Reviewer #1 (Public review):

Summary:

This is a useful study by Angla et al that investigates the basis of observations made from previous studies where loss of Onecut (OC) transcription factors lead to changes in spinal interneuron populations that do not themselves normally express OC. The authors hypothesize that OC expression in spinal motor neurons has non-cell-autonomous effects on pre-motor interneuron (V1, V2a/b/c) population size and distribution. By knocking out OC in the motor neuron lineage (i.e., downstream of Olig2, a motor neuron progenitor marker gene), they indeed show that motor neuron-specific loss of OC expression decreases V2c interneuron number and alters the spatial distribution of V1, V2a, V2b, V2c populations. Using bulk RNA-sequencing of WT and OC conditional knockout (cKO) motor neurons, the authors identify that neurotrophic factor Ntf3 as another factor that is responsible for the non-cell-autonomous effects on the interneuron population; upon knock out of Ntf3 downstream of Olig2 they show that this leads to increased interneuron numbers and alters their spatial distribution, ultimately leading to dysregulation of spinal motor circuits and motor activity.

Strengths:

The authors use sophisticated genetic tools to precisely remove OC and Ntf3 expression in a lineage-specific manner and comprehensively assess the downstream effects across brachial, thoracic, lumbar levels of the spinal cord, as well as at two developmental timepoints, E12.5 and E14.5.

Weaknesses:

The evidence supporting their claims is incomplete. In figure 2 and 4, the control distributions (despite reporting the same populations in the same regions) look different, suggesting large sample-to-sample variance in distribution. The authors explain that this is due to several technical limitations in their study.

Furthermore, the way the Ntf3 results are framed can be misleading; it reads as though one of the main claims of this study is that OC acts through Ntf3.

Reviewer #1 (Public Review):

In this study, the authors set out to investigate spatial RNA processing events, specifically alternative splicing and 3' UTR usage, in mouse brain and kidney tissues using ReadZS and SpliZ methodologies on spatial transcriptomics data. The research contributes to understanding tissue-specific gene expression regulation from a spatial perspective. The study introduces a novel approach for analyzing spatial transcriptomics data, allowing for the identification of RNA processing and regulation patterns directly from 10X Visium data. The authors present convincing evidence supporting the identification of novel RNA processing patterns using their methodology, which holds significant implications for researchers in the field of spatial transcriptomics and the study of alternative splicing and 3' UTR usage.

The conclusions of the study are mostly well-supported by the data; however, certain aspects could be improved to strengthen the findings.

(1) The conclusions of this study would be strengthened by conducting a more extensive tissue sample analysis and including biological replicates. Additionally, appropriate batch effect corrections should be applied when dealing with biological replicates.

(2) The 3' UTR usage and alternative splicing should be compared among clearly labeled clusters for a more comprehensive analysis.

(3) The authors should clarify their rationale for choosing ReadZS and SpliZ approaches and provide comparisons with other methods to demonstrate the advantages and potential limitations of their chosen methodologies.

Reviewer #1 (Public review):

Sensory hair cells of the inner ear convert mechanical sound vibrations into electrical signals through mechano-electrical transduction (MET). While the protein components of the MET machinery have been studied extensively, much less is known about how the surrounding membrane lipid environment contributes to hair cell function. The recent discovery that TMC1 and TMC2 also function as lipid scramblases has brought renewed attention to the importance of membrane lipid asymmetry and the mechanisms that maintain it in sensory hair cells.

In this study, the authors identify the P4-ATPase ATP8B1 and its partner TMEM30B as key regulators of membrane lipid asymmetry in outer hair cells. Using complementary genetic models, HA-tagged knock-in mice, localization analyses, and functional experiments, they show that ATP8B1-TMEM30B is enriched in stereocilia and the apical membrane of outer hair cells and is required to maintain phosphatidylserine asymmetry, support hair cell survival, and preserve normal hearing. The parallels between the ATP8B1/TMEM30B loss-of-function phenotypes and TMC1 deafness-associated mutants with constitutive scrambling support a model in which ATP8B1-TMEM30B flippase activity maintains membrane lipid asymmetry and homeostasis, whereas constitutive TMC1-mediated phospholipid scrambling disrupts this balance and contributes to membrane instability.

The authors have addressed the points raised during the initial review thoroughly. The revised manuscript includes clearer methodological details, additional physiological characterization, improved presentation and quantification of several datasets, and a more balanced interpretation of the localization and mechanistic findings. These changes improve both the clarity and rigor of the study while leaving its main conclusions unchanged.

As with any study that opens a new area of investigation, important mechanistic questions remain. In particular, it will be interesting to determine how disruption of membrane lipid asymmetry ultimately impairs MET function and triggers hair cell degeneration, how flippase and scramblase activities are coordinated in vivo, and how these pathways are integrated with the broader molecular machinery underlying mechanotransduction. These questions highlight the exciting directions that this study opens for the field.

Overall, this work provides evidence that ATP8B1-TMEM30B is a critical regulator of stereocilia membrane lipid asymmetry and represents an important contribution to our understanding of membrane homeostasis in auditory hair cells. I have no further major concerns and support publication.

Comments on second revision:

I appreciate the authors' efforts to address the issue and perform the additional experiment, and I agree that removing these data is reasonable and does not affect the main conclusions of the manuscript.

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 in which 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 in which 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 size, volume and protein compositions such as the nucleus, nucleoli and Golgi. They demonstrate that SPEx can identify proteins enriched or reduced in ROIs.

Strengths:

The general, relatively easy and inexpensive nature of this approach, as well as its potential applicability to 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:

The authors addressed our concerns and technical questions in revision.

A main limitation of the current method is that it relies on isolation of structures of interest in 2D. The authors discuss some alternative approaches that might allow future access to more 3D target isolation.

There remain several unexplored minor extensions of the technical development of the method, including comparing the proteomes of organelles that are visually distinct, and applying different anchoring procedures to try to increase protein retention.

Reviewer #1 (Public review):

Summary:

The study identifies and characterizes a set of amino acid states that make the protein robust to other mutations, to the point of being able to compensate mutations that render wildtype proteins entirely non-functional. The study uses a previously published dataset and uses it to find and study such super-compensators. It then analyzes the biophysics and fitness landscape structure of what may be behind the compensation, identifying stability as an important parameter that, nevertheless, is not sufficient to explain all of the compensatory effect. These findings have important implications for our understanding of protein evolution, with these super-compensators possibly acting in a role of "permissive mutations" and open up evolutionary trajectories that may be closed without them. Perhaps the identification of such super-compensator substitutions can be incorporated into various protein design approaches.

Strengths:

The paper presents a compelling case with a rigorous analysis of the expected error rates of observation. While not unique, the current state-of-the-art in the field typically does include experimental error rate estimation like this work. The paper also does a good job in exploring the issue, including looking at plausible biophysical basis of super-compensators.

Comments on revised version.

The authors have addressed all of my concerns in the revision.

Reviewer #1 (Public review):

Summary:

The authors provide a compelling story linking NAD dependent HSD3b6 gene expression in the eyelid to a viscous cycle involving decreased steroidogenesis and AR signaling, pro-inflammatory cytokine release, inflammation, CD38 activation and further NAD+ decline that induces meibomian gland atrophy leading to dry eye disease. The work follows on studies published by the authors in Nature Aging and uses a previously reported HSD3b6 knockout mouse to try and show meibomian gland atrophy, inflammation and CD38+ cell recruitment. Overall the data presented does not appear to support the authors conclusions, and the described interactions seem to be overstated, in that if HSD3b6 was critical to meibomian gland function, one might expect complete loss of the meibomian gland in knockout mice, which is not the case.

Comments on revised version:

It should be noted that while the authors indicate the CD38 is significantly up-regulated in the HSD3b6 KO mouse, the increase was not sufficient to show a significant adjusted P-value after revision of the manuscript. Bulk RNA sequencing also shows no significant change in meibum lipid gene expression for aged mice that are treated with 78c, an inhibitor of CD38, that the authors indicate increases NAD levels leading to increased meibomian gland size compared to vehicle treated mice. Unfortunately, there was no increase in meibum lipid gene expression with 78c or with 78c+NMN treatment as identified by adjusted P-value.

Class and gender: Anna is a poor woman serving the household, and her kneeling shows the hierarchy.

This passage shows that Maiguru is not completely powerless. Despite the challenges in her marriage, she finally takes action to express her dissatisfaction. I think this demonstrates the importance of women having the courage to stand up for their rights.

This shows how economic differences can affect relationships between women. Mainini feels that her opinions are ignored because she is poor. It reminds me that every woman deserves to be heard regardless of her financial position.

Tambu's mother has never been allowed to choose anything. When she asks Lucia “Since when has it mattered what I want?”, it shows how a woman’s life is controlled by her father and then her husband.

https://www.facebook.com/groups/4770669677posts/posts/10164413651119678/

The Haas Atlas (page 7) would identify the foundry mark as being a Hulse-Typen manufactured Pica face for Smith-Corona: https://archive.org/.../typewriter.../page/n5/mode/2up

Page 31 of the 1954 Smith-Corona Typewriter Type Styles and Keyboards Catalog would indicate it seems to match up solidly with the Pica Gothic No. 4 typeface: https://typecast.munk.org/.../1954-smith-corona-scm.../ Especially look at the Q, the J, and the 8. The '64 NOMDA guide Mark links is the same, but doesn't have as extensive a typesample.

Many machines, and especially the American manufactured ones, don't always have foundry marks which line up with the respective catalogs.

Reviewer #1 (Public review):

This is a very elegant and experimentally convincing study. Using systematic screening of actin tail formation in two bacterial strains and employing a panel of CRISPR-CAS ko cell lines, the authors identify the dynamin-related GTPases GVIN as antibacterial agent, which forms an oligomeric coat around an intracellular Burkholderia strain. The bacterial O-antigen LPS layer is required for the formation of the GVIN coat, which disturbs the polar localization of the bacterial actin-polymerizing BimA protein.

The experiments appear of high quality, figures are well prepared, and clean and statistically significant results are provided. I have no criticism on the presented approaches.

The identification of a novel GBP1-independent pathway targeting intracellular bacteria is not only of fundamental importance for the immunity field, but also of high interest to researchers in other areas, for example, evolutionary or structural biologists.

Comments on revised version.

This is a beautiful study.

Reviewer #1 (Public Review):

[Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers, given the substantial revisions completed since the original submission: these have improved the clarity and transparency of the results in a relatively complex study. These changes largely address those concerns raised in review that do not relate to the underlying experimental design and the control of the distinct task conditions.]

Summary:

In this manuscript, the authors investigate the impact of rare and extreme events on rodents' decision-making under risk, in gain and loss contexts. They describe the behavior of 20 rats performing a four-armed bandit task, where probabilistic gains (sugar pellets) and losses (time-out punishments) can - in some arms - incorporate extremely large - but rare - outcomes. They report that most rats are sensitive to rare and extreme outcomes despite their infrequent occurrence, and that this sensitivity is primarily driven by extreme loss events which they try to avoid, rather than extreme gains that they seek to obtain.

They finally propose a modification of standard reinforcement-learning, which features a specific sensitivity to rare and extreme outcomes and can account for the observed behavior.

Strengths:

The manuscript really taps into a surprisingly neglected but very relevant aspect of decision-making: the effect of rare and extreme events (REE). The authors have developed an experimental setup that seemingly allows investigation of this aspect, which is not trivial given the idiosyncratic properties of rare and extreme events.

The parameters of the experimental setup seem also to be well thought off: basically, in the absence of REE, some options are objectively better than others (because, in expectation, they overall deliver more food, or minimize time-out punishments), but this ordering reverses if REE are taken into account. This allows for a clean test of the integration of REE in the rodent's decision-making model.

The data is presented and analyzed in a very descriptive but exhaustive and transparent way, down to the description of individual rodent's behavior.

Reviewer #1 (Public review):

The authors did very little to address our concerns outside of written clarifications. While they altered wording to describe Gsb from "pioneering" to "chromatin priming" our stance remains that there is no evidence shown to confirm that Gsb plays a role in chromatin remodelling. (IE while ectopic expression of Gsb results in a different chromatin conformation, there is no evidence that the ectopic expression of Gsb alters an already established chromatin state.) Additionally, the phenotypic experiments show loss of NB5-6 reporter and neuronal markers upon loss of Gsb which confirmed the developmental requirement for Gsb within the endogenous NB5-6 domain, but another striking feature that was described was that loss of Gsb also results in loss of NB3-5 identity. The abrupt change to a new NB lineage made this portion of the manuscript feel a bit disjoined and while this is described as an additional reciprocal phenotype, there are still no sequencing data or other evidence as to how this loss has occurred.

We disagree that pioneer or chromatin-priming activity should be inferred strictly from single timepoint binding profile. We also disagree that any "remodeling" was shown. To sufficiently show remodeling, one must express a factor at/after a time where the chromatin conformation has been established to be already open/closed and then show the expression of that factor induces conformation changes to the opposite conformation. Otherwise, it is hard to infer whether these chromatin changes are due to the presence of the factor blocking the normal conformational changes that should occur.

The authors mention they walked back the terminology from "pioneering" to "chromatin priming" and make it clear that Gsb does not act alone to establish the chromatin landscape. We are not sure that this is proper terminology either.

We mention that the phenotypic data do not align with the sequencing data. The authors added text to clarify the rationale behind each experiment - some being to induce outside of the endogenous domain with clean genetic access while others to obtain information within the endogenous spatial domain.

Reviewer #1 (Public review):

Summary:

In this manuscript the authors study the distribution of the KMT2A methytransferase across the human ribosomal gene loci (rDNA) and the effects of its depletion on rDNA chromatin modification using a KMT2A conditional cell line. They used ChIP mapping data and claim to show that KMT2A depletion does not affect H3K4me3 levels but does reduce acetylation of H3K9 and reduces PCAF (KAT2B) binding. They further claim that KMT2A depletion causes stalling of RNA Polymerase I at the upstream "Spacer" promoter duplication on the rDNA.

Weaknesses:

I found the manuscript very confusing. In particular, I found it short on short on experimental detail and I found the data in general to be unconvincing. It is my opinion that the authors overinterpret their data, leading to unrealistic and unsupported claims. The manuscript also avoided discussing relevant recent studies on the regulation of the rDNA and of rDNA chromatin, most citations being to data 15 to 20 or more years old. This is most apparent when discussing the factors such as UBTF and SL1 that determine preinitiation complex assembly and the status of active versus inactive rDNA chromatin. Both have seen significant advances in recent years, and these should be thoroughly discussed.

Specific comments:

The authors do not describe the generation of the KMT2A conditional HEK293T cells or their treatment. Since these cells form the basis for subsequent experiments, they must be explained fully. I found that the conditional cell line had already been used in a previous unrelated publication "Chinchole et al. 2022". This manuscript explained that the KMT2A knockout is induced via Dox controlled Cas9 in a HEK cell clone constitutively expressing guide RNAs to KMT2A. The KMT2A knockout requires a 7 day treatment with Dox, leaving wide open the question of direct versus indirect effects of KMT2A loss, and perhaps explaining some of the discrepancies with published data.

In mapping the interactions of KMT2A across the rDNA by ChIP they use 18 PCR amplicons, some of which the authors inaccurately state as targeting specific rDNA elements. For example, they state Primers (that is, Amplicons) #1-3 target the spacer and core 47S promoters. In fact, amplicon #2 overlaps neither of these promoters and lies centrally within the 1kbp of enhancer repeats between them. Exactly why emphasis is put on KMT2A chipping at these two promoters is anyhow questionable since KMT2A levels (Figure 1) appear equally high at multiple IGS amplicons.

Discrepancies with published data are particularly apparent in their mapping of H3K4me2, H3K4me3 and H3K9ac, all directly relevant to their conclusions. These modifications have been shown in several publications to map to a single major site a few hundred bp upstream of the spacer promoter in multiple human and mouse cell types. Since the authors fail to monitor H3K4 methylation and H3K9 acetylation at this dominant site they also fail to provide significant evidence that KMT2A in fact affects H3 methylation or H3 acetylation on the rDNA.

The KMT2A deletion drastically reduces PolI and UBF (UBTF) engagement across the rDNA, and consistent with this, SL1 and RRN3 engagement is reduced as are 47S levels. Thus, whatever the 7-day KMT2A deletion is doing to the cells it is certainly inducing a very significant shut-down of active rDNA. How such an extreme experimental model can tell anything of KMT2A functions is unclear.

In the last results section, the authors further claim "Loss of KMT2A results in a stalled RNA Pol I complex at the spacer promoter" (Figure 4 and S4). To support this the authors try to map spacer promoter transcripts but mistakenly claim that the amplicon #1 can detect these transcripts. In fact, this amplicon overlaps the Spacer Promoter initiation site and so could not detect transcripts coming from this promoter. Published data, not cited, have demonstrated that PolI stalling at the spacer promoter occurs as a normal phenomenon on the active rDNA. Its loss is associated with suppression, not the opposite as inferred in this manuscript.

Reviewer #2 (Public review):

The authors examine the relative contributions of SNP and transcriptomic information to predicting trait variation and phenotypic plasticity in switchgrass. Using a diversity panel of 426 genotypes evaluated in Texas and Michigan, USA., this study demonstrates that transcriptomic predictors could outperform genomic markers in predicting environmental differences for traits like biomass yield and tiller count, highlighting the trait-dependent advantage of transcriptomic information over SNPs. However, combining SNPs and transcriptomic predictors generally yielded limited improvement in prediction accuracy.

A principal limitation of this study concerns the experimental design and the robustness of transcriptomic predictors. With only one transcriptomic sample per genotype in each environment, within-genotype variation in transcript abundance cannot be estimated, making it difficult to distinguish genotype-specific expression responses from individual-plant variation. Unlike SNP genotypic information, which provides a stable representation of a genotype's inherited genetic variation underlying phenotypic differences, transcript abundance is highly sensitive to environmental conditions, developmental stages, and physiological state at the time of sampling. As a result, the stability of the transcriptomic predictors and their predictive performance across independent environments, years, or sampling conditions remain untested. Furthermore, the stability of feature selection across different training populations has not been established, raising additional questions about the reproducibility of the identified candidate genes. Together, these limitations constrain their generalizability and the biological interpretation of transcriptomic associations with phenotypic plasticity.

Overall, this study provides valuable comparative evidence regarding the predictive performance of genomic and transcriptomic information. Its principal contribution lies in demonstrating that the relative predictive advantages of these information sources are trait-dependent and their integration does not necessarily improve prediction accuracy. While the predictive findings are supported, the biological interpretations extend beyond the evidence presented, as the identified transcriptomic predictors and their interactions do not establish causal genetic effects, epistasis, or direct regulatory relationships. Therefore, the proposed genetic mechanisms and environment-specific interactions should be regarded as hypotheses requiring independent biological validation, rather than established mechanistic findings.

Reviewer #1 (Public review):

Summary:

The authors ask whether Samp1 contributes to the reorganization of the genome and the regulation of gene expression during muscle-cell differentiation. Using C2C12 cells, they find that Samp1 depletion impairs differentiation and is accompanied by altered transcriptional responses and changes in peripheral chromatin and chromosome 8 distribution. These findings support Samp1's involvement in the differentiation program, although the causal relationship between the spatial and transcriptional phenotypes remains unresolved.

Strengths:

The study addresses a biologically significant question: how proteins at the nuclear envelope contribute to the coordinated changes in genome organization and gene expression that accompany cell differentiation. Its main contribution is to extend the established requirement for Samp1 in myogenesis to measurable spatial and transcriptional phenotypes, providing a more specific basis for investigating its function.

The complementary imaging approaches examine chromatin organization at different scales. Fluorescence ratiometric imaging assesses the relative distribution of chromatin reporters across the nucleus, whereas chromosome 8 imaging provides a chromosome-specific measurement. Agreement between these approaches strengthens the observation that Samp1-depleted cells undergo an altered spatial response to differentiation conditions, while leaving the underlying mechanism open.

The four-time-point transcriptional analysis adds temporal information that an endpoint comparison would miss. It allows readers to examine baseline differences and subsequent responses to differentiation conditions, and provides a potentially useful resource for identifying genes and pathways associated with the impaired differentiation program.

The work also builds on earlier validation with an independent depletion construct and rescue of differentiation by RNAi-resistant Samp1. This prior evidence strengthens the biological foundation of the present study. Together, the results identify Samp1 as a useful target for further investigation of the relationship between nuclear organization and muscle development.

Weaknesses:

The study leaves open how the differentiation, spatial, and transcriptional changes are connected. By day three, control cells have formed myotubes, whereas Samp1-depleted cells largely remain myoblasts. This is a consequence of the depletion and does not invalidate the comparison. However, it means that altered chromosome positioning could contribute to the differentiation defect or arise because differentiation has been disrupted. The authors acknowledge that the mechanism remains unresolved; the findings should therefore be interpreted as evidence that these processes depend on Samp1, without establishing that changes in genome positioning drive the transcriptional response.

The earlier rescue of differentiation strengthens the evidence for Samp1's role in myogenesis. The new spatial and transcriptional findings, however, have not been tested by rescue. This leaves some uncertainty about whether these particular effects are specific to Samp1 depletion in the stable cell line used here.

The connection between impaired gene repression and association with the nuclear lamina remains indirect. The published Lamin B1 DamID data describe normal differentiation and do not measure what happens to lamina association after Samp1 depletion. They support the proposed connection but cannot establish it.

The imaging measurements also need a cautious interpretation. The peripheral chromatin index describes relative fluorescence enrichment, rather than directly measuring heterochromatin abundance. The biological meaning of changes such as an index of approximately 1.1 versus 1.3 is therefore not immediately clear. Similarly, the chromosome 8 analysis measures the distribution of chromosome-paint signal within five optical sections rather than the entire chromosome territory. The results support spatial differences, but their magnitude may depend on nuclear geometry, the sampled sections, and image-processing choices whose influence is not fully assessed.

Some conclusions about the transcriptional response go beyond what the statistical comparisons directly test. A significant change during differentiation in control cells and a nonsignificant change in depleted cells do not, by themselves, establish that the responses differ. The comparison between groups at day three provides evidence of expression differences, but does not determine whether the changes from their respective starting points differ.

Reviewer #1 (Public review):

The manuscript combines MspA nanopore measurements with molecular simulations to investigate p53-TAD, its cancer-associated N29K/N30D variant, and its interactions with MDM2 and EGCG. The experiments demonstrate a promising approach for distinguishing sequence- and ligand-dependent single-molecule signals. The manuscript is clearly written, and the use of multiple simulation trajectories is a strength. However, the structural and thermodynamic interpretations extend beyond what the current simulations establish. I recommend addressing the following points.

(1) The assignment of low-current states to compact conformations near the constriction, and intermediate-current states to more expanded conformations higher in the vestibule, is plausible but not directly validated (Figures 1-2; main text, pp. 4-9). Current blockage can depend on protein position, orientation, charge distribution, and pore contacts as well as compactness. The simulations do not calculate ionic conduction, and the four structural clusters in Figure 2f do not have an established correspondence to the six experimental current states. Could the authors provide a validated conductance calculation for representative configurations, or explicitly present these structural assignments as hypotheses? In particular, an EGCG-associated current level overlapping S4 does not establish that the ligand stabilizes the same structural ensemble sampled by apo p53-TAD.

(2) Figure 2c-d presents occupancy-derived profiles and regional differences using free-energy terminology. However, the simulations describe finite-duration, constant-force capture trajectories initiated outside the pore, rather than demonstrated equilibrium sampling. Could the authors establish stationarity, evaluate dependence on the analysis window, and explain how the applied bias and normalization are treated? Otherwise, these results should be described as driven occupancy profiles, without interpreting their differences as equilibrium stabilization energies. Similarly, Figure 5 should be clearly identified as a schematic reconstruction rather than a measured molecular free-energy landscape.<br /> The apparent EGCG affinity of 0.43 {plus minus} 0.49 μM also requires cautious interpretation. This value comes from event-frequency measurements at +75 mV and is poorly constrained. Event frequency depends on capture efficiency as well as solution binding populations. At +200 mV, manual voltage reversal censors spontaneous pore-escape times for long-lived complexes; moreover, pore escape and molecular dissociation are distinct processes. Please clarify which kinetic and thermodynamic quantities the assay can establish. The proposed confinement-mediated reduction in the entropic cost of EGCG binding is reasonable as a hypothesis, but is not demonstrated by these measurements.

