Reviewer #1 (Public review):

Summary:

The manuscript has several strengths, including a technically comprehensive approach that combines mouse genetics, electrophysiology, live imaging in assembloids, and human organoid models, providing a rich and multifaceted dataset. Cross-species validation through the parallel use of mouse and human systems strengthens the generality of the observed phenotypes and increases relevance to human neurodevelopment.

Consistent phenotypic observations across systems show that ARHGEF6 loss affects migration, neurite morphology, growth cone structure, and neuronal survival, supporting a coherent role in cytoskeletal regulation.

There is clear evidence for developmental defects, including reduced interneuron numbers, increased apoptosis in the ganglionic eminences, and migration deficits, all well supported by quantitative analyses. Also, there is a high-quality electrophysiological characterization that demonstrates reduced firing in interneurons, providing a well-controlled functional phenotype.

Strengths:

The manuscript has several strengths, including a technically comprehensive approach that combines mouse genetics, electrophysiology, live imaging in assembloids, and human organoid models, providing a rich and multifaceted dataset. Cross-species validation through the parallel use of mouse and human systems strengthens the generality of the observed phenotypes and increases relevance to human neurodevelopment.

Consistent phenotypic observations across systems show that ARHGEF6 loss affects migration, neurite morphology, growth cone structure, and neuronal survival, supporting a coherent role in cytoskeletal regulation.

There is clear evidence for developmental defects, including reduced interneuron numbers, increased apoptosis in the ganglionic eminences, and migration deficits, all well supported by quantitative analyses. Also, there is a high-quality electrophysiological characterization that demonstrates reduced firing in interneurons, providing a well-controlled functional phenotype.

Weaknesses:

Despite the strengths mentioned above, the study has some conceptual and experimental weaknesses that reduce its impact. The mechanistic insight is limited, as the research does not directly establish how ARHGEF6 regulates downstream signaling pathways.

Also, there is insufficient evidence for interneuron specificity; although the central claim is that ARHGEF6 plays a selective role in interneurons, the data do not adequately exclude the possibility that the observed effects reflect broader neuronal defects. The study lacks critical controls across cell types, as several phenotypes observed in organoids and progenitors, including apoptosis, reduced neuronal output, and altered morphology, could also affect multiple neuronal populations without being directly tested. Furthermore, the data are predominantly descriptive, with many results remaining correlative and failing to establish causal relationships.

Some more comments:

(1) Given that ARHGEF6 is a guanine nucleotide exchange factor for Rac1 and Cdc42, the absence of direct measurements of GTPase activity or downstream signaling represents a significant gap. The interpretation that the observed phenotypes are mediated through specific cytoskeletal pathways, therefore, remains inferential.

(2) The manuscript repeatedly interprets the findings as interneuron-specific. However, several key observations are not demonstrated to be restricted to IN. Without direct comparison to excitatory neurons or other cell types, it is difficult to conclude that ARHGEF6 plays a selective role in interneurons rather than a more general role in neuronal development. The well-done analysis of the transcriptomic dataset is not sufficient to claim IN specificity. This issue is particularly important for the interpretation of the human organoid experiments, where reductions in SOX2⁺ progenitors and NEUN⁺ neurons, as well as increased apoptosis, could reflect global developmental defects. Similarly, in the mouse experiments, the reduction in GAD67⁺ cells is compelling, but it is not shown whether other neuronal populations are also affected.

(3) The study provides a strong phenotypic description but limited causal resolution. For example, migration defects, altered growth cone morphology, and reduced branching are all consistent with impaired cytoskeletal regulation, but the links between these phenotypes are not directly established. Likewise, while the electrophysiological data convincingly show reduced firing in interneurons, the connection between altered cytoskeletal dynamics and intrinsic excitability is not explored.

(4) Several aspects of data presentation could be improved. In multiple figures (e.g., Figure 1A, D; Figure 4 and Video S1, 2), the images are difficult to interpret due to high cellular density, limited magnification, or lack of clear annotation. In some cases, it is not fully clear how quantifications were performed or which regions were analyzed. Improving the visual clarity with arrows, boxes, and high-magnification inserts of the data would strengthen confidence in the conclusions.

Reviewer #1 (Public review):

A triple-transgenic (3xTgAD) mouse model of Alzheimer's disease was exposed to a high-fat diet and assigned to one of three interventions: voluntary physical activity, a low-fat diet, and their combination. A high-fat diet significantly increased body weight and induced widespread neuroanatomical changes, with effects modulated by sex and genotype. The combined intervention led to significant weight loss in males of both genotypes. Neuroanatomical analyses revealed that a high-fat diet significantly reduced hippocampal and cerebellar volumes in wild-type mice but had a less pronounced effect on 3xTgAD mice; nevertheless, interventions, particularly the combined approach, increased localized brain volumes in these regions regardless of genotype. Spatial gene enrichment analysis of this pattern identified glucose homeostasis. Overall, these findings suggest that voluntary physical activity and a low-fat diet can modulate brain structure and behaviour, partially counteracting the effects of a high-fat diet, and potentially recruiting biological processes that may support brain health.

The authors describe studies of the 3xTg mouse model of Alzheimer's disease (AD). They set out to study the interactions of diet and exercise on three outcomes: weight gain, MRI, and either the novel object recognition or Morris water maze tasks of memory.

They conclude there are sex and genotype effects on hippocampal volume.

There are several strengths to the study. First, they start out with a great deal of mice. Once they are divided into groups, the sample sizes are not always strong, however. It would be good to know that they were sufficiently powered.

The data are also interesting. Mice were placed on several different diets during the study, which will be of interest to many who question the role of diet in outcomes. They also add exercise as an intervention, and study not only diet but also the combined effect of diet and exercise. This is relevant to those interested in controlling dementia by diet and exercise. Finally, they perform some very interesting analyses to study the data.

That said, the study also has several limitations. For example, it is quite complex. Mice had a standard diet until 2 months of age, then were switched to either a low-fat or a high-fat diet. Some mice had both a different diet and exercise. MRI was performed at 2, 4, and 6 months, when behavior was tested. A drawback of this design is that no assessment of outcomes relevant to this animal model, such as amyloid-beta or tau phosphorylation, was conducted. Also, they used the novel object recognition task, despite stating in the Discussion that this task does not show impairments until well after 6 months of age. They added exercise, but it is not clear whether the animals used the exercise apparatus equally. Also, the animals were housed "communally", so adding an exercise wheel may have made the cage crowded, adding stress to the study. The diets were not simply low- or high-fat because many constituents besides fat content also changed. Regarding fat, the type of fat also changed between diets. Therefore, the gut microbiome was probably affected differently by factors other than fat intake. There was no measurement of food consumption, so some mice may not have eaten as much of the new diet as they did of the old diet they were used to.

Regarding the data, only the outcomes of complex analyses are shown. One would first want to see the changes in body weight and perhaps later how it is analyzed in a more complex way. For behavior, one would first want to see outcomes as typically presented. For example, learning, recall, platform test results from the Morris water maze, and discrimination indices for object recognition. Note that, at one point, I believe the authors note that some groups did not explore thoroughly, which would make novel object recognition hard to interpret. If there was any difficulty with ambulation, both tasks would be hard to interpret.

Regarding MRI, from what can be seen, structures cannot be distinguished clearly. At least some raw data should be shown to demonstrate this and to determine what the data show. The raw data suggest that some of the larger structures can be distinguished, and we should see the data for these areas, even if all areas can't be assessed. Lifestyle interventions can mitigate the effects of diet-induced obesity on body weight, behaviour, and brain anatomy in mouse models. Using a longitudinal design, wild-type and triple-transgenic (3xTgAD) mouse models of Alzheimer's disease were exposed to a high-fat diet and assigned to one of three interventions: voluntary physical activity, a low-fat diet, and their combination. A high-fat diet significantly increased body weight and induced widespread neuroanatomical changes, with effects modulated by sex and genotype. The combined intervention led to significant weight loss in males of both genotypes. Neuroanatomical analyses revealed that a high-fat diet significantly reduced hippocampal and cerebellar volumes in wild-type mice but had a less pronounced effect on 3xTgAD mice; nevertheless, interventions, particularly the combined approach, increased localized brain volumes in these regions regardless of genotype. Multivariate integration of behavioural and neuroanatomical measures identified a brain pattern linking hippocampal and cerebellar volumes to intervention and behavioural performance. Spatial gene-enrichment analysis of this pattern identified biological processes, including glucose homeostasis, as potential biological mechanisms underlying intervention effects. Overall, these findings suggest that voluntary physical activity and a low-fat diet can modulate brain structure and behaviour, partially counteracting the effects of a high-fat diet, and potentially recruiting biological processes that may support brain health. In the end, the authors focus primarily on the hippocampus and discuss the cerebellum, but it seems that changes occur throughout the brain. The choice to focus on the hippocampus and cerebellum needs to be supported.

To gain further insight, the authors analyze genes across different brain regions using the Allen Brain Atlas. Although this seems reasonable in theory, once one realizes how many genes are shared across diverse brain regions, one wonders how such an analysis was conducted. More understanding of this approach, as well as how it was validated, is important. In the end, the authors conclude that the glucose homeostatic pathways were primarily altered, and one would like to understand whether that is indeed true and whether it is the only set of pathways that were changed.

This raises another point: what occurs in a normal wild-type mouse on the standard diet during the first 6 months of life? Do the glucose homeostatic pathways change simply due to age? Sex? It may be that, with age, the mice become more sedentary, which is why. Once that is resolved, what occurs on the standard diet for the 3xTg mice? Perhaps they are more active or more sedentary, regardless of diet or exercise? Thus, the studies end up raising more questions than answers.

Given so much work has already been done, it seems best to simply reorganize the presentation with raw data first, followed by the analysis. For the second section, the implicit assumptions of the analyses should be very clear so that the analyzed data are understood and believable. Limitations of the assumptions, pooling some groups, etc., need to be clear.

Figures. In Figure 1, the weekly measurements are not shown. The points are connected, so an unbroken line is shown. Around the line are lighter lines indicating errors, but with all the lines and colours, one does not know what standard errors surround the values for any given group. This makes the data hard to interpret. In later figures, significant differences are indicated with asterisks, but this seems to be done inconsistently.

