Reviewer #1 (Public review):

Summary:

In this manuscript, Flamholz and colleagues use metagenomic sequencing to profile the microbiome of individuals with sickle cell disease (SCD), the most common genetic blood disorder in the world. To build on previous studies that found dysbiosis in SCD, this manuscript aims to examine whether changes in either bacterial species or bacteriophages correlate with inflammatory hallmarks of the disease. The authors claim that sickle cell dysbiosis does not correlate with inflammatory hallmarks of the disease, but instead, aged neutrophil numbers and bacteriophages do. Appropriate control subjects and additional analyses are needed to support that conclusion.

Strengths:

The primary strength of this paper is the investigation into disease-associated changes in bacteriophages. This is an entirely novel idea in the sickle cell field, and based on the current results, may be an important, under-recognized disease hallmark. It is unclear, however, if phages are "the chicken or the egg" in terms of sickle cell inflammatory profiles; do these increases in phage number simply result from other disease processes, or are they in any way contributing to disease pathophysiology?

Weaknesses:

A primary weakness of the manuscript is the fact that the majority of individuals included in the control group maintain sickle cell trait (HbAS genotype). Although typically asymptomatic, it is unclear if this genotype is associated with microbial changes that would not be observed in a true control group (HbAA genotype). This is a significant limitation that may limit the ability to draw conclusions from the current data set.

Another key weakness is the lack of beta diversity assessment. Although decreased alpha diversity is observed in individuals with SCD, and specific bacterial taxa are differentially abundant following multivariate analyses, there is no overall comparison of bacterial community composition between individuals with SCD and controls. Prior to drawing conclusions about the relationship (or lack thereof) between the SCD microbiome and inflammatory markers, it is important to know if this study did indeed find disease-associated changes in microbiome composition.

It is unclear which individuals were used for aged neutrophil (AN) and molecular data assessments. For example, were children who were still receiving penicillin prophylaxis included in these specific assessments? Given the authors' previous work demonstrating that antibiotic treatment decreases AN pathology, it seems critical to limit all AN/molecular analyses to older subjects who are not on daily penicillin treatment (if possible).

A minor weakness is the continued use of "disease" vs. "healthy" indicators as primary microbiome metrics that are used for molecular correlations. The lack of metric specificity - and lack of discussion regarding which diseases were used to generate these indicators (how similar/different are they to sickle cell?) - could be said to make these metrics essentially meaningless.

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 satisfactorily addressed the comments raised in the previous round of review with textual revisions.]

Summary:

The manuscript by Singh et al. presents an application of MOA-seq to better define transcriptional control underlying the hypoxia response in human endothelial cells. This group's previously described MOA-seq technique allows for precise, identity-agnostic mapping of occupied sites of DNA-binding proteins across the epigenome and over time. Here, they applied MOA-seq to HUVECs under normal oxygen conditions or variable lengths of hypoxia treatment, comparing changes in occupancy over time and associating these changes with corresponding transcriptome alterations. This approach revealed thousands of dynamically occupied sites comprising 10 major kinetic clusters that appear to define distinct subsets and phases of the hypoxia response. Analysis of DNA motifs in these dynamically occupied regions captured the known major roles of HIF1A in the hypoxia response and also implicated new HIF1A-associated regulators. Importantly, they also identified many potential HIF1A-independent candidate TFs that act at HREs, which has been an outstanding question in the field. Additionally, this study identified ~7K additional sites not previously defined as regulatory elements by ENCODE.

Strengths:

Overall, this study is well executed and described, providing new biological insights as well as a rich data resource for the field. As MOA-seq was previously developed for use in plants, this work demonstrates the application of this method in mammalian cells and highlights its utility in identifying new potential regulatory sites not captured by DNase-seq or ATAC-seq. The conclusions made by the authors are well supported by the results, with the caveat that extensive use of DNA motif identification and ontology analyses invariably leads to some uncertainty regarding factor identity and gene network properties.

Reviewer #2 (Public review):

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

Summary:

This paper is an exciting follow-up to two recent publications in eLife: one from the same lab, reporting that slender forms can successfully infect tsetse flies (Schuster, S et al., 2021), and another independent study claiming the opposite (Ngoune, TMJ et al., 2025). Here, the authors address four criticisms raised against their original work: the influence of N-acetyl-glucosamine (NAG), the use of teneral and male flies, and whether slender forms bypass the stumpy stage before becoming procyclic forms.

Strengths:

We applaud the authors' efforts in undertaking these experiments and contributing to a better understanding of the T. brucei life cycle. The paper is well-written and the figures are clear.

Comments on revisions:

We thank the authors for the revised manuscript and for considering our comments.

Reviewer #1 (Public review):

Summary:

This manuscript reports the discovery and characterization of the first bifunctional degrader of tankyrase. Notably, the tankyrase degrader exhibits stronger β-catenin inhibition and tumor growth suppression compared to conventional tankyrase inhibitors. Mechanistically, while tankyrase inhibitors stabilize tankyrase and promote Axin puncta formation-thereby impairing β-catenin degradation-the degrader avoids this effect, resulting in deeper suppression of β-catenin signaling. These findings suggest that targeted degradation of tankyrase offers a novel therapeutic strategy for β-catenin-driven cancers. Overall, this is a compelling study with significant translational potential.

Strengths:

(1) The manuscript presents a rigorous and well-executed study on a timely and impactful topic.

(2) The biochemical and cellular characterization of the tankyrase degrader is thorough, and the comparative analysis with tankyrase inhibitors is insightful.

(3) The finding that tankyrase stabilization by inhibitors may interfere with Axin function is novel and significant. It aligns with earlier observations (e.g., Huang 2009) that transient tankyrase overexpression can stabilize β-catenin independently of PAR domain activity.

(4) The use of TNKS1/2 knockout cells expressing catalytically inactive tankyrase to demonstrate β-catenin inhibitory activity of the tankyrase degrader is elegant.

(5) The finding that the tankyrase degrader has superior anti-proliferative effects in colorectal cancer models has important therapeutic implications.

Comments on revised version:

I had a favorable opinion of the manuscript in the first round of review. I don't have additional comments on the revised manuscript. The manuscript looks fine to me.

Reviewer #1 (Public review):

Summary:

This "Tools and Resources" submission describes a platform for the modeling of stimulus-response relationships in the retina. It includes a repository for experimental data sets with standardized programmatic access, and a suite of software for constructing stimulus-response models and evaluating them.

Strengths:

(1) The paper is well written.

(2) The platform could serve an integrative function by connecting different research programs and offering a common baseline for evaluating stimulus-response models.

(3) The finding that there is "substantial explainable variance remains uncaptured by current models" is a useful insight to motivate further work and measure progress.

Weaknesses:

(1) The modeling supported by the package focuses on predictive accuracy at the cost of less interpretability.

(2) The article needs to make a stronger argument that this style of modeling is fruitful, especially when applied to the retina.

Main comments:

(1) Abstract machine learning vs mechanistic models. The "Core + Readout" architecture advocated here seems to be divorced from all the neurobiological detail that is already known in the retina. It mostly aims at prediction, not interpretation. Such a black-box modeling framework is useful in brain regions where we know very little about connectivity, or mechanisms, or even about the primary function being performed there, like in the mammalian cortex. In those cases, any model that can deliver a prediction is a step forward, even if it does not connect to biological mechanisms. But that's decidedly not the situation in the retina, where so many mechanistic details are known: from consensus cell types, to synaptic detail, to single-neuron biophysics, to circuit motifs. How can one connect this ML modeling approach with the extensive mechanistic knowledge available in retinal neuroscience? And can the combination somehow lead to a better understanding? The authors seem to recognize this tension (e.g. line 215ff and 370ff) but don't give it much weight. A stronger case needs to be made here for how this kind of modeling will advance the field.

(2) The "gradient field" approach. Figure 4c illustrates a case of this dissonance. The gradient field of the response increases with contrast in multiple directions. This is obvious a priori (see line 274) from the more mechanistic model we already have of this On-Off cell. These are the W3 cells described in www.pnas.org/cgi/doi/10.1073/pnas.1211547109. The circuit-based model from that paper, with rectifying on and off subunits from bipolar cells, gives a much more compact explanation for what the neuron does. Because each of the subunits has a spatio-temporal receptive field, this model can predict the entire dynamics to arbitrary stimuli, rather than just 2 dimensions of static stimuli as in the present analysis. So what is the value added here? Again, a stronger case needs to be made that these "Core + Readout" modeling activities enhance understanding.

(3) The "most exciting input" approach (Line 193ff):

- Presumably, some power constraint must be put on the stimulus? Otherwise, increasing the contrast will make it more exciting. What are these constraints?

- Presumably, this optimal stimulus is computed from the model based on non-optimal stimuli? What are the assumptions going into that?

- The most exciting stimulus is not necessarily the most useful characterization. Near its maximal firing rate, the neuron doesn't discriminate stimuli much, because the slope there is zero (line 237). Instead (or in addition), one would like to know along which stimulus axis the neuron is most sensitive. See e.g. discussion in Dayan & Abbott 2000, Figure 3.11.

Reviewer #1 (Public review):

In this article, the authors investigate how glutamate transporter function regulates excitability and synaptic coding in T-stellate cells in the mouse ventral cochlear nucleus. They test this in acute brain slices using whole-cell electrophysiology and artificially raise the relative local concentration of glutamate via pharmacological inhibition of transporter proteins. The main finding is that when sub-saturating doses of DL-TBOA are applied, cells become much more sensitive to synaptic input, diminishing the normally high fidelity of EPSP-spike coupling in these neurons. Notably, high-frequency stimulation in the presence of DL-TBOA reveals a large and slowly decaying AMPA receptor component that underlies persistent/rebound firing in earlier recordings. These effects are not seen in other ventral cochlear neurons, suggesting that rapid glutamate clearance in T-stellate cells, particularly, is important for auditory intensity coding. Overall, these experiments are well-performed, and the findings are robust, though there are some aspects that could be expanded to make the work more impactful. These include a better understanding of the relative contribution of neuronal vs glial transporters and an ability to separate the relative contributions of tonic glutamate concentrations in the cleft vs changes in membrane potential in action potential output. Additionally, there were some minor issues of clarity in both the figure presentation and the main text language that should be addressed.

Major Points:

(1) Given the dramatic effect of saturating DL-TBOA on tonic leak/RMP and that the sub-maximal concentration used in most of the experiments still varied between 25-50 uM, Figure 1 would be strengthened substantially by a dose-response curve. Ideally, 5 or 6 concentrations, plotting the effect on tonic current or RMP increase.

(2) Examining the contribution of glial (EAAT1/2) vs. neuronal (EAAT3) transporters (Fig 8) is intriguing but comes across as incomplete here, especially given the small number of recordings. Using a different non-selective EAAT inhibitor (TFB-TBOA) to chase the EAAT1/2 blocker combo seems like an odd choice, given that you have already characterized the effects of DL-TBOA well. One could also try a lower concentration (~50-100 nM) of TFB-TBOA since it is somewhat selective itself for glial EAAT1/2. Given the data presented, neuronal transporters (presumably EAAT3) appear to dominate the rapid clearance of glutamate at this synapse, but this point isn't emphasized or explored sufficiently.

(3) Separating the effects of depolarization vs. glutamate clearance was never explored. What effect does depolarizing the cell ~10 mV in control conditions (i.e., without TBOA) have on AP number/fidelity during synaptic stimulation experiments? The authors state that submaximal DL-TBOA generally causes no more than a 5 mV change in RMP, but tonic depolarization could also influence spike fidelity. This experiment could demonstrate that the increase in excitability during/after stimulation is not due to increased engagement of voltage-gated channels.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors apply the AsLOV2 domain to control the localisation and the exposure of two peptides (PMI and PMI-M3) that compete with Mdm2/MdmX for binding to p53, thus freeing p53 from these negative regulators and allowing its levels to rise. The authors follow an established strategy in optogenetics, which is to combine two layers of regulation for tighter control: (1) caging the peptide into the Ja helix of AsLOV2; 2) sequestration of the peptide away from its site of action using the LOVTRAP system.

Strengths:

The authors show that a reporter is activated when cells are exposed to light. A strength is in the lower background that was achieved after adding the second layer of regulation.

Weaknesses:

This study claims to be focused on the control of endogenous p53; however, endogenous p53 levels are not quantified. Moreover, endogenous p53 target genes are also not analysed. Only a synthetic reporter is quantified, which has been placed in the genome of HCT116 cells after the creation of a stable cell line. Microscopy images show only one or a maximum of two cells. Finally, the authors claim their strategy is a general one that can be applied to control other peptides, but they do not show this generality in this paper.

Reviewer #1 (Public review):

Summary:

This is an important and interesting manuscript that uncovers the cross-talk between mitochondrial quality control and phagosome maturation arrest imposed by Mtb.

A broader host pathogen (intracellular) question pertains to evading phagosomal maturation/arrest. While cellular events that culminate in this arrest have been largely elucidated, involvement of other organelles, such as mitochondria, has not been highlighted mechanistically. This manuscript paints a larger picture than the well-known conventional endolysosomal pathway and portrays a larger landscape involving elements of the mitochondrial quality control, such as mitophagy and mitochondrial-derived vesicles' involvement in the host-pathogen tussle.

Strengths:

The systematic characterisation to unravel the interplay between mitochondrial-related pathways and the endolysosomal system allows the authors to unearth some important findings.

Weaknesses:

The conclusions drawn require more robust experimentation and analysis.

Reviewer #1 (Public review):

Summary:

Fang et al. characterize the cellular basis of early ovarian development through a comparative analysis of single-cell transcriptomic data. The authors integrate a novel bovine scRNA-seq dataset, spanning six gestational stages (E38-E112), with stage-matched human (PCW6-16) and mouse (E11.5-E18.5) counterparts. Beyond identifying shared gonadal cell types across these three species, the study uncovers a previously uncharacterized bovine-specific cell population with steroidogenic features. Their analysis highlights conserved, dynamically expressed regulators, including TFAP2C and ZCWPW1 in germ cells and FOS and JUNB in granulosa cells. Furthermore, by employing a machine learning Support Vector Machine (SVM) model, the authors quantify cell-type conservation, demonstrating that while immune and germ cells are highly conserved across species, granulosa cells exhibit substantial evolutionary divergence. This study makes a significant contribution to developmental biology by establishing a comprehensive, cross-species single-cell roadmap of fetal ovarian development. By integrating livestock data with human and rodent models, the authors identify novel cellular states and provide a framework for assessing transcriptional conservation across species.

Strengths:

(1) While human and mouse fetal ovaries have been mapped, the inclusion of a high-resolution bovine dataset (107,930 cells total across the study) provides a critical "large mammal" perspective that is often missing from comparative studies.

(2) The identification of a bovine-specific cell population is an important finding. It suggests that ruminants may have a different developmental timeline for steroidogenic precursors (potentially theca cell ancestors) compared to rodents or humans.

(3) Training a Support Vector Machine (SVM) to quantitatively assess cell-type similarity is a major strength. It moves beyond qualitative UMAP "eye-balling" to provide a statistical probability of conservation.

(4) The study links gene expression to higher-order biological processes like epigenetic reprogramming and cell-cell communication (CellChat), providing a holistic view of the gonadal niche.

Weaknesses:

(1) The authors integrated publicly available scRNA-seq datasets generated across different laboratories and technical platforms. However, the specific methods used to control for and evaluate batch effects are not clearly described. It is critical to clarify whether the observed species-specific differences are purely biological or partly influenced by technical variation between datasets.

(2) A challenge inherent to all single-cell studies is the reliance on manual marker-gene-based annotation. While this is standard practice, it remains unclear how robust these assignments are, particularly for the novel "bovine-specific" population. Further evidence or cross-validation (e.g., through varied clustering resolutions or automated annotation tools) is required to ensure these clusters represent true biological states rather than computational artifacts.

(3) The authors utilized a linear SVM to assess cross-species similarity. However, it is not clear how this model performs compared to established single-cell mapping and comparative tools (e.g., MetaNeighbor or Seurat v5). Providing a justification for this specific SVM-based approach, or a brief comparison with existing benchmarks, would strengthen the methodological rigor of the study.

(4) While the computational evidence is compelling, the study would be significantly enhanced by independent validation of the "unclassified bovine-specific" cell population. To confirm the biological reality and reproducibility of this novel cell state, the authors should provide additional evidence. This could include in situ validation (e.g., immunofluorescence or in situ hybridization) to determine its physical location and morphology within the gonad, or demonstrating the presence of this specific cell population within an independent, non-overlapping bovine dataset.

Reviewer #1 (Public review):

Summary:

This manuscript is a narrative review addressing age-related alterations in SR-mitochondria interactions in skeletal muscle and their contribution to sarcopenia. It synthesizes existing literature on calcium signaling, mitochondrial dynamics, redox balance, and structural remodeling, and discusses potential interventions including exercise and pharmacological strategies. While the topic is timely and relevant, the manuscript largely reiterates established concepts without providing sufficient conceptual novelty, critical synthesis, or mechanistic insight beyond the current literature.

Strengths:

(1) Timely topic: The focus on SR-mitochondria communication in aging muscle is relevant and of growing interest.

(2) Broad coverage: The review compiles a wide range of literature spanning calcium handling, mitochondrial biology, ROS signaling, and exercise physiology.

(3) Clear organization: The manuscript is structured logically with thematic sections (SR, mitochondria, MAMs, aging, interventions).

(4) Didactic value: Could serve as a general overview for non-specialists entering the field.

Weaknesses:

(1) Lack of novelty and conceptual advance: The manuscript does not offer new hypotheses, frameworks, or critical reinterpretation of the field. Most statements summarize already well-established knowledge, and no unifying model or novel perspective is developed to justify publication in a high-impact journal like eLife.

(2) Limited critical analysis: The review is predominantly descriptive rather than analytical. Conflicting findings (e.g., MFN2 roles, MAM density changes, Ca²⁺ overload vs deficiency) are mentioned but not critically evaluated or reconciled. There is little discussion of limitations in the cited studies or gaps in the field.

(3) Overgeneralization and speculative claims: Several assertions are presented with insufficient nuance (e.g., causal links between MAM disruption and sarcopenia, or therapeutic efficacy of interventions). The distinction between correlation and causation is often unclear, reducing scientific rigor.

(4) Insufficient depth for a specialist audience: Despite its length, the manuscript lacks mechanistic depth in key areas (e.g., precise molecular regulation of MAMs in vivo, tissue-specific differences, quantitative aspects of Ca²⁺ flux). It reads more like a textbook summary than a high-level scholarly review.

(5) Redundancy and verbosity: Many sections repeat similar concepts (Ca²⁺ dysregulation, ROS effects, mitochondrial dysfunction) without adding new insight, leading to an unnecessarily long manuscript with limited added value.

(6) Weak integration of recent literature into a coherent narrative: Although many references are cited, they are not effectively synthesized into a cohesive argument. The manuscript lacks a strong central thesis or clearly defined take-home messages.

(7) Limited translational or experimental perspective: The section on therapeutic targeting is largely speculative and does not critically assess feasibility, limitations, or current clinical evidence.

Reviewer #1 (Public review):

Summary:

The question posed on cell-type-dependent relationships to theta-nested gamma rhythms is an important one. The authors use a variety of ontogenetic, imaging, electrophysiology, and computational techniques to show that reciprocal interactions between excitatory neurons and interneurons in the medial entorhinal cortex generate gamma oscillations. They measure LFP gamma, gamma power of postsynaptic currents in different neurons, spike phases with reference to LFP gamma, and spatial correlations of membrane potentials across a large population of neurons. Arguing (correctly) that gamma rhythm in this setting is generated through a pyramidal-interneuron network gamma (PING) mechanism, they demonstrate cell-type-specific differences in gamma phase-locking. While they show spatial dependencies of sub-threshold voltages and even argue for topographic clustering, these could simply be reflections of the synchronous stimulation paradigm that they use.

Overall, I appreciate the methodology and rigor, but would have expected more from the study in terms of relevance to physiological stimulation conditions as well as in terms of mechanisms underlying the differences that they report here..

Strengths:

The authors are rigorous in how they conduct the experiments, report the data, and perform the analyses. The modeling respects the heterogeneities and is truthful to the experimental design. The conclusions on PING mechanisms are fine, but are not unexpected given the circuitry of the mEC.

Weaknesses:

The interpretation of the conclusions, while for the most part is fine, could have been better, especially given the conceptual limitations of the experimental design. The modeling part could have gone beyond simple descriptive matching and addressed mechanistic questions.

Reviewer #1 (Public review):

Summary:

This study investigates how Ca2+ levels inside the RGCs' mitochondria relate to whether these cells survive or die after injury to the optic nerve. The authors used advanced in vivo fundus live imaging techniques in mice to watch these changes unfold in real time, combined with genetic and drug-based tools to alter calcium flow into these compartments. Their central finding is a striking paradox: cells that naturally survive injury tend to have higher baseline calcium levels in these compartments, yet experimentally reducing calcium entry protects the broader population of cells from death.

Strengths:

The authors are applying sophisticated biosensors to track cellular chemistry in living animals over days and weeks. The tools and methods are creative and direct to detect the longitudinal RGC degeneration with mito-Ca2+ imaging. The topic and research aspect are novel and attractive. The results are significant, showing a clear relationship between the mito-Ca2+ regulatory machinery and cell survival.

Weaknesses:

The details of the mitochondrial-located signal of the Ca2+ sensor need to be further proved in the mito-matrix or between the mito-membranes. The study primarily describes a correlation and a surprising experimental outcome without fully explaining the underlying biological reasons for the paradox. While the evidence supporting the phenomenon is good, the mechanistic insight into why high calcium is linked to survival, or why lowering it helps after injury, remains limited.

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:

Chen et al. describe metabolic phenotypes in Dp16 Down Syndrome mice, specifically the Dp(16)1Yey/+ mice - segmental duplication model carrying a majority of the triplicated Hsa21 gene orthologs. The group has performed metabolic phenotyping data in chow and high-fat diets, as well as undertaking a transcriptomic and metabolomic approach in tissues such as white and brown adipose tissues, liver, skeletal muscle, and hypothalamus to reveal both shared and sex-specific differences. The group describes sexual dimorphism in body weight, body temperature, food intake, and physical activity. Core shared features are insulin resistance, glucose intolerance, impaired lipid clearance, and dyslipidaemia in the Dp16 mice. They report tissue signatures of immune activation and a pro-inflammatory state, ER and oxidative stress, fibrosis, impaired glucose and fatty acid catabolism, altered lipid and bile acid profiles, and reduced mitochondrial respiration in Dp16 mice.

Strengths:

Overall, this is a good study with detailed, comprehensive data from an excellent group who have previously published on metabolic phenotyping of 2 other Down Syndrome mouse models. Although somewhat descriptive, it does certainly add to the current field and understanding of strengths and weaknesses of Down Syndrome mouse models, as well as identifying new features whilst strengthening previously suggested mechanisms.

Joint public review:

Summary:

In this study, Stirtz et al., performed a targeted screen of 80 Drosophila strains carrying heterozygous MiMIC insertions in genes that are homologous to human genes that have been linked to autism spectrum disorders (ASD). This is an important and timely topic, as human genetic studies have identified a large number of ASD risk genes, yet the functional characterization of many of these candidates remains limited. The authors identify 48 putative mutants with altered sleep, activity, or social behavior. They then focus on one hit, domino (the orthologue of human SRCAP), for which the heterozygous MiMIC mutants show altered behavior in males but not in females. They show that domino is a candidate regulator of sleep, activity, social behavior, transcriptional programs, and RNA splicing. The authors molecularly validate that the heterozygous MiMIC insertion in domino causes a 50% reduction in gene expression, and use RNA-seq to show that the heterozygous MiMIC males and females have altered gene expression profiles and splicing patterns. Finally, they use immunostaining against the commonly used synaptic marker, Bruchpilot, to show that both males and female heterozygous domino flies express a higher immunosignal compared to the wild-type control.

Strengths:

This work provides potential genetic links between human ASD genes and fly behavioral phenotypes. Overall, it represents an ambitious and technically valuable effort that generates a substantial behavioral dataset across a large number of ASD-associated orthologues and develops quantitative analytical approaches to extract information from complex phenotypes. One strength of this study is its focus on heterozygous mutants, which is more representative of human scenarios. The study also provides a potentially useful resource for the field, particularly through the identification of candidate genes and behavioral signatures that may warrant future mechanistic investigations. The screening experiments and analysis are well conceived, the manuscript is very clearly written and is easily understandable, and the concise, accurate interpretations for each result, aided by clear graphic representation of multiple dimensions in the behaviors tested, allow the reader to understand the paper with ease.