(3) The SMD protocol distributes a constant force over acidic residues, restrains the pore, and confines p53-TAD within a cylindrical potential (Figure S12; Supplementary Methods, lines 174-198). These approximations are acknowledged, but their influence on the WT-mutant comparison remains unresolved. The double substitution approximately preserves net charge while changing its distribution, and the force prescription treats acidic and basic residues differently. Do the positional and conformational differences persist across pulling strengths, restraint widths, and alternative force assignments? Please also confirm that the simulated pore contains the mutations present in the experimental M2-MspA-NNN construct.<br /> The selected p53-TAD and MDM2 fragments omit full-length p53 regulation and interdomain coupling. This is a construct limitation, rather than a necessary consequence of the narrow constriction, because capture occurs within the wider vestibule. The existing discussion of wider pores and tethering could be extended to clarify this distinction. Phosphorylation-dependent TAD dynamics would be an interesting future application, although the present states cannot be assigned to modification-sensitive conformers without additional evidence.

(4) The mutant exhibits increased intramolecular contacts, but the pore-resident radius-of-gyration differences are only approximately 0.2-0.3 Å, with small standardized effects (Figure 2e; Table S1). Several other descriptors have confidence intervals spanning zero, including the bulk Rg difference. Consequently, the simulations support modest sequence-dependent changes more strongly than a pronounced shift in global compactness. Figure S15 also identifies altered protein-pore contacts, which could contribute directly to positional occupancy and current differences. Could the authors provide convergence analyses, trajectory-level uncertainty, and sensitivity to starting ensembles, and distinguish intrinsic compaction from altered pore association?

(5) The replacement of MDM2-complex events by EGCG-associated events is consistent with inhibition, but differential capture could also change the observed event populations (Figures 3-4). The manuscript acknowledges this possibility on p. 12. Could the authors clarify its implications for the inhibition claim and provide an orthogonal measurement or appropriate controls to distinguish reduced complex abundance from preferential detection? Charge, steric geometry, and pore interactions should be considered without assuming that compaction necessarily explains enrichment.

No new simulations directly investigate EGCG binding or MDM2 competition. Ensemble-based ligand modeling could strengthen the interpretation, but docking poses or short trajectories alone would not establish preferential binding to S4, binding affinity, or conformational selection versus induced fit. The discussion should place the proposed mechanism alongside prior EGCG-p53 studies, including the computational predictions involving Trp23-Lys24 and Pro47-Thr55 reported by Bahena Culhuac and Bello, 2024. If additional modeling is beyond the scope of this work, the claims concerning S4 stabilization, compaction, and MDM2 displacement should be qualified accordingly.

Overall, the experimental platform is promising. The principal revision needed is to distinguish the observed nanopore signal changes from their proposed molecular explanations. Targeted robustness tests and appropriately qualified structural and thermodynamic claims would substantially strengthen the manuscript.

Reviewer #1 (Public review):

Summary:

Dila Rizalar, Villaseñor and colleagues report the membrane trafficking steps undergone by CD98hc, a major component of the LAT1 amino acid transporter. They found that CD98hc entered cells through Fast Endophilin-Mediated Endocytosis (FEME) and quickly recycled back to the plasma membrane (PM) together with beta1-Integrin to mediate a stable supply of amino acids and support metabolic homeostasis.

Strengths:

The work was planned, executed and analysed to a high standard. A variety of cell models, techniques and assays were performed and combined with mathematical modeling. Appropriate analyses and statistical tests were used to support their conclusions.

Overall, the evidence provided is convincing.

Weaknesses:

(1) The proposed endosomal recycling pathway(s) (positive for Rab9, Rab11 and Rab14) is/are not believed to support recycling fast enough (c. 10-20 min) to explain the very short timeframe (c. 1 min) reported by the authors. This apparent discrepancy should be clarified by the authors, ideally with small additional work.

Notably, the fast Rab4-dependent recycling of TfR directly from Rab5 sorting endosomes has more consistent kinetics (c. 2-4 min, see the classic papers from the Mellman, Maxfield and Zerial labs) and should be investigated or at least discussed.

(2) LAT1 (and thus possibly CD98hc) were previously reported to enter cells through Clathrin-mediated Endocytosis (CME) upon potent PKC activation (PMA) or entry into cell quiescence (Barthelemy and André, Sci Rep 2019 and Kahlhofer J et al. EMBO J 2025). Interestingly, instead of being recycled back to the PM, LAT1 was then routed to lysosomes for degradation.<br /> Does LAT1 co-traffic with CD98hc along the FEME - fast endosomal recycling loop identified by the authors?

If so, the authors could discuss the physiological context in which the whole transporter shifts between the 2 routes (FEME-recycling in steady-state maintenance and CME-Lysosome upon acute signaling? There is a precedent in EGFR for such variation in endocytic fates upon signal strength).

If not, the authors could discuss why the 2 chains would traffic differently (in such a scenario: CD98hc through the FEME-recycling arm and LAT1 through CME-Lysosome).

(3) The study would benefit from mechanistic insights about the molecular details linking CD98hc to FEME.

Specifically, identifying motif(s) and/or adaptor(s) binding to the main FEME adaptors (Endophilin A1, A2 or Bin1) would be a valuable improvement of the work and would provide a strategy to design a point mutant version of CD98hc that would be unlinked to FEME.

Reviewer #3 (Public review):

This manuscript focuses on the presence/origin of directional memory during epithelial cell migration. It starts by analyzing single cells and then moves to more complex systems (confluent layers and scratch assays). The paper first demonstrates that the migration in all of these systems is well-described by persistent random walks which likely emerge from fractional Brownian motion. This is an important demonstration, as it implies orientation memory in the systems. Then the paper proceeds to attempt to discern the origin of this memory and claims to establish key roles for adherens junctions and vinculin dimerization. While for the most part the manuscript is well-written, there are some signification overinterpretations in experimental results. The largest issue is demonstrating the role for vinculin dimerization, which is not a well-studied phenomena inside living cells, as all data is reliant on a single point mutation (Y1065E).

(1) Likely the most significant issue with the manuscript is the interpretation of the vinculin Y1065E variant and the assumption that the only defect the mutations causes is a lack of dimerization. Vinculin dimerization is mediated by a conformational change in the vinculin tail domain induced by F-actin binding (Thompson, FEBS Letters, 2013). Dimerization of the vinculin tail domain has been clearly demonstrated in in vitro systems involving purified proteins, as the authors point out in the manuscript. However, the dimerization of full-length vinculin has not been well characteristic in living cells. There are several reasons to suspect dimerization is potentially not prevalent in cells. For instance, in the presence of other actin binding proteins, there may not be sufficient available binding sites available on neighboring actin filaments to facilitate dimerization. Additionally, pY1065 vinculin and vinculin Y1065E have been associated with increased vinculin activation (Huang, JBC, 2014), so other effects seem possible. While the Y1065E variant clearly has an effect on the tension sensor readout and vinculin dynamics, further experimental evidence is needed to show that these effects are due to lack of dimerization in living cells. Perhaps most importantly, the vinculin tension sensors used in this study have been used extensively in knock-out systems and intermolecular FRET has not been observed (PMID: 20613844, 33335089, 30523675). To justify the definitive claims made in the manuscript, the authors likely need to develop, or employ, an assay for detecting vinculin dimerization in living cells. The authors could choose between intermolecular FRET, proximity labeling assays (i.e. antibodies with DNA for signal amplification), bimolecular fluorescent complementation (i.e. split GFP) based approaches, or some other approach. It should be noted that working with full length vinculin, not just Vt, and designing assay that can incorporate vinculin Y1065 variants (Y1065E and potentially Y1065A/F) would strengthen results. Also, the authors should be aware that the observation of strong dimerization may invalidate the use of FRET-based tension sensors in this system or at least necessitate intermolecular FRET control experiments.

(2) A major conclusion in the manuscript is that in response to overexpression of a specific vinculin constructs, focal adhesions behave the same in single cells, confluent cells, and collectively migrating cells for all the mutants but Y1065E. However, outside of the FRET measurements, there is not much evidence to support this claim. The authors should perform a greater comparison of the focal adhesions between the systems used in the manuscript (single cell, confluent cells, collectively migrating cells). Key measurements would include focal adhesion number per cell, focal adhesion size, focal adhesion orientation, vinculin dynamics (e.g. FRAP), focal adhesion stability, and some indicators of focal adhesion composition. For the last aspect, focusing on focal adhesion components that also have roles in adherens junctions, such as VASP, seems appropriate. Without such characterization, it is an overinterpretation to assume that focal adhesions are the same in each system and, therefore, effects are due to vinculin behavior in the adherens junctions.

Reviewer #1 (Public review):

Summary:

The authors studied the development of hippocampal connectivity gradients based on open datasets and performed correlation analyses with other MRI features as well as gene expression information from other datasets. Although the main findings are correlational and cross-sectional, the analyses are overall sophisticated and replicated in several datasets.

Strengths:

The hippocampus is a key region in understanding large-scale brain organization and cognition, and the authors applied advanced and suitable analytics to study its development. The paper is overall well-organized and well-written, and the findings are relevant for studying large-scale brain development.

Weaknesses:

While sophisticated, several of the analyses appear mainly correlational, cross-sectional, and rely on cross-dataset contextualization, which should also be stated as a limitation of the current work.

Reviewer #2 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

Summary:

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

Strengths:

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

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

Comments on revised version.

I thank the authors for addressing my concerns in the Introduction and Discussion of their paper. I don't require any further edits at this point.

Reviewer #1 (Public review):

Niu and colleagues tackle several questions concerning the assembly of the heterotetrameric kinesin-1 complex, comprising kinesin heavy chains (KHCs) and kinesin light chains (KLCs), its interaction with adaptor proteins (the mitochondrial adaptor TRAK2), and the implications of these interactions for kinesin autoregulation.

Broadly, the study comprises four parts; strengths and weaknesses are described for each in turn.

First, the authors examine the coiled-coil interface that enables tight binding between KHC and KLC. Binding-site mapping supports previous observations that KHC and KLC interact through the CC3 region of KHC and the amino-terminal coiled-coils of KLC. These regions are modelled using AlphaFold3, and the resulting models are used to inform mutagenesis experiments that identify critical residues within the hydrophobic core of the interface. Although the binding site has previously been reasonably well defined, both biochemically and using computational models, this work extends those findings to individual residues at the interface and provides supporting biophysical analysis. This provides useful experimental validation of the computational models and important tools for the field to enable the selective uncoupling of KHC and KLC.

Second, the authors examine how the mitochondrial adaptor TRAK2 interacts with KHC. Previous studies have defined the critical regions of KHC CC4 and TRAK2 reasonably well, and the authors' biochemical mapping broadly supports these findings. They extend this through biophysical analysis and computational modelling that proposes a 2:2 stoichiometry and an interesting assembly in which CC4 separates to enable formation of a four-helix bundle with TRAK2. This AlphaFold model is supported by biophysical, biochemical, and cell-imaging analyses, including the identification of point mutations that disrupt the interaction. These findings are important because the molecular basis of KHC CC4 interactions with diverse cargo adaptors has remained somewhat opaque, and this study begins to place these interactions on a firmer molecular footing. Some of the principles proposed here may also extend to other KHC-binding adaptors.

Third, and of particular interest, the authors examine the interplay between KLC and TRAK binding to KHC. This is important because previous work has generally favoured the view that mitochondrial transport is independent of KLCs, although some studies disagree. Whether KLCs are excluded from TRAK-containing complexes and vice versa and, if so, how this occurs has remained unclear. The authors provide evidence suggesting that the extended coiled-coil region of KLC, outside the direct high-affinity KHC-binding interface, modulates CC4 to prevent TRAK binding, suggesting that KHC-TRAK and KHC-KLC complexes may be mutually exclusive. Precisely how this mechanism translates into transport regulation within the cell remains unclear, but the effort to establish a structural hypothesis for understanding this interplay is valuable.

Fourth, the authors provide evidence for a novel, weak interaction between CC1 and CC4 that they propose may contribute to kinesin-1 autoinhibition and its regulation by KLC and TRAK-an 'autoinhibitory latch'. I found this part of the work more preliminary because, although the biophysical and biochemical analyses provide some support for such an interaction, unlike other aspects of the study, it is less well supported by computational modelling or mutational analysis. Moreover, there is no direct evidence for how this interaction might function in the context of full-length kinesin-1 autoinhibition, for example through assays of motor activity or conformational state, and the proposed model is not particularly clear with respect to full-length KLC (including its TPR domains) or regulation by MAP7.

Taken together, the study makes a valuable contribution to our understanding of several facets of kinesin-1 assembly, cargo recognition, and regulation. Key strengths lie in the complementary biochemical, biophysical, computational, and cell-imaging approaches used to support most of the key findings, together with the definition of several very useful point mutants that should inform future work.

There are, however, some general weaknesses surrounding unnecessary or overstated claims of novelty and the connection to prior work, and some factual errors. These can be straightforwardly addressed through better integration of the existing literature into the narrative, without detracting from the authors' contributions. Some conclusions appear to extend beyond the available data, and these should be tempered accordingly. Computational models should also be presented more thoroughly and systematically, together with confidence metrics and multiple ranked outputs. The Methods should more clearly describe the constructs and sequences used so that the work can be straightforwardly reproduced, alongside information on replication and additional quantification. Some clarification around measured stoichiometries and the authors' interpretation for some of the short coiled-coil fragments will also be important.

Reviewer #1 (Public review):

Summary:

Nakatani and De Schutter conduct an in-depth computational investigation of astrocyte electrophysiology. Utilizing a conductance-based NEURON model with realistic astrocyte morphology, the study replicates both in vitro and in vivo experimental protocols to characterize local and global depolarizations of the astrocyte membrane. The findings indicate that bath-applied (global) potassium stimuli induce membrane potential changes that follow the potassium equilibrium potential throughout the cell, whereas localized potassium stimulation disrupts this behavior. In the latter case, the local response is governed by the ratio of local conductances. The authors further analyze the observed electrical compartmentalization to identify which molecular components directly affected by synaptic stimuli - such as potassium clearance, glutamate transport, and GABAA and NMDA receptors - could plausibly generate this effect under physiological conditions. The study concludes that perisynaptic astrocyte processes (PAPs) can respond to specific synaptic inputs in an electrically isolated manner, exerting minimal influence on the soma.

The central prediction - that astrocyte processes can be electrically compartmentalized from the soma under local synaptic stimulation, while bath stimulation produces a globally uniform response - is both interesting and surely significant for understanding and measuring astrocyte electrophysiology. However, the manuscript's incomplete model description and disorganized Results section hinder assessment of how well the conclusions are supported. Addressing these issues could render the model a valuable resource for the community and a useful framework for guiding and interpreting voltage-imaging experiments in astrocytes.

Strengths:

(1) The study addresses a timely, long-standing question and may pioneer a multiscale approach to quantifying astrocyte electrophysiology beyond the traditional view of an electrically passive, isopotential glial syncytium. The findings are consistent with recent voltage-imaging evidence of localized depolarization in astrocyte processes.

(2) A key strength of the study is the use of a state-of-the-art, morphologically realistic compartmental model that captures both somatic and peripheral dynamics. This approach addresses spatial and temporal resolutions that are challenging to achieve experimentally.

(3) Simulating both in vitro-like (bath perfusion) and in vivo-like (local, synapse-driven) stimulation protocols within a unified framework may explain discrepancies observed between experimental results obtained under these differing conditions.

(4) The model predictions are benchmarked against experimental voltage-imaging data, and the systematic analysis of individual molecular contributors-including potassium channels, glutamate transporters, and synaptic receptors-yields testable predictions.

(5) The topic is relevant to a broad and multidisciplinary audience, including researchers in glial biology, synaptic physiology, and computational neuroscience.

Weaknesses:

(1) Terminology and clarity

The manuscript relies heavily on jargon and abbreviations, which impedes readability. The central concept of a "Nernstian" depolarization or response, introduced in the title, is unconventional and not explicitly defined. It appears that "Nernstian" refers to a membrane potential that follows the potassium Nernst potential (EK) of the astrocyte. This terminology is ambiguous, as the analysis is restricted to K+ and does not ensure that the astrocyte response excludes contributions from other ion Nernst potentials.

Furthermore, "Nernstian" reflects a modeling choice rather than an alternative such as the Goldman-Hodgkin-Katz formalism. Additional terms in the abstract, including "a variety of biochemicals," "astrocyte peripheries," "isolated peripherals," and "empirical" model, are also ambiguous. Because these terms are central to the main claim, they may confuse readers.

(2) Framing of the research question

The introduction is brief and does not clearly articulate the research question or the manner in which the study addresses it. The motivation, which should draw on cell anatomy (including the gap-junction-coupled syncytium), molecular composition, and stimulation protocol, is underdeveloped, and the knowledge gap is not explicitly identified.

(3) Incomplete model description

Model design, parameters, and stimulation protocols are often not described or justified, and references are missing (e.g., for K2P, TREK1/TWIK1, and the Na+/K+ pumps). In particular:

a) it is not explained how the three ionic species (Na+, K+, Cl−) needed to account for synaptic currents interact and are modeled in the extracellular and intracellular spaces, nor how they contribute to the astrocyte membrane potential;<br /> b) the diffusion formulation (Equation 9) neglects any tangential component of diffusion, an approximation that is left unjustified;<br /> c) gap junctions are modeled as ohmic currents driven by the potential difference between astrocytes, but the network arrangement, location, and distribution of the coupled astrocytes and of the gap junctions in the simulations are not specified;<br /> d) the analytical results (Equations 46-50) are not obviously derived from Equation 16, and the essential derivation steps are missing;<br /> e) it is unclear whether and how the voltage indicator ArcLight is modeled when comparing simulated traces with experimental fluorescence data.

Overall, the simulation setup lacks clarity, which limits reproducibility and impedes evaluation of the study's conclusions.

(4) Organization and rationale of the results

The Results are difficult to interpret owing to unclear organization and an ambiguous rationale. The derivation and application of the scale parameter ΓK (Equation 1) are unclear; in particular, its dependence on axial conductance appears inconsistent with the assumption that intracellular diffusion does not influence PAP responses (cf. van Egeraat et al., Biophys. J. 1993).

(5) Unsupported or missing results

Several statements are not supported by the data presented: some claims could not be matched to any evidence (e.g., L104-106), results described in the text could not be located in the figures (L159-164), comparisons are reported as "data not shown" (L140-143), and the source of the "realistic ECS sizes" (L191-194) is unclear. The authors do not demonstrate the validity of the extracellular K+ perturbation used to mimic action potentials.

(6) Presentation of figures

The figures are often crowded and stylistically inconsistent. Most importantly, in Figures 3, 4, and 6, the rendering of the noisy experimental data obscures the simulation results they are meant to benchmark, making it difficult for readers to assess agreement between model and experiment.

Reviewer #1 (Public review):

Summary

In "Mouse models with human antibody repertoires for inducing multiple lineages of HIV-1 broadly neutralizing antibodies", Tian et al. report the production of 6 humanized mouse models to support HIV-1 vaccine design studies aiming to induce different classes of broadly neutralizing antibodies.

These mice were designed to express immunoglobulin genes of human bNAbs targeting four different conserved epitopes of the HIV-1 Envelope while allowing the development of CDRH3 and CDRL3 diversity during gene rearrangements.

Strengths:

(1) The manuscript is clearly written, and findings are clearly presented with self-explanatory figures. The Introduction and Discussion sections provide very valuable context to understand the relevance of these mouse models and are accessible to a general reader. It is appreciated that the data are presented with transparency, highlighting both strengths and weaknesses of the mouse models.

(2) The characterization of the mouse models is overall thorough and rigorous. Immunization data to confirm functionality are not presented; however, it is understandable that this information will be presented in the context of subsequent studies. The authors indicate that these mice have been distributed to different teams.

(3) These mice will be very valuable tools for the HIV-1 vaccine community to evaluate vaccine candidates in a "closer-to-human" setting that allows a certain level of polyclonality while using human immunoglobulin genes.

Weaknesses:

(1) The authors investigate the use of the knocked-in human immunoglobulin genes in heavy chains and light chains and provide the frequency of productive rearrangements using these genes. However, there is no information about the frequency of the right heavy-light chain pairs. This information would be important to calculate the real frequencies of potential bNAb precursors in these mouse models.

(2) The authors use HTGTS-rep-seq to investigate immunoglobulin gene rearrangements at the genomic DNA level. This method is very useful for analyzing gene recombination because it provides information about both productive and non-productive rearrangements. However, this method does not provide information about BCR expression, and it is not optimal for investigating repertoire composition. BCR expression would provide a more rigorous characterization of the mice and confirm functionality.

(3) The goal of this study was to produce models that could provide a diverse bNAb-like repertoire. Specific quantification of the diversity of the repertoire is not provided. The study does not provide information about the sequences that do not use the intended genes.

Overall, the availability of these humanized mouse models will be very valuable for the HIV-1 vaccine community and will provide valuable pre-clinical models to advance the design and testing of vaccine candidates.

GroupID: 2

DiseaseAssertion: VHL

Group size: 1

GroupNumberMales: N/A

GroupNumberFemales: N/A

GroupEthnicity: N/A

GroupAgeRange: N/A

Group Info: Retrospective analysis of a longitudinal cohort of 21 VHL germline mutation carriers and retinal haemangioblastomas. All patients were categorized in two genotypic categories: missense mutations and truncating mutations.

GroupPresentingHPOs: HP:0009711 (retinal capillary hemangioma)

GroupHPOFreeText: The patient presented with retinal capillary hemangioma.

GroupNotHPOs: N/A

GroupNotHPOFreeText: N/A

GroupPreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Table S1

Variant: Exon 1-2 deletion

CaseProblemVariantFreeText: N/A

LegacyVariant: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: cDNA level information: c.(?_1)_463+?del

MutationType: exon_loss_deletion

CivicName: Exon 1-2 deletion

MultipleGeneVariants: N/A

Reviewer #1 (Public review):

Summary:

This study explores the role of Dynamin 2 (Dnm2)-mediated clathrin-mediated endocytosis (CME) in the fate determination of neural progenitor cells in the embryonic cerebral cortex, with particular attention to its asymmetric regulation of ligand and receptor functions in the Notch signaling pathway. Using conditional knockout mice, in vitro co-culture systems, in utero electroporation, and luciferase reporter assays, the authors propose that Dnm2 is required for neural stem cell maintenance and that CME plays a dual role in Notch signaling.

Strengths:

The authors systematically demonstrate the critical role of Dnm2 in embryonic cerebral cortical development. They show that Dnm2 is the predominant dynamin isoform expressed in the embryonic central nervous system, and that its conditional knockout disrupts ventricular zone (VZ) architecture, causes premature depletion of radial glia (RG), and leads to precocious neurogenesis, accompanied by periventricular hemorrhage and perinatal death.

Weaknesses:

I believe that some conclusions of this study should still be treated with caution. Specific comments follow:

(1) scRNA-Seq analysis of the Dnm2-cKO cortex at E11.5 and E13.5 is needed, or alternatively, additional LGE-MGE scRNA-Seq should be performed, to examine whether the key gene expression changes are conserved across the MGE-CGE-LGE and cortex.

(2) Regarding Figure 5 (Loss of Dynamin 2 leads to abnormal accumulation of Dll1 and altered NICD distribution in the E11.5 cortex), I do not understand why Dll1 accumulation occurs primarily in the medial cortex.

(3) A detailed schematic model is needed to illustrate the function and mechanism of action of Dnm2.

(4) Dnm2-cKO exhibits extensive cell death and vascular abnormalities. The authors should explain the possible specific causes.

(5) Clinical relevance: Are there specific forebrain phenotypes associated with Dnm2 mutations in humans? What are the possible reasons? This study appears to have limited relevance to Dnm2-related human disease.

Reviewer #1 (Public review):

Summary and Strengths:

In this manuscript, the authors continue their efforts to characterize metabolism-related phenotypes and molecular changes in Down syndrome mouse models, with a focus on Ts66Yah in the present study. Because the phenotypes described are relevant to metabolic abnormalities observed in individuals with Down syndrome, these findings are important. In addition, Ts66Yah is a relatively new model generated by removing the triplicated region of Mmu17 from Ts65Dn, which is not syntenic to Hsa21. Thus, Ts66Yah retains the triplicated region of Mmu16 that is syntenic to a region of Hsa21, carried on an extra chromosome.