In the text, more caution is needed for some assertions. For example, it is not clear that a 2- to 6-month-old is an adolescent. Opinions about the ages of mice that correspond to human life stages have always been debated. Another example is indicating that male mice might gain weight differently than females, as if it were an outcome of diet or exercise. This is because male rodents continue to gain weight in adulthood, but females stabilize because estrogen limits appetite. Additionally, females may not show group differences because they are more variable. This can relate to their estrous cycle. If stressed or housed without males nearby, they may not have a regular estrous cycle, which can then affect their outcomes. This may be particularly true for behavior when they may have been tested during different estrous cycle phases, if they had estrous cycles.

Reviewer #1 (Public review):

Summary:

This study presents an Important tool for the study of MR1 antigen binding, opening new possibilities, and cutting-edge techniques. The evidence supporting the claims of the authors is solid, although including some functional experiments using primary T-cells would also provide a more complete physiologic evaluation. The work will be of interest to T cell immunologists, in general, especially those studying unconventional T cells.

Strengths:

In this study, the authors developed a single-chain MR1-derived protein by exchanging the α3 domain and β2-microglobulin for a helical stabilizing domain that they had previously developed. The aim was to generate a more compact structure that would still fold properly, without the risk of losing β2-microglobulin. This overall more robust structure would facilitate ligand exploration using various cutting-edge biophysical techniques.

The authors successfully demonstrated that their construct folds similarly to native MR1 and retains the ability to bind MAIT TCR in solution, as shown by cryo-EM experiments. Its melting temperature was equivalent to that of the native protein. Importantly, the construct enables the use of differential scanning fluorometry and transverse relaxation-optimized spectroscopy, which represent the main strengths of this work. These approaches should greatly facilitate the screening of additional unknown ligands and enable interaction mapping.

Weaknesses:

One possible area for improvement would be to extend the validation to additional known ligands, particularly weaker binders. Furthermore, although the cryo-EM data are highly convincing, including either MAIT cell staining or MAIT activation assays with the generated construct would provide stronger functional validation of its equivalence to the wild-type protein with respect to ligand-binding properties.

Overall, this work is of great interest to the field, as several groups worldwide are seeking to identify endogenous/tumour-derived MR1 ligands. In addition, some pathogens lacking the capacity to produce 5-OP-RU have been shown to activate MAIT cells, raising the possibility that unknown pathogen-derived ligands may also exist.

Reviewer #1 (Public review):

Summary:

P. Izquierdo et al. investigated the genetic determinism of various traits of interest in switchgrass using large-scale genomic and transcriptomic data. More specifically, they worked on a diversity panel comprising 426 genotypes evaluated in common-garden experiments at two locations (Michigan and Texas). The phenotypic and genomic data were already published. In this work, they produced transcriptomic data for each of the 426 genotypes at each site, and they carried out phenotype predictions using genomic and transcriptomic data separately or together. While they were moderately correlated at each location, both omic information appeared to be complementary for the prediction of phenotype. To further exploit the fact that they have data across two locations, they computed differences for phenotypes and transcripts between locations as indicators of trait and transcript plasticity, respectively. They built predictive models of trait plasticity using genomic information and transcript plasticity, which proved to be quite accurate for traits affected by GxE. Finally, they made use of SHAP values from predictive models of flowering time and biomass at each location, as well as for their plasticity, to gain insight into their genetic determinism. These SHAP values provide the importance of the predictive features (SNP and/or transcripts) for trait prediction. This allowed them to confirm some candidate genes and to propose new candidates for both traits.

Strengths:

I found this study interesting and rich. I think the sample size (426 genotypes) is large enough to support the findings. The use of a modern machine-learning approach (XGBoost) together with SHAP indices to find interesting features and get insights into the biological mechanisms underlying flowering time and biomass production is quite original. The methodology employed is globally sound. I also like the fact that the authors accounted implicitly for the population structure by providing a baseline prediction using the first 5 PCs.

Weaknesses:

While the methodology is globally sound, I sometimes had difficulties following exactly what was done. This is partly due to the fact that the authors used 2 omics (SNPs and transcripts) to predict phenotypes, and sometimes, in the results, it is not clear which of the 2 is the focus. This was especially the case for the importance of the features and the interpretability of the models, where I found it sometimes hard to tell whether the analysis was done on SNPs or transcripts.

Also, regarding the methodology, I did not understand why the authors needed to perform a feature selection approach. Maybe it was required to perform the interaction analysis, which could not be deployed on all the features? But regarding the importance of the features, I do not get the added value of the selection over the direct use of SHAP indices when using all features. Maybe this is because I am not a specialist in this kind of approach, but maybe the authors could add more details to explain the rationale behind the feature selection.

Reviewer #1 (Public review):

Summary:

Wang Liao and colleagues aim to provide a comprehensive synthesis of zebrafish circadian research, with particular emphasis on the decentralized photoreceptive architecture that distinguishes teleosts from mammals, and to outline future research directions leveraging emerging technologies for translational applications. The authors frame zebrafish as occupying a "crucial evolutionary and experimental niche" and argue that the model system is uniquely suited to address open questions in chronobiology.

Strengths:

The review is broad in scope and up to date in its citation of recent primary literature. The coverage of physiological outputs - spanning cardiovascular rhythmicity, hepatic metabolism, immune function, reproduction, and gut homeostasis - is more comprehensive than many existing reviews in this area, and researchers seeking an entry point into any of these subfields will find a useful orientation. The figures are well-designed and effectively summarise complex regulatory relationships. The section on immune rhythmicity is a particular strength, providing mechanistic detail on how specific clock components (Clock1a, Per1b, Per2, Cry1a) differentially regulate neutrophil behaviour, bacterial killing, and cytokine expression; this level of molecular specificity distinguishes it from comparable sections in the review. The brief discussion of non-canonical clock gene functions (CLOCK in neuronal connectivity, BMAL1 in stem cell state, vascular calcification) raises genuinely interesting points that are underexplored in the field and might deserve more prominence.

The future perspectives section makes a conceptually interesting move in suggesting that the zebrafish decentralized architecture could reframe a central question in chronobiology - from how a master clock imposes order on passive peripheral oscillators, to how semi-autonomous oscillators achieve coherence. This is the most original conceptual contribution in the manuscript, and it would benefit from much further development.

Weaknesses:

The core limitation of this review is that it functions primarily as an annotated bibliography rather than a critical synthesis. Section after section follows the same pattern: a physiological system is introduced, several findings from recent papers are described in sequence, and the section ends. Missing throughout is an evaluative voice - where does the field agree, where does it disagree, which findings have been replicated versus remain preliminary, and which conceptual questions are genuinely unresolved versus merely unstudied? Readers with expertise in the field will find little that reframes their understanding; readers new to the field will receive information but not the interpretive scaffolding needed to assess its significance.

The framing of zebrafish as occupying a "crucial evolutionary and experimental niche" is asserted but not substantiated. The experimental advantages of zebrafish - optical transparency, external development, genetic tractability - are real, but they apply primarily to larval stages, typically the first two weeks of development. The review does not adequately address whether the key features it highlights, particularly peripheral photosensitivity and autonomous peripheral oscillators, have been demonstrated in adult animals, where optical transparency is lost. Many of the physiological findings described (sleep-wake cycles, cardiovascular function, reproduction, and immune function) are most relevant in adult or juvenile fish, yet the mechanistic underpinnings often come from larval studies. Whether the mechanisms generalise across developmental stages is not discussed, and this is an important gap that the review could acknowledge explicitly.

The claim that zebrafish bridge invertebrate and mammalian models is a conventional framing that appears in most zebrafish review articles; its repetition here adds little. More interesting - and underexplored - is the comparative question of how the decentralised clock architecture of teleosts compares with that of other non-mammalian vertebrates, or indeed with invertebrate systems such as Drosophila, where peripheral tissue clocks and non-visual photoreception have also been studied. The review does not engage with this comparative dimension, which would be the natural intellectual context for the claims being made.

The future perspectives section identifies several promising directions - optogenetic circuit mapping, whole-body longitudinal imaging, inter-organ communication, network modeling - but these are described at a high level of generality. Most are not specific to the questions raised by the zebrafish decentralized clock architecture; they would appear in any forward-looking review of circadian biology. The one conceptually distinctive idea - that zebrafish could be used to ask how distributed oscillators achieve coordinated coherence without hierarchical control - is identified but not developed into concrete experimental questions or testable predictions. The discussion of non-canonical clock gene functions in the Future Perspectives section would benefit from being more directly connected to what zebrafish specifically can offer: given that teleost genome duplication has produced additional paralogues of clock genes, there is a concrete opportunity to dissect canonical from non-canonical functions through comparative analysis of paralogues with diverged expression patterns. This point is hinted at but not made explicitly.

Appraisal of conclusions:

The conclusions are broadly consistent with the evidence cited, and the authors are appropriately cautious in noting that many signalling cascades and inter-tissue communication mechanisms remain incompletely characterised. The conclusion that zebrafish represents a valuable and underexploited model for circadian-disease translational research is well-supported. However, the review would be significantly strengthened if the authors distinguished more clearly between what is firmly established, what is supported by preliminary or single-study evidence, and what remains genuinely speculative.

Likely impact and utility:

This review will be useful as an orientation document for researchers new to zebrafish circadian biology, and the comprehensive treatment of physiological outputs across organ systems is a genuine service to the field. Its impact as an intellectual contribution is limited by the descriptive approach and the absence of original synthesis or conceptual reframing. The most interesting ideas in the manuscript - the reframing of the central/peripheral clock hierarchy question, and the potential of clock gene paralogues for probing non-canonical functions - could be further developed and, if pursued, could form the basis of a more distinctive and impactful contribution.

Reviewer #1 (Public review):

Sheidaei and colleagues report a novel and potentially important role for an early mitotic actomyosin-based mechanism, PANEM contraction, in promoting timely congression of chromosomes located at the nuclear periphery, particularly those in polar positions. The manuscript will interest researchers studying cell division, cytoskeletal dynamics, and motor proteins. Although some data overlap with the group's prior work, the authors extend those findings by optimizing key perturbations and performing more detailed analyses of chromosome movements, which together provide a clearer mechanistic explanation. The study also builds naturally on recent ideas from other groups about how chromosome positioning influences both early and later mitotic movements.

Comments on revised version:

In the revised manuscript, organizational issues have been largely resolved. In addition, the inclusion of new experiments in additional cell lines, along with an expanded discussion that places actomyosin contractility in the broader conceptual context of other mechanisms governing chromosome movement, has significantly strengthened the manuscript.

