significantly improved and is clearly framed as an anatomical resource that identifies circuit motifs consistent with existing models of grooming control.

Reviewer #1 (Public review):

Summary

The strength of this manuscript lies in the behavior: mice use a continuous auditory background (pink vs brown noise) to set a rule for interpreting an identical single-whisker deflection (lick in W+ and withhold in W− contexts) while always licking to a brief 10 kHz tone. Behaviorally, animals acquire the rule and switch rapidly at block transitions and take a few trials to fully integrate the context cue. What's nice about this behavior is the separate auditory cue, which shows the animals remain engaged in the task, so it's not just that the mice check out (i.e., become disengaged in the W- context). The authors then use optical tools, combining cortex-wide optogenetic inactivation (using localized inhibition in a grid-like fashion) with widefield calcium imaging to map what regions are necessary for the task and what the local and global dynamics are. Classic whisker sensorimotor nodes (wS1/wS2/wM/ALM) behave as expected with silencing reducing whisker-evoked licking. Retrosplenial cortex (RSC) emerges as a somewhat unexpected, context-specific node: silencing RSC (and tjS1) increases licking selectively in W−, arguing that these regions contribute to applying the "don't lick" policy in that context. I say somewhat because work from the Delamater group points to this possibility, albeit in a Pavlovian conditioning task and without neural data.

The widefield imaging shows that RSC is the earliest dorsal cortical area to show W+ vs W− divergence after the whisker stimulus, preceding whisker motor cortex, consistent with RSC injecting context into the sensorimotor flow. A "Context Off" control (continuous white noise; same block structure) impairs context discrimination, indicating the continuous background is actually used to set the rule (an important addition!) Pre-stimulus functional-connectivity analyses suggest that there is some activity correlation that maps to the context presumably due to the continuous background auditory context. Simultaneous opto+imaging projects perturbations into a low-dimensional subspace that separates lick vs no-lick trajectories in an interpretable way.

In my view, this is a clear, rigorous systems-level study that identifies an important role for RSC in context-dependent sensorimotor transformation, thereby expanding RSC's involvement beyond navigation/memory into active sensing and action selection. The behavioral paradigm is thoughtfully designed, the claims related to the imaging are well defended, and the causal mapping is strong.

Comments on revisions:

The authors have been responsive to the prior review and I think the manuscript is a valuable and important addition to the literature.

Reviewer #1 (Public review):

Summary:

The study examined the extent to which children's word recognition skill improves across early development, becoming faster, more accurate and less variable, and the extent to which word recognition skill is related to children's concurrent and later vocabulary knowledge.

The main strength of the study comes from the dataset which recycles previously collected data from 24 studies to examine the development of word recognition skill using data from 1963 children. This maximizes the impact of previously collected data while also allowing the study to reliably ask big picture questions on the development of word recognition skill and its relation to chronological age and vocabulary knowledge. Data analysis is rigorous, thought through and very clearly described. Data and code necessary to reproduce the manuscript are shared on the project's Github. The limitations of the study are acknowledged and the manuscript does well to tone down the causal implications of their results.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the role of the microtubule-binding protein EML3 during cortical development through the generation and characterization of an Eml3 mouse mutant. The authors focus mainly on the effects of EML3 loss on brain development, although Eml3 mouse mutants also present with developmental delay and growth restriction, and die perinatally due to respiratory distress caused by delayed maturation of the lungs. The main finding in the developing cortex is the presence of focal neuronal ectopias, which contain neurons from all cortical layers, as revealed by immunostaining. The authors use electron microscopy to show that ectopias seem to be caused by disruption to the pial basement membrane at early stages of development, which allows neurons to breach through it. To find a functional link between EML3 and the observed phenotype, studies are conducted that demonstrate expression of EML3 in radial glia cells and mesenchymal cells, both cell types involved in the formation and maintenance of the pial basement membrane. Furthermore, interaction partners for EML3 are identified through coIP-MS analysis, including tubulin beta-3, 14-3-3 proteins and cytoplasmic dynein light chain. However, mice carrying a mutant EML3 allele engineered to abolish the interaction between EML3 and cytoplasmic dynein light chain do not recapitulate any of the symptoms of complete EML3 loss.

Strengths:

The manuscript offers several important strengths that contribute significantly to the field. This study presents the first characterization of Eml3 knockout animals, providing novel insights into the role of Eml3 in vivo. Information on Eml3 function so far was restricted to cell culture data, so the results in this manuscript start to fill an important gap in our knowledge about this microtubule-binding protein. The experimental approach is carefully designed, with appropriate controls that ensure the reliability of the data. Moreover, the authors have addressed a key challenge in the analysis, namely the developmental delay of the knockout animals. By implementing a strategy to match developmental stages between wild-type and knockout groups, they allow for meaningful and valid comparisons between the two genotypes. Importantly, the authors have successfully generated three different Eml3 mutant mouse lines (knockout, floxed and with disrupted binding to cytoplasmic dynein light chain), which are very valuable tools for the broader scientific community to further study the roles of this gene in development and disease in the future.

Weaknesses:

While the manuscript presents valuable data, there are also several weaknesses that limit the overall impact of the study. Most notably, there is no clear mechanistic link established between the loss of Eml3 function and the observed phenotype, leaving the biological significance of the findings somewhat speculative, as it is not straightforward how a microtubule-associated protein can have an impact on the stability of the pial basement membrane. In this respect, but also in general for the whole manuscript, there seems to be a considerable amount of experimental work that has been conducted but is not presented, possibly due to the negative nature of the results. Additionally, the phenotype reported appears to be dependent on the genetic background, as it is absent in the CD1 strain. This observation raises concerns as to how robust the results are and how much they can be generalized to other mouse strains, but, more importantly, to humans.

Reviewer #1 (Public review):

[Editor's note: this version has been assessed by the Reviewing Editor with further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

Urination requires precise coordination between the bladder and external urethral sphincter (EUS), while the neural substrates controlling this coordination remain poorly understood. In this study, Li et al. identify estrogen receptor 1-expressing neurons (ESR1+) in Barrington's nucleus as key regulators that faithfully initiate or suspend urination. Results from peripheral nerve lesions suggest that BarEsr1 neurons play independent roles in controlling bladder contraction and relaxation of the EUS. Finally, the authors performed region-specific retrograde tracing, claiming that distinct populations of BarEsr1 neurons target specific spinal nuclei involved in regulating the bladder and EUS, respectively.

Strength:

Overall, the work is done with high quality. The authors integrate several cutting-edge technologies and sophisticated, thorough analyses, including opto-tagged single unit recordings, combined optogenetics and urodynamics, particularly those following distinct peripheral nerve lesions.

Comments on revised version:

During the revision, the authors have adequately addressed my concerns and made the suggested changes accordingly. I have no additional comments.

Reviewer #1 (Public review):

The authors point out that the fitness estimates obtained from different experimental assays (monoculture, pairwise competition or bulk competition) are not generally equivalent, not even with regard to the fitness ranking of different genotypes. Using a computational model based on experimentally measured growth phenotypes for knockout strains in yeast, as well as data from Lenski's Long Term Evolution Experiment (LTEE), they derive a set of best practice rules aimed at extracting the optimal amount of information from such experiments.

The study is very complete on a technical level, and the conceptual weaknesses raised in the first round of reviews have been fully addressed in the revision.

Reviewer #1 (Public review):

Summary:

This study presents evidence that addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg+2 ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

Strengths:

This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This represents a significant development in the long-term effort to produce synthetic cells.

Weaknesses:

- The authors carried out additional experiments indicating that ~60% of the reconstituted ribosomes are functional and that a significant proportion are capable of synthesizing GFP from the correct initiation codon to the correct stop codon, and also of producing an enzymatically active protein at appreciable levels. Their SDS-PAGE and MS analyses of N-terminally tagged GFP are also quite useful but did not assess the frequency of initiation at the wrong start codon, termination at the incorrect stop codon, or the frequency of frameshifting during elongation. This would require examining additional reporters designed to examine dependence on a Shine-Dalgarno sequence or the impact of an in-frame stop codon to assess the fidelity of initiation and termination events, respectively, and one with a programmed frameshift site to assess the elongation fidelity of their reconstituted ribosomes.

- Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs that, if successful here, would have eliminated the possibility that one or more unknown ribosome assembly factors that co-purify with native ribosomes was added to their reconstitution reactions.

Reviewer #1 (Public review):

Summary:

This is an important study that describes the consequences of the DNMT3A mutation in human neuronal development for the first time. The selective impact of DNMT3A function on GABAergic interneurons is interesting and an important feature of future therapeutics. The claims made in that manuscript are supported by strong evidence for the most part. And the data are of high quality in general and presented well.

Strengths:

The strengths of the work include: Characterization of multiple DNMT3A loss-of-function alleles, including two misense variants, R882H, P904L, and a deletion allele. The missense mutation lines both include an ideal control with the same genetic background. The CRISPRi-mediated DNMT3A knockdown has also been included. The study identifies the mTOR-PI3K pathway as a factor of overgrowth issues found in the mutant organoid. In bulk mRNA sequencing and whole-genome bisulfite sequencing, identify hypomethylated genomic regions associated with gene expression repression. Again, this is more pronounced in the ventral organoid compared to the dorsal organoid. In addition, the extensive electrophysiological characterizations with a high-density microelectrode array support the more mature status of mutant interneurons.

Weaknesses:

Although a strong study overall, some weaknesses are noted. These include:

(1) The lack of validation data for the generated iPSCs and hESCs, such as the chromosomal contents, ploidy, and pluripotency states.

(2) Other weaknesses relate to data interpretation and insufficient discussion of related matters, as detailed in the recommendations to the authors.

(3) Also, some errors are noted and detailed in the recommendation section.

Reviewer #1 (Public review):

Kawamura et al. investigated the role of circumferential smooth muscle contractions in chick gut tube elongation, addressing the hypothesis that "peristaltic activity generated by the gut promotes its own elongation during embryogenesis". Although not acknowledged in the current manuscript, this interesting premise was, in fact, previously demonstrated.

Indeed, the experiments in the present manuscript closely parallel a previous study (Khalipina et al, 2019: "Smooth muscle contractility causes the gut to grow anisotropically") that also cultured chick gut tissue and performed time-lapse analyses to quantify peristalsis. Both studies showed that inhibiting peristalsis with Ca-channel blockers induces a switch from elongational to radial growth in the gut.

However, one of the main strengths of the current study is the innovative use of optogenetic manipulation to rescue gut lengthening in drug-inhibited gut tissue by re-stimulating peristaltic contractions. In addition, the authors use aphidicolin to show that peristalsis-mediated gut elongation is independent of cell division. They also track individual smooth muscle cells and show that they divide circumferentially, but become redistributed along the length of the gut tube with peristalsis.

While these data are solidly quantitative, they do not provide mechanistic insight into how peristaltic contractions cause smooth muscle cells to be redistributed.

The evidence presented in this manuscript supports the main conclusion that peristalsis plays a critical role in embryonic gut elongation, but this conclusion itself is not novel. In addition to corroborating previous work, this manuscript provides some useful additions to our existing knowledge of the role of mechanical forces in embryonic gut morphogenesis and illustrates the utility of a previously published optogenetic manipulation technique.

Reviewer #1 (Public review):

Summary:

Zhang et al. report on an ambitious study that investigates multiple aspects of the neural and behavioral underpinnings of auditory-motor surprisal in the context of an auditory-motor learning paradigm (piano keyboard). Using an intricate design comprising several sub-parts and control procedures, they report that early ERPs (50-100 ms latency) reflect violations of established key-pitch mappings.

Strengths:

This is a carefully devised and executed study. The paradigm is quite intricate and, at the same time, addresses multiple aspects of auditory-motor learning, and does so in a rigorous way.

Weaknesses:

Perhaps because of the exhaustive approach, it is sometimes difficult to follow which parts of the experimental design the results come from; there are some questions regarding appropriate statistical methods, the inclusion/treatment of musical background in participants, and the nature (latency & extent) of the identified neural components that detect auditory-motor violations.

Reviewer #1 (Public review):

Summary:

Osswald and colleagues aim to show how motor units of the first dorsal interosseous (FDI) are flexibly recruited across two functionally different movements: index finger abduction and index finger flexion. They motivate this by arguing that FDI is the prime mover in abduction but acts as a synergist in flexion, alongside flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS) as the prime movers. This is a worthwhile question because it speaks to how descending neural inputs to the spinal cord flexibly control movement.

The authors claim that recruitment order and recruitment threshold of FDI motor units differ between abduction and flexion, and that beta-band intramuscular coherence is reduced when FDI acts as a synergist. However, there are significant methodological concerns that undermine the results and conclusions.

Strengths:

The study certainly aims to address a central question in motor neuroscience - how flexible recruitment of motor units occurs across movements where the same muscle changes its functional role. They correctly identify the FDI as a multi-functional muscle and use intramuscular high-density EMG arrays to record several motor units simultaneously, which is a major technical strength. They also track individual motor units between conditions and, therefore, have generated a potentially valuable dataset for studying spinal motor control across different movements.

Weaknesses:

The key limitation comes from the authors' interpretation of "neural drive" to FDI. The authors acknowledge that global EMG during flexion is smaller than that during abduction (for the same force), and surmise that the FDI receives different amounts of neural drive between these two movements, which is a potential confound for their analyses. To match the neural drive (i.e., global EMG), the authors ask participants to generate the same global EMG in flexion as in abduction; the forces generated by FDI are significantly different (2-3N for abduction and 1-8-6.2 for flexion). From this, they find changes in recruitment order, recruitment threshold, and beta coherence. However, different FDI motor units (and different muscle fibres) are active during abduction versus flexion. Using global EMG as a proxy for neural drive ignores this spatial separation of EMG generation during abduction and flexion, such that some amount of global EMG generated by one part of FDI (during abduction) is considered the same (from a neural drive perspective) as the same amount of EMG generated by a completely different part of FDI (during flexion). But these two global EMGs (during abduction and flexion) are not biologically equivalent because they are generated by different motor units and muscle fibres. Consequently, neural drive during flexion and abduction is not equivalent, which makes biological interpretation less clear. Furthermore, it is difficult to tell if abduction-versus-flexion differences are due to task role (prime mover vs synergist) or differences in force/mechanical demands, multi-muscle coordination, and spatial sampling limits of intramuscular recordings.

As mentioned, we think that the question asked is a very interesting one and framed appropriately to investigate the behaviour of motor units during prime mover and synergist roles. Simultaneously recording the prime movers for index flexion (FDP and FDS) would significantly improve the completeness of the study and allow for multi-muscle comparisons that are more relevant to how the motor system resolves prime mover vs synergist roles.

The authors use motor unit action potential as a proxy for motor unit size. This is not suitable because muscle fibres closer to the electrode will appear larger, independent of their true size. We advise that the authors remove analyses pertaining to motor unit size if it cannot be accurately measured.

Finally, several mechanistic interpretations in the discussion (e.g., spinal interneuronal suppression, reduced corticospinal input, proprioceptive mechanisms) read as more speculative than the current data can support without added controls or citations.

Reviewer #1 (Public review):

Summary:

In this work, the authors study the migration of isolated cells and of cells in ensembles. They quantify several aspects of the corresponding migration patterns and investigate how these quantities depend on molecules that are known to play an important role in migration. Furthermore, they study the effect of external cues on these migration processes.

Strengths:

The authors provide a clean and uniform setting for comparing the migration of isolated cells and of cells in an ensemble in control and mutant conditions, and in the presence and absence of external cues. This allows for a meaningful comparison between different conditions. In this way, the authors obtain useful data that link the migration of isolated cells to that of cells in collectives.

Weaknesses:

A major weakness of the manuscript is that the authors do not properly introduce the quantities and concepts they are working with. In this way, it is hardly accessible for a reader who does not have a thorough background in cell migration and anomalous transport. In addition, the manuscript uses some notions that are not standard, for example, vinculin or FA stability, which are not properly introduced. Most strikingly, "collective directional memory" is not defined.

The authors infer relationships between different quantities, but they remain qualitative, even though the authors use a language that suggests otherwise. For example, "The combination of Focal Adhesion stability and force transmission from the cytoskeleton predicts the migration speed of single cells" (p 2). I am not sure what is meant by prediction, but this heading suggests that knowledge of FA stability and force transmission yields the migration speed. Reading this line, I expect that if I give you values for FA stability and force transmission, you would give me a value for the migration speed. Such a quantitative mapping is not provided. In fact, it cannot be provided, because - as mentioned before - these quantities are not properly defined, so I would not know how to measure them. I do not even know their units.

Furthermore, the authors do interpret some of their results without explaining or justifying the basis for their interpretation. For example, they use the FRET index of vinculin - another notion that is not properly introduced - to make statements about mechanical stress.

It also seems that the figures could be improved. Some of the sketches are, in my opinion, not helpful. Examples are Figure 3A (how could a cell move while the hexagonal arrangement of the cells is maintained?) or Figures 2F, 4F, and 6F (what do the colored ellipses indicate?). In Figures 1B, 1D, 2A, 2E, 3B, 3D-F, 4A, 4F, 5B-D, it is not clear which lines merely connect data points and which lines are fits to the data.

Reviewer #2 (Public review):

[Editors' note: this version has been assessed by the Senior Editor without further input from the original reviewers. The authors have addressed the minor comments raised in the previous round of review.]

Summary:

This study uses dental traits of a large sample of Chinese mammals to tract evolutionary patterns through the Paleocene. It presents and argues for a 'brawn before bite' hypothesis -- mammals increased in body size disparity before evolving more specialized or adapted dentitions. The study makes use of an impressive array of analyses, including dental topographic, finite element, and integration analyses, which help to provide a unique insight into mammalian evolutionary patterns.

Strengths:

This paper helps to fill in a major gap in our knowledge of Paleocene mammal patterns in Asia, which is especially important because of the diversification of placentals at that time. The total sample of teeth is impressive and required considerable effort for scanning and analyzing. And there is a wealth of results for DTA, FEA, and integration analyses. Further, some of the results are especially interesting, such as the novel 'brawn before bite' hypothesis and the possible link between shifts in dental traits and arid environments in the Late Paleocene. Overall, I enjoyed reading the paper and I think the results will be of interest to a broad audience.

Weaknesses:

For the original draft of the manuscript, I had four major concerns with the study, especially related to the sampling, diet, and evidence for the 'brawn before bite' hypothesis. I still believe that the original issues that I raised may be weaknesses of the study. For example, there is still limited discussion on diets (even though the dental topographic analyses used in the study are designed for inferring diets). And I find the results a little challenging to interpret because teeth of multiple positions are included in the same samples, which seems problematic. That said, the authors have addressed each of my previous concerns and have made major revisions, including running new analyses, and thus I support the paper.

Reviewer #1 (Public review):

The revised manuscript includes several useful additions, and I appreciate the efforts to clarify parts of the analysis. The dataset remains valuable. However, several key issues raised previously are not yet fully resolved and continue to limit the clarity of the main conclusions.

(1) I appreciate that the authors guide the reader to the relevant regions in the analysis of chromosome fusions (Fig. 2b). However, these subtelomeric regions are not clearly visualized, making it difficult to compare fused and unfused profiles, even though the conclusions rely largely on visual inspection of them. A more direct comparison between fused and unfused ends, together with quantitative summaries (e.g., binned Red1 enrichment and comparisons with internal regions), would make this experiment more convincing.