Weaknesses:

The work presents a few important weaknesses, especially with regard to the genetic and molecular validation of the mutants identified.

(1) The authors validate that the MiMIC insertion affects the gene of interest only for the domino gene. The original MiMIC study (PMID: 25824290, eLife) reported that ~8% (5/63) MiMIC lines do not function as strong loss-of-function alleles. Thus, of the 48 hits identified here, one would estimate that ~4 of them may not cause the loss of function of the gene defined by the MiMIC insertion. To strengthen their claim, the authors would need to confirm that all of the MiMIC lines that they consider as hits do indeed significantly reduce the expression of the target genes.

(2) Although the authors document that they validated the phenotype seen in the domino MiMIC line using a second mutant allele (Trojan), these two mutants share the same genetic background because the Trojan line was made from the MiMIC line via recombinase-mediated cassette exchange. Thus, the phenotype seen in the MiMIC and Trojan lines would need to be confirmed using a completely independent mutant in order to demonstrate that the reported behavioral, molecular, and synaptic defects reported can be fully attributed to the partial loss of domino function. Also, while the authors performed an RNA-seq experiment in both the MiMIC and Trojan lines, they do not show whether the Bruchpilot phenotype is also seen in the Trojan allele. Thus, this phenotype would also need to be examined in the Trojan allele or, preferably, in a mutant allele that is independent of the MiMIC line.

(3) The RNA-seq results would benefit from a discussion of potential compensatory or secondary transcriptional effects resulting from the constitutive domino reduction, particularly since the expected global bias toward transcriptional downregulation was not observed. In addition, some neurobiological interpretations appear stronger than currently justified by the literature or the data presented, particularly regarding the Bruchpilot immunoreactivity analyses and their relationship to sleep-regulatory circuits. Additional validation using better-established sleep-related neuronal populations, together with a clearer discussion of sex-specific effects and alternative interpretations of the observed phenotypes, would substantially strengthen the manuscript.

(4) An explanation of the extensive PCA analyses performed would help the naïve reader.

Reviewer #1 (Public review):

Summary:

In the manuscript "A stable cryogenic fluorescence microscope for correlative super-resolution light and electron microscopy," the authors demonstrate a new cryogenic light microscopy design and characterize its temperature and spatial stability. The manuscript does a good job of reviewing the state of the field and highlights the need for improved cryogenic microscope stages. The system avoids challenges associated with vacuum-based designs, particularly vacuum transfer systems that can be difficult to engineer, while also showing minimal ice contamination and drift, which are the primary challenges associated with open cryostat systems.

Strengths:

The key strengths of the manuscript are the simple design and the significant level of detail provided in the description of the cryogenic stage. This represents a valuable step forward for the field by providing a home-built, non-vacuum stage design that others can emulate.

Weaknesses:

There are only minor weaknesses or issues to address, which, if resolved, would strengthen the manuscript overall.

(1) A key element of the design gets little attention, which is the plastic cap for the objective. It is not entirely clear to the reader how this is being used except as something of a thermal break between the cryogen environment and the objective, but there are some questions. Is the objective housing touching the plastic cap? Where is the front of the cap relative to the front objective lens? Is the front objective lens exposed to the cryogenic environment? Could the authors provide some 3D views of that in an SI figure? This would help clarify.

(2) The refilling system is not shown in the diagrams provided in Figure 1 and S1 in sufficient detail. How is the system mechanically coupled to the dewar on the microscope stage? Are there any concerns about coupling vibrations onto the table?

(3) There is a description on page 6 that a rectangular aperture is used to align the excitation with the position and orientation of the sample. I know the authors are using this for excitation of the lamella, but without saying so in this text, it is confusing. I would consider stating that this is for future work involving excitation of lamella and then citing their preprint.

(4) In Figure 2d, the z-drift is shown with the focus lock correction applied. This is highly relevant, but I also think it would be good to plot the z position plus the stage position in an SI figure. This will give a better idea of the mechanical stability of the system. Also, in this figure, I wonder if the authors could comment on the source of the jumps in lateral position. For example, just before 30 minutes. Lastly, I would make the lower plot have a tighter y-axis range. It is hard to see anything, hence the inset.

(5) The ice contamination looks minimal in Figure 3. I think it would benefit the manuscript to have lower magnification images as well, to show the level of ice contamination across a representative square. This would be good, but only if the authors have it in hand.

(6) In Figure 4b, the y-axis is unclear. It looks like it has been normalized. Consider revising.

(7) A fluorescence intensity trace for the data shown in Figures 4c and f would be helpful to show the single-molecule behavior.

Reviewer #1 (Public review):

Summary:

The authors investigate how site-specific acetylation within the histone H3 folded domain affects RNA polymerase II transcription through nucleosomes. They focus on H3K56ac, H3K64ac, and H3K122ac, prepare chemically defined nucleosomes carrying each modification, and compare their effects using an in vitro transcription assay, cryo-electron microscopy structures, and micrococcal nuclease sensitivity assays.

The main finding is that H3K56ac and H3K122ac increase production of full-length run-off transcripts and reduce pausing near the nucleosomal dyad region, whereas H3K64ac has little detectable effect under the same reconstituted conditions. The structural analyses suggest that H3K56ac weakens or destabilizes DNA near the entry/exit region, while H3K122ac alters histone-DNA contacts near the dyad. These observations support a model in which different acetylation sites within the H3 folded domain influence nucleosomal transcription barriers through distinct local effects on histone-DNA interactions.

This is a useful study because it examines histone core-domain acetylation using chemically defined nucleosomes and directly compares several modifications in the same experimental system. However, the broader cellular context of these modifications is not sufficiently developed, and some mechanistic conclusions rely on correlations between static nucleosome structures and endpoint transcription assays rather than direct observation of polymerase passage through modified nucleosomes.

Strengths:

(1) The study uses site-specifically acetylated H3 proteins and reconstituted nucleosomes, allowing direct comparison of H3K56ac, H3K64ac, and H3K122ac under controlled conditions.

(2) The combination of transcription assays, cryo-electron microscopy, and nuclease sensitivity assays provides multiple lines of evidence, particularly for increased DNA end flexibility in H3K56ac nucleosomes.

(3) The authors analyze unmodified, H3K56ac, H3K64ac, and H3K122ac nucleosomes in parallel, with reported structural resolutions of approximately 3 Angstroms and accompanying validation materials.

(4) The negative result for H3K64ac is informative, because it distinguishes the direct effect of this modification in a minimal reconstituted system from prior cellular associations with active chromatin and histone eviction.<br /> The comparison with H3 N-terminal acetylation highlights that acetylation within the folded domain may affect transcription at different positions or by different mechanisms than tail acetylation.

Weaknesses:

The rationale for focusing on H3K56ac, H3K64ac, and H3K122ac has not been developed sufficiently. The manuscript would benefit from a clearer summary of what is known about the abundance of these modifications in cells, the enzymes or histone metabolic pathways that may introduce or remove them, and whether they are thought to occur before histone deposition, on assembled nucleosomes, or during nucleosome remodeling.

The central mechanistic model is based mainly on correlations between structures of free nucleosomes and endpoint transcription assays. The study does not directly observe RNA polymerase II paused at or passing through the relevant nucleosomal positions, so the proposed link between local structural changes and reduced pausing should be stated with appropriate caution.

The H3K56ac interpretation is supported by both structural observations and nuclease sensitivity data, but the map comparison underlying the reduced entry/exit DNA density is still mostly qualitative. The manuscript should more clearly state the map comparison conditions, such as contouring and local map quality, so that non-specialist readers can judge how robust the local density differences are.

The H3K122ac mechanism is plausible, but the evidence for dyad destabilization is more indirect. The main support comes from the orientation of the K122 side chain and its distance from DNA, while an independent biochemical test of dyad-region destabilization is not provided.

The transcription assay appears to include statistical testing, but the figure legend and methods should more clearly state which tests were used, what comparisons were made, how n was defined, and whether multiple-comparison correction was applied.

The relationship between the 198 bp transcription template, the linker DNA, the 9-base mismatched region, and the DNA regions modeled in the cryo-electron microscopy structures is somewhat difficult to follow. This does not necessarily require new experiments, but a clearer explanation would help readers connect the transcription assay design with the structural models.

The use of H3.2 C110A for chemical ligation and the use of the PL2-6 single-chain antibody fragment for cryo-electron microscopy sample stabilization are reasonable technical choices, but their purposes and possible effects on interpretation should be explained more clearly for readers outside structural biology.

Because the work uses a minimal in vitro system with human nucleosomes and Komagataella phaffii RNA polymerase II/TFIIS, the conclusions should be limited to direct physical effects on nucleosome transcription barriers unless cellular cofactors, remodelers, histone chaperones, additional modifications, and nucleosome positioning are addressed or discussed.

Reviewer #1 (Public review):

Summary:

This manuscript uses simulations and MSMs paired with experimental binding assays to examine the binding mechanisms of different antibodies to their targets. The authors argue that contacts in encounter complexes play an important role in determining the association rates and binding affinities that distinguish more mature antibodies from less efficacious antibodies from earlier in the maturation process.

Strengths:

The idea is interesting, and the combination of computational models and experiments is a good direction.

Weaknesses:

The manuscript focuses heavily on kinetics, but it is not clear whether the simulations recapitulate the relative rates of binding of the two antibodies. The relationship between the simulated binding behavior and the experimentally observed kinetic differences is therefore not fully established.

The comparison of committor probabilities or fluxes between the two antibodies may not be appropriate. These properties are related to the barrier height the system has to cross to move forward vs back to the starting state, under the simplifying assumption that the properties of other states aren't critical. Even in this simplified case, the same flux or committor probability could occur with very different barrier heights, e.g., rates or transition probabilities.

Some claims are presented in a very qualitative way that people who aren't experts in MSMs may have difficulty tying to the results in Figure 1.

DOI: 10.1016/j.celrep.2026.117477

Resource: (ZFIN Cat# ZDB-ALT-120117-1,RRID:ZFIN_ZDB-ALT-120117-1)

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-ALT-120117-1

What is this?

Reviewer #2 (Public review):

Summary:

In antibiotic research, accurately measuring decreases in bacterial populations is essential. The authors conducted a comprehensive evaluation of the luminescence assay, a commonly used but previously under-quantified method, benchmarking it against the gold-standard CFU counting approach. They found that luminescence measurements generally aligned with CFU results but sometimes reported slower decline rates for certain antimicrobials. These discrepancies were linked to differences in how the two methods capture biomass and colony formation, which vary with the antimicrobial's mechanism of action. The study demonstrates that luminescence assays can serve as a high-throughput alternative to labor-intensive CFU counting, provided their limitations are understood and corrected.

Strengths:

The authors developed a mathematical model to partially correct luminescence-based measurements, making the approach broadly applicable to several commonly used antibiotics. They also analyzed antibiotic-treated single-cell morphologies and linked filamentation to bulk luminescence signals. This analysis helped define the range of drug conditions under which luminescence assays provide reliable estimates of bacterial dynamics.

They extensively evaluated the method using 20 antibiotics and one antimicrobial peptide, encompassing many of the most commonly used agents and experimental factors (e.g. treatment time) typically considered in antibiotic research.

Comments on revised version:

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

Reviewer #1 (Public review):

Summary:

The authors describe a clever genetic system based on rapamycin-inducible expression of a beta-galactose reporter. The authors compare this spectrophotometer-based readout to the parasite reduction rate version 2 (PRRv2) recently described by some of the same authors and based on incorporation of [3H]-hypoxanthine. The results are generally comparable, with some differences for slower-acting compounds. The authors report that this format is better suited for higher-throughput studies and requires less time to quantify the time-dependent onset of parasiticidal action compared with the PRRv2.

Strengths:

This is a very well-executed and well-described body of work with a comprehensive set of analyses.

Weaknesses:

The authors should revise their text to also describe other methods used to quantify parasite growth. This method saves time compared to the PRRv2 but is too complex for simple screening of antiplasmodial activity of agents tested alone. Its value lies in assessing the speed of action of compounds tested in combination.

There are a number of areas for improvement:

(1) Many antimalarials have quite specific times of action. Are these MULTI-i2 assays, and the comparator PRRv2 assays, conducted with asynchronous cultures? This should be described in the methods and referred to in the text (apologies if I missed some references).

(2) The authors correctly state that flow cytometry-based readouts, such as with MitoTracker alone, can limit throughput and that MitoTracker alone can produce spurious results. The authors should cite work from other labs that combine MitoTracker with a nuclear dye, such as SYBR Green I. I think others have also been used, such as YoYo-1, which overcomes the limitations of using MitoTracker alone. Also, many labs use a nuclear dye such as SYBR Green I in a spectrophotometer-based format that enables rapid processing of plates at scale (96, 384, or even 1536 wells per plate). Luciferase-based screens have also been used in large-scale screening campaigns. The introduction should cite these various approaches, especially as the MULTI-i2 method is quite a complex screen with an initial period of drug exposure (up to 3 days) followed by a five-day phase initiated by rapamycin addition to induce expression of the beta-gal sensor.

(3) It would be helpful for authors to provide some indication of the cost comparison between the PPRv2 and MULTI-i2.

(4) Also, the authors should indicate whether these reagents will be deposited in a repository such as BEI Resources. They should also indicate conditions for other groups to request these materials, such as whether an MTA is required.

(5) The pharmacological models are interesting, but likely well out of the range of expertise of many labs. Has code been deposited into public repositories that make it possible for other labs to implement these analyses?

Reviewer #2 (Public review):

Summary:

The manuscript by Wang and colleagues aims to determine whether hepatic glucose metabolism is differentially regulated by the left and right sides of the LPGi and to reveal decussation of hepatic sympathetic nerves.

The authors used tissue clearing to identify sympathetic fibers in the liver lobes, then injected PRV into the hepatic lobes. Five days post-injection, PRV-labeled neurons in the LPGi, which were identified. The results indicated contralateral dominance of premotor neurons and partial innervation of more than one lobe. Then the authors activated each side of the LPGi, resulting in a greater increase in blood glucose levels after right-sided activation than after left-sided activation, and in changes in protein expression in the liver lobes. These data suggested lobe-specific modulation of HGP. Chemical denervation of a particular lobe did not affect glucose levels due to compensation by the other lobes. In addition, nerve bundles decussate in the hepatic portal region.

Strengths:

The manuscript is timely and relevant. It is important to understand the sympathetic regulation of the liver and the contribution of each lobe to hepatic glucose production. The authors use state-of-the-art methodology.

Weaknesses:

(1) Image clarity was improved in some cases, but not in others. For example, Figure 3I, showing c-Fos expression, is not convincing due to the image quality and lack of orientation.

(2) The methods section states that 8-weeks-old male mice were used in the experiments without specifying the experiments (e.g., brain injection with AAVs or PRV organ inoculation). The authors should include these details.

(3) The authors should use the exact location of pre- and postganglionic neurons as they often refer to neurons in the sympathetic chain. Their findings should be compared with the existing literature on the location of preganglionic cells.

(4) Figure legends should be revised and matched with the text.

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 cannot 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.

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.

Comments on latest version.

Nice to see the added extra analyses. Can't see any more will be achieved via further rounds and happy with the summary to stand as is.

Reviewer #1 (Public review):

Li and Wu, in this article, explore the proliferation of wall-less L-forms derived from Bacillus subtilis as mimics for protocells and report an interesting new mechanism for their proliferation. The authors carry out live-cell imaging of the L-forms and find that the clusters of cells forming proto-colonies proliferate better than the isolated single cells of L-forms. They further examine the causes for this indefinite proliferation of proto-colonies of L-forms, as compared to the isolated cells, which lyse and die out sooner. The authors show that when L-forms exist as isolated single cells, the growth in volume exceeds the rates at which surface area increases, leading to lysis. The authors further quantify the circularity and effective radius in growing proto-colonies, qualitatively estimate membrane tension and suggest that the confined space allows for mechanical shear in these cells. They propose that the mechanical stress on the membranes from adjacent cells in confined spaces deforms membranes and supports cell division to keep the population growing. These findings are also supported by modelling the proto-colonies in quasi-2D planes.

The study is quite interesting and significant as it has implications for both evolutionary aspects as well as clinical importance, given the proliferation of certain pathogens as L-forms. The aspect of carrying out long-term imaging of colonies of L-forms as spatially constrained entities and the findings are fascinating. While the conclusions presented are backed by experiments, I only have a few questions concerning the proposed mechanism of division and proliferation of these proto-colonies.

(1) The authors propose that the growth of neighbours leads to shearing forces in membranes and show that membrane tension increases at the periphery of the proto-colonies. They suggest that the increased membrane tension leads to a greater chance of deformation, enabling cell division. However, it is not quite clear how greater membrane tension could lead to cell division. Studies have suggested that membrane fluidisation is important for the cytokinesis event, which includes FtsZ-based division (Ramirez-Diaz, 2025).

(2) Thus, it becomes quite important to rule out any role for the cytoskeletal proteins in the observed division with an increase in membrane tension. The authors note in line 188 that the division in protocells is independent of FtsZ, but this independence is for protocells that divide by extrusions and resolution, where the membrane is highly fluidised (Mercier et al., 2012).

(3) The authors may use the L-form derivative where the FtsZ protein can be depleted and assess the proliferation of the proto-colonies. Likewise, authors should rule out the role of MreB as well.

(4) Although the growth rates have been shown to be similar for proto-cells and the proto-colonies, and only the membrane tension has been shown to be higher at the periphery, it is also important that the authors rule out any increased lipid synthesis in the fraction of dividing cells in these proto-colonies. Without this, one could also envisage a model where membranes are fluidised due to an increase in lipid biosynthesis in a fraction of cells in these confined spaces, leading to increased vesiculations which experience membrane shear and deform. The authors can also consider examining proto-colonies of L-forms of branched-chain fatty acid-deficient strains.

(5) Lastly, why does CellROX stain the proto-colonies? Are these tightly packed cells experiencing higher oxidative stress, and could that also contribute to membrane tension? This should at least be discussed.

Reviewer #1 (Public review):

Summary:

Flexible natural behavior requires flexible sensory-motor mapping. In the visual domain, a visual stimulus at one location can guide a saccade toward another. How the receptive field (RF) and motor field (MF) properties of oculomotor structures support this flexibility is not known. Dotson and Reynolds address this question in the marmoset, using oblique Neuropixels penetrations across horizontal segments of the frontal eye field+, supplemented by electrical microstimulation. They report that visual RF and saccade MF vector angles each change smoothly with occasional abrupt jumps, that the two maps are organized as mosaics at distinct preferred spatial scales, and that a moiré interference pattern arising from a constrained spatial-scale mismatch between partially correlated mosaics reproduces the empirical distribution of RF-MF angular differences. They conclude that visuomotor flexibility is embedded in the geometry of mismatch and matches between visual and motor maps.

Strengths:

(1) The question is well-motivated. Sensory-motor mapping is known to be flexible, and asking whether the topographic relationship between the two maps itself supports that dissociation is a fresh reframing of a long-standing problem in oculomotor control.

(2) FEF+ lies on the smooth marmoset cortical surface, which permits high-density horizontal sampling that would be difficult in the macaque arcuate sulcus, and oblique penetrations are a sensible way to track tuning across the surface. The dataset is substantial by the standards of the field (39 sites of high-density recordings across two animals, several thousand isolated units).

(3) The data are thorough, and the convergence of three independent lines of evidence is the strongest feature of the paper. Unit recordings, electrical microstimulation, and two architecturally distinct generative models point to the same organization.

(4) The central idea is conceptually novel. The proposal that flexibility can reside in the geometry of the maps, rather than only in time-varying activity, is original, and it generates concrete, testable predictions for tasks that require flexible visuomotor routing.

Weaknesses:

Major concerns

(1) The analysis collapses each oblique penetration onto a single horizontal axis and pools angles across all cortical layers, treating cortical distance as purely tangential. Because the trajectory is angled, horizontal distance and depth are confounded, so some of the apparent RF-MF drift along a penetration could reflect a laminar transition, in addition to tangential mosaic structure.

(2) RFs and MFs are estimated from the same free-viewing sessions in temporally adjacent epochs, leaving each measurement open to contamination by the other. Activity near a saccade can reflect peri-saccadic remapping rather than the stable retinotopic RF, and saccade-aligned activity following a recent flash can carry a residual visual component, given the long-lasting visual responses in FEF+ (>500 ms). Residual cross-contamination of this kind would tend to make RF and MF angles look more similar than they are, inflating the apparent local coupling and biasing the RF-MF difference distribution that the moiré model is fit to.

(3) The paper claims that visual and saccade mosaics occupy distinct spatial scales, but the two preferred spatial frequencies are close, and the separation is summarized by overlapping "failed-test" bands rather than by a statistical test or confidence interval on the preferred frequency itself. The reliability of this separation is not established.

(4) It is not clear whether the moiré model is a better model than the non-mosaic alternative. The moiré models are shown to be consistent with the data through failure to reject a Kolmogorov-Smirnov null, which is a weak form of evidence, and they are not benchmarked against a non-mosaic alternative or null model. The AM/NM convergence demonstrates architecture independence, but not that a mosaic organization is required.

Minor concerns

(1) The link from topography to behavioral flexibility (such as anti-saccades and other context-dependent transformations) is presented as a prediction but is not tested with any task manipulation. The work establishes an organizational principle and a plausible generative mechanism; whether that organization is actually exploited during flexible behavior remains open, and the framing should make this clear so the functional claim is not over-read.

(2) It is unclear how relative depth (depth 0) is defined and how layer boundaries were assigned. The Methods mention common-average re-referencing for CSD and local field analyses, but no CSD or power-depth profile is shown to anchor the layer IV / depth 0 reference across penetrations.

(3) The Discussion is brief relative to the strength of the claims. It would be helpful to address the concerns and alternative explanations above, where these cannot be fully resolved by the data.

Reviewer #1 (Public review):

Summary:

The authors wanted to better understand how the various septin-associated kinases contribute to septin organization and function in budding yeast. This question has been recently addressed by similar kinds of studies but there are still some open questions, particularly as regards to what extent the kinases may interact with and/or modify components of the contractile ring that drives cytokinesis.

Strengths:

This study uses sensitive imaging with good temporal and spatial resolution to monitor the localization of various proteins in living cells. Particularly informative is the use of a GFP/GFP-binding-protein "tethering" approach to ask if the requirement for one protein can be bypassed by physically tethering another protein to a third protein. Results from a yeast two-hybrid assay for measuring protein-protein interactions in vivo are buttressed by direct in vitro binding assays using purified proteins, which is important given the likelihood of "bridging" interactions between yeast proteins in the two-hybrid approach. The authors' conclusions are quite well supported by the data.

Weakness:

Ultimately, while the study provides some interesting and novel insights, we still don't understand which phosphorylation events on which proteins are important for the events occurring at the molecular level, so the advance in knowledge is somewhat incremental.

Reviewer #1 (Public review):

Summary:

The extent P. falciparum liver stage parasites export proteins into the host cell is unclear. Most blood stage exported proteins tested in liver stages were not exported. An exception is LISP2 that is exported in P. berghei but not P. falciparum liver stages. While the machinery for export is present in liver stages, efforts to demonstrate export have so far been mostly unsuccessful. Parasite proteins exported during the liver stage could be presented by MHC and thereby become the target of immune control, incentive to study liver stage export and identify proteins exported during this stage. However, particularly for P. falciparum it is very difficult to study liver stages.

This work studies LSA3 in P. falciparum blood and liver stages. The authors show that this protein is exported into the host cell in blood stages but in liver stages no or only very little export was detected. A disruption of LSA3 reduced liver stage load in a humanized mouse model, indicating this protein contributes to efficient development of the parasites in the liver.

The paper also studied the localization of LSA3 in blood stages and used a known inhibitor to show that it is processed by plasmepsin 5, a protease important for protein trafficking. The work also showed that LSA3 is not needed for passage through the mosquito.

Strengths:

The main strength of this work is the use of the humanized mouse model to study liver stages of P. falciparum, which is technically challenging and requires specialized facilities. The biochemical analysis of LSA3 localization and processing by plasmepsin 5 are thorough and mostly overcame adverse issues such as a cross-reactive antibody and negative influence of the GFP-tag on LSA3 trafficking. The mosquito stage analysis is also notable as these kinds of studies are difficult with P. falciparum. However, there was no evidence for a function of LSA3 in mosquito stages.

Weakness:

The cross-reactivity of the antibody together with the co-infection strategy prevents reliable assessment of LSA3 localization in liver stages. Despite of this it seems LSA3 is not exported in liver stages and the paper does not bring us closer to the original goal of finding an exported liver stage protein.