Reviewer #1 (Public review):

Summary:

This paper tries to address an important outstanding issue, which is the evolutionary origin of the SLC25 family of mitochondrial carrier proteins, which are common to all eukaryotic life, with few exceptions. The authors have carried out phylogenetic analyses and DALI searches of AlphaFold databases of bacterial and archaeal membrane proteins. They identify two bacterial proteins, CysZ and YhiY, and they propose that they are progenitors of SLC25 family members. Whilst the paper addresses an interesting topic, the conclusions are not supported by the data and are not presented in an unbiased manner, as they highlight only features that provide some tentative support for the hypothesis. They do not address the large number sequence and structural properties that refute the hypothesis, such as the asymmetric vs three-fold pseudo-symmetric features, hexamer vs monomer, and the complete lack of any conserved motifs with similar functions. Any resemblances between CysZ/YhiY and mitochondrial carriers thus seem to be superficial and could well be coincidental, as they represent generic properties of membrane proteins rather than specific ones, indicative of an evolutionary relationship.

Strengths:

This paper explores the evolutionary origins of the SLC25 family of mitochondrial carrier proteins, which are found across nearly all eukaryotic organisms. They were likely to be present in the last common ancestor of all eukaryotes, around two billion years ago. The question is whether they are of bacterial, archeal or eukaryotic origin. The authors propose that two bacterial proteins, CysZ and YihY, may represent ancestral forms of these carriers, based on structural comparisons of models, a sequence motif, and phylogenetic analyses. While the research addresses an important and longstanding question, the presented evidence does not convincingly support their hypothesis.

Weaknesses:

A central concern is the reliance on structural similarity searches using predicted protein models, since these models are often built using known protein structures as templates, and thus these searches may produce misleading matches. The reported similarities between CysZ, YihY, and mitochondrial carriers are weak and fall within ranges expected for unrelated membrane proteins, which commonly share general structural features, such as helical bundles. Quantitative measures of similarity are low and do not support a shared evolutionary origin. The case for YhiY is extremely poor as neither structure nor sequence features support the claim. Importantly, the opening of the YihY is towards the membrane rather than the water phase, as is the case for carriers, indicating that it has a very different structure and function. The case for CysZ is somewhat better, as it is a helical bundle with two short helices somewhat resembling the matrix helices of mitochondrial carriers, and a short sequence PXDXXK that is part of one of the known sequence motifs of mitochondrial carriers, but this is where the similarities end.

Mitochondrial carriers have a distinctive threefold pseudo-symmetrical structure and a highly complex transport mechanism involving six structural elements. This paper's hypothesis does not explain how such a high level of threefold pseudo-symmetry could have evolved from entirely asymmetric proteins. To complicate matters further, CysZ is not functional as a monomer but forms a functional hexamer, which also explains why it has two half helices rather than two transmembrane helices. Thus, the hypothesis is that CysZ, which is an asymmetric protomer of a functional hexamer, has evolved into a three-fold pseudo-symmetric protein, which is functional as a monomer. A more convincing explanation is that the threefold pseudo-symmetrical structure arose from gene triplication and fusions, with later mutations introducing asymmetry to support diverse substrate binding. In support of this notion, mitochondrial carriers transporting large molecules, such as ATP, show more asymmetry, whereas those for small molecules remain nearly symmetrical. In general, the vast majority of transport proteins arose from gene duplications and fusions of the domains.

Although mitochondrial carriers have a similar sequence motif as found in CysZ (PXDXXK), their roles are very different. In mitochondrial carriers, this motif is located roughly in the middle of transmembrane helices H1, H3, and H5, where proline creates a pronounced kink, bringing the charged residues inward to form a salt-bridge network in the central water-filled cavity. The formation and disruption of this network is essential for the transport mechanism when switching between inward- and outward-open states. In CysZ, the motif is found at the end of a helix and in the following loop at the end of the transporter, with residues pointing outward toward the water phase. These residues are typical of membrane-water interface regions, where proline acts as a helix breaker and charged residues interact with the water phase. Thus, this motif in CysZ does not match the position or function seen in mitochondrial carriers, and its presence is likely to be coincidental, because these residues often occur in the water-membrane region. Importantly, none of the other important conserved three-fold symmetrical motifs of mitochondrial carriers is found in these bacterial proteins, such as the cytoplasmic network [YF][DE]xx[RK], cardiolipin binding sites, ER-links, and sequences of small amino acids, which are critical for its dynamic mechanism.

The phylogenetic relationship is also overstated, as there is no sequence similarity between these proteins other than that occurring because of similar biophysical properties, such as transmembrane helices. The authors suggest that a specific mitochondrial carrier represents the ancestral member of the family, but this conclusion appears to be inferred rather than rigorously demonstrated. Key aspects, such as tree rooting and taxon sampling, are not sufficiently addressed, weakening confidence in the evolutionary claims. Further, the selection of only a few bacterial and archaeal proteomes for analysis limits the study's scope. Broader searches would be necessary to support claims about conservation and ancestry. Independent sequence searches indicate that CysZ and YihY are not widely conserved in the bacterial groups most closely related to mitochondria, undermining the argument that they are plausible ancestors.

Overall, the presented similarities are superficial and can be explained by general features of membrane proteins rather than by specific adaptations to function. The hypothesis that CysZ and YihY are evolutionary precursors of mitochondrial carriers is not supported by the presented data.

Reviewer #1 (Public review):

Renard, Ukrow et al. applied their recently published computational pipeline (CHROMAS) to the skin of Euprymna berryi and Sepia officinalis to track the dynamics of cephalopod chromatophore expansion. By segmenting each chromatophore into radial slices, and analyzing the co-expansion of slices across regions of the skin, they inferred the motor control underlying chromatophore groups.

Strengths:

- The authors demonstrate that most motor units of cephalopod skin include a subregion of multiple chromatophores, creating "virtual chromatophores" between fixed chromatophores. This is an interesting concept that challenges prevailing models of chromatophore organization, and raises interesting possibilities for how chromatophore arrays may be patterned during development.

- This study introduces new analytical approaches of cephalopod skin that will be valuable for the quantitative study of cephalopod behavior.

Weaknesses:

- The authors use patch-clamp experiments in E. berryi to test their approach for inferring motor units. The stimulations indeed evoke expansions of sub-regions of each chromatophore, creating "virtual chromatophores". However, they were not able to predict these motor units from behavioral analysis before confirming them with patch-clamp, limiting the strength of this validation.

- In S. officinalis, chromatophores are far more numerous than in E. berryi and exhibit frequent spontaneous activity, making it more challenging to distinguish shared motor drive. Patch-clamp experiments in this species would provide important validation and strengthen confidence in the method for inferring motor units.

- Although multiple experimental conditions were tested (e.g., age, size, behavioral context, sedation, head-fixation, lighting), data is only shown from a small subset of experiments. Analyzing pooled data across conditions would allow for more generalizable conclusions.

- Different clustering algorithms were used for the two species (HDBSCAN for E. berryi and Affinity Propagation for S. officinalis). Since Affinity Propagation appeared to better capture correlation structure in S. officinalis, it would be informative to reanalyze the E. berryi data using the same method to assess potential algorithm-dependent biases.

Conclusion:

The CHROMAS tool is likely to be valuable to the field, given the need for quantitative frameworks in cephalopod biology. The predictions outlined here provide a useful foundation for future experimental investigation.

Reviewer #1 (Public review):

Summary:

This paper investigates whether transformer-based models can represent sentence-level semantics in a human-like way. The authors designed a set of 108 sentences specifically to dissociate lexical semantics from sentence-level information and collected 7T fMRI data from 30 participants reading these sentences. They conducted representational similarity analysis (RSA) comparing brain data and model representations, as well as the human behavioral ratings. It is found that transformer-based models match brain representation better than static word embedding baseline which ignores word order but fall short of models that encode the structural relations between words. The main contributions of this paper are:

(1) The construction of a sentence set that disentangles sentence structure from word meaning.

(2) A comprehensive comparison of neural sentence representations (via fMRI), human behavior, and multiple computational models at the sentence level.

Strengths:

(1) The paper evaluates a wide variety of models, including layer-wise analysis for transformers and region-wise analysis in the human brain.

(2) The stimulus design allows precise dissociation between lexical and sentence-level semantics. The RSA-based approach is empirically sound and intuitive.

(3) The constructed sentences, along with the fMRI and behavioral data, represent a valuable resource for studying sentence representation.

Weaknesses:

(1) The rationale behind averaging sentence embeddings across multiple transformer models (with different architectures and training objectives) is unclear. These transformer-based models have different training paradigms and model architectures, which may result in misaligned semantic spaces. The averaging operation may dilute the distinct sentence representations learned by each model, potentially weakening the overall semantic encoding for sentences. Please clarify this choice or cite supporting methodology.

(2) All structure-sensitive models discussed incorporate semantics to some extent. Including a purely syntactic baseline, such as a model based on context-free grammar, would help confirm the importance of syntactic structures.

(3) In Figure 2, human behavioral judgments show weak correlations with neural data, and even fall below those of computational models, suggesting the behavioral judgments may not reflect the sentence structures in a brain-like way. This discrepancy between behavioral and neural data should be clarified, as it affects the interpretation of the results.

(4) To better contextualize model and neural performance, sentence similarity should be anchored to a notion of semantic "ground truth", such as the matrix shown in Figure 1a. Comparing this reference with human judgments, brain responses, and model similarities would help establish an upper bound.

(5) The structure of this paper is confusing. For instance, Figure 5 is cited early but appears much later. Reordering sections and figures would enhance readability.

(6) While the analysis is broad and comprehensive, it lacks depth in some respects. For instance, it remains unclear what specific insights are gained from comparing across brain regions (e.g., whole brain, language network, and other subregions). Similarly, the results of simple-average and group-average RSA appear quite similar and may not advance the interpretation.

(7) While explaining the grid-like pattern due to sentence length is important, this part feels somewhat disconnected from the central question of this paper (word order). It might be better placed in supplementary material.

Comments on revised version:

The new version of the paper has addressed my main concerns, including:

(1) clarification about the methodology of Transformer embeddings

(2) discussion about the purely syntactic models

(3) discussion about the low correlation between behavioural ratings and brain activations

(4) better structure of the paper

(5) clarification about pre-registration

I believe the paper has been substantially improved after revision.