(2) The SK1/S288c comparison (Fig. 2c) is an excellent approach, but is currently presented just as profiles, which again requires substantial effort from the reader to extract the relevant information. A systematic analysis across all informative chromosome ends-for example, comparing Red1 levels in syntenic regions using binned log2 fold-change-would more directly test the proposed in cis effect (L168) and clarify the contribution and range of Y'-associated effects. Other factors (e.g. distance from chromosome ends) could also be assessed within this framework.

Related to this, it is unclear if Y' elements themselves exhibit lower Red1 binding than the genome average. Providing the mean Red1 signal per Y' element would clarify this point and may also aid interpretation of the relationship between coding density and Red1 enrichment.

(3) The Dot1-Sir3 section is now simpler. However, I still find it difficult to follow the underlying rationale. In particular, it is unclear why a Dot1 function dependent on H3K79 methylation is introduced, given that the data in the previous section suggest H3K79 methylation is dispensable for subtelomeric Red1 depletion. A clearer statement of the authors' working model would be helpful.

Reviewer #1 (Public review):

In this paper, Solyga, Zelechowski & Keller study human visuomotor mismatch responses as an alternative instantiation of prediction errors to classic oddball paradigms. Using VR, they created a condition in which participants were moving around thereby creating a visuomotor coupling between physical movement and visual flow. To attempt to isolate the contribution of specifically movement-related predictions in this condition, they contrasted it to a condition in which participants were seated and rewatching their movement trajectory during the 'active' condition. Visuomotor mismatches were created by temporarily decoupling movement and visual experience by halting the VR display as participants continued to move.

The core finding of the paper is that participants exhibit a positively-valenced response to the visuomotor decoupling in the active but not in the passive condition. Since walking speed only insignificantly slows down following decoupling events in the active conditions, the authors argue that this difference can not be accounted for by "changes in participants' behavior or to simple visual offset responses" with the latter being equal across both conditions. The following reinstatement of the coupling in turn does not differ between the two conditions. The authors additionally show that this mismatch response differs from visual onset responses elicited by checkerboard inversions and that it's "qualitatively" stronger than more commonly studied auditory oddball mismatch responses.

The design with its focus on ecological validity is impressive, well-rationalized and the results are well illustrated. I additionally appreciate the control analyses with regards to changes in walking speed and playback DOF and, now added, additional participants who experience the passive condition before the active. I have a couple of questions/comments.

My main question in round 1 regarded the isolation of visuomotor mismatch. Although the comparison with a seated control seems like a very sensible way to control for simple visual responses, there seem to be more differences than just a break in visuomotor coupling between the conditions. I therefore wonder whether the reduced offset response in the seated condition may be, in part, explained differently. For example, given that participants always conduct the active condition before rewatching their movement in the seated condition, it seemed likely that there is a component of learning across the session that flow will sometimes be halted. This is confirmed with the analyses. The explanation that there is a visuomotor component here is given further weight by their conduction of an additional group of participants who perform the conditions in the reverse order, so this has strengthened the manuscript considerably. However, it does of course remain an imperfect control because the visual stimulus is now different between the conditions for these participants. It's the best that can be achieved with this type of paradigm though and of course it yields a great deal of ecological validity.

I was also wondering whether the authors may consider the findings in frontal electrodes more closely given that the title of the paper focuses on a specifically occipital effect. Their further analyses have confirmed that there are likely interesting frontal effects. From a theoretical point of view, the spatial dissociation in adaptation effects, which were stronger in frontal and weaker in occipital areas, seems interesting and perhaps worth discussing, especially given the interpretation that "mismatch processing may initially arise in sensory visual areas before engaging higher-order frontal regions." How come the frontal decrease in responses is not accompanied by an analogous decrease in its supposed occipital source? Could these two responses reflect different kinds of prediction error signals (i.e. objective vs subjective)?

I remain concerned that the authors fight too defensively that they have absolutely isolated visuomotor prediction mechanisms with this paradigm. It's a nice, informative study, but it seems odd to argue there are no other possible explanations. One picks a design to optimize some features but they will always come at some cost to others. Prioritising ecological validity, which is a justifiable aim, necessarily usually weakens some control over confounds.

To outline my reasoning fully: My concerns wrt generic influences of action on perception are reflected in Fig 1. The P1 is smaller when walking than sitting. It seems likely that the mismatch response reflects something about extrapolation or prediction, because it is larger when walking. However, it's not necessarily sensorimotor prediction. Even if you remove action from the equation, the flow can be extrapolated or predicted most of the time in a way it cannot so well when the video is halted. Of course the sitting condition somewhat controls for it, but when it came second the visual flow disruptions were more predictable here. A reduction in effects over time is indeed confirmed with their analyses. They now have conducted a study with the conditions in the reverse order and they find the same thing. But of course this necessitates non-identical visual flow because the sitting condition is playing the previous participant's flow. So it is likely that across all of these comparisons, it is the visuomotor mismatch that is especially salient. It's just that each comparison is a bit messy/confounded. It would strengthen the manuscript if there were some consideration given to the other processes likely at play here.

As a more minor point in response to our previous review, whether particular accounts represent an 'orthodox' view at present does not determine whether they raise logical issues in need of consideration. The authors may have missed that the papers in question consider mechanisms underlying the attenuation of particular pieces of information *from perception*. Not perceptual processing. We have one percept at any one moment in time and must understand how different population types synergistically generate that percept.

Similarly a little strange is the way in which the authors aggressively defend the position that self-generated motion is 'the strongest' type of prediction. Sure, we probably experience the effects of our actions more often than ambulances. But what about objects obeying laws of gravity or others' faces being structured and moving in systematic ways? It is hard to quantify, such that presumably many scientists would be skeptical of such a claim, and it is not needed logically to justify the importance of examining mechanisms enabling action to shape perceptual processing. I'd assume it better to fight the battles you need to (and can) fight, such that the robust claims carry more weight.

Hope these comments are helpful.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. We appreciate the revisions and the authors addressed all of the remaining minor concerns listed by the reviewers. We have no further suggestions for revision.]

Summary:

Rolland and colleagues investigated the interaction between Vibrio bacteria and Alexandrium algae. The authors found a correlation between the abundance of the two in the Thau Lagoon and observed in the laboratory that Vibrio grows to higher numbers in the presence of the algae than in monoculture. Timelapse imaging of Alexandrium in coculture with Vibrio enabled the authors to observe Vibrio bacteria in proximity to the algae and subsequent algae death. The authors further determine the mechanism of the interaction between the two and point out similarities between the observed phenotypes and predator prey behaviours across organisms.

Strengths:

The study combines field work with mechanistic studies in the laboratory and uses a wide array of techniques ranging from co-cultivation experiments to genetic engineering, microscopy and proteomics. Further, the authors test multiple Vibrio and Alexandria species and claim a wide spread of the observed phenotypes.

Comments on revisions:

I thank the authors for their additional work on the manuscript. My comments were addressed to my satisfaction.

Reviewer #1 (Public review):

Summary:

This paper presents an ambitious and technically impressive attempt to map how well humans can discriminate between colours across the entire isoluminant plane. The authors introduce a novel Wishart Process Psychophysical Model (WPPM) - a Bayesian method that estimates how visual noise varies across colour space. Using an adaptive sampling procedure, they then obtain a dense set of discrimination thresholds from relatively few trials, producing a smooth, continuous map of perceptual sensitivity. They validate their procedure by comparing actual and predicted thresholds at an independent set of sample points. The work is a valuable contribution to computational psychophysics and offers a promising framework for modelling other perceptual stimulus fields more generally.

Strengths:

The approach is elegant and well-described, and the data are of high quality. The writing throughout is clear and the figures are clean (elegant in fact) and do a good job of explaining how the analysis was performed. The whole paper is tremendously thorough and the technical appendices and attention to detail are impressive (for example, a huge amount of data about calibration, variability of the stim system over time etc). This should be a touchstone for other papers that use calibrated colour stimuli.

Comments on revised version:

The authors have addressed all the issues I raised to my satisfaction.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate whether glycogen phosphorylase is a potential molecular target of benzoylphenylurea insecticides and examine the physiological consequences of inhibiting glycogen breakdown in the diamondback moth Plutella xylostella. The authors express and characterize recombinant glycogen phosphorylase, test its inhibition by a mammalian glycogen phosphorylase inhibitor and by the insecticide diflubenzuron, and assess the physiological effects of glycogen phosphorylase inhibition through chemical exposure and RNA interference. Based on these experiments, the authors conclude that benzoylphenylurea insecticides do not target glycogen phosphorylase and propose that insects compensate for glycogen phosphorylase inhibition through activation of gluconeogenesis, allowing them to maintain glucose homeostasis and complete development despite strong suppression of the enzyme.

Strengths:

The study addresses an interesting and long-standing question in insect toxicology regarding the mechanism of action of benzoylphenylurea insecticides. The authors combine several complementary approaches, including recombinant enzyme characterization, inhibitor assays, RNA interference, gene expression analyses, and metabolite measurements. The biochemical characterization of the recombinant glycogen phosphorylase and the demonstration that the tested glycogen phosphorylase inhibitor can strongly inhibit enzyme activity represent important technical strengths. In addition, the study integrates biochemical and physiological observations to explore how insects might compensate for disruptions in central carbohydrate metabolism.

Weaknesses:

Several aspects of the central conclusions rely on indirect evidence and would benefit from additional validation. The proposed compensatory mechanism (gluconeogenesis supported by amino acid mobilization) is inferred primarily from transcriptional changes in gluconeogenic genes, reduced protein levels, and changes in metabolite concentrations. While these observations are consistent with increased gluconeogenic activity, they do not directly demonstrate metabolic flux through this pathway. Direct measurements of gluconeogenic flux would be required to confirm that carbon derived from non-carbohydrate substrates contributes to glucose production.

Some interpretations are also speculative. For example, the lack of glycogen accumulation following glycogen phosphorylase knockdown is attributed to alternative glycogen degradation pathways, such as α-amylase or glycogen debranching enzymes, but these possibilities are not experimentally examined. Measuring the expression or activity of these enzymes would help evaluate whether such pathways contribute to the observed metabolic response.

The physiological consequences of the proposed metabolic compensation are also not fully explored. If proteins are mobilized to support gluconeogenesis, this shift might be expected to affect organismal traits such as adult body size, flight capacity, or reproductive performance. Assessing these traits could provide valuable insight into whether the proposed compensatory metabolism carries fitness costs.

Finally, some conclusions extend beyond the direct evidence presented. The study shows that diflubenzuron does not inhibit glycogen phosphorylase in vitro, but broader conclusions regarding the mechanism of action of benzoylphenylurea insecticides as a class may require additional evidence. In addition, some biochemical and cell-based observations would benefit from confirmation in whole insects, given that metabolic regulation can differ substantially between isolated enzyme or cell-based systems and intact larvae, where hormonal signaling, tissue interactions, and nutrient availability influence metabolic responses.

Reviewer #1 (Public review):

Summary:

The authors aimed to determine whether dietary conditioning of fecal microbiota donors can influence the therapeutic efficacy of fecal microbiota transplantation (FMT) in alcohol-associated liver disease (ALD). Specifically, they tested whether donor diets enriched in vegetable or egg-derived proteins alter microbiota composition and function in ways that enhance recovery from alcohol-induced liver injury. Using a murine ALD model, the study integrates microbiome profiling, metabolomics, proteomics, and functional assays to identify mechanisms underlying improved outcomes. The authors propose that vegetable protein-conditioned microbiota promote beneficial microbial remodeling and increased production of caproic acid, which in turn activates hepatic PPARα signaling and enhances fatty acid β-oxidation, thereby reducing steatosis and inflammation.

Strengths:

The study is ambitious and methodologically comprehensive. The central idea, that donor diet can modulate FMT efficacy in ALD, is compelling and potentially impactful. It combines in vivo disease models, microbiome analysis (16S rRNA sequencing), metabolomics and proteomics, pharmacological inhibition experiments, and in vitro validation in hepatocytes. This multi-layered approach is a clear strength and allows the authors to explore the gut-liver axis. The comparison between different protein sources (vegetable vs egg) is very interesting, and the PPARα inhibition experiments provide relatively strong functional support for the involvement of host metabolic signaling pathways in mediating the observed effects.

Weaknesses:

Despite the comprehensive scope of the manuscript, several aspects of the study limit the strength of its mechanistic conclusions. The causal attribution to caproic acid remains incomplete. While caproic acid is identified and functionally tested, there is no direct demonstration that it is necessary for the Veg-FMT phenotype in vivo. The metabolomics data suggest multiple candidate metabolites, but these are not systematically explored. The study identifies specific bacterial taxa and, separately, key metabolites, but does not establish a direct connection between microbial composition and metabolite production. The use of GW6471 supports involvement of PPARα but does not fully establish specificity, as off-target effects cannot be excluded. Finally, it is not fully clear whether effects are exclusively microbiota-driven or could partially reflect the transfer of diet-derived metabolites.

The authors successfully demonstrate that donor dietary conditioning influences the therapeutic efficacy of FMT in a murine model of ALD. The data convincingly show that vegetable protein-conditioned microbiota is associated with improved liver injury, reduced inflammation, and enhanced intestinal barrier integrity compared with controls or an egg protein-enriched diet. While the proteomic and gene expression data suggest activation of pathways related to fatty acid β-oxidation, these measurements do not directly demonstrate increased metabolic flux. The use of the PPARα antagonist GW6471 provides important functional support for the involvement of this pathway, as inhibition attenuates the protective effects of Veg-FMT. However, this approach primarily establishes pathway dependency rather than directly confirming enhanced β-oxidation activity. The authors may therefore wish to moderate their interpretation or clarify this distinction, particularly given the relatively modest fold changes observed in several targets. The role of caproic acid as a central mediator is plausible but not definitively established. Finally, the link between microbiota composition, metabolic function, and host signaling remains partly correlative. Overall, the study achieves its primary aim at a phenotypic level, but some of the mechanistic claims would benefit from more cautious interpretation or additional validation.

Likely impact of the work on the field, and the utility of the methods and data to the community:

The work addresses an important and underexplored question: how donor characteristics influence FMT efficacy. By introducing donor diet as a modifiable variable, the study has potential implications for optimizing microbiota-based therapies. The datasets (microbiome, metabolomics, and proteomics) may also be valuable to the community, as they provide a resource for exploring gut-liver metabolic interactions. The translational impact will, however, depend on validation in human systems and a clearer identification of causal mechanisms.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The review comments were minor and constructive, and the authors have been very responsive.]

Summary:

This brief piece by Swartz and colleagues outlines the complexities surrounding the choice of clinical specialty for physician-scientists. It is, in general, clear and well-written, and it will be useful to research-oriented medical students choosing a path and to the mentors who are guiding them.

Strengths:

The writing is clear. The points made are not profound, but they are important and will be of use to the intended audience.

Reviewer #1 (Public review):

Summary:

This paper describes a deep learning toolbox that can be used to automatically estimate functional topographic maps directly from human brain anatomy. Building on the first author's earlier work, which demonstrated the feasibility of using deep learning for this purpose, the new version of the toolbox now requires only a single anatomical MRI scan to generate predictions, eliminating the need for a myelin scan. This represents a significant practical improvement.

Strengths:

Having such a toolbox is very useful, since manual annotation and delineation of functional visual field maps is a laborious process that also requires deep expertise. The toolbox can save researchers substantial amounts of time and money, and also allows less experienced researchers to now perform this type of analysis. Notably, for certain participants and patients, the time they are able to reside in the scanner might be limited. Being able to focus on the primary research question, rather than the essential yet basic topographic information, could boost data quality and evaluation and might limit the number of participants that need to be included.

Weaknesses:

In the paper, the authors compare the performance of their new version to two previous approaches. Figure 2b shows that the new toolbox performs similarly to the previous deep-learning-based toolbox, but requires only an anatomical scan, which is a significant improvement. They also compare it to an older method that uses an atlas without requiring deep learning. For eccentricity and pRF size predictions, both deep-learning methods perform better than the older approach. For polar angle, a critical parameter for delineating visual field maps, the gain is substantially less. Moreover, the comparison to the atlas method (Benson2014) is not entirely fair, as, to our knowledge, there is also a more advanced atlas version that uses Bayesian fitting methods and already performs better than the old method. To better understand the gain of using deep learning, it would be beneficial if the authors also made the comparison to this more recent atlas-based approach. Moreover, it would be useful to know the correlations for the representative participant. Some examples of relatively "bad" maps would also be useful to have (and could be provided as supplementary information).

Figure 2b shows that the toolbox is quite good at estimating eccentricity and polar angle parameters, but less good at estimating the population receptive field (pRF) size. I will return to this latter point.

An interesting feature is that while the toolbox is trained on a specific data set (HCP), it can, "out-of-the-box", be applied to different existing data sets, without the need to retrain the model. This is quite important for the general utility of the method. The results for this are shown in Figure 3. Again, in panel b, it can be seen that the toolbox does a good job at estimating eccentricity and polar angle values, but performs rather poorly for pRF size: the deepRetinotopy toolbox has a strong tendency to only estimate very small pRFs, particularly when applying it across different datasets. For this reason, at the moment, these estimates appear hardly useful. It would be very helpful for readers if the authors could clarify or elaborate on this point, particularly regarding the limitations of pRF size predictions. They explain that this could be due to the use of different types of stimuli, but even within the same (HCP) dataset, the predictions primarily suggest tiny pRFs, even though the training dataset also contains larger ones (which can be better seen in supplementary Figure 4). Showing the predictions for higher-order brain areas, which have larger pRFs on average, could serve a similar evaluation purpose. Presumably, the underlying reasons are complex and could relate to the use of different stimuli, different analysis toolboxes, and how the deep learning model is currently being trained. Possibly, the abundance of small pRFs at lower eccentricity in the training set (which is usually the case in any empirical analysis) has given the model a very strong bias toward predicting small pRFs.

There would be various ways to verify which of these components is critical. For example, the model could be trained only on the bar stimuli of the HCP dataset, or the pRFs for all stimuli and datasets could be estimated using the same software tool. The latter seems important. For example, Supplementary Figure 4 indicates a high correlation between the Stanford and NYU cohorts that have used the same stimulus and analysis package, despite having different resolutions and scanners. Further investigation into the underlying reasons for these discrepancies would strengthen the paper. It would also provide valuable guidance for users of the toolbox on which toolbox predictions to trust and which not, as well as how well the model generalizes to other stimulus types, scanners, and image resolutions.

An aspect that is not directly apparent from the title, abstract, and introduction is that the deepRetinotopy toolbox does not by itself produce estimates of visual area labels or boundaries. It predicts only polar angle and eccentricity values. To predict labels and boundaries, the authors combine the toolbox with an atlas (the aforementioned Bayesian atlas). For visual areas V1 - V3, it does a very good job, in that the predictions are as good as the empirical ones. Notably, the authors indicate that the predictions for V2 and, in particular, V3 are worse than for V1, but Figure 4 clearly shows that predictions are as good as the empirical ones. More cannot be expected from a model that is trained on such empirical data.

Irrespective of the limitations with respect to predicting pRF size, the toolbox opens up functionally oriented analyses of very large cohorts of healthy participants, of which only anatomical data is available. The authors present an example of this by confirming the existence of differences in horizontal and vertical asymmetries in the field maps of the visual cortex of children and adults. While Figure 5 confirms the existence of differences, the analysis could be expanded to provide deeper insights, such as normalized developmental trajectories for both asymmetries, given the size of the dataset. This would better highlight the true power of their approach.