Weaknesses:

(1) Figure 3 and Figure 10: The authors should offer a possible explanation for why the GTT and ITT differences between Ts66Yah and wild-type males observed under chow conditions are no longer evident under HFD conditions. The statement in the Abstract that "both male and female Ts66Yah mice share a common phenotype of pronounced glucose intolerance and insulin resistance" appears stronger than the data support. Under chow conditions, impaired insulin sensitivity by ITT is observed in males but not females. Moreover, although the Abstract states that glucose intolerance and insulin resistance are further exacerbated by HFD, the GTT and ITT abnormalities under HFD appear to be predominantly observed in females, whereas the genotype differences are no longer evident in males. The Abstract should therefore be revised to more accurately reflect these sex- and diet-dependent effects.

(2) Figure 7B: In the female liver PCA plot, PC1 is labeled as explaining 13.2% of the variance, whereas PC2 explains 21.6%. Since principal components are ordered by decreasing explained variance, PC1 should explain greater variance than PC2. The authors should verify whether the axis labels or percentages are correct.

(3) Figure 14 legend: A cutoff of log2FC > 0 was used, whereas Figure 8 uses a log2FC cutoff of 0.5. The authors should explain the rationale for using different thresholds in the two analyses.

Joint Public Review:

Kitanovski and colleagues compare TCRβ repertoires from paired primary tumors and sentinel lymph nodes from 24 treatment-naive melanoma patients. The paired design and comprehensive repertoire analyses are important strengths, and the study provides solid evidence for differences between these compartments, including reduced diversity and greater clonal dominance in primary tumors.

The principal limitation is that antigen specificity is inferred from bulk TCRβ sequencing rather than directly demonstrated. Database annotation and sequence similarity are useful for generating hypotheses, but neither an annotated β-chain nor membership in a sequence-similar community establishes antigen specificity. In particular, the absence of a database match should not be interpreted as evidence of neoantigen recognition. Claims concerning neoantigen specificity should therefore be moderated or supported by additional evidence.

The ClustIRR analysis is potentially useful for identifying relationships among predominantly private TCR repertoires, but its added value and robustness require further evaluation. Benchmarking against conventional repertoire-overlap or alternative TCR-similarity approaches, testing sensitivity to key clustering parameters, and determining whether the results are driven by highly abundant communities would substantially strengthen the analysis. HLA genotype and the non-independence of pairwise repertoire comparisons should also be considered when interpreting inter-individual repertoire similarity.

Overall, the study provides a valuable dataset and convincing evidence for spatial differences in melanoma TCR repertoire architecture, while the biological interpretation of individual TCRs and communities in terms of defined antigen or neoantigen recognition remains less well supported.

Reviewer #1 (Public review):

Damphousse, Calvin and Redish recorded from dorsal hippocampus in rats running past a robotic predator to reach food. They re-analyzed the cohort from Calvin et al. (2025, PLoS Biol) and added a new cohort. They compared three behaviors: retreats right after an attack, mid-track aborts (MTAs) where the rat turns back without being attacked, and mid-track continues (MTCs) where the rat pauses and then carries on. The authors report that retreats and MTAs, though both end at the nest, differed in speed and in decoded position. During retreats, decoding stayed near the attack zone. During MTAs, it was weighted more toward the nest and the far end of the track. Pauses that ended in an abort showed more decoding of the attack zone than pauses that ended in continuing, and a similar difference was seen on the way out, before the pause.

The main contribution is conceptual. Separating fear-like escape from anxiety-like withdrawal within the same task is a real step beyond showing that the hippocampus represents threat. The task places the robot, the attack threshold and the reward at different points on the track, which makes the decoding easier to interpret. Figs. 5-7 use the same analysis layout, so the comparisons are easy to follow. The main patterns are seen in both cohorts, even though the cohorts differ in several procedural details.

The revision tightens the abstract, clarifies how epochs were defined and normalized, and acknowledges that the results are correlational. Overall, the data support the main conclusion that hippocampal representations differ between reactive and anticipatory responses to threat. The paper should interest people working on hippocampus, defensive behavior and approach-avoidance conflict, and the task offers a good way to study how threat and reward are weighed against each other.

Reviewer #1 (Public review):

Summary:

Charonitakis and co-authors characterize dishabituation in adult flies, where they use olfactory habituation to octanol, then dishabituate the flies with disruptions of electric shock or yeast odors. They systematically investigate the neurotransmitters and neural circuits involved in dishabituation and figure out a lot about how this process works in the brain, as an independent circuit. I like the paper and I like the very structured approach to figuring out the problem.

Strengths:

The introduction nicely sets the stage for the work presented, bringing in knowledge from other organisms and motivating the study.

The results section lays out a logical set of experiments, using a common set of behavioral assays in many flies exposed to thermogenetic or optogenetic manipulation. The paper systematically figures out the necessity and/or sufficiency of specific brain regions and neurotransmitters, culminating in a new understanding of how the important process of dishabituation works.

I like the bar graph representation for the data throughout, with the helpful icons - if a paper figures are going to be 90% bar graphs it helps when they are super clear like this. And I like how the all the parts build up to the conclusion in the last figure, nicely summarizing the thorough characterization of dishabituation.

Weaknesses:

There are no major concerns, and I appreciate the addition of a new figure that details the habituation / induced dishabituation protocol, along with additional discussion of dishabituation, and framing the topic for a more general audience.

Reviewer #1 (Public review):

Summary:

This manuscript describes how defective food grinding due to loss of TGF-beta signalling leads to fat loss via specific neuropeptides and their cognate receptors in C. elegans. They define transcriptional mechanisms from the ADL sensory neuron to neuropeptide relays from neurons to the gut, wherein enzymatic effectors mediate fat metabolism. The genetic evidence presented to support this neuropeptide circuitry is based largely on transcriptional changes detected in bulk mRNA, cell-type-specific rescue, and lipid measurements using Nile Red staining in permeabilized/fixed animals and with lipidomics. The findings are of interest to researchers in neuropeptide regulation of physiology.

Strengths:

(1) The multifaceted analysis of dbl-1 and sma-6 provides good evidence that the primary defect leading to metabolic phenotypes is due to pharyngeal function.

(2) The transcriptional survey of neuropeptide expression under fasting and in TGF-beta pathway mutants will be a useful resource for many labs studying neuropeptides in food sensing.

(3) Outlining the process from dietary restriction to fat metabolism in the gut provides a big-picture view that establishes physiological relevance.

(4) Identifying new functions for specific neuropeptides and their receptors in fat metabolism.

Weaknesses:

Evidence that links NLP-10-NPR-35 signalling to FLP-18-NPR-4 is weak, and primarily rests on overlaps in npr-35 and flp-18 expression, as well as rescue of flp-18 under the npr-35 promoter. It remains possible that FLP-18 acts from those neurons, but is not activated by NPR-35 signalling, since the mechanisms downstream of NPR-35 are unclear in this context. Strengthening this link would require showing a change in FLP-18 activity; for example, showing npr-35-dependent changes in flp-18 transcription in npr-35-expressing neurons.

The expression patterns of the neuropeptides and receptors that guide the sites of cell-specific rescue experiments are from well-fed animals. However, it remains a possibility that starvation alters neuropeptides and their receptors are expressed, which would produce a different circuit.

Different food levels produce different physiological and transcriptional responses. The manuscript largely assumes that starvation is equivalent to food reduction when they are physiologically distinct states.

Reviewer #1 (Public review):

Summary:

The manuscript describes results from a series of experiments designed to test different facets of the gating model and potential alternative explanations. In most respects, the results are squarely inconsistent with the gating model. Some of the authors' attempts to test the model force the authors to define the model in greater detail than even the model's proponents typically do. That is a revealing exercise in itself, which is well worth bringing to the field's attention. The manuscript is well written for the most part, and figures illustrate results clearly.

Strengths:

The gating model states that pauses in the activity of a select subset of SNr neurons disinhibit (i.e., "open the gate" to) a specific desired action, whereas a larger population of SNr neurons increase their firing so as to suppress other potentially conflicting actions. Selection of different actions is performed by the generation of pauses and increases in activity in different subsets of SNr neurons. Contrary to that prediction, the authors report that SNr neurons that show a reduction in firing for one action tend to show reductions in activity for other actions as well and, similarly, increase-type neurons tend to show increases for all of the actions examined. The authors tested the generality of these observations across a series of experiments that compared activity between markedly different actions (moving forelimb vs. tongue), similar actions (different directions of forelimb movement) and several forms of self-directed and spontaneous movement. Moreover, the authors examined the activity of anatomically identified subsets of SNr neurons that innervate forelimb and orofacial sub-regions of the superior colliculus. Combined, the series of experiments described here provide overwhelming evidence against any straightforward "simple" form of the gating model of basal ganglia function.

Weaknesses:

The most important limitations of the manuscript actually arise from additional ambiguities of the gating model - ambiguities that may be worth mentioning in the manuscript. For example, how granular is the action selection process supposed to be? Perhaps the SNr circuits examined in the present study are selecting "actions" defined at a level that the authors did not examine. This is not a major limitation, however, given the concerted effort invested in testing different levels of granularity and contexts. This relates to another ambiguity of the model in that basal ganglia output does not affect actions directly, but only indirectly via its influences on downstream multi-synaptic neural circuits.

Another ambiguity of the model is how to interpret the activity of neurons that don't respond. In other words, are comparisons of decrease- and increase-type responses the appropriate comparisons to make to test the model? The present study compares decrease- vs. increase-type responses, but perhaps no-response and increase-type neurons should be lumped together. After all, the simplest version of the action selection hypothesis (cf. Hikosaka) considers the high baseline tonic activity of SNr neurons to be action suppressive by itself, without an additional task-related increase. A comprehensive action selection model should include an explanation of the relative roles of non-response and increase-type responses. The current manuscript would benefit from a discussion of this ambiguity and a rationale for the decisions the authors made to make the model testable.

A third ambiguity of the model is how to interpret graded changes in SNr firing rate. It comes as no surprise that the authors observed slow ramp-like changes in SNr firing rate prior to movement initiation in the absence of an immediate sensory trigger. The authors interpret these ramp-like changes as being equivalent (from the perspective of the gating model) to the abrupt changes in firing rate observed around the time of initiation for movements that are sensory-triggered. But the simple gating model presents basal ganglia output as having only three states: no-change, pause, and increase. Implied in the model is that these changes in firing rate influence not only what action is performed but also when it is performed. It is not clear that slow ramping responses qualify as "open the gate" disinhibitory temporally-abrupt signals. The manuscript could provide more explanation concerning the position the authors take to make ramping responses fit into the gating model.

Reviewer #1 (Public review):

Summary:

Movements, including reflexes, can undergo changes during development. This is the case for the angular vestibulo-ocular reflex in many species, including the Xenopus laevis frog. This study aimed to study the underlying mechanisms by which this reflex matures during development of larval Xenopus laevis using electrophysiology and electron microscopy. Their electrophysiological recordings of the motor axons of the abducens motor nerve during evoked angular vestibulo-ocular reflex reveal that a subpopulation of abducens motoneurons, those that are silent rather than spontaneously active, increase their firing rate and modulation during the angular vestibulo-ocular reflex during development. Furthermore, electron microscopy imaging of the developing abducens motor nerve shows increased myelination. Light microscopy reveals increased innervation of the lateral rectus muscle

Strengths:

Recordings during the evoked angular vestibulo-ocular reflex enable assessment of activity of motoneurons during the movement. Recordings done at specific developmental stages reveal convincing changes in modulation of firing of silent motor units that are distinguished by their high-amplitude spikes in nerve recordings. Microscopy, both electron and light, provide strong evidence for increased myelination of the abducens motor nerve and innervation of the lateral rectus muscle.

Weaknesses:

The authors did not provide data showing why the silent motor units became more responsive with development. Is it related to maturation of the sensory inputs to these neurons or changes in intrinsic properties?

Reviewer #1 (Public review):

The authors investigate a previously described behavior of sexually immature female Drosophila melanogaster, wherein immature females flick their wings in response to male courtship. They record, analyze, and characterize the auditory signals generated by these wing-flicks and show that these sounds reduce male courtship song and copulation attempts. The study combines several techniques including behavioral observations, acoustic analysis, developmental experiments, sensory manipulations, and neuronal manipulations to investigate the stimulus dependence and neural basis of this behavior. Amidst all the literature on sexual communication, this study uniquely addresses an interesting question on communication during sexual maturation, providing valuable insights on why and how sexual maturation could be signaled.

A major strength of the study is its behavioral and acoustic measurements with manipulations of sensory input and identified neurons. Comparative analysis of immature wing-flick, male-male aggression, and male courtship song was equally interesting. The developmental analysis also provides a clear temporal relationship between female maturation, wing flicking, and mating receptivity. Together, these experiments provide compelling evidence that immature female wing flicking is a reproducible behavior in response to male courtship, and that it significantly reduces male copulation attempts. Identifying a defined population of neurons that coordinate this response behavior further supports this study.

However, it must be pointed out that some of the broader conclusions extend beyond what is directly demonstrated. In particular, while the evidence supports the interpretation that wing flicking functions as a rejection or deterrence signal from sexually immature females, the designation of this behavior as a signal of "asexuality" and the proposed evolutionary relationship between female wing flicking and male agonistic wing flicking are less firmly established. Furthermore, I think the similarity in acoustic structure and the involvement of related neuronal populations is not sufficient to conclude a) that the two behaviors share an evolutionary origin or b) that one behavior evolved through modification of the other. Similarly, proposed explanations concerning energetic costs, predation risk, and adaptive benefits should be presented as hypotheses rather than conclusions unless directly tested. These interesting points can be included in a subsection "Ideas and Speculation", to clearly distinguish directly demonstrated effects and evolutionary or mechanistic interpretations.

In conclusion, the study contributes to our understanding on communication during sexual maturation.

Reviewer #1 (Public review):

Proctor et al. developed the COB (chloroplast outer-envelope beta-barrel) database, a resource for identifying, classifying and structurally analysing chloroplast outer envelope beta-barrel proteins, built by combining sequence search, machine-learning classification, structural prediction and similarity-based database expansion. The scale and accessibility of the resource is a genuine strength, and it will be useful to researchers working on chloroplast protein import, outer envelope membrane biology and membrane protein evolution. However, several of the manuscript's central claims are not adequately supported by the data presented.

First, the classifier's negative training set consists entirely of non-transmembrane beta-barrel proteins, so its reported accuracy does not demonstrate reliable discrimination from other protein classes (like alpha-helical membrane proteins). The similarity-based expansion that substantially grew the database demonstrates only internal consistency among related sequences, not independent validation, and errors in the seed set will propagate through it. By design, this approach also misses divergent beta-barrels, as the author's own recovery of OEP40 and TOC159 outside the classifier illustrates. This is important because the authors identify the evasion of divergent barrels from these types of tools as a gap in the field. For these reasons, the database should not be described as "comprehensive".

Second, the structural conclusions and the biological implications made substantially outpace the evidence. The reported prevalence of "open" barrel conformations is, for now, a feature of the AlphaFold predictions, not a demonstrated biological phenomenon, and the authors themselves note these may be artefactual. Similarly, the hybrid-barrel analysis is built almost entirely around TOC159-family proteins, which are heavily overrepresented in the Arabidopsis input set. The manuscript does not test whether this apparent "universal" interaction is anything more than a compositional artefact of that enrichment, and most of the novel interactions have only moderate confidence scores. As framed, both the title and multiple statements in the text assert a general topology of "open and hybridized" for this group of proteins that only experimental data can establish.

Third, the evolutionary comparisons between Streptophyta and Chlorophyta, and between chloroplast and bacterial beta-barrels, are presented as robust differences but are not supported by any formal statistical treatment of proteome size, taxonomic sampling or phylogenetic non-independence.

Overall, the database and classifier are a valuable contribution for further exploration and experimental validation of chloroplast outer envelope membrane beta-barrel proteins, but the manuscript's broader structural and evolutionary claims need to be substantially tempered, and the title changed, to match what the data actually show.

Reviewer #1 (Public review):

Maeda et al. propose an algorithm for meta reinforcement learning (Meta RL) that they term "hybrid deep reinforcement learning" (HDRL), which combines two distinct learning mechanisms: one that occurs through continual synaptic plasticity (via backpropagation-through-time), and the other that occurs through recurrent dynamics (i.e., in-context learning). The standard approach to Meta-RL, which I will refer to here as "RL2", is one that involves learning only through recurrent dynamics; this model has found recent application to understanding meta reinforcement learning in the brain. One downside of RL2 as a model of meta-learning in the brain is that, in learning only through recurrent dynamics, RL2 essentially throws the baby out with the bathwater by assuming synaptic plasticity does not have a role in moment-by-moment learning. This study therefore makes a valuable contribution in showing how both synaptic plasticity and recurrent dynamics can be combined in a single model. Importantly, this model does not require an explicit mechanism for mediating between these two learning mechanisms; rather, the authors show that the task (and meta-task) itself determines which mechanisms the model comes to rely on. They then show that their HDRL model can better explain differences in behavioral strategies across conditions of a perceptual decision-making task, and use in silico perturbations to establish that these differences in strategies are due to the distinct contributions of their model's two learning mechanisms. Finally, the authors provide evidence that the neural population activity of mice recording during each task has a consistent structure as the activity of artificial neurons in their HDRL model, but not to the standard RL2 model, which always relies on learning through recurrent activity. This study offers an important extension of the standard method for Meta RL, to one that is both more biologically plausible (i.e., because it seems unreasonable for plasticity to only occur periodically as it does in RL2) and more computationally interesting (i.e., as it explains how these two distinct learning strategies may be deployed automatically based on task demands).

The paper's conclusions seem fairly well supported by their analyses; however, there were a few details that need to be clarified.

(1) There are many architectural differences between the H-DRL vs RL2 model, which precludes a true comparison between the two models solely on the addition of Weight RL. The implementation of H-DRL is relatively stripped down compared to RL2, in terms of using Elman rather than LSTM, standard RL vs A2C, only positive activations allowed, and Q-learning instead of policy gradient. Because of these implementation details (none of which seem essential to the 'real' difference between these models), it is difficult to know exactly how these models differ. E.g., if the differences in behavior are in part due to the differences in architecture between HDRL and RL2 rather than to the differences in learning mechanisms.

(2) The conclusion that mouse behavior is more consistent with HDRL than with RL2 seems likely but is not sufficiently supported by the analyses. One thing I wondered was whether all model instances were truly converged; RL2 will of course take much longer than HDRL to learn since it is only updated per session. The authors' claim that mouse behavior is more like HDRL than RL2 seems to be based on the BIC comparison in Figure 2f, but insufficient detail is given to understand what this analysis is. Figure 2e, by comparison, seems to present all three models as similar (though this comparison is difficult to make given the different y-axes).

(3) The interpretation of the two-step task feels incomplete, and potentially in conflict with the interpretation of HDRL in the rest of the paper. When trained on the two-step task, HDRL is presented as reproducing a mixture of model-based and model-free behavior. What does the RL2 model do in this context? Later, on the perceptual decision-making task, HDRL seems to be presented as being model-based-like (and using recurrent activity) in one context and model-free-like (and using plasticity) in the other. Is it the case that in the two-step task, HDRL is simply doing both? As is, it's unclear to me how I should interpret the analyses related to the two-step task.

Reviewer #1 (Public review):

Summary:

"A comparative single-cell transcriptomic atlas for diverse populations of vertebrate hair cells" by Basu et al. aims to define pan-hair cell enriched (HCE) genes across hair cell subtypes from four vertebrate species through integration of published scRNA-seq, SMART-seq, and single-nucleus RNA-seq datasets. Pan-HCE genes are weighted using AUC values, where AUC is used to identify genes enriched in hair cells relative to supporting cells. The resulting dataset is made publicly available, allowing researchers to compare gene expression across species. The dataset integration and creation of a public resource are clear strengths for the inner ear community.

Strengths:

This manuscript presents a valuable cross-species resource and a substantial integration effort.

Weaknesses:

Weaknesses addressed by the authors in the text include the lack of human cochlear scRNA-seq comparisons to mouse/chicken cochlear hair cells. While the rationale that the datasets were not available at the time of investigation is acceptable, the use of human cochlear datasets would be the ideal experimental comparison to mouse and chicken cochlear hair cells.

Reviewer #1 (Public review):

Summary:

This study develops a large-language-model implementation of the complementary learning system (CLS) framework for continual episodic learning. The authors employ a pretrained open-weights model to serve as a neocortex-like semantic knowledge store and introduce a novel hippocampus-like memory system in which each episode is assigned its own sparse rank-2 masked LoRA adapter, eliminating interference at encoding. A separate semantic embedding model provides a retrieval key for each episode, and winner-take-all routing selects the adapter whose stored embedding best matches an incoming cue. The reported storage efficiency of approximately 2-3 trainable parameters per token suggests that this one-adapter-per-episode scheme could scale to large numbers of memories, although its total storage requirements also include the embeddings used for content-addressable retrieval.

A key finding is that successful encoding of an episode does not guarantee flexible access to its contents. Although a correctly selected adapter can reconstruct its stored episode, the model performs poorly when asked to answer questions about that episode directly. The iRAG procedure addresses this by inserting the reconstruction into the base model's context. Consolidation through replay of the original passages enables reconstruction without adapters, but direct question answering remains substantially less successful, even with richer training objectives. Overall, the paper provides a technically impressive implementation of episodic memory in LLMs. However, several aspects of its learning and consolidation procedures significantly limit its correspondence to CLS, as discussed below.

Strengths:

A major strength of this work is the concrete parametric implementation of a CLS-like division of labor within a modern transformer network. The episodic memory system incorporates key properties of the biological hippocampus, including pattern-separated encoding and content-addressable retrieval. The interaction between the selected adapter and the LLM provides an appealing functional analogue of hippocampal-cortical reinstatement, in which activation of a sparse episodic trace biases distributed cortical processing so as to reconstruct the mnemonic content of an episode.

A second strength is that the model cleanly separates interference during episodic storage from interference during retrieval. By assigning memories to non-overlapping parameter traces, individual episodes remain faithfully stored even at large memory loads. Performance declines for more strongly overlapping memories can therefore be attributed largely to competition during retrieval. This distinction shows that successful pattern separation does not by itself guarantee reliable access to stored memories.

A final strength is the careful optimization and evaluation of the episodic memory system. The authors introduce a masked LoRA that substantially reduces parameter cost relative to standard LoRA approaches, systematically identify the most parameter-efficient placement of these adapters, and show that storage cost scales approximately linearly with episode length, with similar results across two base models. They also test the framework on two datasets with quite different memory structures: largely independent, topically diverse Wikipedia passages and more strongly correlated autobiographical episodes with recurring people, places, and themes.

Weaknesses:

(1) The model implements the two learning regimes motivated by CLS: pattern-separated episodic learning and slower learning in shared weights. However, it does not implement the central proposed interaction between them, whereby hippocampal traces reinstate experiences that serve as training events for neocortical consolidation during offline periods (e.g., Káli & Dayan 2004, Nat. Neuro.; Singh et al. 2022, PNAS; Sun et al. 2023, Nat. Neuro). Instead, consolidation here relies on replaying the retained ground-truth passages and therefore does not depend on the episodic memory system at all. The authors could either demonstrate consolidation driven by replay from the episodic adapters or more explicitly discuss this simplification and its implications for the CLS comparison.

(2) The authors characterize the episodic system as implementing "fast learning" or "rapid storage". However, encoding involves repeated optimization on the same passage, with up to 1,000 epochs per attempt and potentially multiple retraining attempts. This raises questions about its correspondence to the rapid, single-shot learning that characterizes the hippocampal component of CLS and is supported by evidence in the trisynaptic pathway (e.g., Nakazawa et al. 2003, Neuron; Nakashiba et al. 2008, Science). The authors should (a) report the distribution of optimization steps, retraining attempts, and mask sizes required for successful encoding on the two datasets and (b) clarify this distinction when relating the model to biological hippocampal learning. They could also examine whether larger adapters reduce the optimization required, clarifying the trade-off between storage efficiency and encoding speed.

(3) Relatedly, it is unclear whether there is meaningful variability in the number of optimization steps required across episodes and whether characteristics of the passages explain this variability. For example, do passages containing less predictable content require more training and/or larger adapters? One way to examine this would be to consider the frozen base model's pre-learning perplexity for each passage and test whether this predicts optimization steps to criterion or final parameter count, controlling for passage length. Such an analysis could help clarify what determines the difficulty and resource cost of forming a new episodic memory.