Reviewer #1 (Public review):

This rigorous and creative study uses an elegant combination of metabolomics, transcriptomics, and budding yeast molecular genetics to discover that (i) activating AMPK to maintain mitochondrial respiration fueled by cytosolic Acetyl CoA and (ii) increasing fatty acid synthesis independent of respiration drive independent pathways that increase the fitness of replicatively-aged budding yeast cells, albeit without increasing their lifespan. This work will be of interest to scientists in the field of aging and metabolism. Some clarifications in the text would address the following concerns, which would increase the impact of the study:

(1) What does activation of AMPK (via PGDP-Sak1 expression) do to the replicative lifespan? How many bud scars, in general, do the subpopulations that are older - yet have less Tom70 (increased mitochondrial fitness) - have, after the 48 hrs time point that they are examining? How many divisions occurred in this 48hr time period - i.e. is it long enough to have all cells reach the end of their replicative lifespan? This information is important to rule out that a subset of the mutant cells just divided faster and hence had more divisions within 48 hrs (growing faster and living longer are different things). Having identical growth curves doesn't indicate per se that they all divide at the same rate, as there may be a subpopulation that divides faster and a subpopulation that doesn't grow so well.

(2) A2A cells do not have an extended replicative lifespan (RLS) but show an increase in the "low senescence" population (Figure 2). If the cells are not becoming senescent, why don't they have longer RLS? Not having a longer lifespan seems inconsistent with the statement that "bud scar counting confirmed that A2A cells reach a higher age than wild type", which comes back to how many times the cells can divide in the 48hr timepoint studied and their rate of cell division? Also, the lifespan curve shown is plotted against time, not cell division number, which does not take into account different division times of cells within the population (described above). It would be much more useful to show standard lifespan curves showing cell division numbers per lifespan per cell.

(3) Increased "fitness" of the old cells is implied from the increased size of the colonies that the old cells can make. However, this is a measure of the fitness of the daughters per se, not the old mother cells. Are the old mothers just passing on healthier mitochondria and more lipids to the daughters, such that they can divide more times? If the aged cells have an "increased fitness", why don't they divide more times themselves (i.e. live longer?).

(4) The statement is made that "these experiments define two classes of aging cells with distinct metabolic needs, coherent with the model of two aging trajectories previously proposed (referencing Nan Hao's work)". However, the big difference here is that in Nan Hao's work, their two aging trajectories influenced the length of lifespan, but that does not appear to be the case here. That distinction should be made clear. Perhaps the authors could also speculate as to why the A2A yeast stops dividing after presumably the same number of cell divisions, even though they have an activated AMPK and activated fatty acid synthesis pathway.

(5) I am a bit confused by the use of the word "senescence" by this lab here and in their previous growth on galactose studies. If yeast don't senesce, which is usually defined as an irreversible arrest of the cell cycle where cells stop dividing, shouldn't the yeast that do not senesce still be dividing and hence have a longer lifespan? Should a different term be used rather than senescence? Such as "fitness late in life". The authors giving their definition of senescence may help reduce this apparent contradiction.

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 the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole to pole oscillation whereby a time average minimum of the Min proteins at mid cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites, and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.

Strengths:

In the current study, the authors perform a detailed biochemical characterization of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations was nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

In the revised manuscript, the authors now demonstrate localization and tracking data for minC and minJ deletion strains, which suggest that MinJ impacts MinD membrane cycling, but MinC does not. Additional in vitro work showed that the PDZ domain of MinJ modifies MinD ATP hydrolysis rates, and the authors propose that MinJ may promote MinD dimer formation.

Weaknesses of the revised version: No major weaknesses.

Reviewer #1 (Public review):

Summary:

The study by the Obata group characterizes the dynamics of the canonical malate dehydrogenase-citrate synthase metabolon in yeast.

Strengths:

The study is well-written and appears to give clear demonstrations of this phenomenon.

Studies of the dynamics of metabolon formation are rare; if the authors can address the concern detailed below, then they have provided such for one of the canonical metabolons in nature.

Weaknesses:

There is a fundamental issue with the study, which is that the authors do not provide enough support or information concerning the split luciferase system that they use. Is the binding reversible or not? How the data is interpreted is massively influenced by this fact. What are the pros and cons of this method in comparison to, for example, FLIM-FRET? The authors state that the method is semi-quantitative - can they document this? All of the conclusions are based on the quality of this method. I know that it has been used by others, but at least some preliminary documentation to address these questions is required.

Comments on revised version:

I feel that the authors have adequately addressed my prior concerns. I have no further critiques of their work.

Reviewer #1 (Public review):

Summary:

This study investigated how visuospatial attention influences the way people build simplified mental representations to support planning and decision-making. Using computational modeling and virtual maze navigation, the authors examined whether spatial proximity and the spatial arrangement of obstacles determine which elements are included in participants' internal models of a task. The study developed and tested an extension of the value-guided construal (VGC) model that incorporates features of spatial attention for selecting simpler task mental representation.

Strengths:

(1) Original Perspective: The study introduces an explicit attentional component to established models of planning, offering an approach that bridges perception, attention, and decision-making.

(2) Methodological Approach: The combination of computational modeling, behavioral data, and eye-tracking provides converging measures to assess the relationship between attention and planning representations.

(3) Cross-validated data: The study relies on the analysis of three separate datasets, two already published and an additional novel one. This allows for cross-validation of the findings and enhances the robustness of the evidence.

(4) Focus on Individual Differences: Reports of how individual variability in attentional "spillover" correlates with the sparsity of task representations and spatial proximity add depth to the analysis.

Appraisal of Aims and Results:

The study sets out to determine how spatial attention shapes the construction of task representations in planning contexts. The authors provide evidence that spatial proximity and arrangement influence which environmental features are incorporated into internal models used for navigation, and that accounting for these effects improves model predictions. There is clear documentation of individual variation, with some participants showing greater attentional spillover and more sparse awareness profiles.

Comments on revised version:

The authors did a great job and I am very happy with the revised manuscript.

Reviewer #1 (Public review):

Summary: This study investigated how visuospatial attention influences the way people build simplified mental representations to support planning and decision-making. Using computational modeling and virtual maze navigation, the authors examined whether spatial proximity and the spatial arrangement of obstacles determine which elements are included in participants' internal models of a task. The study developed and tested an extension of the value-guided construal (VGC) model that incorporates features of spatial attention for selecting simpler task mental representation.

Strengths:

(1) Original Perspective: The study introduces an explicit attentional component to established models of planning, offering an approach that bridges perception, attention, and decision-making.

(2) Methodological Approach: The combination of computational modeling, behavioral data, and eye-tracking provides converging measures to assess the relationship between attention and planning representations.

(3) Cross-validated data: The study relies on the analysis of three separate datasets, two already published and an additional novel one. This allows for cross-validation of the findings and enhances the robustness of the evidence.

(4) Focus on Individual Differences: Reports of how individual variability in attentional "spillover" correlates with the sparsity of task representations and spatial proximity add depth to the analysis.

Weaknesses:

(1) Clarity of the VGC model and behavioral task: The exposition of the VGC model lacks sufficient detail for non-expert readers. It is not clear how this model infers which maze obstacles are relevant or irrelevant for planning, nor how the maze tasks specifically operationalize "planning" versus other cognitive processes.

The method for classifying obstacles as relevant or irrelevant to the task and connecting metacognitive awareness (i.e., participants' reports of noticing obstacles) to attentional capture is not well justified. The rationale for why awareness serves as a valid attention proxy, as opposed to behavioral or neurophysiological markers, should be clearer.

(2) Attention framework: The account of attention is largely limited to the "spotlight" model. When solving a maze, participants trace the correct trail, following it mentally with their overt or covert attention. In this perspective, relevant concepts are also rooted in attention literature pertaining to object-based attention using tasks like curve tracing (e.g., Pooresmaeili & Roelfsema, 2014) and to mental maze solving (e.g., Wong & Scholl, 2024), which may be highly relevant and add nuance to the current work. This view of attention may be more pertinent to the task than models of simultaneously tracking multiple objects cited here. Prior work (notably from the Roelfsema group) indicates that attentional engagement in curve-tracing tasks may be a continuous, bottom-up process that progressively spreads along a trajectory, in time and space, rather than a "spotlight" that simply travels along the path. The spread of attention depends on the spatial proximity to distractors - a point that could also be pertinent to the findings here.

Moreover, the tracing of a "solution" trail in a maze may be spontaneous and not only a top-down voluntary operation (Wong & Scholl, 2024), a finding that requires a more careful framing of the link to conscious perception discussed in the manuscript.

Conceptualizing attention as a spatial spotlight may therefore oversimplify its role in navigation and planning. Perhaps the observed attentional modulation reflects a perceptual stage of building the trail in the maze rather than a filter for a later representation for more efficient decision making and planning. A fuller discussion of whether the current model and data can distinguish between these frameworks would benefit readers.

(3) Lateralization of attention: The analysis considers whether relevant information is distributed bilaterally or unilaterally across the visual display, but does not sufficiently address evidence for attentional asymmetries across the left and right visual fields due to hemispheric specialization (e.g., Bartolomeo & Seidel Malkinson, 2019). Whether effects differ for left versus right hemifield arrangements is not made explicit in the presented findings.

(4) Individual differences: Individual differences in attentional modulation are a strength of the work, but similar analyses exploring individual variation in lateralization effects could provide further insight, and the lack of such analyses may mask important effects.

(5) Distinction between overt and covert attention: The current report at times equates eye movement patterns with the locus of attention. However, attention can be covertly shifted without corresponding gaze changes (see, for example, Pooresmaeili & Roelfsema, 2014).

The implications for interpreting the relationship between eye movement, memory, and attention in this setting are not fully addressed. The potential dynamics of attention along a maze trajectory and their impact on lateralization analysis would benefit from further clarification.

Appraisal of Aims and Results:

The study sets out to determine how spatial attention shapes the construction of task representations in planning contexts. The authors provide evidence that spatial proximity and arrangement influence which environmental features are incorporated into internal models used for navigation, and that accounting for these effects improves model predictions. There is clear documentation of individual variation, with some participants showing greater attentional spillover and more sparse awareness profiles.

However, some conceptual and methodological aspects would be clearer with greater engagement with the broader literature on attention dynamics, a more explicit justification of operational choices, and more targeted lateralization analyses.