While the authors address limitations with respect to studying experience-dependent atypical functional organization, they do not address how the deepRetinotopy toolbox would handle (acquired) brain lesions. Addressing this, even if only speculative, would be welcome. Another welcome addition would be to see the predictions for additional brain areas, even if those would (presumably) be worse at present. Such information would nevertheless be essential for users considering applying this toolbox. Moreover, this could be a valuable resource serving as a benchmark for future iterations of either deepRetinotopy or other approaches.

Reviewer #1 (Public review):

Summary:

In this manuscript, Emperador-Melero et al. seek to determine whether recruitment of endocytic machinery to the periactive zone is activity-dependent or tethered to delivery of active zone machinery. They use genetic knockouts and pharmacological block in two model synapses - cultured mouse hippocampal neurons and Drosophila neuromuscular junctions - to determine how well endocytic machinery localizes after chronic inhibition or acute depolarization by super-resolution imaging. They find acute depolarization in both models have minimal to no effect on the localization of endocytic machinery at the periactive zone, suggesting that these proteins are constitutively maintained rather than upregulated in response to evoked activity. Interestingly, chronic inhibition slightly increases endocytic machinery levels, implying a potential homeostatic upregulation in preparation for rebound depolarization. Using genetic knockouts, the authors show that localization of endocytic machinery to periactive zones occurs independently of proper active zone assembly, even in the absence of upstream organizers like Liprin-α.

Overall, they propose that the constitutive deployment of endocytic machinery reflects its critical role in facilitating rapid and reliable membrane internalization during synaptic functions beyond classical endocytosis, such as regulation of the exocytic fusion pore and dense-core vesicle fusion. Although many experiments reveal limited changes in the localization or abundance of endocytic machinery, the findings are thorough, and data substantially supports a model in which endocytic components are organized through a pathway distinct from that of the active zone. This work advances our understanding of synaptic dynamics by supporting a model in which endocytic machinery is constitutively recruited and regulated by distinct upstream organizers compared to active zone proteins. It also highlights the utility of super-resolution imaging across diverse synapse types to uncover functionally conserved elements of synaptic biology.

Strengths:

The study's technical strengths, particularly the use of super-resolution microscopy and rigorous image analyses developed by the group, bolster their findings.

Weaknesses:

One limitation, acknowledged by the authors, is the persistence of spontaneous activity at these synapses, which could still impact the organization of these regions.

Comments on revisions:

The authors have addressed all of my previous comments.

Reviewer #3 (Public review):

In this manuscript, the authors use HiC to study the 3D genome of CD14+ CD16+ monocytes from the blood of healthy and those from patients with Alcohol-associated Hepatitis.

Overall, the authors perform a cursory analysis of the HiC data and conclude that there are a large number of changes in 3D genome architecture between healthy and AH patient monocytes. They highlight some specific examples that are linked to changes in gene expression. The analysis is of such a preliminary nature that I would usually expect to see the data from all figures in just one or two figures.

In addition, I have a number of concerns regarding the experimental design and the depth of the analyses performed that I think must be addressed.

(1) There is a myriad of literature that describes the existence of cell-type-specific 3D genome architecture. In this manuscript, there is an assumption by the authors that the CD14+ CD16+ monocytes represent the same population from both the healthy and diseased patients. Therefore, the authors conclude that the differences they see in the HiC data are due to disease-related changes in the equivalent cell types. However, I am concerned that the AH patient monocytes may have differentiated due to their environment so that they are in fact akin to a different cell type and the 3D genome changes they describe reflect this. This is supported by published articles, for example: Dhanda et al., Intermediate Monocytes in Acute Alcoholic Hepatitis Are Functionally Activated and Induce IL-17 Expression in CD4+ T Cells. J Immunol (2019) 203 (12): 3190-3198, in which they show an increased frequency of CD14+ CD16+ intermediate monocytes in AH patients that are functionally distinct.

I suggest that if the authors would like to study the specific effects of AH on 3D genome architecture then they should carefully FACsort the equivalent monocyte populations from the healthy and AH patients.

(2) The analysis of the HiC data is quite preliminary. In the 3D genome field, it is usual to report the different scales of genome architecture, for example, compartments, topologically associated domains (TADs) and loops. I think that reporting this information and how it changes in AH patients in the appropriate cell types would be of great interest to the field.

Comments on revisions:

In the revision the authors did not respond to my concerns which I believe still remain valid and compromise the author's conclusions of AH-specific effects on genome architecture.

Reviewer #1 (Public review):

Summary:

This manuscript by Wang et al. describes the development of an optimized soluble ACE2-Fc fusion protein, B5-D3, for intranasal prophylaxis against SARS-CoV-2. As shown, B5-D3 conferred protection not only by acting as a neutralizing decoy, but also by redirecting virus-decoy complexes to phagocytic cells for lysosomal degradation. The authors showed complete in vivo protection in K18-hACE2 mice and investigated the underlying mechanism by a combination of Fc-mutant controls, transcriptomics, biodistribution studies, and in vitro assays.

Strengths:

The major strength of this work is the identification of a novel antiviral approach with broad-spectrum and beyond simple neutralization. Mutant ACE2 enables broad and potent binding activity with the S proteins of SARS-CoV-2 variants, while the fused Fc part mediates phagocytosis to clear the viral particles. The conceptual advance of this ACE2-Fc combination is convincingly validated by in vivo protection data and by the completely abrogated protection of Fc LALA mutant.

Additionally:

The authors include a discussion (in Discussion part) about a previously reported ACE2 decamer (DOI: 10.1080/22221751.2023.2275598) and compared with the ACE2-Fc fusion protein developed in this study. The authors also tested the off-target activity and showed no evidence of toxicity in vivo.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have appropriately addressed the comments raised in the previous round of review.]

Summary:

The study by Lemen et al. represents a comprehensive and unique analysis of gene networks in rat models of opioid use disorder, using multiple strains and both sexes. It provides a time-series analysis of Quantitative Trait Loci (QTLs) in response to morphine exposure.

Strengths:

A key finding is the identification of a previously unknown morphine-sensitive pathway involving Oprm1 and Fgf12, which activates a cascade through MAPK kinases in D1 medium spiny neurons (MSNs). Strengths include the large-scale, multi-strain, sex-inclusive design, the time-series QTL mapping provides dynamic insights, and the discovery of an Oprm1-Fgf12-MAPK signaling pathway in D1 MSNs, which is novel and relevant.

Reviewer #1 (Public review):

Summary:

Many studies have investigated adaptation to altered sensorimotor mappings or to an altered mechanical environment. This paper asks a different but also important question in motor control and neurorehabilitation: how does the brain adapt to changes in the controlled plant? The authors addressed this question by performing a tendon transfer surgery in two monkeys during which the swapped tendons flexing and extending the digits. They then monitored changes in task performance, muscle activation and kinematics post-recovery over several months, to assess changes in putative neural strategies.

Strengths:

(1) The authors performed complicated tendon transfer experiments to address their question of how the nervous system adapts to changes in the organisation of the neuromusculoskeletal system, and present very interesting data characterising neural (and in one monkey, also behavioural) changes post tendon transfer over several months.

(2) The fact that the authors had to employ to two slightly different tasks -one more artificial, the other more naturalistic- in the two monkeys and yet found qualitatively similar changes across them makes the findings more compelling. After all these are very challenging experiments!

(3) The paper is well written, the analyses are sound, and the authors interpret the data appropriately, acknowledging the key limitations.

Weaknesses:

None of note.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors set up a pipeline to predict insect repellents that are pleasant and safe to humans. This is done by daisy chaining a new classification model based predicting repellents with a published model on predicting human perception. Models use a feature-engineered selection of chemical features to make their predictions. The predicted molecules are then validated against a proxy humanoid (heated brick) and its safety is tested by molecular assays of human cells. The humanistic approach to modeling these authors have taken (which consider cosmetic/aesthetic appeal and safety) is novel and a necessary step for consumer usage. However, the importance of pleasantness over effectiveness is still up for debate (DEET is unpleasant but still used often) and the generalization of safety tests is unknown and assumed. The effectiveness of the prediction models is also still warranted. They pass the authors own behavioral tests, but their contribution to the field is unknown as both models (new and published) have not been rigorously bench-marked to previous models. Moreover, the author's breadth of literature in this field is sparse, ignoring directly related studies.

Strengths:

Humanistic approach to modeling consider pleasantness and safety. Chaining models can help limit the candidate odorants from the vastness of odor space.

Weaknesses:

The current models need to be bench-marked against leading models predicting similar outcomes. Similarly, many of these papers need to be addressed and discussed in the introduction. The authors might even consider their data sources for model training to increase performance and lexical categorization for interoperability. For instance, the Dravnikes data lexicon, currently used in the human perception lexicon, has been highly criticized for its overlapping and hard to interpret descriptive terms ("FRAGRANT", "AROMATIC").

Human Perception<br /> Khan, R. M., Luk, C. H., Flinker, A., Aggarwal, A., Lapid, H., Haddad, R., & Sobel, N. (2007). Predicting odor pleasantness from odorant structure: pleasantness as a reflection of the physical world. Journal of Neuroscience, 27(37), 10015-10023.

Keller, A., Gerkin, R. C., Guan, Y., Dhurandhar, A., Turu, G., Szalai, B., ... & Meyer, P. (2017). Predicting human olfactory perception from chemical features of odor molecules. Science, 355(6327), 820-826.

Gutiérrez, E. D., Dhurandhar, A., Keller, A., Meyer, P., & Cecchi, G. A. (2018). Predicting natural language descriptions of mono-molecular odorants. Nature communications, 9(1), 4979.

Lee, B. K., Mayhew, E. J., Sanchez-Lengeling, B., Wei, J. N., Qian, W. W., Little, K. A., ... & Wiltschko, A. B. (2023). A principal odor map unifies diverse tasks in olfactory perception. Science, 381(6661), 999-1006.<br /> Related cleaned data: https://github.com/BioMachineLearning/openpom

Insect Repellents:<br /> Wright, R. H. (1956). Physical basis of insect repellency. Nature, 178(4534), 638-638.

Katritzky, A. R., Wang, Z., Slavov, S., Tsikolia, M., Dobchev, D., Akhmedov, N. G., ... & Linthicum, K. J. (2008). Synthesis and bioassay of improved mosquito repellents predicted from chemical structure. Proceedings of the National Academy of Sciences, 105(21), 7359-7364.

Bernier, U. R., & Tsikolia, M. (2011). Development of Novel Repellents Using Structure− Activity Modeling of Compounds in the USDA Archival Database. In Recent Developments in Invertebrate Repellents (pp. 21-46). American Chemical Society.

Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

The current study assumes that insect repellents repel via its odor valence to the insect, but this is not accurate. Insect repellents also mask the body odor of humans making them hard to locate. The authors need to consult the literature to understand the localization and landing mechanisms of insects to their hosts. Here, they will understand that heat alone is not the attractant as their behavioral assay would have you believe. I suggest the authors test other behaviors assays to show more convincing evidence of effectiveness. See the following studies:

De Obaldia, M. E., Morita, T., Dedmon, L. C., Boehmler, D. J., Jiang, C. S., Zeledon, E. V., ... & Vosshall, L. B. (2022). Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels. Cell, 185(22), 4099-4116.

McBride, C. S., Baier, F., Omondi, A. B., Spitzer, S. A., Lutomiah, J., Sang, R., ... & Vosshall, L. B. (2014). Evolution of mosquito preference for humans linked to an odorant receptor. Nature, 515(7526), 222-227.

Wei, J. N., Vlot, M., Sanchez-Lengeling, B., Lee, B. K., Berning, L., Vos, M. W., ... & Dechering, K. J. (2022). A deep learning and digital archaeology approach for mosquito repellent discovery. bioRxiv, 2022-09.

Comments on revisions:

The revisions made to the manuscript do not fully address the concerns raised in the previous round of review. The authors are encouraged to consider the following points to strengthen the work.

The benchmarking of the human perception models against Keller et al. (2017) and Gutiérrez et al. (2018) is insufficient, as the field has progressed considerably in the last five years with newer approaches using larger data sources. Benchmarking against more recent models would better situate the contribution of this work.

The exclusion of human repellency data from preprint Boyle et al. (2016) is worth reconsidering. For a study that takes an explicitly human-centric modeling approach, human behavioral data on repellency, pleasantness, and usage intent would directly support the central claims of the manuscript.

The key claims regarding repellency and consumer acceptability would be considerably strengthened by the addition of these data.

Reviewer #1 (Public review):

Summary:

The paper describes a biologically plausible version of JEPA using recurrent neural networks called RPL for recurrent predictive learning. Given an embedding z_t, a recurrent neural network processes these inputs with the form: c_t+1 = RNN(c_t, z_t). Then the predictive network f is predicting the future inputs with the format: min || f(c_t) - stop_grad(z_t+delta t) ||^2. I understand that a prediction error is defined as: e = z_t+delta t - f(c_t) to model cortical measurements in the oddball task.

The RPL model is also shown to build an internal world model, with "real-world" data like the movement of moving animals or speech signals. The representation is then compared to V1 data and expected prediction error signals in an oddball setting. In a stacked hierarchy of RNN learning with RPL, the higher layers appear to learn high-level latent variables, although gradients are not propagated downward to the lower layers.

Strengths:

(1) The paper tackles an open question: Self-supervised learning is thought to be a fundamental principle to explain how computation is structured in the brain. Cortical data suggest qualitatively that prediction error is a core principle of representation learning in the brain, but the field is still looking for a simple yet expressive model that would explain how the cortex learns its representations. RPL contributes in that direction by making a useful link between cortical representation learning in RNN models and the JEPA learning algorithm that was demonstrated to scale to large world model learning from video data by Lecun's group. It is very useful to connect this popular deep learning algorithm to cortical data.

(2) The model formalism is relatively elegant and simple: Simple next input prediction objectives are conceptually simple but not necessarily trivial to build at scale. There is a clear benefit in comparison with contrastive or IL methods because they are free from dataset-specific data augmentation and negative samples. Thereby moving the comp neuro field towards conceptually simpler models of representation in the cortex. Yet predictive only models (and in particular predictive models in latent space instead of pixel space) are not easy to build in a stable fashion. JEPA family is basically intended to solve this question; it is very nice and timely to bring this to comp neuro.

(3) The methodology combining comp neuro and deep learning makes sense: The conceptual and qualitative analogy with cortical prediction errors is relevant and consistent with what is expected as a model of self-supervised learning in cortical models. The methodology to compare RPL with IL and CL is methodologically meaningful and grounded: showing, for instance, how some of the models fail to represent some latent structure in some toy datasets is interesting.

(4) h-RPL: The h-RPL is perhaps the most creative departure from the JEPA model family. It would be interesting to say more about what was particularly difficult to see in the latent variables emerging in the hierarchical model. I often find it magical that layer-wise learning rules of this type are not learning redundant representations. Any insights why this is not the case here would be potentially insightful.

Weaknesses:

In general, I fully support the type of question and ideas that the paper is putting forward. It is, however, very hard in this research field to gain insight into specific conceptual contributions or specific bits of experimental data that the model puts forward. In pointing to the following weaknesses, I am encouraging the authors to lay out more clearly what the unique hypothesis is or the contribution of the RPL model that we should remember it for.

(1) The devil is in the details:

1a) Comparison with JEPA variants: JEPA variants are integrating different details into the learning algorithm. Integrating, for instance, "masking" of the latent encoder targets, or EMA in the style of BYOL or Siamese networks, for the predicted representations. It is great that RPL does not seem to need any of those (next input prediction is a natural implementation of masking, and EMA does not seem to be used). It is notoriously hard for the JEPA model to work without these features. Since some of these details are sometimes surprisingly crucial for a simulation to work, it would be good to report which of the other important details were key to live without EMA and masking. Is it the difference in learning rate, for instance? Or maybe the tasks considered are simply easy enough for any model to work; if so, it could be useful to acknowledge to what extent this is true.

1b) Comparison with IL and CL: On a high level, the comparison with IL and CL algorithms is written as conclusive. I suspect that the failure modes of IL and CL that are described are not due to the algorithms themselves, but rather to the construction of invariance statistics or the choice of negative sample sets (the sets of samples among which variance 1 is requested by VICreg). For instance, if variance (or negative sample set) is taken only across time, the variance object identity is expected to collapse. Similarly, if the variance is taken across the object identity, the variance across time can collapse. So I wonder if the failure of IL and CL is induced by the construction of the variance definition.

(2) Prediction error: When compared to the recording of cortical activity in Figure 7. It is not obvious from the figure which latent space we are talking about mathematically. Is the vector z, c or the prediction error e? This is rather important from a neuroscientific point of view, because the prediction error e is expected to explain the neuronal data. On the other hand, the prediction error e is only used in the learning algorithm to define the loss function, but it is not the communication medium between the RNN units c (or with the encoder z).

In the brain, since the measurements are recorded as neural activity, they are communication channels between specific units (z or c). It is probably c or z that would already explain the oddball prediction error. I believe that other models, like Forward-forward of Nejad et al., have tried quite hard to address this apparent tension. Whether or not this is resolved by RPL, it thinks it would be beneficial to state the problem and clarify how the algorithm addresses or ignores the issue.

(3) Successor representation without value? I believe the term successor representation is historically relevant in a reinforcement learning (RL) setting and has a precise mathematical definition. Without RL, I feel that learning successor representation is conceptually identical to learning a transition matrix (aka, a primitive world model). I therefore wonder if the pitch for high-level framing of the successor representation is appropriately described or trivial.

(4) Learning in RNN: Learning with recurrent networks appears to be a key in this model presented here (it is in the algorithm name). Yet, this aspect of the model and the literature on biologically plausible learning rules for RNN is not really discussed.

Reviewer #1 (Public review):

The authors demonstrate an innovative approach to investigate the effect of cone dropout on visual acuity using their newly developed olo system. By systematically reducing the coverage of real-world input to the cone photoreceptor mosaic ("cone dropout condition"), the authors are able to assess how having fewer cones leads to reduced vision, in comparison to existing approaches ("pixel dropout condition").

The capture of a rich dataset, including cone imaging and eye motion, is valuable. Benchmarking with the prior literature, suggesting that good visual acuity can be maintained despite a 50% loss in cone density, is impressive. However, it is known that cone density varies dramatically from the peak cone density location in the foveal center to even a location a few degrees outside of the fovea. In addition, there is a high degree of subject-to-subject variation in peak cone density. Given that the C stimulus is hollow in the middle, the stimulus does not actually hit the location of the peak cone density but must land slightly outside of it. Therefore, considering the actual cone density of where the stimulus lands will be important to discuss and/or analyze.

The observation of visual acuity maintenance with cone dropout has been a longstanding mystery since the 2013/2018 papers by Ratnam and Foote. The authors should be commended for their approach to addressing this important question. However, there are some simplifications and assumptions being applied to make this jump (i.e., that a 50% reduction in cone stimulation in a healthy eye is comparable to a 50% reduction in cone density in a patient). It seems unlikely that, in a patient's eye, with cone dropout, there will be gaps in the mosaic. Not considering any other non-photoreceptor-related reasons for visual acuity loss, which can occur in patients, the cone aperture acceptance angle may be different due to changes in cone size or packing; the sensitivity of individual cones may also be reduced due to deficits in the visual cycle recovery, which could be affected in disease. Some of these limitations could be addressed and acknowledged more explicitly.

