Reviewer #1 (Public review):

Summary:

The researchers examined how individuals who were born blind or lost their vision early in life process information, specifically focusing on the decoding of Braille characters. They explored the transition of Braille character information from tactile sensory inputs, based on which hand was used for reading, to perceptual representations that are not dependent on the reading hand.

They identified tactile sensory representations in areas responsible for touch processing and perceptual representations in brain regions typically involved in visual reading, with the lateral occipital complex serving as a pivotal "hinge" region between them.

In terms of temporal information processing, they discovered that tactile sensory representations occur prior to cognitive perceptual representations. The researchers suggest that this pattern indicates that even in situations of significant brain adaptability, there is a consistent chronological progression from sensory to cognitive processing.

Strengths:

By combining fMRI and EEG, and focusing on the diagnostic case of Braille reading, the paper provides an integrated view of the transformation processing from sensation to perception in the visually deprived brain. Such a multimodal approach is still rare in the study of human brain plasticity and allows to discern the nature of information processing in blind people early visual cortex, as well as the timecourse of information processing in a situation of significant brain adaptability.

Weaknesses:

ROI and searchlight analyses are not completely overlapping, although this might be due to the specific limits and strengths of each approach. Moreover, the conclusions regarding the behavioral relevance of the sensory and perceptual representations in the putatively reorganized brain, although important, are limited due to the behavioral measurements adopted.

Reviewer #1 (Public review):

Summary:

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers. Compared to other types of breast cancer, TNBC exhibits highly aggressive clinical characteristics, a greater likelihood of metastasis, poorer clinical outcomes, and lower survival rates. Immunotherapy is an important treatment option for TNBC, but there is significant heterogeneity in treatment response. Therefore, it is crucial to accurately identify immunosuppressive patients before treatment and actively seek more effective therapeutic approaches for TNBC patients.

Strengths:

In this work, the authors collected and integrated data from single cells and large volumes of RNA sequencing and RNA-SEQ to analyze the TME landscape mediated by genes associated with iron death. On this basis, the prediction model of prognosis and treatment response of 131 patients was constructed using a machine learning algorithm, which is beneficial to provide individualized and precise treatment guidance for breast cancer patients.

Weaknesses:

However, there are still some issues that need to be clarified:

(1) The description of the research background is too brief and concise, and it is necessary to add some information about the limitations of existing methods and the differences and advantages of this study compared with other published relevant studies, so as to better highlight the necessity and research value of this study.

(2) This study is a retrospective analysis of a public data set and lacks experimental validation and prospective experiments to support the results of bioinformatics analysis. This should be added to the acknowledgment of limitations in the study.

Reviewer #3 (Public review):

Summary:

Juan Liu et al. investigated the interplay between habitat fragmentation and climate-driven thermophilization in birds in an island system in China. They used extensive bird monitoring data (9 surveys per year per island) across 36 islands of varying size and isolation from the mainland covering 10 years. The authors use extensive modeling frameworks to test a general increase of the occurrence and abundance of warm-dwelling species and vice versa for cold-dwelling species using the widely used Community Temperature Index (CTI), as well the relationship between island fragmentation in terms of island area and isolation from the mainland on extinction and colonization rates of cold- and warm-adapted species. They found that indeed there was thermophilization happening during the last 10 years, which was more pronounced for the CTI based on abundances and less clearly for the occurrence based metric. Generally, the authors show that this is driven by an increased colonization rate of warm-dwelling and an increased extinction rate of cold-dwelling species. Interestingly, they unravel some of the mechanisms behind this dynamic by showing that warm-adapted species increased while cold-dwelling decreased more strongly on smaller islands, which is - according to the authors - due to lowered thermal buffering on smaller islands (which was supported by air temperature monitoring done during the study period on small and large islands). They argue, that the increased extinction rate of cold-adapted species could also be due to lowered habitat heterogeneity on smaller islands. With regards to island isolation, they show that also both thermophilization processes (increase of warm and decrease of cold-adapted species) was stronger on islands closer to the mainland, due to closer sources to species populations of either group on the mainland as compared to limited dispersal (i.e. range shift potential) in more isolated islands.

The conclusions drawn in this study are sound, and mostly well supported by the results. Only few aspects leave open questions and could quite likely be further supported by the authors themselves thanks to their apparent extensive understanding of the study system.

Strengths:

The study questions and hypotheses are very well aligned with the methods used, ranging from field surveys to extensive modeling frameworks, as well as with the conclusions drawn from the results. The study addresses a complex question on the interplay between habitat fragmentation and climate-driven thermophilization which can naturally be affected by a multitude of additional factors than the ones included here. Nevertheless, the authors use a well balanced method of simplifying this to the most important factors in question (CTI change, extinction, colonization, together with habitat fragmentation metrics of isolation and island area). The interpretation of the results presents interesting mechanisms without being too bold on their findings and by providing important links to the existing literature as well as to additional data and analyses presented in the appendix.

Weaknesses:

The metric of island isolation based on distance to the mainland seems a bit too oversimplified as in real-life the study system rather represents an island network where the islands of different sizes are in varying distances to each other, such that smaller islands can potentially draw from the species pools from near-by larger islands too - rather than just from the mainland. Although the authors do explain the reason for this metric, backed up by earlier research, a network approach could be worthwhile exploring in future research done in this system. The fact, that the authors did find a signal of island isolation does support their method, but the variation in responses to this metric could hint on a more complex pattern going on in real-life than was assumed for this study.

Comments on revisions:

I'm happy with the revisions made by the authors.

Reviewer #1 (Public review):

Summary:

The Avrillon et al. explore the neural control of muscle by decomposing the firing activity of constituent motor units from the grid of surface electromyography (EMG) in the Tibialis (TA) Anterior and Vastus Lateralis (VL) during isometric contractions. The study involves extensive samples of motor units across the broadest range of voluntary contraction intensities up to 80% of MVC. The authors examine rate coding of the population of motor units, which describes the instantaneous firing rate of each motor unit as a function of muscle force. This relationship is characterized by a natural logarithm function that delineates two distinct phases: an initial phase with a steep acceleration in firing rate, particularly pronounced in low-threshold motor units, and a subsequent modest linear increase in firing rate, more significant in high-threshold motor units.

Strengths:

The study makes a significant contribution to the field of neuromuscular physiology by providing a detailed analysis of motor unit behavior during muscle contractions in a few ways.

(1) The significance lies in its comprehensive framework of motor unit activity during isometric contractions in the broad range of intensities, providing insights into the non-linear relationship between the firing rate and the muscle force. The extensive sample of motor units across the pool confirms the observation in animal studies in which the the spinal motoneuron exhibits a discharge consists of the distinct phases in response to synaptic currents, under the influence of persistent inward currents. As such, it is now reasonable to state the human motor units across the pool are also under control of gain modulation via some neuromodulatory effects in addition to synaptic inputs arising from ionotropic effects.<br /> (2) The firing scheme across in the entire motoneuron pool revealed in this study reconciles the discrepancy in firing organization under debate; i.e., whether it is 'onion skin' like or not (Heckman and Enoka 2012). The onion skin like model states that the low threshold motor units discharge higher than high threshold motor units and has been held for long time because the firing behaviors were examined in a partial range of contraction force range due to technical limitations. This reconciliation is crucial because it is fundamental to modelling the organization of motor unit recruitment and rate coding to achieve a desired force generation to advance our understanding of motor control.<br /> (3) The extensive data collection with a novel blind source separation algorithm on the expanded number of channel of surface EMG signal provides a robust dataset that enhances the reliability and validity of findings, setting a new standard for empirical studies in the field. \par<br /> Collectively, this study fills several knowledge gaps in the field and advances our understanding the mechanism underlying the isometric force generation.

Reviewer #2 (Public review):

Summary:

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Strengths:

The methods applied are challenging and appropriate and appear to be well executed. Open questions about the physiological underpinnings of M-cell function are addressed using sound experimental design and methodology, and convincing results are provided that advance our understanding of how two streams of sensory information can interact to control behavior.

Weaknesses:

Our concerns about the manuscript are captured in the following specific comments, which we hope will provide a useful perspective for readers and actionable suggestions for the authors.

Comments relevant to the revised manuscript:

Our general assessment (above) stands unchanged from the original version. All of our comments and concerns about the original manuscript have been addressed except for two, one very minor and one quite important:

Original Comment 1 (Minor):<br /> "Line 124. Direct stimulation of the tectum to drive M-cell-projecting tectal neurons not only bypasses the retina, it also bypasses intra-tectal processing and inputs to the tectum from other sources (notably the thalamus). This is not an issue with the interpretation of the results, but this description gives the (false) impression that bypassing the retina is sufficient to prevent adaptation. Adding a sentence or two to accurately reflect the complexity of the upstream circuitry (beyond the retina) would be welcome."

The authors have replied:<br /> "The reviewer is right in that direct tectal stimulation bypasses all neural processing upstream, not only that produced in the retina and that the tectum does not exclusively process visual information. The revised version now acknowledges (lines 245-252, revised manuscript) the complexity of the system."

We think that this is sufficient to address our concern. Some citations may be in order to underpin the new text.

Original Comment 5 (Major):<br /> Figure 4C and lines 398-410.<br /> "These are beautiful examples of M-cell firing, but the text suggests that they occurred rarely and nowhere close to significantly above events observed from single modalities. We do not see this a valid result to report because there is insufficient evidence that the phenomenon shown is consistent or representative of your data."

The authors have replied:<br /> "Our experimental conditions required anesthesia and paralysis, conditions designed to reduce neuronal firing and suppress motor output. We think it is valuable to report that we still see that simultaneous presentation subthreshold unisensory stimuli can add up to become suprathreshold, paralleling behavioral observations. We do not claim and acknowledge that those examples are representative of our recording conditions, but are likely to be more representative of the multisensory integration process taking place in freely moving fish. The revised manuscript adds context to these example traces to justify their inclusion (lines 420-426)."

We do not feel that this important concern has been addressed. The stats are definitively negative. There is no statistical evidence from these data that multisensory integration is occurring in this assay. The aesthesia, paralysis, and low n may provide explanations for this negative result, but it is still a negative result (p=0.5269). To show two examples of multisensory integration for subthreshold stimuli fits the narrative, but this result is not supported. Examples where individual stimuli caused APs (and combined stimuli did not) also occurred, presumably, and at a rate that is statistically indistinguishable to the examples shown in Figure 5. As such, if results from this assay are going to be in the manuscript, acoustic-only and tectum-only examples should be shown as well, although they would not fit the narrative. To be meaningful, this experiment would have to show that multisensory integration is happening in this circuit. Frustrating though it must be, the experiment has given a negative result to that question.

Reviewer #1 (Public review):

Summary

A novel statistical model of neural population activity called the Random Projection model has been recently proposed. Not only is this model accurate, efficient, and scalable, but also is naturally implemented as a shallow neural network. This work proposes a new class of RP model called the reshaped RP model. Inheriting the virtue of the original RP model, the proposed model is more accurate in terms of data fitting and efficient in terms of lower firing rate than the original, as well as compatible with various biological constraints. In particular, the authors have demonstrated that normalizing the total synaptic input in the reshaped model has a homeostatic effect on the firing rates of the neurons, resulting in even more efficient representations with equivalent accuracy. These results suggest that synaptic normalization contributes to synaptic homeostasis as well as efficiency in neural encoding.

Strength

This paper demonstrates that the accuracy and efficiency of the random projection models can be improved by extending the model with reshaped projections. Furthermore, it broadens the applicability of the model under biological constraints of synaptic regularization. It also suggests the advantage of the sparse connectivity structure over the fully connected model for modeling spiking statistics. In summary, this work successfully integrates two different elements, statistical modeling of the spikes and synaptic homeostasis in a single biologically plausible neural network model. The authors logically demonstrate their arguments with clear visual presentations and well-structured text, facilitating an unambiguous understanding for readers.

Discussions

The authors have clearly responded to most of our questions in the revised manuscript and we are happy to recommend publishing the final version of the article as it is. Below, we would like to present some alternative interpretations of the results. These comments are not exclusive with the claims made in the articles; it is rather intended to enhance the understanding of readers by providing additional perspectives.

As summarized above, the main contribution of the work consists of two parts; (1) the reshaped RP model achieved higher performance in explaining the statistics of the spiking activity of cortical neurons with more efficient representations (=lower firing rate), (2) synaptic homeostatic normalization in the reshaped RP model yields even more efficient representations without impairing the fitting performance.

For part (1),<br /> Suppl. Fig. 1B compares reshaped RP models with RP and RP with pruning and replacement (R&P). The better performance of RP with P&R might imply the advantage of pruning over gradient descent in this setting, reflecting the non-convexities of the problem. Alternatively, it might suggest the benefit of the increased number of parameters, since pruning allows the network to explore the broader parameter space during the learning process. This latter view might partially explain the superiority of the reshaped RP model over the original RP model.<br /> It is interesting that the backprop model has higher firing rate than the reshaped model (Fig. 1D). This trend is unchanged when optimization of the neural threshold is also allowed (Supp. Fig. 2A). Since backprop model overperforms reshaped model slightly but robustly, high firing rates in the backprop model might be a genuine feature of the spike statistics.

For part (2),<br /> We note that λ regulates the average firing rate, since maximizing the entropy <-ln p(x)> with a regularization term -λ <\sum _i f(x_i)> results in λ_i = λ for all i in the Boltzmann distribution of eq. 2. Suppl. Fig. 2B could be understood as demonstrating this "homeostatic" effect of λ.<br /> Suppl. Fig. 3 could be interpreted as demonstrating the interaction of two different homeostatic mechanisms: one at the firing-rate level (as regulated by λ) and the other at the synaptic level (as regulated by φ). It shows that the range of synaptic constraints where the fitting performance is not impaired differs by the value of λ. For example, if lambda is small (\lambda = 0.25), synaptic constraint can easily deteriorate the performance; on the other hand, if lambda is large (\lambda = 4), performance remains unchanged under strict synaptic constraint. Considering that practically we are most interested in the regime where the model performs best (λ = 2.0), an advantageous feature of the homeostatic model is that homeostatic constraint is harmless at λ=2.0 for the wide range of constraints.

Reviewer #1 (Public review):

Summary:

The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on visual cortex. By correlating mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in he visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.

While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

Strengths:

(1) Well-designed study addressing a timely question in the field.<br /> (2) Successful transition from previous work focused on visual cortex to auditory cortex, demonstrating generic principles in mismatch responses.<br /> (3) Correlation between mouse locomotion speed and acoustic feedback levels provides evidence for prediction signal in the auditory cortex.<br /> (4) Coupling of visual and auditory feedback show putative multimodal integration in auditory cortex.

Weaknesses:

(1) Lack of quantification of animal behavior upon mismatches, potentially leading to alternative interpretations of recorded signals.<br /> (2) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.<br /> (3) Discrepancies in reported values in a few figure panels raise questions about data consistency and interpretation.<br /> (4) Ambiguity regarding the identity of the [AM+VM] MM neurons.

Comments on revisions:

I am satisfied with all clarifications and additional analyses performed by the authors.<br /> The only concern I have is about changes in running after [AM+VM] mismatches.<br /> The authors reported that they "found no evidence of a change in running speed or pupil diameter following [AM + VM] mismatch (Figures S5A)" (line 197).<br /> Nevertheless, it seems that there is a clear increase in running speed for the [AM+VM] condition (S5A). Could this be more specifically quantified? I am concerned that part of the [AM+VM] could stem from this change in running behavior. Could one factor out the running contribution?

Reviewer #1 (Public review):

Summary:

This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

Strengths:

Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

Reviewer #1 (Public review):

Summary:

Tobón and Moser reveal a remarkable amount of presynaptic diversity in the fundamental Ca dependent exocytosis of synaptic vesicles at the afferent fiber bouton synapse onto the pilar or mediolar sides of single inner hair cells of mice. These are landmark findings with profound implications for understanding acoustic signal encoding and presynaptic mechanisms of synaptic diversity at inner hair cell ribbon synapses. The paper will have an immediate and long-lasting impact in the field of auditory neuroscience.