While the localization analysis in blood stages is well done and thorough, the advance is somewhat limited. LSA3 may be in structures like J dots, but this hypothesis was not tested. Although parasites with a disrupted LSA3 were generated, the function of this protein was not explored. However, this was now done in a separate study focussing on blood stage parasites (PMID: 41135800).

Due to the difficulty of working with humanised mice, it was not possible to refine some of the conclusions and questions remain:<br /> The impact on liver stage development is interesting, but which phase of the liver stage is affected, and the phenotype remain largely unknown. The co-infection used (WT together with LSA3 mutant) has the advantage of a direct comparison of the mutant with the control in the same liver but complicates phenotypic analysis if the LSA3 antibody is also cross-reactive in liver stages. This issue adds a question mark to the shown localization and precludes phenotypic comparisons. It was also not possible to determine if the cross-reactive protein is expressed at that stage. While this might have been evident from the mixed WT/mutant infection (if all cells are positive for LSA3 there is cross-reaction; if about half of the cells are negative, there isn't) but assessing this failed.

Significance:

It is important information that LSA3 contributes to efficient liver stage development. However, neither LISP2 nor LSA3 seem to be exported in P. falciparum liver stages and can't confirm the potential of vaccines with proteins exported in this stage. LSA3 is still important and may still be the target of the immune response, but based on this work, probably not due to export in liver stages.

Reviewer #1 (Public review):

Summary:

The study by Wang et al. investigates cardiac electromechanical modeling and simulation techniques, focusing on the calibration and validation of ventricular models according to ASME V&V40 standards. The researchers aim to calibrate model parameters to align with key biomarkers such as QRS duration and left ventricular ejection fraction and validate the model against independent measurements such as displacement and strain metrics. The authors also examine the impact of parameter variations on deformation, ejection fraction, strains and other biomarkers. The overarching aim of the study is to give credibility to the underlying computational electromechanics framework as a step towards the cardiac Digital Twin vision.

Strengths:

(1) The study presents a solid validation strategy for cardiac models based on independent data.

(2) It integrates electrophysiological, mechanical, and hemodynamic biomarkers for sensitivity analysis and calibration.

Weaknesses and Limitations:

(1) Model Assumptions: The study relies on several simplified modeling assumptions that do not reflect the current state-of-the-art:

a) Isotropic scaling of the ventricular mesh to generate an unloaded reference geometry.

b) Simplified afterload and preload models that do not consistently capture the full range of physiological responses.

c) Simplified epicardial boundary conditions.

These limitations are appropriately acknowledged and discussed by the authors in a dedicated Limitations section.

(2) Numerical Framework:

a) The numerical framework used for the mechanical part of the model may be susceptible to locking effects that could contribute to artificially stiff and less contractile behavior. This is indicated by a ten-fold scaling of the peak active contractile force parameter relative to literature values and notable sensitivity of the model to the tissue compressibility parameter. While - as acknowledged by the authors - part of this can be attributed to simplified modeling choices, other comparable studies have not reported similar issues.

b) The human electrophysiology model is not described in enough detail. Currently, it is not mentioned in the manuscript that an Eikonal model was used to compute activation times on the endocardial surface to be robust against coarse mesh resolutions.

(3) Geometrical model and digital twin: The model presented combines anatomical data, electrical measurements, and physiological reference values from different individuals or population averages, rather than being derived from a single patient. The authors have appropriately moderated their claims in the revision, framing the work as a step towards the cardiac Digital Twin vision rather than asserting that the model itself constitutes a digital twin.

(4) Calibration procedure: The description of the calibration procedure has been substantially improved in the revision. The authors now provide explicit rationale for each calibration step and clarify that the procedure targets multiple physiological biomarkers in sequence. Verification that the calibrated model produces physiological cellular dynamics, including intracellular calcium transients, is now provided. The revised manuscript also shows the simulated electrocardiogram alongside population reference ranges, which partially addresses the question of calibration quality. However, a direct comparison of the simulated electrocardiogram with the individual measured signal used for calibration is not provided, which would give a more stringent and direct assessment of how well that specific calibration target was achieved.

Comments on revised version.

The revision represents a genuine improvement. The calibration procedure is now more transparently described, physiological cellular dynamics are verified, the digital twin framing has been appropriately moderated to reflect a step towards that vision rather than a claim of having achieved it, and an expanded limitations section identifies where the framework falls short.

Several of the concerns raised in the first round have been addressed, but some issues remain:<br /> The model still requires a ten-fold scaling of the peak active contractile force relative to literature values, and the imbalance between left and right ventricular output persists, along with non-physiological right ventricular pressures and ejection fraction. These are partly fundamental limitations of the current modelling approach that may not be fully resolvable within the scope of this paper, and the authors are to be credited for acknowledging them. However, they do constrain the conclusions that can be drawn about the credibility of the framework for reproducing healthy cardiac physiology.

The population-averaged reference dataset and the calibration and validation framework remain contributions of value to the community, and the revised limitations section adds useful transparency about the current state of the art.

Reviewer #1 (Public review):

Wang et al., recorded concurrent EEG-fMRI in 107 participants during nocturnal NREM sleep to investigate brain activity and connectivity related to slow oscillations (SO), sleep spindles, and in particular their co-occurrence. The authors found SO-spindle coupling to be correlated with increased thalamic and hippocampal activity, and with increased functional connectivity from the hippocampus to the thalamus and from the thalamus to the neocortex, especially the medial prefrontal cortex (mPFC). They concluded the brain-wide activation pattern to resemble episodic memory processing, but to be dissociated from task-related processing and suggest that the thalamus plays a crucial role in coordinating the hippocampal-cortical dialogue during sleep.

The paper offers an impressively large and highly valuable dataset that provides the opportunity for gaining important new insights into the network substrate involved in SOs, spindles, and their coupling.

Comments on latest version:

The authors have substantially revised their manuscript and sufficiently addressed all of my previous concerns. I have no further comments.

Reviewer #1 (Public review):

Summary:

The manuscript titled "Petabase-scale papillomavirus discovery" capitalizes on Logan assemblages to identify novel and known PVs.

This is a brilliant use of Logan assemblages, and this paper highlights the use of this, plus also shows that one person's trash is another one's gold. Superb paper and I applaud the authors for starting with the PVs as easier to identify due to their set of genes coupled with the conserved L1 protein and associated typing for PVs (10% pairwise identity threshold for identification of new PV types).

Strengths:

This study highlights the hidden gems in public resources, especially if mined properly. Thanks to Logan assemblages, this is possible, and this manuscript highlights this with their data mining of papillomaviruses, identifying known and novel PVs in pangolins, lizards, fish, and white rhinos.

Weaknesses:

None identified

Reviewer #1 (Public review):

The manuscript by Tang et al. characterizes the expression dynamics and functional roles of aldehyde dehydrogenase 1 activity in uterine physiology. Using a combination of in vivo lineage tracing and cell ablation coupled with organoid culture, the authors propose that Aldh1a1 lineage-marked cells contribute to uterine gland development and epithelial regeneration. The descriptive data will be of interest to reproductive biologists and clinicians and builds on established hypotheses in the field. The manuscript is well written and scientifically sound and the important experimental limitations and interpretation caveats are presented throughout.

The majority of my initial comments have been adequately addressed within the text.

Remaining limitations include:

(1) The impact of tamoxifen injection directly on Aldh1a1 expression in the developing uterus.

(2) It would be beneficial to demonstrate the degree of cell death following diphtheria toxin treatment 24-48 hours after injection in Tam-treated mice at PND 10. It is not clear as to why the 4-day timepoint was selected. Cells expressing the DTR should begin undergoing apoptosis within several hours after treatment.

Reviewer #1 (Public review):

This revised paper investigates how heparan sulfate (HS) engagement functions in the cellular entry of SARS-CoV-2. The authors used a series of microscopy techniques, labeled pseudoviruses and authentic SARS-CoV-2 strains, and cells lacking or expressing HS and/or hACE2 to re-examine the specific stage(s) HS and hACE2 function in the entry process. They suggest that HS mediates SARS-CoV-2 cell-surface attachment and endocytosis, and that hACE2 functions downstream of this to facilitate productive infection. Their results also suggest that SARS-CoV-2 binds clusters of HS molecules projecting 60-410 nm, which act as docking sites for viral attachment. The authors conclude their work establishes a revised entry paradigm in which HS clusters mediate SARS-CoV-2 attachment and endocytosis, with ACE2 acting at some stage downstream. They speculate this idea might apply broadly to other viruses known to engage HS and has translational implications for developing antiviral agents that target HS interactions.

The strengths of the study include the use of multiple high-resolution microscopy modalities, the tracking of labelled viruses, the use of both pseudoviruses and authentic SARS-CoV-2, and use of primary airway cells. While some studies were performed in the revision to address the Reviewer concerns, which improved the paper clarity, others were cursorily addressed, which limit the impact of the studies. Particularly. experiments were not performed to account for TMPRSS2 expression and plasma membrane fusion. Moreover, addition of studies in which hACE2 is expressed in cells genetically lacking HS were not designed. Thus, it the picture remains unclear picture exactly where downstream hACE2 functions and how this might differ given new structural models of TMPRSS2 activation (PMID: 42050172), which occur after ACE2 recognition of spike on the cell surface.

Reviewer #1 (Public review):

Summary:

The manuscript examines the factors that restrict the induction of IL-17-producing T cells during Mycobacterium tuberculosis (Mtb) infection. The authors show that neither infectious route, nor duration of infection are responsible. But they do show that mice that lack the Th1-defining transcription factor, a finding consistent with prior reports in the field of immunology. They also show that 2 highly attenuated Mtb mutants in ESX-1 and PDIM, two well-known Mtb virulence factors, do induce IL-17 producing T cells. In contrast, Mtb mutants in mmpl4 are also similarly attenuated, but do not induce IL-17-producing T cells, suggesting that this property is not simply a result of attenuation but due to specific properties of ESX-1 and PDIM-deficient mutants.

Strengths:

(1) It is interesting that mice infected with ESX-1 and PDIM mutants have increased induction of Th17 cells.

(2) Data is solid and convincing throughout.

Weaknesses:

There are two main criticisms:

(1) B6 mice, compared to humans are known to be very Th1 skewed and the Th1 transcription factor T-bet is known to be a strong inhibitor of Th17 responses. Thus, these Th17 inhibitory factors may be stronger in B6 mice than humans, as many humans do make Th17 responses to Mtb infection.

(2) The molecular insights about how Th17 induction is somewhat limited. Tbet induction is known to restrict Th17 development and this is a t cell intrinsic mechanism. In contrast, the IL-23 association revealed seems to be extrinsic to T cells and to act on T cells. It is not clear these factors related to each other in restricting Th17 induction.

Additional points:

(1) The manuscript states, "Under the conditions where Th17s are highly induced, mice infected with either ΔESX-1 or PDIM lacking Mtb, the Il17a-/- mice had ~3-5 fold higher CFU than WT mice (Figures 3F-G). These results indicate that the induction of Th17s is not dependent on the attenuation of Mtb in general, but instead Mtb utilizes ESX-1 and PDIM to suppress the induction of a Th17 response that enhances protection against Mtb infection." One consideration, however, is that ESX-1, PDIM, and mmpl4 mutants all have similarly reduced CFUs in the lung, but have different CFUs in the lung-draining LN where T cell priming occurs? The bacterial burden in the LN may be more important for regulating T-bet, IL-23, and Th17 differentiation, since the LN is where T cell priming occurs, than the CFU in the lung. Perhaps ESX-1 and PDIM mutants have reduced CFU in the LN, but mmpl4 does not. This difference in LN burdens may be the primary driver of Th17 priming, as high avidity interactions are thought to be an important driver of T-bet induction. Thus, without examining the LN, some questions remain regarding the conclusion that the altered Th17 response in the attenuated strains is not due to the attenuation itself. However, I agree the CFU in the LN probably reflects that in the lung, and if so, the author's conclusions would be sound.

(2) Do LN cDC1 and high levels of IL-12 p35 manifest in mice infected with the mmpl4 mutant? Likewise do LN cDC2's express low levels of IL-12 p19 (akin to those infected with WT Mtb). If these observations for ESX-1 and PDIM mutants are mechanistically linked to the increased numbers of Th17 cells, then you would expect mice infected with mmpl4 mutants to be more like those infected with WT Mtb than to those infected with ESX-1 and PDIM mutants. These experiments would help provide more convincing evidence that the identified mechanisms are due specifically to outcomes regarding Th17 induction. However, I agree the author's conclusions are the most likely explanation given the current data.

Reviewer #1 (Public review):

Summary:

Animal behavior is continuously influenced by the internal state moment by moment, including emotion primitives as the authors pointed out. Although emotion is a more human-related state, evolutional conservation is undeniable, which can be inferred by the behavioral manifestation. To further elaborate the neuronal mechanisms of emotion primitives, the simplest behavioral parameter related to emotional primitives should be well characterized. In this study, the authors described in detail of wall-following behavior (WAFO) and the total walking distance (TOWA) using flies after subjecting them to various conditions or flies being genetically manipulated according to the previous reports that could affect emotion primitives. Overall, the study is well designed and structured. In addition, the discussion on emotion primitives will be of value to the field.

Strengths:

The strength of this study is its use of a simple behavioral parameter, TOWA, and also a simple design of behavior, WAFO. The importance of the behavioral assay is reproducibility and comparability. In fact, the author demonstrated a summary of comparisons where different treatments result in scalable behavioral changes in WAFO and TOWA.

Conceptual concerns:

My suggestion to strengthen the authors' conclusion that "TOWA can be interpreted as a behavioral proxy for exogenously induced arousal" was to show that an increase in TOWA after stress exposure can be observed in a small (1-cm) arena that acts as an exogenous arousing stimulus, but not in a larger arena (>6.6 cm) where such arousing effects are absent. This comparison would demonstrate that basal locomotor activity measured in larger arenas is not altered by stress, whereas the additional component observed in smaller arenas reflects stress-induced internal state. Therefore, the authors would be able to distinguish clearly the effects of stressors or experiences on either simple locomotion or an emotion-like internal state. Then the future works can follow this protocol using smaller and larger arena to assess emotion-like internal state.

I appreciate the significant authors' efforts to monitor TOWA using arenas with different diameters up to 6 cm. However, the conclusion was unfortunately the same as that obtained using the 1-cm arena. As the authors commented, flies do not show persistent and quantifiable wall-following in arenas larger than 5.8 cm, which limits further examination of this question. I personally agree with the authors' interpretation, but I hope that the authors obtain more definitive experimental contrasts to support this claim in the future study.

Reviewer #1 (Public review):

Summary:

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

Strengths:

This study provides compelling evidence that mtDNA variants influence TL through a mechanism involving mitochondrial-derived ROS, potentially causing telomeric oxidative damage. The data are robust, and the manuscript is well written. However, the study could be strengthened by addressing the following questions and minor weaknesses below.

Weaknesses:

(1) Introduction. Line 81, the relationship between TL and the risk of lymphoid and myeloid leukemia is not straightforward. POT1 variants associated with long TL increase the risk for lymphoid and myeloproliferative neoplasms (see PMID: 41564438 for example).

(2) Figure 1. Since sex also influences TL, it would be good to know the sex of the selected individuals or explain why this is not necessary.

(3) Please include a description of the 143B cells that were used for cybrid formation in the Results section when introducing the cybrids.

(4) Lines 155-156. The authors note that cybrids from donors 1 and 2 show "pronounced" telomere damage. This result indicates an increase in 53BP1-positive telomeres, which could be indicative of telomere dysfunction or damage. Quantification of the increased chromosome end fusions for cybrids 1 and 2 would strengthen the result. Do the increased fusions correlate with an increase in telomere signal-free ends? These should be apparent in the telomere FISH images of metaphase chromosomes.

(5) Lines 168-169. What is the evidence that the "in vitro metabolic shift" causes acute oxidative stress?

(6) Why did the elevated ROS in cybrid #3 (Figure 4C) not translate to shorter telomeres in the cybrid (Figure 2A)? Perhaps there is a difference between factors that determine TL in the cybrid vs the donor's lymphocytes? In Figure 4B, it appears that the statistical comparisons for mitochondrial superoxide are all relative to Cyb3. If so, why are the comparisons not with the parental 143B rho0 cell line? Please clarify.

(7) Given the heterogeneity in TL and mtDNA variants in the human population, the conclusions could be further strengthened by increasing the number of donors and cybrids analyzed. However, there are admittedly practical factors. Overall, these findings are compelling and provide a solid foundation for expanding this analysis in the future. This is more of a comment than a weakness.

Reviewer #1 (Public review):

Summary:

The predominant view on CHOP's functions during ER stress is that it promotes cell death. This is in contrast to a handful of reports in the literature that claim that CHOP is a positive regulator of protein synthesis during chronic ER stress, and therefore is part of the adaptation program to ER stress. These previous studies were performed in tissue culture cells. Velarde and co-authors have used a mouse model of induction of mild ER stress to study the function of CHOP in hepatocytes.

Major strengths and weaknesses of the methods and results:

The authors use state-of-the-art mice to manipulate (i) CHOP and (ii) ATF6, a protective factor of ER proteostasis, and address the hepatocyte responses to mild ER stress in vivo and in cultures. Validated gene expression programs are well correlated to liver pathology in the mouse models. This is a very well-done study.

The authors clearly show that CHOP transitions hepatocytes under mild ER stress to a chronic ISR state, which is phenocopied by ATF6-depleted hepatocytes. So the conclusion that CHOP exacerbates ER stress in hepatocytes during mild ER stress is correct. It is also clear that CHOP targets negatively the transcription of hepatocyte identity genes, which opens a new direction of studies on the function of CHOP in secretory cells in general.

Conclusion:

This is a significant study that will benefit different research fields, and specifically studies on proteostasis, as was recently highlighted in Nat. Str. Mol. Biol. by experts in the field.

To this reviewer, the importance of the study is that it links the function of a transcription factor (CHOP) to stress intensity (mild versus severe) in a physiological experimental model (hepatocyte function and pathology).

Reviewer #1 (Public review):

Summary:

This study aims to reveal the contribution of individual gap junction proteins to the signal transmission and connectivity of living C. elegans animals in a completely non-invasive way through all-optical electrophysiology. The authors achieve this by simultaneous expression of bipoles, an excitatory/inhibitory light-activated actuator and Quasar2, a genetically encoded voltage dye. With this study, the authors extend their previous efforts to leverage the strength of optogenetic neurophysiology and set a new standard in this domain. In addition, they adapted their established methods to perform cell-specific optogenetic voltage clamp and revealed changes in gap junction connectivity. They also find that increasing excitability in innexin mutants is indicative of a reduction in gap-junction connectivity and current leaks.

Strengths:

This is an extremely strong manuscript, a technical feat and tour de force to infer junctional coupling through all-optical electrophysiology. The establishment of the voltage clamp method is powerful and allows researchers to obtain not only tight control over voltage signals but also permits the investigation of gap junction function in response to positive and negative voltage steps in a completely non-invasive fashion. This will be a new paradigm for investigating muscle electrophysiology in future.

Weaknesses:

This is a strong pioneering study, and I found very few technical weaknesses. The correlation quantification is relatively weak to establish connective causality, as a shared upstream input may lead to a similar perceived correlation. This is especially concerning for an average lag time of ~0, and the authors may want to investigate if there is unchanged connectivity in an unc-31 or unc-13 mutant. Conceptually, the local connectivity is scaled to account for behaviour: future studies may wish to perform this method on moving animals, and in specific neuronal populations, where a non-invasive optogenetic voltage clamp method will truly shine.

Reviewer #1 (Public review):

Summary:

This is a very interesting and well-done study of the effects of selective lesions to the sensorimotor cortex and the red nucleus on control of upper limb movements. The findings that the red nucleus may subserve recovery of upper limb motor function after cortical lesions in macaques and the different motor functions of different cortical sensorimotor areas are significant findings of considerable interest to sensorimotor neuroscientists, neurologists and neurosurgeons. The methods are mostly excellent, but there are some questions about the use of endothelin lesions in cortical areas and the use of trajectory variability as a marker of movement quality and fine motor control. Furthermore, it is questionable that increased trajectory variability in reaching a target reflects reduced movement quality, reduced ability to independently control muscles, and is a proximal analog of reduced dexterity.

Strengths:

The rationale that rubrospinal projections onto spinal neurons may subserve the good recovery of upper limb movements observed after lesions of sensorimotor cortex is compelling. The methods involving complete lesions of the red nucleus followed by recovery prior to lesions affecting various sensorimotor cortical areas are a strength. The excellent interpretations offered in the Discussion section are also a strength.

Weaknesses:

There are weaknesses in the Methods, including:

(1) no information on dimensions of the cup containing the food reward or types of food rewards,

(2) recording 3D hand movements with a single camera,

(3) cortical endothelin lesions were not very precise,

(4) the use of trajectory variability as a measure of movement quality and reduced ability to independently control muscles.

Some interpretations presented in the Discussion are not well supported. The discussion related to movement quality should be modified to focus on trajectory variability. The suggestion that rubrospinal projections onto motor neurons are apparently irreplaceable is not well justified because one monkey receiving a complete red nucleus lesion showed nearly full recovery of maximum movement speed, while the other monkey did not. The nearly full recovery of one monkey was probably due to new corticospinal connections onto motor neurons, whereas it is possible that the other monkey would have recovered better given more time before the 2nd lesion to cortical areas.

Reviewer #1 (Public review):

Summary:

Regional differences in the brain's waste-clearance system may interact with neural activity to influence where amyloid-B accumulates. Using intrathecal GBCA administration to produce "Glymphatic MRI" in 96 subjects, the authors mapped cortical glymphatic influx and clearance and found distinct spatial patterns, with transcriptomic analyses linking better glymphatic function to neuronal cell types (through genes). In a subgroup with resting-state fMRI, regions with stronger resting-state activation generally showed higher contrast clearance, indicating a positive coupling between these processes. Notably, cortical regions where neural activity and glymphatic clearance were mismatched showed greater amyloid-β burden in a separate, publicly available PiB-PET dataset, suggesting that activity-clearance decoupling may contribute to regional vulnerability and neurodegeneration.

Strengths:

This is a rare and valuable dataset. Intrathecal contrast injection in ~100 subjects is quite a remarkable accomplishment alone, but the addition of resting-state fMRI, a correlative PiB cohort, and gene-expression pattern data is impressive.

Weaknesses:

This is a cross-sectional study, and we can't determine whether neural activity drives glymphatic clearance, whether glymphatic dysfunction alters neural activity, or whether both are shaped by a third factor. Language describing "flow", "influx", and "clearance" could be made more specific so the reader can more easily follow the methodological approach.

Reviewer #1 (Public Review):

This work provides a new dataset of 71,688 images of different ape species across a variety of environmental and behavioral conditions, along with pose annotations per image. The authors demonstrate the value of their dataset by training pose estimation networks (HRNet-W48) on both their own dataset and other primate datasets (OpenMonkeyPose for monkeys, COCO for humans), ultimately showing that the model trained on their dataset had the best performance (performance measured by PCK and AUC). In addition to their ablation studies where they train pose estimation models with either specific species removed or a certain percentage of the images removed, they provide solid evidence that their large, specialized dataset is uniquely positioned to aid in the task of pose estimation for ape species.

The diversity and size of the dataset make it particularly useful, as it covers a wide range of ape species and poses, making it particularly suitable for training off-the-shelf pose estimation networks or for contributing to the training of a large foundational pose estimation model. In conjunction with new tools focused on extracting behavioral dynamics from pose, this dataset can be especially useful in understanding the basis of ape behaviors using pose.

Since the dataset provided is the first large, public dataset of its kind exclusively for ape species, more details should be provided on how the data were annotated, as well as summaries of the dataset statistics. In addition, the authors should provide the full list of hyperparameters for each model that was used for evaluation (e.g., mmpose config files, textual descriptions of augmentation/optimization parameters).

Overall this work is a terrific contribution to the field and is likely to have a significant impact on both computer vision and animal behavior.

Strengths: - Open source dataset with excellent annotations on the format, as well as example code provided for working with it. - Properties of the dataset are mostly well described. - Comparison to pose estimation models trained on humans vs monkeys, finding that models trained on human data generalized better to apes than the ones trained on monkeys, in accordance with phylogenetic similarity. This provides evidence for an important consideration in the field: how well can we expect pose estimation models to generalize to new species when using data from closely or distantly related ones? - Sample efficiency experiments reflect an important property of pose estimation systems, which indicates how much data would be necessary to generate similar datasets in other species, as well as how much data may be required for fine-tuning these types of models (also characterized via ablation experiments where some species are left out). - The sample efficiency experiments also reveal important insights about scaling properties of different model architectures, finding that HRNet saturates in performance improvements as a function of dataset size sooner than other architectures like CPMs (even though HRNets still perform better overall).