Overall, this is an impressive study incorporating state-of-the-art technology to probe the fundamental limits of human vision.

Reviewer #1 (Public review):

Summary:

Fujita and colleagues investigated two selective peripheral nerve voltage-gated sodium channel inhibitors targeting either Nav1.7 or Nav1.8 on the excitability of human dorsal root ganglion neurons. The authors discovered that Nav1.8 inhibition is more effective at suppressing repetitive firing of DRG neurons, and this may explain the greater clinical efficacy observed for suzetrigine.

Strengths:

The study is interesting, and the findings are conceptually satisfying in that they may explain one aspect of Nav1.7 vs Nav1.8 targeting success.

Weaknesses:

(1) The use of postmortem human DRG neurons provides translational relevance, but the use of these cells is also a liability, given their high degree of variability. Of note are the 10 to 20-fold differences in baseline properties among cells, which dwarf the effects of the test compounds. The experiments may suffer from undersampling.

(2) A potential confounder when using post-mortem human DRG neurons is heterogeneity of cell types. The methods clearly state that the cells selected for recording were of 'generally' small size, but specific criteria for what constitutes 'small' or other unstated selection criteria were not provided. A table of individual cell capacitance and input resistance values, along with information about individual donors (age, sex, ethnicity), is important to include. Additionally, some discussion of how DRG neuron heterogeneity impacts the findings. This relates to concern #1 about sample size determination and how cell heterogeneity factored into this calculation.

Reviewer #1 (Public review):

The manuscript shows that different traits of adults and larvae correlate with Red List status. The authors argue that this shows a big gap in the conservation of amphibians and that the traits of all life stages should be taken into account in amphibian conservation. Specifically, amphibian conservation should do more for the habitats where the larvae live.

The manuscript is well written and easy to understand. The methods are sound.

While the study will make an interesting contribution to conservation science, there are many things that I disagree with.

I don't think that amphibian larvae and their requirements are a "blind spot" as the title suggests. When reading the manuscript, I didn't learn how conservation practice should change in response to the results.

I wonder whether the relationship between species traits and extinction risk is of great importance for conservation. If a species is Data Deficient on the IUCN Red List, then species traits could be used to predict its Red List category. However, for other conservation projects, I don't see how this would work. How would traits be linked to captive breeding, conservation translocation, pond construction or habitat management in general? In some cases, I can envision a link between species traits and pond hydroperiod.

Species traits are body size and morphological traits. That makes sense. However, one of the species traits was microhabitat. I find it far-fetched to call habitat a species trait. This is standard habitat ecology. It is well known that habitats matter and that different habitat types face different threats, and consequently, the species that live in those habitats. Furthermore, habitat and morphology may be confounded. For example, tadpoles in lentic and lotic habitats have very different morphologies. So is it habitat or morphology?

I don't know how the threat status of Chinese amphibians is determined. IUCN has multiple reasons why a species can be Red Listed. One reason is range size, and another reason is population decline. Personally, I don't think they should be pooled in an analysis because they are fundamentally different reasons why a species has a high extinction risk. A reduction in population size of greater than 30% in 10 years or 3 generations is not the same thing as a small distribution range. Another issue is that IUCN developed the Green Status of species. The Green Status shows that even a species which is LC on the Red List may be significantly depleted.

The species traits in Table 1 are mostly functional/morphological and body size related (and microhabitat). While there may be correlations between traits and Red List status, it is unknown whether this is correlation or causation. In addition, it is difficult to know the conservation interventions that may be necessary now that we know that relative head with and Red List status are correlated.

In the discussion, the authors explain why body size and other traits may affect extinction risk and whether there is a causal relationship. I agree that body size may have a direct effect because larger species are harvested more frequently (it was interesting to learn that tadpoles are harvested as well). However, as macroecological studies show, smaller species often have larger populations than larger species. Abundance may matter.

I found it much harder to understand why relative head length and tympanum size correlated with Red List status. I wasn't convinced by the arguments in the discussion. Typanum size may be related to hearing and anthropogenic noise. Several studies are cited which show that frogs alter their calling behaviour in response to noise. Crucially, however, they describe changes in behaviour or properties of the advertisement call, yet none show that noise has effects on population viability. If some anthropogenic stressor affects individuals, then this does not mean that it will cause a population decline. When IUCN published the second global amphibian assessment, did they list noise as a major threat to amphibians?

There are statements that the tadpole stage is the most important stage: "a critical period for amphibian survival" (line 78-79). While there is high mortality in the tadpole stage, tadpole survival is rather unlikely to affect population survival. Many population models show this. See, for example, Biek et al. 2002 in Conservation Biology. Other papers have argued that the postmetamorphic juvenile stage is most important (Petrovan and Schmidt 2009 Biological Conservation).

The authors repeatedly make the statement that amphibian conservation should focus more on the tadpole stage. I don't understand why this statement is made. For example, a major activity in amphibian conservation is the restoration and de novo construction of ponds (see Calhoun et al. 2014 PNAS, Moor et al. 2022 PNAS). Ponds are habitats for tadpoles. Others removed fish from amphibian breeding sites because fish prey on tadpoles (and adults; see Vredenburg 2004 PNAS). Semlitsch (2002 in Conservation Biology) argued that the management of pond hydroperiod is a critical element of amphibian recovery plans. Ponds should be temporary because this effectively removes predators that consume tadpoles. Clearly, the tadpole stage is not a neglected stage in amphibian conservation.

Reviewer #1 (Public review):

Summary:

This manuscript provides a comprehensive and mechanistic analysis of how tsetse flies feed on blood across a wide range of host skin types. The authors combine detailed anatomical characterization of the feeding apparatus with quantitative measurements of mechanical properties, probing forces, and blood uptake, complemented by experiments using artificial skin. They show that tsetse flies do not rely on extreme forces or uniquely specialized structures, but instead on subtle and highly efficient structural and mechanical adaptations (such as the toothed labellum and coordinated proboscis movements) to achieve effective blood pool feeding. The study successfully moves beyond descriptive anatomy to a quantitative, functional analysis that explains how feeding is accomplished across diverse substrates.

Strengths:

A major strength of the work is the impressive integration of multiple complementary approaches. Advanced imaging tools provide a convincing three-dimensional view of the proboscis, labellum, and associated structures, while direct force measurements and blood intake quantification place these observations on a solid quantitative footing. The use of artificial skin with different mechanical properties is particularly powerful, as it allows structure-function relationships to be tested under controlled and reproducible conditions. Together, these datasets provide strong and coherent support for the authors' central conclusions. The quantitative treatment of feeding mechanics represents a significant advance over largely descriptive prior work by others (e.g., Gibson W et al 2017) and establishes a valuable mechanistic insight for studying blood feeding in insect vectors more broadly.

Weaknesses:

The study focuses almost entirely on uninfected flies and does not address how infection might alter feeding mechanics or performance. Previous work has shown that trypanosome infection can affect salivary gland function and feeding time (Van Den Abbeele et al 2010), and even cause damage to mouthparts, all of which can influence feeding behavior and efficiency. While this does not detract from the technical quality or the core findings of the study, a more explicit discussion of these biological variables would help place the results in a broader transmission-relevant context and clarify how generalizable the conclusions are to natural infection settings.

Overall, this is an outstanding and carefully executed study that will have a significant impact on the fields of vector biology and parasite transmission.

Reviewer #1 (Public review):

In this manuscript, the authors investigate the relationship between genetic codes and their robustness to single-point mutations. They construct ten alternative genetic codes by reassigning nine codons to Leu, Ser, or Ala, and assess mutational robustness using three reporter proteins subjected to error-prone PCR. This represents an interesting experimental approach to addressing the hypothesis that the standard genetic code is optimized for mutational robustness.

Major comment:

While I find the experimental design valuable, I am not fully convinced by the authors' conclusion that "alterations of the genetic code within the ranges explored in this study have no significant effect on mutational robustness". The current analysis is based on the functional output of three individual reporter proteins. Given that cellular systems involve far more complex interactions, it would be more appropriate to limit this conclusion to mutational robustness at the level of individual protein activity, rather than making broader generalizations.

Specific comments:

(1) tRNA modification and expression efficiency (Page 5, line 131).

The authors attribute the observed inefficiency to the lack of chemical modifications in the tRNAs used. However, gene expression efficiency can also be strongly influenced by DNA sequence design. To better support this claim, it would be helpful to compare luciferase activity when expressed using native E. coli tRNAs. This comparison could clarify whether the observed effects are due to tRNA modification status or other sequence-dependent factors.

(2) Discrepancy between expression level and activity (Figure S7 vs Figure S8).

Although GAL expression levels appear similar across different genetic codes (Figure S7), their activities differ substantially (Figure S8), even in the low-mutation library. This discrepancy warrants further investigation. Possible explanations include differences in protein folding efficiency or translational error rates, as mentioned by the authors in the main text.

To address this, the authors could analyze the protein products using mass spectrometry. If this is not feasible due to low expression levels, alternative approaches such as SDS-PAGE (e.g., with radiolabeling or Western blotting) would still provide valuable information. Additionally, comparing activity after in vitro refolding could help distinguish between folding defects and sequence-level errors. While I understand that the primary aim of this study is to compare mutational robustness across genetic codes, discussing these observations would significantly enhance the mechanistic insight of the work.

(3) Protein expression analysis for additional reporters.

Since protein expression levels are critical for interpreting reporter activity, similar analyses should also be performed for luciferase (Luc) and mSG in both high- and low-mutation libraries. This would ensure that differences in activity are not confounded by variations in protein abundance.

Reviewer #2 (Public review):

Summary:

This study uses dental traits of a large sample of Chinese mammals to tract evolutionary patterns through the Paleocene. It presents and argues for a 'brawn before bite' hypothesis -- mammals increased in body size disparity before evolving more specialized or adapted dentitions. The study makes use of an impressive array of analyses, including dental topographic, finite element, and integration analyses, which help to provide a unique insight into mammalian evolutionary patterns.

Strengths:

This paper helps to fill in a major gap in our knowledge of Paleocene mammal patterns in Asia, which is especially important because of the diversification of placentals at that time. The total sample of teeth is impressive and required considerable effort for scanning and analyzing. And there is a wealth of results for DTA, FEA, and integration analyses. Further, some of the results are especially interesting, such as the novel 'brawn before bite' hypothesis and the possible link between shifts in dental traits and arid environments in the Late Paleocene. Overall, I enjoyed reading the paper and I think the results will be of interest to a broad audience.

Weaknesses:

For the original draft of the manuscript, I had four major concerns with the study, especially related to the sampling, diet, and evidence for the 'brawn before bite' hypothesis. I still believe that the original issues that I raised may be weaknesses of the study. For example, there is still limited discussion on diets (even though the dental topographic analyses used in the study are designed for inferring diets). And I find the results a little challenging to interpret because teeth of multiple positions are included in the same samples, which seems problematic. That said, the authors have addressed each of my previous concerns and have made major revisions, including running new analyses, and thus I support the paper.

Reviewer #2 (Public review):

Summary:

The authors pair analysis of replication timing and allele-specific expression in clonal populations of primary human cells. They combine these data with previously published data on clones from transformed human cell lines. They identify a number of genomic regions that display asynchronous replication timing in at least one clone and correlate these regions with allele-specific expression of genes within them. They also observe that several interesting gene sets, including genes that are associated with human diseases, map to asynchronously replicating regions. This is a good experimental approach that builds on already published data demonstrating the connection between allelic imbalance and replication timing.

- This is a research topic that touches on a few sub-fields of biology, and thus to make the paper more approachable we would recommend a careful edit of the text for clarity and precision of language.

- Authors point out that this is a decades-old field; we would suggest to use terminology established within the field is possible. Allelic imbalance has been referred to as AI, MAE (monoallelic expression), RMAE (random monoallelic expression) etc. The paper whose mouse data the authors make use of uses Asynchronous Stochastic Replication Timing (ASRT) instead of VERT to refer to the same phenomenon.

- Methods do not provide fully sufficient detail to fully evaluate or reproduce these experiments.

- It is helpful to show representative loci as the authors do in Fig 1F and G and Fig 2 but these panels are very densely rendered and thus difficult to process visually - even the cartoon version (1D) is thick with overlapping lines. The point that allelic imbalance is enriched in VERTs would be enhanced if the authors could present the allelic ratio for all genes found in all VERTs, demonstrating how replication timing on either chromosome affects the allelic ratio.

- The authors make the important point that VERTs are unlikely to be shared among different cell types and tissues (Fig 1i), but then find an enrichment for neuronal and immune genes in VERT regions identified in ACPs. It follows that these same genes are unlikely to be in such regions in the tissues where they are relevant. Some of the GO terms presented are too broad to suggest any biological significance to the result, even if there is statistical significance (for example, the top term for LCL clones 'Cytoplasm' is associated with 12,000 genes, and the second term for mouse clones 'Membrane' is associated with 10,000). It would be helpful to focus on GO terms lower in the GO hierarchy.

- Figure 3 highlights the association of related gene clusters with VERTs but the VERTs are assigned based on variable replication timing in just 1 or 2 clones. This is an interesting observation, but to make the point that "VERT regions frequently coincide with gene clusters in the human genome" there needs to be a systematic assessment of replication timing at all gene clusters across all clones, and a statistical test for significance.

- It is an interesting hypothesis that VERTs are conserved between species at syntenic loci. If such regions are really conserved, one would expect that replication timing at these sites would be consistently asynchronous. However the data presented shows that in human clones these VERTs can be specific to an individual donor (as in 5A) or an individual clone (as in 5H).

- The finding that VERTs coincide with neurodevelopmental disease genes in immune and cartilage cells is at odds with the previous statements and data about the tissue specificity of VERTs. In order to support the claim that neurodevelopmental disease associated genes reside in asynchronously replicating regions, and are thus more prone to allelic imbalance, it would be helpful if the authors demonstrated this phenomenon in neuronal cells.

- The authors consistently lean on sparse samples (i.e. a single clone) within a modestly sized dataset (4 clones from 2 donors each) to propose a new model for haploinsufficiency in human disease. It may well be but the consistent focus on limited elements in the data and perhaps an overreach in the interpretation makes it difficult to appreciate the very good experiments presented.

- This section refers to the revised version of the paper.

We would like to thank the authors for the changes and explanations offered. Although we don't fully agree with a few answers offered, overall the answers and changes in the manuscript have significantly improved the work presented. As such it should be of interest to many readers.

Reviewer #1 (Public review):

Summary:

The study by Zatulovskiy et al. examined how cell size influences cell susceptibility to ferroptosis. The authors found a size dependence specifically for ferroptosis-inducing drug Era2, but not for other drugs. Using various human cell lines (HMEC, HT 1080, RPE 1), the authors generated populations of small and large G1 cells by FACS, CDK4/6 inhibition (palbociclib), or inducible cyclin D1 knockdown, and measured cell susceptibility to ferroptosis. Larger cells were more resistant than smaller cells. Mechanistically, larger cells showed reduced plasma membrane lipid peroxidation, higher glutathione concentrations, and changes in relevant cellular proteins levels, as analyzed using previously published data. Deleting ACSL4, which is involved in ferroptosis, partly eliminated the size dependence of ferroptosis. The work concludes that cell size is a key determinant of ferroptosis susceptibility. Overall, this work expands our understanding of how cell size is correlated with functional properties of cells, which can have implications for biomedical sciences.

Strengths:

The study establishes a credible link between cell size and susceptibility to ferroptosis, as induced by Era2. Experimental replication is sufficient, and key conclusions rely on data from multiple cell lines and on multiple approaches to manipulate cell size. This suggests that the conceptual findings made in this paper could reflect a more fundamental feature of mammalian cells. In addition, this work provides an interesting contrast to another recent study about size-dependency of ferroptosis (https://doi.org/10.1016/j.isci.2025.112363), where increased cell size heightened sensitivity to the GPX4 inhibitor RSL3.

Original Weaknesses:

Disentangling cell size effects from other confounding factors, such as the cell cycle or overall metabolic rate, is challenging, and the authors have managed to qualitatively prove that cell size influences Era2-induced ferroptosis. However, the quantitative nature of this link between cell size and susceptibility to ferroptosis remains somewhat unclear due to the confounding factors that are present in many of their experiments. Notably, the quantitative nature of this link could also be cell type and growth condition -dependent, which remain to be investigated in detail. It should also be noted that this work focused on cell culture studies, and it remains unclear how much the findings of this paper could influence therapeutic strategies in vivo.

Comments on revised version:

I would first like to emphasize that I find this work solid, and I think the authors have done good work with the revisions.

My only remaining recommendation is that the authors aim to more carefully examine the magnitude of the observed cell size-dependency in ferroptosis susceptibility. Their manuscript contains several experiments where the quantitative nature of this link remains unclear due to confounding factors, such as the cell cycle. For example, in Fig 2B&C, it seems that accumulation of cells in G1 (from ~60% to ~95%) decreases ferroptosis equally to the effect caused by cell volume doubling (from day 2 to day 4 of palbo treatment), suggesting that cell cycle has a much more pronounced effect on ferroptosis than cell size (especially when considering the size change from day 0 to day 2). However, the magnitude of the cell size effect is not consistent between all experiments shown. This is not surprising, as the authors use different approaches to changing cell size and different cell lines, but it makes the work more qualitative than quantitative. Notably, another confounding factor is the cell's metabolic/biosynthetic rate. It seems reasonable to assume that prolonged palbociclib treatment will decrease metabolic and protein synthesis rates (normalized to cell size), and this could make the cells less susceptible to ferroptosis. The rapamycin treatment results shown by the authors also support this notion. One approach to examining this could be to grow cells in various growth conditions to manipulate their growth & metabolic rate.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Senior Editor without further input from the original reviewers. The authors have moderated their claims and discussed the limitations of their experimental design more transparently. The previous reviews are included for reference.]

Comments on previous version:

The authors investigated tactile spatial perception on the breast using discrimination, categorization, and direct localization tasks. They reach four main conclusions:

(1) The breast has poor tactile spatial resolution.<br /> This conclusion is based on comparing just noticeable differences, a marker of tactile spatial resolution, across four body regions, two on the breast. The data compellingly support the conclusion; the study outshines other studies on tactile spatial resolution that tend to use problematic measures of tactile resolution, such as two-point-discrimination thresholds. The result will interest researchers in the field and possibly in other fields due to the intriguing tension between the finding and the sexually arousing function of touching the breast.

The manuscript incorrectly describes the result as poor spatial acuity. Acuity measures the average absolute error, and acuity is good when response biases are absent. Precision relates to the error variance. It is common to see high precision with low acuity or vice versa. Just noticeable differences assess precision or spatial resolution, while points of subjective equality evaluate acuity or bias. Similar confusions between these terms appear throughout the manuscript.

A paragraph within the next section seems to follow up on this insight by examining the across-participant consistency of the differences in tactile spatial resolution between body parts. To this aim, pairwise rank correlations between body sites are conducted. This analysis raises red flags from a statistical point of view. 1) An ANOVA and its follow-up tests assume no variation in the size of the tested effect but varying base values across participants. Thus, if significant differences between conditions are confirmed by the original statistical analysis, most participants will have better spatial resolution in one condition than the other condition, and the difference between body sites will be similar across participants. 2) Correlations are power-hungry, and non-parametric tests are power-hungry. Thus, the number of participants needed for a reliable rank correlation analysis far exceeds that of the study. In sum, a correlation should emerge between body sites associated with significantly different tactile JNDs; however, these correlations might only be significant for body sites with pronounced differences due to the sample size.