(4) The consolidation results raise concerns about the model's capacity for stable lifelong learning, as incorporating new memories comes at a cost to existing knowledge. The presented consolidation for 1000 memories produces substantial degradation of the base model's existing capabilities, particularly under the conditions that most improve direct access to consolidated knowledge. In a lifelong setting, subsequent consolidation would presumably continue to cause interference not only for pre-existing semantic knowledge but also for memories incorporated during earlier consolidation cycles. The alternation between NREM and REM sleep has been proposed as one biologically motivated solution to this problem, with NREM supporting integration of recent hippocampal memories into cortex and REM helping to reactivate and protect pre-existing cortical knowledge (Norman et al. 2005, Neural Networks; Singh et al. 2022, PNAS). The authors already note that a lifelong system would need to interleave encoding and consolidation and determine which memories to replay; it would also be valuable to discuss how previously consolidated knowledge and broader base-model capabilities could be protected across repeated cycles of consolidation.

(5) It appears that the consolidation conditions reported in Fig. 9 might differ substantially in training exposure. As described in Supplementary Note 5, "passage-only" uses one example per episode, "paraphrase augmentation" uses ten, and the "knowledge instruction" and "masked reconstruction" conditions add many further training examples. Because training is specified in epochs, could the authors clarify whether these conditions are matched for optimizer steps or training tokens? If not, it would be helpful to determine how much of the improvement in direct QA and accompanying loss of base-model capabilities reflects the specific objectives versus greater training exposure. A step- or token-matched control would help disentangle these possibilities.

(6) The reported storage efficiency of approximately 2-3 parameters per token appears to refer only to the masked LoRA adapters, despite each episode also requiring a stored 1,024-dimensional embedding for routing. The authors appropriately note that this index is part of the memory system's storage cost and is in fact substantially larger than the adapter store. It would therefore be useful to incorporate the routing embeddings into the reported per-token storage estimate, or otherwise report the total storage cost per episode/token alongside the adapter-only figure.

Reviewer #1 (Public review):

Summary:

This study describes targeting Poltheta Helicase with a new drug RP-3467. The properties of the drug and a clinical response in a gBRCA1 Ovarian Cancer Patient with a mutation in TP53BP1 are described.

Strengths:

This is an important and interesting set of observations.

Weaknesses:

(1) The response of the patient with a TP53BP1 mutant tumor is of interest but requires more clarification before solid conclusions can be drawn. The presence of the TP53BP1 mutation after but not before olaparib treatment is suggestive of selection, but in the absence of demonstration of a biallelic loss, this is conjecture. It could be a co-selected passenger mutation. The authors should report the TP53BP1 variant allele fraction, LOH, and orthogonal validation should be performed by targeted deep sequencing or ddPCR. This would establish the prevalence before and after selection and enhance the interpretation of the functional effects of the mutation observed.

(2) To further strengthen the proposed mechanism, the authors should consider knocking in the mutation (or a similar LOF mutation) into an appropriate cell line and establish resistance to olaparib and sensitivity to the combination, as well as each drug individually. This will address the latter question of whether it is the poltheta inhibitor or the combo that is responsible for the clinical response.

(3) In the animal experiments, "synergy" is overstating the situation. Without an interaction analysis, this is an enhanced combination activity. Also, several of the animal experiments have a small N. It would be better to show the growth of tumors in individual mice.

(4) The authors propose that Poltheta inhibition could prevent the emergence of reversion mutations. This is not addressed in the paper, and if they want to suggest this, they should provide some evidence.

Reviewer #1 (Public review):

This manuscript by Zhou, Williams, Millner, and colleagues reports findings from a large-scale, multicenter study of Klebsiella pneumoniae (Kp) isolates. These isolates were sequenced using short-read sequencing and extensively characterized using a variety of field-appropriate tools, such as Kleborate, AMRFinderPlus, and PlasmidFinder. Then, the authors measured the growth of these Kp isolates across many (>200) environmental conditions they pre-determined as important for making human-centric fitness conclusions. These genotype and phenotype data were integrated, and the authors deployed an association-based strategy to identify genetic determinants of the observed fitness effects. Finally, the authors built models to predict fitness outcomes using the associated genetic determinants. From these data, the authors conclude that fitness is "an emergent property" of a given Kp isolate's genetic background and environmental constraints, with a nod toward the future development of biomarkers to predict fitness outcomes using genotype alone.

The primary strength of this study is the integration of two large datasets, one genotypic and the other phenotypic. This approach is novel in its scale (genotypic, geographic, etc.), which adds to the strength of the overall conclusions. These conclusions, for the most part, are consistent with the data. As such, this study can act as an example to other studies tying genotype to phenotype, especially in the context of careful genetic analysis and rational phenotypic design. A secondary strength is that the findings are supported by other, smaller-scale studies such that there is a growing consensus as to the general fitness determinants employed by Kp that are potentially driving its increased global importance as a bacterial pathogen.

The primary weakness of this study is that some confounding factors are not taken into account or are deemphasized. The primary confounding factor is that the associations between genetic signatures of fitness are not fully considered. This is especially important given that an important driver of Kp fitness is its accessory genome, which is heavily influenced by mobile genetic elements and plasmids. The use of short-read sequencing leads to an inherent inability to tie fitness effects to such genetic elements, which may lead to masking or overinterpretation of particular genotype-phenotype relationships. For example, nearly all of the gene-level determinants of condition-specific fitness and pleiotropic genes are known plasmid-associated genes; however, it is unclear if these specific genes are driving fitness or if they are merely signatures of other associated genetic elements. This broadly weakens the conclusions and translatability, and in the absence of long-read sequencing to resolve genetic architecture, it is an unresolvable weakness. A second is the over-interpretation of the variables used to assess bacterial growth, which is the primary fitness metric. Colony opacity can be influenced by Kp capsule production, which is a highly variable trait that is associated with environmental conditions. Finally, it is unclear whether clonal isolates are excluded to avoid overinterpretation of fitness outcomes.

Reviewer #2 (Public review):

Summary:

This is an interesting study that explores how human RPE could be used as source for new retinal neurons. This is a welcome addition to the field of retinal regeneration which is currently focused almost exclusively on the regenerative capacity of Muller glia cells. The line of inquiry is firmly rooted in findings from amphibian and embryonic chick model systems and advances a fetal human retina RPE based screening system as a rich resource for insights into human RPE biology, including as a potential stem cell source.

The authors investigate the potential of fetal human RPE cells to be reprogrammed into retinal neurons using overexpression of pro-neural factors. While this is a critical knowledge gap in the field of retinal regeneration with significant promise for developing regenerative therapies, several methodological concerns impact the interpretation of results. Firstly, while the authors sought to evaluate factors that enhance RPE reprogramming when co-expressed with ASCL1, nearly all co-expression constructs tested failed to achieve appreciable expression of ASCL1, leaving a central hypothesis of this study largely untested (Major concern 1). Second, although the authors were able to detect a cluster of photoreceptor-like cells in their screen, they were unable to identify which reprogramming construct generated this cluster (Major concern 2). Finally, an essential control that definitively demonstrates the value of combinatorial transcription factor reprogramming is missing (Major concern 3). In summary, the authors establish a valuable new paradigm for culturing and reprogramming fetal human RPE, and even more importantly, demonstrate successful reprogramming to neural fates. However, the discussion and interpretation of results needs to be modified significantly to make it clear that (i) the outcome of many co-expression paradigms remains effectively unknown/untested due to failed over-expression of ASCL1, and that (ii) the reprogramming construct giving rise to photoreceptor-like cells could not be conclusively identified from their initial screen.

Strengths:

(1) Powerful new screening system advanced for exploring the regenerative potential of human feta RPE cells.

(2) Co-expression vector system for testing additive effects of proneural transcription factors

Weaknesses:

Major concerns:

(1) The authors executed a screen for combinations of factors that can enhance ASCL1-mediated reprogramming of RPE into retinal neurons. However, the expression level of ASCL1 was remarkably low in virtually all co-expression paradigms (see Fig. 3C). Notably, the reprogramming combination with the highest potency (ASCL1 + NEUROD1) was also the one exhibiting the highest level of ASCL1 expression. The "failed" reprogramming of most of the co-expression constructs (ASCL1+LMO1, ASCL1+EZH2, and ASCL1+RAX2) is potentially a false negative resulting from low transgenic expression of ASCL1.

(2) The authors interpretation is that the overexpression of NeuroD1 and Ascl1 generated a new cluster that expressed markers of photoreceptors such as RXRG and RCVRN (see Figure 3D). However, there does not actually appear to be any overlap between the ASCL1+NEUROD1 cluster (orange dots, left panel) and the cells expressing markers of photoreceptors (yellow/green/purple?/black? dots, right panel; yellow being ASCL1-EZH2, green being ASCL1, purple being ASCL1-and black being control - though color coding here is admittedly somewhat confusing). Thus, the photoreceptor-like cluster of interest actually seems to correspond to gray cells that were unmapped / exposed to an unknown programming cocktail. So, it remains completely unknown which reprogramming construct generated this cluster.

(3) To conclusively establish the additive role of NEUROD1 in reprogramming, it would be prudent to compare ASCL1 + FA directly to ASCL1+NeuroD1+FA. This control was not included but is needed for a more complete interpretation of results.

Comments on revised version.

We thank the authors for their thoughtful responses to our concerns and for modifying a figure to improve clarity. However, we were disappointed to see that the responses to our major concerns were not incorporated into the revised manuscript, as we believe they provide context that is essential to proper interpretation of the data. Minimal revisions of the current manuscript would address our first two major concerns. This could be achieved by adding two additional sentences, for example incorporating the sentences below which are excerpted from the authors response to our concerns:

The following excerpt from the authors response would address major concern 1 if incorporated into the relevant section in the main text of the manuscript:

"Insufficient ASCL1 expression in the combinations might have led to lack of reprogramming, representing a false-negative result."

And this excerpt would address major concern 2:

"The farthest subcluster with highest RCVRN/RXRG expression was not assigned ASCL1-NEUROD1 identity."

Reviewer #2 (Public review):

Summary:

The authors investigate chemical strategies to disrupt the interaction between the SARS-CoV-2 accessory protein Orf9b and the host mitochondrial receptor Tom70, an interaction implicated in suppression of type-I interferon responses. They employ two discovery approaches: a crystallographic fragment screen against the Orf9b homodimer and a high-throughput fluorescence polarization screen for compounds that compete with Orf9b binding to Tom70. The study identifies fragment-binding hotspots on Orf9b, develops lipidated analogs that stabilize the Orf9b homodimer, and discovers Tom70-binding compounds with low micromolar activity that inhibit Orf9b binding in vitro.

Strengths:

Impressive amount of work using a variety of complementary approaches and methods to validate binding (FP, SPR, and computational modelling and SAR). The combination of crystallographic fragment screening on Orfb9 and HTS on Tom70 provides two independent routes for perturbing the Orf9b-Tom70 interaction. The structural work seems to be of very high-quality. The fragment campaign is extensive, yielding a substantial number of fragment-bound structures and identifying biologically meaningful binding hotspots on Orf9b.

Finally, the screen results in reporting useful chemical starting points. Although potency remains modest, the study provides tractable scaffolds and a clear framework for future optimization.

Comment on revised version.

I don't have any further comments. The authors have addressed my suggestion by adding a statement for future work in the discussion.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

Summary:

The main goal of this manuscript is to develop a mathematical model of the regulation of cortical dynamics by Cdk1 activity to explain why, in some embryos (e.g., Xenopus), surface contraction waves are believed to move in the same direction as Cdk1, while in other embryos (e.g., starfish) they are believed to move in the opposite direction.

Strengths:

(1) The paper addresses a very important question.

(2) The mathematical model is sensible and suggests that the different relationship between Cdk1 and surface contraction waves might arise from the different behavior of the mitotic entry wave and the mitotic exit wave.

(3) The authors propose a mechanism by which the wave observed at mitotic exit might not passively follow the trigger wave observed at mitotic entry'

(4) The proposed mechanism is a potential explanation of the observed differences.

(5) The proposed mechanism is centered on different dynamics between the nucleus and the cytoplasm, highlighting the potential importance of the nucleus (and nuclear size) in organizing cortical dynamics.

Weaknesses:

(1) The proposed mechanism works if the activity in the nucleus is much higher than the high activity (high state of the bistable system) of the cytoplasm. So, as the wave propagates across the cytoplasm, the activity around the nucleus remains higher, which potentially causes a delay in the onset of Cyclin B-Cdk1 degradation in the region around the nucleus compared to the surrounding cytoplasm. This effect happens over a typical length scale, and if such a length scale is comparable to embryo size, this becomes the predominant mechanism. However, such a mechanism should exist near the nucleus independently of embryo size. So, it seems that for embryos where the wave back and wave front should travel together, nuclear activity must be adjusted not to be much higher than cytoplasmic activity. Is there any experimental evidence that the overall activity of Cdk1 is higher in the nucleus than in the cytoplasm?

(2) The authors give the existence of trigger waves as a fact. While the predominant view is that such waves exist in the first cycle of the Xenopus embryos (however, this is from measurement of the cortical contractions, so a bit circular for this paper), it is unclear if waves exist in the starfish embryo, so the potential explanation that the starfish embryo simply has different Cdk1 dynamics cannot be ruled out.

Reviewer #1 (Public review):

[Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

The study provides a robust bioinformatic characterization of the evolution of pT181. My main criticism of the work is the lack of experimental validation for the hypotheses proposed by the authors.

Previous comments on the study:

(1) One potential reason for the decline in pT181 copy number over time may be a high cost associated with the multicopy state. In this sense, it would be interesting if the authors could use (or construct) isogenic strains differing only in the state of the plasmid (multicopy/integrated). With this system, the authors could measure the fitness of the strains in the presence and absence of tetracycline, and they could be able to understand the benefit associated with the plasmid transition. The authors discuss these ideas, but it would be nice to test them.

(2) It would be interesting to know the transfer frequencies of the multicopy mobilizable pT181 plasmid, compared to the transfer frequency of the plasmid integrated into the SSCmec element (which can be co-transferred, integrated in conjugative plasmids, or by transduction).

(3) One important limitation of the study that should be mentioned is that inferring pT181 PCN from whole-genome data can be problematic. For example, some DNA extraction methods may underestimate the copy number of small plasmids because the small, circular plasmids are preferentially depleted during the process (see, for example, https://www.nature.com/articles/srep28063).

Comment from the Reviewing Editor:

In the revised version the authors have addressed comment 3 from Reviewer #1 through additional analysis, finding that the decline in copy number over time remains significant after controlling for instrument used, type of DNA extraction technique, and sequencing centre. The suggestion in Comment 1 from Reviewer #1 has not been acted upon as the authors state that this additional experimental work "goes beyond the scope of the paper" and is being explored for a future study. Similarly, the suggestion in Comment 2 from Reviewer #1 has not been acted upon as the authors state that "inferring these rates from natural sequence data is not feasible in this case given the low heterogeneity of the plasmid sequence. A laboratory-based experimental study could not address the real transfers we observe over the course of decades, as in vitro S. aureus transfer rates are often not good proxies for in vivo".

Reviewer #1 (Public review):

Summary:

This manuscript reports exceptionally preserved, millimeter-scale segmented bilaterian microfossils from the Fortunian Kuanchuanpu Formation in South China. The authors propose that these non-biomineralized animals were preserved through microbial pseudomorphs rather than direct phosphatization, filling a critical taphonomic and evolutionary gap in early animal segmentation.

Strengths:

This paper filled the critical taphonomic and evolutionary gap in early animal segmentation.

Weaknesses:

Extremely similar work has already been published by Xian et al., 2026, PNAS, with specimens from absolutely the same formation, and they also proposed that these animals are segmented. So in this context, this work's significance is eclipsed.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors employed Cryo-ET to visualize the structural organization of the tripartite attachment complex or the TAC, which is a unique structure connecting the flagellar basal body to the mitochondria in Trypanosoma brucei, a protozoan parasite causing human sleeping sickness in sub-Saharan Africa. The TAC is essential for segregation of the mitochondrial DNA, or kDNA, via the connection to the segregating basal bodies during the cell cycle. Although molecular characterization of the TAC and its components has yielded insights into the function of the TAC, the structural organization of the TAC, especially the largest protein p197 within the TAC, remains poorly defined. Using intact zoid cells, the authors provided, for the first time, a high-resolution image of the cytoplasmic part of the TAC in its native context, resolved the basal body, the mitochondrial membranes, the exclusion zone filament connecting the BB to the outer mitochondrial membrane, and quantified the geometry of the region surrounding the TAC. These observations led to a proposed role of p197 as a length-variable connector to reconcile stable kDNA anchoring with the mechanical demands of BB movement. Overall, the work advances our understanding of the structure and function of p197 within the TAC in trypanosomes.

Strengths:

A major strength of the manuscript is the application of Cryo-ET in imaging the structure of the cytoplasmic part of the TAC, with a focus on the largest protein p197, which provided novel insights into its molecular function. Trypanosome cells are too thick for Cryo-ET, and the authors adopted intact zoid cells for Cryo-ET, allowing the resolution of the TAC structure in native conditions.

Weaknesses:

While this is the first attempt to image the TAC by Cryo-ET, which provided new insights into the potential role of p197 in mediating the connection between the BB and the mitochondria, there is a lack of mechanistic understanding of how p197's length is regulated and how the varying length of p197 coordinates with basal body movement. Also, it is unclear why the cell needs to regulate the length of p197, given that the main function of p197 is to connect the BB and the mitochondria for the segregation of kDNA via the BB.

The proposed length variation for p197 is an interesting idea, but it is unclear what exactly the authors meant by saying that p197 is length variable. Do they mean that the central repeats in p197 can fold/extend to variable lengths, or do they mean that p197 with variable lengths contains different numbers of the central repeats? This should be clarified.

The current work can be strengthened by performing some molecular and/or biochemical experiments to assess the function of p197 to complement the Cryo-ET-based structural characterization reported in the manuscript. Expression of some smaller-sized p197 proteins with varying numbers of central repeats in p197 RNAi cells will provide evidence for the proposed function of p197 length variation.

Reviewer #1 (Public review):

Summary:

Ribeiro et al. investigate the molecular mechanisms affecting the efficiency of synthetic cell-penetrating peptide (CPP) uptake by cultured mammalian cells. CPPs are frequently coupled to cargoes that can change cell function and are highly useful to investigators studying cell biology. Moreover, CPPs are potentially amenable to delivery of cargoes that can affect cells in clinically useful ways, with several under investigation as potential anti-cancer treatments. A challenge to the therapeutic use of CPPs is their inefficient delivery to their intended molecular targets, with poor cellular uptake being a major barrier.

The present study presents new insights into the cellular and plasma membrane properties that can affect CPP uptake. In the studied cell line, the authors find that cell cycle-dependent changes in the plasma membrane potential correlate with uptake of a CPP conjugated to a fluorescent dye. These changes correlate with mRNA abundance of a voltage-gated potassium channel, KCNH1. RNA silencing of KCNH1 expression or pharmacological blockade of KCNH1 currents impairs CPP uptake, supporting the proposed model.

Strengths:

(1) Identified KCNH1 expression and function as a specific modulator of CPP uptake in U2OS cells and LAN1 cells.

(2) Data show a clear correlation between the plasma membrane potential, cell cycle stage, and efficiency of intracellular CPP delivery in these cells.

Weaknesses:

(1) Some of the claims are more generalized than warranted by the presented evidence. Is KCNH1-dependent modulation of cell cycle-dependent changes in the plasma membrane potential and ensuing effect on CPP uptake a niche property of this channel in some cell lines? Or a more broadly generalized function? I think this can be addressed merely through more careful or caveated language in the manuscript. However, an additional experiment that would be potentially informative and complementary to the chronic high-potassium depolarization experiments would be to examine the impact of heterologous expression of a hyperpolarizing potassium channel (for example, Kir2.1).

(2) Please provide more details on how endosomes were identified and measured.

Reviewer #1 (Public review):

Summary:

The authors have carried out a study of lipid membrane permeabilities on different nucleoside/tide isomers. The study primarily relied on PAMPA as the experimental technique for assessing permeabilities, although interestingly, the authors found that there was a correlation between permeability and retention time on a C18 column. The authors specifically looked at the permeabilities of furano and pyrano alpha/beta nucleosides/tides, since a major question is why biochemistry uses only the beta furano isomers, when three other isomers based on anomeric configuration and ring size are also possible. The authors found that for all A, U, G or C nucleoside isomers, the beta-furanoside is consistently the most lipid-permeable. These findings were also supported by computational (DFT and MD) studies. The authors conclude that such a property could plausibly have been a chemical selection mechanism that favoured these isomers when they first started interacting with protocell membranes.

Strengths:

There are two major strengths to this study: (1) it offers an alternative/additional explanation backed by experimental and computational evidence as to how the canonical ribonucleotides (namely, the beta-furanoside isomers) were selected by interactions with protocell membranes; (2) it makes the important observation that retention time on a C18 column is correlated with permeabilities determined by PAMPA, the "industry standard", suggesting that LC could also be a convenient way to assess relative permeabilities of analytes of interest. With this report, there are many promising follow-up studies that can be carried out in the context of synthetic lipid protocells in order to further support the hypothesis that selective permeability played a major role in early chemical/Darwinian evolution.

Weaknesses:

The biggest weakness is that there needs to be more of a critical discussion of chemical vs Darwinian selection. There are two basic hypotheses for how the canonical ribonucleotides came to be: they were the products of prebiotic synthesis and chemical selection pressures, or they emerged as a product of natural selection after Darwinian evolution already became established. While the experimental evidence in terms of ribonucleotide prebiotic synthesis is discussed well, the work of Eschenmoser in particular, which supports the alternative hypothesis that RNA is the product of Darwinian evolution, should be discussed in more detail on page 4.

The authors should also acknowledge somewhere in the discussion/conclusion that there is a certain amount of bias that the field has towards extant biology, and this bias could be influencing how certain results are interpreted as beneficial for ribonucleotide selection, when the same result might be used to argue against it. For example, the fact that the 2',3'-cyclic phosphates are the most permeable among the phosphate regioisomers might be used to argue these were the preferred substrates for oligomerization in a protocell, given it had a catalyst which could do it, like a short peptide. While the differences in permeabilities are quite solid, what it means in terms of selection could be applied to multiple, specific scenarios, each with its own interpretations and implications.

Reviewer #1 (Public review):

Summary:

The authors studied the development of hippocampal connectivity gradients based on open datasets and performed correlation analyses with other MRI features as well as gene expression information from other datasets.

Strengths:

The hippocampus is a key region in understanding large scale brain organization and cognition, and the authors applied advanced and suitable analytics to study its development. The paper is overall well organized and well written, and the findings are relevant for studying large-scale brain development.

Weaknesses:

While sophisticated, several of the analyses appear mainly correlational, cross-sectional, and rely on cross-dataset contextualization, which should also be stated as a limitation of the current work.

Comments on revised version.

I thank the authors for their revisions which have improved the paper.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors build a connectome-scale resource for studying mitochondria in the Drosophila Hemibrain. The authors segment and classify millions of mitochondria, quantify their size, abundance, appearance, and spatial relationship to synapses across thousands of neuron types, and extend neuPrint so that these organelles can be queried together with neuronal connectivity. The analyses identify substantial mitochondrial variation across cell types, systematic differences between pre- and postsynaptic mitochondrial organization, associations with neurotransmitter class, and changes in mitochondrial properties with synaptic fan-out.

Strengths:

The scale and accessibility of the resource are the major strengths of this work. The mitochondrial annotations are integrated with an established connectomic dataset and can be queried through neuPrint, which should make the study useful to researchers beyond the immediate biological questions addressed here. The manuscript also describes the underlying data model and how mitochondrial information is linked to neurons and synapses.

The manuscript compares mitochondrial organization across multiple levels, including individual cells, cell types, brain regions, synaptic polarity, neurotransmitter class, and synaptic fan-out. Several descriptive results are particularly clear, including the tendency for mitochondria to be closer and larger at presynaptic than postsynaptic sites and the identification of cell-type-specific patterns.

The biological interpretations are generally placed in the context of prior experimental literature on synaptic energy demand, calcium handling, and mitochondrial positioning. In many places, the authors appropriately use cautious language such as "may," "possible," and "consistent with," which helps distinguish mechanistic hypotheses from direct measurements. The cited literature therefore provides a reasonable biological framework for interpreting the structural observations, even where the corresponding physiological variables are not measured in this specimen.