Weaknesses: - More details on training hyperparameters used (preferably full config if trained via mmpose). - Should include dataset datasheet, as described in Gebru et al 2021 (arXiv:1803.09010). - Should include crowdsourced annotation datasheet, as described in Diaz et al 2022 (arXiv:2206.08931). Alternatively, the specific instructions that were provided to Hive/annotators would be highly relevant to convey what annotation protocols were employed here. - Should include model cards, as described in Mitchell et al (arXiv:1810.03993). - It would be useful to include more information on the source of the data as they are collected from many different sites and from many different individuals, some of which may introduce structural biases such as lighting conditions due to geography and time of year. - Is there a reason not to use OKS? This incorporates several factors such as landmark visibility, scale, and landmark type-specific annotation variability as in Ronchi & Perona 2017 (arXiv:1707.05388). The latter (variability) could use the human pose values (for landmarks types that are shared), the least variable keypoint class in humans (eyes) as a conservative estimate of accuracy, or leverage a unique aspect of this work (crowdsourced annotations) which affords the ability to estimate these values empirically. - A reporting of the scales present in the dataset would be useful (e.g., histogram of unnormalized bounding boxes) and would align well with existing pose dataset papers such as MS-COCO (arXiv:1405.0312) which reports the distribution of instance sizes and instance density per image.

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 fuelled 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. The reviewers have achieved their aims and the results support their conclusions. This work provides important insight into molecular mechanisms that allow aging without loss of fitness and will be of interest to scientists in the field of aging and metabolism.

Reviewer #1 (Public review):

Summary:

The authors scrutinized difference in C terminal region variant profiles between Rett syndrome patients and healthy individuals and pinpointed that subtle genetic alternation can cause benign or pathogenic output, which harbors important implication in Rett syndrome diagnosis and proposing therapeutic strategy. This work will be beneficial to clinicians and basic scientists who work on Rett syndrome and carries potential to be applied to other Mendelian rare diseases.

Strengths:

Well-designed genetic and molecular experiments, translating genetic differences into functional and clinical changes. This is a unique study resolving subtle changes in sequences give rise to dramatic phenotypic consequences.

Comments on revised version.

Improvements were made during the revision.

Reviewer #1 (Public review):

Summary:

This manuscript by Noirot-Gros et. al. presents a herculean effort to map the protein-protein interactome of the c-di-GMP signaling network in Pseudomonas fluorescens (Pf). C-di-GMP, the key driver of biofilm formation in bacteria, is controlled by a highly complex network of synthesis, degradation and effector proteins. Pf is no exception as it encodes dozens of such proteins. The authors use a Yeast Two-Hybrid approach genome-wide screen with 10 diguanylate cyclase (DGC) enzymes as bait to assess protein-protein interactions in this network. The results identify over one hundred such interactions with several different hubs, including c-di-GMP signaling, other signaling systems, membrane proteins, etc. The authors then explore the original bait proteins as well as identify interactors on biofilm formation-related phenotypes and swarming using a high-throughput CRISPRi expression knockdown approach. The amount of data generated is quite impressive. Much of the manuscript uses statistical-based network analysis to group different proteins based on their interactions or impact on phenotypes, which is a high-level analysis that can catalyze further study into this system. The authors chose three specific proteins to assess their impact on cell morphology, DNA repair, and protein localization. Overall, in my view, this is perhaps the best analysis of a c-di-GMP protein-protein interactome, and it provides a multitude of hypotheses to be tested. However, therein lies the weakness of the manuscript in that very few of these hypotheses are actually tested. But such is not the goal of this network analysis type of approach. Overall, I think the work will be highly impactful to those in the c-di-GMP field, and it provides a template for others attempting such analyses of protein-protein interactions.

Strengths:

The manuscript is impressive in the sheer scale of the protein-protein interactions identified, network analysis, and phenotypic analysis of specific proteins in the network. It is an impressive amount of work that could be very useful to the field. It is also statistically rigorous in its analysis of significant interactions or network nodes.

Weaknesses:

The weakness of the manuscript is that, with three exceptions, very few of the hypotheses are actually tested. For example, BifA is shown to be a network hub protein that interacts with many other diguanylate cyclases, and this is hypothesized to be through GGDEF heterodimerization. I appreciate that experimentally testing such a hypothesis is probably another entire manuscript, but some early forays into such ideas could be undertaken using AlphaFold structural modeling of protein-protein interactions compared with GGDEFs that don't form heterodimers. Also, an inherent weakness is that such detailed analyses of a c-di-GMP signaling network, in which each diguanylate cyclase and phosphodiesterase may respond to a unique cue, is that the network identified and the conclusions made are highly specific to the experimental conditions in which the work was done. Therefore, it is unclear how broadly these conclusions (i.e. BifA is the central regulator of c-di-GMP signaling) apply to other conditions. But it is impossible to get around such a limitation, and this work can lead to testing the robustness of the identified network in other environments.

Reviewer #1 (Public review):

This manuscript by Hall et al. uses a multi-omic approach to investigate how distinct members of the Mycobacterium tuberculosis complex (MTBC: M. bovis, M. tuberculosis, the attenuated M. bovis BCG vaccine strain, and gamma-irradiated M. bovis) affect bovine alveolar macrophage epigenetic and transcriptional responses after 24 hours. The investigators used RNA-Seq, ATAC-Seq, and ChIP-Seq to assess differential gene expression in each complex type and integrated gene transcription with chromatin accessibility and epigenetic modifications, highlighting key immune response genes/pathways upregulated in response to infection and pathogen-specific host adaptation mechanisms. The analysis also revealed that the most pronounced transcriptional and epigenetic responses were in the M. bovis-infected cells compared with the other complex types. Comparing top genes associated with M. bovis infection of macrophages to a GWAS data set revealed 4 key genes associated with increased susceptibility to infection.

Overall, this is a technically sound manuscript that contains highly interesting and useful data on bovine innate immune responses to different types of Mycobacterium tuberculosis, which are important to the immunology and infectious disease community as well as the livestock industry. However, in its current format, the manuscript presents the data/figures in a way that is not particularly informative (despite the rich data set) and is too descriptive. We also have some general concerns and suggestions listed below.

Reviewer #1 (Public review):

Summary:

In this manuscript, Sun et al. investigate the hemispheric lateralization of functional brain networks during verbal versus nonverbal working memory tasks. Utilizing state-of-the-art precision neuroimaging in highly sampled individuals, the authors define a set of distributed association networks and examine their task-evoked responses. The authors report a "generalized laterality effect," wherein multiple association networks appear to functionally split across the hemispheres, with left hemisphere components exhibiting a relative preference for verbal stimuli and right hemisphere components preferring nonverbal stimuli.

Strength:

The use of dense-sampling fMRI is a major strength of this study, allowing for a highly accurate, individual-specific mapping of network topologies that group-averaging typically obscures. Despite the interpretational concerns raised below, this high-quality, within-subject imaging dataset represents a valuable resource for the community. Furthermore, the inclusion of an independent prospective replication dataset provides valuable confidence in the robustness of the core imaging metrics. However, while the data quality is exceptionally high, the conceptual interpretations regarding "network splitting", "preferential recruitment", and the generalizability of the verbal/nonverbal dichotomy require significant refinement. Several methodological and statistical clarifications are needed to fully support the authors' claims.

Weaknesses:

Major:

(1) The manuscript relies heavily on task contrast values (e.g., Face > Word) to conclude that networks functionally "split" their profiles, with specific hemispheres being "preferentially recruited" by either verbal or nonverbal materials. While the data clearly demonstrate relative hemispheric differences, claiming absolute bidirectional specialization and active recruitment appears to overstate the findings in two key ways:

First, statistical evidence for true bidirectional "splitting" is scarce. A significant hemispheric difference confirms a relative shift in processing, but it does not permit claims about absolute preference. When examining the face>word effects against zero in the discovery dataset (Figure 3), the right hemisphere of the LANG, FPN-B, CG-OP, and SAL networks shows no significant preference for faces over words. In the replication dataset (Figure 6), two of the four targeted networks (FPN-A, CG-OP) similarly fail to show a significant right-hemisphere preference. Furthermore, it is unclear whether these tests against zero were corrected for multiple comparisons (e.g., 18 individual tests in Figure 3). Networks reporting significance at the uncorrected p < 0.05 level (such as the left hemisphere of LANG and FPN-B) might not survive standard correction, suggesting that even the left hemisphere's preference for words may be statistically marginal. Second, contrast differences in networks exhibiting negative signals may reflect relative deactivation rather than active recruitment. A mathematically positive contrast value derived from two negative activation states (e.g., Face [-6] > Word [-8]) does not indicate active "recruitment" for face processing. Instead, it merely reflects a relative difference in deactivation. Characterizing this dynamic as "preferential recruitment" misleads the reader regarding the actual physiological state of the network. Furthermore, such asymmetric suppression is frequently driven by generalized differences in task difficulty or cognitive effort, rather than true stimulus-specific processing.

(2) Building on the previous point, it would be highly beneficial to include the behavioral data (such as accuracy and reaction times) for the N-Back conditions, which do not currently appear to be reported in the manuscript. This information is important because if one condition (e.g., the Face N-back) was significantly more challenging or required greater cognitive effort than the other (e.g., the Word N-back), the observed hemispheric dissociations might reflect differences in arousal, effort, or attentional deployment rather than stimulus-specific processing. ion

(3) The manuscript claims a "generalized" hemispheric laterality effect. However, the supplemental figures suggest this effect may be highly sensitive to the specific stimuli used in the main text (unfamiliar Faces vs. rhyming Words), which represent extreme ends of visuospatial and phonological processing. As we talked about earlier, a true functional "split" implies that the hemispheres respond in opposite directions. However, visual inspection of the supplemental graphs reveals that for the vast majority of networks, both hemispheres are actually driven in the exact same direction (Figures S8 and S9).

In the Face > Letter contrast (Figure S8), true bidirectional splits largely disappear. With the exception of FPN-A, the bars for both the left and right hemispheres point in the exact same visual direction for every network (e.g., both hemispheres are visually positive in DN-A and dATN-B, and visually negative in CG-OP and dATN-A). This indicates that the hemispheres actually share the same categorical preference and merely differ in magnitude. Strikingly, the LANG network shows no significant difference between Faces and Letters in either hemisphere, suggesting that the robust leftward shift observed in Figure 3 was possibly driven by the heavy semantic and phonological demands of the rhyming task, rather than a generic preference for "verbal" processing.

Similarly, in the Scene > Word contrast (Figure S9), the hemispheres do not visually diverge in their response direction for most networks. For example, both hemispheres are visually negative (indicating a shared preference for Words) in the LANG, FPN-B, CG-OP, and SAL networks. Conversely, both hemispheres are visually positive (indicating a shared preference for Scenes) in the dATN-B and DN-A network.

Because the hemispheres do not visually diverge in their response direction for most networks across these supplementary contrasts, the claim of a robust, generalized hemispheric "split" is unsupported.

Reviewer #1 (Public review):

Summary:

This paper examines potential sex differences in the conflict between exploitation, pursuing food and rewards in previously-associated locations/paradigms, or exploration of new locations that might result in better outcomes. Dysregulation of this conflict may be an underlying behavioral modality of psychiatric diseases. They used four distinct tasks: a two-armed Bandit 100:0 task, a standard fixed ratio 1 task, a two-armed Bandit 80:20 task, and a closed-loop economy PR1 task that allows for the assessment of motivational breakpoint.

Male mice show significantly higher accuracy under conditions of high probability known rewards, sticking with an action that just resulted in a reward or "win-staying". This was demonstrated in multiple paradigms, and there was a predictive nature of this behavior that could predict animal sex with modest accuracy. Under probabilistic environments, males were no longer more accurate than females but still used a higher win-stay strategy. A closed-loop PR1 task showed that there were no inherent differences in motivational breakpoint between sexes. Finally, the authors use simulations to determine an appropriate number of animals needed to detect these differences.

Strengths:

The manuscript attempts to resolve inconclusive sex differences that have heretofore been neglected or inconclusive due to insufficient power. The most impressive aspect of this paper is its scale, assaying 62 female mice and 74 male mice in identical exploration-exploitation tasks using high-throughput and noninvasive operant feeding via FED3. Very few labs can achieve this scale, which is necessary to detect sex differences with a small effect size.

The authors use some sophisticated modeling approaches and analysis of data from the 136 mice to investigate the significance of these sex differences and interrogate other conditions. They also use simulations to model the likelihood of replicating these differences given a sample size. This is extremely helpful for other researchers as they consider sex as a biological variable.

Weaknesses:

The study is largely descriptive in nature and does not pursue any mechanism of the underlying differences, like hormones, neuromodulators, or circuits. The lack of estrous cycle tracking is acknowledged as a limitation.

Reviewer #1 (Public review):

Summary:

The authors claim that bacteria are guided by diffusiophoresis. They perform experiments of bacterial motility in microfluidic channels with salt gradients. The data show that P. pudita bacteria swim towards higher sodium chloride concentrations, but there is no evidence that this is due to a diffusiophoresis.

Weaknesses:

It is well known that bacteria perform chemotaxis in salt gradients (see e.g., PNAS 86, pp. 8358-8362, 1989). The underlying mechanism based on chemoreceptors is widely accepted, but the authors do not mention this possibility. I recommend a control experiment where the chemotaxis genes are knocked out. Even if this mechanism can be ruled out, the current data show no evidence for a mechanism based on diffusiophoresis.

Reviewer #1 (Public review):

Summary:

This paper presents a creative physics-based model of an ATPase-like molecular machine using mechanically coupled linkages to mimic allosteric cycles. The authors construct the complete reaction network by enumerating all mechanically allowed binding configurations and transitions between them. The resulting system contains hundreds of microstates connected through node-level binding, dissociation, intramolecular rearrangements, cleavage, and ligation reactions. Stochastic simulations are then used to study how the machine cycles between ligand-bound and substrate-bound states. Overall, the manuscript presents an interesting and creative mechanochemical framework for modeling ATPase-like allosteric cycles integrating multivalent binding, geometric exclusion through rigidity arising from binding of substrates and ligands, with stochastic simulations.

Strengths:

(1) The manuscript presents a creative mechanochemical framework that combines multivalent binding, geometric exclusion, rigidity-based coupling, stochastic kinetics, and catalysis within a unified model.

(2) The use of geometric exclusion and rigidity to generate negative allosteric coupling is elegant and provides an intuitive physical mechanism for coordinated molecular behavior.

(3) The interpretation of catalysis as a transient release of mechanical constraints is conceptually interesting and offers a novel perspective on how energy-consuming reactions can regulate state transitions.

(4) The distinction between productive and futile cycles is insightful and provides a useful framework for understanding pathway selection in molecular machines.

(5) The explicit construction of a stochastic state network allows the authors to connect microscopic binding events with emergent cyclic behavior.

(6) The work provides a conceptual platform for exploring how simple mechanical principles may give rise to allosteric regulation and mechanochemical transduction in synthetic molecular systems.

Weaknesses:

(1) The manuscript is very dense and difficult to follow. The notation and microstate labels (e.g., {S/L}:{10,5}) obscure the central ideas, and the stochastic model is not explained clearly enough. The authors should provide a simpler schematic, a mapping of state labels, and a step-by-step example of a productive cycle. The supplementary videos would also benefit from additional explanation.

(2) The framework appears most applicable to mechanically gated motor proteins and may not generalize to allosteric enzymes that operate through conformational ensembles, dynamic coupling, or entropy-driven regulation. The scope of the model should be discussed more carefully.

(3) The reported behavior appears highly dependent on specific parameter choices and rate hierarchies. A broader sensitivity analysis is needed to demonstrate robustness.

(4) The binary treatment of states as either rigid or flexible oversimplifies the continuous energy landscapes and fluctuations observed in real biomolecules. The limitations of this approximation should be discussed.

(5) The role of nonequilibrium thermodynamics is underdeveloped. The relationship between the model, ATP chemical potential, free-energy dissipation, entropy production, and the energetic cost of futile cycles should be discussed more explicitly.

(6) Although a large number of microstates are enumerated, it remains unclear which states and pathways dominate the dynamics. A more coarse-grained analysis highlighting the key states and transitions would improve interpretability and facilitate comparison with experimental systems.

Reviewer #1 (Public review):

The authors aim to reconstruct a multi-layer metabolic regulatory network in Alzheimer's disease by integrating transcriptomic, proteomic, and metabolomic datasets from human brain tissue. By linking transcription factors, enzyme expression, metabolic reactions, and metabolites, the study seeks to provide a systems-level understanding of disease-associated metabolic dysregulation.

The revised manuscript has improved in clarity and includes additional analyses in response to prior comments, particularly the incorporation of cell-type proportion estimates derived from matched single-cell datasets. This represents a meaningful step toward addressing concerns about the interpretation of bulk tissue and strengthens the study's descriptive rigor.

However, several key limitations remain. First, although cell-type proportions are now estimated, this information is not integrated into downstream analyses. As a result, it remains unclear whether the observed metabolic changes reflect cell-intrinsic regulation or shifts in cellular composition. This distinction is critical for interpreting the inferred regulatory network and limits the strength of the conclusions.

Second, the biological conclusions remain largely confirmatory. The reported downregulation of energy-related pathways, including the TCA cycle and oxidative phosphorylation, is consistent with prior literature. The trans-omic framework provides a structured representation of these changes, but the manuscript does not convincingly demonstrate that this approach yields new mechanistic insight beyond existing knowledge. In particular, the network is not sufficiently leveraged to identify novel regulatory relationships or generate testable hypotheses.

Third, while the framework integrates multiple molecular layers, the contribution of upstream transcriptional regulation remains unclear. The most compelling findings appear to arise from protein and metabolite layers, and the manuscript does not clearly demonstrate how transcription factors or mRNA-level changes contribute to the interpretation of metabolic dysregulation. This weakens the claim that the study provides a fully integrated trans-omic perspective.

Fourth, concerns regarding network robustness remain. The analysis relies on partially overlapping cohorts across omics modalities, and although this limitation is acknowledged, no formal sensitivity or robustness analyses are presented. This reduces confidence in the stability and generalizability of the inferred network structure.

Finally, the handling of covariates remains limited. While the authors justify this based on data availability and prior studies, the lack of consistent adjustment for known confounders introduces uncertainty in attributing observed differences specifically to disease-related biology.

Overall, the study presents a technically sound application of a trans-omic integration framework and provides a coherent overview of metabolic dysregulation in Alzheimer's disease. However, the findings are primarily descriptive and confirmatory, and the current analyses do not fully demonstrate that the approach yields novel biological insight. The work will be of interest to researchers in systems biology and multi-omics integration, but its impact on advancing understanding of disease mechanisms is likely to be moderate.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors did an excellent job with their resubmission, politely and elegantly answering the comments from the reviewers.]

Summary:

Large language models (LLMs) have been developed rapidly in recent years and are already contributing to progress across scientific fields. The manuscript tries to address a specific question: whether LLMs can accurately infer signaling networks from gene lists.

Strengths:

The manuscript raises a good question: whether current LLMs can accurately generate signaling networks from gene lists.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revised version.

Authors have worked on most of the revisions stated in previous feedback and included in the newer version, which has been significantly improved. Other comments have been described to be out of scope from this study.

Reviewer #1 (Public review):

Summary:

This manuscript aims to test the idea that visual recognition (of faces) is hierarchically organized in the human ventral occipital-temporal cortex (VOTC). The paper proposes that if VOTC has a hierarchical organization, this should be seen in two independent features of the VOTC signal. First, hierarchy assumes that signals along the hierarchy increase in representational complexity. Second, hierarchy assumes a progressive increase in the onset time of the earliest neural response at each level of the hierarchy. To test these predictions, the authors extract high-frequency broadband signals from iEEG electrodes in a very large sample of patients (N=140). They find that face selectivity in these signals is distributed across the VOTC with increasing posterior-anterior face selectivity, hence providing evidence for the first prediction. However, they also find broadband activity to occur concurrently, therefore challenging the view of a serial hierarchy.

Strengths:

(1) The hypothesis (that VOTC is hierarchically organized) and predictions (that hierarchy predicts increases in representational complexity and increases in onset time) were clearly described.

(2) The number of subjects sampled (140) is extremely large for iEEG studies that typically involve <10 subjects. Also, 444 face selective recording contacts provide a very nice sampling of the areas of interest.

Comments on revised version:

The authors have performed additional analyses and checks and I would now rate the findings as important and compelling.

Reviewer #1 (Public review):

Summary:

This manuscript describes the results of phylogenetic and epidemiological modeling of the PopART community cohorts in Zambia.

Comments on revised version:

Thank you for the opportunity to re-review this interesting paper.

This reviewer struggled to follow along with the author's response letter. It was challenging because responses were brief and did not list the specific changes made, leaving the reviewer to search for changes in the text. As best I could tell, there were no changes made that matched some of the highest-priority suggestions.

Critique #1 - This reviewer did not find the presentation of confidence intervals in the Abstract and other sections, which were suggested. Please note the format that was suggested in the original critique from Reviewer 1.

Critique #2 - regarding removal of unsubstantiated claims and use of a p-value to compare analysis to a null hypothesis - it seems the authors skipped over this critique and did not address it.

Regarding bias: the authors answered a different question than the one asked. The reviewer asked what proportion of transmissions were sampled; the authors stated that only communities from which phylo data was acquired were modeled. Was sampling 100% in those communities? Please provide the percentage and provide analysis that shed light on how sampling bias could impact the analysis.

Regarding "cherries" - the reviewer did not understand the author's response. The query was regarding what percent of the total number of phylogenetic pairs (denominator) were the 355 that had high confidence in directionality (numerator). The response could be expressed be a proportion.

The expectation of ART reducing the age of sources of transmission seems unrealistic to this reviewer. People on ART are not always adherent and can still transmit during gaps in adherence. ART dramatically increases life expectancy with HIV, which would have the opposite effect.

Reviewer #1 (Public review):

Summary:

This manuscript describes the development of an expression system enabling up to 12 transgenes using the alternatively spliced fourth exon of *Drosophila* *Dscam* gene under the control of a UAS element. This will be a useful tool if expression is needed in *Drosophila* cells (in culture or in vivo). where *Dscam* splicing machinery is active, which limits its use.

Strengths:

The tool developed is based on a well-established genomic element. The underlying idea is relatively simple yet effective.

Weaknesses:

The authors describe the weaknesses of their system well, most importantly, depending on the presence of adequate levels of Dscam splicing factors in targeted cells. This likely limits effective use of the methodology to some cell lines (e.g., S2) and certain tissues (nervous system and innate immune system). The manuscript could do a better job in showing protein expression levels more quantitatively, either in comparison to other methods or as absolute values (transcript numbers, protein molarity, etc.).

Reviewer #1 (Public review):

Summary:

In their submitted work, Lee and colleagues examine the correlation between electrophysiological activity as measured by SEEG, and layer-specific activation patterns, as measured through 7T fMRI. This analysis was performed using patients undergoing monitoring for epilepsy surgery guidance, as well as healthy controls, as they both listened to music.

They find that, in general, higher-frequency SEEG activity correlated positively with the fMRI signal, while lower frequencies correlated negatively. Across cortical depth, higher-frequency activity correlated positively with middle-to-upper layers, whereas lower frequencies showed their strongest negative correlations in superficial layers.

Strengths:

This is an interesting physiological study in that, to the best of this reviewer's knowledge, it has not been done before with auditory stimuli using the combination of iEEG (as opposed to scalp EEG) and fMRI. The framework fits well with models of layer-specific feedforward versus feedback processing (e.g., Bastos et al., 2012).

Weaknesses:

Its main limitations are a lack of specificity to the acoustic stimuli, the absence of correction for venous draining, and the fact that it is largely a replication/port of prior work.

Reviewer #1 (Public review):

Summary:

This manuscript examines the expression of putative chemoreceptors in CSF-contacting neurons of the larval zebrafish spinal cord. Using in situ hybridization, the authors show that sstr2a is preferentially expressed in ventral CSF-cNs, whereas grm2a, ptprna, and ldlrad2 are detected in both ventral and dorsolateral CSF-cNs, with additional expression in neighboring cells around the central canal.

Strengths:

The study provides useful anatomical information on the expression of putative chemoreceptors in CSF-contacting neurons. The experiments appear to be carefully performed, and the results are clearly presented with high-quality illustrations and informative schematics.