(2) Larger breasts are associated with lower tactile spatial resolution<br /> This conclusion is based on a strong correlation between participants' JNDs and the size of their breasts. The depicted correlation convincingly supports the conclusion. The sample size is below that recommended for correlations based on power analyses, but simulations show that spurious correlations of the reported size are extremely unlikely at N=18. Moreover, visual inspection rules out that outliers drive these correlations. Thus, they are convincing. This result is of interest to the field, as it aligns with the hypothesis that nerve fibers are more sparsely distributed across larger body parts.

(3) The nipple is a unit<br /> The data do not support this conclusion. The conclusion that the nipple is perceived as a unit is based on poor tactile localization performance for touches on the nipple compared to the areola. The problem is that the localization task is a quadrant identification task with the center being at the nipple. Quadrants for the areola could be significantly larger due to the relative size of the areola and the nipple; the results section seems to suggest this was accounted for when placing the tactile stimuli within the quadrants, but the methods section suggests otherwise. Additionally, the areola has an advantage because of its distance from the nipple, which leads to larger Euclidean distances between the centers of the quadrants than for the nipple. Thus, participants should do better for the areola than for the nipple even if both sites have the same tactile resolution.

To justify the conclusion that the nipple is a unit, additional data would be required. 1) One could compare psychometric curves with the nipple as the center and psychometric curves with a nearby point on the areola as the center. 2) Performance in the quadrant task could be compared for the nipple and an equally sized portion of the areola and tactile locations that have the same distance to the border between quadrants in skin coordinates. 3) Tactile resolution could be directly measured for both body sites using a tactile orientation task with either a two-dot probe or a haptic grating.

Categorization accuracy in each area was tested against chance using a Monte Carlo test, which is fine, though the calculation of the test statistic, Z, should be reported in the Methods section, as there are several options. Localization accuracies are then compared between areas using a paired t-test. It is a bit confusing that once a distribution-approximating test is used, and once a test that assumes Gaussian distributions when the data is Bernoulli/Binomial distributed. Sampling-based and t-tests are very robust, so these surprising choices should have hardly any effect on the results.

A correlation based on N=4 participants is dangerously underpowered. A quick simulation shows that correlation coefficients of randomly sampled numbers are uniformly distributed at such a low sample size. This likely spurious correlation is not analyzed, but quite prominently featured in a figure and discussed in the text, which is worrisome.

(4) Localization of tactile events on the breast is biased towards the nipple<br /> The conclusion that tactile percepts are drawn toward the nipple is based on localization biases for tactile stimuli on the breast compared to the back. Unfortunately, the way participants reported the tactile locations introduces a major confound. Participants indicated the perceived locations of the tactile stimulus on 3D models of these body parts. The nipple is a highly distinctive and cognitively represented landmark, far more so than the scapula, making it very likely that responses were biased toward the nipple regardless of the actual percepts. One imperfect but better alternative would have been to ask participants to identify locations on a neutral grey patch and help them relate this patch to their skin by repeatedly tracing its outline on the skin.

Participants also saw their localization responses for the previously touched locations. This is unlikely to induce bias towards the nipple, but it renders any estimate of the size and variance of the errors unreliable. Participants will always make sure that the marked locations are sufficiently distant from each other.

The statistical analysis is again a homebrew solution and hard to follow. It remains unclear why standard and straightforward measures of bias, such as regressing reported against actual locations, were not used.

Null-hypothesis significance testing only lets scientists either reject the null hypothesis or not. The latter does NOT mean the Null hypothesis is true, i.e., it can never be concluded that there is no effect. This rule applies to every NHST test. However, it raises particular concerns with distribution tests. The only conclusion possible is that the data are unlikely from a population with the tested distribution; these tests do not provide insight into the actual distribution of the data, regardless of whether the result is significant or not.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous rounds of review.]

Giordano et al. demonstrate that yeast cells expressing separated N- and C-terminal regions of Tfb3 are viable and grow well. Using this creative and powerful tool, the authors effectively uncouple CTD Ser5 phosphorylation at promoters and assess its impact on transcription. This strategy is complementary to previous approaches, such as Kin28 depletion or the use of CDK7 inhibitors. The results are largely consistent with earlier studies, reinforcing the importance of the Tfb3 linkage in mediating CTD Ser5 phosphorylation at promoters and subsequent transcription.

Notably, the authors also observe effects attributable to the Tfb3 linker itself, beyond its role as a simple physical connection between the N- and C-terminal domains. These findings provide functional insight into the Tfb3 linker, which had previously been observed in structural studies but lacked clear functional relevance. Overall, I am very positive about the publication of this manuscript.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have carefully considered all the reviewers' comments. The newly added analyses, figures, and text sections are of high quality, and we commend the authors for their in-depth revision of the manuscript.]

This manuscript presents a high-quality, chromosome-level genome assembly of the European cuttlefish (Sepia officinalis), a representative species of the cephalopod lineage. Using state-of-the-art sequencing and scaffolding technologies -including PacBio HiFi long reads and Hi-C chromatin conformation capture - the authors deliver a genome assembly with exceptional contiguity and completeness, as evidenced by high BUSCO scores. This genome resource fills a significant gap in cephalopod genomics and offers a valuable foundation for studies in neurobiology, behavior, and evolutionary biology. However, there are several major aspects that need to be strengthened.

Reviewer #1 (Public review):

Summary:

This manuscript by Alonso-Caraballo et al, is a novel piece of work that examines the impact of oxycodone self-administration on neural plasticity within paraventricular thalamic (PVT) to nucleus accumbens shell (Shell) pathway - two regions shown to play a key role in cue-induced drug seeking on their own, and whether this plasticity varies based on abstinence period and biological sex.

Strengths:

The authors show using a clinically relevant long-access model of opioid self-administration promotes dependence and acute withdrawal in both male and female rats. During subsequent cue-induced relapse tests at 1 or 14-days following the conclusion of self-administration, data show that while both male and females demonstrate drug-seeking behavior at both time points, females show a further elevation in responding on day 14 versus day 1 that is not observed in the males. When accounting for past work showing elevations in drug seeking in males after 30 days, these data indicate that craving-induced relapse for opioids may develop faster and may be more pronounced in females compared to males.

These behavioral findings were paralleled by use of ex vivo acute slice electrophysiology and circuit-specific ex vivo optogenetics to examine the impact of oxycodone self-administration on synaptic strength within the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway(s). Data support a time-dependent but sex independent strengthening of glutamatergic signaling at PVT-to-NAcSh medium spiny neurons (MSNs) that is only present following a relapse test at 14 days post abstinence in males versus females, providing the first evidence that opioid self-administration and/or cue-induced drug-seeking augments this pathway. Using an extensive set of physiological measures, the authors show that this increased synaptic strength reflects a upregulation of presynaptic release probability. Further, this upregulation of excitatory signaling aligned temporally with an increase in MSN excitability, as assessed by increases in action potential firing frequency. Finally, the authors provide the first evidence that similar to other inputs to the NAcSh, PVT projections innervate both MSN as well as local interneurons, promoting a GABA-A specific feedforward inhibitory circuit. Interestingly, unlike direct excitatory inputs to MSNs, no changes were observed ostensibly within this feedforward circuit, highlighting a selective enhancement of excitatory drive and output of MSNs with protracted abstinence.

Overall, these data highlight a potential role for heightened synaptic strength within the PVT-NAcSh pathway in cue-induced relapse behavior during protracted abstinence and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals.

Weaknesses:

Overall, the experimental approach and data provided appear rigorous and support their overall conclusions and achieve their goal of understanding how opioid self-administration impacts synaptic strength within the PVT-NAcSh pathway. Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that reexposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

With regard to interpretation of electrophysiology findings, the lack of inclusion of an abstinence only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug induced adaptations in the NAc requires a post drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exopsure -- an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input-output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

Overall, these findings provide novel insight into how the PVT-NAcSh pathway is altered by opioid self-administration and whether this is unique based on abstinence period and sex. Importantly, these were the primary objectives stated by the author. Data highlight a potential role for the observed adaptations in relapse behavior and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals. However, it should be noted that no causal link is demonstrated without experiments to reduce/prevent relapse.

Comments on revisions:

The authors addressed previous concerns brought up, specifically by clarifying data interpretation as well as text modifications related to potential caveats of these interpretations. However, I recommend that the title be changed to not focus on sex differences to avoid misunderstanding. The authors should also address the lack of difference physiologically compared to the behavior as a caveat more clearly in the discussion (i.e. likely suggests this isn't the pathway driving the difference).

Reviewer #1 (Public review):

The manuscript by Ho and Schock investigates the role of the Z-disc protein Zasp52 during Drosophila flight muscle development. It was known before, mainly by findings from this group, that Zasp52 is required for normal sarcomere morphogenesis, specifically Z-disc morphogenesis in indirect flight muscles. But the exact molecular mechanism by which Zasp52 contributes, apart from the fact that it is localised there and is somehow involved in multimerization/cross-linking, was not clear. This paper proposes that an intrinsically disordered region (IDR) in Zasp52 is needed for some of its functions, by stabilising Zasp52 localisation at the Z-disc. Specifically, the IDR in Zasp52 is proposed to be required for Z-disc maintenance during the mechanical challenges of flight, while being dispensable for the initial morphogenesis during development. This hypothesis is supported by strong genetic evidence and behavioural tests, deleting Zasp's IDR impairs flight from mid-age onwards, while a block in flight activity lifts the phenotype.

However, some of the phenotypic analysis, in particular the bending of the sarcomere, likely upon mechanical challenge by muscle contractions, needs more detailed investigations to be fully convincing.

Strengths:

(1) The linker in the alternatively spliced exon 15 of Zasp52 was deleted with a state-of-the-art genetic editing strategy. Surprisingly, flies are homozygous viable, showing that this long part of the Zasp52 protein is not essential for animal survival or sarcomere morphogenesis.

(2) The observed sarcomere phenotypes with age, especially the bending Z-discs, are new and exciting.

(3) The displayed EM images document interesting phenotypes.

(4) Most of the observed phenotypes can be rescued by re-expression of the long Zasp52 isoform, which does contain the IDR region, but not by a shorter one without it, suggesting that IDR is important.

(5) FRAP data measure the local turnover of a short-ZaspGFP and show that this increased in the Zasp mutant lacking the IDR domain, suggesting that Zasp-IDR might stabilise Zasp at the Z-disc.

(6) Interestingly, flight and sarcomere morphology phenotypes can be rescued by preventing the flies from flying, suggesting that they are mechanically induced.

Weaknesses:

(1) The western blot quantifications of Zasp isoform expression are weak. No error bars are indicated in the quantifications; the quantifications appear to be more qualitative than quantitative. According to band intensities, the long Zasp isoforms seem to be less present compared to the shorter ones, even in the flight muscles.

(2) The phenotypic analysis of the sarcomere appears somewhat superficial throughout the paper. Only Zasp52 and phalloidin are shown; no other Z-disc or thick filament proteins. At least myosin stainings and overview images are important to better judge the phenotypic variations. Are the variants between individuals or regional in the same muscle?

(3) EM images would benefit from better quantification.

(4) Other proteins were not analysed with the FRAP-based turnover assay for comparison in wild type and mutant. All Z-proteins might turn over faster in the mutant with the defective Z-disc.

Reviewer #1 (Public review):

The study by Escamilla del Arenal et al. utilized a conditional knockout mouse model to study the role of Mex3a in immature olfactory sensory neurons (OSN). Mex3a is a dual-functional protein that has RNA-binding function and ubiquitin-E3 ligase activity. The results revealed that Mex3a expression is critical for proper OSN differentiation and contributes to cell surface protein trafficking and translation, cilia structure, and planar cell polarity in mature neurons. Moreover, Mex3a enforces lineage fidelity, selectively repressing sustentacular programs in neurons and neuronal programs in sustentacular cells.

In addition, the authors established an in vivo HyperTRIBE mouse model to identify Mex3a RNA targets and incorporated UbiFast into the Mex3a conditional knockout (cKO) model to find its protein targets to investigate how Mex3a regulates OSN differentiation. The experimental systems are laborious and comprehensive, which allowed the authors to identify new Mex3a putative targets in OSN.

The phenotypic results derived from the conditional Mex3a cKO mice are solid. Mechanistic findings also revealed that, in addition to facilitating protein degradation, Mex3a may confer K27 ubiquitin linkage on its target proteins, which has a non-proteolytic role but affects target protein activity, other post-translational modifications, or protein-protein interactions. However, among all Mex3a putative targets, the authors decided to emphasize on the Mex3a-mediated K27 ubiquitination on stress granule protein Serbp1 and ribosome protein Rps7, and the association between Mex3a expression and Serbp1 and p-eEF2 ribosome recruitment. This Mex3a-Serbp1-p-eEF2 ribosome recruitment axis, although it can be important in Unfolded Protein Response (UPR) signaling, seems rather general and cannot explain the striking lineage-specific phenotypes observed in the mouse model. The authors need to provide more solid evidence to demonstrate that K27-Ubiquitinylation of Serbp1 is a key step of Mex3a function in OSN differentiation to strengthen the relation between the phenotypes and mechanism presented in this study.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The revised version adequately addresses the relatively minor comments from the previous round of review.]

Summary:

This interesting paper probes the problematic relationships between the classical "spiralian" taxa, i.e., annelids, molluscs, brachiopods, platyhelminths and nemerteans, and shows that the branches leading to them are so short as to be unreliable guides to their relationships. This, in turn, has important implications for how we view the origin of the animal phyla.

Strengths:

A very careful analysis of a famous old problem with quite significant results. The results seem to be robust and support their conclusions.

It often passes uncommented that many different trees are published about animal relationships, yet some parts of the tree seem extremely difficult to resolve; the spiralians are perhaps the most difficult case. More recently, problems about sponges or ctenophores as sister groups to the rest of the animals have alerted us to major areas of uncertainty in large-scale phylogenetic reconstruction; this paper is a welcome reminder that other, perhaps even harder, problems exist which may be difficult to ever resolve with the (molecular) data we have.

Reviewer #1 (Public review):

Summary:

In this manuscript from the Levy lab, the authors investigate whether SETD6 regulates hepatic lipid accumulation through direct methylation of PPARγ. They show that SETD6 binds and mono-methylates PPARγ at K170, and provide evidence that this modification enhances PPARγ occupancy at target promoters, promotes expression of lipid metabolism genes, as well as facilitates lipid droplet accumulation in HepG2 cells. The authors also find a positive feedback loop or circuit in which PPARγ activates SETD6 transcription in a methylation-dependent manner, thereby reinforcing this lipogenic program. Overall, the work presents a novel SETD6-PPARγ regulatory axis linking lysine methylation to transcriptional control of lipid storage genes, with possible relevance to NAFLD-associated biology.

In all, I find this to be an important paper that describes and advances a new regulatory pathway that has significance to human health and disease. It would also be of interest to a broad audience. That said, there are also some concerns that the authors should address, as outlined below.

Major concerns (pertains to rigor - highest priority)

(1) Overall, the work presented is of high quality, and the data nicely support the conclusions; however, a few panels should be strengthened that have missing controls or information:<br /> a. The co-IP panel in Figure 1B lacks a lane where HA SETD6 is expressed without PPARγ. This control is needed to verify that the SEDT6-HA signal depends on PPARγ.<br /> b. In Figure 1C, the authors should show that the co-IP works in both directions (include IP for PPARγ/blot for SETD6). I am a bit confused also over the labeling with IP on the left and on top of the panel next to the beads label. More importantly, the data would be stronger if the authors took advantage of a deletion line to validate that the co-IP is specific to the presence of both.<br /> c. The same IP labeling issue exists for Figure 3B (label is on the same and on top).<br /> d. Antibody information (e.g., where the pan-methyl Ab comes from and at what dilutions they are used at) is missing.

Nice to have experiments (medium priority - strongly consider)

(2) A missing gap is how K170me1 contributes to DNA binding and gene transcription. One possibility is that methylation enhances the DNA-binding activity of PPARγ. Given that the authors have all of the reagents, it would be possible to perform a gel shift assay (or other approach) with and without SETD6-mediated methylation. Is DNA binding affected/enhanced?

(3) Along these lines, I wonder if there is another possibility: could SETD6-mediated methylation of PPARγ drive SETD6-PPARγ interaction? In other words, in the K170R, is SETD6 still even associated with PPARγ, and this interaction is required for promoter recruitment? Alternatively, would a catalytic dead version of SETD6 fail to associate with PPARγ? Currently, no experiments test the impact of an unmethylatable version of PPARγ or a catalytic dead version of SETD6 on SETD6-PPARγ interaction or SETD6 recruitment to promoters.

Minor concerns (text and figure display)

(4) The text has multiple typos and grammatical errors, and there are some issues with the figure display.

Reviewer #1 (Public review):

Summary:

In this manuscript, Yang et al expand on their previous work showing that platelet recruitment to the liver via liver macrophages is important for APAP-induced liver injury. Here, they show that platelets induce a glycolytic switch in liver non-parenchymal cells, including Kupffer cells, and that this is mediated by the protein Aldolase A produced by platelet-derived extracellular vesicles (PEV). They show that targeting Aldolase A may be a valid therapeutic strategy for severe APAP injury.

Strengths:

(1) They nicely showed that platelet effects in APAP are mediated by Aldoa via platelet-derived extracellular vesicles.

(2) Their data show that one of the effects of platelets in APAP liver injury is inducing metabolic switch to the glycolytic pathway, including in KCs.

(3) Their data points to the therapeutic potential of targeting ALDOA in severe APAP liver injury.

Weaknesses:

(1) They have not shown that the platelet-induced glycolytic switch is only in KCs.

(2) They also have not shown that KC's role in APAP injury is primarily mediated by their interaction with platelets and the subsequent glycolytic switch.

Reviewer #1 (Public review):

Summary:

Kim and Parsons present a timely overview of the NTR/prodrug system and its applications in regenerative biology research, with particular emphasis on tissue-specific cell ablation. The system has substantially advanced the field by enabling non-invasive, conditional cell elimination, and has proven especially powerful in zebrafish, though applications in other classical model organisms are also noted. The review covers the historical origins of the NTR system, its use in regeneration studies, small-molecule screening, and genetic and CRISPR-based screening, as well as future directions including the development of the highly efficient NTR2 enzyme variant.

Strengths:

This is a useful and well-structured contribution. The manuscript is a valuable resource for the regeneration biology community.

Weaknesses:

The revised manuscript shows significant improvements; however, two points remain insufficiently addressed and should be resolved in the final version.

(1) The term 'suicide gene'

As noted in my first round of revisions, the term 'suicide gene' as applied to bacterial nitroreductase remains unaddressed in the revised manuscript, despite being scientifically inappropriate and a potential source of confusion regarding the NTR/Mtz mechanism.

'Suicide' implies an intrinsic, cell-autonomous programme of self-destruction. This is incompatible with the NTR/Mtz system, in which cell death is experimentally induced through exogenous administration of metronidazole (Mtz) by the investigator. While the 'suicide gene' framing may have utility in the cancer therapy literature, likely to aid communication with non-specialist and clinical audiences, however, it is not standard in the zebrafish field, where NTR is more accurately described as a conditional toxigene. Since this review focuses predominantly on zebrafish models, its terminology should reflect that of the relevant literature.