The authors also acknowledge important limitations of the Hemibrain sample, including incomplete neurons, regional differences in reconstruction quality, and the fact that the analysis represents a single physiological snapshot. The public code and data infrastructure further increase the utility and reproducibility of the study.

Weaknesses:

The dataset has extraordinary cellular coverage but derives from one Hemibrain specimen. Claims about "natural ranges," generality across Drosophila neurons, or organizational principles extending beyond this specimen should therefore be phrased with some caution. This is particularly relevant for cell types represented by only one or a few instances. For example, MBON13 is represented by a single cell in this dataset, so its unusually high mitochondrial investment is well supported for that instance, but variability of this property within the cell type cannot be assessed here. This is consistent with the authors' own acknowledgement in the Discussion that detailed conclusions for individual cell types may require larger numbers of cells.

The mitochondrial appearance classes support several biological conclusions, particularly the differences associated with brain region and neurotransmitter class. The manuscript describes how dark, medium, and light training labels were generated and reports strong validation of mitochondrial detection and nearby-mitochondrion recall, but I could not find a corresponding independent validation of the three appearance labels themselves. Some assessment of classification reproducibility, for example using a held-out manually assessed set, a confusion matrix, or inter-annotator agreement, would strengthen confidence in conclusions that depend on these categories.

Reviewer #1 (Public review):

This study addresses an important question: whether dopaminergic activity in Drosophila functions as a general value signal across behavioral contexts. Its main strength is the breadth of the experimental program, combining multiple DAN driver lines, several operant paradigms, rescreening, and more targeted follow-up experiments. The cross-paradigm design is well suited to test whether self-stimulation effects generalize.

The main weakness is that several conclusions extend beyond what the experiments directly establish. The genetic tools define populations rather than the same identified neurons at single-cell resolution. In the key time-course experiments, the data more clearly support a reduction of initial avoidance toward neutrality than a consistent aversive-to-appetitive sign reversal. Similarly, the non-contingent comparison uses continuous pre-exposure rather than a stimulation-history-matched open-loop condition; it therefore shows that simple non-contingent exposure is insufficient, but does not isolate agency itself as the causal variable.

The general claims are also constrained by incomplete overlap among the initial screens and by design changes between the original and follow-up joystick experiments. Overall, the study provides useful evidence that the behavioral effects of DAN activation are weak, context-dependent, and time-dependent, while the claims about neuronal identity, positive valence, agency, and a general dopamine "common currency" require more cautious framing.

Reviewer #1 (Public review):

Olmstead et al. present a single-cell nuclear sequencing dataset that interrogates how hippocampal gene expression changes in response to distinct physiological stimuli and across circadian time. The authors perform single nucleus RNA sequencing on mouse hippocampal tissue after (1) kainic acid-induced seizure, (2) exposure to an enriched environment, and (3) at multiple circadian phases.

The dataset is rigorously collected, and a major strength is the use of the previously established ABC taxonomy from Yao et al. (2023) to define cell types. The authors further show that this taxonomy is largely independent of activity-driven transcriptional programs. Using these annotations, they examine activity-regulated gene expression across neuronal and glial subclasses. They identify ZT12, corresponding to the transition from the light to the dark period, as transcriptionally distinct from other circadian time points, and show that this pattern is conserved across many cell types. Finally, they test how circadian phase influences activity-dependent gene expression by exposing mice to an enriched environment at different times of day, and report no significant interaction between circadian phase and enriched environment exposure.

Comments on revised version.

The authors have addressed the comments from the first round of review.

Reviewer #1 (Public review):

Summary:

Doronin et al. performed wide-field calcium imaging to investigate the cortex-wide activity dynamics in mice after the administration of the psychedelic DOI.

Strengths:

The lab has extensive experience with this approach. The analyses are not trivial, but here the authors progressively step through the rich data to identify salient effects. The observed effects at the level of cortical networks will be of great interest to the field, both for basic scientists interested in circuit mechanisms of psychedelics and for human researchers who may want to reverse translate from humans to mice.

Weaknesses:

The analyses were extensive, but the choices made for the analyses are quite specific, and there are places where other analyses may be more appropriate and logical. For example, instead of comparing activity with serotonergic axonal density, compare with Htr2a transcript because the receptor is the drug target. Moreover, data were combined for day 1 and day 5 for an unclear reason. Furthermore, a few statements in the text did not seem fully supported by the results and may be over-interpreted.

Reviewer #1 (Public review):

Summary:

This study by Weber et al. investigates how artificial activation or inhibition of dopaminergic neurons innervating the Drosophila larval mushroom bodies in the context of learning paradigms influences individual properties of acquired memories. Similar approaches have been undertaken in the past (e.g., in the adult); however, the reduced complexity of the larval system makes this study valuable, especially as it eases the possibility for informed modelling approaches, as done here. A special aspect shown here is that artificial training overrides the need for context typically observed for recall of larval aversive memories.

Strengths:

This study adds insights into how the larval mushroom bodies store memories. The behavioral data using genetically precise manipulation of identified neurons is combined with computational modelling. Information on the physiological basis of the underlying mechanism is not provided. In light of the claim of ‚temporal precise optogenetic manipulation' and the authors ‚manipulating the intrinsic physiology' of DANs, this is important and should be stated clearly. While this reviewer agrees that the ‚temporal precision' is provided for memory phases, this might not be true for physiological parameters, especially when using tools like ChR2-XXL.

Thus, this study offers interesting behavioral and cellular insights; however, this reviewer also recommends specifying claims on temporal and physiological precision more carefully.

Weaknesses:

(1) The authors should be more precise about temporal and physiological windows/properties. It is recommended to discuss the published properties of the optogenetic tools used and how this might influence the obtained results, especially when comparing the results to 'naturally' reached learning after pairing stimuli in the absence of optogenetic intervention.

(2) The authors should comment more directly as to why pairing odors with activation or inhibition of DANs does not lead to measurable learning (Figure 1).

(3) A key finding is that DL DAN activation leads to context-independent memories (as also somewhat indicated in the title). To make this claim, the 'natural' training experiments should be conducted in parallel, or these potential limitations should be mentioned. However, if the authors state that the results using regular conditioning approaches are so clear that this is not necessary, the should state that.

(4) Potential caveats of activating chloride channels over prolonged time periods should be mentioned.

(5) The appropriate literature showing odor-evoked synaptic depression to MBONs (e.g., cited in lines 431/432) in the context of learning should be cited throughout.

Reviewer #1 (Public review):

Summary:

This study investigates the structural basis of neuromodulatory transmission in the cerebral cortex. Using genetic labelling, correlative light and electron microscopy (CLEM), and focused ion beam scanning electron microscopy (FIBSEM), the authors reconstruct cholinergic axons originating from the basal forebrain and serotonergic axons from the raphe nuclei in the mouse somatosensory cortex, and compare their ultrastructural organisation with that of glutamatergic and GABAergic axons.

The study provides detailed quantitative evidence that cholinergic and serotonergic axons have an axonal architecture fundamentally different from that of classical fast-transmitting glutamatergic and GABAergic axons. In particular, vesicles in cholinergic and serotonergic axons are distributed more diffusely along the axon and are only weakly associated with morphologically identifiable synaptic specialisations. These findings provide strong structural support for the idea that volume transmission represents a major mode of signalling by these neuromodulatory systems in the cortex.

Although the paucity of conventional synaptic contacts made by cortical neuromodulatory axons has been reported previously, including in classical work by Descarries and colleagues, the present study provides substantially more detailed and quantitative three-dimensional evidence. The study also reveals characteristic differences among neurotransmitter systems in vesicle density, vesicle distribution relative to synaptic membranes, and vesicle morphology.

Strengths:

A major strength of this study is the combination of genetic labelling, CLEM, and high-resolution FIBSEM, which allows molecularly identified cholinergic and serotonergic axons to be reconstructed without the electron-dense reaction products used in conventional immunoelectron microscopy. This enables detailed analysis of vesicles and membrane specialisations along extended axonal segments.

Another important strength is that the analysis is not restricted to individual boutons or varicosities. Instead, the authors analyse vesicle distributions continuously along reconstructed axons and compare them quantitatively with glutamatergic and GABAergic axons. This provides a more comprehensive assessment of the relationship between vesicle distribution and morphologically identifiable synaptic sites.

Overall, the data are convincing and support the authors' main conclusions. In particular, the finding that most vesicles in cholinergic and serotonergic axons are spatially dissociated from identifiable synaptic specialisations provides strong structural evidence for predominantly non-synaptic or volume transmission. The work should therefore be of broad interest to researchers studying neuromodulation, cortical circuitry, and connectomics.

Weaknesses:

I do not identify any major methodological weakness. The manuscript is well prepared, and the experimental results generally support the authors' conclusions. I have only a few points that could further improve the interpretation and discussion.

First, in the Introduction, ionotropic receptors are emphasised in the context of rapid actions of acetylcholine and serotonin. However, these neuromodulators can also produce relatively rapid effects on cortical neurons through G-protein-coupled receptors. This could be acknowledged, for example, with reference to Gulledge et al. (2009) and Tanaka & North (1993).

Second, the implications of the findings for volume transmission could be discussed somewhat more explicitly. If acetylcholine and serotonin are released extrasynaptically and are not immediately degraded or removed, they may diffuse through the extracellular space and activate receptors located at some distance from the release site. Thus, their actions need not be restricted to cellular elements immediately adjacent to vesicle-rich regions of the axon. A brief discussion of the sequence from action-potential-dependent Ca²⁺ entry and transmitter release to extracellular diffusion and activation of spatially distributed receptors would strengthen the interpretation.

Reviewer #1 (Public review):

Summary and strengths:

This manuscript examined whether realized thermal and aridity niche limits were associated with extinction risk across more than 23,000 terrestrial vertebrate species. The authors found that climatic niche limits were phylogenetically conserved and that species with lower upper thermal limits were more likely to be threatened, while relationships with aridity niche limits were weaker and less consistent. They then used simulations based on current extinction-risk patterns and found taxon-specific changes in the distribution of climatic niche limits and loss of phylogenetic diversity under simulated future extinction scenarios.

The study addresses an important question at an impressive taxonomic scale, and the underlying dataset and analyses have the potential to provide valuable insights into how climatic niche limits relate to patterns of extinction risks.

Weaknesses:

(1) One main concern is a disconnect between the central framing of the study and what the analyses can establish. The manuscript proposes that climatic niche limits increase species' vulnerability to climate and land-use change, yet extinction risk is measured using overall IUCN threat status, which integrates pressures from many different causes, including exploitation (e.g. hunting; pet trade), disease, and invasive species. For several of these threats, there is no clear mechanistic basis for why climatic niche limits should influence extinction risk. Consequently, the observed association does not establish that climate or land-use change is the underlying driver of the relationship between climatic niche limits and extinction risk. Analysing whether climatic niche limits are associated with extinction risk from climate- and habitat-related threats would provide a more direct test of the proposed mechanism.

(2) The treatment of Data Deficient species is another important concern. The authors classified Data Deficient species as Not Threatened in their analysis. However, previous studies have shown that many may in fact be threatened (Borgelt et al., 2022, Communications Biology; Jetz and Freckleton, 2015, Philosophical Transactions of the Royal Society B). This issue is particularly important here given the large number of Data Deficient species, especially among reptiles and amphibians, for which extinction risk may itself be underestimated (Caetano et al., 2022, PLoS Biology; González-del-Pliego et al., 2019, Current Biology). Treating these species as Not Threatened could therefore influence the reported relationships and should be evaluated through additional analyses.

(3) The role of the phylogenetic diversity analysis in addressing the central question is also unclear. The primary question concerns the relationship between climatic niche limits and extinction risk, whereas the subsequent analysis examines phylogenetic diversity loss following simulated extinctions. Disproportionate losses of phylogenetic diversity following non-random extinction are already well studied (e.g. Hughes et al., 2022, Current Biology; Oliveira et al., 2020, Global Ecology and Biogeography; Davis et al., 2018, PNAS). The authors should therefore clarify what this analysis adds beyond existing work and how it provides a meaningful extension of the climatic niche-extinction framework.

(4) The interpretation of the future extinction scenarios is also stronger than warranted by the simulations. The authors appropriately acknowledge that these scenarios do not explicitly incorporate climate or land-use change, but subsequently interpret the results as suggesting that climatic niche constraints shape future extinction pathways and that climatically vulnerable species will be disproportionately affected. Identifying species with lower climatic niche limits as climatically vulnerable is reasonable within the framework of the study. However, because the simulations are based on present-day extinction-risk transition probabilities rather than future climate- or land-use-driven processes, they do not establish that future climate or land-use change will preferentially drive the extinction of these species.

Overall, the manuscript addresses an important question and provides potentially valuable data and analyses. However, several of the central conclusions currently extend beyond what the analyses can directly support. Addressing the issues above would substantially strengthen the manuscript and clarify the scope of its conclusions.

Reviewer #1 (Public review):

Summary:

Sensory preconditioning protocols have been used to understand how the brain controls aversive or appetitive behavioral responses towards stimuli that have never been directly reinforced. In this study, the authors combine pharmacological NMDA blockade in a specific brain region previously involved in sensory preconditioning (i.e., perirhinal cortex) and different versions of a light-tone sensory preconditioning task in rats (i.e., different number of associations during preconditioning, distinct temporal separation between stage 1 and stage 2, stimuli order) to better understand how the integration between separated stimuli occurs. Overall, the main conclusion of the study is that the number, order, and time determine how distinct experiences are encoded and stored in the brain, which clearly advances the understanding of how these complex cognitive processes are regulated by the brain.

Strengths:

(1)The manuscript is very well written, and the methodology is well detailed, fully aligned with previous work by the research group.

(2) Results from each pharmacological manipulation and/or protocol configuration are clear, demonstrating the robustness of their findings.

(3) The overall conclusions are very interesting and clearly advance our understanding of higher-order conditioning processes.

Weaknesses:

(1) The Discussion is overly redundant with the Results and should be rewritten to place the findings more clearly within the existing literature and add specific important topics for the field (e.g., type of stimulus presentation, other brain circuits involved, etc).

(2) Before-after and sex-disaggregated data should be presented to better show statistical differences between periods with or without cue during the test and between sexes.

(3) The authors should broaden their coverage of recent literature on light-tone higher-order conditioning beyond their own previous work.

I never imagined that people in the past were so more open-minded than people today.

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 reviewers' suggestions.]

Summary:

This paper presents a toolkit for the transformation of Blastocystis. The authors have screened a number of selectable agents, promoters and reporter genes and present their findings. This resource will be of immense use to those in Blastocystsis field, as well as those seeking to establish transformation tools in other species where such tools do not yet exist. Establishing new transformation tools is extremely challenging, and the authors have done an excellent job.

Strengths:

The authors have carried out a systematic screen of promoters, reporter genes and selectable agents. They have screened numerous for each, and all the data is presented. It is good to see when things did not work as well as when things did - so this data set is extremely useful indeed.

Comments on previous version.

The authors have revised their manuscript to clarify that molecular analyses have not yet occurred and have resolved the technical/publication issues with the figures. I look forward to seeing these tools used in future publications to answer important questions in Blastocystsis research.

Reviewer #1 (Public review):

Summary:

This is an interesting study that addresses whether mitochondrial DNA (mitoDNA) variants impact telomere length (TL), which may be relevant to potential maternal inheritance of TL in offspring. The study addresses this question using an innovative cybrid model approach in which mitochondria from donor platelets from 7 individuals that vary in TL and differ in mitoDNA variants are fused with 143B cells that lack mitochondria. MitoDNA variants that exhibited reduced complex I activity showed telomere shortening in cybrids, and increased telomere dysfunction. Interestingly, these phenotypes could be reduced with NAC antioxidant and NAD+ supplementation, suggesting that ROS and oxidative DNA damage at telomeres contributed to the telomere shortening. They further showed that cybrids with lower levels of ROS correlated with longer TL in the lymphocytes of the mitochondrial donors.

This study has many strengths, including the use of cybrid technology, which allows for analysis of how mitochondria and mtDNA variants from various human donors influence telomere length when introduced into the same genetic background of 143B mitochondria deficient cells. Other strengths include the use of human donors that vary in leukocyte telomere length, and analysis of multiple endpoints of telomere integrity and mitochondrial function in the resulting cybrids. This study provides convincing evidence that mtDNA variants influence TL, through a mechanism involving mitochondrial derived ROS, potentially causing telomeric oxidative damage. These results may help explain prior work demonstrating a link between mitochondrial function and telomere length regulation during embryogenesis and development. The data are robust, and the manuscript is well written. Overall, the results support the conclusions and will likely have an impact on the field of mitochondrial function and telomere length regulation, with important implications for development, aging, and aging-related diseases.

Given the heterogeneity in TL and mtDNA variants in the human population, the conclusion could be further strengthened by increasing the number of donors and cybrids analyzed. Other minor weaknesses stem from the limited number of samples. For example, the study presented limited telomere FISH metaphase analysis of chromosome end fusions, telomere damage, and telomere attrition rates due to low sample numbers. Furthermore, the proposal that acute oxidative stress was induced by a metabolic shift from glycolysis to OXPHOS in vitro is based primarily on assumptions from prior work, rather than direct demonstration in the current study. However, results with the NAC antioxidant combined with NR strongly support this assumption. Overall, the findings from this study are convincing and provide a solid foundation for expanding this analysis in the future to include a greater number of samples from donors for cybrid analysis.

Reviewer #1 (Public review):

Summary:

Epileptogenesis involves deficits in GABAergic inhibition mediated by chloride-permeable GABA(A) channels, which are caused by elevated intracellular chloride. These changes in chloride homeostasis result from increased activity of the NKCC1 chloride importer and loss of function of the KCC2 chloride extruder. The authors used organotypic hippocampal slice preparations from mice expressing the genetically encoded chloride indicator Clomeleon as a model of posttraumatic epileptogenesis to show that these changes are mediated by the WNK-SPAK/OSR1 pathway. Blockade of the WNK-SPAK/OSR1 pathway with WNK463 alleviated epileptic activity. These findings provide important insights into the mechanisms of epileptogenesis and open new avenues for treating epilepsy.

Strengths:

The conclusions are well supported by the detailed pharmacological analysis.

Comments on revised version.

The authors addressed all the recommendations in their revised manuscript.

Reviewer #1 (Public review):

[Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers.]

Leukemia-driving NUP98 oncofusion proteins form chromatin-associated biomolecular condensates in the nucleus, and these structures are important for oncogenic transformation. Most NUP98 fusions do not contain domains that mediate the recognition of specific DNA elements. Instead, they entail domains that are important for chromatin regulation. For instance, the NUP98::KDM5A fusion features a fusion of the NUP98 N-terminus with the third PHD domain of the histone demethylase KDM5A. As PHD domains are critical for the recognition of methylated histones without any sequence specificity, it is not clear what controls the condensation and chromatin binding of NUP98::KDM5A, leading to the induction of oncogenic transcriptional programs.

In this work, the authors use a combination of cellular and in vitro studies to show that biomolecular condensation of NUP98::KDM5A is dependent on H3K4me3 binding. Their model proposes that concentration-dependent chromatin-associated condensation of NUP98::KDM5A depends on local densities of H3K4me3 on chromatin and the levels of the fusion oncoprotein. In line with this, the analysis of gene expression data from NUP98::KDM5A-positive AML cells shows a positive correlation between differentially expressed genes and H3K4me3 levels.

This is an interesting manuscript that aims to dissect the molecular mechanisms underlying biomolecular condensation of the NUP98::KDM5A oncoprotein. The work is solid, and the results are well explained and presented in a logical order.

Reviewer #1 (Public review):

Summary:

Jacobs et al. report how acute psilocybin influences value-based decision-making and associated neural correlates in the medial prefrontal cortex (mPFC) of rats performing a probabilistic two-choice task with reward probability varying in blocks. The authors highlight that acute psilocybin does not influence basic outcome-sensitive choice strategies (i.e., win-stay/lose-shift), but find that psilocybin increases choice variability and optimal choices in low-uncertainty contexts. Using RL modeling, the authors chose one of five models examined because it represented the behavior around transitions. This recapitulates their behavioral results, showing changes in choice variability (entropy) specifically under high-uncertainty conditions and improving asymptotic performance under low uncertainty. Single-unit recording from mPFC during the task revealed that psilocybin may enhance the representation of reward-preferring neurons and suppress non-reward-preferring neurons, with no changes to outcome encoding and total/relative value signals largely intact. The authors interpret these findings as evidence that psilocybin rebalances mPFC processing to favour learning from positive outcomes, potentially offering a candidate neural coding mechanism for psilocybin's purported ability to increase cognitive flexibility acutely. While the research question is highly significant, the experimental design, analysis, and interpretations have critical shortcomings.

Strengths:

Understanding how psilocybin influences behavioral effects like reward learning and cognitive flexibility is critical to understanding its potential use in the clinic. The behavioral, neural, and modeling studies use a powerful set of approaches that are useful for understanding this complex problem.

Weaknesses:

(1) Design and sample size were major concerns. Only 8 rats were used, and the primary comparison is within-subject psilocybin vs. the immediately preceding "baseline" day rather than a fully counterbalanced or randomized drug/vehicle design. Since psilocybin sessions always followed baseline sessions (never the reverse), order effects, practice effects, or day-to-day drift cannot be fully excluded. Additionally, a saline control was only available for 5 of 8 animals, on a haphazard schedule ("due to unanticipated technical issues"), so the claim that effects are pharmacologically specific to psilocybin rather than to injection/novelty stress rests on a small, incomplete control dataset.

(2) There are a number of major concerns regarding behavioral confounds. Results about the number of trials performed in each condition seem buried in the methods instead of being provided with the rest of the behavioral data. Critically, the results show a 40% reduction in the number of trials completed. This is important to highlight and consider in the context of reduced motivation/hunger, and also in terms of the associated neural activity. Additionally, the percentage of rewarded trials is not reported - this is important for the interpretation of the behavioral results. For example, is the increased choice variability following psilocybin treatment measured by entropy advantageous? Does this result in more or fewer rewarded trials?

(3) Any differences in neural data are greatly diminished by the behavioral differences. Given the extraordinary long latency (averages of up to 20s) to reward receipt, it is unclear how the neural data in Figure 3 referencing reward is meaningful given the time frame is a maximum of 1.5s post-response. How can neurons be labeled non-reward-preferring based on their activity well before they retrieved the reward? Further, it is unclear how the post-response neural activity should be interpreted in the context of this long and variable latency to retrieve reward. The abnormally long latency to retrieve a reward is not discussed, especially in the context of response latencies on the order of 2 to 4s. Furthermore, the difference between groups (7 versus 20 s) should be discussed because this may represent a difference in motivation/hunger/response drive between groups and influence the interpretation of both behavioral and neural data.

Additionally, it is unclear how to interpret the neural data given the offset timing between groups because of the large behavioral differences. For example, given the decrease in latency to respond following the psilocybin treatment, the neural data shown (aligned to the response; e.g., Figure 6A, B) is largely offset from the time of trial initiation. There is no discussion of what is occurring behaviorally during the latency, which may be the cause of the different neural signatures.

(4) For the RL Modeling, there is circularity in the author's approach to choosing a model - namely, based on the "posterior predictive ability to recover psilocybin effects". The author's process for arriving at Model 3 as the specific "preferred" model over the intermediate options (particularly Model 4 with stickiness) is not well-described. The validation step (of recovering the key psilocybin behavioral transition signatures) is circular and not sound evidence for model adequacy rather than independent confirmation. I would want to see the actual PSIS-LOO comparison table (with SEs and Pareto k diagnostics) from Supplemental Figure 2A before fully accepting that Model 3 was clearly the best-justified choice rather than simply the most convenient one given convergence constraints. There was no prior predictive check with the parameters being chosen from the literature. Additionally, the Bayesian parameter effects rely on a relatively lenient 80% HDI threshold (rather than 95%).

(5) Some minor concerns:

a) The order of the Type I and II neurons appears to be reversed in this sentence based on the rest of the results/figures. "These neurons had distinct temporal patterns, showing reward-related increases immediately at action or later in the action/outcome epoch and are depicted as Type I and Type II, respectively".

b) Connecting results from Figures 3 and 4 would be interesting; in other words, showing how Type I/II neurons look in Figure 4 plots.

c) In Figure 1H, are the post-transition trials collapsed across high and low next blocks, since it seems from the depicted trial order that low uncertainty blocks can be followed by either a high uncertainty block or another (reversed) low uncertainty block.

d) The methods by which the authors arrived at the binned value neurons are not well-described, and more clarity on how authors arrived at relative and total value estimates would be helpful.