Weaknesses:

This work remains largely descriptive and based on mRNA expression. Therefore, the proposed roles in chemoreception, ligand sensing, lipid capture, or long-range CSF signaling remain speculative without protein-level or functional validation.

Reviewer #1 (Public review):

Summary:

The Voltage-Dependent Anion Channel 1 (VDAC1) is the most abundant β-barrel protein in the outer mitochondrial membrane and the main conduit for metabolite and ion exchange between the cytosol and mitochondria. Its oligomerization has been proposed to control mitochondrion-mediated apoptosis, making it a prime target for therapeutic intervention in diseases associated with excessive cell death, such as neurodegenerative disorders and autoimmunity. VBIT-4 is a small molecule developed to inhibit VDAC oligomerization and has shown therapeutic potential in various preclinical models. Despite its widespread use, the mechanism of action of VBIT-4 has not yet been fully elucidated. In this paper, Ravishankar et al. combine a suite of biophysical approaches with computer simulations to demonstrate that VBIT-4 forms water-permeable defects in membrane bilayers without any detectable effects on VDAC1 channel properties or oligomerization. Furthermore, cytotoxicity assays revealed identical VBIT-4 IC50 values in wild-type and VDAC1-KO cells, indicating that its activity does not depend on VDAC1. Collectively, these findings cast significant doubt on the widely held assumption that VBIT-4 is a specific inhibitor of VDAC1 oligomerization. Instead, it appears that VBIT-4 functions as a membrane-active compound.

Strengths:

This is a carefully conducted and well-written study that highlights potential side effects of VBIT-4, a compound that has been used to study the role of VDAC1 in a range of physiological and pathological conditions. The work is of interest to a broad readership by showcasing the importance of a systematic assessment of drug-membrane interactions to identify potential off-target membrane-driven effects of small molecules that may be mistakenly attributed to the inhibition of specific proteins. Its strength lies in the variety of complementary approaches the authors used to rigorously challenge the effect of VBIT-4 on VDAC1 organization and function. Overall, the experimental data are compelling and of high quality.

Weaknesses:

The authors used high-speed atomic force microscopy (HS-AFM) to study the impact of VBIT-4 on VDAC1 oligomerization in real time at nanoscale resolution. Toward this end, they adsorbed POPC:POPE:cholesterol membranes reconstituted with or without VDAC1 on mica. This revealed that the addition of VBIT-4 produced small perforations in the bilayer that were independent of VDAC1. In the absence of VBIT-4, VDAC1 showed the characteristic honeycomb topography that the authors described in a previous study (Reference 17). To quantitatively assess whether VBIT-4 affects VDAC1 organization, they analyzed protein compaction within clusters using inter-protein distance measurements. This analysis revealed no significant difference in VDAC1 organization between control conditions, 1 uM and 10 uM VBIT-4, supporting a model in which VBIT-4 primarily perturbs the lipid matrix rather than VDAC1 assemblies. This conclusion is based on the assumption that VDAC channels retain some lateral mobility in bilayers adsorbed onto mica. Do the authors have evidence that this is indeed the case? Did they also perform HS-AFM on VDAC1-containing membranes treated with VBIT-4 prior to adsorption onto mica?

Reviewer #1 (Public review):

Summary:

Many previous studies have reported inter-item biases in visual working memory tasks. These biases can be either attractive or repulsive, depending on the particular experiments. It has been difficult to explain these biases in a unifying theoretical framework. Recently, Chetverikov (the first author of the current manuscript) proposed a demixing model for explaining these biases in Ref 22. That paper shows that both attractive and repulsive biases could emerge in the demixing framework depending on the noise properties. The current manuscript seeks to test the predictions of the demixing model experimentally in a series of new experiments and find evidence supporting the demixing model.

Because previous modeling results described in reference 22 (which is a preprint) are essential in interpreting the results reported in the current manuscript, I also studied that preprint and used the results reported in that paper to help interpret the results in this paper. My comments below will also contain discussions of that modeling paper.

Strengths:

Overall, the computational model tested in the paper is novel and interesting.

The demixing framework represents an appealing hypothesis that deserves further investigation.

The current paper provides new empirical data showing that the target stimuli with the same absolute noise level can be either repelled from or attracted to non-target items, depending on the relative noise levels. The observation that biases depend on the relative noise levels is by itself an interesting one, and is consistent with the prediction of the demixing model.

Weaknesses:

While this manuscript contains interesting new experimental observations and theoretical ideas, it has several substantial problems in its current form, which limit the conclusions that can be drawn. The description of the computational model is too brief. The key modeling assumptions need to be better motivated and explained. As the computational models generate different predictions in different regimes, it is a bit difficult to evaluate how well the experimental data support the model at a more quantitative level. Also, the results focused on studying the biases in the behavior; it is unclear whether the model can fully explain the behavior data (such as error distributions or behavioral precision).

Major concerns:

(1) Concerns/suggestions regarding the computational modeling

The current paper seeks to test the predictions of the demixing-based computational model proposed in reference 22. There are several problems with the modeling component in the current paper.

(1a) The description of the model is too brief and difficult to understand. Although the model was proposed in reference 22, it would still be beneficial to provide more details of the model so that readers can understand and appreciate the strengths/limitations of the model.

The generative model and the inference procedure could be better explained to better link the model to the behavior. In particular, how was the observer's behavioral report in each trial modeled? This requires more explanation because currently the demixing procedure estimates four parameters for a given trial, yet for a given trial, only one behavioral report was produced (e.g., current Experiment 1), or two reports were produced sequentially (e.g., current Experiment 2).

(1b) Key modeling assumptions need better justification.

One such key assumption is that on a given trial, each stimulus triggers many samples (or approximately, an entire response distribution), rather than a single sample. This assumption deviates substantially from prior work on ideal observer models. It was not clear whether this assumption is realistic. For the type of stimuli used in the current experiments, perhaps one can argue that each pixel corresponds to one sample of brain activity, thus collectively each stimulus should trigger many samples of activity in the brain. If this were to be the case, it would have two implications. First, the noise parameter in the model should be directly related to the magnitude of the stimulus noise. Thus, one should be able to plug these experimentally-controlled parameter values into the model to directly generate predictions about the biases. Second, when using stimuli with no stimulus variability (e.g., simple grating stimuli), the predicted biases should change. However, it wasn't clear whether this would hold experimentally, i.e., using gratings would lead to different biases or no biases.

If the variability of the samples for a given stimulus involves neural noise, it would be useful to justify why it is reasonable to consider that many samples were generated per stimulus.

(1c) As mentioned in (1b), the model assumes that on each trial, a large number of samples was generated. It would be useful to study and report how the prediction would change when the number of samples generated per stimulus is small. In particular, what happens when each stimulus only generates one measurement? This might be useful for interpreting previous experiment results with grating stimuli.

(1d) Reference 22 studies how the predicted biases depend on the d-prime of the identifying dimension and found that the pattern of the biases varies substantially depending on the information available for the identifying dimension. However, the current paper didn't really discuss this important point. It is also unclear what parameters the authors used for the d-prime of the identifying dimension. Was it fitted directly to the data? The Methods section has some description on the "identifiability dimension", but it was a bit obscure.

Intuitively, when the d-prime of the identifying dimension is very large, the demixing problem becomes irrelevant. In this case, there should not be any biases induced by demixing. In the case of the d-prime for the identifying dimension is 0, the problem should reduce to the simplified 1-d problem studied in reference 22. If my reading of reference 22 was correct, they reported different conclusions. It would be useful to clarify these points.

In any case, the d-prime of the identifying dimension appears to be a key parameter. It would be great to constrain this parameter using the empirical data. When the d-prime of the identifying parameter is small, the observer would easily confuse the probed stimulus with the other stimulus in a given trial. This should lead to poor task performance. Thus, it may be possible to directly estimate the value of the d-prime of the identifying dimension based on the observer's performance, and then use this parameter to generate model predictions accordingly.

(1e) The current model assumes that a large number of samples are generated per stimulus and the brain can manipulate this information to perform the demixing task. It was well documented that visual working memory has a capacity limit (i.e., it can only hold information about a few items); this discrepancy needs to be clarified or addressed.

(2) How well the computational model can explain the experimental data remains not entirely clear

The authors show that there exists a parameter regime that can qualitatively explain the experimental finding. They also show that it is possible to fit the model to the data to explain the bias patterns. However, given that the model is flexible, it would be stronger if the authors could show that the same parameters that explain the biases could also explain other aspects of the behavior, for example, the magnitude of the errors.

In other words, the model is not well constrained in the way it was tested in the paper. But it should be possible to improve it. First, if the noise parameter in the model is determined by the stimulus variability, one can determine it directly based on the external noise in the stimuli (discussed also in 1b) and see what prediction it leads to. Second, from the behavioral data, it may be possible to estimate the noise for the identifying dimension. Doing so will help better constrain the model.

It would also help if the authors could report the best-fitted parameters from the experimental data. From these parameters, one can simulate synthetic data and apply the demixing model to see if the error distribution of the simulated observers is indeed similar to the experimentally measured error distribution. That way, one can check whether the fitted parameter explains the observer's behavioral performance beyond the biases.

Other comments:

(1) How does the model account for the swap errors? I am not sure I understood the way how the swap errors were treated in the paper. To me, substantial swap errors seem to be a consequence of having low d-prime values for the identifying dimension; that is, if there is only little information to discriminate the identity of the two stimuli, swap errors would be large. However, this possibility didn't seem to be mentioned in the paper.

(2) Since the solution of the demixing problem was obtained using a numerical procedure based on EM. It would be useful to check whether the initialization has affected the biases obtained.

Reviewer #1 (Public review):

Summary:

This paper provides interesting observations about the effects of a classical mutation in the daf-2 insulin-like receptor in male C. elegans. The observations are a contribution in and of themselves; however, the conclusions reached about these observations are not supported by the work presented. Most importantly, male-specific effects on healthspan measures are asserted without direct comparison to hermaphrodites. Perhaps more fundamentally, essential features of the methods and experimental design are lacking, which makes formal assessment of the results impossible, especially given our knowledge of negative male-male interactions, which have gone completely unacknowledged here. Indeed, there is a general lack of context for known sex differences in C. elegans, especially in terms of the core elements of longevity, which are presented here as entirely novel but in fact are not.

Major comments:

(1) The main overall criticism of the premise of the paper is that it lacks a clear hypothesis that would lead to explicit experimental tests. Instead, many of the results are observational, and the conclusions reached go beyond the actual experiments conducted. The goal should be explicit and consistent between the introduction/ discussion, and the findings should directly address the goal.

The overall focus appears to be that daf-2 males have an extended lifespan for reasons that are different from hermaphrodites. This conclusion is apparently based on the observation of lipid reserves in mutant animals. However, none of the healthspan measures were conducted in parallel with identical measures in hermaphrodites. How can the authors then claim that males are unique? This is especially problematic since other studies have demonstrated that daf-2 hermaphrodites also have altered lipid composition (Vrablik 2015 Biochim Biophys Acta; Horikawa 2010 Mol Cell Endocrin).

(2) The authors make unwarranted claims about causation from observational data that is correlative in nature. Again, they claim that male longevity is caused by increased lipid reserves. This may in fact be the case, but there is no evidence to show that this is causal, only that lipid reserves are increased in mutant animals. Causation requires an actual experiment, in this case, disrupting lipid maintenance in daf-2 males (e.g., Lapierre 2013 Autophagy). Their conclusions are consistent with their results, but their conclusions are much too strong given the nature of the evidence, especially given the concerns about proper comparisons to hermaphrodites.

(3) With these concerns in mind, all conclusions related to male-specific effects should be statistically tested using a sex-by-treatment interaction term in the statistical model. This is obviously impossible for the healthspan data, but for lifespan, this can be directly tested using (genotype x sex interaction in the CPH analysis). Further, it is unclear why each of the replicates is shown separately in Figure 1.

It is nice that the authors do not directly pool them, as most longevity studies do, but the replicate effects can be included in a more comprehensive model, which would yield an appropriate "average" effect curve.

(4) There is an inadequate review of pre-existing literature and findings that predate the observations presented here. While this is not an issue in general, the authors present their work as entirely novel when it is not.

In addition to Gems and Riddle (2000), which is tangentially cited in the discussion, the following papers should be cited and discussed in the introduction to clarify what is currently known and what remains to be explored:

Partridge and Gems (2002) Mechanisms of aging: public or private?

McCulloch and Gems (2007) Sex‐specific Effects of the DAF‐12 Steroid Receptor on Aging in Caenorhabditis elegans

Hotzi et al (2018) Sex‐specific regulation of aging in Caenorhabditis elegans

Al-Saadi et al (2025) Disruption of the insulin signaling pathway in C. elegans dramatically increases male longevity and enhances reproductive health late in life

In addition, the authors assert that the study of sex differences is unstudied. If the authors are specifically referring to the sex differences in aging research, they should explicitly state that and revise their language to reflect that it is "understudied" rather than "unstudied". But as stated below, there are many studies that look at sex-specific differences in behavior, physiology, development, etc. This is most important in the context of sexual conflict, of which there are many studies that are directly relevant to the work presented here. The authors are encouraged to review some of these papers.

(5) This is particularly important in the context of how the experiments presented here were actually conducted. The methods are inadequate to assess this, and the results would therefore be impossible to replicate in the absence of additional details. Exactly how many individuals were raised on each plate during the longevity assays (and other work) is critical to understanding the results of this study. This is because males have direct, chemically and physically mediated negative impacts on one another (see many papers from the Brunet and Murphy labs). Further, it is not even clear whether males and hermaphrodites were reared separately from one another. Males are known to leave plates without hermaphrodites, which requires appropriate inclusion of censoring criteria in studies such as these. It is unclear whether and how this was handled. Censoring is an essential feature of any longevity study and so needs to be explicitly described in the statistical methods.

The methods describe the use of heat shock to induce the production of males, but it is unclear which generation is being used here. Ordinarily, males would be induced, and then male populations would be maintained by forced mating (picking to ensure that there is a high relative frequency of males) for several generations to eliminate any carryover effects of the heatshock itself. Were the heatshock males put directly into the longevity assays? If so, were hermaphrodites subject to identical treatment? This is confusing, and a potentially critical confound is not performed correctly.

Reviewer #1 (Public review):

Summary

In this paper, the authors provide a systematic investigation of structural brain differences associated with congenital aphantasia (self-reported lifelong absence of voluntary visual imagery). Specifically, the authors analysed a structural neuroimaging dataset involving 18 individuals with aphantasia and 18 visualizers to test two competing hypotheses: (1) that aphantasia reflects alterations in visual pathways and early visual cortex, and (2) that it instead reflects differences in higher-order frontotemporal and cingulate systems. To test these hypotheses, the authors employed multiple analysis approaches (e.g., cortical morphometry, tractometry, graph-theoretic network analysis).

They report structural differences between the two groups in frontotemporal and cingulate systems. In contrast, they found no reliable group differences in early visual cortex or major visual tracts. On this basis, they propose that aphantasia is primarily associated with differences in higher-order systems supporting integration and conscious access to internally generated representations, rather than with deficits in sensory visual representations themselves.

Strengths

(1) The present work addresses an important gap in the mental imagery literature, providing a systematic investigation of structural neuroimaging differences in congenital aphantasia. By showing that structural differences between aphantasics and visualizers are mainly concentrated in frontotemporal and cingulate systems (rather than in visual cortex), it makes an important step toward a better understanding of individual differences in mental imagery and provides a set of candidate regions for future mechanistic work.

(2) A key strength of the study is the multimodal approach employed to address the main research question, integrating tractometry, functional region-of-interest (fROI)-based tractography, graph-theoretic network analysis, and surface-based cortical morphometry, which provide a converging assessment of structural differences between aphantasics and visualizers.

(3) The complementary use of Bayesian analyses alongside NHST to assess evidence for null results is a further strength of this work.

Weaknesses:

(1) A weakness of this work is related to aspects of the framing and, in particular, what can be confidently inferred from the results. The framing of existing accounts of aphantasia in the Introduction appears limited in that it reduces the views on aphantasia to two options (sensory strength account versus conscious access account) without acknowledging a third distinct position, namely that aphantasia reflects a specific deficit in the voluntary generation of imagery (Milton et al., 2021; Zeman et al., 2015, 2020; Whiteley, 2021; Cavedon-Taylor, 2022). Like the conscious access account, the view that aphantasia involves a deficit in the generation of sensory representation also speaks against the hypothesis of reduced sensory strength of internally generated representations. This third view could be acknowledged/discussed as it also maps quite well onto the presented results.

(2) Relatedly, I think the main weakness of the paper concerns the interpretation of results being restricted to a lack of "conscious access". The paper frames its findings as mainly evidence for a conscious access failure, the view that visual representations are generated by aphantasics but cannot be consciously accessed. However, the structural findings are equally consistent with a voluntary generation failure, especially since the same higher-order regions examined can also be implicated in the top-down generation and control of imagery. The authors themselves initially define aphantasia as "lifelong absence of voluntary visual imagery". Given the nature of structural imaging data (as opposed to functional data), it is not possible with the present study to distinguish between a lack of generation versus a lack of conscious access. As such, examining this alternative interpretation appears appropriate, and it would considerably strengthen the paper. Structural MRI alone is not sufficient to dissociate imagery generation from conscious access, as these are fundamentally functional questions.

(3) Some inconsistency and lack of clarity around the specific choice of regions/networks, which could be better motivated and explained. E.g., the "core imagery network" analysed in the white-matter connections analysis was derived from a previous 7T study (with which the sample partially overlaps) and is not necessarily the network most commonly associated with visual imagery in the literature (e.g., see Dijkstra et al., 2019; Pearson, 2019). It is, for instance, unclear why V1 was examined in the cortical thickness analysis but not in the previous one, given that both analyses are related to the visual pathway hypothesis. Related to this, in the graph-theoretic analysis, the rationale for network selection is inconsistently established in the Introduction. The attention and salience networks do have some grounding in the Introduction through the mention of specific regions such as FEF and anterior insula, though these are discussed as individual regions rather than as networks. However, the default mode network receives no motivation in the Introduction. More explicit elaboration on these choices would be appropriate.

(3) The interpretation provided in the Discussion tends to oversimplify what is in fact a heterogeneous and rich set of structural findings into a relatively coherent mechanistic account. The observed differences are spatially and directionally variable across tracts, cortical regions, and metrics: e.g., FA is reduced in the UF and posterior interparietal corpus callosum but increased in the dorsal cingulum; cortical thickness is reduced in aPFC but increased in medial temporal regions, and so forth. The Discussion acknowledges this in part (e.g., proposing increased dorsal cingulum FA as potentially compensatory) but does not address the directional heterogeneity systematically. The authors could discuss more explicitly what the opposing directions of effects mean for their overall interpretation. Relatedly, some parts of the Discussion link specific structural findings to specific imagery processes in ways that go beyond what the current data can support. The authors could more clearly distinguish between what the structural data show and what functional interpretations are taken from prior work.

Reviewer #1 (Public review):

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

Overall, the main conclusions are sound but interpreting several lines of experiments and results remain complicated. Consequently, the overall picture of the role of Gcn5 in Drosophila larval lymph gland development, and its relationship to mTOR and autophagy, remains unclear.

Reviewer #1 (Public review):

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

Summary:

This study examines whether gaze direction actively shapes choice during food preference decisions or whether gaze and choice evolve largely independently until the moment of commitment. The established framework in this context, the aDDM, assumes that gaze causally biases the accumulation of evidence in favour of the fixated item. The authors show convincingly that this model fails to fit key behavioural patterns across several datasets, as do other published models that make the same assumption. The authors propose an alternative model (Post-Decision-Gaze or PDG) in which gaze and decision formation are decoupled: gaze does not influence the decision process, nor is it drawn toward the ultimately chosen item, until after the decision threshold is reached. Only during the motor execution period (after commitment) is gaze directed to the chosen option. They demonstrate that this model fits several observed patterns better than the aDDM and related variants.

Strengths:

The work thoroughly considers multiple models and datasets. It advances an interesting alternative perspective on gaze-decision interactions and highlights meaningful shortcomings in existing models. The authors take the time to explain how modelling assumptions produce specific patterns in the data, which is certainly insightful to readers interested in the modelling of value-based decision making.

Weaknesses:

It is unclear to what extent the model's success relies on the way non-decision time is formalised in the model. In the proposed PDG model, non-decision time is decomposed into separate visual encoding, saccadic execution, and manual execution components. Several values (assumed or recovered) do not match known physiological or behavioural ranges. This is a common issue in the literature, and the authors may want to address it in light of broader work discussing what non-decision time consists of in both manual and saccadic actions (e.g., Bompas et al., 2024, Non decision time: the Higgs boson of decision, Psychological Review).

Reviewer #1 (Public review):

McGaughey and Gold ask where in the decision process the flexibility of evidence accumulation arises, proposing that it is not solely a property of downstream integrators but is also supported by stimulus-specific sensory adaptation in the middle temporal area (MT). Recording single-unit activity in rhesus macaques during a motion direction-discrimination task in which an adapting stimulus of varying temporal stability precedes an identical test stimulus, they find that more rapidly changing contexts produce weaker and less discriminable MT responses to the test stimulus, which they argue accounts in part for context-dependent changes in decision-making behavior. Through session-level correlations they further identify pupil-linked arousal as a parallel, apparently separable contributor.

The main strength is the shift of perspective toward the encoding stage: rather than treating MT as a static input to flexible downstream integrators, the authors show that early sensory cortex can itself contribute adaptive, context-dependent signals that shape behavior. The conceptual advance is supported by a well-designed paradigm-total exposure to each motion direction is matched across conditions and the test stimulus is held identical-together with single-unit recordings and simultaneous pupillometry. The behavioral effect is consistent across three animals, and the fact that context-dependent differences emerge over repeated stimulus presentations within a trial, rather than as a sustained baseline offset across blocks, ties the effect convincingly to stimulus-specific adaptation.

The behavioral effect constrains the temporal dynamics of decision formation but does not uniquely identify its algorithmic basis: a leak, a saturating non-linearity, or a reduction in the gain of integration are all compatible with a shallower rise of accuracy with viewing time, and the reduced MT discriminability is itself an encoding-stage efficiency effect of this kind. The manuscript appropriately treats the algorithmic basis as unresolved, noting that distinguishing these accounts would require analyses not available here, such as reverse-correlation or motion-energy kernels with lower-coherence test stimuli.

The inference that the adaptation- and arousal-related signals operate independently rests on the absence of session-wise correlations between the neural and pupil measures and their behavioral contributions. Given the noise in the trial-wise estimates, this is best read as consistent with, rather than demonstrating, true independence, as the authors note.

Overall, the authors largely achieve their aim of showing that sensory adaptation in MT shapes the evidence available for time-dependent perceptual decisions. The evidence for a sensory-encoding contribution is convincing, while the claim of independence between adaptation and arousal is more tentative and is framed as such.

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:

Kashiwagi et al. undertook a population analysis of dendritic spine nanostructure applied to the objective grouping of 8 mouse models of neuropsychiatric disorders. They report that spine morphology in cultured hippocampal neurons shows a higher similarity among schizophrenia mouse models (compared with autism spectrum disorder (ASD) mouse models) and identify an effect of Ecrg4 (encoding small secretory peptides) on spine dynamics and shape in these models.

Strengths:

The study developed a method for objectively comparing spine properties in primary hippocampal neuron cultures from 8 mouse models of psychiatric disorders at the population level using high-resolution structured illumination microscopy (SIM) imaging. This novel technique identified two distinct groups of mouse models according to the population-level spine properties: those with ASD-related gene mutations and those with schizophrenia-related gene mutations. Functional studies, including gene knockdown and overexpression experiments, identified an effect of Ecrg4 on the spine phenotype of the schizophrenia model mice.

Weaknesses:

The main weakness is that the study is wholly in vitro, using cultured hippocampal neurons. The authors present this as an advantage, however, arguing that spine morphology as measured in a reduced culture system can demonstrate direct effects of gene mutations on neuronal phenotypes in the absence of indirect influences from nonneuronal cells or specific environments.

Reviewer #1 (Public review):

Summary:

This important study examines how antibiotic-resistant bacterial cells can protect neighboring sensitive cells in mixed populations that occupy both surface-associated and freely growing states. Using experiments in Enterococcus faecalis together with a mathematical model, the authors test the hypothesis that protection would be stronger in biofilm-associated populations, but instead find that resistance-mediated protection extends broadly across both population types. The work provides evidence that antibiotic efficacy depends strongly on community composition, population density, and density-dependent detoxification dynamics.