A further conceptual problem with the 'suicide gene' framing is that it obscures the pharmacological nature of Metronidazole. Mtz is a pharmaceutical agent with intrinsic baseline toxicity: extended exposure or modestly elevated concentrations cause toxic side effects and lethality even in non-transgenic (wild-type) zebrafish (PMID: 24428354). NTR-expressing cells do not self-destruct; rather, they are rendered selectively hypersensitive to Mtz relative to other eukaryotic cells by virtue of expressing the enzyme. This distinction is mechanistically important and should be reflected in the language used throughout the manuscript.

In summary, the term 'suicide gene' does not accurately capture enzyme-mediated bioactivation of an exogenous prodrug and should be removed from the manuscript.

(2) Barriers to using the NTR/Mtz system in non-aquatic model organisms

In response to my suggestion that the title should include "zebrafish" to accurately convey the scope of the review to prospective readers, the authors stated that "there is no intrinsic barrier to adopting this technique more broadly in other systems," citing the example that "NTR was first developed in mice, but with a prodrug that proved difficult to use, and it was not widely pursued." These two statements are, however, contradictory: if the prodrug proved difficult to use, this constitutes precisely the kind of practical barrier the authors claim does not exist. The authors should clarify and reconcile this inconsistency, and provide a more thorough discussion of why the NTR/Mtz system has seen limited adoption in classical model organisms, such as mice and Drosophila.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

This manuscript by Zhao et. al investigates the canonical hedgehog pathway in testis development of Nile tilapia. They used complementary approaches with genetically modified tilapia and transfected TSL cells (a clonal stem Leydig cell line) previously derived from 3-mo old tilapia. The approach is innovative and provides a means to investigate DHH and each downstream component from the ptch receptors to the gli and sf1 transcription factors. They concluded that Dhh binds Ptch2 to stimulate Gli1 to promote an increase in Sf1 expression leading to the onset of 11-ketotesterone synthesis heralding the differentiation of Leydig cells in the developing male tilapia.'

Strengths of the methods and results:

- The use of Nile tilapia is important as it is an important aquaculture species, it shares the genetic pathway for sex determination of mammalian species, and molecular differentiation pathways are highly conserved<br /> - The approach is rigorous and incorporates a novel TSL, clonal stem Leydig cell model that they developed that is relatively faithful in following endogenous developmental steps and can produce the appropriate steroid.<br /> - Tilapia are relatively amenable to CRISPR/Cas9 targeting and, with their accelerated developmental time frame, provide an excellent model system to interrogate specific signaling pathways.<br /> - The stepwise analysis from dhh-gli-sf1 is thoughtful and well done.

Achieved Aims: The authors set out to test the hypothesis that the canonical Dhh signaling pathway for Leydig cell differentiation and steroidogenic activity is mediated via ptch2 and gli1 regulation of sf1. The results are strong, there are additional steps needed to verify that redundancy/compensation is not contributing to the outcomes.

This work is important in better understanding of nuanced commonalities and differences in developmental pathways across species. Specific to Leydig cell differentiation and steroidogenesis, their work with tilapia supports conservation of the canonical Dhh pathway; however, there appear to be some differences in downstream mediators compared to mouse. Specifically, they conclude that ptch2/gli1 stimulates sf1 and steroidogenesis in tilapia where gli1 is dispensable in mouse. Instead, Gli3 has recently been shown to play an important role to stimulate Sf1 and support the hedgehog pathway.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Summary:

The authors describe co-regulated gene modules underlying stage differentiation in Leishmania donovani through a system-level analysis of multiple molecular layers. Using amastigotes isolated from infected hamster spleens and corresponding culture-derived promastigotes, they analyzed genomic variation, transcript abundance, protein levels, phosphorylation states, and metabolite profiles. By combining these, the study identified potential regulatory mechanisms associated with parasite differentiation and generated hypotheses regarding how gene expression is coordinated across different levels.

Strengths:

A major strength of the study is the breadth of the dataset generated. The integration provides an unusually comprehensive view of molecular changes associated with Leishmania differentiation in vitro. Such multi-layer datasets involving bona fide vertebrate host stages remain relatively rare in parasitology and will likely become a valuable resource for the molecular parasitology community. In addition, the use of amastigotes isolated from infected hamsters rather than relying on axenic models provided a biologically relevant framework for the analyses.

The revised manuscript improved several aspects of the original. The RNA-seq analysis is described with a clearer pipeline, and several claims regarding causal regulatory feedback associations have been appropriately toned down. Among the observations reported, the association between parasite differentiation and proteasome-mediated protein degradation is particularly remarkable. The combination of quantitative proteomics with pharmacological inhibition of the proteasome with lactacystin provides support for a role for protein turnover in developmental transitions and paves the way for future mechanistic studies.

Weaknesses:

Most regulatory interpretations remain largely inferential or indirect. The integration identifies correlations between different levels, but direct functional validation is limited/absent. Many of the descriptions should not be interpreted as validated. As highlighted by the authors in this revised version, the mechanistic studies will be part of future work and are beyond the scope of the current work. Of note, the attempt to confirm lactacystin-induced inhibition of proteasomal activity via anti-polyUb immunoblotting did not demonstrate the expected outcome of increase in overall poly-ubiquitylation.Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Reviewer #1 (Public review):

The manuscript by Tassan-Lugrezin et al. confirms the existence of the MICOS complex in the causative agent of malaria Plasmodium falciparum. Prior to this study, only one of the two core MICOS subunits, Mic60, was found by homology search to be encoded in the apicomplexan parasite's genome. This study demonstrates the absence of the other core subunit, Mic10. It also identifies another MICOS subunit, Mic19, which co-migrates with Mic60 in a very large molecular weight complex upon blue native polyacrylamide gel electrophoresis. The authors then demonstrate that expression of both Mic60 and Mic19 is considerably upregulated during the differentiation of P. falciparum from the pathogenic asexual blood stage (ABS) to gametocytes, which correlates with the activation of oxidative phosphorylation during this process. While gene deletion of Mic19, Mic60 and both simultaneously does not affect this transition, the crista are nevertheless deformed. More significantly, crista junctions are significantly reduced, indicating that MICOS serves the same function in apicomplexans as it does in opisthokonts: maintaining crista junctions. Furthermore, the genetic interaction of mic60 and mic19 observed by augmented crista deformation when both are deleted is further evidence of their biochemical interaction, further supporting their similar complexome profiles. This study represents an important contribution to our understanding of MICOS evolution. Furthermore, the study shows that proper cristae formation is not essential for Plasmodium life cycle progression under in vitro conditions. Moreover, mutant gametocytes are still able to mate in the mosquito vector, albeit with lower efficiency.

Strengths:

The study is a result of a lot of technically challenging work in the model Plamsodium. The technically difficult life cycle progression experiments are well performed as far as I can tell. The electron microscopy is very well done and rigorously analyzed to obtain information about crista parameters. In particular, the authors were able to quantify the occurrence and diameter of crista junctions, which is very challenging in small mitochondria with small cristae. Finally, the authors provide convincing support that Mic60 and the newly discovered Mic19 act to shape crista junctions and MICOS can apparently carry out this function without the core subunit Mic10.

Weaknesses:

In its current form, there are conceptual weaknesses. The authors focus on the development of crista from a highly likely acristate state. This is true. But there can be more insight by considering their result in light of discovering the first functioning MICOS complex without one of its two core proteins, Mic10. The surprisingly large size of is also not really considered by the authors. This brings me the second weakness in my opinion. While I think the study represents a lot of work utilizing appropriate and crucial experiments, it seems the Complexome data was not explored enough. This data revealed Mic19, but what other potential subunits are co-migrating with Mic60 and Mic19 that can explain the large size of Plasmodium MICOS? Also, what is the consequence of the loss of Mic60 and Mic19 on the mitoproteome? Perhaps other MICOS subunits can be identified by their depletion in the knockouts versus the parental cell line.

Comments on latest version:

I am reviewing this manuscript again after reviewing it for Reviewers Commons. I appreciate the author's responses to my comments. The new version is improved but, in my opinion, still needs more work.

These revisions are changes to text, interpretations and obtaining more data from existing data or databases. I do still think one experimental control is necessary to substantiate the authors claim about membrane potential.

Reviewer #1 (Public review):

Summary:

In this manuscript, the role of the insulin receptor and the insulin growth factor receptor was investigated in podocytes. Mice, where both receptors were deleted, developed glomerular dysfunction and developed proteinuria and glomerulrosclerosis over several months. Because of concerns about incomplete KO, the authors generated and studied podocyte cell lines where both receptors were deleted. Loss of both receptors was highly deleterious with greater than 50% cell death. To elucidate the mechanism of cell death, the authors performed global proteomics and found that spliceosome proteins were downregulated. They confirmed this directly by using long-read sequencing. These results suggest a novel role for insulin and IGF1R signaling in RNA splicing in podocytes.

This is primarily a descriptive study and no technical concerns are raised. The mechanism of how insulin and IGF1 signaling regulates splicing is not directly addressed but implicates potentially the phosphorylation downstream of these receptors. In the revised manuscript, it is shown that the mouse KO is incomplete potentially explaining the slow onset of renal insufficiency. Direct measurement of GFR and serial serum creatinines might also enhance our understanding of progression of disease, proteinuria is a strong sign of renal injury. An attempt to rescue the phenotype by overexpression of SF3B4 would also be useful but may be masked by defects in other spliceosome genes. As insulin and IGF are regulators of metabolism, some assessment of metabolic parameters would be an optional add-on.

Significance:

With the GLP1 agonists providing renal protection, there is great interest in understanding the role of insulin and other incretins in kidney cell biology. It is already known that Insulin and IGFR signaling play important roles in other cells of the kidney. So, there is great interest in understanding these pathways in podocytes. The major advance is that these two pathways appear to have a role in RNA metabolism.

Latest comments:

The new reviewer raised two major points, whether the KO effect on splicing is specific to IGF1 and whether the interpretation could be developmental rather than due to splicing. The reviewer raises some important issues but the evidence to suggest that this is specific is data in the literature that IR/IGF signaling is already known to regulate splicing and that splicing defects were not detected in other models that they have analyzed. I agree with the reviewer (and authors) that the incomplete floxing of the genes is a major complication. The point that there could be a developmental defect with mice being born with fewer podocytes and perhaps the authors should caveat this point. The fact that they mice are born with normal function, that renal function can be maintained with up to 80% loss of podocytes suggest that they are likely born with a good number of podocytes and the dysfunction that occurs at 6 months is due to a process, induced by the loss of IR/IGF signaling that is detrimental to the podocyte.

Reviewer #1 (Public review):

Summary:

Johnston and Smith used linear electrode arrays to record from small populations of neurons in the superior colliculus (SC) of monkeys performing a memory-guided saccade (MGS) task. Dimensionality reduction (PCA) was used to reveal low-dimensional subspaces of population activity reflecting the slow drift of neuronal signals during the delay period across a recording session (similar to what they reported for parts of cortex: Cowley et al., 2020). This SC drift was correlated with a similar slow-drift subspace recorded from the prefrontal cortex, and both slow-drift subspaces tended to be associated with changes in arousal (pupil size). These relationships were driven primarily by neurons in superficial layers of the SC, where saccade sensitivity/selectivity is typically reduced. Accordingly, delay-period modulations of both spiking activity and pupil size were independent of saccade-related activity, which was most prevalent in deeper layers of the SC. The authors suggest that these findings provide evidence of a separation of arousal- and motor-related signals. The analysis techniques expand upon the group's previous work and provides useful insight into the power of large-scale neural recordings paired with dimensionality reduction. This is particularly important with the advent of recording technologies which allow for the measurement of spiking activity across hundreds of neurons simultaneously. Together, these results provide a useful framework for comparing how different populations encode signals related to cognition, arousal, and motor output in potentially different subspaces.

Comments on revised manuscript:

The authors have done a very good job of responding to all of the reviewers' concerns.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the weaknesses noted above, which were raised in the previous round of review.]

Summary:

The manuscript investigates how exogenous attention modulates spatial frequency sensitivity within the foveola. Using high-precision eye-tracking and gaze-contingent stimulus control, the authors show that exogenous attention selectively improves contrast sensitivity for low- to mid-range spatial frequencies (4-8 cycles/degree), but not for higher frequencies (12-20 CPD). In contrast, improvements in asymptotic performance at the highest contrast levels occur across all spatial frequencies. These results suggest that, even within the foveola, exogenous attention operates through a mechanism similar to that observed in peripheral vision, preferentially enhancing lower spatial frequencies.

Strengths:

The study shows strong methodological rigor. Eye position was carefully controlled, and the stimulus generation and calibration were highly precise. The authors also situate their work well within the existing literature, providing a clear rationale for examining the fine-grained effects of exogenous attention within the foveola. The combination of high spatial precision, gaze-contingent presentation, and detailed modeling makes this a valuable technical contribution.

Weaknesses:

The manipulation of attention raises some interpretive concerns. Clarifying this issue, together with additional detail about statistics, participant profiles, other methodological elements, and further discussion in relation to oculomotor control in general, could broaden the impact of the findings.

Reviewer #1 (Public review):

Summary:

In their manuscript entitled "Terminal tracheal cells of Drosophila are immune privileged to maintain their Foxo-dependent structural plasticity", Bossen and colleagues determine that the terminal cells of the tracheal system differ from other larval tracheal cells in that they do not typically show an Imd-dependent immune response to fungal and viral infections. Authors reach this conclusion based on the expression of a reporter line, Drs-GFP. The authors speculate that this difference may reflect differential expression of an immune pathway component, as tracheal terminal cells (ttcs) do not respond to forced expression of PRGP-LS. The authors then go on to show that, unlike the other cells of the tracheal system, terminal cells do not express PGRP-LC as reported by a GAL4 enhancer trap. Forced expression of PGRP-LC in terminal cells resulted in reduced branching, cell damage and features of the cell death program. These effects could be suppressed by depletion of AP-1 or Foxo transcription factors. Authors show that Foxo plays a negative role in branching of ttcs, with ectopic branching occurring upon RNAi (or under hypoxic conditions). The authors speculate that immune privilege of the ttcs may have evolved to permit Foxo regulation of ttc branching.

Strengths:

The authors provide compelling genetic data that support their overall conclusions.

Weaknesses:

FC do not appear to express DRS reporter in Figure 1 or elsewhere, raising the question of whether fusion cells are also immune privileged.<br /> Fig 5, TRE_RFP expression, is convincing in wt ttc, but not in ttc o/x PGRP-LCx

Reviewer #3 (Public review):

Agarwal et al identified the small molecule semapimod from a chemical screen of repurposed drugs with specific antimycobacterial activity against a leucine-dependent strain of M. tuberculosis. To better understand the mechanism of action of this repurposed anti-inflammatory drug, the authors used RNA-seq to reveal a leucine-deficient transcriptomic signature from semapimod challenge. The authors then measured a decreased intracellular concentration of leucine after semapimod challenge, suggesting that semapimod disrupts leucine uptake as the primary mechanism of action. Unexpectedly however, resistant mutants raised against semapimod had a mutation in the polyketide synthase gene ppsB that resulted in loss of PDIM synthesis. The authors believe growth inhibition is a consequence of decreased accumulation of leucine as a result of an impaired cell wall and a disrupted, unknown leucine transporter. This study highlights the importance of branched-chain amino acids for M. tuberculosis survival and the chemical genetic interactions between semapimod and ppsB indicate that ppsB is a conditionally essential gene in a medium deplete of leucine.

The conclusions regarding the leucine and PDIM phenotypes are moderately supported by experimental data. The authors do not provide experimental evidence to support a specific link between leucine uptake and impaired PDIM production. Additional work is needed to support these claims and strengthen this mechanism of action.

A mechanistic gap still exists for the model of semapimod antitubercular activity. The basis for semapimod activity is that the leucine auxotroph strain cannot acquire leucine from its environment, and thus the bug ceases to grow. Under normal growth conditions, the leucine auxotroph strain produces PDIM and acquires exogenous leucine through some mechanism (either through a transporter or through PDIM). Semapimod binding to PpsB causes the cell to alter its PDIM profile (lacking experimental for this), and now with the altered PDIM profile the cell cannot acquire enough exogenous leucine to sustain growth (either because the altered PDIM profile interferes with the leucine transporter activity or through PDIM uptake). Acquiring a mutation in ppsB results in cells unable to produce PDIM (some evidence supporting this) but can now acquire enough exogenous leucine to sustain growth. I cannot find the connection between cells that have normal PDIM with normal leucine uptake and cells that are missing PDIM with normal leucine uptake.

(1) The manuscript would benefit from adding additional antibiotic controls to experiments. With the current experimental approaches, it is unclear if these signatures are the result of semapimod specifically or the effect of an antimicrobial agent. Adding additional strains to the 2D TLC experiments could provide more confidence in the absence or modifications of the PDIM band.

(2) The intriguing observation that wild-type H37Rv is resistant to semapimod but the leucine-auxotroph is sensitive should be further explored. If the authors are correct and semapimod does inhibit leucine uptake through a specific transporter or modified PDIM profiles, testing semapimod activity against the leucine-auxotroph in various concentrations of BCAAs could highlight the importance of intracellular leucine. Cells might recover growth in the presence of semapimod treatment if enough leucine is provided in the media and some fraction is able to enter the cell through the impaired PDIM barrier.

Reviewer #1 (Public review):

Summary:

The manuscript by Rayan et al. aims to elucidate the role of RNA as a context-dependent modulator of liquid-liquid phase separation (LLPS), aggregation, and bioactivity of the amyloidogenic peptides PSMα3 and LL-37, motivated by their structural and functional similarities.

Strengths:

The authors combine extensive biophysical characterization with cell-based assays to investigate how RNA differentially regulates peptide aggregation states and associated cytotoxic and antimicrobial functions.

Weaknesses:

While the study addresses an interesting and timely question with potentially broad implications for host-pathogen interactions and amyloid biology, some aspects of the experimental design and data analysis require further clarification and strengthening.

Space

Reviewer #2 (Public review):

Summary:

This study presents a detailed single-cell transcriptomic analysis of the post-natal development of mouse anterior chamber tissues. The dataset is robust, consisting of ~130,000 cells collected across seven time points from early post-natal development to adult. Analysis focused on the development of cells that comprise Schlemm's Canal (SC) and trabecular meshwork (TM).

Comments on revisions:

My critiques have been adequately addressed.

Reviewer #1 (Public review):

Summary:

The authors show that if they generate a weighted multi-conformer ensemble of structural models to fit crystallographic electron density data, the application of statistical mechanical methodologies to that ensemble can provide reasonable estimates of configurational entropy terms related to protein-ligand binding.

Strengths:

A fair range of proteins (12) and ligands (70) is included in the study. The analytical methodologies are well described. Both successful and less successful analytical approaches are discussed, and the latter are frequently as insightful as the former.

Weaknesses:

Compared to the universe of protein-ligand complexes, this dataset is inevitably very limited, so the generality of the observations made here remains speculative. Though a fair range of proteins is studied, the dynamic range in the binding affinity data is limited. The practical utility of the approach is never really commented on.