Reviewer #1 (Public review):

Summary:

In this manuscript the authors derive a mean-field model for a network of Hodgkin-Huxley neurons retaining the equations for ion exchange between the intracellular and extracellular space.<br /> The mean-field model derived in this work relies on approximations and heuristic arguments that, on the one hand, allow a closed-form derivation of the mean-field equations, and on the other hand restrict its validity to a limited regime of activity corresponding to quasi-synchronous neuronal populations. Therefore, rather than an exact mean-field representation, the model provides a description of a mesoscopic population of connected neurons driven by ion exchange dynamics.

Strengths:

The idea of deriving a mean-field model which relates the slow-timescale biophysical mechanism of ion exchange and transportation in the brain to the fast-timescale electrical activities of large neuronal ensembles.

Weaknesses:

The idea underlying this work is not completely implemented in practice.

The derived mean field model do not show a one-to-one correspondence with the neural network simulations, except in strongly synchronous regimes. The agreement with the in vitro experiment is hardly evident, both for the mean-field model and for the network model. The assumptions made to derive the closed-form equations of the mean field model have not been justified by any biological reason, they just allow for the mathematical derivation. The final form of the mean-field equations do not clarify whether or not microscopic variables are used together with macroscopic variables in an inconsistent mixture.

Comments on revisions:

The main weaknesses I listed in the first report are still present, since the authors did not answer my questions on a solid basis. I report the list for completeness:

(1) It seems that the reduction methodology that is employed is not the most suitable one for the single-neuron model they are considering.<br /> (2) The formulation of the mean-field derivation is unnecessarily complicated. It could be heavily simplified by following previously published approaches to derive biologically realistic neural masses.<br /> (3) The model seems to work only for highly synchronized situations and not for the standard asynchronous evolution usually observed in neural circuits.

Therefore, my statement remains unchanged.

Reviewer #1 (Public review):

Pyne and Pandey et al. report the observation of early DNA degradation at the phagocytic cup during macrophage engulfment. Using an elegant experimental system that combines actin staining to visualise cup formation with direct monitoring of DNA degradation, the authors identify rapid recruitment of the membrane-bound nuclease DNase X (DNase1L1) to nascent phagocytic cups. This recruitment occurs within minutes of cup formation, is independent of DNA presence at the substrate, and appears to originate from intracellular membrane structures rather than from the extracellular environment. The results support the conclusion that DNase X activity is present at the phagocytic cup and that DNA digestion can begin prior to phagolysosomal maturation.

The study is technically strong. The experimental system is clean, specific, and allows precise spatial and temporal detection of DNA degradation. The imaging-based approaches are carefully executed and enable convincing visualisation of DNase X recruitment and activity. The use of an alternative substrate beyond the primary SNS system strengthens the core observation, and the data broadly support the authors' central claim.

However, several limitations temper the physiological interpretation. The system relies largely on short, free DNA substrates, leaving open how efficiently DNase X processes more complex or physiologically relevant DNA structures, such as nucleosome-bound DNA or neutrophil extracellular traps (NETs). It remains unclear whether DNase X deficiency would alter macrophage responses to larger nucleic acid structures, influence engulfment efficiency, or modify downstream inflammatory signalling pathways such as TLR9 or STING activation. Moreover, the experimental setup prevents full phagocytic cup closure, potentially prolonging DNase activity compared with physiological phagocytosis, which typically proceeds rapidly to cargo internalisation. For example, the peak signal observed in Figure 5 occurs approximately 90 minutes after phagocytic cup formation, a time point at which many phagocytic cups would be expected to have already closed under physiological conditions. Additional work using fully engulfed cargo in more physiological contexts would clarify whether early DNase X activity meaningfully contributes to overall DNA clearance kinetics.

Mechanistically, the signal that triggers DNase X recruitment remains unresolved. Although actin rearrangement was excluded as the primary driver, the upstream cues that direct DNase X-containing membrane structures to the forming cup are not yet defined.

In the broader context, early DNase X activity at the phagocytic cup could represent an additional safeguard against inflammatory signalling by limiting extracellular or surface-associated DNA before phagolysosomal degradation by DNase II. This mechanism may be particularly relevant in settings where DNA fragmentation before engulfment is incomplete, such as necroptosis or NET formation. Determining whether DNase X deficiency exacerbates inflammatory responses, alters DNA clearance efficiency in vivo, or contributes to immune pathology will be critical for establishing its physiological and disease relevance.

Overall, this is a compelling study that introduces a novel concept of pre-phagolysosomal DNA digestion. The conclusions are well supported within the in vitro system used, but further investigation using diverse DNA substrates and physiologically relevant models will be required to fully define the impact of this mechanism on immune regulation and disease.

Reviewer #1 (Public review):

Summary:

During erythroid differentiation, hematopoietic progenitors relinquish multipotency and activate lineage programs. The switch from GATA2 to GATA1 is particularly important in this process, yet GATA2 chromatin‑binding kinetics remain undefined. The authors investigated GATA2-chromatin interaction dynamics during erythroid differentiation in three different cell systems using single‑molecule live‑cell imaging, and they also used CUT&Tag to profile GATA2 chromatin occupancy.

By single‑molecule imaging, the authors report two interaction modes for GATA2: short‑lived (<1 s) and long‑lived (>5 s) binding. The proportion of long‑lived molecules, the number of binding events, and the duration of long‑lived binding change (or are maintained) during differentiation. Notably, long‑lived chromatin engagement by GATA2 increases during early erythroid differentiation and decreases at the late stage. CUT&Tag identifies regulatory elements selectively occupied by GATA2 during the early transition stage. Together, these results support a model in which transcription factor kinetics form a dynamic chromatin‑engagement profile that characterizes the GATA2‑to‑GATA1 transition.

Strengths:

(1) Characterizing transcription‑factor binding kinetics during the GATA2->GATA1 transition addresses a fundamental mechanism in erythroid differentiation.

(2) Combining single‑molecule live imaging with CUT&Tag provides both dynamic and locus‑specific perspectives.

(3) Single-molecule analysis across three different cell systems strengthens the potential generalizability of the findings and highlights biological variability.

Weaknesses:

I agree that single‑molecule imaging is a powerful approach for investigating GATA2 kinetics, but the single‑molecule data are the most important part of the paper and need improvement. The analyses focus on three measures: (i) duration of long binding, (ii) proportion of short‑ and long‑binding molecules, and (iii) total binding events. However, several methodological and control issues limit confidence in the kinetic interpretations. The authors should address the following major concerns.

(1) Two binding states: justification and controls

The authors propose two states of GATA2 binding. Are there only two states? Studies that separate short‑ and long‑lived binding (e.g., Chen et al., 2014, PMID: 25342811) address two states of transcriptional factors very carefully. Some long‑binding duration distributions here are very long‑tailed (e.g., Figure 2D middle), suggesting a possible third state. The authors must explain how they determined that two states provide the "best fit" to the data and how they classified "short" versus "long" binding.

Controls should be included for long‑lived and short‑lived binding (e.g., histone proteins, HaloTag‑NLS, or a binding‑deficient GATA2 mutant) as in other studies. These controls are essential to exclude alternative explanations (see points below).

(2) Exclude photophysical and focal‑plane artifacts

The authors should exclude contributions from (i) photobleaching, (ii) blinking, and (iii) Z‑axis motion (disappearance from the focal plane). Although photobleaching correction is mentioned in the Methods, no details are provided. Describe and quantify the photobleaching correction and demonstrate that it was applied across all cell types and conditions.

Some spots in the supplementary movies appear to blink or to move substantially between frames. Provide analyses or controls that distinguish true dissociation events from photophysical blinking/bleaching or axial motion.

(3) HILO illumination and nuclear region sampled

HILO is powerful but sensitive to illumination angle: slight changes sample different nuclear regions (e.g., nuclear interior versus periphery). The nuclear periphery is enriched in heterochromatin and may bias binding statistics. Explain how the authors controlled the HILO angle and confirmed that comparable nuclear regions were imaged across cells and conditions.

(4) Quantification of event counts and long‑binding durations

The number of binding events and measured long‑binding durations are strongly affected by imaging conditions (labeling/staining, bleaching, nucleus size, cell cycle state, focal plane, spot detectability, etc.). Imaging clarity appears to differ among cells/conditions in the supplementary movie. Provide more careful analysis describing how these variables were controlled or corrected for, and assess the sensitivity of results to choices in detection and tracking parameters.

(5) Evidence that spots are single molecules

The authors state that spots represent single molecules but do not provide supporting evidence. Spot brightness varies considerably in the movies. Brightness differences may reflect axial position. Provide evidence supporting single‑molecule assignment (e.g., single‑step photobleaching traces, brightness distributions compared to a known single‑molecule control, or photon count analysis).

(6) Description of spot‑analysis pipeline

The manuscript lacks a sufficient description of the spot‑analysis method. I reviewed the STRAP pipeline paper cited (Haque and Coleman 2025 bioRxiv) and the GitHub code, but the Methods in the current manuscript should include a detailed STRAP pipeline. This would enable readers to evaluate and reproduce the analyses.

(7) Differences among cell systems

The three cell systems yield notably different results (e.g., Figure 2C vs 4C and Figure 2D/3D vs 4D). Provide a more detailed explanation for these differences and discuss how biological variability, technical differences, or imaging biases might account for the discrepancies.

Reviewer #1 (Public review):

This manuscript is very interesting and timely. By introducing the critical effects of desolvation barriers and solvent (water)-separated minima into the implicit-solvent potentials (of mean force, PMFs) for coarse-grained molecular dynamics simulations of biomolecular liquid-liquid phase separation (LLPS), this work fills a gap that should be apparent to researchers of protein folding in the past couple of decades but has so far escaped deserved attention such that these basic features of aqueous solvation have seldom, though not never, been invoked in recent studies of biomolecular condensates. Although the present paper deals almost exclusively with homopolymers, this work can be a foundation for the future development of a new, more physical coarse-grained interaction scheme for simulating amino acid sequence-dependent effects, which I presume is the authors' ongoing or next endeavor. The results presented in this manuscript are highly valuable.