Strengths:

A major strength of the study is the close integration of experimental measurements with a relatively simple quantitative model that captures many of the observed population dynamics. In particular, the work highlights how interactions between antibiotic detoxification, cellular growth, and saturation at carrying capacity can generate nonintuitive behavior, including the reported population inversion effect. The agreement between the well-mixed model and the experimental observations is convincing, and the spatial analyses suggest that cells within the biofilm are sufficiently intermixed that large-scale spatial segregation is unlikely to dominate the observed behavior.

Weaknesses:

The mechanistic interpretation could, however, be clarified further by more explicitly emphasizing the competing timescales associated with detoxification, growth, and resource limitation. The current results suggest that when resistant cells are initially abundant, detoxification occurs rapidly relative to growth, allowing the population to approach carrying capacity after relatively few doublings, whereas slower detoxification at lower resistant fractions may permit greater expansion of sensitive cells once antibiotic concentrations decline. Additional direct measurements of antibiotic concentrations over time would also strengthen the connection between the experimental system and the modeling framework by testing whether the detoxification dynamics assumed in the model are quantitatively appropriate, although this seems very plausible.

The study also raises interesting questions regarding the role of spatial structure and exchange between planktonic and biofilm-associated populations. It would be informative to explore whether biofilm-specific protection becomes more pronounced at lower antibiotic concentrations, where local detoxification may compete more directly with antibiotic penetration into the biofilm, and in this context, the dynamics of exchange between biofilm and planktonic populations would be interesting to understand. Overall, the evidence supporting the central conclusions is convincing, and the study will likely be of broad interest to researchers studying microbial communities, antibiotic resistance, and collective population dynamics.

Reviewer #1 (Public review):

Summary:

Lituma and colleagues investigate the role of NMD in astrocytes, an underexplored question given that prior work on NMD in the brain has focused exclusively on neurons. Using a tamoxifen-inducible, astrocyte-specific Upf2 conditional knockout (cKO) mouse, they report that loss of astrocytic NMD causes: (1) reductions in astrocyte cell volume and surface area across hippocampus, visual cortex, and prefrontal cortex; (2) decreased excitatory synapse density, reduced dendritic spine density, and impaired synaptic engulfment; (3) deficits in basal synaptic transmission and LTP, with selective impairment of mGluR-LTD; (4) elevated spontaneous calcium transients in astrocytes; and (5) anxiety-like behavior in the elevated plus maze (EPM) and contextual fear conditioning paradigms. Transcriptomic analysis of FACS-isolated astrocytes identifies 277 differentially expressed genes, ~40% of which carry canonical NMD-inducing features, implicating pathways linked to calcium signaling, phagosome formation, and glial development. A rescue experiment using the CalEx calcium extrusion pump demonstrates partial restoration of synaptic strength and anxiety behavior when astrocytic calcium is normalized.

The study addresses an important gap in our understanding of RNA regulation in glial cells, and the overall conceptual framework is well described. The experimental design is generally appropriate, and the multi-pronged approach lends the main claims a degree of validity.

Strengths:

(1) Novelty: This is the first study to systematically examine NMD function in astrocytes in vivo. The identification of astrocytic NMD targets via RNA-seq combined with an NMD-inducing feature classifier is a meaningful methodological contribution.

(2) Multi-method approach: The authors combine morphological analysis (Imaris 3D reconstruction), synaptic markers (PSD-95, LAMP2 engulfment assay), spine density measurements, acute slice electrophysiology, two-photon calcium imaging, behavioral testing, and transcriptomics. The convergence across these methods strengthens confidence in the claims.

Weaknesses:

(1) While the transcriptomic analysis is a valuable addition, the connection between specific NMD targets and the observed calcium phenotype remains largely correlational. The authors identify Gabbr2 and Adora1 as upregulated candidates with canonical NMD features and speculate that their elevated expression drives aberrant calcium signaling. However, no validation (e.g., qRT-PCR or protein-level confirmation) of these candidates is presented. The mechanistic pathway between NMD disruption and elevated calcium is thus inferred from pathway analysis rather than demonstrated. This is a significant gap between the transcriptomic and physiological arms of the study, and the authors should be more explicit about this limitation or, ideally, provide at least one validated target.

(2) The reduction in astrocyte surface area in cKO mice is interpreted as contributing to reduced synapse contact and engulfment capacity. This is a reasonable hypothesis, but the study does not directly demonstrate that reduced astrocyte territory correlates with reduced synaptic coverage at the level of individual cells or brain regions. The temporal sequence of these events is unknown. Do morphological deficits precede synaptic changes? Clarification and qualification of this causal chain in the Discussion would strengthen the manuscript.

(3) LFS-induced LTD is unaffected, while mGluR-LTD is reduced. This is intriguing and potentially informative about astrocyte contributions to distinct LTD mechanisms, but the difference receives limited discussion. Given the relevance of mGluR signaling to calcium dynamics and the identified pathway enrichments (GPCR signaling), this specificity deserves more attention.

(4) The CTRL + CalEx condition is included in the EPM experiment but not in the electrophysiology or calcium imaging experiments, making it difficult to fully assess whether CalEx itself has off-target effects on synaptic transmission or anxiety in wild-type animals. The CTRL + CalEx EPM data (Figure 7F) appears to show a modest reduction in open arm time relative to CTRL, which, if robust, would suggest that excessive calcium reduction in astrocytes is also anxiogenic. This finding would be physiologically relevant and deserves comment.

Reviewer #1 (Public review):

Summary:

The article "Nanoscale organization of beta-II spectrin within segments of the membrane-associated periodic skeleton in mouse sciatic nerve axons" by Gazal et al. looks into the organization of the spectrin scaffold in mouse sciatic nerves using super-resolution microscopy. It is now well established that axons, across species, contain a membrane-associated periodic scaffold mainly composed of circumferential actin filaments and longitudinally arranged spectrin tetramers. While super-resolution imaging of neurons in cell culture is relatively easy, exploring the ultrastructure of myelinated axons in intact nerve fibers is a daunting task. Nevertheless, the authors have attempted this by fixing and preparing cross-sections of sciatic nerves. They have then tried to quantify the fluorescence intensity patterns of specific components, especially that of labeled beta-II spectrin and have analysed its distribution.

One of the main findings is that spectrin is distributed along the axonal periphery and along the outer part of the myelin sheath. By labelling multiple cellular components and using intensity analysis, the authors show the sequence of structural organization of a few key components. They see that, unlike in the case of axons in culture, the axonal cross-sections within the sciatic nerve deviate significantly from a circular shape. They then use 3D-dSTORM to investigate the distribution of beta-II spectrin along the axonal circumference. They see that this distribution is very heterogeneous, both in the sizes of spectrin puncta and their arrangement along the periphery. The amount of spectrin scales linearly with axonal circumference.

Strengths:

Super-resolution imaging of axons of intact nerve fibers to investigate the organization of beta-II spectrin.

Weaknesses:

While most of the findings, like the spatial distribution of spectrin and related components, are reasonably well supported by data, I have concerns regarding the subsequent claims made in the article. The detection of axial periodicity based on the observation of a peak in the inter-tetramer spacing distribution is not very convincing, and a 3D representation (or a video of 3D reconstruction) would have been better. And so are the claims on characteristic spectrin spacing of 200 nm along the axonal circumference. A peak in the distribution does not imply a periodic arrangement.

Reviewer #1 (Public review):

In the wild, bacteria can be found in a wide range of metabolic states, including states in which they are resource limited. Because phages heavily rely on the infected cell's molecular machinery to replicate, it is natural to wonder how phage-bacteria interactions depend on the metabolic state of the cell. In this work, Marantos et al. investigate specifically how the rate of infection of 5 different phages changes between cells grown in energy-rich conditions and cells grown in energy-depleted conditions. Their results clearly show that 4 out of the 5 phages studied display a significant reduction in infection rate in cells that are energetically depleted and provide a potential explanation for this observation by looking into the mechanisms that these phages use to irreversibly infect their host cells.

The work also tries to explain the observation using a mathematical/mechanistic model that describes infection as the sequence of two steps, where a phage first needs to bind to a cell receptor, from which it can potentially unbind, and then irreversibly infects by injecting its genome. The mechanistic interpretation offered by the model highlights an interesting trade-off between adsorbing to a metabolically active host and discriminating between active and inactive hosts that, somehow, a phage has to optimize. It would be interesting, in the future, to investigate how different phages optimize this task.

Comments on revised version.

I am happy with how the authors have addressed all the comments. The manuscript is much clearer and more readable and the previous overstated claims have been removed/clarified.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The findings are reported by reporter gene assay (microscopy). No evidence is given using genetics. The authors claim that the DNA is maintained episomally. However, could it be possible that there is integration? No PCRS/RT-PCRs are shown (although it can safely be assumed that the DNA/RNA is present where the transformation was successful), nor are any Western blots. These would have been useful to show that the P2A ribosomal skipping had occurred, and that proteins were expressed individually rather than as a polyprotein.

Comments on revised version.

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

Reviewer #2 (Public review):

Summary:

The manuscript titled "Latent gene network expression underlies partial re-evolution of a polyphenic trait in the worker caste of ants" by Vasquez-Correa et al. aimed to study genetic mechanisms underlying developmental plasticity, especially binary polyphenism in queen vs worker ant castes. This is an interesting question regarding the extent to which phenotypic traits were altered, lost or regained, and how molecular pathways (upstream vs. downstream) can facilitate this process.

In ants, reproductive castes (queens and males) develop wings as well as 3 ocelli for mating flights and other activities, while worker castes are wingless, and in some species, they have either no or a reduced number of ocelli. The phylogenetic analysis showed that in the Camponotini ant clade, the one-ocellus phenotype re-evolved in three species independently. The authors analyzed the conserved developmental pathways between Drosophila (well-established) and ants using HCR (a high-quality in situ hybridization technique). They found that although upstream genes for the development of ocelli (otd and hh) showed similar expression between castes, downstream genes (toy, eya, and so) had reduced or no expression in workers of C. floridanus, and this differential expression may lead to partial or complete loss of ocelli. Consistently, workers develop rudimentary tissues, suggesting that they initiate the ocellus developmental process but somehow stop it before adulthood.

Strengths:

Evo-devo approaches to reveal conserved molecular pathways of ocellus development. High-quality HCR provided convincing evidence of the expression of key genes in ocelli, eyes and antenna throughout larval development.

Using HCR, the authors showed differential expression of downstream genes in males vs. soldiers vs. minor workers of C. floridanus, which might explain phenotypic differences between castes.

Comments on revised version.

The authors have addressed the concerns in the revision. No further comments.

Reviewer #1 (Public review):

Summary:

Cisplatin, a platinum-based chemotherapeutic agent, induces intra- and interstrand crosslinks, thereby blocking DNA replication and transcription and triggering apoptosis. The authors aim to demonstrate that DNA polymerase κ (Polκ), traditionally seen as a translesion synthesis (TLS) polymerase, able to synthesize DNA through DNA lesions, plays a non-catalytic, structural role in stabilizing replication forks and protecting cells from cisplatin-induced cytotoxicity. A key finding of this work is the identification of two novel molecular axes: PCNA-Polκ-Polδ, which facilitates efficient DNA replication; PCNA-Polκ-USP18, which stabilizes DNA damage response proteins. These findings provide actionable therapeutic targets for overcoming head and neck squamous cell carcinoma chemoresistance, a cancer with rising incidence and limited treatment options.

Strengths:

The study relies on a robust experimental design, including Polk allegedly CRISPR-Cas9 knockout, siRNA knockdown, and rescue experiments with wild-type, catalytically dead, and PCNA-interaction-deficient Polκ variants, supporting a non-catalytic role of Polκ. The work also reports a strong implication of Polk in cisplatin resistance, the identification of USP18 as a possible Polk partner and the consequences of Polk depletion on post-translational stabilisation of DNA damage response proteins.

Weaknesses:

The findings reported in this manuscript cannot be generalized to all cisplatin resistance mechanisms, as cells may develop multiple adaptive strategies to survive chemotherapy. Polκ's role varies across cancer types. For example, it is downregulated in stomach and colorectal cancers but upregulated in HNSCC, lung, and ovarian cancers. Thus, its use as a biomarker or drug target may be context-dependent.

Acute cisplatin exposure is sufficient to trigger Polκ upregulation to levels similar to those in resistant cells. However, it remains unclear how long this upregulation persists and to what extent it contributes to survival. Further, the sensitivity of cisplatin-naïve H357 or SCC9 cells (H357-S and SCC9-S) to Polκ knockdown has not been addressed. This is a critical question, as acute cisplatin exposure induces Polκ expression to levels similar to those in resistant cells. This could argue against a direct role for Polκ in mediating resistance and instead suggest indirect mechanisms (like Polκ-dependent mutations during adaptation).

The experimental design and results aimed at demonstrating the existence of a PCNA-Polκ-USP18 axis (Figure 9A) do not fully support the conclusion that these proteins form a stable complex. This set of experiments also lacks essential controls, such as the immunoprecipitated bait and the amount of immunoglobulins precipitated in all conditions. This also applies to the colocalization experiments in cells shown in Figure 9B. Images are poor and lack quantification. Further, Polk is seen mainly cytoplasmic in the upper panel, while it is nuclear in the lower panel. Discrepancies in Polk subcellular localization are also evident in the Supplementary data. USP18 is known to deubiquitinate ISG15-modified proteins (not just ubiquitin). The study does not rule out ISGylation as a contributing mechanism. The experimental design involving analysis of DNA synthesis dynamics at a single-molecule level is not appropriate. Overinterpretation of the data in several parts of the manuscript and lack of rigor in performing the experiments. Inappropriate consideration and absence of discussion of previously published literature directly related to the subject studied in this manuscript. Discrepancy with a previous report regarding the role of Polk in Chk1 phosphorylation (Tonzi et al., eLife 2018). Synergic effect of T2AA inhibitor and Cisplatin have been already described in « naive » cancer cells (Inoue et al, 2014). Another critical point is that the proliferation rate of Polk-depleted cells is slower than that of wild-type cells. Hence, the colony formation assay shown in Figure 2B can be misleading, since the observed differences can be interpreted only as a proliferation problem.

Reviewer #1 (Public review):

Summary:

This is an important paper examining LTP induced by theta-burst stimulation in hippocampal slices from macaques and rats. While both species show theta-burst-late-LTP, only the non-human primate theta-burst-late-LTP showed synaptic tagging and capture that converts early-LTP into late-LTP in an independent synaptic pathway.

Strengths:

Synaptic tagging is a fundamental feature of repeated 100 Hz-tetanus-induced LTP, whereas theta-burst induction is arguably more physiologically relevant. Thus, synaptic tagging during theta-burst may differ in the two species, a distinction that may prove important in the mechanisms underlying the cognitive differences between the species.

Weaknesses:

Bursts repeated at the frequency (~5 Hz) of the endogenous theta rhythm induce strong LTP, primarily because this frequency disables feed-forward inhibition and allows sufficient postsynaptic depolarization to activate voltage-sensitive NMDA receptors. Therefore, the species differences may be due to differences in inhibition, rather than in molecular mechanisms of maintenance. One way to assess the relative strengths of this early induction mechanism in rats and macaques is to examine the "depolarization envelope" during the sequential bursts, which may be determined from the recordings already obtained. (Larson and Munkácsy, Theta-burst LTP, Brain Res 2015 Sep 24:1621:38-50. doi: 10.1016/j.brainres.2014.10.034)

Another issue is that the PKMzeta-antisense oligodeoxynucleotides block the synthesis of the kinase. However, Mei F, Nagappan G, Ke Y, Sacktor TC, Lu B (2011), BDNF Facilitates L-LTP Maintenance in the Absence of Protein Synthesis through PKMzeta. PLoS ONE 6(6):e21568, provided evidence that BDNF and theta-burst stimulation can act to increase PKMzeta by a protein synthesis-independent mechanism, presumably through decreased degradation. Therefore, the absence of an effect of the PKMzeta-antisense does not exclude the possibility that persistently increased PKMzeta is the mechanism of theta-burst-late-LTP maintenance in mice or macaques. This issue is worth discussing.

Reviewer #1 (Public review):

Summary:

This article describes a very ambitious metascience project aimed at testing the reproducibility of a corpus of publications conducted in Brazil. The strength of the approach lies in its systematic, multicenter replication design. The authors focus on three commonly used experimental paradigms in biology: the MTT assay, RT-PCR, and the elevated plus maze.

The effort is commendable and reveals a rather low rate of reproducibility, in line with findings from fields considered less reproducible in the life sciences, such as cancer biology.

Strengths:

The study is supported by a substantial dataset, incorporating multiple independent replication attempts and the use of stringent, well-defined protocols, which strengthens confidence in the overall conclusions.

Weaknesses:

(1) Being neither an expert in metascience nor in statistics, I cannot fully judge the methodological aspects of the article or its extensive supplementary material. I will therefore focus my comments on readability. I found the manuscript difficult to digest. The authors should improve readability if they wish to reach a broad audience of experimental biologists. In particular, they should simplify the description of protocols and highlight the key findings more clearly, using accessible language. See specific points below

(2) The article appears to oscillate between:

i) a description of the approach and the inherent challenges of such a multicenter replication program.

ii) an estimation of reproducibility.

These could potentially form two separate articles: one aimed at a broad audience emphasizing key results, and another focused on methodological aspects for a more specific metascience audience. The Results section currently contains redundancies and is difficult to follow for non-experts in statistics. I also find it challenging to extract the main findings.

A possible improvement would be to include an initial section clearly describing the protocol (replication of a single experiment, across several labs, for three types of assays), followed by a concise presentation of the main results regarding reproducibility in Brazilian science with subsections. Methodological details could be moved either to a Supplementary Information or to a more specific article, while being summarized in the Discussion.

(3) This study evaluates the reproducibility of a single experiment from each article, taken out of its broader context. While this provides an estimate of reproducibility, it does not directly contribute to resolving uncertainties within a specific field. This may represent a limitation compared to other reproducibility projects that attempt to replicate multiple key claims within a given study (e.g., in cancer biology or Drosophila immunity). I found that a weakness is that it does play a role in cleaning a field of wrong statements.

(4) The observation that external observers can predict which experiments are likely to be reproducible is interesting and should be more clearly emphasized.

(5) The manuscript frequently refers to future publications. It would be helpful to clarify what is included in the present article versus what is deferred to subsequent papers

Reviewer #1 (Public review):

Summary:

The authors used a large dataset evaluating gut carriage of Enterobacterales and ESBL organisms from children aged 6-24 months as the basis for a modeling study to investigate what factors are most important for determining the prevalence of ESBL resistance. The modeling incorporated travel, a simple model of carriage duration (short and long), fitness cost of resistance on transmission and clearance, and antibiotic use. They found that antibiotic use is the primary driver of resistance prevalence, with transmissibility of resistant strains also important for setting the prevalence. Travel, while important when prevalence is very low, plays less of a role in maintaining prevalence once it is established (in keeping with other recent work). They estimated the fitness cost of resistance (terming a reduction of 14% on the rate of transmission and an increase of 23% on the rate of clearance as "low"). While the extent of assumptions and simplifications makes me skeptical of the quantitative conclusions, the qualitative ones seem reasonable and reinforce the long-held principles of the field--reducing antibiotic pressure and interrupting transmission--and highlight the importance of understanding the biological factors that shape the duration of carriage and the likelihood of colonization.

Strengths:

This study incorporates many of the factors that might influence the carriage prevalence of ESBL Enterobacterales. This builds on the work led by this group, both in primary data collection and in theory. Overall, it's such a tough problem that I commend the authors for trying to tackle it. The authors take a thoughtful, rigorous approach, acknowledging simplifications and assumptions where they need to, so as to evaluate the various factors shaping ESBL prevalence.

Weaknesses:

Part of the reason it's such a tough problem is that we have limited data to structure and parameterize a complex model.

(1) The data are not sufficiently described.

The primary data source for this modeling exercise comes from a study of 6-24-month-old children who underwent rectal swabs and evaluation of the carriage prevalence of Enterobacterales, and then whether these Enterobacterales were ESBL; moreover, the study included data on travel and on antibiotic use. Could the authors please direct us to these primary data? Could the authors also justify the parameters in their models from these data--for example, could they please provide the distribution of antibiotic use and the associated timing? Could they also explain why they decided to treat all Enterobacterales as if they were E. coli (line 307)? Is there evidence that all Enterobacterales occupy the same niche and compete with each other?

(2) The model should be more fully described and the limitations explored/explained.

- The authors should point to the code and the ODEs.<br /> - I understand the focus on the pediatric population; the authors argue that this is reasonable because ESBL colonization is similar across age groups. But presumably, antibiotic use differs across age groups, and there is colonization pressure from within households.<br /> - The authors only consider resistance to extended-spectrum beta-lactams and use of beta-lactam antibiotics, but ESBL Enterobacterales are often resistant to other antibiotics as well. How much does the use of other antibiotics also select for Enterbacterales that happen to carry ESBL resistance? "One bug/one drug" modeling, as done here, neglects the complexities of the actual patterns of resistance and range of antibiotic use.<br /> - Do the data support the T3 or S3 compartments, which, if I understand correctly, means no exposure to antibiotics can happen during three months after either treatment or travel? What do the data say about the patterns of antibiotic use? I'd imagine that the likelihood of antibiotic use is not homogenous, but instead, there are some who use repeated rounds of antibiotics.<br /> - Why do the authors exclude individuals who used antibiotics in the prior 7 days? What justifies that cutoff? The authors speculate that the impact of excluding these individuals is likely to be minimal; why exclude them, then? Did the authors evaluate the results if they were included?<br /> - What is the basis of "niche differentiation", as described starting on line 221? Why should clearance of one strain be slower when the strain co-occurs in a host with a strain of another type?

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

The methods and discussion sections are lacking enough detail to fully evaluate the findings and put them into perspective, respectively.

Reviewer #1 (Public review):

Summary:

In this study, the authors describe an early diverging vertebrate KCNE gene present in jawless lampreys that they denote KCNE0.

Three forms of the protein are isolated from different lampreys, which have 95% homology to each other, but only moderate homology to KCNE1-6.

Co-expression with lamprey KCNQ1 produced a non-inactivating current, whereas co-expression with mammalian KCNQ1 resulted in less modulation. Introduction of a tetra-leucine motif from KCNE4 into KCNE0 reduced current on co-expression with KCNQ1, conferring an inhibitory effect.

Strengths:

This is an interesting and uncontroversial report of a new KCNE isoform from lower vertebrates that gives insight into the evolutionary progression of the sequence and functional properties of the accessory protein.

Weaknesses:

(1) No error bars visible for lamprey Q1 isoforms (open symbols) in Figure 2G. No statistical comparison was provided to indicate whether lamprey Q1 isoform V1/2s are significantly different (nor in Supplementary Table 1).

(2) There is the same issue in Figures 3 and 4. No appropriate statistical comparison is made between V1/2s for different truncations of PmKCNE0 (Figure 3), or between KCNQ1 species isoforms with and without PmE0.

CTLA4 Alteration and Neurologic Manifestations: A New Family with Large Phenotypic Variability and Literature Review

Reviewer #1 (Public Review):

The manuscript by Boudjema et al. describes the cellular events underlying centriole amplification and apical migration to allow the assembly of hundreds of motile cilia in multi-ciliated cells. For this, they use cell culture models in combination with fixed and live cell imaging using antibody staining and fluorescence from endogenously tagged centriole and deuterostome markers, respectively. The work is largely descriptive and functional analyses are restricted to treatment with the microtubule depolymerizing drug nocodazole. The imaging is state-of-the-art including confocal microscopy, live imaging with optical sectioning and high optical and temporal resolution, as well as super-resolution imaging by ultra-expansion microscopy.

The study does a good job of providing a very detailed description of the dynamics of centrioles and deuterostomes that lead to centriole amplification and apical migration in multiciliated cells. This detailed view was missing in previous work. It also reveals the involvement of microtubules at multiple steps: the formation of a cloud of deuterostome precursors, the nuclear envelope tethering of newly formed centrioles, their separation, and their migration to the apical surface.

It would have been useful to expand the analysis of the role of microtubules by including analyses of the requirement for specific microtubule motors, for a better understanding and additional evidence that microtubule-based transport is involved. A weak point is that there is no visualization of microtubules together with deuterosomes and centrioles at the different steps of centriole amplification and migration, to directly address how these structures may interact with and move along microtubules.

Overall, apart from experimental aspects and since this is largely a descriptive study, the manuscript would benefit from more precise language and a better description of the complex events underlying centriole amplification and movements.

Comments on revised version.