Main findings: 1) Synaptic delays and jitter of masker responses are significantly shorter (synaptic delay: 1.19 ms) at high SR fibers (pilar) than at low SR fibers (mediolar; 2.57 ms). 2) Masked evoked EPSC are significantly larger in high SR than in low SR. 3) Quantal content and RRP size are 14 vesicles in both high and low SR fibers. 4) Depression is faster in high SR synapses suggesting they have a higher release probability and tighter Ca nanodomain coupling to docked vesicles. 5) Recovery of master-EPSCs from depletion is similar for high and low SR synapses, although there is a slightly faster rate for low SR synapses that have bigger synaptic ribbons, which is very interesting. 6) High SR synapses had larger and more compact (monophasic) sEPSCs, well suited to trigger rapidly and faithfully spikes. 7) High SR synapses exhibit lower voltage (~sound pressure in vivo) dependent thresholds of exocytosis.

Great care was taken to use physiological external pH buffers and physiological external Ca concentrations. Paired recordings were also performed at higher temperatures with IHCs at physiological resting membrane potentials and in more mature animals than previously done for paired recordings. This is extremely challenging because it becomes increasingly difficult to visualize bouton terminals when myelination becomes more prominent in the cochlear afferents. In addition, perforated patch recordings were used in the IHC to preserve its intracellular milieu intact and thus extend the viability of the IHCs. The experiments are tour-de-force and reveal several novel aspects of IHC ribbon synapses. The data set is rich and extensive. The analysis is detailed and compelling.

Reviewer #1 (Public review):

Summary:

The study investigates protein-protein interactions (PPIs) within the nuage, a germline-specific organelle essential for piRNA biogenesis in Drosophila melanogaster, using AlphaFold2 to predict interactions among 20 nuage-localizing proteins. The authors identify five novel interaction candidates and experimentally validate three of them, including Spindle-E and Squash, through co-immunoprecipitation assays. They confirm the functional significance of these interactions by disrupting salt bridges at the Spn-E_Squ interface. The study further expands its scope to analyze approximately 430 oogenesis-related proteins, validating three additional interaction pairs. A comprehensive screen of around 12,000 Drosophila proteins for interactions with the key piRNA pathway player, Piwi, identifies 164 potential binding partners. Overall, the research demonstrates that in silico approaches using AlphaFold2 can link bioinformatics predictions with experimental validation, streamlining the identification of novel protein interactions and reducing the reliance on extensive experimental efforts. The manuscript is commendably clear and easy to follow; however, areas for improvement should be addressed to enhance its clarity and rigor.

Major Concerns:

(1) While AlphaFold2 was developed and trained primarily for predicting protein structures and their interactions, applying it to predict protein-protein interactions is an extrapolation of its intended use. This introduces several important considerations and risks. First, it assumes that AlphaFold's accuracy in structure prediction extends to interactions, despite not being explicitly trained for this task. Additionally, the assumption that high-scoring models with structural complementarity imply biologically relevant interactions is not always valid. Experimental validation is essential to address these uncertainties, as over-reliance on computational predictions without such validation can lead to false positives and inaccurate conclusions. The authors should expand on the assumptions, limitations, and risks associated with using AlphaFold2 for predicting protein-protein interactions.

(2) The authors experimentally validated three interactions, out of five predicted interactions, using co-immunoprecipitation (co-IP). They attributed the lack of validation for the other two predictions to the limitations of the co-IP method. However, further clarification on the potential limitations of the co-immunoprecipitation behind the negative results would strengthen the conclusions. While co-IP is a widely used technique, it may not detect weak or transient interactions, which could explain the failure to validate some predictions. Suggesting alternative validation methods such as FRET or mass spectrometry could further substantiate the results. On the other hand, AlphaFold2 predictions are not infallible and may generate false positives, particularly when dealing with structurally plausible but biologically irrelevant interactions. By acknowledging both the potential limitations of co-IP and the possibility of false positives from AlphaFold2, the authors can provide a more balanced interpretation of their findings.

(3) In line 143, the authors state that "This approach identified 13 pairs; seven of these were already known to form complexes, confirming the effectiveness of AlphaFold2 in predicting complex formations (Table 2). The highest pcScore pair was the Zuc homodimer, possibly because AlphaFold2 had learned from Zuc homodimer's crystal structure registered in the database." While the authors mentioned the presence of the Zuc homodimer's crystal structure, they do not provide a systematic bioinformatics analysis to evaluate pairwise sequence identity or check for the presence of existing structures for all the proteins or protein pairs (or their homologs) in databases such as the Protein Data Bank (PDB) or Swiss-Model. Conducting such an analysis is critical, as it significantly impacts the novelty and reliability of AlphaFold2 predictions. For instance, high sequence identity between the query proteins could lead to high-scoring models for biologically irrelevant interactions. Including this information would strengthen the conclusions regarding the accuracy and utility of the predictions.

(4) While the manuscript successfully identifies novel protein interactions, the broader biological significance of these interactions remains underexplored. The manuscript could benefit from elaborating on how these findings may contribute to understanding the piRNA pathway and its implications on germline development, transposon repression, and oogenesis.

Reviewer #1 (Public review):

Summary:

Through a series of CRISPR-Cas9 screens, the GPX4 antioxidant pathway was identified as a critical suppressor of cold-induced cell death in hibernator-derived cells. Hamster BHK-21 cells exposed to repeated cold and rewarming cycles revealed five genes (Gpx4, Eefsec, Pstk, Secisbp2, and Sepsecs) as critical components of the GPX4 pathway, which protects against cold-induced ferroptosis. A second screen with continuous cold exposure confirmed the essential role of GPX4 in prolonged cold tolerance. GPX4 knockout lines exhibited complete cell death within four days of cold exposure, and pharmacological inhibition of GPX4 further increased cell death, underscoring the necessity of GPX4's catalytic activity in cold conditions.

An additional CRISPR screen in human cold-sensitive K562 cells identified 176 genes for cold survival. The GPX4 pathway was found to confer significant resistance to cold in hibernators and human cells, with GPX4 loss significantly increasing cold-induced cell death.

Comparing hamster and human GPX4, overexpression of GPX4 in human K562 cells, whether hamster or human GPX4, dramatically improved cold tolerance, while catalytically dead mutants showed no such effect. These findings suggest that GPX4 abundance is a key limiting factor for cold tolerance in human cells, and primary cell types show strong sensitivity to GPX4 loss, highlighting that differences in cold tolerance across species may be due to varying GPX4-mediated protection.

Strengths:

(1) Innovative Approach: The study employs a series of unbiased genome-wide CRISPR-Cas9 screens in both hibernator- and non-hibernator-derived cells to investigate the mechanisms controlling cellular cold tolerance. Notably, this is the first genome-scale CRISPR-Cas9 screen conducted in cells derived from a hibernator, the Syrian hamster.

(2) Identification of the GPX4 Pathway: Identifying glutathione peroxidase 4 (GPX4) as a critical suppressor of cold-induced cell death significantly contributes to the field. Recently, GPX4 was also reported as a potent regulator of cold tolerance through overexpression screening (Sone et al.) in hamsters, which further supports this finding.

(3) Improved Cold Viability Assessment: The study identifies an important technical artifact in using trypan blue to assess cell viability following cold exposure. It reveals that cells stained immediately after cold exposure retain the dye, inaccurately indicating cell death. By introducing a brief rewarming period before viability assessment, the authors significantly improve the accuracy of detecting cold-induced cell death. This refinement in methodology ensures more reliable results and sets a new standard for future research on cold stress in cells.

Weaknesses:

(1) Mechanisms Regulating GPX4 Levels: While the study highlights GPX4 levels as a major determinant of cellular cold tolerance, it does not discuss how these levels are regulated or why they differ between hibernators and non-hibernators. This omission leaves an important aspect of GPX4's role in cold tolerance unexplored.

(2) Generalizability Across Species: Although the study demonstrates the role of GPX4 in several mammalian species, it does not investigate whether this mechanism extends to other vertebrates (e.g., fish and amphibians) that also face cold challenges. This limitation could restrict the broader evolutionary claims made by the study.

(3) Variability in Cold Sensitivity Across Human Cell Lines: The study observes significant variability in cold tolerance among different human cell lines but does not explain these differences clearly. This leaves a key aspect of human cell cold sensitivity insufficiently addressed.

Reviewer #1 (Public review):

Summary:

This is an important and very well-presented set of experiments following up on prior work from the lab investigating knock-down (KD) of EMC10 in the restoration of neuronal and cognitive deficits in 22q11.2 Del models, including now both human iPSCs and a mouse model in vivo now with ASOs.

The valuable progress in this current manuscript is the development of ASOs, and the proof of efficacy in vivo in mice of the ASO in knock-down of EMC10 and amelioration of in vivo behavioral phenotypes.

The experiments include iPSC studies demonstrating elevations of EMC10 in a solid collection of paired iPSC lines. These studies also provide evidence of manipulation of EMC10 by overexpression and inhibition of miRNAs that exist in the 22q11 interval. The iPSC studies also nicely demonstrate the rescue of impairments with KD of EMC10 in neuronal arborization as well as KCl-induced neuronal activity. The major in vivo contributions reflect an impressive demonstration of the efficacy of two ASOs in vivo on both KD of EMC10 in vivo and through improvement in behavioral abnormalities in the 22q11 mouse in a range of different behaviors, including social behavior and learning behaviors.

Overall, there are many strengths reflected in this study, including in particular the synergy between in vitro studies in human cell models and in vivo studies in the well-characterized mouse model. The experiments are generally rigorously performed, well-powered, and nicely presented. The claims with regard to the mechanisms of EMC10 elevations and the importance of restoration of EMC10 expression to neuronal morphology and behavior are well supported by the data. The work may be further supported in future studies, by investigation of rescue by ASOs of circuit dysfunction in vivo or ex vivo through electrophysiology in the mouse model. Also, in future studies, investigation of the mechanism by which EMC10, an ER protein involved in protein processing, may function in the observed neuronal abnormalities; however, these studies are clearly for future investigations.

The potential impact of the work is found in the potential value of the ASO approach to the treatment of 22q11, or the pre-clinical evidence that knock-down of this protein may lead to some amelioration of cognitive symptoms. Overall, a very convincing and complementary set of experiments to support EMC10 KD as a therapeutic strategy.

Reviewer #1 (Public review):

Summary:

Chang and colleagues used tetrode recordings in behaving rats to study how learning an audiovisual discrimination task shapes multisensory interactions in the auditory cortex. They found that a significant fraction of neurons in the auditory cortex responded to visual (crossmodal) and audiovisual stimuli. Both auditory-responsive and visually-responsive neurons preferentially responded to the cue signaling the contralateral choice in the two-alternative forced choice task. Importantly, multisensory interactions were similarly specific for the congruent audiovisual pairing for the contralateral side.

Strengths:

The experiments were conducted in a rigorous manner. Particularly thorough are the comparisons across cohorts of rats trained in a control task, in a unisensory auditory discrimination task, and the multisensory task, while also varying the recording hemisphere and behavioral state (engaged vs. anesthesia). The resulting contrasts strengthen the authors' findings and rule out important alternative explanations. Through the comparisons, they show that the enhancements of multisensory responses in the auditory cortex are specific to the paired audiovisual stimulus and specific to contralateral choices in correct trials and thus dependent on learned associations in a task-engaged state.

Weaknesses:

The main result is that multisensory interactions are specific for contralateral paired audiovisual stimuli, which is consistent across experiments and interpretable as a learned task-dependent effect. However, the alternative interpretation of behavioral signals is crucial to rule out, which would also be specific to contralateral, correct trials in trained animals. Although the authors focus on the first 150 ms after cue onset, some of the temporal profiles of activity suggest that choice-related activity could confound some of the results.

The auditory stimuli appear to be encoded by short transient activity (in line with much of what we know about the auditory system), likely with onset latencies (not reported) of 15-30 ms. Stimulus identity can be decoded (Figure 2j) apparently with an onset latency around 50-75 ms (only the difference between A and AV groups is reported) and can be decoded near perfectly for an extended time window, without a dip in decoding performance that is observed in the mean activity Figure 2e. The dynamics of the response of the example neurons presented in Figures 2c and d and the average in 2e therefore do not entirely match the population decoding profile in 2j. Population decoding uses the population activity distribution, rather than the mean, so this is not inherently problematic. It suggests however that the stimulus identity can be decoded from later (choice-related?) activity. The dynamics of the population decoding accuracy are in line with the dynamics one could expect based on choice-related activity. Also the results in Figures S2e,f suggest differences between the two learned stimuli can be in the late phase of the response, not in the early phase.

First, it would help to have the same time axis across panels 2,c,d,e,j,k. Second, a careful temporal dissociation of when the central result of multisensory enhancements occurs in time would discriminate better early sensory processing-related effects versus later decision-related modulations.

In the abstract, the authors mention "a unique integration model", "selective multisensory enhancement for specific auditory-visual pairings", and "using this distinct integrative mechanisms". I would strongly recommend that the authors try to phrase their results more concretely, which I believe would benefit many readers, i.e. selective how (which neurons) and specific for which pairings?

Reviewer #1 (Public Review):

The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further.

After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revisions.

Reviewer #1 (Public review):

Summary:

The manuscript the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteosomal function in an ATP independent manner. Although there are multiple works that have highlighted the role of this protein in tumour, this study specifically tried to develop a correlate with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.

Strengths:

The observations of the authors hint that beyond PA28y association with proteasome, it might also stabilize certain proteins such as C1QBP which influences the energy metabolism.

Weaknesses:

The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.

In most of the experiments the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex which is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in more convincing manner.

In all of the assays C1QBP has been detected as doublet. However, the expression pattern of the two bands vary depending on the experiment. In some cases the upper band is intensely stained and in some the lower bands. Does C1QBP isoforms exist and whether they are differentially regulated depending on experiment conditions/tissue types?

Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of literatures that have dealt with metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, significance of this study and how it will extend our understanding of OSCC needs to be elaborated.

Review of Revised Version:

Although the authors have partly corrected the manuscript by removing the mislabeling in their Co-IP experiments, my primary concern on the actual functional connotations and direct interaction between PA28y and C1QBP still remains unaddressed. As already mentioned in my previous review, since the core idea of the work is PA28y's direct interaction with C1QBP, stabilizing it, the same should be demonstrated in a more convincing manner.

My other observation on the detection of C1QBP as a doublet has been addressed by usage of anti-C1QBP Monoclonal antibody against the polyclonal one used before. C1QBP doublets have not been observed in the present case.

The authors have also worked on the presentation of the background by suitably modifying the statements and incorporating appropriate citations.

However, the authors are requested to follow the recommendations provided to them by the reviewers to address the major concerns.

Reviewer #1 (Public Review):

Summary:

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crest-derived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

Comments on latest version:

Author response: We disagree that the datasets from previous studies provide additional insights that are relevant to the current study. It must be appreciated that Wnt1Cre and Pax2Cre are genetic lineage tracers and that migratory ENS progenitor cells labeled with these reagents do not maintain expression of Wnt1 and Pax2 mRNA or protein. The Wnt1 and Pax2 genes are only transiently expressed within their distinct regions of the ectoderm, and their expression turns off as cells delaminate and begin migration. Thus, Pax2Cre-labeled ENS progenitor cells are not Pax2-positive thereafter. The single cell RNA-Seq studies suggested by the reviewer were collected from older embryos and postnatal mice, and do not represent the E10.5-E11.5 period that accounts for genesis of Ret-mediated and Ednrb-mediated Hirschsprung disease pathology. Even with the most recent work by Zhou et al (Dev Cell, 2024) that included E10.5 cells, this analysis only evaluated neural crest-derived Sox10Cre lineage cells, which does not include the placode-derived Pax2Cre lineage (as we show explicitly in Fig. 2-figure supplement 2). Consequently, it would not be possible to find the "Pax2-positive cells" in these datasets. Performing a new transcriptomic analysis by isolating Pax2Cre-lineage and Wnt1Cre-lineage cells at the appropriate developmental time points could be the basis of future studies, but we think these are beyond the scope of the present paper.