However, there is room for improvement in the authors' description of (i) the broader impact of effects of desolvation barrier and solvent-separated minimum in the thermodynamics of biomolecular condensates, especially with regard to the ramifications on hydrostatic pressure-dependent effects; (ii) the physical implication of using a 20-parameter hydropathy scale rather than a 210-parameter pairwise amino acid interaction scheme; and (iii) temperature-dependent effects, including the authors' discussion of "enthalpic" and "entropic" contributions. In all these aspects, the authors' discussion should be put in a more comprehensive context of the existing literature. At a few other places, the description of the methods and results should be clarified as well. Accordingly, the authors should revise the manuscript to address the following items thoroughly within the revised manuscript (not merely in the response letter) with the additional references mentioned below included in the revised discussion:

(1) In several places, e.g., on line 77 (p.2), the authors appear to suggest that "implicit-solvent representation" is the origin of the deficiency in commonly utilized coarse-grained potentials that this study is aiming to rectify. But desolvation barriers and solvent-separated minima are also features of implicit-solvent representations; they are just features that should be incorporated in more accurate implicit-solvent potentials. This point is stated quite clearly and accurately in the Abstract (p.1) but not consistently in the rest of the text. The authors should check the entire text carefully to ensure that a coherent, accurate perspective is presented.

(2) In the discussion of the importance of desolvation barriers and solvent-separated minima in the Introduction (pp.1-3), connections should be drawn to recent works that utilize these PMF features to rationalize hydrostatic pressure (P)-modulated effects on biomolecular LLPS, including the P-dependent reentrant phase separation of alpha elastin; see Cinar et al. (2019) Chem Eur J 25:13049 (https://chemistry-europe.onlinelibrary.wiley.com/doi/full/10.1002/chem.201902210) and references therein, especially discussions around Figures 10, 11 & 13 in this reference.

(3) In the lower panels of Figures 2D, E (p.5), what do the differently colored small circles in the double-minimum free energy profiles represent? Does the color shading have the same meaning as that in the upper panels? If so, what do the positions of the circles on the free energy profile represent? The authors should clarify this.

(4) The discussion regarding entropy and enthalpy around Figure 2 is quite confusing as it stands. What do the authors mean exactly by the association of entropy or enthalpy with the desolvation barrier of the solvent-separated minimum? Are they referring to conformational entropy?

(5) Do the authors assume that the PMF (effective implicit-solvent potential) is a purely enthalpic term? It appears to be the authors' assumption. If so, the assumption has to be stated clearly in their discussion of "entropy" vs "enthalpy" around Figure 2.

(6) Closely related to points 3-5 above, it should be stated clearly that the "temperature" used in the authors' simulations does not represent experimental temperature if the authors are using purely enthalpic effective potentials because PMFs are in fact temperature-dependent. This clarification is necessary to avoid misunderstanding. In this regard, it should be noted that temperature-dependent effective interactions have been used for modeling biomolecular condensates in analytical theory (Lin, Song, Forman-Kay & Chan, J Mol Liq 2017, already in the citation list) as well as in coarse-grained molecular dynamics simulations [Dignon et al. (2019) ACS Cent Sci 5:821-830 (https://pubs.acs.org/doi/10.1021/acscentsci.9b00102); Chakravarti & Joseph (2025) Protein Sci 34:e70284 (https://onlinelibrary.wiley.com/doi/10.1002/pro.70284)]. The latter two studies, not cited currently, are particularly relevant and thus should be cited because the authors may wish to incorporate temperature-dependent features in their ongoing or future effort in constructing a more comprehensive coarse-grained interaction scheme for biomolecular LLPS simulation.

(7) In tackling "entropy" vs "enthalpy", it should be noted that the temperature dependence of the effective interactions entails an entropic contribution (which is itself temperature dependent) in addition to conformational entropy. As for the effective potential with desolvation barrier and solvent-separated minimum, it should be noted that the decomposition into entropic and enthalpic contributions at the direct contact, desolvation barrier, and solvent-separated minimum can be dramatically different, see, e.g., MaCallum et al. (2007) PNAS 104:6206-6210 (https://www.pnas.org/doi/full/10.1073/pnas.0605859104) and references therein.

(8) P.7, line 340: The proportionality relation follows directly from the standard Flory-Huggins result T_c = T chi(T)/chi_c, thus the proportionality constant is exactly 1/chi_c. Is this the standard relation that the authors are invoking here? The authors should clarify this.

(9) The study on dynamic consequences on pp.8-11 is interesting, but clarifications are necessary:

(i) The vertical schematic in Figure 4A should be explained in detail in its entirety. As it stands, no explanation is provided either in the figure caption or in the text. In particular, what does "elasticity driven" refer to?

(ii) The top snapshot in Figure 4A is labeled t_sim = 0 ns. Does it mean that the snapshot shown is the only chain configuration that the authors used to start the simulation, and that the snapshot does NOT represent the result of any time evolution, no matter how short the duration is? However, if that is the case, why is this snapshot identified with spinodal decomposition if it is not the product of a time evolution from a more homogeneous configuration?

(iii) Related to (ii) - do the rectangular boxes shown represent the entire simulation box or just part of the box containing the polymer chains? One would imagine that if the top snapshot represents spinodal decomposition, the simulation would have been started at a more uniform distribution a short time prior? Why is this not the case?

(iv) What precisely do the small yellow beads and black-colored springs in the zoom-in image of Figure 4E represent?

(10) In discussing dynamic effects, it is useful to draw connections to related works on the effect of chain flexibility on "aging" of condensate [Biswas & Potoyan (2024) PRX 45:9222-9245 (https://journals.aps.org/prxlife/abstract/10.1103/PRXLife.2.023011)] and characterization of viscoelasticity in simulations of biomolecular condensates [Tejedor et al. (2023) J Phys Chem B 127:4441-4459 (https://pubs.acs.org/doi/10.1021/acs.jpcb.3c01292)], as the effects of desolvation can be explored further based on these prior works.

(11) Much of the present study is based on the original HPS formulation of Dignon et al. (2018). In this regard and also in anticipation of future development of improved interaction schemes, several issues should be stated and discussed, even if briefly:

(i) The original HPS model has a basic shortcoming in accounting for the relative interaction strengths of, among others, arginine vs lysine residues [Das et al. (2020) PNAS 117:28795-28805 (https://www.pnas.org/doi/10.1073/pnas.2008122117)].

(ii) Compared to 210-parameter pairwise interaction schemes, such as KH in Dignon et al. (2018) and Joseph et al. (2021), the 20-parameter interaction scheme is likely too restrictive to account for pairwise amino acid residue interactions [Wessén et al. (2022) J Phys Chem B 45:9222-9245 (https://pubs.acs.org/doi/10.1021/acs.jpcb.2c06181)].

(iii) The height of the desolvation barrier may vary significantly for different amino acid residue pairs, see, e.g., Figure 11 of Cinar et al. (2019) mentioned above (and references therein). The authors should discuss these nuances in the revised version. They may also wish to take them into consideration in future investigations.

Reviewer #1 (Public review):

Summary:

This manuscript presents an original quantitative approach for tracking the online formation and updating of prior beliefs. In an Alternating Serial Reaction Time task, participants were exposed to probabilistic visual streams, and their pre-stimulus saccadic behavior (i.e., the first eye movement after the previous stimulus disappeared) was monitored via eye-tracking. Since the stimuli followed an alternating probabilistic sequence, upcoming events did not appear with full certainty: some stimuli had a higher, some a lower probability. By comparing anticipatory oculomotor behavior between high and low probability events, the authors dissociated between learning/belief updating and general oculomotor noise. Noise-driven errors were more frequent than learning-dependent errors, which nonetheless triggered more belief updating (i.e., a change in oculomotor behavior in a subsequent encounter of the same event). Interestingly, updating depended more strongly on whether a prior belief was consistent with the task's probabilistic structure than on prediction errors. These findings suggest that incidental, implicit statistical learning may rely on conservative updating with a relatively low learning rate, or on errorless algorithms, rather than prediction errors per se.

Strengths:

By applying a fine-grained analysis of anticipatory oculomotor behavior, this work establishes new continuous metrics to quantify the gradual learning and refinement of prior expectations during statistical learning. These metrics provide convincing evidence of the dynamics of anticipatory oculomotor behavior.

The method is paradigm-independent, offering generalizable metrics for tracking the dynamic formation and refinement of predictive models in any task involving probabilistic stimulus streams. In the future, computational modeling may leverage these continuous metrics to better dissect the mechanisms underlying statistical learning.

Weaknesses:

The authors subscribe to the idea that statistical learning is not a unified concept but rather is implemented via multiple underlying mechanisms. However, it remains unspecified what these different mechanisms could be, and how eye movements could contribute to distinguishing between them.

The authors claim that they developed a novel methodological approach to probe whether anticipatory eye movements directly reflect priors, thereby filling an outstanding gap. However, this claim ignores mounting relevant work on structure learning using eye-tracking in the developmental field.

The authors claim that their framework quantifies trial-by-trial oculomotor dynamics, while in fact the analyses use epochs (i.e. groups of multiple trials) as predictors. Why not use trial number as a predictor to truly investigate trial-by-trial dynamics that directly reflect anticipation, surprisal, and revision?

Reviewer #1 (Public review):

This paper reports an auditory-directed analysis of the HCP 7T short movie dataset. It has the goal of using the film audio to create tonotopic (pRF) maps and combine these with other HCP-provided data (e.g., T1/T2 ratio) to improve understanding of auditory cortex organization and relative functional segregation, particularly in reference to speech processing.

The paper is ambitious, uses well-founded existing tools for combining data across subjects, and in the Discussion in particular, makes a lot of careful points about interpretation. The paper shows that, at least for a very large dataset on 7T (and for at least a few individual participants) good quality cross-subject-average tonotopic maps can be extracted from fMRI movie datasets via basic spectral modelling of the movie soundtracks. It also suggests ways that these movie-based maps can be combined to come up with potential models of cortical organization. The PCA analysis is a creative way of combining maps (see below for comments)

These are valuable tools for the field in exploiting/exploring existing data, and I look forward to trying them out myself. I want to emphasize that this is not 'damning with faint praise' - a concrete demonstration of this approach with freely available tools/examples is not only the product of a lot of effort (thank you!), but will be an impetus to research going forward.

In terms of the contribution to our understanding of auditory cortex organization, using this large N cohort, they replicate a number of findings in the literature from the last couple of decades, including the overlap of low frequency preference with greater speech stimulus preference (e.g. Moerel, de Martino, & Formisano, 2012, J Neuro), patterns of BF width across cortex (Moerel et al., various; Thomas et al. 2015), use of shorter and longer natural sounds (Moerel et al., 2012, 2014; Dick et al., 2012), the importance/influence of sustained spectral attention for tonotopic mapping (da Costa et al., 2013; Dick et al., 2017; Riecke et al. 2017), the use of tonotopy and 'myelin' mapping to establish areal or regional boundaries (Dick et al., 2012; de Martino et al., 2015; Besle et al., 2018, etc) and the overall shape and consistency of tonotopic maps (e.g., Talavage et al., 2004, Humphries et al., 2010 and many others). To my knowledge/memory, this is the first tonotopy paper that has used the cross-subject cortical-surface-based averaging techniques that are driven by more than curvature/sulcal alignment.