PatientID: 12

KindredID: 6

Case: M, 12Y0M, Ethnicity Unknown

DiseaseAssertion: Hemangioblastom

FamilyInfo: A family pedigree is provided in figure 1. The patient’s father carried the same mutation as patient 12, but the father’s occurred de novo, as neither of the father’s parents carried the mutation. The patient’s father had cerebellar, retinal and bulbar hemangioblastomas, and retinal detachment. The patient’s 14-year-old brother (patient 11) was asymptomatic.

CasePresentingHPOs: HP:0010797 (hemangioblastoma)

CaseHPOFreeText: Patient had a CNS hemangioblastoma.

CaseNotHPOs: HP:0009711 (retinal capillary hemangioma)

CaseNotHPOFreeText: Patient did not have Retinal capillary hemangiomas.

CasePreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: N/A

Variant: c.341+1_341–1(463*1_?)

LegacyVariant: N/A

CaseProblemVariantFreeText: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: N/A

MutationType: delins

CivicName: Null (c.341+1_341–1)

MultipleGeneVariants: N/A

Reviewer #1 (Public review):

Summary:

The paper presents an elegant task designed to investigate humans' ability to generalize knowledge of learned graph structures to new experiences that share the same structure but are built from different stimuli. Using behavior and MEG recordings, the authors test evidence for neural representation and application of structural knowledge.

Comments on revised version.

The authors have addressed my concerns and have largely toned-down the overstatement of their findings which was pervasive in the previous version of the manuscript. However, the revised title still seems rather misleading given the findings presented. I suggest the authors revise this to: 'A neural mechanism for transfer of abstract structural knowledge in humans'.

Reviewer #2 (Public review):

Summary:

In this manuscript, Malfatti et al. study the role of Chrna2 Martinotti cells (Mα2 cells), a subset of SST interneurons, for motor learning and motor cortex activity. The authors trained mice on a forelimb prehension task while recording neuronal activity of pyramidal cells using calcium imaging with a head-mounted miniscope. While chemogenetically increasing Mα2 cell activity did not affect motor learning, it changed pyramidal cell activity such that activity peaks become sharper and differently timed than in control mice. Moreover, co-active neuronal assemblies become more stable with a smaller spatial distribution. Increasing Mα2 cell activity in previously trained mice did increase performance on the prehension task and led to increased theta and gamma band activity in the motor cortex. On the other hand, genetic ablation of Mα2 cells affected fine motor movements on a pasta handling task while not affecting the prehension task. While overall this study addresses an important and timely question, limitations in the design of the motor learning task and data analysis significantly weaken the conclusions drawn in this manuscript.

Strengths:

The proposed question of how Chrna2-expressing SST interneurons affect motor learning and motor cortex activity is important and timely. The study employs sophisticated approaches to record neuronal activity and manipulate the activity of a specific neuronal population in behaving mice over the course of motor learning. The authors analyze a variety of neuronal activity parameters, comparing different behavior trials, stages of learning, and the effects of Mα2 cell activation. The analysis of neuronal assembly activity and stability over the course of learning by tracking individual neurons throughout the imaging sessions is notable, since technically challenging, and yielded the interesting result that neuronal assemblies are more stable when activating Mα2 cells.

Overall, the study provides compelling evidence that Mα2 cells regulate certain aspects of motor behaviors, likely by shaping circuit activity in the motor cortex.

Weaknesses:

I appreciate the authors' efforts to address the remaining issues raised by the reviewers and agree that it becomes clearer by refocusing the study away from learning towards effects of Ma2 cell activation on motor execution. I also appreciate added clarifications throughout the manuscript.

That said the study still relies heavily on measuring changes throughout "performance refinement", which is just another word for learning. Given their task design, it is hard to assess any learning or refinement over the course of training. This makes it hard to interpret why certain changes in neuronal activity occur at the defined sessions. The main conclusion thus is that there is "a complex interplay between Mα2 activity, oscillatory brain dynamics, and motor function", as the authors state as well in their discussion. I would encourage a further focus or restriction of the data presentation and interpretation on motor execution and limit any plasticity findings on training session 6, where the reaching task properly starts, such as in the study the authors reference (10.1016/j.neuron.2021.07.030).

Relatedly, the authors cannot say that "enhancing Mα2 cell excitability improved the performance of a motor task relying on an already refined skill (Figure 6C)" (lines 1134-1136). Since there is no improvement in success ratio throughout the training prior to CLZ injection (Supp Fig. 7A), there is no evidence for learning or skill refinement in the period where the pellet is presented on the plate.

Regarding the Ca2+ image analysis, the baseline subtraction approach the authors use only appropriately reduces part of the technical variations seen with 1p Ca2+ imaging. It only partially accounts for differences in GCaMP expression or different illumination across cells, or slower task-independent fluctuations in neuronal activity. Given the goal of the analysis is to show activity modulation in the peri-trial interval around prehension onset, some version of trial-wise baseline normalization (which is commonly used) is necessary and would likely result in less confusing results.

Minor:

- Unclear which session traces in figure 1I are from.

- Label in Supp Fig. 7A should say "switch from spoon to plate".

Reviewer #1 (Public review):

In the previous revision the authors addressed some of the points, but they also make some technical errors. My overall view of the manuscript hasn't changed since the original evaluation.

Previously I noted that SC sparsity presents an issue when comparing to full FC matrices. They authors misinterpreted the Honey et al paper. They resampled ALL entries of the SC matrix (including zeros) from a Gaussian distribution. In effect, this assigns zeros small (but uniform) weights. In Honey et al, the authors resampled only existing edge weights from a gaussian distribution (the rationale at the time was that there might be pushback against the extremely heavy-tailed edge weight distribution). In other words, the zeros are still zeros following this resampling procedure.

That said, I agree that the log transform is likely useful or necessary given edge weight distributions.

In short, I still think that the approach is interesting and meritorious, I just don't think the execution is correct.

Comments on revised version.

In this revision the authors addressed my remaining points.

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 authors have substantially strengthened the manuscript in response to my comments. In particular, they have improved the treatment of non-canonical calls, added further illustrative examples at the level of individual RNA variants, repeated pathway enrichment using an appropriate expression-matched background, assessed treatment-associated cell viability, and moderated several biological claims. The revised manuscript is now most compelling as a technical benchmarking and best-practice resource for high-confidence prediction of a common RNA chemical modification from nanopore direct RNA sequencing.

The matched-input comparison of two methods, use of unmodified RNA controls, and inhibitor treatment are important strengths. Together, these experiments provide solid support for the central conclusion that careful filtering and appropriate controls can substantially reduce likely false-positive calls and yield a reproducible set of high-confidence predictions. The added analyses also provide useful illustrations of the capacity of long-read sequencing to assign predictions to individual RNA variants, although they do not establish broad superiority over the short-read comparison method.

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.

Comments on revised version.

I thank the authors for their detailed comments on the reviewers' comments. There however remain some issues that should be properly addressed to make the manuscript stronger.

(1) The authors state that Tie1 and Tek levels are noticeably higher in atrial vs ventricular endocardial cells. Can the authors provide the appropriate statistics to give strength to this claim, since the Supplemental Figure 1F does not indicate robust chamber-specific differences? As normal statistics (t-test, Mann-Whitney, ANOVA) cannot be applied to raw RNAseq data, the authors should collect independent samples and perform qRT-PCR analyses on these.

(2) When Tek is depleted in endothelial cells at E12.5, ventricular defects are also observed at later stages. Presumable, this could mean that earlier depletion of Tek, prior to initiation of ventricular trabeculation, might have resulted in equally severe atrial and ventricular defects. Similarly, they observe that, when Tie1 is deleted early on (tamoxifen administration at E10.5), combined with the heterozygous deletion of Tek, both atrial and ventricular defects are observed. While their postnatal studies do suggest a differential impact of Tie2 on atrial vs ventricular trabeculation (or the maintenance thereof), a more thorough analysis is needed to truly support the authors' claim that Tie1 and Tie2 synergistically impact atrial trabeculation more dramatically than ventricular trabeculation. However, to give weight to this claim, they should perform EC-specific depletion of Tie1 and Tie2 (or at least 3 out of 4 alleles) at different developmental stages, where tamoxifen is administered prior to ventricular or atrial trabeculation.

(3) Earlier reports indicate that a part of Tie1 null mutants (app. 15%) die in utero. Did the authors also observe this phenomenon in their mutant lines, and did these animals have more severe atrial defects?

(4) Although the reviewers did not request detailed mechanistic insights into the molecular mechanisms that drive Tie1-mediated atrial trabeculation, several of the reviewers' requests (e.g. YAP/TAZ staining) were unfortunately not performed. This is a shame, as peer review is instrumental to strengthen manuscripts and increase their validity. It would have therefore been nice if the authors had at least made an attempt to address some of outstanding reviewers' questions.

DOI: 10.64898/2026.10.20.26366003

Resource: RRID:ZFIN_ZDB-ALT-070406-1

Curator: @dhovakimyan1

SciCrunch record: RRID:ZFIN_ZDB-ALT-070406-1

What is this?

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:

There is prior literature showing a robust relationship between sulcal interruptions in the posterior occipital temporal sulcus (pOTS) and reading ability. The goals of this study were to extend these findings to children examined longitudinally as they become better readers, and to examine the underlying white matter properties in individuals with and without pOTS sulcal interruptions. To do this, the authors collected longitudinal structural, diffusion, and behavioral data in 51 children (TP1 age 5.5, TP3 age 8.2 years).

First, the authors found that the gyral gap was consistent across time within the subject. This is expected, as they state in the introduction that sulcal patterns are typically established in utero. Next, they found that children with an interrupted pOTS have higher reading scores (across a variety of measures) at timepoint (TP) 3 than children with continuous pOTS, and this was specific to the pOTS, as no associations emerged for the anterior OTS or MFS; this is again expected from prior literature. They then found that the binary presence of this gap, but not anterior OTS or MFS predicted T3 reading performance. Further, they found that a subsample of the lowest readers at TP1 did not have differences in reading score by gyral gap, but that this difference emerged at TP3. Additionally, the gyral gap at TP1 is similar to variance TOWRE 3 reading skills as some behavioral measures at TP1. Examining underlying white matter in a smaller subset of children, the authors found higher MD in children with an interrupted pOTS vs. those with a continuous pOTS, which was contrary to their hypothesis, and higher local connectivity for interrupted, aligning with their hypothesis, but this difference was no longer present when accounting for TP3 reading scores. The authors conclude that structural properties, in this case, the gyral gap, may guide neural plasticity for reading.

Strengths:

This paper has an interesting set of longitudinal data to examine the perhaps changing relationship between sulcal interruptions in the pOTS with reading scores. I commend the authors on data collection and attention to detail in the anatomical analyses.

Reviewer #1 (Public review):

[Editor's 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:

In this work, T. Wijerathne et al. investigated and reported the agonistic effect of Yoda1 and Yoda2 over PIEZO2 function using patch clamp electrophysiology, Ca2+ imaging, and molecular dynamics. They find that Yoda1 sensitizes PIEZO2 to membrane tension, can induce Ca2+ influx, and decreases its inactivation to a lesser degree than it does to PIEZO1 channels. Additionally, their data shows that Yoda2 sensitizes PIEZO2 channels to membrane indentation to a greater extent, but it has a weaker effect on channel inactivation than Yoda1. Interestingly, they report that a mutation in a conserved arginine between PIEZO channels can be used to abolish PIEZO1-mediated Ca2+ flux in response to Yoda molecules. As a whole, the results presented here should be put into perspective against previous and future works involving systems where both PIEZO1 and PIEZO2 might be expressed. This is especially true for works where Yoda1 has been used as a basis for determining the absence of PIEZO2.

Strengths:

The authors use multiple techniques to investigate how Yoda molecules affect the three most important biophysical aspects of PIEZO channels that, when changed, result in pathophysiological responses: a) sensitivity to mechanical stimuli, b) Ca2+ entry, and c) channel inactivation. Lastly, they find a specific amino acid/region that could be exploited for drug design and/or development.

Reviewer #1 (Public review):

Summary:

The study investigates the Drosophila non-visual light receptor rhodopsin7 with regard to its role in light information processing and resulting consequences for behavioral patterns and circadian clock function. Using behavioral, in situ staining, and receptor activation assays together with different fly mutants, the authors show that rhodopsin7 is an important determinant of activity under and response to darkness, which likely signals via a pathway distinct from other, visual Drosophila rhodopsins. Based on phylogenetic analysis, the authors further discuss a potentially conserved functional role of non-visual photoreceptors like rhodopsin7 and the mammalian melanopsin light information processing and circadian clock modulation.

Strengths:

The manuscript follows a very clear structure with all investigations logically building onto each other. Background information and methodology are provided in appropriate detail so that readers can fully understand why and how experiments were conducted. It is further praiseworthy that the authors provide the details that allow also non-experts in the field to fully understand their approaches. Experimental work was conducted in a highly standardized manner, and also considered potential "side-aspects" like the consequences of temperature cycles and changed photoperiods. The detailed and clear description of the obtained results makes them very convincing, with (almost) all observable patterns being addressed.

By highlighting the evolutionary old phylogenetic position of rhodopsin7 and its conservation across numerous clades, the authors provide strong reasoning for the relevance of their work, also pointing out the similarities to the mammalian melanopsin. The postulated hypothesis regarding protein structure and functioning, as well as the role in light information processing and behavioral and circadian clock modulation are well based on the authors' observations, and speculative aspects are correctly pointed out.

Weaknesses:

Where the manuscript still has potential for improvement is the discussion, which in its current form does seem slightly self-contained and does not fully integrate the findings of previous studies on Drosophila rhodopsin7. As the introduction specifically points out that previous findings have been contradictory, this seems like a missed opportunity. Further details on this are provided in the recommendations below.

Similarly, the manuscript currently lacks a discussion of the possible relevance of rhodopsin7 (and other non-visual light receptors in other organisms) in the context of a species' environment and lifestyle, i.e., what is the relevance/benefit of having rhodopsin7 in the fly's everyday life? While this clearly involves speculation, when done carefully, it can elevate the paper's relevance from a primarily academic to a societal one.

An additional point concerns the title and abstract, which postulate rhodopsin7 roles in contrast vision as well as motion and brightness perception. Contrast remains poorly defined in the text, leaving it ambiguous whether it refers to bright/dark contrasts, e.g., along edges, or the temporal contrast that results from dark pulses (startle response). While the latter seems to apply here, the former is likely more intuitive. Thus, this aspect should be rephrased (also in the title) or properly clarified early on. Regarding motion detection, this is backed up by the optomotor response results, but the findings stand somewhat isolated from the other results, lacking a clear connection aside from general visual processing. Lastly, brightness perception is mentioned in the abstract, but never again, possibly due to inconsistent phrasing throughout the manuscript.

Reviewer #1 (Public review):

Summary:

The authors have addressed many of the concerns. In sum, this study examined how ErbB4 signalling in parvalbumin-positive (PV) interneurons contributes to olfactory bulb (OB) microcircuit function and olfactory behaviour in mice. Using RNAscope, single-cell RT-PCR, conditional Erbb4 deletion, awake in vivo recordings, slice electrophysiology and optogenetics, the authors report that ErbB4 was expressed in PV interneurons of the EPL and IPL, and that its loss impaired odour discrimination and dishabituation, reduced GABAergic transmission onto mitral cells, and elevated ongoing LFP power. The experiments were well-designed, but several central claims were not yet adequately supported by the data as presented.

Strengths:

Pretty comprehensive set of experiments.

Weaknesses:

(1) Figure 1. The investigation time of the AAV-Cre-GFP group varied considerably across isoamyl acetate concentrations. Were there any differences between the AAV-GFP and AAV-Cre-GFP groups at the 0 (mineral oil) condition? Since the threshold was defined by a comparison against mineral oil, a baseline difference would propagate directly into the threshold estimate and could reduce exploratory drive rather than an olfactory deficit.

(2) Previous papers have reported that ErbB4 is expressed in neuroblasts of the SVZ and RMS, and that disruption of NRG1-ErbB4 signalling may decrease GABAergic neuron number and alter distribution (Anton et al., Nat Neurosci 2004; Ghashghaei et al., PNAS 2006). The authors showed in Figure 2L that ErbB4 expression in the OB was already decreased at P7, suggesting reduced neuron number and disrupted neuronal distribution. The authors should therefore quantify the number and laminar distribution of the different neuronal types in the OB. Cresyl violet staining showed that gross lamination was preserved, but could detect changes in cell density or in the relative proportions of interneuron subtypes.

(3) Figure 4C. First of all, there were no indicators marking the sessions, so we had no idea what state the mice were in. Also, why did the odour-evoked MFRs of PV-Erbb4⁺/⁺ and PV-Erbb4⁻/⁻ mice show opposite trends during the 0-2 s window in the first session - were there any differences in the behaviour of the two groups during this session? As the mice were head-fixed on a floating ball, locomotion and sniffing should have been monitored, since both strongly influence M/TC firing.

(4) Figure 4K-L. The authors did not explain why spontaneous and odour-evoked MFR were so much different among the "no response" M/TCs. Did this mean that ErbB4 knockout in PV interneurons disrupts the function? This would imply a tonic rather than a stimulus-locked disinhibition, which is a different claim from the one made in the Discussion. In addition, the authors stated that panel 4L indicates "the distribution of types of odour-evoked responses was significantly altered in PV-Erbb4⁻/⁻ mice compared with PV-Erbb4⁺/⁺ mice, suggesting compromised odour detection" - what exactly does this mean? A shift in the proportions of response categories is also compatible with a changed baseline firing rate altering the classification itself.

(5) Figure 5. The power of all frequency bands in PV-Erbb4⁻/⁻ mice was higher than in PV-Erbb4⁺/⁺ mice, including the gamma band. However, Tan et al. (2022) reported that inhibition of ErbB4 kinase activity decreases LFP power and GABA release, which is inconsistent with the present data. This discrepancy should be addressed explicitly. A uniform elevation across all four bands was atypical for a PV-dependent mechanism and could reflect a global scaling difference; separating periodic from aperiodic components and reporting normalised power would help.

(6) Photocurrent amplitude and mCherry⁺ PV cell density was not compared across genotypes; Figure 9-figure supplement 1B-C showed photocurrents for only one group. Since the authors themselves reported in Figure 7-figure supplement 1 that PV interneurons in knockout mice reduced mEPSC frequency and amplitude and increased AP frequency, the presynaptic population was clearly not in an equivalent state. The reduced oIPSC could therefore result from differences in ChR2 expression level or in the number of labelled cells. Photocurrent curves and mCherry⁺ cell density must be provided for both groups.

Reviewer #1 (Public review):

[Editor's note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The inclusion of a summary schematic in this revision significantly improves the manuscript. We have also modified the last sentence of the original editorial assessment.]

Summary:

The authors demonstrate the stereoselective role of D-serine in 1C metabolism showing that D-serine competes with L-serine and inhibits mitochondrial L-serine transport. They observe expression of 1C metabolites in their metabolomics approach in primary cortical neurons treated with L-serine, D-serine and mixture of both. Their conclusions are based on the reduction in levels of glycine, polyamines and their intermediates and formate. Single cell RNA sequencing of N2a cells showed that cells treated with D-serine enhanced expression of genes associated with mitochondrial functions such as respiratory chain complex assembly and mitochondrial functions with downregulation of genes related to amino acid transport, cellular growth and neuron projection extension. Their work demonstrates that D-serine inhibits tumor cell proliferation and induces apoptosis in neural progenitor cells highlighting the importance of D-serine in neurodevelopment.

Strengths:

D-amino acids do not merely function as ligands at receptors but have underlying roles in signaling and metabolism. These roles are just beginning to be uncovered. The authors elucidate the metabolic role of D-serine in the context of neuronal maturation by its suppression of mitochondrial L-serine availability for SHMT2 and 1C flux. This is the strength of the manuscript. The implications for the metabolic role of D-serine in neurons is a highlight and underlines its roles in neuronal metabolism.

Comments on previous version.

This reviewer is satisfied by the effort made by the authors based on the prior comments raised.

Reviewer #1 (Public review):

Summary:

This study by Tsuji et al. explores a mechanical threat model in Drosophila using air puffs as a stimulus. The authors first establish the paradigm and show that air puffs induce cardiac deceleration along with increased locomotion. They then identify dopamine as a key regulator of this response and go on to map the underlying circuit. In doing so, they pinpoint two pairs of DA-WED neurons as critical players. They carefully used intersectional strategies to achieve relatively clean labeling of these neurons, which helps ensure that the observed effects can be attributed specifically to DA-WED neurons. They further show that DA-WED neurons are both required and sufficient to drive cardiac deceleration, and that their activity increases in response to air puff stimulation. These neurons also contribute to the locomotor response. Directly inducing cardiac deceleration via optogenetic manipulation of cardiomyocytes also increases locomotion, suggesting a link between cardiac state and behavioral output.

Strengths:

Overall, the experiments are thoughtfully designed, well controlled, and clearly presented. The figures are easy to follow, and the conclusions are generally well supported by the data. The manuscript is also clearly written, with a discussion that acknowledges potential caveats and outlines future directions. The genetic tools, behavioral paradigm, heart rate measurement approaches, and stimulation methods introduced here will be valuable resources for the community. few minor comments:

Comments on revised version:

I thank the authors for responding to my questions, they have thoroughly addressed my comments. I am therefore supportive of publication of the manuscript.

Reviewer #1 (Public Review):

Summary:

This study by Z. Moradi, S. Jahedazad, and colleagues presents a refined two-choice assay paradigm for investigating thermotactic behavior in groups of Drosophila melanogaster. The authors developed an experimental setup that maintains a stable temperature profile and utilized supervised classifiers based on extracted locomotion parameters to distinguish between temperature conditions within the arena as well as antennal ablation status. The integration of a Braitenberg vehicle model to simulate fruit fly exploration patterns and U-turn behaviors at thermal boundaries represents a valuable contribution to the study of collective thermotaxis.

Strengths:

(1) The detailed description of the behavioral assay and the rigorous calibration methods make the experimental setup reproducible.

(2) The incorporation of a state-of-the-art animal tracking system enables the precise extraction of multiple locomotion parameters, providing valuable insights into the fruit fly exploration as well as for predicting temperature-dependent behavioral responses. The methodology could be potentially adopted in other studies.

(3) The authors' decision to study thermotactic behavior in groups is a noteworthy addition. The finding that overall staccato movement patterns remain largely unaffected by antennal ablation, while the frequency and duration of pairwise encounters increase with temperature, offers valuable insights into individual sensory processing vs the influence of collective behavior.

Weaknesses:

(1) The study investigates groups of fruit flies, which is an interesting contrast to previous single-fly studies. While group dynamics are mentioned as potentially important, no direct comparison or analysis was conducted between individual vs group, which would have been a straightforward and valuable addition. The strength of intergroup interactions remains unclear, as the pairwise encounter parameters appeared to have low significance according to the importance factor analysis.

(2) The manuscript does not clearly articulate the novelty of the work; the innovative aspects are present but not effectively highlighted.

(3) The descriptions of the locomotor parameters and Braitenberg modeling, which constitute the most critical components of the study, are not optimally presented in the main text. The authors should provide a more detailed rationale for why certain locomotion parameters emerged as more influential (i.e., with higher importance scores) than others. Additionally, clearer explanations from a physical or mechanistic perspective would help readers better appreciate and interpret the significance of these findings.

Reviewer #1 (Public review):

The authors show that during prophase I of male meiosis, nucleoli disassemble and nucleolar components relocalize to the sex chromosome (XY) body. They further demonstrate that this process is regulated by the ATR-dependent signaling pathway that mediates meiotic sex chromosome inactivation (MSCI). Pharmacological disruption of pre-rRNA synthesis using the RNA polymerase I inhibitor BMH-21 leads to the recruitment of RNA polymerase II to the sex chromosomes and ectopic expression of sex chromosome-linked genes. These findings uncover a previously unrecognized role for pre-rRNAs in maintaining transcriptional silencing during meiosis. The study employs a combination of cell biology, genetics, and genomics approaches, and the conclusions are supported by compelling, well-organized data.

Comments on revised version.

I have read the revision and I have no additional questions.

Reviewer #1 (Public review):

[Editor's 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.]