Reviewer comment: Since these cells are a completely new discovery, additional validation would be beneficial. Whole early GI tract datasets are available, such as human 6-week fetal gut data (PMID: 29802404) and whole mouse embryo studies spanning development that include ENS (PMID: 38355799). If the authors believe that none of these existing datasets can detect these cells in their developmental state and that targeted cell studies with specific Cre drivers would be required, they should make this explicitly clear.

A key advantage of discovering a new cell type, particularly in the relatively understudied field of ENS, is the opportunity for the broader community to leverage this finding to inform their own research. If these cells are absent from current datasets, even those covering the whole GI tract, this should be clearly communicated.

I aim to support the authors here. New discoveries in science require robust validation to enhance their impact. The authors have generated an important reagent with great potential for broader use, and addressing these straightforward requests would strengthen the study and make it more valuable to the scientific community.

Author response: The observation that human mutations in RET and EDNRB both cause Hirschsprung disease is decades old, and of course numerous studies in human, mouse, and cells have addressed the relation between the two signaling pathways. We did not mean to imply that we were the first to discover that Ret and Ednrb signaling pathways interact. The reviewer cites a number of papers all from the Chakravarti lab that address this phenomenon; while these are a valuable contribution to the field, there is still more to be learned. The model elaborated in PMID: 31313802, in which Ret and Ednrb are both enmeshed in a common gene regulatory network, does not readily explain why each has a different phenotypic manifestation and doesn't take into account the importance of the placodal lineage. The main new contributions of our paper are the existence of a new cell lineage that contributes to the ENS, and that the placodal and neural crest lineages utilize Ret and Ednrb signaling differently. The clarification of how these elements are differentially used by the two lineages explains long-segment and short-segment Hirschsprung disease (Ret and Ednrb mutants, respectively) far better than in past studies. The reviewer unfortunately dismisses these insights and seems to feel that a biochemical exploration of one specific component of the signaling interaction (Y1015 phosphorylation) would be more relevant. This should be the basis of future studies and are beyond the scope of the new findings reported in the present paper

Reviewer comment: The authors completely miss the point. There is no association between phenotypic severity (L-HSCR, S-HSCR, or TCA) and mutations in a given gene in HSCR. EDNRB, for example, has a syndromic association with Waardenburg-Shah syndrome (WS4-A), which includes pigmentation anomalies due to EDNRB expression in neural crest cells that give rise to pigment cells.

The authors' discovery reinforces the current paradigm that nearly all HSCR is mediated by mutations in genes within the GRN, accounting for 72% of the population attributable risk. This is valuable; reinforcing established paradigms with new data is crucial, and the authors should appreciate the significance of this contribution.

The discovery of the signaling interaction is particularly important, as it offers a potential explanation for disease severity and provides a basis for classifying patients in future sequencing studies. It is surprising that the authors seem reluctant to highlight this novel finding, as it could greatly benefit future research, including the development of specific mouse mutants and advancing human genetics studies.

Author response: The reviewer overlooked that one of the review articles that we cited (Chen, Hsu, & Hung, 2020) has a dedicated paragraph for RET (section 3.14), which summarizes the work by Barheri-Yarmand et al (PMID: 25795775) which is the very paper noted by the reviewer in the comment above. The reviewer also somewhat misstated the results of the Barheri-Yarmand et al study. By immunostaining, this paper showed nuclear localization of endogenous Ret, albeit a version of Ret with a disease-associated mutation that makes it constitutively active by constitutive autophosphorylation. Nonetheless, this was endogenous Ret. The paper also used overexpression of GFP-tagged RET in HEK293 cells to show that wildtype RET can behave in a similar manner, at least under these circumstances. Our point is simply that Ret (and other receptor tyrosine kinases) can be found in the nucleus in certain biological contexts, and our observations are consistent with this precedent. The reviewer also suggests a biochemical follow-up analysis related to this observation, which we agree would be of interest. Such an investigation however is beyond the scope of the present study.

Reviewer comment: As the authors themselves now highlight from the cited paper that any evidence of RET entering the nucleus is of a mutant RET protein, How does this align with their discovery for wildtype protein?

This finding of nuclear localization of RET is both intriguing and unprecedented. Despite extensive biochemical studies on RET, given its role as an oncogene, this feature has not been identified before. If validated, this discovery could significantly advance the field and improve interpretation of future studies. I reiterate my previous point: a novel finding that challenges the current paradigm requires additional evidence.

Reviewer #1 (Public review):

The manuscript describes comprehensive structure-function studies combining structural studies, Alphafold2-based modelling, and extensive structural validation by mutagenesis and biochemical experiments. Consequently, a sophisticated activation mechanism of Mical1 as a representative of the MICAL family is elucidated at the molecular level. Since MICAL proteins are important regulators of membrane trafficking and cytoskeleton dynamics, the study is of high relevance for many groups. Structural data are of high quality, the modelling data appear to be sound, and the subsequent biochemical analyses are carried out in great detail, yielding a complete story. I have little to criticize on this beautiful work.

Reviewer #1 (Public review):

The manuscript "Osterix Facilitates Osteocytic Communication by Targeting Connexin43" investigates the role of Osterix (Osx) in osteocytes using a Col1α1-CreER;Osxfl/fl mouse model and cultured cells. The study reveals that Osx is expressed in osteocytes, and its deletion in vitro leads to a significant reduction in osteocyte dendrite formation, highlighting its critical role in maintaining cellular communication. Through ChIP-seq analysis, the authors identified Connexin43 (Cx43) as a direct downstream target of Osx. Moreover, treatment with all-trans retinoic acid (ATRA), a known agonist of Cx43, was able to rescue the dendritic network in osteocytes, restoring their communication capabilities in vitro.

This research provides valuable insights into the molecular mechanisms by which Osx influences osteocyte function, particularly through its regulation of Cx43. However, despite these findings, the study does not fully elucidate all the mechanisms involved in Osx-mediated osteocytic communication. Several conclusions, particularly those related to the broader signaling pathways, require additional experimental evidence and further investigation to be fully substantiated. This study provides a new aspect in understanding the complex role of Osx in bone biology but leaves open questions regarding the intricacies of its regulatory network.

Major Comments:

(1) In the Col1a1-CreER;tdTomato mice, the number of tdTomato+ cells in the cortical bone appears lower compared to Osx+ cells. The overlap between tdTomato+ and Osx+ cells in Figure 1 is limited. Could this affect the knockout efficiency? Can the authors provide data on Osx knockout efficiency in vivo? While immunostaining of Osx is shown in both control and mutant mice in Figure 2A, the Osx expression pattern differs from Figure 1A. Osx expression is relatively low in the bone marrow in Figure 1A, but much stronger in Figure 2A.

Additionally, Osx+ cells in Figure 1A seem confined to the bone surface, whereas Figure 2A shows a broader distribution. What developmental stage of mice was used in Figure 1? Could the authors also provide co-staining with other osteocyte markers alongside Osx?

(2) The authors mentioned using both siRNA and Lenti-Osx to modulate Osx expression. What was the specific purpose of these experiments? If the authors aim to demonstrate that Osx plays a critical role in osteocytes, they should provide data on downstream targets or markers relevant to osteocyte function. Additionally, did these treatments affect processes like differentiation or cell viability in osteocytes? The current results only demonstrate that siRNA and Lenti-Osx can successfully modulate Osx expression in vitro, but further evidence is needed to support broader functional conclusions.

(3) Osx knockout mice exhibited a decreased osteocyte dendritic network both in vivo and in vitro. To better understand how this affects overall bone health, could the authors provide additional parameters, such as bone thickness, bone strength, and other relevant metrics? Furthermore, to determine whether these phenotypes are primarily due to defects in the osteocyte dendritic network or a reduction in osteocyte numbers, the authors should also assess the number of osteocytes in the knockout mice Figure 2.

(4) Regarding the Lucifer Yellow Dye Transfer Assay in Figure 3, the authors should provide data on cell density and cell viability for both control and mutant groups. Additionally, although less dye is observed in the mutant group, the migration distance appears comparable to the control group. Could the authors explain this result? Furthermore, how was transmission speed between the groups evaluated in Figure 3D? More details on the method used to assess transmission speed would be helpful.

(5) For a more comprehensive and unbiased analysis of Osx function in osteocytes, the authors should present a full analysis of differentially expressed genes, rather than focusing solely on integrins. Additionally, it would be beneficial to include an analysis of the knockdown group alongside the other groups, considering the animal model used in this study involves knockout mice.

(6) In the immunofluorescence staining of integrin αvβ1 in the si-Osx and Lenti-Osx groups, the cellular localization of integrin αvβ1 appears altered. Unlike the control group, where it is mainly localized in the cytoplasm, positive signals are observed in the nucleus of the si-Osx and Lenti-Osx groups. Additionally, since integrin αvβ1 is a membrane protein, shouldn't it primarily be observed on the cell membrane rather than in the cytoplasm? Could the authors clarify this observation?

(7) The results regarding Cx43 expression after Lenti-Osx treatment are questionable. It appears that the images for the Lenti-GFP and Lenti-Osx groups have been misrepresented. The merged images for the Lenti-GFP control group seem to belong to the Lenti-Osx group, and vice versa. If the images were presented in their correct order, the conclusions would contradict the authors' claims. This issue needs to be addressed to ensure an accurate interpretation of the data.

(8) The authors demonstrated that ATRA treatment elevates Cx43 protein levels in the control group, where Osx function is normal. However, can ATRA also restore Cx43 protein levels in the si-Osx treated group, where Osx transcriptional function is impaired? Theoretically, Cx43 protein levels should not be restored in the si-Osx group. Could the observed rescue phenotype be due to effects downstream of Cx43? This possibility should be considered and clarified.

(9) Does the Cx43 mutation of knockout cause similar phenotypes in the animal model? Can restoration of Cx43 rescue the bone phenotype?

Reviewer #1 (Public review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single cell RNA-seq.

Reviewer #1 (Public review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single cell RNA-seq.

Reviewer #1 (Public review):

The revision by Wang et al is a much more clear and readable manuscript than the original version, which I think was a bit too terse and hard to parse. In this version, I think I basically understand all the analyses that the authors undertake and how they argue that those analyses support their conclusions.

The fundamental claim of the manuscript is that rRNA genes experience substitutions much too quickly, given that they are a multi-copy gene system. As clarified by the authors in their response, and as I think is relatively clear in the manuscript, they are collapsing all copies of the rRNA array down. They first quantify polymorphism (in this expanded definition, where polymorphism means variable at a given site across any copy). The authors find elevated levels of heterozygosity in rRNA genes compared to single copy genes, which isn't surprising, given that there is a substantially higher target size; that being said, the increase in polymorphism is smaller than the increase in target size. They then look at substitutions between mouse species and also between human and chimp, and argue that the substitution rate is too fast compared to single copy genes in many cases.

I think that this is an interesting problem and one that obviously occupies some space in the literature. As the authors point out, one possibility for explaining the elevated fixation rate is that there is some kind of positive selection in these putatively non-functional regions. The authors, instead, argue that the elevated rate of evolution is due to neutral homogenizing processes. I'm sympathetic to this argument, I'm a neutralist myself :)

That being said, I find the whole analysis and the connection with the WFH model very strange. As I stated in my previous review, it feels very odd to chalk everything up to variance in reproductive success, rather than explicitly modeling the molecular processes that may lead to the homogenization. For example, the authors bring up gene conversion, and even do a small test of gene conversion. But a force like biased gene conversion is perhaps better modeled as a deterministic force, rather than a stochastic force. Indeed, I think that explicit modeling of mutation dynamics has been very helpful in understanding the role of replicative vs damage-related mutation in humans, as seen in Gao et al (2016) and Spisak et al (2024). I realize, as the authors say in their cover letter, that this is hard! But a major concern with this manuscript is that it's about whether drift can plausibly explain the pattern, but then it's basically impossible to know if it really can, because we have no way to compare the estimated parameters with biophysical or biochemical measurements of the rates of homogenizing forces, because the homogenizing forces are just wrapped up under "variance in reproductive success". I think a much more interesting manuscript would have a more explicit model of homogenizing forces.

I also have some concerns about the data analysis, echoing some concerns of the other reviewer. The biggest issue is that traditional read mapping and SNP calling pipelines for highly duplicated loci don't really make sense. I don't fully understand the variant calling pipeline. The authors state that "All mapping and analysis are performed among individual copies of rRNA genes." which makes it sound like the reads mapping to different copies were somehow deconvolved, which is what you'd need to do to use "normal" variant calling approaches that call look for homozygotes and heterozygotes. But I don't know enough about this literature to understand how they did that and if it makes any sense. If, instead, they called variants against collapsed rRNA copies, then using a standard variant calling approach does not make sense. If you have a variant in 2 out of 100 copies, a standard variant calling algorithm would very likely call that a homozygous ancestral site. Conditional on the variant calls being reasonable, however, I'm basically okay with their use of read counts to estimate "allele frequencies" within individuals.

I have some more minor comments:

(1) In the paragraph starting line 61, the authors say that WF models are unable to handle things like viral epidemics and transposons. I don't think that's really fair: the issue here isn't WF dynamics or not, it's that there is fundamentally evolution on two levels (which is also the case in the rRNA case considered in this manuscript). I certainly agree with the authors that you can't just naively apply standard pop gen theory in these systems, but I think the arrow at the WF model is misaimed, as the real issue is drift and selection on multiple levels.

(2) Line 268-269: The authors argue that the long term rate of evolution in rRNA genes is roughly similar to single copy genes, suggesting not a big influence of increased mutation rate. I'm not sure I understand where this number comes from, as opposed to the divergence numbers they look at in Table 3. These seem to be two different conclusions from roughly the same measurement? Surely I am misunderstanding something.

References:

Gao, Z., Wyman, M. J., Sella, G., & Przeworski, M. (2016). Interpreting the dependence of mutation rates on age and time. PLoS biology, 14(1), e1002355.

Spisak, N., de Manuel, M., Milligan, W., Sella, G., & Przeworski, M. (2024). The clock-like accumulation of germline and somatic mutations can arise from the interplay of DNA damage and repair. PLoS biology, 22(6), e3002678.

Reviewer #1 (Public review):

Summary:

The authors set out to measure the diffusion of small drug molecules inside live cells. To do this, they selected a range of fluorescent drugs, as well as some commonly used dyes, and used FRAP to quantify their diffusion. The authors find that drugs diffuse and localize within the cell in a way that is weakly correlated with their charge, with positively charged molecules displaying dramatically slower diffusion and a high degree of subcellular localization.

The study is important because it points to an important issue related to the way drugs behave inside cells beyond the simple "IC50" metric (a decidedly mesoscopic/systemic value). The authors conclude, and I agree, that their results point to nuanced effects that are governed by drug chemistry that could be optimized to make them more effective.

Strengths:

(1) The work examines an understudied aspect of drug delivery.

(2) The work uses well-established methodologies to measure diffusion in cells

(3) The work provides an extensive dataset, covering a range of chemistries that are common in small molecule drug design

(4) The authors consider several explanations as to the origin of changes in cellular diffusion

Comments on revised version:

In general, my comments were addressed, new discussions were added, and the paper has been improved significantly, which is great.

However, despite providing very clear instructions, a lot of my comments re statistical treatment were disregarded. Bar charts still do not show the repeats as individual points. Errors bars still represent SEM, which gives a wrong idea about the spread of the data. FRAP lines are still averages, and still do not show the spread of the data.

Significance assignments are done based on average and SEMs, as opposed to the full dataset. There is nothing technically wrong with this, but it generally creates an impression that things are more reproducible/rigorous/significant than they would be if the data was shown completely.

Reviewer #1 (Public review):

Summary:

This manuscript by Shan, Guo, Zhang, Chen et al., shows a raft of interesting data including the first cryo-EM structures of human PIEZO1. Clearly the molecular basis of PIEZO channel inactivation is of great interest and as such this manuscript provides some valuable extra information that may help to ultimately build a molecular picture of PIEZO channel inactivation. However, the current manuscript though does not provide any compelling evidence for a detailed mechanism of PIEZO inactivation.

Strengths:

This manuscript documents the first cryo-EM structures of human PIEZO1 and gain of function mutants associated with hereditary anaemia. It is also the first evidence showing that PIEZO1 gain of function mutants are also regulated by the auxiliary subunit MDFIC.