The paper focuses in particular on creating new sets of ROIs based on the various maps derived from the data. Despite being quite familiar with this body of work, I found it difficult to follow how the ROIs were derived, and how and why they were different and/or an improvement over existing parcellation schemes (see for instance Sereno, Sood, & Huang, 2022 for a comprehensive parcellation framework across modalities including auditory, based on combined receptive surface mapping, myelin estimates, and other metrics).

Given the hour of fast(ish) fMRI data on a 7T with pretty big voxels (so high SNR), one aspect of the results that I found surprising - and potentially informative - was the lack of reliable tonotopic 'mappability' in the majority of participants. The authors' analytic approach to computing the pRFs seems completely reasonable (and shows good average maps), and yet individual maps seem unreliable except for the very best examples. I wondered if this might be due to problems in data collection with earbuds becoming slightly uncoupled and therefore delivering a lot less lower-frequency response and also not preventing scanner noise from getting to the ear; this is often a problem with any in-scanner earbud system (including the Sensimetrics). I wondered if the robustness of the 'speech maps' was associated with that of tonotopy; if they are highly associated, that would suggest that either there were huge individual differences in auditory attention, or perhaps that there was some variability in the acoustic signal delivered to each participant.

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.

Weaknesses:

A few minor points to add to the clarity of the manuscript:

(1) The DA-WED driver (R48A08-AD ∩ VT008692-DBD ∩ TH-FLP) appears quite clean in the brain. However, since the study focuses on cardiac function and locomotion, it would be helpful to check expression in cardiomyocytes and the ventral nerve cord. This would help rule out any off-target expression that might contribute to the phenotypes and further support the idea of a descending pathway from brain dopaminergic neurons.

(2) Since DA-WED>Kir2.1 abolishes the puff-induced locomotor response (Figure 5b), suggesting that DA-WED neurons are directly involved in mediating locomotion. In the model (Figure 5L), it might make more sense for the pathway from mechanical threat to locomotion to pass through DA-WED neurons. The authors could consider adjusting the schematic if they agree.

(3) In line 408, Figure 5K should be 5L as it's a discussion of the model.

(4) In Figure 5j, the x-axis is missing time labels. Even if it matches Figure 5h, adding labels would make it easier to interpret at a glance.

(5) In line 312, it would be helpful to briefly explain why a 28 ms light pulse was used, compared to other pulse durations elsewhere in the paper.

(6) The cardiac deceleration seems to recover quickly after the air puff ends, whereas the locomotor response persists longer (around 10-15 seconds; see Figure 1 and Figure 5). This difference might suggest that DA-WED neurons influence locomotion through an additional or partially independent pathway, beyond their role in cardiac regulation. It could be worth briefly discussing this possibility.

Reviewer #1 (Public review):

Summary:

The current study is a follow-up to a previously published study by the same research group (Nold et al. 2025). In the previous study, the authors had included a set of exploratory analyses which assessed the effects of fitness level (denominated by a relative FTP), sex, and drug treatment (Naxolone versus placebo). In this previous study, the authors state that "exploratory analysis showed a significant main effect of fitness level on differences in pain ratings in the [saline] condition... suggesting increased hypoalgesia with increasing fitness levels, pooled across all stimulus intensities".

In the current study, the authors have recruited an additional 22 female participants (21 included in analysis) from local cycling clubs to assess if fitness level does indeed impact exercise-induced hypoalgesia responses to experimental thermal and pressure pain models.

Strengths:

The current study has the potential to present a convincing argument about the effect of fitness level and potentially other factors (e.g., sex) on exercise-induced hypoalgesia responses. Combining data across two of their primary studies would be highly fruitful to the research community interested in this area. Specifically, it has the potential to inform sports medicine practitioners and how they administer exercise protocols to help those experiencing pain with a further consideration for the fitness level (and maybe sex) of their patients.

Weaknesses:

However, the current study makes several bold claims about the role of fitness level and sex on exercise-induced hypoalgesia, which I do not believe that this study on its own - or in conjunction with the previously published study by the same authors - can make at present. Namely, the current study does not appear to conduct any specific analyses between the cohorts from either study (current and present). The results mention a difference in the group mean values in "fitness level" between cohorts, but the analysis itself on pain responses/exercise-induced hypoalgesia is limited only to the cohort from the current study. If the authors wanted to provide a convincing argument that fitness level has an effect on exercise-induced hypoalgesia, then the analysis of this study would have to include an analysis between the groups considered to be of "high" and "low" fitness level. I do not think the current study does this. Instead, it makes an assumption from the previous study (Nold et al. 2025) which only states that "exploratory analysis showed a significant main effect of fitness level on differences in pain ratings in the [saline] condition... suggesting increased hypoalgesia with increasing fitness levels, pooled across all stimulus intensities". The analysis of this study would have to include fitness level "high fitness" versus "low fitness" of participants across both studies in its statistical model to properly discern if fitness level has an impact on exercise-induced hypoalgesia.

A similar comment can be made with respect to sex differences, as these have not been assessed in the analysis of this study either.

Another area of weakness in this study is how "fitness level" has been demarcated across participants. One issue is how authors have assumed that the current cohort is 'fit', whereas the previous cohort was 'less fit', meaning that the authors could be coming to false conclusions about fitness level. In detail, figures within the current study show a large overlap between the 'fit' and 'less fit' cohorts, where some participants have a higher relative functional threshold power (FTP) in the 'less fit' cohort than the 'fit' cohort and vice versa. Therefore, I believe the authors should better demarcate between those that are in the 'more fit' and 'less fit' groups according to a validated and well-established criterion from the kinesiology and sport science literature. That being said, I think this may be problematic in some ways as FTP is considered a relatively poor measure to denote fitness levels, a limitation highlighted in the previous study's review.

Altogether, whilst I commend the researchers on their body of work across the two studies, the current methods and analysis provide an incomplete assessment of their primary research question, and therefore, I would urge the authors to reconsider some of their methods/analysis and the framing of their results to better reflect the main research question they have attempted to answer. Likewise, I would recommend that readers ensure they consider the current results with caution until the authors have addressed some areas of concern which currently limit their main conclusions.

Reviewer #1 (Public review):

Summary:

This manuscript investigates how cellular NAD/NADH ratios are controlled in cancer cell lines in vitro. The authors build on previous work, which shows that serine synthesis is sensitive to NAD/NADH ratios and PHGDH expression. Here, the authors demonstrate that serine synthesis is variable across a panel of cell lines, even when controlling for expression of serine synthesis enzymes such as PHGDH. The authors show that cellular NAD/NADH ratios correlate with the ability to synthesize serine and grow in serine-deprived environments when PHGDH levels remain constant. Investigating this variability in NAD/NADH ratios, the authors find that the cells that can positively respond to serine deprivation are able to increase oxygen consumption and cellular NAD/NADH ratios. Cells that do not increase oxygen consumption in response to serine deprivation do not increase NAD/NADH ratios and cannot grow well without serine. The authors go on to show that in cells with the ability to increase oxygen consumption upon serine deprivation, PHGDH expression alone is sufficient to fully restore growth-serine; in cells that cannot increase oxygen consumption, both PHGDH expression and interventions to increase NAD/NADH ratios are required to increase growth. Thus, cells need both PHGDH and NAD/NADH increases to maximize serine synthesis in response to serine deprivation. The authors previously showed that lipid synthesis likewise requires NAD regeneration. Interestingly, one cell line that does not increase oxygen consumption in response to serine limitation tends to increase oxygen consumption in response to lipid deprivation; accordingly, depriving this cell line of lipids increases the synthesis of serine. Together, these findings show that how cells respond to nutrient deprivation is highly variable and that the response to nutrient deprivation (for example, whether or not oxygen consumption is increased) will determine how well cells tolerate depletion of nutrients with related biosynthetic constraints. This work sheds light on the complexity of cancer cell metabolism and helps to explain why it is difficult to predict which nutrients will be limiting to any cancer cell type or environment.

Strengths:

(1) The authors use multiple interventions to manipulate NAD/NADH ratios in cells.

(2) Experiments are well controlled and appropriately interpreted.

Comments on revised version:

The authors thoughtfully and thoroughly responded to all reviewer comments. The revised manuscript addresses the critiques.

Reviewer #1 (Public review):

The authors conducted a comprehensive benchmarking and evaluation of co-folding platforms, including AlphaFold3, Boltz-2, Chai-1, and the docking algorithm Dock3.7, which employs a physics-based scoring function that incorporates van der Waals interactions, electrostatics, and ligand desolvation energies. The system of interest was the SARS-CoV-2 NSP3 macrodomain (Mac1), an increasingly popular antiviral target, and the ligand sets comprised 557 unseen ligand poses (keeping the training for these co-folding platforms in mind). Additionally, the authors investigated whether the co-folding models could distinguish true ligands from non-binding small molecules. The study is thorough, with extensive statistical support and consensus across multiple metrics (chemoinformatics for quantifying ligand similarity and efficacy). The questions that the authors aim to address are whether the co-folding models struggle with memorization, whether they can distinguish between a true and a false binder, whether they replicate experimental binding affinities and efficacy, and how they compare to the physics-based docking algorithm (Dock3.7).

Strengths:

Overall, this is a scientifically solid paper.

The work is highly detailed and well executed, featuring thorough data analysis and statistical assessment.

Comments on revised version:

The authors have adequately addressed my concerns.

Reviewer #1 (Public review):

Summary:

Eroglu and Hobert demonstrate that injecting CRISPR guides and repair constructs to target three genes at a time, tagging each with a different fluorescent protein, and selecting which gene to tag with which fluorophore based on genes' expression levels, can improve efficiency of gene tagging.

Strengths:

This manuscript demonstrates that three genes can be targeted efficiently with three different fluorophores. It also presents some practical considerations, like using the fluorophore least complicated by agar/worm autofluorescence for genes with low expression levels, and cost calculations if the same methods were used on all genes.