Reviewer #1 (Public review):

Matsumoto et al. identify Com2, a C2H2-type zinc finger transcription factor not previously linked to sphingolipid metabolism, as a regulator of this pathway in budding yeast. They show that depletion of sphingolipids by myriocin, an inhibitor of serine palmitoyl transferase, increases Com2 expression. This, in turn, promotes the expression of the protein kinase Ypk1 and enhances TORC2-dependent phosphorylation of Ypk1. The authors identify a Com2-binding site in the YPK1 promoter and provide evidence that Com2 functions upstream of Ypk1 to regulate its<br /> expression. They further report that Com2 abundance is controlled by the ubiquitin-proteasome system: degradation of Com2 is inhibited by myriocin treatment and enhanced by phytosphingosine. Mutational analyses of putative phosphorylation and ubiquitination sites support a role for these modifications in regulating Com2 stability. Based on these findings, the authors propose that Com2 acts as a transcriptional regulator of sphingolipid metabolism that responds to sphingolipid levels and promotes Ypk1 expression.

Strengths:

This study provides a valuable finding on the regulation of sphingolipid synthesis by the transcription factor Com2 in budding yeast. The evidence supporting the authors' claims is solid, although additional evidence clarifying the mechanisms and biological significance of ubiquitin-proteasome-mediated degradation of Com2 would strengthen the work. This work will be of interest to microbiologists studying budding yeast.

Weaknesses:

The biological significance of Com2 degradation is not sufficiently clear, which represents an important limitation of the study. It would also be important to determine whether Com2 is actively degraded under normal growth conditions, such as during logarithmic growth in the absence of drug treatment.

Reviewer #1 (Public review):

Summary:

This paper asks how the NK cell receptor KIR2DL4 binds HLA-G and undergoes endocytosis. The authors propose that an allosteric disulfide-bond switch controls whether the receptor is in a ligand-binding or non-binding state, and they support this model using mutagenesis, imaging, mass spectrometry, and structural prediction.

Strengths:

A major strength is the use of diverse, complementary approaches to validate the central claim. The authors combined unbiased random mutagenesis to identify key residues, confocal microscopy to track cellular localization , and mass spectrometry to quantify the redox states of specific disulfide bonds. These methods consistently support a single model: an allosteric disulfide switch. The transition between a Cys10-Cys28 bond and a Cys28-Cys74 bond serves as a functional switch that controls whether the receptor resides at the plasma membrane to bind ligand or remains inactive in endosomes.

Weaknesses:

The core model is interesting, but some of the strongest mechanistic claims still rely heavily on structure prediction rather than direct structural evidence, especially the proposed HLA-G contact surface in Figure 6.

The paper supports an effect of the disulfide state on trafficking and uptake, but the case for direct KIR2DL4-HLA-G binding still feels somewhat indirect. The manuscript itself notes that direct binding had not been previously shown, and the current explanation partly depends on inference about which disulfide state is present.

Most of the main experiments are done in transfected 293T cells, so it is still not fully clear how strongly this mechanism carries over to the more relevant NK-cell setting discussed in the paper.

The cellular evidence for the PDI story is not specific, since it depends a lot on inhibitor and blocking experiments that could affect the broader extracellular redox environment.

Reviewer #1 (Public review):

Summary:

Sun et al. generated germline-specific cKO mice for the Znhit1 gene and examined its effect on male meiosis. The authors found that the loss of Znhit1 affects the transcriptional activation of pachytene. Znhit1 is a subunit of the SRCAP chromatin remodeling complex and a depositor of H2AZ, and in cKO spermatocytes, H2AZ is not deposited into the gene region. The authors claim that this is why the PGA was not activated. These findings provide important insights into the mechanisms of transcriptional regulation during the meiotic prophase.

Strengths:

The authors used samples from their original mouse model, analyzing both the epigenome and the transcriptome in detail using diverse NGS analyses to gain new insights into PGA. The quality of the results appeared excellent.

Comments on revisions:

Sun et al. have responded to each comment with great care and sincerity, and substantial improvements are evident.

In particular, the addition of scRNA-seq data from P35 samples appears to play an important role in supporting the authors' claims.

However, there is still room for improvement in the reanalysis of the data and in the Discussion section.

From the data perspective, for example, the authors state in line 347 of the revised manuscript that "We found that Znhit1-deficient spermatocytes phenocopied abnormal meiotic phenotypes observed in A-MYB mutants." However, the corresponding descriptions in the main text and figure legends are not sufficiently detailed, and therefore do not fully support or substantiate this interpretation. Incorporating a statistical comparison between DEGs in Znhit1-sKO and A-myb KO would likely strengthen this point.

Regarding the overall structure of the Discussion, the connections among delayed DSB repair, MSCI, and PGA regulation via H2A.Z remain somewhat descriptive and difficult to follow. This may reflect a lack of direct evidence linking these processes; however, a more logically structured and clearly articulated Discussion would improve clarity.

Reviewer #1 (Public review):

Summary:

Al Asafen and colleagues here apply a set of scanning fluorescence correlation spectroscopic approaches (Raster Image Correlation Spectroscopy (RICS), cross-correlation RICS, and pair correlation function spectroscopy) to address the nucleo-cytoplasmic kinetics of the Dorsal (Dl) transcription factor in early Drosophila embryos. The Toll/Dl system has long been appreciated to establish dorsal-ventral polarity of the embryo through Toll-dependent control of Dl nuclear localization, and represents one of a handful of model morphogen gradients produced with high enough precision to yield robust biophysical measurements of general transcription factor activity and function. By measurement of GFP-tagged Dl protein, either in wild-type embryos, or in mutant embryos with low/medium/high levels of Toll signaling, the authors report diffusivity of Dl in nuclear and cytoplasmic compartments, as well as the fraction of mobile and immobile Dl, which can be correlated with DNA binding through cross-correlation RICS. A model is presented where Cactus/IkB is implicated in preventing Dl from binding to DNA.

Strengths:

The study uses raster image correlation spectroscopy approaches to measure biophysical components of the Dl gradient in Drosophila embryos. It convincingly demonstrates a positive correlation between Toll pathway activity and the fraction of bound Dl in the nucleus. RICS methodology has widespread potential applications in cell and developmental biology, and this study will contribute to its adoption.

Weaknesses:

The study seeks to test a hypothesis for how the Toll pathway may limit Dl DNA binding in the nucleus. This experiment, while producing initial support for a role of nuclear Cactus, is confounded by co-expression of wild-type Dl, thus limiting the interpretation of the experimental results.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chen, Tu, and Lu focused on how brain-wide dopamine release dynamically changes during sleep/wake state transitions. Using multi-site fiber photometry to monitor DA release, alongside simultaneous EEG and EMG recordings, the authors show distinct DA dynamics during transitions from NREM to WAKE, REM to WAKE, WAKE to NREM, and NREM to REM. Next, they analyze temporal coordination between regions using cross-correlation analysis. Finally, chemogenetic activation of VTA or DRN but not SNc dopamine neurons is shown to promote wakefulness.

Strengths:

The manuscript addresses an interesting question: how brainwide dopamine activity evolves across sleep/wake transitions. The combination of multi-site DA recordings with simultaneous EEG/EMG monitoring is technically sophisticated. The experimental logic is generally clear, and the dataset is rich. The result has several interesting observations.

Weaknesses:

The authors used the GRAB-DA2m sensor to monitor dopamine release. Although DA2m exhibits higher affinity for dopamine compared to NE (around 15-fold difference in EC50 in HEK cell assays), it is still possible that NE contributes to the recorded signals, particularly during sleep/wake transitions when locus coeruleus activity is strongly modulated. Given the widespread and state-dependent dynamics of NE, this potentially needs to be addressed.

Similarly, the chemogenetic experiments rely on CNO to activate hM3Dq-expressing dopamine neurons. However, it is well established that CNO can be converted to clozapine in rodents, and clozapine itself is known to influence sleep/wake. Although the authors included non-hM3Dq-expressing mice as controls, the potential confounding effects of clozapine on sleep regulation remain a concern.

Midbrain dopamine neurons exhibit both tonic and phasic firing patterns. In Figure 1, most reported dopamine transitions appear relatively slow. However, some faster, phasic-like components are observable. For example, in NAc-L during REM-to-WAKE transitions, there are 2 phasic-like decreases between −20 and 0 s. The authors used laser-evoked stimulation experiments in the VTA and DRN and showed that 2 s versus 10 s stimulation produces distinct dopamine kinetics, suggesting that different firing patterns generate distinct DA dynamics. Moreover, the temporal profiles vary not only across regions but also across transitions within the same region. For example, in CeA, the NREM-to-WAKE transition shows a relatively rapid decrease, whereas REM-to-WAKE displays a much slower decline. Similarly, some regions (e.g., NAc-L NREM-to-WAKE, DRN REM-to-WAKE) show faster changes, while others (e.g., mPFC WAKE-to-NREM, VTA NREM-to-WAKE) show slower kinetics. These observations argue against a simple region-specific explanation and instead suggest that distinct firing modes may differentially contribute depending on transition type.

While cross-correlation analysis provides insight into the temporal coordination of DA signals across regions, several limitations should be considered. Sleep/wake transitions are inherently non-stationary events, whereas cross-correlation assumes relatively stable signal properties within the analysis window. This mismatch may bias lag estimates and obscure transient lead-lag relationships. Moreover, the temporal resolution of fiber photometry and the kinetics of genetically encoded DA sensors limit the precision with which timing relationships can be interpreted, particularly for sub-second lags.

In the Introduction, the authors state that they aim to address 'which dopaminergic populations causally drive these patterns.' However, the chemogenetic approach used operates on a relatively slow timescale: CNO-induced activation takes 15-30 minutes to produce effects, and the induced changes are long-lasting. In contrast, the dopamine transitions described in Figure 1 occur on a much faster timescale compared to CNO manipulation. Thus, while chemogenetic activation demonstrates that stimulating VTA or DRN dopamine neurons promotes wakefulness, it does not directly establish that these populations causally drive the rapid transition-related DA dynamics observed in the photometry recordings.

Reviewer #1 (Public review):

Summary:

Pecak et al have deciphered the conformational dynamics of a heterodimeric model ABC transporter, TmrAB, a functional homolog of the human antigen transporter TAP, using single-molecule Forster resonance energy and fluorophores attached to residues at either nucleotide binding domains or periplasmic gate. The analysis not only differentiated ATP-free and bound states but also enabled the real-time monitoring of protein conformational changes, precisely dissecting transport cycles and resolving transient intermediates. This study is absolutely significant in providing and establishing a general pipeline delineating the conformational dynamics in heterodimeric ABC transporters.

Strengths:

The scientific study is very well documented for experimental design, results, and conclusions supported by the experimental data. The authors have determined the conformational dynamics of TmrAB across different ATP concentrations, including physiological ones, and resolved an outward open state and other conformational states consistent with previous cryoEM and DEER studies.

Weaknesses:

The scientific study needs a bit of in-depth analysis with respect to consistency in Kd and its implications on the mechanism.

Reviewer #1 (Public review):

Summary:

This is an interesting study describing intensity changes of lamellipodial Arp2/3 complex incorporation dependent on the substratum the cells are spreading on (PLL vs fibronectin), but also on manipulation of either contractility or osmotic pressure or even external mechanical load exerted onto cells, e.g., by increasing medium viscosity. The authors use quite fancy cell systems for their studies, first of all, a CRISPR-engineered fibroblast cell line in which both endogenous loci of the Arp2/3 complex subunit Arpc2 are tagged with mScarlet, but at the same time, conditionally removable using tamoxifen. These lines, optionally also harboring Pxn-GFP and Lifeact-miRFP670, have previously been described by the authors (Chandra et al, 2022, PMID: 34861242). In addition, they use cells allowing local photoactivation of Rac signalling through a Tiam1 activation module combined with Halo-tagged Arpc2, apparently stably co-expressed in tamoxifen-treated Arpc2-KO fibroblasts. These cells may or may not have been published previously.

Overall, the study provides convincing evidence that Arp2/3 complex accumulation in the lamellipodium negatively correlates with its width and perhaps the mechanical load these actin networks are exposed to at the leading edge membrane, shown initially through allowing cells to spread on substrates in which the formation of integrin-based adhesions is poor (PLL) or stimulated (through fibronectin). In the latter case, lamellipodia are comparably narrow, perhaps reasonably well clutched, and thus feel sufficient counter-force at the leading edge membrane to build a dense, Arp2/3-dependent actin network. Albeit interesting and important to show as the authors did, these results are not entirely surprising given the literature published on actin remodeling in cells in conditions similar to those used by the authors (i.e., on PLL). Thus, the results should be better embedded into the context of this previous literature to more precisely reveal which aspects are new and interesting and which ones are more or less intuitive and expected.

However, the authors also show yet another result, which is quite spectacular indeed, revealing dramatic local protrusion of a Rac-dependent lamellipodium on PLL only in the presence of methylcellulose, but not on PLL alone. Although the authors cannot fully explain the mechanisms causing these results, they are thought-provoking and will certainly stimulate future, relevant research on this topic and new insights. Altogether, I think this is an interesting study that can be shared rapidly, given that the authors provide more experimental detail and transparency concerning their used cell model systems. Aside from a few other suggestions for amendments and corrections, I would also recommend citing classical literature that has provided the basis for the interpretation of the results shown here, as specified below.

Specific criticism and comments:

(1) I feel the paper is interesting for actin remodeling and Arp2/3 complex aficionados, but quite difficult to read and to understand in places for non-experts in the field, so I think the text requires more detailed explanation of specific terms, model systems used, and overall correction of either grammatical or semantic errors, or colloquial language.

(2) In general, I think the characterization of Arp2/3 complex incorporation into the lamellipodia of cells spreading on PLL versus FN is interesting, as it has not been done previously in such a systematic fashion to my knowledge. However, I think the authors could emphasize better how this relates to previously established structural features of actin filament networks, published on PLL. So more than 3 decades ago, Hotchin & Hall published clear evidence that starved fibroblasts can only form focal complexes or adhesions downstream of PDGF or LPA-stimulation if seeded on FN, but not on PLL (see Figure 1 in PMID: 8557752). Around the same time, Flinn and Ridley showed this virtual absence of classical, Rac-dependent focal complexes to be accompanied by the formation of beautiful, broad lamellipodia (see Fig. 1A in PMID: 8743960), which only formed in the absence of excess RhoA activity and thus contractility by the way (see also below). A few years later, Small et al summarized all these phenotypes in a comprehensive review and also showed that cells on PLL (similar to the rapidly migrating keratocytes) combined large, flat lamellipodia with tiny, nascent adhesions scattered throughout these structures (see Figure 2 in PMID: 10047522). These authors also noted that the sole inhibitor-mediated reduction of contractility could switch FN-phenotypes with narrow, ruffling lamellipodia and peripheral focal complexes back to a PLL-type phenotype of broad lamellipodia (see Figure 1 in PMID: 10047522). In the following decade then, different labs (Verkhovsky, Bershadsky, Vavylonis, Watanabe et al) showed beautiful phase contrast or fluorescence movies illustrating that the broad lamellipodial phenotype of cells plated on PLL was accompanied by low frequency membrane ruffling and instead a rapid, continuous rearward flow of continuously assembling actin filament networks, partly also directly shown with actin networks labeled with both LifeAct and Arp2/3 complex subunits (see e.g. PMIDs 18800171 and 22500749). In Alexandrova et al, 2008 (PMID 18800171), authors showed that the formation of adhesions in spreading cells triggers the transition from fast to slow flow (which is of course relevant to the current study and conclusions), whereas Ryan et al, 2012 (PMID 22500749) already established the broad incorporation of actin and Arp2/3 complex into the very broad lamellipodia formed on PLL by Xenopus fibroblasts and the rapid flow of both components from distal to proximal lamellipodial regions. None of these seminal studies has been cited, although they are highly relevant for the interpretation and conclusions of the results presented. I would strongly recommend specifically referring to these studies, as this will actually support the conclusions and interpretations drawn.

(3) On the subject of literature, on the second page of the intro, end of 2nd paragraph, the authors describe Rac signaling to Arp2/3 complex through WRC considered essential for Arp2/3-mediated actin assembly at lamellipodial leading edges, but aside from one of their own papers cite none of the seminal studies by Insall, Scita, Stradal, Rottner, Bogdan labs having published seminal aspects on this pathway.

Considering the rapid F-actin flow in lamellipodia, obviously accompanied by admittedly sparse but continuous Arp2/3 complex incorporation, it is not so surprising that the latter will be obligatory here, and also the accumulation of its prominent activator WRC, as well as the branch stabilizer cortactin. Thus, the data described on page 3 of the Results section could also be framed in the context of all this previously published knowledge, providing a more comprehensive and realistic view of the relevance and novelty of the described data.

(4) In the abstract, the authors state in the context of the force-feedback mechanism established in vitro for the formation of Arp2/3 complex-dependent actin networks that "this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells". In my view, this is not correct, as in their prominent Cell paper, the Sixt laboratory has done exactly that (Mueller et al, 2017, PMID: 18800171). Although Mueller et al have not looked at Arp2/3 complex dynamics as far as I recall, they have still connected the extent and hence intensity of actin networks at the leading edges of keratocyte lamellipodia with the forces exerted onto them, including direct experimental manipulation of those forces. Although the study has been cited in an independent context, this point should be made clear, and the corresponding sentence in the abstract should be amended.

(5) One point that struck me a little bit was the authors' detailed description of cell spreading on PLL and the quite strong variability of Arp2/3 incorporation dependent on the timing after spreading (as for instance the very strong and quite narrow Arp2/3 leading edge intensity at 2 hours post-seeding in Figure 3S2D). In the authors' view, they have worked with a very clean system, as they emphasized to even have eliminated the FN-locus in their cells, excluding the secretion of endogenous FN (PMID: 34861242), but how about ECM components potentially present in serum, such as, for instance, vitronectin? Indeed, it looks like the authors have done all experiments in the presence of 10% serum as far as I can see, although most of the classical PLL-experiments mentioned above have been performed with starved cells in the absence of serum. I think it would generate a more complete picture of the phenotypes and results as compared to the literature if the authors performed a subset of the key experiments on PLL without serum. I don't think the starving of cells as such is important and could be counteracted by simply lamellipodia-inducing growth factors adding into the spreading medium, traditionally perhaps PDGF or EGF (dependent on the receptor distribution of those fibroblasts), but the absence of serum would have two advantages: it would not only exclude any potential impact of serum-containing ECM components, but also alleviate the hyperstimulation of the Rho-pathway through LPA-bound BSA, the major serum-protein, which has previously been shown to counteract the "undisturbed" formation of PLL-type lamellipodia (see Figure 1B in Flinn & Ridley, PMID: 8743960).

(6) Regarding the scanning EM-images shown in the Supplement, currently called Figure 3S2A and -B (in the text erroneously termed Figures 3S1A and-B, see above). I am not sure how representative these individual EM-images of the cell plated on PLL are, given the data of rapid rearward flow of actin and Arp2/3 complex subunits, at least at early stages of spreading. Again, the classical literature on PLL-type lamellipodia and, in particular, previously published movies of such lamellipodia suggest broad lamellipodia with few ruffles, and the opposite with cells plated on FN. So in this context, the scanning EM-data shown on both PLL and FN do neither fit the authors' own data very well nor the literature, and I would recommend making sure that the individual cells selected were (i) correctly annotated and (ii) representative of a specific time point of spreading actually fitting the previously described data.

(7) It also surprised me to see that the authors describe the spreading process on PLL to actually be much slower than on FN (see Figure 3S2C - in the text Figure 3S1C). It is tempting to speculate that this might change if plating the cells in serum-free medium, as traditionally, full spreading and lamellipodia formation downstream of PDGF-stimulation (at least in 3T3 fibroblasts) is described to occur in the range of 10-30 minutes at maximum, and not several hours as shown here. This point could also be considered, or at least discussed.