Weaknesses:

While the structures are interesting and clear differences can be seen in the presence of the auxiliary subunit MDFIC the major conclusions and central tenets of the paper, especially a role for pore lipids in inactivation, lack data to support them. The post translational modification of PIEZOs auxiliary subunit MDFIC is not modelled as a covalent interaction.

Comments on revisions:

The revisions do absolutely nothing to allay any of the major concerns documented in my initial review of this manuscript.

(1) Mouse vs Human inactivation<br /> Not only is a quantification not provided the literature on this point is still not at all referenced or discussed.<br /> (2) MDFIC -lipidation<br /> Even if they are not assigned in the PDB for illustration they can at least be modelled correctly as covalently bound acyl chains.<br /> (3) Pore lipids and inactivation<br /> None of the explanations are consistent with the data shown.<br /> (4) Cytosolic plug<br /> There is not even any extra discussion provided on this point.<br /> (5) Reduced sensitivity of PIEZO1 in the presence of MDFIC and its regulatory mechanism<br /> No quantification is provided.<br /> (6) Both referencing of the PIEZO1 literature and prose could be improved.<br /> There is little to no attempt to improve the referencing.

Reviewer #1 (Public review):

Summary:

Mehmet Mahsum Kaplan et al. demonstrate that Meis2 expression in neural crest-derived mesenchymal cells is crucial for whisker follicle (WF) development, as WF fails to develop in wnt1-Cre;Meis2 cKO mice. Advanced imaging techniques effectively support the idea that Meis2 is essential for proper WF development and that nerves, while affected in Meis2 cKO, are dispensable for WF development and not the primary cause of WF developmental failure. The study also reveals that although Meis2 significantly downregulates Foxd1 in the mesenchyme, this is not the main reason for WF development failure. The paper presents valuable data on the role of mesenchymal Meis2 in WF development. However, further quantification and analysis of the WF developmental phenotype would be beneficial in strengthening the claim that Meis2 controls early WF development rather than causing a delay or arrest in development. A deeper sequencing data analysis could also help link Meis2 to its downstream targets that directly impact the epithelial compartment.

Strengths:

(1) The authors describe a novel molecular mechanism involving Mesenchymal Meis2 expression, which plays a crucial role in early WF development.

(2) They employ multiple advanced imaging techniques to illustrate their findings beautifully.

(3) The study clearly shows that nerves are not essential for WF development.

Weaknesses:

(1) The authors claim that Meis2 acts very early during development, as evidenced by a significant reduction in EDAR expression, one of the earliest markers of placode development. While EDAR is indeed absent from the lower panel in Figure 3C of the Meis2 cKO, multiple placodes still express EDAR in the upper two panels of the Meis2 cKO. The authors also present subsequent analysis at E13.3, showing one escaped follicle positive for SHH and Sox9 in Figures 1 and 3. Does this suggest that follicles are specified but fail to develop? Alternatively, could there be a delay in follicle formation? The increase in Foxd1 expression between E12.5 and E13.5 might also indicate delayed follicle development, or as the authors suggest, follicles that have escaped the phenotype. The paper would significantly benefit from robust quantification to accompany their visual data, specifically quantifying EDAR, Sox9, and Foxd1 at different developmental stages. Additionally, analyzing later developmental stages could help distinguish between a delay or arrest in WF development and a complete failure to specify placodes.

(2) The authors show that single-cell sequencing reveals a reduction in the pre-DC population, reduced proliferation, and changes in cell adhesion and ECM. However, these changes appear to affect most mesenchymal cells, not just pre-DCs. Moreover, since E12.5 already contains WFs at different stages of development, as well as pre-DCs and DCs, it becomes challenging to connect these mesenchymal changes directly to WF development. Did the authors attempt to re-cluster only Cluster 2 to determine if a specific subpopulation is missing in Meis2 cKO? Alternatively, focusing on additional secreted molecules whose expression is disrupted across different clusters in Meis2 cKO could provide insights, especially since mesenchymal-epithelial communication is often mediated through secreted molecules. Did the authors include epithelial cells in the single-cell sequencing, can they look for changes in mesenchyme-epithelial cell interactions (Cell Chat) to indicate a possible mechanism?

(3) The authors aim to link Meis2 expression in the mesenchyme with epithelial Wnt signaling by analyzing Lef1, bat-gal, Axin1, and Wnt10b expression. However, the changes described in the figures are unclear, and the phenotype appears highly variable, making it difficult to establish a connection between Meis2 and Wnt signaling. For instance, some follicles and pre-condensates are Lef1 positive in Meis2 cKO. Including quantification or providing a clearer explanation could help clarify the relationship between mesenchymal Meis2 and Wnt signaling in both epidermal and mesenchymal cells. Did the authors include epithelial cells in the sequencing? Could they use single-cell analysis to demonstrate changes in Wnt signaling?

(4) Existing literature, including studies on Neurog KO and NGF KO, as well as the references cited by the authors, suggest that nerves are unlikely to mediate WF development. While the authors conduct a thorough analysis of WF development in Neurog KO, further supporting this notion, this point may not be central to the current work. Additionally, the claim that Meis2 influences trigeminal nerve patterning requires further analysis and quantification for validation.

(5) Meis2 expression seems reduced but has not entirely disappeared from the mesenchyme. Can the authors provide quantification?

Reviewer #1 (Public Review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single-cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single-cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single-cell RNA-seq.

Weaknesses:

The manuscript is written in a very compressed style and many technical details of the evaluations conducted are unclear and processed data has not been made available for evaluation, limiting the ability of the reader to independently judge the merits of the method.

Reviewer #1 (Public review):

Summary:

In this study, the authors address whether the dorsal nucleus of the inferior colliculus (DCIC) in mice encodes sound source location within the front horizontal plane (i.e., azimuth). They do this using volumetric two-photon Ca2+ imaging and high-density silicon probes (Neuropixels) to collect single-unit data. Such recordings are beneficial because they allow large populations of simultaneous neural data to be collected. Their main results and the claims about those results are the following:

(1) DCIC single-unit responses have high trial-to-trial variability (i.e., neural noise);<br /> (2) approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth;<br /> (3) single-trial population responses (i.e., the joint response across all sampled single units in an animal) encode sound source azimuth "effectively" (as stated in the title) in that localization decoding error matches average mouse discrimination thresholds;<br /> (4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus (as stated in the Abstract);<br /> (5) evidence of noise correlation between pairs of neurons exists;<br /> and 6) noise correlations between responses of neurons help reduce population decoding error.<br /> While simultaneous recordings are not necessary to demonstrate results #1, #2, and #4, they are necessary to demonstrate results #3, #5, and #6.

Strengths:

- Important research question to all researchers interested in sensory coding in the nervous system.<br /> - State-of-the-art data collection: volumetric two-photon Ca2+ imaging and extracellular recording using high-density probes. Large neuronal data sets.<br /> - Confirmation of imaging results (lower temporal resolution) with more traditional microelectrode results (higher temporal resolution).<br /> - Clear and appropriate explanation of surgical and electrophysiological methods. I cannot comment on the appropriateness of the imaging methods.

Strength of evidence for the claims of the study:

(1) DCIC single-unit responses have high trial-to-trial variability -<br /> The authors' data clearly shows this.

(2) Approximately 32% to 40% of DCIC single units have responses that are sensitive to sound source azimuth -<br /> The sensitivity of each neuron's response to sound source azimuth was tested with a Kruskal-Wallis test, which is appropriate since response distributions were not normal. Using this statistical test, only 8% of neurons (median for imaging data) were found to be sensitive to azimuth, and the authors noted this was not significantly different than the false positive rate. The Kruskal-Wallis test was not reported for electrophysiological data. The authors suggested that low numbers of azimuth-sensitive units resulting from the statistical analysis may be due to the combination of high neural noise and a relatively low number of trials, which would reduce the statistical power of the test. This is likely true and highlights a weakness in the experimental design (i.e., a relatively small number of trials). The authors went on to perform a second test of azimuth sensitivity-a chi-squared test-and found 32% (imaging) and 40% (e-phys) of single units to have statistically significant sensitivity. However, the use of a chi-squared test is questionable because it is meant to be used between two categorical variables, and neural response had to be binned before applying the test.

(3) Single-trial population responses encode sound source azimuth "effectively" in that localization decoding error matches average mouse discrimination thresholds -<br /> If only one neuron in a population had responses that were sensitive to azimuth, we would expect that decoding azimuth from observation of that one neuron's response would perform better than chance. By observing the responses of more than one neuron (if more than one were sensitive to azimuth), we would expect performance to increase. The authors found that decoding from the whole population response was no better than chance. They argue (reasonably) that this is because of overfitting of the decoder model-too few trials were used to fit too many parameters-and provide evidence from decoding combined with principal components analysis which suggests that overfitting is occurring. What is troubling is the performance of the decoder when using only a handful of "top-ranked" neurons (in terms of azimuth sensitivity) (Fig. 4F and G). Decoder performance seems to increase when going from one to two neurons, then decreases when going from two to three neurons, and doesn't get much better for more neurons than for one neuron alone. It seems likely there is more information about azimuth in the population response, but decoder performance is not able to capture it because spike count distributions in the decoder model are not being accurately estimated due to too few stimulus trials (14, on average). In other words, it seems likely that decoder performance is underestimating the ability of the DCIC population to encode sound source azimuth.

To get a sense of how effective a neural population is at coding a particular stimulus parameter, it is useful to compare population decoder performance to psychophysical performance. Unfortunately, mouse behavioral localization data do not exist. Instead, the authors compare decoder error to mouse left-right discrimination thresholds published previously by a different lab. However, this comparison is inappropriate because the decoder and the mice were performing different perceptual tasks. The decoder is classifying sound sources to 1 of 13 locations from left to right, whereas the mice were discriminating between left or right sources centered around zero degrees. The errors in these two tasks represent different things. The two data sets may potentially be more accurately compared by extracting information from the confusion matrices of population decoder performance. For example, when the stimulus was at -30 deg, how often did the decoder classify the stimulus to a lefthand azimuth? Likewise, when the stimulus was +30 deg, how often did the decoder classify the stimulus to a righthand azimuth?

(4) DCIC can encode sound source azimuth in a similar format to that in the central nucleus of the inferior colliculus -<br /> It is unclear what exactly the authors mean by this statement in the Abstract. There are major differences in the encoding of azimuth between the two neighboring brain areas: a large majority of neurons in the CNIC are sensitive to azimuth (and strongly so), whereas the present study shows a minority of azimuth-sensitive neurons in the DCIC. Furthermore, CNIC neurons fire reliably to sound stimuli (low neural noise), whereas the present study shows that DCIC neurons fire more erratically (high neural noise).

(5) Evidence of noise correlation between pairs of neurons exists -<br /> The authors' data and analyses seem appropriate and sufficient to justify this claim.

(6) Noise correlations between responses of neurons help reduce population decoding error -<br /> The authors show convincing analysis that performance of their decoder increased when simultaneously measured responses were tested (which include noise correlation) than when scrambled-trial responses were tested (eliminating noise correlation). This makes it seem likely that noise correlation in the responses improved decoder performance. The authors mention that the naïve Bayesian classifier was used as their decoder for computational efficiency, presumably because it assumes no noise correlation and, therefore, assumes responses of individual neurons are independent of each other across trials to the same stimulus. The use of a decoder that assumes independence seems key here in testing the hypothesis that noise correlation contains information about sound source azimuth. The logic of using this decoder could be more clearly spelled out to the reader. For example, if the null hypothesis is that noise correlations do not carry azimuth information, then a decoder that assumes independence should perform the same whether population responses are simultaneous or scrambled. The authors' analysis showing a difference in performance between these two cases provides evidence against this null hypothesis.

Minor weakness:<br /> - Most studies of neural encoding of sound source azimuth are done in a noise-free environment, but the experimental setup in the present study had substantial background noise. This complicates comparison of the azimuth tuning results in this study to those of other studies. One is left wondering if azimuth sensitivity would have been greater in the absence of background noise, particularly for the imaging data where the signal was only about 12 dB above the noise.

Reviewer #1 (Public review):

This study by Alejandro Rosell et al. reveals the immunoregulatory role of the RAS-p110α pathway in macrophages, specifically in regulating monocyte extravasation and lysosomal digestion during inflammation. Disrupting this pathway, through genetic tools or pharmacological intervention in mice, impairs the inflammatory response, leading to delayed resolution and more severe acute inflammation. The authors suggest that activating p110α with small molecules could be a potential therapeutic strategy for treating chronic inflammation. These findings provide important insights into the mechanisms by which p110α regulates macrophage function and the overall inflammatory response.

The updates made by the authors in the revised version have addressed the main points raised in the initial review, further improving the strength of their findings.

Reviewer #1 (Public review):

Summary:

Tian et al. describes how TIPE regulates melanoma progression, stemness, and glycolysis. The authors link high TIPE expression to increased melanoma cell proliferation and tumor growth. TIPE causes dimerization of PKM2, as well as translocation of PKM2 to the nucleus, thereby activating HIF-1alpha. TIPE promotes the phosphorylation of S37 on PKM2 in an ERK-dependent manner. TIPE is shown to increase stem-like phenotype markers. The expression of TIPE is positively correlated with the levels of PKM2 Ser37 phosphorylation in murine and clinical tissue samples. Taken together, the authors demonstrate how TIPE impacts melanoma progression, stemness, and glycolysis through dimeric PKM2 and HIF-1alpha crosstalk.

The authors manipulated TIPE expression using both shRNA and overexpression approaches throughout the manuscript. Using these models, they provide strong evidence of the involvement of TIPE in mediating PKM2 Ser37 phosphorylation and dimerization. The authors also used mutants of PKM2 at S37A to block its interaction with TIPE and HIF-1alpha. In addition, an ERK inhibitor (U0126) was used to block the phosphorylation of Ser37 on PKM2. The authors show how dimerization of PKM2 by TIPE causes nuclear import of PKM2 and activation of HIF-1alpha and target genes. Pyridoxine was used to induce PKM2 dimer formation, while TEPP-46 was used to suppress PKM2 dimer formation. TIPE maintains stem cell phenotypes by increasing expression of stem-like markers. Furthermore, the relationship between TIPE and Ser37 PKM2 was demonstrated in murine and clinical tissue samples.

The evaluation of how TIPE causes metabolic reprogramming can be further assessed using isotope tracing experiments.

Reviewer #1 (Public Review):

Summary:

The work by Joseph et al "Impact of the clinically approved BTK inhibitors on the conformation of full-length BTK and analysis of the development of BTK resistance mutations in chronic lymphocytic leukemia" seeks to comparatively analyze the effect of a range of covalent and noncovalent clinical BTK inhibitors upon BTK conformation. The novel aspect of this manuscript is that it seeks to evaluate the differential resistance mutations that arise distinctly from each of the inhibitors.

Strengths:

This is an exciting study that builds upon the fundamental notion of ensemble behavior in solutions for enzymes such as BTK. The HDX-MS and NMR experiments are adequately and comprehensively presented.

Comments on the revised version:

I am satisfied with the revisions and the clear explanations.

Reviewer #1 (Public Review):

Summary:

The authors want to understand fundamental steps in ligand binding to muscle nicotinic receptors using computational methods. Overall, although the work provides new information and support for existing models of ligand activation of this receptor type, some limitations in the methods and approach mean that the findings are not as conclusive as hoped.

Strengths:

The strengths include the number of ligands tried, and the comparison to the existing mature analysis of receptor function from the senior author's lab.

Weaknesses:

The weakness are the brevity of the simulations, the concomitant lack of scope of the simulations, the lack of depth in the analysis and the incomplete relation to other relevant work. The free energy methods used seem to lack accuracy - they are only correct for 2 out of 4 ligands.

Reviewer #1 (Public review):

Summary:

Zhang et al. describe a delicate relationship between Tet2 and FBP1 in the regulation of hepatic gluconeogenesis.

Strengths:

The studies are very mechanistic, indicating that this interaction occurs via demethylation of HNF4a. Phosphorylation of HNF4a at ser 313 induced by metformin also controls the interaction between Tet2 and FBP1.