Ma et al. use human-chimpanzee tetraploid cells, across different cell types, to identify the genetic causes and then transcriptomic consequences of divergence in DNA methylation. They conclude that the evolution of DNA methylation is driven primarily by cis-regulatory changes, and that the evolution of CpG sites contributes to cis-regulation while transcription factor expression underlies some trans changes. They then argue that divergence in DNA methylation is associated with changes in gene expression and may contribute to human phenotypes.

The tetraploid model is able to provide compelling evidence that most regulatory evolution occurs due to cis-regulatory changes, and that sites that have diverged in DNA methylation level cluster together into DMRs which share similar divergence patterns and genetic architecture. This stands in intriguing contrast to trans-mechanisms which explain most variation within modern human populations. The authors proceed to show that trans-effects can be explained, in part, by nearby TF binding motifs and many cis-regulatory changes are explained by CpG-disrupting variants. While these mechanisms clearly contribution to divergence in DNA methylation, the degree to which they explain cis- and trans-regulatory divergence would be valuable.

Next, the authors seek to show that differences in DNA methylation are functionally relevant. Consistent with previous results, they show that differences in DNA methylation are (weakly) associated with changes in gene expression. They hypothesize that genes with concordant regulatory should exhibit great methylation-expression coupling than other genes and show that cis-expression/cis-methylation pairs are more strongly correlated than trans/trans pairs. I think that looking at cis/trans or trans/cis changes would also be useful. Another limitation is that this analysis is limited to promoter regions. It is not clear how many divergent DMRs that includes and how many of those genes have differences in expression. The key question is whether differences in DNA methylation are functionally important, and the answer provided by these analyses is "sometimes".

Finally, the authors make a case for lineage-specific selection on DNA methylation that is connected to human traits. Using a sign-test, they identify sets of genes which have likely experienced selection in humans and may underlie human phenotypes.

In conclusion, I think this study provides a valuable resource for differences in DNA methylation between humans and chimpanzees across tissues and provides important insight into the relative abundance of cis and trans regulatory divergence. Additional research is necessary to investigate the underlying regulatory mechanisms and more care needs to be taken in exploring the functional consequences.

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 study describes the migration of epidermal keratinocytes through porous membranes and observed a unique size selection whereby only on 3-micron membrane are keratinocytes able to migrate and reform an intact epidermis. The authors propose that the model replicates three cell states of intact epidermis, EMT, and MET. They also show that this response depends on the actin cytoskeleton, keratin 6, and Piezo1, and the migration could be stimulated with TGFbeta ligands.

Strengths:

Strengths of the study include the establishment of a simple yet robust in vitro model that capture all three cell states, which could be useful for future investigation of wound healing or metastasis across a range of epithelia. There is also good quantitative characterisation of the pore size effects, providing some interesting observations about the physical regulation of keratinocyte migration, and the study provides some initial insights into the regulatory mechanisms. The images and presentation are clear.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors describe the use of BindCraft computational protein design to create a series of binders to the chemokine CCL25. This chemokine normally mediates CCR9-dependent trafficking of immune cells to the gut, making it a potential target for the treatment of inflammatory bowel disease and related conditions. Importantly, CCL25 also binds a scavenging receptor, ACKR4. The computational protein design approach used does not involve defining particular epitopes to be targeted, allowing a free search for any potential interaction surface.

Among four designs tested, three were predicted to interact at a similar site on the chemokine, while a fourth clone, VUP25111, was predicted to bind to a different site. All four designs showed binding to CCL25, with similar high-nM KD values in all cases. The first three clones showed evidence of direct competition with the receptor for CCL25 binding, while VUP25111 showed incomplete inhibition of binding. In functional assays, all clones acted as antagonists except for VUP25111, which inhibited arrestin recruitment by CCR9, but did not affect G protein activation by CCR9 or arrestin recruitment by ACKR4 (which signals exclusively through arrestin and not G protein).

Strengths:

The work is completed to a high technical standard, and the functional diversity of the clones is intriguing. It is exciting to see pathway-selective modulation of signaling, and this basic paradigm is likely to generalize to other chemokine/receptor systems. The exceptional complexity of chemokine signaling makes this an excellent area to explore the development of modulators that can restrict signaling to a specific subset of receptors.

Weaknesses:

No major weaknesses were noted by this reviewer.

Comments on revised version:

The revision looks fine to me.

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.]

In this study, Hossain et al. investigated the role of Interleukin-2-inducible T cell kinase (ITK) in autoimmune lung injury, demonstrating that ITK-deficient (Itk-/-) mice are protected against pristane-induced pulmonary hemorrhage (PH). Authors suggest that this protection correlates with a significant remodeling of the T cell compartment in Itk-/- mice, including increased frequency of memory-like CD4+ and CD8+ T cells (CD44⁺CD62L⁺) as well as higher frequency of Treg populations. Furthermore, adoptive transfer of ITK-deficient Treg isolated from injured ITK-deficient mice confers protection against pulmonary hemorrhage in WT recipients.

Strengths:

The adoptive transfer of congenically marked wildtype and Itk-/- Treg populations clearly demonstrates that ITK-deficient Treg can actively rescue pre-existing lung injury and reverse systemic secondary metrics like proteinuria in wild-type recipients, providing proof-of-concept validation for the therapeutic utility of the ITK-Treg axis.

Reviewer #1 (Public review):

Summary:

The manuscript presents a study combining human functional MRI (fMRI), electroencephalography (EEG), and human judgement data within a two-step regression framework to determine when several regions of interest (ROIs) along the so-called third visual pathway for social perception are activated, and which aspects of visual representations of social interactions they encode. The goal is to determine whether social interactions are encoded through a hierarchy of representation along the third pathway, and whether this hierarchy is implemented in a feedforward manner.

The first analysis linking EEG to fMRI data identifies early activation (within 60 ms) in early visual cortex (EVC) and later (onset between 112 and 310 ms) activations in selected, more anterior ROIs along the pathway, i.e., motion-selective MT, extrastriate body area (EBA), object-selective lateral occipital cortex (LOC), and posterior and anterior social-interaction superior temporal sulcus regions (pSTS and aSTS). The second analysis linking EEG-fMRI data to stimulus features derived either computationally from the images or from human judgments shows that EVC activity arises early (within 68 ms) and is predicted by low-level visual features and motion energy. Activity in all other ROIs emerges after 140 ms (or later) and is predicted by motion energy (in MT, EBA and LOC), arousal (in MT), spatial expanse (in LOC, pSTS and aSTS), agent distance (in LOC, pSTS and aSTS) and communication (in aSTS). No effects of facingness, object-directedness, joint action, or valence are found in any ROI.

Two observations are highlighted: features important for recognizing social interactions (i.e., agent distance) are encoded with a similar latency in the mid-level LOC and in STS; the communication content is selectively encoded in anterior STS.

It is concluded that social interactions are processed in a hierarchical way, with more abstract information (i.e., communication) encoded in more anterior regions. Moreover, because this higher-level information is encoded at similar or even shorter latencies than mid-level features in mid- and high-level ROIs, these findings are taken to challenge a strictly feedforward hierarchy within the third visual pathway.

Strengths:

This is a timely manuscript concerning a newly discovered and now extensively investigated brain network, purportedly dedicated to social perception. In the manuscript, the selectivity of this network for the perception of social interactions is assumed rather than demonstrated. Instead, the study addresses two related questions that are critical for understanding how the network (or pathway) is organized and functions: (i) whether representations are hierarchically organized, progressing from more specific information (lower-level visual features) to increasingly abstract information (interaction category, e.g., communication), and (ii) whether processing follows a feedforward mechanism with prior and rapid encoding of lower-level features of social interactions, followed by encoding of mid-level and higher-level features.

Addressing these questions is important because it goes beyond the knowledge of where social information is represented, toward understanding what kinds of visual input and transformations support the recognition of social interactions, and how the processing stages are organized (e.g., in a feedforward manner or with recurrent interactions). Establishing both aspects would define the particular computational architecture supporting the human ability to understand visual social interactions.

The manuscript is also methodologically valuable, as it introduces a variation of more conventional fMRI-EEG fusion analyses. This method establishes fMRI and EEG correspondences, thus revealing spatiotemporal dynamics of brain activity (i.e., where activity occurs in the brain, at which time point), and further links these dynamics to stimulus features (i.e., which aspects of the stimuli predict activity in a given brain area at a given time). The description and discussion of the method in the manuscript are clear and transparent. This facilitates replication and allows appreciation of the rationale of the approach and its potential for future research.

Weaknesses:

The method introduced in the manuscript appears to be a valuable addition to the range of non-invasive approaches to study the spatiotemporal dynamics of representations in the human brain. The results presented in the manuscript, however, do not make a strong case for the sensitivity of the method.

The study fails to replicate several effects that have demonstrated reliability across previous studies. In particular, motion energy in the stimuli only poorly predicts - relatively late -activity in motion-sensitive MT. Moreover, unlike several previous studies, including work by McMahon, Isik, and colleagues, the study finds no effects of facingness (or any other mid-level features) in EBA, object-directedness in LOC, joint actions in pSTS, and valence in pSTS. The manuscript does not discuss discrepancies, raising concerns about the sensitivity and validity of the method. In particular, the absence of previously reported effects is difficult to interpret without considering whether the method is sufficiently sensitive to detect the relevant stimulus dimensions.

Another related and unexplored issue concerns the potentially limited correspondence between EEG and fMRI signals, and between stimuli used to derive the stimulus features for the analyses and stimuli used in the actual EEG experiment. EEG and fMRI data were collected in different samples, and different individuals may assign different social value to the same events, potentially affecting neural processing in regions such as the pSTS (see e.g., work by Varrier, Finn et al.). Furthermore, the stimuli used in the EEG experiment were derived from, but differed from those used in the fMRI experiment. Although this is not entirely clear from the manuscript, it appears that stimulus features were measured for the original stimuli used in the fMRI experiment and assumed to be valid also for stimuli in the EEG experiment. The manuscript does not acknowledge these issues, or other potential sources of noise arising from them, either on their own right or in relation to the above null effects. This makes it difficult to determine whether the absence of several expected effects reflects genuine characteristics of neural representations or limitations of the measurement and analysis pipeline.

Beyond the methodological concerns, the results themselves are less straightforward than the manuscript suggests. Evidence for hierarchical representation emerges from the effect of low-level visual features in EVC and the encoding of communication in aSTS. Activity in all ROIs 'in the middle', from MT, EBA and LOC to pSTS, is predicted by a mixture of low- and mid-level stimulus features, whose effects in some cases come online after the encoding of the interaction category in aSTS (at 195 ms). This raises important questions concerning how a higher-level representation of an interaction category can emerge before the encoding of the lower- or mid-level information that is presumably diagnostic of that category. The manuscript would benefit from a more thorough discussion of this issue, particularly because the temporal ordering of these effects is central to the authors' claims about hierarchical and feedforward processing.

Reviewer #1 (Public review):

In their manuscript Arjun et al. investigate the role of the histone acetyl transferase Gcn5 in controlling Drosophila blood cell homeostasis in the larval lymph gland. Using gcn5 zygotic mutants as well as targeted knock-down and over-expression of Gcn5 in various lymph gland cell populations, they show that these manipulations impact niche cell number, blood cell progenitor maintenance, plasmatocyte differentiation, crystal cell differentiation, DNA damage accumulation. Their results indicate that Gcn5 controls autophagy and show that reducing the expression of the autophagy machinery affect blood cell differentiation. By using drugs as well as genetic approaches to modulate the mTOR pathway, they found that Gcn5 levels are regulated by mTOR, but that the impact of this pathway on blood cell homeostasis can override Gcn5 function.

Overall, the main conclusions are sound. They bring interesting insights into the regulation of autophagy by Gcn5 and mTOR and lay the ground for further dissections of Gcn5 function and mode of action during Drosophila lymph gland development.

Comments on revised version.

The authors have modified the title, abstract and discussion to tune down some of their conclusion and provide a more nuanced interpretation of their data. They have also modified some figure panels as requested.

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.]

In this paper, Pal and colleagues propose a mechanistic unification of two influential accounts of inter-areal communication: communication through coherence and communication subspaces. A major strength of the paper is that it does not treat coherence and communication subspaces as independent phenomena, as typically done, but instead derives both from the same circuit with divisive normalization. In this framework, noise-driven fluctuations around the normalized fixed point determine covariance and cross-power structure (which, in retrospect, makes so much sense to be related). Then, they show how these determine linear prediction performance and the effective dimensionality of the communication subspace. They also show (however not very visually, see recommendation below for a figure) how divisive normalization is crucial to shape inter-areal coherence and the dimensionality of communication.

I found this conceptual contribution potentially very influential, but somewhat obscured by the technical complexity of the model. The central intuition (I think) is that recurrent normalization can organize cross-area fluctuations, both frequency-specific correlations and cross-covariances. Took me a while to grasp this insight, mostly because I was stuck with the model details. Note that I have some experience with network dynamics, but not with this particular model.

Reviewer #1 (Public review):

This study was designed to manipulate and analyze the effects of chemosensory cues on visuomotor control. They approach this by analyzing how eye-body coordination and brain wide activity are altered with specific chemosensation in larval zebrafish. After analyzing the dynamics of coupled saccade-tail coordination sequences - directionally linked and typically coupled to body turns - the authors investigated the effects of sensory cues shown to be either aversive or appetitive on freely swimming zebrafish on the eye-body coordination. Aversive chemicals lead to an increase of saccade-tail sequences in both number and dynamics, seemingly facilitating behaviors like escape. Brain-wide imaging led the authors to neurons in the telencephalic pallium as a target to study eye-body coordination. Pallium neuron activity correlated with both aversive chemicals and coupled saccade-tail movements.

Comments on revised version.

The revised manuscript addresses my previous concerns.

Reviewer #2 (Public review):

Summary:

Mione et al. aim to resolve a long-standing question in comparative neuroscience: whether the macaque brain contains a functional analogue to the distributed human multiple-demand (MD) network. To address this, they employ a direct cross-species fMRI comparison using a multi-step saccadic maze task in humans and a simplified two-step version in macaques. By contrasting goal-directed navigation against a control condition that requires similar motor responses but no strategic planning, the study isolates the neural signatures of cognitive control across species.

Strengths:

The most compelling aspect of this work is its methodological alignment. Previous attempts to compare these systems often relied on comparisons of human BOLD signals and macaque single-unit recordings. By running parallel fMRI protocols, the authors establish a shared measurement basis that allows for a more direct comparison. The resulting activation maps demonstrate conserved network topology in dorsolateral and dorsomedial frontal cortex and insula. Individual-level data are presented alongside a conjunction map across the two animals, with the threshold used to define common activations stated explicitly, and supplementary analyses restricted to correctly completed trials or using a smaller smoothing kernel leave the main features intact. This strengthens confidence in the frontal correspondences that appear robust.

Weaknesses:

A design asymmetry between the human and monkey tasks remains. In humans, 2-, 4- and 6-step problems were intermixed within blocks, whereas macaques solved only two-step trials. Because participants never know when a harder problem will occur, they may stay in a state of proactive cognitive control (Braver, 2012) throughout the block, which could inflate MD activation even during simpler trials and complicate direct comparison with the monkey data. The authors acknowledge this limitation but do not quantify or correct for the effect.

Individual-level consistency for some claimed MD regions is limited. The two monkeys differ considerably in parietal cortex, and a conjunction analysis shows no common activation there even at a permissive threshold of t > 1.5. Strong dorsal/medial parietal and ventrolateral prefrontal activations were each seen in only one animal, and the abstract presents them accordingly. This is an honest presentation, but it leaves the evidence for these regions resting on one subject, so whether they can be regarded as part of a macaque MD network remains uncertain.

In the Discussion, the authors conclude that results from single animals "tell against" genuine species differences in these regions. The implied reasoning appears to be that if macaques truly lacked parietal and ventrolateral prefrontal nodes, neither animal would have shown activation there. Since one monkey did show it, they take this as evidence that the regions are not genuinely different between species, attributing the other animal's absence to limited power or insufficient task demands. This inference is difficult to justify with only two animals and no common activation across them at any meaningful threshold. A single positive result does not establish that a region reliably belongs to the macaque MD network.

Overall, this manuscript provides solid evidence for functional correspondence between human and macaque cognitive control networks in dorsomedial frontal cortex and insula. Individual-level data are presented transparently, with conjunction thresholds stated explicitly and limitations acknowledged throughout. What remains is largely interpretive. Given only two animals and no cross-animal convergence in parietal or ventrolateral prefrontal regions, claims that these form part of a macaque MD network should be read as provisional.

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:

Naina Gour and colleagues provide a detailed observational study in which they demonstrate that MRGPRX4, a human G-protein coupled receptor (GPCR), is expressed exclusively in human melanomas and, when expressed in mouse melanocytes, drives the development of melanomas in mice. These findings provide evidence that MRGPRX4 has the properties of an oncogene, at least in certain cellular environments.

Strengths:

A strength of this work is the nice historical note in which overexpression of MAS1, a GPCR, led to classic studies of transformed fibroblasts in culture and tumors in nude mice. Cloning of MAS1 led to the identification of the MRGPR family of receptors, now known to be key players in neuroimmune and neurosensory phenomena. Here, the story comes full circle with a member of the MRGPR family being linked to a tumor, specifically melanoma. Perhaps the story is not entirely surprising given that the neural crest serves as a precursor for both nerves and melanocytes. But it is nice to see.

Additional strengths include the vast array of tools and techniques employed, from public databases to engineered mice, to establish firmly that MRGPRX4 is expressed in melanomas, although not in every malignant cell.

Reviewer #1 (Public review):

Summary:

This manuscript describes a chemical screen for activators of the eIF2 kinase GCN2 (EIF2AK4) in the integrated stress response (ISR). Recently, reported inhibitors of GCN2 and other protein kinases have been shown at certain concentrations to paradoxically activate GCN2. The study uses CHO cells and ISR reporter screens to identify a number of GCN2 activator compounds, including a potent "compound 20." These activators have implications for the development of new therapies for ISR-related diseases. For example, although not directly pursued in this study, these GCN2 activators could be helpful for the treatment of PVOD, which is reported for patients with certain GCN2 loss-of-function mutations. The identified activators are also suggested to engage with the GCN2 directly and can function devoid of GCN1, a co-activator of GCN2.

Strengths:

The manuscript appears to be a largely rigorous study that flows in a logical manner. The topic is interesting and significant.

Weaknesses:

Portions of the manuscript are not fully clear. There are some experimental presentation and design concerns that should be addressed to support the stated conclusions.

Reviewer #1 (Public review):

The manuscript by Kang et al. examines Gasdermin family member localization in keratinocytes and further reports that the PP2A inhibitor caliculyn A induces a pyroptosis-like morphology, presumably due to cytoskeletal disruption. Gasdermin A and B then translocate from the cytoskeleton to the membrane, where they enable keratin secretion without cleavage. The manuscript is well-controlled, and the data are convincing. The findings could be triangulated more effectively with additional microscopic studies to complement the fractionation experiments.

My specific comments are as follows:

(1) Nitrogen cavitation experiments and fluorescence microscopy have shown that the N-terminus of Gasdermin A first localizes to the mitochondrial membrane and then to the plasma membrane (Kondolf et al., JBC, 2024). The same was shown for GSDMD by Judy Lieberman's group in a 2024 Immunity paper. It is surprising that the fractionation experiments didn't include mitochondrial localization.

(2) It appears that the blots in Figure 2 showing purity of fractionation (Lamin, LDHA, KRT19) are the same as those in Figure 1. I am assuming the experiment was performed at the same time. I think this should be made crystal clear, and it also raises the question of why not just include GSDMC in Figure 1, since they already have the other Gasdermins in that figure.

(3) There's negative data in this manuscript, but I consider it important. Gasdermin antibodies are problematic, and caspase cleavage of GSDMC has been inconsistent. This negative data is important to share.

(4) In Figure 1, why do GSDMD and GSDME show cleavage products without stimulation (minor point for curiosity)?

(5) The fractionation experiments would be more convincing if they were complemented by microscopic localization. Antibodies might be an issue, but others have used fluorescent tags successfully (need to tag the Gasdermins internally).

(6) PP2A is a broad serine-threonine phosphatase with hundreds of substrates. It's hard to rule in a cytoskeletal role to the exclusion of all the other changes caliculyn will cause - especially after 3 hours of exposure. A paragraph discussing this could be added to the Discussion. It's surprising that the authors don't disrupt the cytoskeleton in other ways.

(7) I'm confused by the data in Figure 5C. Knocking down GSDMA and GSDMB causes no change in keratin release (and may even increase it). If the point of the manuscript is that these cause protein secretion, shouldn't there be a change? Do they affect secretion?

(8) Along these lines, which GSDMB isoforms are present in the cell? It's well established that exon structure affects function.

(9) Minor point for curiosity: N/TERT cells can be differentiated and can form organoids. Have the authors attempted to determine if differentiation affects their findings? This would be especially interesting given the different localizations of GSDMA and GSDMB in the layers of the skin.

Overall, this manuscript provides valuable data for the field. Although it includes negative results, I believe sharing them is crucial. The experiments are well-executed and mostly convincing, though some aspects, particularly Figure 5C, appear contradictory. The manuscript would benefit from this reputable lab addressing the earlier points, especially the microscopic testing of the hypothesis.

Reviewer #1 (Public review):

Summary:

Badarnee and colleagues analyse fMRI data collected during an associative threat-learning task. They find evidence for parallel processes mediated by the mediodorsal, LGn and pulvinar nuclei of the thalamus. The evidence for these conclusions is promising, but limited by a lack of clarity regarding the preprocessing and statistical methods.

Strengths:

The approach is inventive and novel, providing information about thalamocortical interactions that are scant in the current literature.

Comments on revised version.

No further comments. The authors have adequately addressed my concerns.

Reviewer #1 (Public review):

Summary:

The study focuses on the characterization of drug‑tolerant persisters (DTPs), a subset of tumor cells that survive therapeutic elimination, in an experimental melanoma model (SKMEL5 cell line) under BRAF‑targeted therapy. Using single‑cell RNA sequencing combined with barcode‑based lineage tracing, the authors interrogate DTP heterogeneity and lineage origins. The major conceptual novelty claimed is a shift from an asserted "consensus view" in which DTPs are treated as distinct, uniform cell states, toward the inference that DTPs comprise a heterogeneous population of sub‑states within a single treatment‑defined regime. They also demonstrate, in this model, a lack of strong lineage bias in the emergence of DTPs. Finally, the authors refine and corroborate prior observations of shifts in calcium/ion‑channel regulation and increased ferroptosis sensitivity in DTPs.

Strengths:

The work offers a conceptually interesting reframing of DTPs, emphasizing population‑level heterogeneity and the existence of sub‑states. Even if the "consensus view" it challenges is somewhat overstated, this perspective is likely to stimulate further discussion and more nuanced DTP conceptualization.

The combination of single‑cell transcriptomics with barcode‑based lineage tracing provides strong evidence, in the SKMEL5/PLX4720 model, that DTP origin is not limited to rare pre‑existing subpopulations. The observation that most lineages contribute to the idling DTP population, with relatively modest changes in abundance, provides a useful contrast to observations in other experimental systems.

Integrated bulk RNA‑seq and bulk ATAC‑seq analyses showing concordant signatures across transcriptional and chromatin accessibility data provide useful systems‑level insights into how prolonged BRAF inhibition enriches ion‑transport and calcium‑handling pathways in idling DTP populations.

Direct calcium‑flux experiments demonstrate reduced store‑operated calcium entry in idling DTPs despite similar ER Ca2+ content in untreated and treated cells. This functional assay provides a convincing, mechanistically interpretable readout that supports the proposed ion‑channel remodeling in the idling state.

The use of a well‑characterized, widely used BRAFV600E melanoma line (SKMEL5) facilitates cross‑comparison with prior work on BRAF inhibitor responses in melanoma.