(8) The movies are of very high quality and beautiful to look at, but it would help the reader to get a bit more information in the legends (like the meaning of the time-stamps, which will display elapsed time in minutes:seconds I assume, but this info is missing from the legends as far as I can see. Also, it would help the reader to better mark in the movies when a specific treatment kicks in. For instance, in movie 10, the legend states treatment starts at 10:00 (minutes:seconds?), but it would help very much if the authors could paste the term "blebbistatin" directly into the movie, beginning with the frame of treatment start.

Reviewer #1 (Public review):

Summary:

Yuan and colleagues present a thorough study of gene activation before and during metamorphosis in sponge larvae, combining in-depth analyses of staged transcriptomes and chromatin accessibility profiling (ATACseq). Amongst several very interesting findings, the study reveals that the acquisition of settlement competence, which arises in response to decreasing light at sunset, is characterized by changes in chromatin accessibility that anticipate strong transcriptional shifts occurring as metamorphosis starts. Another notable finding is a set of transcription factors amongst the genes strongly up-regulated at the onset of metamorphosis. In addition, larvae exposed to constant light, a condition that stalls metamorphosis, were found to activate metabolic pathways that are not normally expressed in swimming larvae. Together, the findings provide a rare level of understanding into how environmental conditions can promote deployment of alternative developmental programs in planktonic larvae.

Strengths:

This is a very comprehensive, well-documented and rigorous study of a phenomenon of wide interest. It will inspire researchers working on other species to look for similar, environmentally-driven "anticipatory" epigenetic mechanisms. It also provides a wealth of detailed information on genes, notably transcription factors, that are candidates for involvement in regulating specific metamorphosis transitions - and beyond. The data presented here are thus undoubtedly a rich and valuable resource.

Weaknesses:

I see no significant weaknesses; however, the documentation of the data is very compressed, with all the findings contained in 4 multi-panel figures with succinct legends. It is not always straightforward to connect the conclusion statements in the text to the figures. Although the relevant data is available in supplementary files, I would appreciate more help in navigating the data to assess the support for key conclusions, if possible, illustrating each text conclusion explicitly in the main figures.

Reviewer #1 (Public review):

Objectives of the study and impact of the work:

The authors of this article primarily aim to reconstruct the evolutionary history of the insect odorant receptor (OR) family, which is responsible for the detection of odorant signals by olfactory neurons. Due to the lack of phylogenetic signal present in the sequences of this multigene family, which evolves very rapidly, phylogenetic analyses have so far never made it possible to precisely retrace how ORs diversified prior to the appearance of present-day insect orders, and what the drivers of this diversification were. For example, one may suspect that the adaptation of ORs to odors emitted by plants constituted a critical step in insect evolution during the "angiosperm terrestrial revolution," which occurred at the end of the Cretaceous, but nothing currently allows this to be asserted.

There are very nice examples, notably in Drosophilids, derived from comparisons between closely related species and documenting mechanisms of OR adaptation to certain signals. However, what the authors attempt to do in this work is to produce a macroevolutionary analysis at the scale of insects as a whole, based almost exclusively on bioinformatic analyses. To do this, they annotated OR genes in about one hundred insect species and developed pipelines for analyzing sequence similarity, structural similarity, and functional similarity, the latter being estimated through a molecular docking approach. An important feature in the evolution of insect ORs is the emergence of a unique co-receptor, called Orco, which appears to be an OR that has lost the ability to bind odorants. In addition to the large-scale bioinformatic analysis, the authors also aim to explore more specifically the factors that favored the emergence of Orco and the selective advantage conferred by the existence of OR-Orco complexes.

Given the importance of odorant receptors in insect biology and in their adaptation to different environments and lifestyles, retracing their evolutionary history is indeed a major question in evolutionary biology. In principle, this type of work therefore has the potential to become a reference in the field and to provide a basis for significant scientific advances.

Major strengths and weaknesses:

The sampling chosen for collecting OR sequences is very impressive, with more than 100 insect families represented, covering most of the major orders. This sampling appears appropriate for the question being addressed. The analysis pipeline used to collect the sequences makes sense, relying on homology-based annotation tools coupled with a structure-based filter. Nevertheless, one can note aberrant numbers of ORs for certain species (much lower than reality), which indicates that the pipeline probably did not function correctly for all genomes. In the absence of a validation step comparing the results with already known OR repertoires, it is difficult to estimate the overall quality of the data. The authors chose to apply a fairly stringent filter on sequence quality (based on predicted 3D structure), which reduces the number from 14,000 to 9,000. This choice seems logical given the subsequent use of these data, but it inevitably leads to data loss. The fact that some OR genes may be missing and that the total number may not be exact for each species is not prohibitive for studying the evolution of the family at a broad scale; however, it calls into question certain results that rely on this total number, such as the correlation between the number of ORs and genome size, lifestyle, and diet.

From the dataset collected, the authors attempted to categorize ORs in several ways, starting with the reconstruction of sequence similarity networks. The approach is interesting, but in the end, the results do not seem to be sufficiently exploited, and it is not obvious what the advantage of this approach is compared with the "classical" phylogenetic approach, which generally fails to reveal homology relationships between ORs from species belonging to different insect orders. Here again, the majority of the clusters identified are "order-specific," and when this is not the case, the authors did not attempt to exploit the results. For example, clusters SeqC26 or SeqC28, which appear to be shared by many insects, are potentially very interesting. It might have been relevant to combine this similarity-based clustering approach with phylogenetic reconstructions within each shared cluster.

The clustering based on structure also leads to the identification of a majority of "order-specific" clusters, but once again, the clusters shared by several orders are not truly exploited, which does not provide new insight into the evolution of ORs. However, the authors highlight a group of ORs in flies that appear to possess an unusual intracellular region. This is interesting, although it is a result more relevant to OR structure than to their evolution. The function of these ORs in Drosophila melanogaster, if it is known, is not discussed.

The analysis of structural diversity then leads the authors to focus on the Orco co-receptors, which are characterized by modifications of the binding pocket and the emergence of an extracellular loop that could explain the loss of the ability to bind odorant molecules. This part, which relies on in vitro experiments, is interesting and constitutes the most striking result of the study, which could in itself have been the subject of a separate manuscript. However, the molecular dynamics modelling does not add anything in the way it is conducted (5 ns is too short).

The rest of the manuscript is based on the prediction of OR response spectra using molecular docking. The work that has been carried out is extremely substantial, and the objective of linking clusters based on sequence similarity or 3D structural similarity with functional categories is entirely relevant. Nevertheless, I see two major problems with this in silico functional analysis:

(1) The docking score threshold used was chosen thoughtfully, which is very good, and according to the calculation performed, should ensure a true positive rate of more than 20%, which is excellent in such a docking analysis. But in the absence of functional validation, this 20% true positive rate is not sufficient to extrapolate OR function as the authors do in the remainder of the manuscript. The risk of error remains too high to compare in such detail the function of ORs from insects with different lifestyles or diets.

(2) The six functional clusters identified are only slightly different from one another, with similar detection of all chemical families except acids and amines (which was expected, given that these families are a priori detected by IRs rather than ORs). This shows that even though the approach is relevant and deserves to be tested, it cannot be used to establish a link between groups/lineages of ORs and response spectra at the scale of insects as a whole. This is reflected in the final analysis by the fact that there is no visible link between sequence or structural clusters and functional clusters. Given the uncertainty surrounding the docking results, the entire subsequent analysis of the relationship between the Binding Breadth Index and ecological variables is highly questionable.

Finally, the evolutionary analysis proposed to conclude that the work suffers from an incorrect interpretation: ORs of non-holometabolous insects cannot be considered equivalent to those of species that existed before the Permian-Triassic extinction. The fact that a locust or a cockroach has more narrowly tuned ORs than holometabolous insects does not mean that this was also the case for ancestral insects. To advance this type of conclusion, it would be necessary to conduct a phylogenetic analysis and reconstruct ancestral states, which is not the case here.

In summary, despite the large number of analyses performed, the authors do not succeed in achieving the stated objective of reconstructing the evolutionary history of insect ORs, and the results obtained do not sufficiently support the conclusions regarding the links between OR repertoires and environment or lifestyle.

Reviewer #1 (Public review):

Summary:

Dancausse et al. investigate behavioral responses to nicotine exposure in Drosophila larvae. They discover that high concentrations of nicotine lead to less movement and twitching, which recover slowly after several hours. Exposure to lower concentrations, however, increases locomotion and leads to hyperactive behavior. The authors also perform pharmacological and genetic manipulations to address the role of dopamine for these behavioral changes. Additionally, they test the role of MB intrinsic neurons by genetic silencing. Both Dopamine and MB manipulations affect responses to nicotine exposure. Finally, they investigate how larvae respond to repeated exposures to nicotine and find that they do not habituate. Additionally, repeated exposure to nicotine leads to a preference towards higher concentrations in a gradient assay.

Strengths:

The authors use rigorous behavioral analysis and discover interesting concentration and experience-dependent effects of nicotine exposure on locomotion in fly larvae, which will be worth investigating in the future to decipher the underlying neural mechanism.

Weaknesses:

As the manuscript currently stands, the results of genetic manipulations are hard to interpret and rather inconclusive. The genetic manipulations have been performed using broadly expressing genetic driver lines, which weakens the conclusions drawn by the authors. Thus, no specific neural populations or brain regions have been discovered, and there is little insight into the underlying neural mechanism.

Based on gradient experiments, the authors suggest that fly larvae could serve as a model organism for addiction. This claim is quite strong, but no control experiments are shown for shorter exposure or a single exposure with a longer resting period before the gradient test. To compare this to addiction-like behaviors, more control experiments should be performed.

The authors should clarify better how experiments were performed in Materials and Methods. Generally, the authors perform novel behavioral analysis, which is not explained in enough detail. The nicotine concentration that has been used for most experiments is this a relevant concentration comparable to other studies? This information would be useful to put into context with other findings.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigate how the anterior claustrum may integrate temporally separated task-relevant signals to guide behavior in a delayed escape paradigm. Because in vivo neural recordings from claustrum during this task are extremely limited-comprising single-trial data with small neuronal samples-the authors adopt a modeling-driven approach. They train recurrent neural networks (RNNs) using only behavioral data (escape latency) to reproduce task performance and then analyze the internal dynamics of the trained networks. Within these networks, they identify a subset of units whose activity exhibits persistent responses and strong correlations with behavior, which the authors label as "claustrum-like." Using dimensionality reduction, decoding, and information-theoretic analyses, they argue that these units dynamically integrate conditioned stimulus (CS) and door-opening signals via nonlinear, trajectory-based population dynamics rather than fixed-point attractor states.

To bridge model predictions and biology, the authors complement the modeling with in vitro slice experiments demonstrating recurrent excitatory connectivity and prolonged activity in the anterior claustrum that depends on glutamatergic transmission. They further compare latent neural trajectories derived from previously published in vivo claustrum recordings to those observed in the RNN, reporting qualitative similarities. Based on these results, the authors propose that the claustrum implements temporal signal integration through recurrent excitatory circuitry and dynamic population trajectories, potentially supporting broader theories of integrative brain function.

Strengths:

This study addresses an important and challenging problem: how to infer population-level computation in a brain structure for which in vivo data are sparse and experimentally constrained. The authors are commendably transparent about these limitations and seek to overcome them through a principled modeling framework. The integration of behavioral modeling, RNN analysis, and slice electrophysiology is ambitious and technically sophisticated.

Several aspects stand out as strengths. First, the behavioral RNN is carefully trained and interrogated using a rich set of modern analytical tools, including cross-temporal decoding, trajectory analysis, and partial information decomposition, providing multiple complementary views of network dynamics. Second, the slice experiments convincingly demonstrate recurrent excitatory connectivity in anterior claustrum, lending biological plausibility to the model's reliance on recurrent dynamics. Third, the manuscript is clearly written, logically organized, and conceptually engaging, and it offers a coherent mechanistic hypothesis that could guide future large-scale recording experiments.

Importantly, the work has significant heuristic value: rather than merely fitting data, it attempts to generate testable computational ideas about claustral function in a regime where direct empirical access is currently limited.

Weaknesses:

Despite these strengths, the manuscript suffers from a recurring and substantial conceptual issue: systematic over-interpretation of model-data correspondence. While the modeling results are potentially insightful, the extent to which they are presented as recapitulating real claustral neural mechanisms goes beyond what the available data can support.

A fundamental limitation is that the RNN is trained solely on behavioral output, without being constrained by neural data at either single-unit or population levels. As a result, the internal network dynamics are underdetermined and non-unique. Many distinct internal solutions could plausibly generate identical behavior. However, the manuscript frequently treats the specific internal solution discovered in the RNN as if it were a close approximation of the actual claustrum circuit.

This issue is compounded by the sparse nature of the in vivo data used for comparison. The GPFA-based trajectory analyses rely on pseudo-populations and single-trial recordings, yet are interpreted as evidence for robust population-level dynamics. Because neurons were not recorded simultaneously, the inferred trajectories necessarily lack true population covariance and shared trial-to-trial variability, limiting their interpretability as genuine population dynamics. Similarly, conclusions about trajectory-based versus attractor-based computation are drawn almost exclusively from model analyses and then generalized to the biological system.

Overall, while the modeling framework is appropriate as a hypothesis-generating tool, the manuscript repeatedly crosses the line from proposing plausible mechanisms to asserting explanatory or even causal equivalence between the model and the brain. This undermines the otherwise strong contributions of the work.

Below are several specific points that warrant further clarification or revision:

(1) Tone of model-data correspondence

Numerous statements describe the RNN as "closely mimicking," "recapitulating," or being "nearly identical" to claustral neural dynamics, sometimes extending to claims about causal relationships between neural activity and behavior. Given that neural data were not used to train the model, and that only a small subset of trained networks showed the reported dynamics, these statements should be substantially softened throughout the manuscript. The RNN should be framed as providing one possible computational realization consistent with existing data, not as a close instantiation of the biological circuit.

(2) Non-uniqueness of RNN solutions

The fact that only a small fraction of trained networks exhibited "claustrum-like" clusters deserves deeper discussion. This observation raises the possibility that the identified solution is fragile or highly specific rather than canonical. The authors should explicitly discuss the non-uniqueness of internal solutions in behavior-trained RNNs, including the range of alternative network dynamics that can reproduce the same behavior. In particular, it should be clarified why the specific network exhibiting "claustrum-like" clusters is informative about claustral computation, rather than representing one arbitrary solution among many.

(3) GPFA trajectory comparisons

The qualitative similarity between RNN trajectories and GPFA-derived trajectories from sparse in vivo data is interesting but insufficient to support claims of robustness or population-level structure. Statements suggesting that these patterns are unlikely to arise from noise or random fluctuations are not justified given the single-trial, pseudo-population nature of the data. Either additional quantitative controls should be added, or the interpretation should be substantially tempered.

(4) Scope of functional claims

The discussion connecting the findings to broad theories of claustral function, global workspace, or consciousness extends well beyond the data presented. These speculative links should be clearly labeled as such and significantly reduced in strength and prominence.

The manuscript repeatedly describes the delayed escape task as an "inference-based behavioral paradigm" and states that animals "infer that a value-neutral alternative space is likely to be safer" when the CS is presented in a novel environment. While I appreciate that the US-CS association was established in a different context and that the CS is then presented in a new environment, I am not convinced that the current behavioral evidence uniquely supports an inference interpretation.

First, it is not clear that this task is widely recognized in the literature as a canonical inference task, in the sense of, for example, sensory preconditioning, transitive inference, or model-based inference paradigms. Rather, the observed effect-that CS animals escape faster to a neutral compartment than neutral-CS controls-can be parsimoniously interpreted in terms of generalized threat value, heightened fear/anxiety, or a bias toward avoidance/escape under elevated threat, without requiring an explicit inferential step about the specific safety of the alternative compartment. The fact that no prior training is needed is compatible with flexible generalization, but does not by itself demonstrate inference in a more formal computational sense.

Second, the inference claim becomes central to the manuscript's conceptual framing (e.g., the idea that rsCla supports "inference-based escape"), yet the behavioral analyses presented here and in the cited prior work do not clearly rule out simpler accounts. Clarifying this distinction would help avoid overstating both the inferential nature of the behavior and the specific role of rsCla and the RNN's "claustrum-like" cluster in supporting inference per se, as opposed to more general integration of threat-related signals with an opportunity for escape.

This manuscript presents an interesting and potentially valuable modeling-based framework for thinking about temporal integration in the claustrum, supported by solid slice physiology. However, in its current form, it overstates the degree to which the proposed RNN dynamics reflect actual claustral neural mechanisms. With substantial revision-especially a more cautious interpretation of model-data similarity and a clearer articulation of modeling limitations-the study could make a meaningful contribution as a hypothesis-generating work rather than a definitive mechanistic account.

Comments on revisions:

The authors have carefully addressed the concerns raised in the initial review. In particular, the manuscript has been substantially improved in terms of tone, conceptual clarity, and the interpretation of the modeling results. The revised version now presents a well-balanced and appropriately framed account of the work.

The study offers a compelling and useful hypothesis-generating framework for understanding temporal integration in the claustrum, and I support its publication. As a minor point, given the acknowledged limitations of pseudo-population and single-trial data, it would be preferable to slightly soften a few remaining statements that describe trajectory structure as directly "reflecting" population-level dynamics (e.g., using "consistent with" instead).

Reviewer #1 (Public review):

Summary:

This study presents a novel toolkit for visualizing and manipulating neurotransmitter-specific vesicles in C. elegans neurons, addressing the challenge of tracking neurotransmitter dynamics at the level of individual synapses. The authors engineered endogenously tagged vesicular transporters for glutamate, GABA, acetylcholine, and monoamines, enabling cell-specific labeling while maintaining physiological function. Additionally, they developed conditional knockout strains to disrupt neurotransmitter synthesis in single neurons. The study reveals that over 10% of neurons in C. elegans exhibit co-transmission, with a detailed case study on the ADF sensory neuron, where serotonin and acetylcholine are trafficked in distinct vesicle pools. The approach provides a powerful platform for studying neurotransmitter identity, synaptic architecture, and co-transmission.

Strengths:

(1) This toolkit offers a generalizable framework that can be applied to other model organisms, advancing the ability to investigate synaptic plasticity and neural circuit logic with molecular precision.

(2) The use of this toolkit, the authors uncover molecular heterogeneity at individual synapses, revealing co-transmission in over 10% of neurons, and offers new insights into neurotransmitter trafficking and synaptic plasticity, advancing our understanding of synaptic organization.

Weaknesses:

(1) While the article introduces valuable tools for visualizing neurotransmitter vesicles in vivo, the core techniques are based on previously established methods. The study does not present significant technological breakthroughs, limiting the novelty of the methodological advancements.

(2) The article does not fully explore the potential implications or the underlying mechanisms governing this process, while the discovery of co-transmission in over 10% of neurons is an intriguing finding. A deeper investigation into the functional uniqueness and interactions of neurotransmitters released from individual co-transmitting neurons-perhaps through case study example-would strengthen the study's impact.

Comments on revisions:

I have no further questions regarding this work. I would like to congratulate the authors on the forthcoming publication of their manuscript. This study presents a versatile methodological framework with strong potential to advance the field of neuroscience, particularly in dissecting neural circuit function and neurotransmission dynamics in vivo.