Weaknesses:

The results are briefly described, and oftentimes, the necessary information is not provided to interpret the data. Similarly, the methods section is not well developed to inform the reader about how these experiments were performed. While the findings are interesting, the results section needs to be better developed to increase confidence in the interpretation of the results.

Reviewer #1 (Public review):

The authors presented a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. With improved resolution and sensitivity, they explored the spatial connectivity of active promoters and identified the potential candidates for establishing/maintaining E-P interactions. Finally, with published CRISPRi screens, they found that most functionally-verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.

While the study's experimental approach and findings are interesting. However, several issues need to be addressed:

(1) The authors described that "the lack of interaction between experimentally-validated enhancers and their cognate promoters in some studies employing C-methods has raised doubts regarding the classical promoter-enhancer looping model", so it's intriguing to see whether the MChIP-C could indeed detect the E-P interactions which were not identified by C-methods as they mentioned (Benabdallah et al., 2019; Gupta et al., 2017). I agree that they identified more E-P interactions using MChIP-C, but specifically, they should show at least 2-3 cases. It's important since this is the main conclusion the authors want to draw.

(2) The authors compared their data to those of Chen et al. (Chen et al., 2022), who used PLAC-seq with anti-H3K4me3 antibodies in K562 cells and standard Micro-C data previously reported for K562, concluding that "MChIP-C achieves superior sensitivity and resolution compared to C-methods based on standard restriction enzymes.". This is not convincing since they only compared their data to one dataset. More datasets from other cell lines should be included.

(3) The reasons to choose Chen's data (Chen et al., 2022) and CRISPRi screens (Fulco et al., 2019; Gasperini et al., 2019) should be provided since there are so many out there.

(4) The authors identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions, but not RNA polymerase II, mediator complex, YY1 and BRD4. More explanation is needed for this point since they're previously suggested to be associated with E-P interactions.

(5) The limitations of the method should be discussed.

Reviewer #1 (Public review):

Summary:

The manuscript proposes that 5mC modifications to DNA, despite being ancient and widespread throughout life, represent a vulnerability, making cells more susceptible to both chemical alkylation and, of more general importance, reactive oxygen species. Sarkies et al take the innovative approach of introducing enzymatic genome-wide cytosine methylation system (DNA methyltransferases, DNMTs) into E. coli, which normally lacks such a system. They provide compelling evidence that the introduction of DNMTs increases the sensitivity of E. coli to chemical alkylation damage. Surprisingly they also show DNMTs increase the sensitivity to reactive oxygen species and propose that the DNMT generated 5mC presents a target for the reactive oxygen species that is especially damaging to cells. Evidence is presented that DNMT activity directly or indirectly produces reactive oxygen species in vivo, which is an important discovery if correct, though the mechanism for this remains obscure.

Strengths:

This work is based on an interesting initial premise, it is well-motivated in the introduction and the manuscript is clearly written. The results themselves are compelling.

Weaknesses:

I am not currently convinced by the principal interpretations and think that other explanations based on known phenomena could account for key results. Specific points below.

(1) As noted in the manuscript, AlkB repairs alkylation damage by direct reversal (DNA strands are not cut). In the absence of AlkB, repair of alklylation damage/modification is likely through BER or other processes involving strand excision and resulting in single stranded DNA. It has previously been shown that 3mC modification from MMS exposure is highly specific to single stranded DNA (PMID:20663718) occurring at ~20,000 times the rate as double stranded DNA. Consequently, the introduction of DNMTs is expected to introduce many methylation adducts genome-wide that will generate single stranded DNA tracts when repaired in an AlkB deficient background (but not in an AlkB WT background), which are then hyper-susceptible to attack by MMS. Such ssDNA tracts are also vulnerable to generating double strand breaks, especially when they contain DNA polymerase stalling adducts such as 3mC. The generation of ssDNA during repair is similarly expected follow the H2O2 or TET based conversion of 5mC to 5hmC or 5fC neither of which can be directly repaired and depend on single strand excision for their removal. The potential importance of ssDNA generation in the experiments has not been considered.

(2) The authors emphasise the non-additivity of the MMS + DNMT + alkB experiment but the interpretation of the result is essentially an additive one: that both MMS and DNMT are introducing similar/same damage and AlkB acts to remove it. The non-additivity noted would seem to be more consistent with the ssDNA model proposed in #1. More generally non-additivity would also be seen if the survival to DNA methylation rate is non-linear over the range of the experiment, for example if there is a threshold effect where some repair process is overwhelmed. The linearity of MMS (and H2O2) exposure to survival could be directly tested with a dilution series of MMS (H2O2).

(3) The substantial transcriptional changes induced by DNMT expression (Supplemental Figure 4) are a cause for concern and highlight that the ectopic introduction of methylation into a complex system is potentially more confounded than it may at first seem. Though the expression analysis shows bulk transcription properties, my concern is that the disruptive influence of methylation in a system not evolved with it adds not just consistent transcriptional changes but transcriptional heterogeneity between cells which could influence net survival in a stressed environment. In practice I don't think this can be controlled for, possibly quantified by single-cell RNA-seq but that is beyond the reasonable scope of this paper.

(4) Figure 4 represents a striking result. From its current presentation it could be inferred that DNMTs are actively promoting ROS generation from H2O2 and also to a lesser extent in the absence of exogenous H2O2. That would be very surprising and a major finding with far-reaching implications. It would need to be further validated, for example by in vitro reconstitution of the reaction and monitoring ROS production. Rather, I think the authors are proposing that some currently undefined, indirect consequence of DNMT activity promotes ROS generation, especially when exogenous H2O2 is available. It would help if this were clarified.

Reviewer #1 (Public review):

Summary:

The authors introduced neutron crystallography coupled with room temperature X-ray crystallography to exam the redox properties of the BtFt [4Fe-4S] cluster expressed in E. coli. Neutron structure allowed the authors to exam the influence of Asp64 on the redox properties of the [4Fe-4S] cluster. The neutron structure also allowed for the identification of the hydrogen network around the [4Fe-4S] structure. This work was followed by density functional theory calculation to examine different redox states which also pointed to the role of Asp64 in affecting or dictating redox function of the [4Fe-4S] cluster. Based on the DFT work the authors examine the redox properties under oxic and anoxic conditions in wild type enzymes and in a D64N mutant again showing the role of Asp64 on the redox kinetics and redox potential of the [4Fe-4S] cluster. Lastly, the authors examined similar [4Fe-4S] ferredoxins from several organisms and with a Asp64 or Glu64 observed a similar role of Asp64 on the low potential state of the [4Fe-4S] cluster. The major conclusion of the study was to identify the role of specific amino acids, in this case Asp64, in controlling the redox state and kinetics of [4Fe-4S] clusters. The authors also demonstrate the strength of neutron crystallography when combined with classical X-ray crystallography and classical spectral/redox studies.

Strengths:

In general, the experimental design is logical and the results are convincing demonstrating the role of Asp64 on the redox properties of [4Fe-4S] clusters in ferredoxins.

Weaknesses:

The role(s) of coordinating amino acids on the redox properties of a functional group is not surprising, this reviewer believes this is a novel result in ferredoxins and does make a nice contribution to the field.

Reviewer #1 (Public review):

Summary:

A description of small phosphatised fossils from the Kuanchuanpu, formations that are claimed to represent unequivocal early segmented bilaterians with limbs, ie annelids or panarthropods. All material from the Kuanchuanpu is of interest, and the mode of preservation is certainly striking.

However, few details apart from bilateral symmetry and paired protrusions are present. In addition, fragments of potential progenitors such as anabaritiids cannot be entirely ruled out. In addition, the broader claims about the nature of the Cambrian explosion, the gap between the fossil record and molecular clocks, and what various authors have said about them are either inadequate or incorrect. For example, Budd and Jackson did not at all make the claim that the earliest bilaterians were soft-bodied and tiny. Glaessner (1958) is a very out-of-date reference to use. We know that bilaterians certainly existed by the time of Kuanchuanpo.

Even so, it is possible that these fragments do represent internal moulds of taxa such as lobopod-like organisms, even if the evidence is not totally persuasive.

Reviewer #1 (Public review):

Summary:

In this manuscript, Corso-Diaz et al, focus on the NRL transcription factor (TF), which is critical for retinal rod photoreceptor development and function. The authors profile NRL's protein interactome, revealing several RNA-binding proteins (RBPs) among its components. Notably, many of these RBPs are associated with R-loop biology, including DHX9 helicase, which is the primary focus of this study. R-loops are three-stranded nucleic acid structures that frequently form during transcription. The authors demonstrate that R-loop levels increase during photoreceptor maturation and establish an interaction between NRL TF and DHX9 helicase. The association between NRL and RBPs like DHX9 suggests a cooperative regulation of gene expression in a cell-type-specific manner, an intriguing discovery relevant to photoreceptor health. Since DHX9 is a key regulator of R-loop homeostasis, the study proposes a potential mechanism where a cell-type-specific TF controls the expression of certain genes by modulating R-loop homeostasis. This study also presents the first data on R-loop mapping in mammalian retinas and shows the enrichment of R-loops over intergenic regions as well as genes encoding neuronal function factors. While the research topic is very important, there is some concern regarding the data presented: there are substantial data supporting the interaction between NRL and DHX9, including pull-down experiments and proximity labeling assay (PLA), however, the data showing an interaction between NRL and DDX5, another R-loop-associated helicase, are inadequate. Importantly, the data supporting the claim that NRL interacts with R-loops are absolutely insufficient and at best, correlative. The next concerns are regarding the R-loop mapping data analysis and visualization.

Strengths:

There is compelling evidence that the NRL transcription factor interacts with several RNA binding proteins, and specifically, sufficient data supporting the interaction of NRL with DHX9 helicase.<br /> A major strength is the use of the single-stranded R-loop mapping method in the mouse retina.

Weaknesses:

(1) Figure S1A: There is a strong band in GST-IP (control IP) for either HNRNPUI1 or HNRNPU, although the authors state in their results that there is a strong interaction of these two RBPs with NRL. Both DHX9 and DDX5 samples have a faint band in the GST-IP. There is an extremely faint band for HNRNPA2B1 in the GST-NRL IP lane. Given this is a pull-down with added benzonase treatment to remove all nucleic acids, these data suggest, that previously observed NRL interactions with these particular RBPs are mediated via nucleic acids. Similarly, there is a loss of band signal for HNRNM in this assay, although it was identified as an NRL-interacting protein in three assays, which again suggests that nucleic acids mediate the interaction.

(2) The data supporting NRL-DDX5 interaction in rod photoreceptor nuclei is very weak. In Figure 2D, the PLA signal for DDX5-NRL is very weak in the adult mouse retina and is absent in the human retina, as shown in Figure 2H. Given that there is no NRL-KO available for the human PLA assay, the control experiments using single-protein antibodies should be included in the assay. Similarly, the single-protein antibody control PLA experiments should be included in the experimental data presented in Figure 2J.

(3) The EMSA experiment using a probe containing NRL binding motif within the DHX9 promoter should include incubation with retina nuclear extracts depleted for NRL as a control.

(4) There is a reduced amount of DHX9 pulled down in NRL-IP in HEK293 cells, but there is no statistically significant difference in the reciprocal IP (DHX9-IP and blotting for NRL) (Figure 4C).

(5) The only data supporting the claim that NRL interacts with R-loops are presented in Figure 5A. This is a co-IP of R-loops and then blotting for NRL, DHX9, and DDX5. Here, there is no signal for DDX5, quantification of DHX9 signal shows no statistically significant difference between RNase H treated and untreated samples, while NRL shows a signal in RNase H treated sample. These data are not sufficient to make the statement regarding the interaction of NRL with R-loops.

(6) Regarding R-loop mapping, the data analysis is quite confusing. The authors perform two different types of analyses: either overall narrow and broad peak analysis or strand-specific analysis. Given that the authors used ssDRIP-seq, which is a method designed to map R-loops strand specifically, it is confusing to perform different types of analyses. Next, the peak analysis is usually performed based on the RNase H treated R-loop mapping; what does it mean then to have a pool of "Not R-loops", see Figure 6B? In that regard, what does the term "unstranded" R-loops mean? Based on the authors' definition, these are R-loops that do not fall within the group of strand-specific R-loops. The authors should explain the reasons behind these types of analyses and explain, what the biological relevance of these different types of R-loops is.

(7) It would be more useful to show the percent distribution of R-loops over the different genomic regions, instead of showing p-value enrichment, see Figure 6C.

(8) Based on the model presented, NRL regulates R-loop biology via interaction with RBPs, such as DHX9, a known R-loop resolution helicase. Given that the gene targets of NRL TF are known, it would be useful to then analyze the R-loop mapping data across this gene set.

Reviewer #1 (Public review):

Summary:

This impressive study presents a comprehensive scRNAseq atlas of the cranial region during neural induction, patterning, and morphogenesis. The authors collected a robust scRNAseq dataset covering six distinct developmental stages. The analysis focused on the neural tissue, resulting in a highly detailed temporal map of neural plate development. The findings demonstrate how different cell fates are organized in specific spatial patterns along the anterior-posterior and medial-lateral axes within the developing neural tissue. Additionally, the research utilized high-density single-cell RNA sequencing (scRNAseq) to reveal intricate spatial and temporal patterns independent of traditional spatial techniques.

The investigation utilized diffusion component analysis to spatially order cells based on their positioning along the anterior-posterior axis, corresponding to the forebrain, midbrain, hindbrain, and medial-lateral axis. By cross-referencing with MGI expression data, the identification of cell types was validated, affirming the expression patterns of numerous known genes and implicating others as differentially expressed along these axes. These findings significantly advance our understanding of the spatially regulated genes in neural tissues during early developmental stages. The emphasis on transcription factors, cell surface, and secreted proteins provides valuable insights into the intricate gene regulatory networks underpinning neural tissue patterning. Analysis of a second scRNAseq dataset where Shh signaling was inhibited by culturing embryos in SAG identified known and previously unknown transcripts regulated by Shh, including the Wnt pathway.

The data includes the neural plate and captures all major cell types in the head, including the mesoderm, endoderm, non-neural ectoderm, neural crest, notochord, and blood. With further analyses, this high-quality data promises to significantly advance our understanding of how these tissues develop in conjunction with the neural tissue, paving the way for future breakthroughs in developmental biology and genomics.

Strengths:

The data is well presented in the figures and thoroughly described in the text. The quality of the scRNAseq data and bioinformatic analysis is exceptional.

Weaknesses:

No weaknesses were identified by this reviewer.

Reviewer #1 (Public review):

This paper focuses on secondary structure and homodimers in the HIV genome. The authors introduce a new method called HiCapR which reveals secondary structure, homodimer, and long-range interactions in the HIV genome. The experimental design and data analysis are well-documented and statistically sound. However, the manuscript could be further improved in the following aspects.

Major comments:

(1) Please give the full name of an abbreviation the first time it appears in the paper, for example, in L37, "5' UTR" "RRE".

(2) The introduction could be strengthened by discussing the limitations of existing methods for studying HIV RNA structures and interactions and highlighting the specific advantages of the HiCapR method.

(3) Please reorganize Results Part 1.

(4) Is there any reason that the authors mention "genome structure of SARS-CoV-2" in L95?

(5) L102: Please clarify the purpose of comparing "NL4-3" and "GX2005002." Additionally, could you explain what NL4-3 and GX2005002 are? The connection between NL4-3, GX2005002, and HIV appears to be missing.

(6) Figure 1A is not able to clearly present the innovation point of HiCapR.

(7) Please compare the contact metrics detected by HiCapR and current techniques like SHAPE on the local interactions to assess the accuracy of HiCapR in capturing local RNA interactions relative to established methods.

(8) The paper needs further language editing.