The authors have significantly improved the manuscript, by refocusing it, introducing text and figure changes, and by adding new data including functional analyses. The revised version now has convincing data that support the claims. All my remaining concerns have been addressed.

Reviewer #1 (Public review):

This work convincingly shows that, rather than gradually "evolving" throughout interphase, global chromatin architecture undergoes unexpectedly sharp remodeling at G1-S (and to a lesser extent, S-G2) transitions. By applying "standard" Hi-C analyses on carefully sorted cells, the authors provide an excellent temporal view of how global chromatin architecture is changed throughout the cell cycle. They show a surprisingly abrupt increase in compartmentation strength (particularly interactions between the "active" A compartments) at G1-S transition, which is slightly weakened at S-G2 transition. Follow-up experiments show convincingly that the compartment "maturation" does not require the DNA synthesis accompanying S phase per se, but the authors have not identified the responsible factors (work for future publications). The possible biological ramifications of these architectural changes (setting up potential replication "factories", and/or facilitating transcription-replication conflict resolution, both more pertinent for the active A compartments, which are most affected) have been well discussed in the article, but still remain speculative at this stage.

My major criticism of this article is aimed more at the state of the field in general, rather than this specific article, but it should be discussed to give a more balanced view: what actually is a chromatin compartment? Chromosomal tracing and live tracking experiments have shown that the majority of "structures" identified from Hi-C experiments are statistical phenomena, with even "strong" interactions only being infrequent and transient. A-B compartments are "built up" from multiple very low-frequency "interactions", so ascribing causal effects for genome functions is even tougher. As a result, I have very little confidence in the results of the authors' polymer simulations and their inferred "peninsula" A compartment structures without any other supporting experimental data.

Comments on revised version.

The authors have included orthogonal DNA FISH evidence to support their claims which greatly strengthens the manuscript. Their further precisions within the discussion have answered all of my previous concerns with the manuscript.

Reviewer #1 (Public review):

Summary:

The manuscript by Lu and colleagues demonstrate convincingly that PRRT2 interacts with brain voltage-gated sodium channels to enhance slow inactivation in vitro and in vivo. The work is interesting and rigorously conducted. The relevance to normal physiology and disease pathophysiology (e.g., PRRT2-related genetic neurodevelopmental disorders) seems high. Some simple additional experiments could elevate the impact and make the study more complete.

Strengths:

Experiments are conducted rigorously including experimenter blinding and appropriate controls. Data presentation is excellent and logical. The paper is well written for a general scientific audience.

Comments on revised version.

The manuscript by Lu and colleagues has been revised sufficiently to address all my prior concerns.

Experiments are conducted rigorously including experimenter blinding and appropriate controls. Data presentation is excellent and logical. The paper is well written for a general scientific audience.

Reviewer #1 (Public review):

Summary:

This study offers a careful and technically strong look at how surface stickiness changes whisker-surface interactions and how that information reaches peripheral sensory neurons. The authors use 3D whisker tracking to capture bending, twisting, rolling, and tip motion during contact with surfaces that differ in stickiness, coarseness, and position. They show that sticky surfaces, especially silicone, broaden the range of whisker deformation, produce stronger but less frequent stick-slip events, and change firing rates in some trigeminal ganglion neurons. Overall, the study is valuable because it goes beyond standard 2D tracking and shows that out-of-plane motion and roll are important for understanding how whiskers encode texture.

Strengths:

The study is technically strong and well motivated. Its main strength is the use of 3D whisker tracking to show that surface stickiness affects whisker deformation in ways that standard 2D tracking would miss, including torsion, roll, out-of-plane motion, and stick-slip dynamics. The authors also connect these mechanical effects to TG activity, providing evidence that stickiness information is available in peripheral sensory responses. Overall, the work expands the study of whisker-based texture sensing beyond coarseness and provides a richer biomechanical framework for understanding tactile encoding.

Weaknesses:

The main weakness is that stickiness is not formally defined early in the manuscript, even though it is the central experimental variable. Several methodological choices also need clearer justification or validation, including the use of 2D measures as comparators for torsion and roll, the thresholds used for stick-slip detection, the degree-5 polynomial fit, the reference ROI, and aspects of the 3D surface reconstruction. The neural evidence should also be interpreted cautiously because the TG sample is small, only a subset of units discriminated silicone, and the correlation between strain sensitivity and silicone discrimination is suggestive rather than definitive.

Reviewer #1 (Public review):

Summary:

Using Mendelian randomisation on available GWAS data, the investigators identified eGenes associated with prostate cancer and applied the data to define relevant immune cell types involved. Additional analysis was performed to explore potential candidate targets and agents from licensed medicines.

This is an interesting approach as the investigators have expertise in other research fields, applied here to prostate cancers. The use of three different datasets is significant, and the approach to further analyse implicated eGenes in drug target analysis is relevant and timely.

A particular strength is taking putative genes from Mendelian randomisation analysis to target and potential drug agents.

Some aspects of the study would need to be clarified to enable interpretation of the findings in the context of the prostate gland and prostate cancers: expanding the descriptions of the supporting Supplementary Data and Tables, explanations of the analysis for the general reader, and clarification of the selection of eGenes (Figure 5).

Reviewer #1 (Public review):

Summary:

The manuscript- "Cell cycle-dependent variation in endocytosis drives phenotypic diversity in M. tuberculosis" by Subhash et al. demonstrates how host cell heterogeneity shapes intracellular pathogen phenotypes. The central and novel finding of this study (G2-phase cells have higher endocytic capacity and harbour more oxidised Mtb) highlights that a host cell cycle (interphase-driven) changes in endocytic capacity regulate bacterial redox states.

Strengths:

Overall, the study is well-executed and conceptually rich, establishing a causal link between host cell cycle progression, endocytic heterogeneity, and M. tuberculosis phenotypic diversity.

The combination of multiple modalities, including live-cell imaging, flow cytometry, scRNA-seq, and redox-sensitive bacterial reporters, supports these findings and substantially strengthens the biological relevance of the work.

The writing is generally clear, and the figures are well-organised.

This work will be of interest to readers across cell biology, microbiology, and infection biology

Weaknesses:

However, several central claims are only partially supported, the mechanistic depth is limited, and several experimental and analytical concerns need to be addressed.

Major Comments:

(1) The authors demonstrate a correlation between the G2 phase and elevated endocytic capacity. However, the mechanistic link (upstream molecular mechanism) between the cell cycle and endocytic upregulation remains largely unaddressed. The authors speculate that membrane biogenesis during volumetric expansion may drive increased endocytosis and note that lipid biosynthesis genes are upregulated in high-endocytic cells. It would substantially strengthen the paper to test this directly, by examining whether inhibition of lipid biosynthesis (e.g., with fatostatin or cerulenin) selectively reduces the G2-associated increase in endocytic capacity. Alternatively, cyclin-CDK axis perturbations (e.g., CDK1 inhibition with RO-3306 to specifically block G2/M entry) could be used to ask whether cells arrested in G2 maintain elevated endocytosis, helping distinguish cell-cycle-position-dependent from cell-cycle-progression-dependent effects.

(2) The current data show a clear association between high endocytic capacity and more oxidised Mtb, and the authors (consistent with their prior work) hint at lysosomal delivery as the likely mechanism. However, direct evidence for this in the current paper is limited. An experiment examining phagosomal pH or lysosomal fusion (e.g., using a pH-sensitive reporter or lysotracker) specifically in high- and low-endocytic-capacity cells after infection would help confirm this.

(3) Temporal resolution of Mtb redox dynamics. The plasticity experiment (Figure 6C-D) is elegant and shows that Mtb redox states revert as host cells divide and daughters enter G1. However, the experiment compares day 0 and day 3 post-sorting, which spans multiple cell divisions. While a finer time resolution (spanning 24h) would establish the causal relationship, the authors could discuss the possibility and consequences of multiple cell divisions between day 0 and day 3 used in the present study.

(4) Relevance of G2 percentages in differentiated macrophages. In Figure 7 and Supplementary Figure S7, only 4.4-5.7% of THP-1-derived macrophages and 5.7% of BMDMs are in G2. While the authors demonstrate statistically significant differences in Mtb redox states between G1 and G2 macrophages, the biological significance of such a small G2 fraction in a non-dividing population deserves discussion specifically with respect to: a) Are these cells re-entering the cycle? b) Is the G2 designation capturing a distinct functional state rather than active cycling? The authors should include additional markers (e.g., phospho-histone H3 for mitotic cells or BrdU incorporation to test for active S-phase) to characterise this population and clarify its identity and origin in differentiated macrophages, thereby meaningfully informing interpretation.

In conclusion, this is an important mechanism-driven study that highlights an important link in host-driven bacterial phenotypic heterogeneity. The experiments are thorough, the model is well-supported, and the study has implications for infection biology.

Reviewer #1 (Public review):

Summary:

Torpor can be induced by chemogenetic activation of the medial preoptic area. This activation leads to protection from myocardial infarction in an isolated heart preparation despite normalization of the ambient temperature, thus, in principle, uncoupling hypothermia from torpor-induced neuroprotection. Putative pathways of protection are suggested by proteomic studies.

Strengths:

(1) Elegant strategy for inducing torpor in rats.

(2) Appropriate controls for verifying the neuron transducer.

(3) Cardiac protection is significant and appears independent of hypothermia.

(4) Interesting omic strategy to begin to find established and novel pathways mediating organ autonomous torpor-induced protection.

Weaknesses:

(1) The study would benefit from using inhibitory chemogenetics of the same neurons to demonstrate that this might make cardiac response to ischemia worse.

(2) Infecting an area of the brain not known to be involved in torpor would be a useful control.

(3) In vivo cardio protection seems essential as the validation of the strategy requires support that is in the intact animal.

(4) The assumption that the positive effects of torpor are mediated via a phosphoproteomic change rather than a translational or transcriptional control mechanism is not established.

(5) A 40 percent reduction in infarct size may work for genetically identical rats with no co-morbidities, but is unlikely to be significant enough to weather the variability that emerges in humans because of these differences and more. The question is not what the mechanism is, but how do we make it more robust? Overall, this is at best a preliminary data set that requires more experiments to deliver on its immense promise.

Reviewer #1 (Public review):

The work corroborates the idea, recently suggested by Rosenthal et al. (2025), that spreading depolarization is involved in the mechanisms of electroconvulsive therapy. Using a mouse model of electroconvulsive therapy and various sophisticated approaches to visualize cortical activity, the authors provide an extensive description of traveling calcium waves induced by electroconvulsive stimulation. The study confirms that the calcium events have properties typical of cortical spreading depolarization and seeks to show that the calcium/SD waves mediate therapeutic and neuroplastic effects of electroconvulsive therapy. The authors find that after electroconvulsive stimulation associated with calcium/SD waves, Fos expression increases widely; in the cortex, this increase is localized to the hemisphere affected by calcium waves. They show that some EEG predictors of the beneficial effects of electroconvulsive therapy correlate with the occurrence of calcium/SD waves. Despite the solid methodology and the study's interesting, its conclusions are not fully supported by the data.

In particular:

(1)The title of the paper claims that "electroconvulsive stimulation drives cortical spreading depolarization dependent immediate early gene expression". However, immunohistochemical staining shows that Fos expression increases not only in the cortex but also in many subcortical regions, including the hippocampus and amygdala (Figure 5A). Really, conventional electroconvulsive therapy stimulates nearly the entire brain volume and induces generalized seizure activity that can trigger SD not only in the cortex but also in other brain sites. Therefore, regions beyond the cortex can also drive the effects of electroconvulsive therapy. Next, the authors use Fos staining as a marker of neuronal plasticity. However, Fos is also a marker of preceding neuronal activation. As electroconvulsive stimulation, seizures, and SD are associated with high neural activity, it is unclear whether the observed Fos upregulation results from the prior activation or heralds the subsequent plastic changes. Other markers of neuroplasticity (e.g., BDNF) should also be examined.

(2) Postictal EEG suppression is one of the most promising correlates of positive clinical outcomes after electroconvulsive therapy. Cortical SD is also tightly coupled with suppression of neuronal activity in affected regions. Although the authors report that postictal suppression is stronger after stimulations with cortical SDs than without SDs, the cortices affected (ipsi) and unaffected (contra) by unilateral cortical calcium/SD events exhibit identical suppression (Figure 6F). The result contradicts established knowledge in the field. If the calcium events are cortical SDs, they should induce EEG suppression only in the affected hemisphere.

(3) The study states a beneficial role of calcium/SD waves in ECS effects. However, SD alters numerous aspects of brain function, leading to a range of effects that can underlie side effects as well. Assessment of the behavioral effects of stimulation with and without calcium/SD waves can help clarify the issue.

The results of the work suggest that cortical SD can contribute to electroconvulsive therapy-related mechanisms and help to optimize the stimulation parameters to achieve maximal therapeutic effect.

Reviewer #1 (Public review):

[Editors' note: all three reviewers confirm that all initial concerns have been fully resolved through comprehensive revisions and supplementary analyses.]

Summary:

By imaging the dynamics of synaptic proteins in cultured neurons, this study presents significant findings regarding the dynamics of excitatory and inhibitory synaptic proteins during development. The evidence shows that the ratios of excitatory and inhibitory synaptic proteins are stable during synapse development. This discovery advances our understanding of the complex mechanisms governing synapse formation. The strength of the evidence is robust, as it is supported by a combination of biological assays and endogenous labeling.

Strengths:

This research sheds light on the dynamics of the excitatory and inhibitory synapses during development. It is crucial to understand that while excitatory synapses and inhibitory synapses are developed independently, the ratio of their number is relatively stable during development, maintaining a stable excitatory/inhibitory ratio.

Important findings and implications in the research include:

(1) Persistent Synapse Dynamics: Excitatory and inhibitory synapses remain highly dynamic even in mature neurons (DIV12-14), challenging the dogma that synaptic structures are stable after the synaptogenesis stage.

(2) Maintained E/I Balance: Despite ongoing synapse turnover (formation/elimination) and presynaptic terminal reduction, the overall density and ratio of excitatory-to-inhibitory synapses remain relatively stable during circuit maturation (Figure 7).

(3) Developmental Shifts: While presynaptic compartments decrease over time, postsynaptic sites increase, suggesting independent regulation of pre- and postsynaptic elements within a stable E/I framework.

Weaknesses:

This study focuses on specific synaptic proteins within synapses, which may not fully represent the dynamics of other synaptic machinery; also, whether similar observations exist in vivo is still unknown. Further research is needed to explore the implications of these findings in more complex neuronal environments.

Comments on revised version:

The authors have addressed all my questions/comments. No further questions for this manuscript.

Reviewer #1 (Public review):

Summary:

This study investigates how human temporal voice areas (TVA) respond to vocalizations from nonhuman primates. Using functional MRI during a species-categorization task, the authors compare neural responses to calls from humans, chimpanzees, bonobos, and macaques while modeling both acoustic and phylogenetic factors. They find that bilateral anterior TVA regions respond more strongly to chimpanzee than to other nonhuman primate vocalizations, suggesting that these regions are sensitive not only to human voices but also to acoustically and evolutionarily related sounds.

The work provides important comparative evidence for continuity in primate vocal communication and offers a strong empirical foundation for modeling how specific acoustic features drive TVA activity.

Strengths:

(1) Comparative scope: The inclusion of four primate species, including both great apes and monkeys, provides a rare and valuable cross-species perspective on voice processing.

(2) Methodological rigor: Acoustic and phylogenetic distances are carefully quantified and incorporated into the analyses.

(4) Neuroscientific significance: The finding of TVA sensitivity to chimpanzee calls supports the view that human voice-selective regions are evolutionarily tuned to certain acoustic features shared across primates.

(4) Clear presentation: The study is well organized, the stimuli well controlled, and the imaging analyses transparent and replicable.

(5) Theoretical contribution: The results advance u

Comments on revised version.

I thank the authors for having carefully considered and implemented my remarks on the first version.

Reviewer #1 (Public review):

Summary:

Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains in the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. The authors attribute this divergence to differences in short-term synaptic plasticity, particularly within somatostatin-expressing (SST⁺) interneurons. This interpretation is supported by 1) the increased density of SST+ neurons in L4 of the septa compared to barrel domain, 2) the stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation and 3) the reduced functional difference in single- versus multi-whisker response ratios across barrel and septal domains in Elfn1 KO mice, which lack a synaptic protein that confers characteristic short-term plasticity, notably in SST+ neurons. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain, suggesting that septal and barrel circuits may differentially route information about single vs multi-whisker stimulation downstream of S1.

Strengths:

This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view these two domains contribute distinctly to the processing single versus multi-whisker inputs and highlight the role of SST+ neuron and their short-term plasticity. Together, this study suggests that the barrel cortex multiplexes whisker-derived sensory information across its domains, enabling parallel processing within S1.

Weaknesses:

Although the divergence in responses to repeated single- versus multi-whisker stimulation between barrel and septal domains is consistent with a role for SST⁺ neuron short-term plasticity, the evidence presented does not conclusively demonstrate that this mechanism is the critical driver of the difference. The lack of targeted recordings and manipulations limits the strength of this conclusion: SST⁺ neuron activity is not measured in L4, nor is it assessed in a domain-specific manner. The Elfn1 knockout manipulation does not appear to selectively affect either stimulus condition, domain or interneuron subtype. Finally, all experiments were performed under anesthesia, which raises concerns about how well the reported dynamics generalize to awake cortical processing.

Reviewer #1 (Public review):

The paper uses a passive whisker detection task in mice to identify a behavioral phenomenon that can reasonably be interpreted as spatial attentional capture. The attentional effect occurs transiently after a successful whisker stimulus detection yields reward, and lasts for a few trials before subsiding. The attentional effect is to the right or left whiskers, depending on whether right or left whiskers are rewarded; no finer spatial resolution for attention was tested. By recording whisker-evoked spiking from single units in S1, the authors show that this form of spatial attention increases the gain of whisker-evoked neuronal responses in S1 for a large subset of S1 units. In contrast, neural responses are not modulated by overall task engagement. Together, these findings show a neural signature of spatial attention in S1 cortex. Because whisker or facial movements were not tracked, it is not clear whether this represents covert attention or whisker movement in response to previously rewarded stimuli, which would be a form of overt attention.

Substantial attentional modulation of neural responses was observed for a subset of whisker-responsive S1 units, but the effect size was small on average for the total unit population. The top 25% of units showed a ~12% attentional response modulation (relative to firing rate range for each unit), but the median unit showed only a 1.3% response modulation. It would have been useful to analyze the magnitude or prevalence of attentional modulation across layers or in fast-spiking vs. regular spiking units, but this was not reported.

Major

(1) It is hard to interpret the underlying causes of the attentional modulation of neural activity without having measured whisker and facial movement. This is a particular issue in S1, where whisker movement against the stimulation grid can alter the mechanical efficiency of stimulus delivery. Such movements would represent overt attention, which would engage an entirely different neural mechanism than covert attention.

(2) An interesting debate is whether the behavioral phenomenon is best described as attention or as dynamic learning of the stimulus-response association for that block. In Posner-type cued attention tasks, and also in many block-type attention tasks in rodents, animals receive reward for successfully detecting either cued or uncued stimuli, and thus attention (higher response probability or improved psychometric sensitivity for cued stimuli) is at least partially dissociated from the stimulus-reward contingency. That is not the case here. The fact that mice have difficulty learning the contingency reversal suggests that the phenomenon is better explained by attention than by learning the contingency; however, to prove this clearly, the existence of the attentional effect on neural activity in Block 1 vs. Block 2 would have to be shown.

(3) Some of the graphical representations of the attentional modulation of neural activity are unclear. The single-unit example of attentional modulation is quite strong (Figure 3d). The mean response for the top 25% of units is also visually clear (Figure 3f). But the effect is not apparent at all in Figure 3e, which the figure legend says shows every unit. What is the yellow point and line in this figure? Why isn't the attentional effect visible in this panel? Perhaps I am misunderstanding Figure 3e, but it is not clear to me why it compares Pref>0.5 to Pref<0.5, when the intended analysis suggests it should be Pref>0 to Pref<0? Also in Figure 3, it is critical for the reader to know whether panels 3g-3h represent the top 25% of units or all units. Neither the results text nor the legend is clear on this.

(4) There is a missed opportunity to quantify attentional modulation across cortical layers, since laminar probes and Neuropixels probes were used for the recordings. In addition, there is no separation of fast-spiking from regular-spiking units, and no quantitative metrics are provided to assess the quality of single units. This could reveal key aspects of cortical processing of attentional signals.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

Mudunuri et al. investigate the foraging response of Drosophila larvae in response to patchy resources of distinct value (concentration of nutrient or valence). They show that larvae adjust their behavior according to both the quality and valence of available resources. Interestingly, previous exposure to resources of lower value increases the permanence time in resources of greater value. This suggests that larvae can value, remember and adapt their behaviour in response to previous foraging experience.

They perform a simple integration model that recapitulates the larval behaviour.

Strengths:

This paper uses a very well-controlled foraging set-up where larvae are tested individually and for 3 hours, allowing for a good statistical analysis of their behaviour.

They investigate for the first time the ability of Drosophila larvae to perceive, remember and compare the quality and valence of distinct resources. It is very exciting, as it will open up the field of foraging decision studies using the fruitfly larvae.

Weaknesses:

(1) Most of the analysis depends on the thresholding, but it is not clear what increasing the radius of analysis means in terms of foraging. There are two issues here:

a) What is the behaviour of the larvae on the edges of the patch? It is obvious that the fructose or the NaCl will diffuse at the edge, so are they remaining in the proximity because they are actively feeding (exploiting) on this decaying concentration, or are they sensing the lower gradient and they are actually looking (chemosensing) for the higher concentration? The behaviour at the edge is really different (check sucrose in Wosniack et al. 2022), and there might be a way of avoiding the diffusion by actually adding a plastic ring and pouring the agar + resource in there. The effect of the ring, per se, would still have to be tested.

b) How was the threshold selected? It is very likely that the concentration at the patch boundary will be very different for 1M and 0.1 M. Could the authors explain why they chose such a distance? What does majority of larvae mean? Is the "majority" the same for 0.1M and 1M? Is there a relationship between the threshold chosen and the diffusion of fructose and NaCl?

(2) The word exploitation is used in the paper, but there are many instances where it is unclear whether that is the case. This should be clarified since there are no controls for exploitation.

(3) In the experiments analysing the adaptation of foraging behaviour, it is not clear if the first and second patch means that only 2 patches were analysed per larva or the first and second in a sequence of patches visited. I think it is the second option (because of Figure S3D), but the authors should clarify this. Also, we do not know how many animals were tested. The number of data points in 4C (4G) compared to 4D (4H) seems very different.<br /> Regarding the results, which are very interesting, why aren't the larvae spending less time in the 0.1M sucrose patch after having fed on a 1M patch, while they spend more time in a 1M after a 0.1M? Could it be that the difference in residence time is correlated with their hunger rather than the comparison between conditions?

(4) I am not an expert in this type of model, and I would appreciate it if the authors could explain how the values of the drift and leak have been fitted in Figure 5H. If possible, I would recommend adding a graph showing the parameter exploration of distinct possible combinations of values.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors address the question of working memory maintenance, starting from the experimental observation that recordings of neural activity during the delay period of working memory tasks are sometimes observed to be dynamic. They introduce a new combination of metrics (noise-robustness and energy efficiency) to quantify the performance of various network mechanisms of memory maintenance, in linear networks. They compared attractor networks, feed-forward networks, and networks trained with a loss that includes a robustness and an energy-efficiency component. They show, by plotting state-space trajectories, that networks optimized with this loss exhibit a form of rotational dynamics. They analyzed the data recorded during the delay of a working memory task in PFC, and observed state-space trajectories similar to those of the trained networks.

The comparison with other network mechanisms is interesting in principle, but limited by the fact that only linear networks are considered. This led to counter-intuitive and misleading statements, like the fact that attractor networks are not robust to noise, or that feed-forward networks have energy consumption that is exponential in the number of neurons.

Strengths:

(1) The idea to use both robustness to noise and energy efficiency to assess the performance of networks on working memory tasks is interesting.

(2) The manuscript is clearly written.

(3) There is an interesting combination of methodologies: theory on simple models, network training, and data analysis.

Weaknesses:

(1) Linear networks only.

The main feature of attractor networks is their robustness to noise, which is typically allowed by the non-linearity of neural responses. To fit their modeling framework, the authors focused only on continuous attractor neural networks (e.g., Seung 1996) and ignored point-attractor models such as the Hopfield model, which are typically used to model WM tasks, and which would presumably lead to very different results, e.g., in Figure 1D.