Weaknesses:

The central "state vs sub‑state" conceptual claim has two major issues. First, the "consensus view" that the authors assert (that DTPs are distinct, uniform cell states) is a straw man. The discreteness of phenotypic states depends on the resolution and interpretive framework (discrete categories versus continuum). I do not think there is a clear consensus on the discreteness of the DTP cell state. Much like the "clone vs subclone" dichotomy, "state vs substate" is typically used operationally rather than in reference to strictly defined biological entities. Therefore, the distinction the authors draw between "states" and "sub‑states" is a subtle reframing of the resolution hierarchy rather than a genuinely new challenge to a deeply held field‑wide belief. The shaky grounds of this distinction are reflected by the internal inconsistencies in the authors' own verbiage: in the introduction, the authors state the inference "idling DTP....are still composed of multiple phenotypic states," which directly contradicts the substate distinction. Second, the experimental evidence is insufficient for a meaningful distinction between states and substates. The distinction primarily relies on low‑dimensional PCA/UMAP representations in Figure 1, where therapy-naive cells form 2 clearly distinct clusters, while BRAFi collapses cells into a single cluster with 2 distinct subclusters. It is unclear if the structure will hold if additional dimensions are considered. Further, as the authors themselves note, the data are essentially snapshots rather than trajectories. Without explicit analysis of higher‑dimensional structure or time‑resolved state transitions, it is unclear that a meaningful distinction can be made.

The findings are derived entirely from a single experimental model: one BRAFV600E melanoma cell line (SKMEL5) treated with a single research‑grade BRAF inhibitor (PLX4720). However, authors frequently use phrasing that implies broader generalizability to DTPs across cancers and therapies. This can be misleading, especially given convincing experimental evidence for distinct alternative scenarios in other systems. For example, DTPs emerging predominantly from specific rare subpopulations have been well documented in multiple experimental models, including melanoma models and BRAF inhibitors. The authors should more carefully delimit the scope of their conclusions to this particular melanoma/BRAFi model system context and more explicitly acknowledge where other systems have been shown to behave differently.

Interpretation of the lack of an apparent bias in lineage tracing studies strongly depends on the magnitude of the selection bottleneck before the cells enter the idling DTP state. If a substantial fraction of cells do not make it to the DTP, then the finding poses a challenge to generalizations from other experimental systems. If most cells survive, then the lack of selection at the barcode level is trivial.

The discussion of DTP heterogeneity and rare cell states would benefit from a more precise and balanced engagement with the existing literature. Some cited papers (including several reviews) are only loosely connected to the specific claims, while several highly relevant primary single‑cell studies on rare‑state variability and drug‑induced reprogramming (for example, work from the Shafer and Raj groups) is not explicitly discussed. This weakens the positioning of the proposed conceptual framework within the broader DTP/systems biology fields.

Impact:

In my opinion, the evidence presented in this work, in its current form, is insufficient to support the strong conceptual claims about the cell‑state architecture of DTPs. Nonetheless, the study makes a meaningful contribution by bringing a population‑level, dynamic perspective to understanding DTP biology. Even if one disagrees with the authors' interpretation, the work is valuable in pushing the field beyond the entrenched "gene A is a critical regulator of hallmark B in cancer C" mold and encourages more explicit consideration of phenotypic heterogeneity, trajectories, and treatment‑defined cell‑state landscapes when thinking about overcoming persistence and resistance.

Reviewer #1 (Public review):

Summary:

Cai et al. investigated the role of ripples in the hippocampus and coupled between hippocampus and neocortex in visual short-term memory (VSTM) using a similar lures match-to-sample task. The main findings are that hippocampal, but not neocortical ripples, ramp up during the maintenance period, peaking shortly before the memory response is given. This ramping up effect was stronger for correct compared to incorrect trials. Furthermore, the authors show that stimulus category could be better decoded during coupled hippocampo-neocortical ripples compared to uncoupled ripples. These results provide compelling novel evidence for a role of ripples in supporting human visual short-term memory.

Strengths:

(1) State-of-the-art intracranial EEG in 13 patients during a well-designed visual short-term memory task, with simultaneous hippocampal and neocortical recordings.

(2) Thorough analysis pipeline with validation to detect ripple events and distinguish them from spurious ripple activity (i.e. as induced by IEDs).

(3) Use of multi-variate classifiers to resolve neural representation of the stimuli.

Comments on the latest version:

I am fully satisfied with their responses and clarifications and I have no further comments.

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:

A fundamental technique for the identification of peptide-specific CD8 T cells is the use of fluorophore-conjugated and peptide loaded MHC tetramers. Classically, refolding of specific peptides with MHC monomers can be labour intensive, and not optimal for screening large numbers of different peptides. Hence, UV-exchanged tetramers have been developed to upscale this, however, still has some associated challenges such as UV-mediated damage to peptide complexes. Here, Pothast, C.R. et al demonstrate the efficacy of using temperature exchanged tetramers for the prevalent alleles HLA-A*03:01, A*11:01, B*07:02, and C*07:02. Building upon their previous work with HLA-A*02:01, H-2Kb, and HLA-E. They first demonstrate the complex stability of tetramers with different affinity peptides at high temperature, showing complex destabilisation can be rescued with higher affinity peptides. This is followed by an optimisation of peptide exchange temperatures, tailored for each allele. The authors then demonstrate successful binding to clonal T cell lines, and then a step further with viral peptides against PBMCs from individuals with confirmed infection history. For the latter they compare to conventional tetramers and demonstrate comparable signal.

Due to the prevalence of these 4 alleles, the ease-of-handling, and short time requirements, these tetramers are likely to show high utility.

Strengths:

The manuscript is well-written and results are solid, although more detail may add clarity to some of the results, in particular figure 1 and 2. Other than the points reported below, the study uses accurate controls to demonstrate the specificity of the tetramers, and the data is convincing.

Overall, their interpretation of the results is accurate and the discussion thorough, additional comments may be included to cover potential tetramer batch variability, and differences in the stability of different alleles. Specifically, whether certain alleles require higher affinity peptides to be stable, compared to others.

Comments on revised version.

The authors addressed the weaknesses highlighted in the previous version, specifically addressing that they do not conclude that thermal peptide exchange is not superior to existing technologies, but may be more feasible, as well as including a section on the stability of the different alleles and template peptides. The authors also updated the labelling and description of figures, making them much more interpretable. Addition of the LILRB1 reporter cell line to demonstrate successful peptide exchange and stability adds extra validity to the results.

Reviewer #1 (Public review):

The authors have substantially strengthened the manuscript in response to my comments. In particular, they have improved the treatment of non-canonical calls, added further illustrative examples at the level of individual RNA variants, repeated pathway enrichment using an appropriate expression-matched background, assessed treatment-associated cell viability, and moderated several biological claims. The revised manuscript is now most compelling as a technical benchmarking and best-practice resource for high-confidence prediction of a common RNA chemical modification from nanopore direct RNA sequencing.

The matched-input comparison of two methods, use of unmodified RNA controls, and inhibitor treatment are important strengths. Together, these experiments provide solid support for the central conclusion that careful filtering and appropriate controls can substantially reduce likely false-positive calls and yield a reproducible set of high-confidence predictions. The added analyses also provide useful illustrations of the capacity of long-read sequencing to assign predictions to individual RNA variants, although they do not establish broad superiority over the short-read comparison method.

A few claims remain insufficiently supported. First, the discussion of non-DRACH sites should cite Cancer mutations rewire the RNA methylation specificity of METTL3-METTL14 (Science Advances, DOI: 10.1126/sciadv.ads4750). In particular, Figure 3C of that study shows that the normal METTL3-METTL14 enzyme retains measurable activity on non-DRACH substrates; this provides a useful precedent for describing the inhibitor-sensitive GGAUU calls as plausible candidates rather than treating non-DRACH calls, especially those detected only by GLORI, as presumptively artefactual. The revised manuscript appropriately notes technical and biological explanations for non-canonical calls and identifies the GGAUU sites as candidates requiring further validation, but this additional context would improve interpretation.

Second, the comparison between Dorado and GLORI remains overinterpreted. The matched-input design and inhibitor perturbation are important strengths, but the current analysis does not establish that sites detected only by GLORI are false positives or that Dorado is a reference standard. This would require, for example, direct comparison of inhibitor responsiveness among GLORI-only and shared sites, while accounting for coverage, conversion efficiency, and other method-specific biases; therefore, phrases such as "GLORI exhibits reduced specificity," "largely spurious," and "validated by an orthogonal approach" are not fully supported by the data presented.

Similarly, the thresholding approach should not be described as "unbiased." The analysis is useful and empirically informed, but it relies on selected parameters, a manually specified penalty, and predetermined decision criteria. Describing the procedure as "data-driven" or "empirically calibrated against matched unmodified RNA controls" would more accurately convey both its value and its limitations.

The additional RNA-variant examples are helpful, but they demonstrate an advantage of long-read data rather than broadly proving that Dorado "outperforms" GLORI for RNA-variant-level quantification. A more accurate formulation would be: "Direct RNA sequencing provides RNA-variant-resolved predictions that are not readily assignable using short-read GLORI data, as illustrated at several loci."

Finally, the authors now acknowledge that the functional significance of cell-type differences remains unresolved, but the abstract still presents the results as evidence of "finely tuned" regulation, and the discussion links them to neuronal function. The data provide solid evidence for differences in predicted modification proportions between cell types, but the evidence is incomplete for claims about functional consequences; these statements should therefore be framed as context or hypotheses for future study rather than as inferences from the present experiments.

Reviewer #1 (Public review):

Summary:

The authors aim to study mutational paths connecting WW domains with different binding specificities. Their approach combines an unsupervised sequence generative model based on RBMs with a path-sampling algorithm. The key result is that most intermediate sequences along the designed transition paths retain measurable binding activity in wet-lab assays, whereas paths containing the same mutations introduced in a randomized order are largely non-functional. This difference is attributed to epistatic interactions captured by the RBM model.

Strengths:

Exploring mutational paths in high-dimensional protein sequence space is a challenging problem. The computational framework used here is state-of-the-art and is strengthened by systematic experimental characterization of binding activity. The study is comprehensive in scope, including multiple transition paths both within and across WW specificity classes, and the integration of modeling with high-throughput experimental validation is a clear strength.

Reviewer #1 (Public review):

Summary:

The study describes the migration of epidermal keratinocytes through porous membranes and observed a unique size selection whereby only on 3-micron membrane are keratinocytes able to migrate and reform an intact epidermis. The authors propose that the model replicates three cell states of intact epidermis, EMT, and MET. They also show that this response depends on the actin cytoskeleton, keratin 6, and Piezo1, and the migration could be stimulated with TGFbeta ligands.

Strengths:

Strengths of the study include the establishment of a simple yet robust in vitro model that capture all three cell states, which could be useful for future investigation of wound healing or metastasis across a range of epithelia. There is also good quantitative characterisation of the pore size effects, providing some interesting observations about the physical regulation of keratinocyte migration, and the study provides some initial insights into the regulatory mechanisms. The images and presentation are clear.

Weaknesses:

While there are some insights into the underlying mechanisms, there could be more detailed analysis of the key signalling pathways and functional relationships between actin, keratins, Piezo, and EMT genes. Although the extent of new biological insight is somewhat limited, the study still demonstrates the utility of the model, and these points may be important questions to address in future studies.

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 goal of this study was to generate a library of new enhancer-driven AAVs in order to selectively and efficiently target astrocytes and oligodendrocytes in rodents. The implied criteria are that such viral vectors should have high specificity for the intended cell type and effectively express in all astrocytes/oligos in the brain or, alternatively, be specific for defined brain regions, layers, or subtypes of astrocytes/oligos. In addition, they should be compatible with intravenous retro-orbital delivery to facilitate experimentation and brain-wide targeting (i.e., show organ specificity and high efficiency in the brain). Ideally, these new AAVs would also maintain their characteristics across disease contexts and show applicability in non-human primates. Tools with such characteristics are generally lacking in studying glial cells and would be extremely useful to scale up and accelerate glial research, allowing targeting of astrocytes/oligos with distinct molecular identity and intersectional strategies.

At present, however, none of the enhancer-AAVs presented in the study seems to meet this combination of criteria, at least not with the level of stringency typically expected in the field. The main reason is that, in its current form, the study does not present one candidate AAV iteratively improved to meet all these criteria; instead, it presents a catalogue of new AAVs with various degrees of specificity, completeness, and mixed characteristics. Therefore, their utility should be interpreted cautiously. Moreover, the way specificity and completeness are intermixed in the analysis makes it difficult to evaluate the actual utility of any given AAV. The study might have been strengthened by focusing on a small set of the most promising candidates (i.e., AiE0890m_3x2C) and validating them thoroughly for expression specificity, completeness, effective cargo expression, ability to allow specific pan-astrocyte or astrocyte-subtype targeting in vivo, and preserved properties in NHPs and in disease, as this would encourage their adoption by the community. Currently, too many AAVs are assessed inconsistently against the desired criteria, with none being evaluated through and through.

The impact of the catalogue is also greatly diminished by the fact that a suite of AAVs with outstanding specificity and efficiency is already available for the study of astrocytes (e.g., 4x6T AAVs) and was not utilized as a standard to benchmark the new library, making it difficult to appreciate the relative benefits of the new AAVs. The inclusion of expression data in NHPs is very significant, but benchmarking against established AAVs would also be needed to fully appreciate their value.

Importantly, readers should also be aware that the study seems noticeably limited in its literacy with glial biology. The introduction and discussion frame the field in a way that seems outdated, creating the impression that the diverse roles of glia in health and disease have not yet been studied, which may inadvertently be perceived as dismissive and stigmatizing.

In summary, the paper introduces potentially useful viral tools and lays the foundations for future multiplexed targeting of distinct glial cell subpopulations in rodents and in non-human primates, which are extremely important directions. Some of the regionally restricted or even sparsely expressed AAVs may prove valuable in enabling subpopulation-specific targeting or molecular profiling strategies, but currently lack full benchmarking. At present, the promises over the utility of the new tools seem overstated, and the library may not yet represent an actionable resource for targeting astrocytes and oligodendrocytes.

Reviewer #1 (Public review):

Summary:

In this study, Liang et al. characterize the genomic composition of transposable elements (TEs) in 167 sauropsid species, with the aim of testing whether increased TE accumulation is modulated by reduced selection efficacy (Mutational Hazard Hypothesis, MHH), and whether this relationship is recursively observed at multiple scales within the group. By employing life history traits (body mass and generation time) and genetic parameters (dN/dS) as proxies of effective population size, they observe a positive relationship between drift levels and TE content across all species. This relationship, however, fades out in most of the clades within sauropsids.

On one hand, this study highlights that diverging patterns of TE accumulation are modestly but still appreciably driven by differences in selection efficacy across sauropsids, providing support for the MHH within this long-diverged vertebrate group. On the other hand, this same general principle fails to explain TE content variation when closely related species within the same clade are considered. The data presented suggest that TE accumulation is dictated by other lineage-specific mechanisms, and/or that differences in effective population size that emerged later in these clades are not relevant enough to drive divergent TE contents.

Overall, the study provides a further empirical test of the MHH, joining an increasing collection of studies that reject its global validity at macroevolutionary scales. The analysis leverages the plethora of genomic data now available for a previously poorly sampled vertebrate group, and adds insightful points of view on the nearly-neutral evolution of the genome in sauropsids.

Strengths:

The paper is well written, and the dataset is appropriate to test the predictions of the MHH. Sauropsids are characterized by highly diversified life history traits and strategies, likely very diverse effective population sizes, and very wide genome size and TE content ranges. This makes them an ideal group to investigate whether the divergent TE contents and landscapes of different lineages emerged due to differences in purging selection.

A few TE-focused comparative studies already covered squamates and birds separately (Pasquesi et al. 2018; Ji et al. 2022), or included a limited number of sauropsid species in analyses of much wider phylogenetic breadth (Weinstein & Roy, 2026; Lewin & Eyre-Walker, 2026). This work is a first explicit attempt to incorporate them together in one analysis to investigate the determinants of TE dynamics and genome size variation. Given the large comparative dataset, it also represents a considerable effort in terms of data compilation, annotation and analysis, integrating life-history and genomic traits together, and can serve as a resource for future studies.

While most of the methods employed are appropriate, the robustness of some aspects of the analysis could be improved, and interpretation of some results would benefit from a reformulation more aligned with what the data suggest (see just below).

Weaknesses:

Conclusions made on the pooled dataset are partially, sometimes solely, based on Pearson's correlations. This is not a solid practice, as results are affected by the phylogenetic structure of the data. Mentioning that a correlation is initially strong but loses signal when accounting for phylogeny is fine if used to highlight the need for accounting for phylogenetic non-independence. However, phylogenetic correlations should be those on which the conclusions are drawn, and the authors should be clear in their formulations that there is no association when PGLS or SEM suggests so, even though Pearson's correlations would suggest otherwise. For instance, the abstract reads that a correlation is found between generation time and TE content across all species, although this is not supported by PGLS. Or the claim that there is a strong positive association between dN/dS and TE proportion is, although still present, significantly downsized under phylogenetic correlation. These are just a couple of examples, but several conclusions throughout the text should be reformulated or rescaled to fit phylogenetically corrected results.

This is particularly pertinent given the problem of scale dependence that the authors highlight: the significance of a given predictor (selection efficacy) of TE accumulation can not only change according to the phylogenetic scale under consideration, but is also sensitive to compositional differences between clades, which are very pronounced here given the strong phylogenetic inertia displayed by both life-history traits and TE amounts. The purpose of phylogeny-aware methods is precisely to "discriminate conditions under which pooled correlations may arise from compositional differences rather than from repeated within-lineage evolutionary processes", so should not be regarded as a secondary result.

Since the study is based on the quantification of multiple Ne proxies, the authors could expand a bit more on that and check their expected reciprocal relationships. For example, in the SEM, body mass and generation time are untied from dN/dS variation, but an explicit test outside of it would be required. If these different indicators actually go in the same direction, it would add a support layer to them, capturing differences in selection effectiveness. In non-avian sauropsids, for example, body mass was not a predictor of dN/dS and longevity (Figuet et al. 2016): the wider dataset gathered here notably increases the sampling power to assess whether/which LHTs are concordant with dN/dS.

Connected to the point above, retrieval of expected correlations between dN/dS and life-history traits in birds has been more challenging, but this has been attributed to the presence of a substitution bias (GC-biased gene conversion). Because of this, dN/dS can become an imprecise Ne proxy if some methodological precautions are not integrated (see Bolivar et al. 2016; https://doi.org/10.1186/s13059-018-1613-z). At the same time, the avian dataset used in Marino et al. does display some expected correlations between dN/dS and several LHTs, while still calculating the metric without correction for substitution biases, but across a much higher number of species (~200). So I wonder whether the combination of substitution noise in dN/dS and less abundant sampling (and maybe less contrast in Ne?) in birds might contribute to confounding the relationships between Ne proxies and a potential correlation of those with TE content.

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:

Negreira, G. et al clearly presented the challenges of conducting genomic studies in unicellular pathogens and of addressing questions related to the balance between genome integrity and instability, pivotal for survival under the stressful conditions these organisms face and for their evolutionary success. This underlies the need for powerful approaches to perform single-cell DNA analyses suited to the small and plastic Leishmania genome. Accordingly, their goal was to develop such a novel method and demonstrate its robustness.

In this study, the authors combined semi-permeable capsules (SPCs) with primary template-directed amplification (PTA) and adapted the system to the Leishmania genome, which is about 100 times smaller than the human genome and exhibits remarkable plasticity and mosaic aneuploidy. Given the size and organization of the Leishmania genome, the challenges were substantial; nevertheless, the authors successfully demonstrated that PTA not only works for Leishmania but also represents a significantly improved whole-genome amplification (WGA) method compared with standard approaches. They showed that SPCs provide a superior alternative for cell encapsulation, increasing throughput. The methodology enabled high-resolution karyotyping and the detection of fine-scale copy number variations (CNVs) at the single-cell level. Furthermore, it allowed discrimination between genotypically distinct cells within mixed populations.

Strengths:

This is a high-impact study that will likely contribute to our understanding of DNA replication and the genetic plasticity of Leishmania, including its well-documented aneuploidy, somy variations, CNVs, and SNPs - all key elements for elucidating various aspects of the parasite's biology, such as genome evolution, genetic exchange, and mechanisms of drug resistance.

Overall, the authors clearly achieved their objectives, providing a solid rationale for the study and demonstrating how this approach can advance the investigation of Leishmania's small, plastic genome and its frequent natural strain mixtures within hosts. This methodology may also prove valuable for genomic studies of other single-celled organisms.

Reviewer #1 (Public review):

In this study, Hossain et al. investigated the role of Interleukin-2-inducible T cell kinase (ITK) in autoimmune lung injury, demonstrating that ITK-deficient (Itk-/-) mice are protected against pristane-induced pulmonary hemorrhage (PH). Authors suggest that this protection correlates with a significant remodeling of the T cell compartment in Itk-/- mice, including increased frequency of memory-like CD4+ and CD8+ T cells (CD44⁺CD62L⁺) as well as higher frequency of Treg populations. Furthermore, adoptive transfer of ITK-deficient Treg isolated from injured ITK-deficient mice confers protection against pulmonary hemorrhage in WT recipients.

Strengths:

The adoptive transfer of congenically marked wildtype and Itk-/- Treg populations clearly demonstrates that ITK-deficient Treg can actively rescue pre-existing lung injury and reverse systemic secondary metrics like proteinuria in wild-type recipients, providing proof-of-concept validation for the therapeutic utility of the ITK-Treg axis.

Weaknesses:

The molecular analysis of Tregs isolated from unchallenged wild-type ITK-deficient mice has limited value to understand how the ITK-deficient Tregs protect against autoimmune pulmonary hemorrhages in lungs. Detailed molecular analysis of Tregs isolated from the lung will be helpful in understanding the mechanistic regulation and identifying further therapeutic targets.

GroupID: 1

DiseaseAssertion: VHL

Group size: 1

GroupNumberMales: N/A

GroupNumberFemales: N/A

GroupEthnicity: N/A

GroupAgeRange: N/A

Group Info: Retrospective analysis of a longitudinal cohort of 21 VHL germline mutation carriers and retinal haemangioblastomas. All patients were categorized in two genotypic categories: missense mutations and truncating mutations. 

GroupPresentingHPOs: HP:0009711 (retinal capillary hemangioma)

GroupHPOFreeText: The patient presented with retinal capillary hemangioma.

GroupNotHPOs: N/A

GroupNotHPOFreeText: N/A

GroupPreviousTesting: N/A

PreviouslyPublished: N/A

SupplementalData: Table S1

Variant: Exon 1-2 deletion

CaseProblemVariantFreeText: N/A

LegacyVariant: N/A

ClinVarID: N/A

CAID: N/A

gnomAD: N/A

VariantEvidence: Chr3(GRCh37): g.10182301_10188764del; c.-1231_463+444del6464

MutationType: exon_loss_deletion

CivicName: Exon 1-2 deletion

MultipleGeneVariants: N/A

Case: 9, F, 21, Italian

DiseaseAssertion: VWD type 3

FamilyInfo: Consanguinity: no

ParentalGenotype: n/a

CasePresentingHPOs: n/a

CaseHPOFreeText: VWF:Ag (IU/dL): <1, FVIII:C (IU/dL): 3, Bleeding Score: 26

CaseNotHPOs: n/a

CaseNotHPOFreeText: Anti-VWF alloantibodies: no

CasePreviousTesting: n/a

Gene: VWF

Variant:

c.1–30071_220+3445del (p.Met1_Gly74delX8), Exon 1-3

c.3940delG (p.Val1314SerfsX33), Exon 28

Compound Heterozygous

Reviewer #1 (Public review):

Summary:

The authors investigate whether the brain uses the same neural representations for "absence" when it comes to seeing nothing versus thinking of zero. To do this, they recorded MEG while participants performed two types of tasks: (1) a perceptual detection task where subjects reported the presence or absence of a faint visual stimulus, and (2) numerical comparison tasks where subjects saw streams of numbers or dot patterns (both including "0" or empty sets) and decided which of two color-coded streams had a larger average. Multivariate decoders were trained to distinguish "present" vs "absent" in the detection task and "zero" vs "non-zero" in the numerical tasks. As a sanity check, the authors first replicate that symbolic (digit "0") and non-symbolic (empty dot sets) zeros share a common neural code (cross-format generalization). Crucially, they find that this numerical "zero" code does not overlap with the code for perceptual absence: cross-decoding between the detection task and number tasks yields Bayes factors strongly favoring distinct representations. In other words, the brain's pattern for "no grating was seen" cannot decode the pattern for "the number zero was shown," and vice versa. A small brief cross-decoding effect around 300 ms was observed, which the authors attribute to low-level visual confounds (and which they test with an additional control decoder for stimulus presence), but overall, the evidence supports a dissociation.

Comments on revised version.

Thank you for the detailed response to my comments. The authors have adequately addressed my concerns, and I have no further comments at this stage.