Reviewer #1 (Public review):

Summary:

This study seeks to identify a molecular mechanism whereby the small molecule RY785 selectively inhibits Kv2.1 channels. Specifically, it sought to explain some of the functional differences that RY785 exhibits in experimental electrophysiology experiments as compared to other Kv inhibitors, namely the charged and non-specific inhibitor tetraethylammonium (TEA). This study used a recently published cryo-EM Kv2.1 channel structure in the open activated state and performed a series of multi-microsecond-long all-atom molecular dynamics simulations to study Kv2.1 channel conduction under the applied membrane voltage with and without RY785 or TEA present. While TEA directly blocks K+ permeation by occluding ion permeation pathway, RY785 binds to multiple non-polar residues near the hydrophobic gate of the channel driving it to a semi-closed non-conductive state. This mechanism was confirmed using an additional set of simulations and used to explain experimental electrophysiology data,

Strengths:

The total length of simulation time is impressive, totaling many tens of microseconds. The study develops forcefield parameters for the RY785 molecule based on extensive QM-based parameterization. The computed permeation rate of K+ ions through the channel observed under applied voltage conditions is in reasonable agreement with experimental estimates of the single-channel conductance. The study performed extensive simulations with the apo channel as well as both TEA and RY785. The simulations with TEA reasonably demonstrate that TEA directly blocks K+ permeation by binding in the center of the Kv2.1 channel cavity, preventing K+ ions from reaching the SCav site. The conclusion is that RY785 likely stabilizes a partially closed conformation of the Kv2.1 channel and thereby inhibits the K+ current. This conclusion is plausible given that RY785 makes stable contact with multiple hydrophobic residues in the S6 helix. This further provides a possible mechanism for the experimental observations that RY785 speeds up the deactivation kinetics of Kv2 channels from a previous experimental electrophysiology study.

Weaknesses:

The study, however, did not produce this semi-closed channel conformation and acknowledges that more direct simulation evidence would require extensive enhanced-sampling simulations. The study has not estimated the effect of RY785 binding on the protein-based hydrophobic pore constriction, which may further substantiate their proposed mechanism. And while the study quantified K+ permeation, it does not make any estimates of the ligand binding affinities or rates, which could have been potentially compared to the experiment and used to validate the models.

Reviewer #1 (Public review):

Summary:

In this paper, the authors have leveraged Single-cell RNA sequencing of the various stages of the evolution of lung adenocarcinoma to identify the population of macrophages that contribute to tumor progression. They show that S100a4+ alveolar macrophages, active in fatty acid metabolic activity, such as palmitic acid metabolism, seem to drive the atypical adenomatous hyperplasia (AAH) stage. These macrophages also seem to induce angiogenesis promoting tumor growth. Similar types of macrophage infiltration were demonstrated in the progression of the human lung adenocarcinomas.

Strengths:

Identification of the metabolic pathways that promote angiogenesis-dependent progression of lung adenocarcinomas from early atypical changes to aggressive invasive phenotype could lead to the development of strategies to abort tumor progression.

Weaknesses:

(1) Can the authors demonstrate what are the functional specialization of the S100a4+ alveolar macrophages that promote the progression of the AAH to the more aggressive phenotype? What are the factors produced by these unique macrophages that induce tumor progression and invasiveness?

(2) Angiogenic factors are not only produced by the S100a4+ cells but also by pericytes and potentially by the tumor cells themselves. Then, how do these factors aberrantly trigger tumor angiogenesis that drives tumor growth?

(3) It is not clear how abnormal fatty acid uptake by the macrophages drives the progression of tumors.

(4) Does infusion or introduction of S100a4+ polarized macrophages promote the progression of AAH to a more aggressive phenotype?

(5) How does Anxa and Ramp1 induction in inflammatory cells induce angiogenesis and tumor progression?

(6) For the in vitro studies the authors might consider using primary tumor cells and not cell lines.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors consider the effects of eugenol (EUG), a plant-produced substance known to reduce oxidative stress in various cellular contexts via Nrf2, in alleviating the effects of streptozotocin (STZ), a known rodent beta cell toxin. They claim that EUG treatment would be useful for T1D therapy.

Strengths:

The experiments shown are sufficiently clear and rather convincing in documenting that eugenol can revert the effects of streptozotocin on animal physiology as well as beta cell oxidative stress and cell death via activation of Nrf2.

In the revised manuscript the authors corrected/explained most of the specific inconsistencies/mistakes pointed out.

However, they did not address the opening paragraph that points out major concerns. I summarize them below, together with some that were dealt with in their response but still remain unaddressed or not commented upon.

- STZ treatment cannot be used as a T1D model for the reasons I outlined in my previous letter. I would have been happy to see a response on that but they did not provide any. The manuscript is misleading in this important respect.

- Mechanistically, the manuscript remains at a rather superficial level. I highlighted some possibilities to enrich the manuscript but none was addressed even in the discussion.<br /> (a) How is eugenol penetrating the cell, is there a receptor that could be potentially targeted?<br /> (b) Are there intermediary proteins that convey the effect to the Nrf2/Keap1 complex or is eugenol directly disrupting their interaction?<br /> (c) What are direct downstream Nrf2 effectors?<br /> (d) Besides, streptozotocin is also a powerful DNA alkylating agent, are such effects relieved by eugenol?

- It is puzzling that all molecular analyses show a gradual reversion effect with increasing doses of eugenol but this gradual effect is apparently missing in many of the physiological parameters assessed in Figure 1, including the all-important OGTT assays. Can the authors interpret this? In the high eugenol group in the OGTT assays there is a group of mice that are clearly outliers. Most likely the STZ treatment for these mice was not efficient and their inclusion skews the results. Besides, it is important to assess differences among eugenol groups (one way ANOVA). The statistical tests provided are incomplete and sometimes not done correctly.

- Given that medical research is still heavily biased in favor of analyses in males and given that the authors have analyzed in Figure 1 a very large number of animals what are the results stratified by sex?

Reviewer #1 (Public review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

Reviewer #1 (Public Review):

In this manuscript, the authors employed direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. They conclude that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Contrary to the literature, they found that, unlike typical RNA decay models in normal conditions, stress-induced RNA decay is dependent on XRN1 but does not depend on the removal of the poly(A) tail. The findings presented are interesting and the authors fully established these paradigm-shifting findings using cutting-edge technologies.

Reviewer #1 (Public review):

Summary:

The authors report an fMRI investigation of the neural mechanisms by which selective attention allows capacity-limited perceptual systems to preferentially represent task-relevant visual stimuli. Specifically, they examine competitive interactions between two simultaneously-presented items from different categories, to reveal how task-directed attention to one of them modulates the activity of brain regions that respond to both. The specific hypothesis is that attention will bias responses to be more like those elicited by the relevant object presented on its own, and further that this modulation will be stronger for more dissimilar stimulus pairs. This pattern was confirmed in univariate analyses that measured the mass response of a priori regions of interest, as well as multivariate analyses that considered the patterns of evoked activity within the same regions. The authors follow these neuroimaging results with a simulation study that favours a "tuning" mechanism of attention (enhanced responses to highly effective stimuli, and suppression for ineffective stimuli) to explain this pattern.

Strengths:

The manuscript clearly articulates a core issue in the cognitive neuroscience of attention, namely the need to understand how limited perceptual systems cope with complex environments in the service of the observer's goals. The use of a priori regions of interest (and a control region), and the inclusion of both univariate and multivariate analyses as well as a simple model, are further strengths. The authors carefully derive clear indices of attentional effects (for both univariate and multivariate analyses) which makes explication of their findings easy to follow.

Weaknesses:

Direct estimation of baseline responses may have improved the validity of the modelling. The presentation of transparently overlapping items has some methodological advantages, but somewhat limits the ecological validity of connections to real-world visual "clutter".

Reviewer #1 (Public review):

Summary:

This article investigated the relationship between different intensities of exercise training and intestinal barrier dysfunction, and further explores the possible mechanisms, including the contribution of stress response, inflammatory response, gut microbiota alterations, and derived metabolites.

Strengths:

This article mainly focused on different aspects of the phenotypes and the morphology of intestinal barrier dysfunction induced by exercise training.

Weaknesses:

This article lacks the verification of the association of causality among various phenotypes and lacks a comprehensive understanding of the underlying mechanisms of how exercise contributes to intestinal barrier dysfunction.

(1) For example, the author claimed that heat shock and ischemia are the causes of intestinal epithelial damage caused by exercise, and it is not only evidenced by detecting the expression of a few regulators, such as HSF and HSP70 after exercise; and by Immunohistochemical analysis of intestinal morphology and inflammation.

(2) Many kinds of intestinal bacteria could produce short-chain fatty acids, such as Faecalibacterium Prausnitzii, did the authors check their abundance in the intestine after exercise training?

(3) How to define exercise intensity? Was VO2 Max testing used in this study?

(4) As the strict control, it is recommended to set 4 groups of exercise training groups: daily vigorous exercise training, daily moderate exercise training, daily vigorous exercise training with intermittent rest days, and daily moderate exercise training with intermittent rest days.

(5) Are there any differences in diet and metabolism between different groups of mice, which may affect the phenotypes, especially the composition and the the diverstiy of gut microbiota?

Reviewer #1 (Public review):

In this study, Sarver and colleagues carried out an exhaustive analysis of the functioning of various components (Complex I/II/IV) of the mitochondrial electron transport chain (ETC) using a real-time cell metabolic analysis technique (commonly referred as Seahorse oxygen consumption rate (OCR) assay). The authors aimed to generate an atlas of ETC function in about 3 dozen tissue types isolated from all major mammalian organ systems. They used a recently published improvised method by which ETC function can be quantified in freshly frozen tissues. This method enabled them to collect data from almost all organ systems from the same mouse and use many biological replicates (10 mice/experiment) required for an unbiased and statistically robust analysis. Moreover, they studied the influence of sex (male and female) and aging (young adult and old age) on ETC function in these organ systems. The main findings of this study are (1) cells in the heart and kidneys have very active ETC complexes compared to other organ systems, (2) the sex of the mice has little influence on the ETC function, and (3) aging undermined the mitochondrial function in most tissue, but surprisingly in some tissue aging promoted the activity of ETC complexes (e.g., Quadriceps, plantaris muscle, and Diaphragm).

Comments on the second revision:

My previous concern remains unaddressed in the new revision. As I mentioned earlier, it is crucial for the authors to include a detailed discussion on the limitations of their method, specifically how maximal respiration does not accurately reflect the actual ATP production rate. Additionally, the authors should highlight the fact that data provided in the manuscript should be interpreted with caution.

Reviewer #1 (Public review):

Summary:

In this study, the authors used both the commonly used neonatal hyperoxia model as well as cell-type-specific genetic inactivation of Tgfbr2 models to study the basis of BPD. The bulk of the analyses focus on the mesenchymal cells. Results indicate impaired myofibroblast proliferation, resulting in decreased cell number. Inactivation of Etc2 in Pdgfra-lineaged cells, preventing cytokinesis of myofibroblasts, led to alveolar simplification. Together, the findings demonstrate that disrupted myofibroblast proliferation is a key contributor to BPD pathogenesis.

Strengths:

Overall, this comprehensive study of BPD models advances our understanding of the disease. The data are of high quality.

Comments on latest version:

In the revision, the authors addressed all critiques.

Reviewer #1 (Public review):

Malaria parasites detoxify free heme molecules released from digested host hemoglobins by biomineralizing them into inert hemozoin. Thus, why malaria parasites retain PfHO, a dead enzyme that loses the capacity of catabolizing heme, is an outstanding question that has puzzled researchers for more than a decade. In the current manuscript, the authors addressed this question by first solving the crystal structure of PfHO and aligning it with structures of other heme oxygenase (HO) proteins. They found that the N-terminal 95 residues of PfHO, which failed to crystalize due to its disordered nature, may serve as signal and transit peptides for PfHO subcellular localization. This was confirmed by subsequent microscopic analysis with episomally expressed PfHO-GFP and a GFP reporter fused to the first 83 residues of PfHO (PfHO N-term-GFP). To investigate the functional importance of PfHO, the authors generated an anhydrotetracycline (aTC) controlled PfHO knockdown strain. Strikingly, the parasites lacking PfHO failed to grow and lost their apicoplast. Finally, by chromatin immunoprecipitation (ChIP), quantitative PCR/RT-PCR and growth assays, the authors showed that both the cognate N-terminus and HO-like domain were required for PfHO function as an apicoplast DNA interacting protein.

The authors systemically performed multidisciplinary approaches to address this difficult question: what is the function of this enzymatically dead PfHO? I enjoyed reading this manuscript and its thoughtful discussion. This study is not only of clinical importance for antimalarial treatments but also deepens our understanding of protein function evolution.

The authors proposed that PfHO interacts with apicoplast genome DNA via the electropositive N-terminus. Interestingly, these positively charged residues are not conserved between Plasmodium, Theileria and Babesia. I will be curious to follow the authors' future work to investigate the function of this electropositive N-terminus, possibly by comparative and mutagenesis analysis?

Reviewer #1 (Public review):

Mohseni and Elhaik have critically examined the widespread use of principal component analysis (PCA) in phylogenetic inferences within the discipline of physical anthropology. The authors present compelling evidence that PCA underperforms compared to machine learning (ML) classifiers. This excellent work not only challenges the reliability of PCA-based taxonomic inferences, but also adds to a growing body of literature questioning the application of PCA in physical anthropology, thereby initiating a fruitful discussion in our field. Moreover, it underscores the crucial need of external validation methods in such studies.

The authors have addressed nearly all of my comments, and my questions have been fully answered. The revised manuscript represents a significant improvement.

The new title more effectively conveys the central message emerging from this research; The revised introduction more precisely addresses the methodological challenges currently facing the discipline.<br /> I am equally amazed by the profound susceptibility of the PCA results, as demonstrated by the alterations introduced by the authors, and by the contrasting robustness of the ML classifiers. I trust that this contrast will spark a fruitful discussion about the application of both methods in our field. It should also inspire further research conducted by physical anthropologists to study the role of ML in this discipline.<br /> Lastly, and importantly, I believe the authors should be commended for addressing the broader implications of their work, particularly in relation to public perceptions of science (pp. 20-21).

Reviewer #1 (Public review):

Summary:

The authors examine CD8 T cell selective pressure in early HCV infection using. They propose that after initial CD8-T mediated loss of virus fitness, in some participants around 3 months after infection, HCV acquires compensatory mutations and improved fitness leading to virus progression.

Strengths:

Throughout the paper, the authors apply well-established approaches in studies of acute to chronic HIV infection for studies of HCV infection. This lends rigor the to the authors' work.

Weaknesses:

(1) The Discussion could be strengthened by a direct discussion of the parallels/differences in results between HIV and HCV infections in terms of T cell selection, entropy, and fitness.

(2) In the Results, please describe the Barton model functionality and why the fitness landscape model was most applicable for studies of HCV viral diversity.

(3) Recognize the caveats of the HCV mapping data presented.

(4) The authors should provide more data or cite publications to support the authors' statement that HCV-specific CD8 T cell responses decline following infection.

(5) Similarly, as the authors' measurements of HCV T and humoral responses were not exhaustive, the text describing the decline of T cells with the onset of humoral immunity needs caveats or more rigorous discussion with citations (Discussion lines 319-321).

(6) What role does antigen drive play in these data -for both T can and antibody induction?

(7) Figure 3 - are the X and Y axes wrongly labelled? The Divergent ranges of population fitness do not make sense.

(8) Figure S3 - is the green line, average virus fitness?

(9) Use the term antibody epitopes, not B cell epitopes.

Reviewer #1 (Public review):

Summary:

In this manuscript, De La Forest Divonne et al. build a repertory of hemocytes from adult Pacific oysters combining scRNAseq data with cytologic and biochemical analyses. Three categories of hemocytes were described previously in this species (i.e. blast, hyalinocyte, and granulocytes). Based on scRNAseq data, the authors identified 7 hemocyte clusters presenting distinct transcriptional signatures. Using Kegg pathway enrichment and RBGOA, the authors determined the main molecular features of the clusters. In parallel, using cytologic markers, the authors classified 7 populations of hemocytes (i.e. ML, H, BBL, ABL, SGC, BGC, and VC) presenting distinct sizes, nucleus sizes, acidophilic/basophilic, presence of pseudopods, cytoplasm/nucleus ratio and presence of granules. Then, the authors compared the phenotypic features with potential transcriptional signatures seen in the scRNAseq. The hemocytes were separated in a density gradient to enrich for specific subpopulations. The cell composition of each cell fraction was determined using cytologic markers and the cell fractions were analysed by quantitative PCR targeting major cluster markers (two per cluster). With this approach, the authors could assign cluster 7 to VC, cluster 2 to H, and cluster 3 to SGC. The other clusters did not show a clear association with this experimental approach. Using phagocytic assays, ROS, and copper monitoring, the authors showed that ML and SGC are phagocytic, ML produces ROS, and SGC and BGC accumulate copper. Then with the density gradient/qPCR approach, the authors identified the populations expressing anti-microbial peptides (ABL, BBL, and H). At last, the authors used Monocle to predict differentiation trajectories for each subgroup of hemocytes using cluster 4 as the progenitor subpopulation.