Weaknesses:

Eroglu has demonstrated in a previous publication that single-stranded DNA injection can increase efficiency of CRISPR in C. elegans, while inserting two fluorescent proteins and a co-CRISPR marker into three loci, and Paix et al 2015 demonstrated simultaneous insertion of two fluorescent tags. The current work is valuable and incremental advance. In general, I applaud the authors' willingness to strategize about how whole proteome tagging might be accomplished. I predict that the advance here will be one of many small advances that will get the field to that goal. The title oversells the advance presented, in my view, since seems like one among many key advances, and the first sentence of the Discussion seems a more apt summary of the key advance here.

Some injections targeted genes on the same chromosome together, which will create unnecessary issues when doing crossing that will be useful for some future experiments. This made me wonder if injecting 3 together really is helpful vs targeting each gene separately, since only 5 worms need to be injected. It cuts time down by 2/3, but perhaps avoiding targeting the same chromosome with two tags would be useful.

The limited utility of current blue fluorescent proteins makes me wonder if it's worth using at this stage, before there are better blue fluorescent proteins, or better yet, far red, to avoid issues with live imaging under phototoxic UV or near-UV illumination.

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:

The authors aimed to uncover novel therapeutic vulnerabilities in APC-mutant colorectal cancer (CRC), which constitutes the majority of CRC cases. They hypothesized that modulating oxygen-sensing pathways (via PHD inhibition) could disrupt adaptive stress responses in these tumours.

Strengths:

The study employs a powerful, two-pronged approach to identify Molidustat's targets. By using both Thermal Proteome Profiling (TPP) and an orthogonal chemical proteomic competition assay, the authors provide compelling evidence that GSTP1 is a genuine, direct off-target, effectively addressing the common limitation of indirect effects in proteomic screens.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

I only see the following relatively minor weaknesses, namely:

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

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

Impact:

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

Reviewer #1 (Public review):

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

(1) The text is well written.

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

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

Weaknesses:

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

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

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Suggested Analysis:

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

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

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

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

Suggested Analysis:

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

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

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

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

Suggested Analysis:

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

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

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

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

Suggested Analysis:

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

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

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

(5) Lack of justification of polydispersity of polyQ:

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

(6) Lack of initiative to connect to Experiments:

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

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

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

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

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

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

Strengths:

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

Weaknesses:

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

(1) General:

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

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

(2) Technical:

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

(2) Missing Rationale for Classification Analysis

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

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

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

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

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

(4) Questionable Assumptions Regarding Strategy

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

(5) Confounding Factors in GVS Interpretation

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

Reviewer #1 (Public review):

Summary:

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

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

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

Strengths:

The biochemical analysis seems solid.

Weaknesses:

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Comments on revised version:

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

Comment on revised version:

No comments - congratulations to the authors!

Reviewer #1 (Public review):

Summary

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

Strengths

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

Weaknesses

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

(1) Distinguishing NCCs from their prerequisites or consequences

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

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

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

(2) Drawing misleading conclusions from certain studies

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

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

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

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

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

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

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

(3) Justifying single-neuron cortical correlates of consciousness

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

(4) No mention of combined fMRI-EEG research

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

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

Weaknesses:

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

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

Assessment of Claims:

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

Impact and Utility:

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

Context:

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

Reviewer #1 (Public review):

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

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

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

Weaknesses:

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

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

Future directions

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

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

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

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

Reviewer #1 (Public review):

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

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

Comments on previous round of revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

The following issues remain unresolved:

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

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

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

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

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

Reviewer #1 (Public review):

Summary

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

Strengths

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

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

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

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

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

Weaknesses

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

The authors addressed all my concerns.

Reviewer #2 (Public review):

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

Comments on revisions:

No further comments.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

Weaknesses:

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

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

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

Reviewer #1 (Public review):

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

Major Comments:

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

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

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

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

(3) IMQ and systemic effects.

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

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

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

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

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

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

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

Joint Public Review:

Summary:

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

Strengths:

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

Weaknesses:

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

Closing statement:

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

Reviewer #1 (Public review):

Summary

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

Summary:

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

Strength:

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

Comments on revised version:

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

Reviewer #1 (Public review):

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #2 (Public review):

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

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

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

Reviewer #1 (Public review):

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

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

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

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

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

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

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

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

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

Hope these comments are helpful.

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Comments on revisions:

The authors have addressed all of my previous comments.

Reviewer #3 (Public review):

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

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

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Additionally:

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

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

None of note.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

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

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

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

Comments on revisions:

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

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

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

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

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

Weaknesses:

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

(1) The devil is in the details:

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

The authors combine discriminative auditory fear conditioning with longitudinal in vivo calcium imaging to ask how prelimbic (PL) representations of learned and generalized threat evolve across recent and remote memory time points. Using two different CS+ frequencies and a no-shock control group, they report that PL population activity tracks graded behavioral generalization, that population similarity is highest for tones eliciting strong threat responding, and that distinct subnetworks can be identified that appear to encode tone-specific sensory features versus learned threat-related response structure.

To my knowledge, this may be the first study to comprehensively examine neural encoding of fear generalization in prelimbic cortex (PL). The manuscript is ambitious and technically interesting, and several aspects are potentially important. In particular, the suggestion that neurons showing graded, learning-related response patterns become selectively stabilized over time is intriguing. The inclusion of two CS+ training conditions and a no-shock control also strengthens the case that at least some of the reported effects are related to associative learning rather than simple sensory differences. However, in its current form, the manuscript does not yet fully support the strength of the conceptual claims. Several issues limit confidence in the interpretation, including the possibility that repeated testing itself contributes to changes across days, uncertainty about the relationship between neural activity and freezing behavior, limited quantitative documentation of longitudinal cell registration, and a number of problems in figure clarity and statistical framing. Overall, the study contains promising observations, but the claims should be narrowed, and several analyses or controls would be needed to fully support the proposed framework.

Detailed Comments

(1) A general concern is that the repeated test procedure itself may contribute to extinction. Because the animals are exposed to multiple CS frequencies across multiple test days, and each tone is presented three times per session, some of the reported changes in behavior and neural activity across days could reflect extinction or repeated nonreinforced retrieval rather than the passage of time per se. This is especially relevant given that the manuscript makes claims about recent versus remote representations and representational drift over 30 days. At a minimum, the authors should discuss this limitation explicitly and temper claims about time-dependent changes. Ideally, they would include a control group in which animals are tested only once or twice (e.g., at an early and later time point with fewer CS frequencies), or a reduced-frequency testing design that minimizes extinction while still allowing evaluation of recent versus remote memory.

(2) More generally, some of the reported learning-related neural differences may be driven by behavioral differences, particularly freezing, rather than by learning or generalization per se. For example, animals that freeze more to certain frequencies may show corresponding neural response differences simply because freezing alters PL activity. The authors should examine this possibility more directly. Analyses testing whether recorded cells encode freezing behavior, or whether tone frequency-related neural differences remain robust when comparing high- and low-freezing epochs, would help determine whether the reported effects reflect learned stimulus value rather than behavioral state differences.

(3) A central feature of the manuscript is the analysis of neural response properties over an extended period of time, up to 30 days after learning. However, aside from a brief mention in the Methods that spatial registration was used, the manuscript provides very little quantitative information about this critical aspect of the study. The paper would be strengthened by including explicit metrics describing longitudinal cell tracking, such as the number and proportion of ROIs retained across all sessions, distributions of spatial-footprint correlations or centroid distances across days, and representative examples of matched imaging fields over time. Without this information, it is difficult to assess how strongly the longitudinal claims are supported.

(4) The text states that "Figs. 1c and 1d show GCaMP6f expression in PL, representative calcium footprints, and activity traces". However, the figure as presented does not clearly show all of these elements, at least not in a way that matches the description in the Results. The correspondence between text and figure should be corrected.

(5) The labeling of Figure 2a is insufficient for interpretation. The legend states that the panel shows raster plots of sound responsiveness, but the axes and scaling are not clearly defined. It is not clear from the figure what the x-axis represents, whether the y-axis corresponds to individual neurons, where the CS period occurs, or what the activity scale at the right denotes. Also, the term 'rasters' implies that spikes were analyzed. It seems that the spike inference approach (CASCADE) was only used for later analyses. Perhaps 'heat-plot' would be more accurate here? Generally, this figure should be annotated more clearly so that the reader can understand it without referring back to the Methods.

(6) In relation to Figure 3, the analysis of population-averaged responses across tone frequencies is useful, but the manuscript would be stronger with additional statistical analyses across time and across groups. For example, if the authors want to argue that learning induces graded changes in neural responses and that these evolve across time, they should directly compare within-group responses across days and also compare matched frequencies between the conditioned groups and the no-shock controls. These analyses would help establish whether the observed differences are genuinely learning dependent and whether they change significantly over time.

(7) The inclusion of two different CS+ frequencies and a no-shock control is a strength of the study and substantially improves the interpretation that graded neural responses are related to learning and generalization rather than to simple sensory processing or passage of time. That said, I am not entirely comfortable with the use of the term "inference" throughout the manuscript. What is being measured here appears closer to sensory generalization than inference in a stronger cognitive sense. The current task does not clearly require that animals infer hidden structure or stimulus value through abstract reasoning; rather, the generalized stimulus may simply be treated as similar to the conditioned cue. The terminology should therefore be reconsidered or softened.

(8) I also found the use of the term "valence" somewhat problematic. The manuscript appears to use valence to refer to graded responding across tones with different aversive significance, but valence typically refers more broadly to distinctions between appetitive and aversive value. Here, terms such as "threat value," "aversive value," may be more precise. The authors should consider revising this language throughout.

Reviewer #1 (Public review):

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #1 (Public review):

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #1 (Public review):

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

Major comment:

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

Specific comments:

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

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

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

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

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

(3) Protein expression analysis for additional reporters.

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Original Weaknesses:

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

Comments on revised version:

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

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

Reviewer #1 (Public review):

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

Comments on previous version:

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

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

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

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

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

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

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

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

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

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

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

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

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

Reviewer #1 (Public review):

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

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

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

Reviewer #1 (Public review):

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

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

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

Strengths:

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

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

(3) The displayed EM images document interesting phenotypes.

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

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

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

Weaknesses:

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

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

(3) EM images would benefit from better quantification.

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

Reviewer #1 (Public review):

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

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

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

Reviewer #1 (Public review):

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

Summary:

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

Strengths:

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

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