The linearity assumption is also problematic for the comparison with feed-forward models. It seems that the authors obtained runaway firing rates, explaining Figure 1F middle, which are typically prevented in non-linear networks.

The choice of parameters for the attractor network in Figure 1 is not explained. Why is t_slow = 10^4 chosen, and what does it correspond to? We expect in linear networks that activity goes back to zero or diverges as an exponential, but in principle, the time constant can be chosen to be of the same order as the time delay, with approximately linearly decreasing SNR.

Regarding the comparison of the different mechanisms, it would have been nice to better define the notion of rotational dynamics, beyond only considering state-space analysis, which is limited to providing mechanistic interpretations.

(2) Fixed duration of delay periods.

I have understood that for a given network, the duration of the delay period is fixed, as opposed to a delay duration that would fluctuate from trial to trial. This would be an important assumption to relax as well, to better match common experimental paradigms, as well as to expose a fairer comparison with other network mechanisms. See Orhan and Ma (2023) for such a discussion.

(3) Relationship with previous works

Many other works addressed the question of dynamic firing rates during maintenance periods of WM tasks; they should be discussed and compared to the mechanism proposed here. This includes: Barak et al, Progress in Neurobio. 2013, Pereira-Obilinovic, Aljadeff, Brunel, PRX 2023, Hansel, Mato, 2013, or works pertaining to the activity-silent neural states (allowed by short-term plasticity), the framework in which the data of Panichello et al are interpreted in the original publication.

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:

In this manuscript, the authors present comprehensive experimental observations and a theoretical framework to explain the heterogeneous behaviour of sarcomeres in cardiomyocytes. They show that a stochastic component exists in their contractile activity, which may act as a feedback mechanism regulating physiological function.

Strengths:

Experiments and data analysis are robust and valid. The rigorous statistical analysis and unbiased methods enable the authors to draw well-supported conclusions that go beyond the existing literature. Their outcomes inform about cellular activity at the individual level and the authors explain how the transient dynamics of single sarcomeres are governed by a force-velocity relationship and lead to the complex contractile patterns. The similarity of the results to the study cited in [24] demonstrates the validity of the in vitro setup for answering these questions and the feasibility of such in-vitro systems to extend our knowledge of out-of-equilibrium dynamics in cardiac cells.

Very interesting the suggestion that the interplay between intrinsic fluctuations and the dynamic instability are part of a feedback mechanism for maintaining structural and functional homeostasis.

The addition of the theoretical model and the new text of the manuscript improves the clarity of the study.

Reviewer #1 (Public review):

Summary:

Dad et al. explored the roles of cytosolic carboxypeptidase 5（CCP5）in the development of ependymal multicilia in the brain. CCP family are erasers of polyglutamylation of ciliary-axoneme microtubules. The authors generated a new mutant mouse of Agbl5 gene, which encodes CCP5, with deletion of its N-terminus and partial carboxypeptidase (CP) domain (named AGBL5M1/M1).

Strengths:

The mutant mice revealed lethal hydrocephalus due to degeneration of ependymal multicilia. Interestingly, this is in contrast with the phenotype of Agbl5 mutants with disruption solely in the CP domain of CCP5 (named AGBL5M2/M2) that did not develop hydrocephalus despite increased glutamylation levels in ependymal cilia as observed for AGBL5M1/M1 mutants. The study has been well-performed and the findings suggest a unique function of the N-domain of CCP5 in ependymal multicilia stability.

Weaknesses:

The content of this article is relatively descriptive and lacks molecular insights, regarding the function of the CCP5 N-domain.

Comments on revised version.

The authors have appropriately revised the manuscript in response to most of my comments.

Reviewer #1 (Public review):

Summary:

Gruskin and colleagues use twin data from a movie-watching fMRI paradigm to show how genetic control of cortical function intersects with the processing of naturalistic audiovisual stimuli. They use hyperalignment to dissect heritability into the components that can be explained local differences in cortical-functional topography and those that cannot. They show that heritability is strongest at slower-evolving neural time scales, and more evident in functional connectivity estimates than in response time series.

Strengths:

This is a very thorough paper that tackles this question from several different angles. I very much appreciate the use of hyperalignment to factor our topographic differences and found the relationship between heritability and neural time scales very interesting. The writing is clear and the results are compelling. In general, I don't have many complaints after a couple reads through the manuscript; most of my comments below are relatively minor suggestions and points of clarification.

Weaknesses:

The only "weaknesses" I identified were some points where I think the methods, interpretation, or visualization could be clarified:

On page 16, you compare heritability in functional connectivity (FC) and response time series and find that the heritability effect is larger in FC. In general, I agree with your diagnosis that this is in large part due to the fact that FC captures the covariance structure across parcels, whereas response time series only diverge in terms of univariate time-point-by-time-point differences. Another important factor here is that (within-subject) FC can be driven by intrinsic fluctuations that occur with idiosyncratic timing across subjects and are unrelated to the stimulus (whereas time-locked metrics like ISC and time-series differences cannot, by definition). This makes me wonder how this connectivity result would change if you used intersubject functional connectivity (ISFC) analysis to specifically isolate the stimulus-driven components of functional connectivity (Simony et al., 2016). This, to me, would provide a closer comparison to the ISC and response time series results, and could allow the authors to quantify how much of the heritability in FC is intrinsic versus stimulus-driven. I'm not asking that the authors actually perform this analysis, as I don't think it's critical for the message of the manuscript-but it could be an interesting future direction. As the authors discuss on page 17, I also suspect there's something fundamentally shared between response time series and connectivity as they relate to functional topography (Busch et al., 2021) that drives part of the heritability effect.

The observation that regions with intermediate ISC have the largest differences between MZ, DZ, and UR is very interesting, but it's kind of hard to see in Figure 1B. Is there any other way to plot this that might make the effect more obvious? For example, I could imagine three scatter plots where the x- and y-axes are, e.g., MZ ISC and UR ISC, and each data point is a parcel. In this kind of plot, I would expect to see the middle values lifted visibly off the diagonal/unity line toward MZ. You could even color the data points according to networks like in Figure 3C. (You also might not need to scale the ISC axis all the way to r = 1, which would make the differences more visible.)

On page 9, if I understand correctly, you regress the vector of ISC values across parcels out of the vector of heritability values across parcels and then plot the residual heritability values. Do you center the heritability values (or include some kind of intercept) in the process? I'm trying to understand why the heritability values go from all positive (Figure 2A) to roughly balanced between positive and negative (Figure 2B). Important question for me: How should we interpret negative values in this plot? Can you explain this explicitly in the text? (I also wonder if there's a more intuitive way to control for ISC. For example, instead of regressing out ISC at the parcel/map level, could you go into a single parcel and then regress the subject-level pairwise ISC values out when computing the heritability score?)

On page 4 (line 155), you say "we shuffled dyad labels"-is this equivalent to shuffling rows and columns of the pairwise subject-by-subject matrix combined across groups? I'm trying to make sure your approach here is consistent with recommendations by Chen et al., 2016. Is this the same kind of shuffling used for the kinship matrix mentioned at line 189?

I found panel A in Figure 4 to be a little bit misleading because your parcel-wise approach to hyperalignment won't actually resolve topographic idiosyncrasies across a large cortical distance like what's depicted in the illustration (at the scale of the parcels you're performing hyperalignment within). Maybe just move the green and purple brain areas a bit closer to each other so they could feasibly be "aligned" within a large parcel. Worth keeping in mind when writing that hyperalignment is also not actually going to yield a one-to-one mapping of functionally homologous voxels across individuals: it's effectively going to model any given voxel time series as a linear combination of time series across other voxels in the parcel.

References:

Busch, E. L., Slipski, L., Feilong, M., Guntupalli, J. S., di Oleggio Castello, M. V., Huckins, J. F., Nastase, S. A., Gobbini, M. I., Wager, T. D., & Haxby, J. V. (2021). Hybrid hyperalignment: a single high-dimensional model of shared information embedded in cortical patterns of response and functional connectivity. NeuroImage, 233, 117975. https://doi.org/10.1016/j.neuroimage.2021.117975

Chen, G., Shin, Y. W., Taylor, P. A., Glen, D. R., Reynolds, R. C., Israel, R. B., & Cox, R. W. (2016). Untangling the relatedness among correlations, part I: nonparametric approaches to inter-subject correlation analysis at the group level. NeuroImage, 142, 248-259. https://doi.org/10.1016/j.neuroimage.2016.05.023

Simony, E., Honey, C. J., Chen, J., Lositsky, O., Yeshurun, Y., Wiesel, A., & Hasson, U. (2016). Dynamic reconfiguration of the default mode network during narrative comprehension. Nature Communications, 7, 12141. https://doi.org/10.1038/ncomms12141

Comments on revised version.

The authors have adequately addressed my previous comments. This is a strong contribution: the methods are sophisticated, the statistical treatment is rigorous, and the results are quite interesting/compelling. I'm happy to endorse the revised manuscript as a finalized version.

Just to confirm: The subjects watched all different movies across the two days, right? For a moment I was wondering "are Day 1 and Day 2 repetitions of the same movies?" Given that Day 1 and Day 2 are an organizational feature of several figures, it might be worth making this very explicit in the Methods and reminding the reader in the Results section.

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 some of comments raised in the previous round of review and have opted to proceed to a Version of Record without additional review.]

Summary:

This is an excellent and strong paper. The authors not only show the mechanisms of action of destabilizing mutations in VHL, but notably, they also go on to computationally design and experimentally test an inhibitor that restores wild-type pVHL function, offering starting points for a new class of kidney cancer drugs. The approach that the authors take here can be used to target destabilizing mutations in repressor proteins, common in diseases, including cancer.

Strengths:

This paper is the culmination of an extraordinary amount of work, over years, including method development and testing by a broad range of tools and experiments. It is thorough and comprehensive. It is also well-written and easy to follow.

Reviewer #1 (Public review):

Summary:

The authors quantified and compared the 3D kinematics of bill and tongue movements between two seed-eating bird species: one that specializes on soft seeds, and one that is more adapted to feeding on hard seeds. Their goal was to determine specifically what the role of the tongue was for processing (e.g., dehusking) seeds, and to understand how differences in biting strength between species affect other aspects of seed processing. The authors provided intricate (visual) details of seed processing movements, and showed how coordination between the tongue and cranial kinesis (i.e., mobility of the upper bill relative to the cranium) is both critically important for properly positioning seeds to enhance feeding efficiency. Many studies have detailed how seed-eating birds process seeds, but this study has elevated those to a new level of quantification and visualization for readers to fully experience firsthand. Furthermore, the authors established that the force-velocity trade-off that has been observed between bill functions (e.g., feeding and singing) is largely driven by the contractile properties of the muscles. The conclusions are well supported by the results, and the authors placed the results more broadly into the context of manual grasping, making the argument that these birds achieve high levels of dexterity with far fewer degrees of freedom, which could have potential biomimetic applications.

Strengths:

This study builds upon - and advances - our understanding of the feeding mechanics of seed-eating birds using cutting-edge 3-dimensional modeling and kinematics. Their quantitative analyses of upper and lower bill, tongue, and seed displacements are complemented by elegant visualizations of seed processing in each species. Their comprehensive Bayesian modeling statistical framework tackles the issue of small sample sizes (i.e., few subjects) with volumes of data for each (i.e., lots of sequential kinematic variables) that plague comparative biomechanics studies, principally because (a) it is difficult to gather these high resolution XROMM and muscle contractile data on more than just a few subjects, and (b) these data streams are inherently very large, as they are gathered at high frame and sampling rates. Furthermore, I believe their approach to statistically testing for differences between species sets a new standard for our field that could (perhaps should?) be implemented in other similar types of studies. Another strength is in how the results were packaged: each subsection indicated how the objectives were addressed, and there were concluding statements trailing each subsection that helped deliver the key takeaways.

Weaknesses:

A potential weakness is one that the authors themselves mentioned, regarding the body (and skull) size differences between species. Because gape size limits bite force, and given the force-velocity tradeoff in muscle function, there could be limitations on the rapid manipulation of relatively large seeds for similar reasons in the smaller finches. I see that the small finches appear to overcompensate in their beak rotations, but it's not clear how those compensatory movements might affect their seed processing kinematics with their preferred seed sizes. This does not nullify the authors' conclusions, but the results for the smaller finches might not be entirely representative of seed processing mechanics in smaller species.

Reviewer #1 (Public review):

Summary:

The authors seek to understand and identify the neural plasticity that underlies recovery from precise unilateral hemi-pyramidotomy. The corticospinal tract is severed on one side in the pyramids below the exit of corticoreticular projections. Recovery from the injury is achieved with an intensive wheel running rehabilitation regime. The anatomical sites of plasticity, the importance of plasticity in different reticular areas<br /> to recovery, and the impact of the degree of plasticity observed on recovery as correlated predictors, are shown.

Strengths:

Refined anatomical analysis using mouse line and genetic and viral intersectional tracing identifies specific reticular targets of likely enhanced cortical control that correlate with recovery of locomotor skill.

Weaknesses:

(1) The study is correlational at this time. This does not undercut the value of the data and the identification of targets of plasticity achieved in the work.

(2) Generalization of motor gains beyond locomotion was not tested. Reach-to-grasp tasks for feeding were not tested.

(3) Some discussions and use of the terms fine motor and skilled motor are fuzzy, and the limitations of the study are not sufficiently clearly stated.

Reviewer #1 (Public review):

Summary:

Microbialization (bacterial overgrowth) is a recognized component of degraded, eutrophied coral reefs where there is a shift from coral to algal dominance on the benthos. In addition, previous work has demonstrated that virus communities shift from a lytic strategy dominated (kill-the-winner) to a temperate (lysogenic) strategy dominated with reef microbialization. Kelman et al. sought to leverage previously published virus metagenomes produced from the water column of healthy and degraded coral reefs to assess virus community metabolic shifts. The authors also produce a conceptual model to demonstrate the potential impact of the observed metabolism shifts on reef fates.

Strengths:

The main strength of the manuscript is the findings from their metagenomic analyses and results. The virus metagenomes were produced using established approaches in the field and yield sufficient data per sample for their analyses. Interesting results regarding the shift in the types of genes from anaplerotic to cataplerotic provide the foundation for testable hypotheses to determine the magnitude of impact virus strategies have on reef health. The introduction is also well written and sets up the scene very well.

Weaknesses:

(1) The methods text currently omits important information related to the sampling design. It is not clear how many metagenomes are from healthy and degraded communities. This impacts the interpretability and robustness of the statistical results. Furthermore, it is unclear if analyses are based on assembled contigs or read-based alignments. Improving the clarity and organization of the Methods is essential for reproducibility.

(2) Regarding the bioinformatics approach, normalization using the "percent known" approach within samples may not fully account for discovery bias related to sequencing depth. While Supplementary Table 1 shows variability in read counts, the lack of community-level metadata makes it difficult to determine if sequencing depth covaries with community type (healthy vs. degraded). The study would benefit from a rarefaction analysis or subsampling to ensure that gene frequency trends and Spearman correlations are biological signals rather than artifacts of sequencing effort.

(3) The qualitative model in Figure 5 is positioned as evidence for the role of viruses in reef health, but it does not provide independent support for the authors' hypotheses. Since the model is parameterized using "arbitrary units" to reflect the authors' assumptions rather than being derived from the empirical metagenomic data, it serves as a helpful illustration of a hypothesis but not as a validation of the findings.

(4) Results and discussion require revisions to improve readability and connectivity across sections. Ensuring a clear distinction between empirical data and model-based speculation would help the audience better appreciate the science.

Reviewer #1 (Public review):

Summary:

The review by Dorrell and Whittington synthesizes the progress made over the past few years with respect to a normative theory of grid cells. The core question addressed by normative frameworks of grid cells is what primary computational function grid cells serve. The review discusses evidence from mechanistic models and experimental data that point to path integration as the computational function of grid cells, consistent with results from normative models. The main goal of the review is to clarify the normative grid cell theory literature. However, the current version of the article reads at times more like a perspective or opinion article in support of the path integration hypothesis rather than a critical review of normative frameworks in the grid cell literature that contrasts the benefits and limitations, as well as pitfalls and caveats, with other modelling approaches.

Some specific comments are as follows:

(1) Abstract: "The first question quickly attracted an answer: grid cells subserve path integration ..." - I am not sure if this statement is correct. The first grid cell paper by Hafting and Fyhn in 2005 suggested that grid cells are part of a path integration-based map, and the paper emphasizes the map part. It remained unclear, and is still debated, whether grid cells are part of a system performing path integration or whether grid maps reflect the output/result of a path integration process. Other theories about the function of grid cells were brought forward as well. Although the main competing theory is discussed in this review, this review article at times appears more as a perspective or opinion article with a clear bias toward the path integration hypothesis rather than objectively discussing the evidence.

(2) Grid cells may serve multiple functions. What would be the implications for our understanding of grid cells and for interpreting the results of normative models? In general, the review could discuss some pitfalls or caveats of normative models in more detail.

(3) A normative framework can be helpful in two ways: (a) Given sufficient details on biological constraints, a normative model can help identify the computational function of grid cells. If a computational function is given and - under the given simulated biological constraints - grid cells were part of the solution, the results of the model would support the hypothesis that grid cells serve the computational function in question. (b) If a computational function were identified beyond any doubt (e.g., assume experimental data demonstrated that grid cells are necessary and sufficient for path integration), a normative model would help identify biological parameters necessary to produce grid cell firing. Unfortunately, the review falls short in making this clear distinction between (a) and (b) and in discussing important caveats regarding mixing up these two ways. E.g., the neural network model approaches by Sorscher et al. and others have been criticized because they try to achieve two things at the same time: find support for the computational function of grid cells and identify optimal parameters that result in grid cells. But doing both at the same time provides a strong bias in tweaking the parameters in exactly the way you need for the model to produce grid cells as a solution (other solutions may be possible given other parameters), preventing strong conclusions regarding the computational function of grid cells and preventing conclusions about what the parameter choices mean for biological connectivity motifs. These caveats in setting up normative models and interpreting them could be discussed in greater detail.

(4) A common assumption underlying most grid cell models is that head direction is viewed as identical to movement direction. However, head direction can differ at times from movement direction, and entorhinal head direction cells code head direction rather than movement direction (Raudies et al., 2015; 10.1016/j.brainres.2014.10.053). This missing link in how movement direction signals reach and inform grid cells could be discussed.

(5) "Knowing that one neuron in a module is active and that you make a movement north uniquely determines which neuron in that module should be active next" - I agree that this rule follows from the fact that grid cells within one module differ in phase but share spacing and orientation. However, I am surprised that the authors do not also make the argument here for the value of a normative model. Rebecca R.G. et al. (10.7554/eLife.96627) use exactly the rule cited above as a normative function. They demonstrate that this rule begets grid cells. Isn't this a prime example of how a normative approach can contribute to scientific inquiry? First, a hypothesis about a computational function is derived from experimental data. And in turn, using a normative framework, the experimental data are derived from the computational function (under appropriate biological results). The paper is discussed later together with Nicolai Waniek's work (10.1162/neco_a_01255). However, in my opinion, their work seems to be somewhat misrepresented in that later paragraph. E.g., velocity is still required as an input to determine which neuron should be active next, neurons do not need to be binary units, and space is not discretized beyond the fact that space is encoded by neurons with spatial firing fields.

Reviewer #1 (Public review):

Summary:

Extracellular electrophysiology datasets are growing in both number and size, and recordings with thousands of sites per animal are now commonplace. Analyzing these datasets to extract the activity of single neurons (spike sorting) is challenging: signal to noise is low, the analysis is computationally expensive, and small changes in analysis parameters and code can alter the output. The authors address the problem of volume by packaging the well-characterized SpikeInterface pipeline in a framework that can distribute individual sorting jobs across many workers in a compute cluster or cloud environment. Reproducibility is ensured by running containerized versions of the processing components.

The authors apply the pipeline in two important examples. The first is a thorough study comparing the performance of two widely used spike-sorting algorithms (Kilosort 2.5 and Kilosort 4). They use hybrid datasets created by injecting measured spike waveforms (templates) into existing recordings, adjusting those waveforms according to the measured drift in the recording. These hybrid ground truth datasets preserve the complex noise and background of the original recording. Similar to the original Kilosort 4 paper, which uses a different method for creating ground truth datasets that include drift, the authors find Kilosort 4 significantly outperforms Kilosort 2.5. The second example measures the impact of compression of raw data on spike sorting with Kilosort 4, showing that accuracy, precision, and recall of the ground truth units is not significantly impacted even by lossy compression. As important as the individual results, these studies provide good models for measuring the impact of particular processing steps on the output of spike sorting.

Strengths:

The pipeline uses the Nextflow framework, which makes it adaptable to different job schedulers and environments. The high-level documentation is useful, and the GitHub code is well organized. The two example studies are thorough and well-designed and address important questions in the analysis of extracellular electrophysiology data.

Weaknesses:

There are no major weaknesses in the revised manuscript. While no data analysis pipeline can cover the needs of all experiments, the authors have added and significant flexibility in the pipeline. Even experimenters who might opt for a simpler pipeline will benefit from this work as a model.

Reviewer #1 (Public review):

Summary:

This manuscript examines how Streptococcus pyogenes regulates expression of the virulence factor SpeB in response to both bacterial and host-derived cues. The authors propose that Vfr acts as a repressor of speB expression and that degradation of Vfr by SpeB or by neutrophil-derived proteases relieves this repression. This creates a model in which S. pyogenes can sense proteolytic activity during infection and use that information to tune virulence factor expression.

Strengths:

The main strength of the study is the bacterial regulatory mechanism. The dual reporter system provides a useful way to follow speB and hasABC expression, and the genetic analysis of known regulators helps validate the system. The media-swap experiments, recombinant Vfr experiments, and SpeB-mediated degradation of Vfr support the conclusion that Vfr represses speB and that proteolysis can relieve this repression. The finding that SpeB can degrade Vfr is particularly interesting because it suggests an autoregulatory mechanism that could reinforce SpeB expression once it has been initiated.

Weaknesses:

The host side of the model is less completely supported. The authors show that neutrophil lysates and protease-containing fractions can induce the speB reporter and degrade Vfr, which supports the idea that neutrophil-derived proteases can affect this circuit. However, the in vivo interpretation relies heavily on PAD4-deficient mice to implicate neutrophil extracellular traps. PAD4 deficiency is a useful perturbation, but it does not by itself distinguish loss of extracellular trap formation from changes in neutrophil recruitment, survival, degranulation, phagocytosis, oxidative killing, or other neutrophil death pathways. As a result, the current data support a role for neutrophil-associated proteolytic activity more strongly than they support a specific role for extracellular traps. This distinction is important for interpreting the central model. The bacterial circuit is well developed, but the host-derived cue remains somewhat underdefined. If the relevant signal is extracellular protease activity more broadly, then the model is still interesting, but the conclusion should be framed around neutrophil-derived proteolytic stress rather than extracellular traps specifically. If extracellular traps are the key in vivo source of protease exposure, then additional evidence would be needed to separate that mechanism from other neutrophil effector functions that remain intact in PAD4-deficient cells.

Overall:

This is a valuable study with solid evidence for a bacterial protease-sensing regulatory mechanism controlling SpeB expression. The work should be useful to investigators interested in bacterial virulence regulation, host-pathogen interactions, and how pathogens integrate immune-derived cues during infection. The impact of the study would be stronger if the host-derived signal were defined more precisely, but the bacterial Vfr-SpeB circuit provides a compelling framework for thinking about how S. pyogenes links proteolytic activity to virulence gene expression.

Reviewer #1 (Public review):

Summary:

The authors presented a simplified E. coli cell-free protein synthesis (eCFPS) system reduces core reaction components from 35 to 7, improving protein expression levels. They also presented a "fast lysate" protocol that simplifies extract preparation, enhancing accessibility and robustness for diverse applications.

Strengths:

The authors present a valuable new protocol for eCFPS, which simplifies its application.

Weaknesses:

The authors provide data for optimization but offer insufficient explanation of the fundamental mechanisms underlying the phenomenon based on data.

Comments on revised version.

The authors have satisfactorily addressed the concerns raised by the reviewers. However, the mechanistic basis of the observed performance gain remains insufficiently substantiated. The attribution of this improvement to enhanced transcription is currently speculative. This point could be directly tested by quantifying mRNA levels, for example, using real-time PCR, in both the initial and optimized systems. Such analysis would significantly strengthen the mechanistic interpretation of the results.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revised version.

Authors have worked on most of the revisions stated in previous feedback and included in the newer version, which has been significantly improved. Other comments have been described to be out of scope from this study.