The manuscript provides a comprehensive characterisation of the diversity of circulating immune cells found in Pacific oysters.

Strengths:

The combination of the two approaches offers a more integrative view.

Hemocytes represent a very plastic cell population that has key roles in homeostatic and challenged conditions. Grasping the molecular features of these cells at the single-cell level will help understand their biology.

This type of study may help elucidate the diversification of immune cells in comparative studies and evolutionary immunology.

Weaknesses:

The study should be more cautious about the conclusions, include further analyses, and inscribe the work in a more general framework.

Reviewer #1 (Public review):

Summary:

Chemotherapy-induced chronic kidney injury is a significant and growing concern, as it can lead to long-term renal damage and compromised kidney function. The authors have highlighted an important aspect of this issue by evaluating the potential protective effects of OPCs against cisplatin-induced kidney injury. They propose that OPCs may mitigate renal damage by reducing NET formation, which could improve kidney function.

Strengths:

The study addressed a significant issue in the field of chemotherapy-induced kidney injury. The use of multiple markers and experimental methods provided a comprehensive exploration of the impact of OPCs on kidney damage. This approach allowed for a nuanced understanding of how OPCs might mitigate renal injury by reducing NET formation and improving kidney function.

Weaknesses:

The hypothesis is intriguing and relevant. However, the study encounters challenges, such as incomplete evidence and discrepancies between the text and data. Addressing these issues is crucial to improving the overall study's conclusions. The paper can potentially advance the understanding of therapeutic strategies for chemotherapy-induced kidney injury. Nonetheless, a clearer presentation of the data is necessary for it to have a substantial impact.

Reviewer #1 (Public Review):

Kainov et al investigated the prevalence of mutations in 3'UTR that affect gene expression in cancer to identify noncoding cancer drivers.

The authors used data from normal controls (1000 genome data) and compared it to cancer data (PCAWG). They found that in cancer 3'UTR mutations had a stronger effect on cleavage than the normal population. These mutations are negatively selected in the normal population and positively selected in cancers. The authors used PCAWG data set to identify such mutations and found that the mutations that lead to a reduction of gene expression are enriched in tumor suppressor genes and those that are increased in gene expression are enriched for oncogenes. 3'UTR mutations that reduce gene expression or occur in TSGs co-occur with non-synonymous mutations. The authors then validate the effect of 3'UTR mutations experimentally using a luciferase reporter assay. These data identify a novel class of noncoding driver genes with mutations in 3'UTR that impact polyadenylation and thus gene expression.

This is an elegant study with fundamental insight into identifying cancer driver genes. The conclusions of this paper are mostly well supported by data, but some aspects of data analysis need to be extended.

Comments on revisions:

The authors addressed most of my comments.

Reviewer #1 (Public review):

Summary:

It is well known that autophagosomes/autolysosomes move along microtubules. However, because previous studies did not distinguish between autophagosomes and autolysosomes, it remains unknown whether autophagosomes begin to move after fusion with lysosomes or even before fusion. In this manuscript, the authors show, using fusion-deficient cells, that both pre-fusion autophagosomes and lysosomes can move along the MT toward the minus end. By screening motor proteins and Rabs, the authors found that autophagosomal traffic is primarily regulated by the dynein-dynactin system and can be counter-regulated by kinesins. They also show that Rab7-Epg5 and Rab39-ema interactions are important for autophagosome trafficking.

Strengths:

This study uses reliable Drosophila genetics and high-quality fluorescence microscopy. The data are properly quantified and statistically analyzed. It is a reasonable hypothesis that gathering pre-fusion autophagosomes and lysosomes in close proximity improves fusion efficiency.

Weaknesses:

(1) To distinguish autophagosomes from autolysosomes, the authors used vps16 RNAi cells, which are supposed to be fusion deficient. However, the extent to which fusion is actually inhibited by knockdown of Vps16A is not shown. The co-localization rate of Atg8 and Lamp1 should be shown (as in Figure 8). Then, after identifying pre-fusion autophagosomes and lysosomes, the localization of each should be analyzed. It is also possible that autophagosomes and lysosomes are tethered by factors other than HOPS (even if they are not fused). If this is the case, autophagosomal trafficking would be affected by the movement of lysosomes.

(2) The authors analyze autolysosomes in Figures 6 and 7. This is based on the assumption that autophagosome-lysosome fusion takes place in cells without vps16A RANi. However, even in the presence of Vps16A, both pre-fusion autophagosomes and autolysosomes should exist. This is also true in Figure 8H, where the fusion of autophagosomes and lysosomes is partially suppressed in knockdown cells of dynein, dynactin, Rab7, and Epg5. If the effect of fusion is to be examined, it is reasonable to distinguish between autophagosomes and autolysosomes and analyze only autolysosomes.

(3) In this study, only vps16a RNAi cells were used to inhibit autophagosome-lysosome fusion. However, since HOPS has many roles besides autophagosome-lysosome fusion, it would be better to confirm the conclusion by knockdown of other factors (e.g., Stx17 RNAi).

(4) Figure 8: Rab7 and Epg5 are also known to be directly involved in autophagosome-lysosome tethering/fusion. Even if the fusion rate is reduced in the absence of Rab7 and Epg5, it may not be the result of defective autophagosome movement, but may simply indicate that these molecules are required for fusion itself. How do the authors distinguish between the two possibilities?

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated how partial loss of SynGap1 affects inhibitory neurons derived from the MGE in the auditory cortex, focusing on their synaptic inputs and excitability. While haplo-insufficiently of SynGap1 is known to lead to intellectual disabilities, the underlying mechanisms remain unclear.

Strengths:

The questions are novel

Weaknesses:

Despite the interesting and novel questions, there are significant issues regarding the experimental design and potential misinterpretations of key findings. Consequently, the manuscript contributes little to our understanding of SynGap1 loss mechanisms.

Major issues in the second version of the manuscript:<br /> In the review of the first version there were major issues and contradictions with the sEPSC and mEPSC data, and were not resolved after the revision, and the new control experiments rather confirmed the contradiction.<br /> In the original review I stated: "One major concern is the inconsistency and confusion in the intermediate conclusions drawn from the results. For instance, while the sEPSC data indicates decreased amplitude in PV+ and SOM+ cells in cHet animals, the frequency of events remains unchanged. In contrast, the mEPSC data shows no change in amplitudes in PV+ cells, but a significant decrease in event frequency. The authors conclude that the former observation implies decreased excitability. However, traditionally, such observations on mEPSC parameters are considered indicative of presynaptic mechanisms rather than changes of network activity.‎ The subsequent synapse counting experiments align more closely with the traditional conclusions. This issue can be resolved by rephrasing the text. However, it would remain unexplained why the sEPSC frequency shows no significant difference. If the majority of sEPSC events were indeed mediated by spiking (which is blocked by TTX), the average amplitudes and frequency of mEPSCs should be substantially lower than those of sEPSCs. Yet, they fall within a very similar range, suggesting that most sEPSCs may actually be independent of action potentials. But if that was indeed the case, the changes of purported sEPSC and mEPSC results should have been similar."<br /> Contradictions remained after the revision of the manuscript. On one hand, the authors claimed in the revised version that "We found no difference in mEPSC amplitude between the two genotypes (Fig. 1g), indicating that the observed difference in sEPSC amplitude (Figure 1b) could arise from decreased network excitability". On the other hand, later they show "no significative difference in either amplitude or inter-event intervals between sEPSC and mEPSC, suggesting that in acute slices from adult A1, most sEPSCs may actually be AP independent." The latter means that sEPSCs and mEPSCs are the same type of events, which should have the same sensitivity to manipulations.

Concerns about the quality of the synapse counting experiments were addressed by showing additional images in a different and explaining quantification. However, the admitted restriction of the analysis of excitatory synapses to the somatic region represent a limitation, as they include only a small fraction of the total excitation - even if, the slightly larger amplitudes of their EPSPs are considered.

New experiments using pari-pulse stimulation provided an answer to issues 3 and 4. Note that the numbering of the Figures in the responses and manuscript are not consistent.

I agree that low sampling rate of the APs does not change the observed large differences in AP threshold, however, the phase plots are still inconsistent in a sense that there appears to be an offset, as all values are shifted to more depolarized membrane potentials, including threshold, AP peak, AHP peak. This consistent shift may be due to a non-biological differences in the two sets of recordings, and, importantly, it may negate the interpretation of the I/f curves results (Fig. 5e).

Additional issues:<br /> The first paragraph of the Results mentioned that the recorded cells were identified by immunolabelling and axonal localization. However, neither the Results nor the Methods mention the criteria and levels of measurements of axonal arborization.

The other issues of the first review were adequately addressed by the Authors and the manuscript improved by these changes.

Reviewer #3 (Public review):

Summary:

The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.

Strengths:

The strengths of this manuscript include the use of multiple model systems and follow up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.

Weaknesses:

As in previous revisions, there remains concerning ambiguity in the methodology used for microbiota sequence analysis and it would be difficult to replicate the analysis in any meaningful way. In this revision, concerns about the rigor and reproducibility of this component of the manuscript have been increased. Readers should be cautious with interpretation of this data.

(1) In previous versions of the manuscript it would appear the correct bioproject accession was listed but, the actual link went to an unrelated project. The updated accession link appears to contain raw data; however, the authors state they used an Illumina HiSeq 2500. This would be an unusual choice for V3-V4 as it would not have read lengths long enough to overlap. Inspection of the first sample (SRR19164796) demonstrates that this is absolutely not the raw data, as there is a ~400 nt forward read, and a 0 length reverse read. All quality scores are set to 30. There is no logical way to go from HiSeq 2500 raw data and read lengths to what was uploaded to the SRA and it was certainly not described in the manuscript.

(2) No multiple testing correction was applied to the microbiome data.

Reviewer #1 (Public Review):

The authors analyse droplet size distributions of multiple protein condensates and fit to a scaling ansatz to highlight that they exhibit features of first-order and second-order phase transitions. While the experimental evidence is solid, the text lacks connection and contextualization to the well-understood expectations from the coupling of percolation and phase separation in protein condensates - a phenomenon that is increasingly gaining consensus amongst the community. The evidence supports the percolatoin+phase separation model rather than being close to a true critical point in the liquid-gas phase space. Overall, the work is useful to the community.

Strengths:<br /> The experimental analysis of distinct protein condensates is very well done and the reported exponents/scaling framework provides a clear framework to help the community help deconvolve signatures of percolation in condensates.

Weaknesses:

The principal concern this reviewer has is that the reviewers adopt a framing in this paper to present a discovery of second-order features and connections to criticality - however they ignore/miss the connections to percolation (a well-understood second-order transition that is expected to play a major role in protein condensates). I believe this needs to be addressed and the paper suitably revised to help connect with these expectations.

- Protein condensates have been increasingly understood to be described as fluids whose assembly is driven by a connection of density (phase separation, first-order) and connectivity (percolation, second-order) transitions. This has been long known in the polymer community (Flory, Stockmayer, Tanaka, Rubinstein, Semenov and others) and recently repopularized in the condensate community (by Pappu and Mittag, in particular, amongst others). The authors make no connections to any of this frameworks - which actually seem to be the essence of what they are describing.

- Percolation theory, which has been around for more than half-a-century, has clear-cut scaling laws that have essentially similar forms to the ansatz adopted by the authors and the commonalities/differences are not discussed by the authors - this is essential since this provides a physical basis for their ansatz rather than an arbitrary mathematical formulation. In particular, percolation models connect size distribution exponents to factors like dimensionality, valence, etc. and if these connections can be made with this data, that would be very powerful.

- The connections between spinodal decomposition and second-order phase transitions are very confusing. Spindal decomposition happens when the barriers for first-order phase transitions are zero and systems can phase separate without crossing nucleation barriers. Further, the "criticality" discussed in the paper is confusing since it more likely refers to a percolation threshold and much less likely to a "critical temperature" (Tc -where spinodal and binodals become identical). I would recommend reframing this argument.

It's unlikely, in this reviewer's opinion, that the authors are actually discussing a "first-order" liquid-gas critical point - because saturation concentrations of these proteins can be much higher with temperature and the critical point would thus likely be at much higher concentrations (and ofc temperature). Further the scaling exponents don't fall in that class naturally. However, if the authors disagree, I would appreciate clear quantitative reasons (including through the scaling exponents in that universality class) and be happy to be convinced to change my mind. As provided, the data does not support this model.

Reviewer #1 (Public review):

Summary:

Bennion and colleagues present a careful examination of how an earlier set of memories can either interfere with or facilitate memories formed later. This impressive work is a companion piece to an earlier paper by Antony and colleagues (2022) in which a similar experimental design was used to examine how a later set of memories can either interfere with or facilitate memories formed earlier. This study makes contact with an experimental literature spanning 100 years, which is concerned with the nature of forgetting, and the ways in which memories for particular experiences can interact with other memories. These ideas are fundamental to modern theories of human memory, for example, paired-associates studies like this one are central to the theoretical idea that interference between memories is a much bigger contributor to forgetting than any sort of passive decay.

Strengths:

At the heart of the current investigation is a proposal made by Osgood in the 1940s regarding how paired associates are learned and remembered. In these experiments one learns a pair of items, A-B (cue-target), and then later learns another pair that is related in some way, either A'-B (changing the cue, delta-cue), or A-B' (changing the target, delta-target), or A'-B' (changing both, delta-both), where the prime indicates that item has been modified, and may be semantically related to the original item. The authors refer to the critical to-be-remembered pairs as base pairs. Osgood proposed that when the changed item is very different from the original item there will be interference, and when the changed item is similar to the original item there will be facilitation. Osgood proposed a graphical depiction of his theory in which performance was summarized as a surface, with one axis indicating changes to the cue item of a pair and the other indicating changes to the target item, and the surface itself necessary to visualize the consequences of changing both.

In the decades since Osgood's proposal, there have been many studies examining slivers of the proposal, e.g., just changing targets in one experiment, just changing cues in another experiment. Because any pair of experiments use different methods, this has made it difficult to draw clear conclusions about the effects of particular manipulations.

The current paper is a potential landmark, in that they manipulate multiple fundamental experimental characteristics using the same general experimental design. Importantly, they manipulate the semantic relatedness of the changed item to the original item, the delay between the study experience and the test, and which aspect of the pair is changed. Furthermore, they include both a positive control condition (where the exact same pair is studied twice), and a negative control condition (where a pair is only studied once, in the same phase as the critical base pairs). This allows them to determine when the prior learning exhibits an interfering effect relative to the negative control condition, and also allows them to determine how close any facilitative effects come to matching the positive control.

The results are interpreted in terms of a set of existing theories, most prominently the memory-for-change framework, which proposes a mechanism (recursive reminding) potentially responsible for the facilitative effects examined here. One of the central results is the finding that a stronger semantic relationship between a base pair and an earlier pair has a facilitative effect on both the rate of learning of the base pair and the durability of the memory for the base pair. This is consistent with the memory-for-change framework, which proposes that this semantic relationship prompts retrieval of the earlier pair, and the two pairs are integrated into a common memory structure that contains information about which pair was studied in which phase of the experiment. When semantic relatedness is lower, they more often show interference effects, with the idea being that competition between the stored memories makes it more difficult to remember the base pair.

This work represents a major methodological and empirical advance for our understanding of paired-associates learning, and it sets a laudably high bar for future work seeking to extend this knowledge further. By manipulating so many factors within one set of experiments, it fills a gap in the prior literature regarding the cognitive validity of an 80-year-old proposal by Osgood. The reader can see where the observed results match Osgood's theory and where they are inconclusive. This gives us insight, for example, into the necessity of including a long delay in one's experiment, to observe potential facilitative effects. This point is theoretically interesting, but it is also a boon for future methodological development, in that it establishes the experimental conditions necessary for examining one or another of these facilitation or interference effects more closely.

The authors were exceptionally responsive to the suggestions of the reviewers, and the revisions have improved the theoretical clarity of the paper. I think the value of this work will grow with time, as memory researchers and theorists use it as a benchmark for new theory development. For example, the data from these experiments will undoubtedly be used to develop and constrain a new generation of computational models of paired-associates learning.

Weaknesses:

One minor weakness of the work is that the overarching theoretical framing does not necessarily specify the expected result for each and every one of the many effects examined. For example, with a narrower set of semantic associations being considered (all of which are relatively high associations) and a long delay, varying the semantic relatedness of the target item did not reliably affect the memorability of that pair. However, the same analysis showed a significant effect when the wider set of semantic associations was used. The positive result is consistent with the memory-for-change framework, but the null result isn't clearly informative to the theory. However, research is never done; comparing the results with the two sets of semantic associations is informative from a methodological perspective, in that it establishes the degree to which semantic relatedness must be altered to affect behavioral performance in a paired-associates task.

Reviewer #1 (Public review):

Somasundaram and colleagues explore the role of transcription factors in retinal ganglion cell (RGC) death and axonal regeneration after a disease relevant insult (mechanical axonal injury). The work significantly extends our knowledge of the role of MAPK and integrated stress response (ISR) in controlling RGC fate after injury. Specifically, the manuscript shows that after axonal injury PERK-activated ISR acts through Atf4 to drive a prodeath transcriptional response in RGCs, in part by crosstalk with the prodeath JUN transcriptional program. Also, and perhaps most interesting, the work shows that PERK-ATF4 pathway activation is pro-regenerative for RGC axons. A major plus of the manuscript is that many new RNA-seq datasets are generated that describe the major prodegenerative and proregenerative gene networks altered after axonal injury. A limitation of the study is that it does not directly compare the effect of inhibiting the PERK-ATF4 pathway with inhibiting JUN and/or JUN-CHOP double deficient animals. It would also be useful, for the cell survival experiments shown in Figure 1, to examine a longer time point than 14 days to understand the long-term consequence of manipulating the PERK-ATF4 pathway.

Reviewer #1 (Public Review):

Summary:

The authors are interested in the developmental origin of the neurons of the cerebellar nuclei. They identify a population of neurons with a specific complement of markers originating in a distinct location from where cerebellar nuclear precursor cells have been thought to originate that show distinct developmental properties. The cerebellar nuclei have been well studied in recent years to understand their development through an evolutionary lens, which supports the importance of this study. The discovery of a new germinal zone giving rise to a new population of CN neurons is an exciting finding, and it enriches our understanding of cerebellar development, which has previously been quite straightforward, where cerebellar inhibitory cells arise from the ventricular zone and the excitatory cells arise from the rhombic lip.

Strengths:

One of the strengths of the manuscript is that the authors use a wide range of technical approaches, including transgenic mice that allow them to disentangle the influence of distinct developmental organizers such at ATOH.<br /> Their finding of a novel germinal zone and a novel population of CN neurons is important for developmental neuroscientists, cerebellar neuroscientists.

Weaknesses:

One important question raised by this work is what do these newly identified cells eventually become in the adult cerebellum. Are they excitatory or inhibitory? Do they correspond to a novel cell type or perhaps one of the cell classes that have been recently identified in the cerebellum (e.g. Fujita et al., eLife, 2020)? Understanding this would significantly bolster the impact of this manuscript.

The major weakness of the manuscript is that it is written for a very specialized reader who has a strong background in cerebellar development, making it hard to read for eLife's general audience. It's challenging to follow the logic of some of the experiments as well as to contextualize these findings in the field of cerebellar development.

Reviewer #1 (Public review):

Summary:

Fats and lipids serve many important roles in cancers, including serving as important fuels for energy metabolism in cancer cells by being oxidized in the mitochondria. The process of fatty acid oxidation is initiated by the enzyme carnitine palmitoyltransferase 1A (CPT1A), and the function and targetability of CPT1A in cancer metabolism and biology has been heavily investigated. This includes studies that have found important roles for CPT1A in colorectal cancer growth and metastasis.

In this study, Chen and colleagues use analysis of patient samples and functional interrogation in animal models to examine the role CPT1A plays in colorectal cancer (CRC). The authors find that CPT1A expression is decreased in CRC compared to paired healthy tissue and that lower expression correlates with decreased patient survival over time, suggesting that CPT1A may suppress tumor progression. To functionally interrogate this hypothesis, the authors both use CRISPR to knockout CPT1A in a CRC cell line that expresses CPT1A, and overexpress CPT1A in a CRC cell line with low expression. In both systems, increased CPT1A expression decreased cell survival and DNA repair in response to radiation in culture. Further, in xenograft models CPT1A decreased tumor growth basally and radiotherapy could further decrease tumor growth in CPT1A expressing tumors. As CRC is often treated with radiotherapy, the authors argue this radiosensitization driven by CPT1A could explain why CPT1A expression correlates with increased patient survival.

Lastly, Chen and colleagues sought to understand why CPT1A suppresses CRC tumor growth and sensitizes the tumors to radiotherapy in culture. Antioxidant capacity of cells can increase cell survival, so the authors examine antioxidant gene expression and levels in CPT1A expressing and non-expressing cells. CPT1A expression suppresses expression of antioxidant metabolism genes and lowers levels of antioxidants. Antioxidant metabolism genes can be regulated by the FOXM1 transcription factor, and the authors find that CPT1A expression regulates FOXM1 levels and that antioxidant gene expression can be partially rescued in CPT1A expressing CRC cells. This leads the authors to propose the following model: CPT1A expression downregulates FOXM1 (via some yet undescribed mechanism) which then leads to decreased antioxidant capacity in CRC cells and thus suppressing tumor progression and increasing radiosensitivity. This is an interesting model that could explain suppression of CPT1A expression in CRC, but key tenets of the model are untested and speculative.

Strengths:

• Analysis of CPT1A in paired CRC tumors and non-tumor tissue using multiple modalities combined with analysis of independent datasets rigorously show that CPT1A is downregulated in CRC tumors at the RNA and protein level.<br /> • The authors use paired cell line model systems where CPT1A is both knocked out and overexpressed in cells lines that endogenously express or repress CPT1A respectively. These complementary model systems increase the rigor of the study.<br /> • The finding that a metabolic enzyme generally thought to support tumor energetics actually is a tumor suppressor in some settings is theoretically quite interesting.

Weaknesses:

• The authors propose that CPT1A expression modulates antioxidant capacity in cells by suppressing FOXM1 and that this pathway alters CRC growth and radiotherapy response. However, key aspects of this model are not tested. The authors do not show that FOXM1 contributes to regulation of antioxidant levels in CRC cells and tumors or if FOXM1 suppression is key to inhibition of CRC tumor growth and radiosensitization by CPT1A. Thus, the model the authors propose is speculative and not supported by the existing data.<br /> • The authors propose two mechanisms by which CPT1A expression triggers radiosensitization: decreasing DNA repair capacity (Fig. 3) and decreasing antioxidant capacity (Fig. 5). However, while CPT1A expression does alter these capacities in CRC cells, neither is functionally tested to determine if altered DNA repair or antioxidant capacity (or both) are the reason why CRC cells are more sensitive to radiotherapy or are delayed in causing tumors in vivo. Thus, this aspect of the proposed model is also speculative.<br /> • The authors find that CPT1A affects radiosensitization in cell culture and assess this in vivo. In vivo, CPT1A expression slows tumor growth even in the absence of radiotherapy, and radiotherapy only proportionally decreases tumor growth to the same extent as it does in CPT1A non-expressing CRC tumors. The authors propose from this data that CPT1A expression also sensitizes tumors to radiotherapy in vivo. However, it is unclear that CPT1A expression causes radiosensitization in vivo or if CPT1A expression acts as independent tumor suppressor to which radiotherapy has an additive effect. Additional experiments would be necessary to differentiate between these possibilities.<br /> • The authors propose in Figure 3 that DNA repair capacity is inhibited in CRC cells by CPT1A expression. However, the gH2AX immunoblots performed in Figure 3H-I that measure DNA repair kinetics are not convincing that CPT1A expression impairs DNA repair kinetics. Separate blots are shown for CPT1A expressing and non-expressing cell lines, not allowing for rigorous comparison of gH2AX levels and resolution as CPT1A expression is modulated.

Reviewer #1 (Public review):

The paper by Chen et al describes the role of neuronal themo-TRPV3 channels in the firing of cortical neurons at a fever temperature range. The authors began by demonstrating that exposure to infrared light increasing ambient temperature causes body temperature to rise to a fever level above 38{degree sign}C. Subsequently, they showed that at the fever temperature of 39{degree sign}C, the spike threshold (ST) increased in both populations (P12-14 and P7-8) of cortical excitatory pyramidal neurons (PNs). However, the spike number only decreased in P7-8 PNs, while it remained stable in P12-14 PNs at 39 degrees centigrade. In addition, the fever temperature also reduced the late peak postsynaptic potential (PSP) in P12-14 PNs. The authors further characterized the firing properties of cortical P12-14 PNs, identifying two types: STAY PNs that retained spiking at 30{degree sign}C, 36{degree sign}C, and 39{degree sign}C, and STOP PNs that stopped spiking upon temperature change. They further extended their analysis and characterization to striatal medium spiny neurons (MSNs) and found that STAY MSNs and PNs shared the same ST temperature sensitivity. Using small molecule tools, they further identified that themo-TRPV3 currents in cortical PNs increased in response to temperature elevation, but not TRPV4 currents. The authors concluded that during fever, neuronal firing stability is largely maintained by sensory STAY PNs and MSNs that express functional TRPV3 channels. Overall, this study is well designed and executed with substantial controls, some interesting findings, and quality of data. Here are some specific comments:

(1) Could the authors discuss, or is there any evidence of, changes in TRPV3 expression levels in the brain during the postnatal 1-4 week age range in mice?

(2) Are there any differential differences in TRPV3 expression patterns that could explain the different firing properties in response to fever temperature between the STAY- and STOP neurons?

(3) TRPV3 and TRPV4 can co-assemble to form heterotetrameric channels with distinct functional properties. Do STOP neurons exhibit any firing behaviors that could be attributed to the variable TRPV3/4 assembly ratio?

(4) In Figure 7, have the authors observed an increase of TRPV3 currents in MSNs in response to temperature elevation?

(5) Is there any evidence of a relationship between TRPV3 expression levels in D2+ MSNs and degeneration of dopamine-producing neurons?

(6) Does fever range temperature alter the expressions of other neuronal Kv channels known to regulate the firing threshold?

Reviewer #1 (Public review):

In this study, Ma et al. aimed to determine previously uncharacterized contributions of tissue autofluorescence, detector afterpulse, and background noise on fluorescence lifetime measurement interpretations. They introduce a computational framework they named "Fluorescence Lifetime Simulation for Biological Applications (FLiSimBA)" to model experimental limitations in Fluorescence Lifetime Imaging Microscopy (FLIM) and determine parameters for achieving multiplexed imaging of dynamic biosensors using lifetime and intensity. By quantitatively defining sensor photon effects on signal-to-noise in either fitting or averaging methods of determining lifetime, the authors contradict any claims of FLIM sensor expression insensitivity to fluorescence lifetime and highlight how these artifacts occur differently depending on the analysis method. Finally, the authors quantify how statistically meaningful experiments using multiplexed imaging could be achieved.

A major strength of the study is the effort to present results in a clear and understandable way given that most researchers do not think about these factors on a day-to-day basis. The model code is available and written in Matlab, which should make it readily accessible, although a version in other common languages such as Python might help with dissemination in the community. One potential weakness is that the model uses parameters that are determined in a specific way by the authors, and it is not clear how vastly other biological tissue and microscope setups may differ from the values used by the authors.

Overall, the authors achieved their aims of demonstrating how common factors (autofluorescence, background, and sensor expression) will affect lifetime measurements and they present a clear strategy for understanding how sensor expression may confound results if not properly considered. This work should bring to awareness an issue that new users of lifetime biosensors may not be aware of and that experts, while aware, have not quantitatively determined the conditions where these issues arise. This work will also point to future directions for improving experiments using fluorescence lifetime biosensors and the development of new sensors with more favorable properties.

Reviewer #1 (Public review):

Summary:

Diarrheal diseases represent an important public health issue. Among the many pathogens that contribute to this problem, Salmonella enterica serovar Typhimurium is an important one. Due to the rise in antimicrobial resistance and the problems associated with widespread antibiotic use, the discovery and development of new strategies to combat bacterial infections is urgently needed. The microbiome field is constantly providing us with various health-related properties elicited by the commensals that inhabit their mammalian hosts. Harnessing the potential of these commensals for knowledge about host-microbe interactions as well as useful properties with therapeutic implications will likely remain a fruitful field for decades to come. In this manuscript, Wang et al use various methods, encompassing classic microbiology, genomics, chemical biology, and immunology, to identify a potent probiotic strain that protects nematode and murine hosts from S. enterica infection. Additionally, authors identify gut metabolites that are correlated with protection, and show that a single metabolite can recapitulate the effects of probiotic administration.

Strengths:

The utilization of varied methods by the authors, together with the impressive amount of data generated, to support the claims and conclusions made in the manuscript is a major strength of the work. Also, the ability to move beyond simple identification of the active probiotic, also identifying compounds that are at least partially responsible for the protective effects, is commendable.

Weaknesses:

Although there is a sizeable amount of data reported in the manuscript, there seems to be a chronic issue of lack of details of how some experiments were performed. This is particularly true in the figure legends, which for the most part lack enough details to allow comprehension without constant return to the text. Additionally, 2 figures are missing. Figure 6 is a repetition of Figure 5, and Figure S4 is an identical replicate of Figure S3.

Reviewer #1 (Public review):

In this manuscript, the authors recorded cerebellar unipolar brush cells (UBCs) in acute brain slices. They confirmed that mossy fiber (MF) inputs generate a continuum of UBC responses. Using systematic and physiological trains of MF electrical stimulation, they demonstrated that MF inputs either increased or decreased UBC firing rates (UBC ON vs. OFF) or induced complex, long-lasting modulation of their discharges. The MF influence on UBC firing was directly associated with a specific combination of metabotropic glutamate receptors, mGluR2/3 (inhibitory) and mGluR1 (excitatory). Ultimately, the amount and ratio of these two receptors controlled the time course of the effect, yielding specific temporal transformations such as phase shifts.

Overall, the topic is compelling, as it broadens our understanding of temporal processing in the cerebellar cortex. The experiments are well-executed and properly analyzed.

Strengths:

(1) A wide range of MF stimulation patterns was explored, including burst duration and frequency dependency, which could serve as a valuable foundation for explicit modeling of temporal transformations in the granule cell layer.

(2) The pharmacological blockade of mGluR2/3, mGluR1, AMPA, and NMDA receptors helped identify the specific roles of these glutamate receptors.

(3) The experiments convincingly demonstrate the key role of mGluR1 receptors in temporal information processing by UBCs.

Weaknesses:

(1) This study is largely descriptive and represents only a modest incremental advance from the previous work (Guo et al., Nat. Commun., 2021).

(2) The MF activity used to mimic natural stimulation was previously collected in primates, while the recordings were conducted in mice.

(3) Inhibition was blocked throughout the study, reducing its physiological relevance.

Reviewer #1 (Public review):

Summary:

The authors examined whether aberrantly projecting retinal ganglion cells in albino mice innervate a separate population of thalamocortical neurons, as would be predicted for Hebbian learning rules. The authors find support for this hypothesis in correlated light and electron microscopy (CLEM) reconstructions of retinal ganglion cell axons and thalamocortical neurons. In a second line of investigation, the authors ask the same question about retinal ganglion cell innervation of local inhibitory interneurons of the mouse LGN. The authors conclude that these connections are less specific.

Strengths:

The authors make good use of CLEM to test a circuit-level hypothesis, and they find an interesting difference in RGC synaptic innervation patterns for thalamocortical neurons vs. local interneurons.

Weaknesses:

The conclusions about the local interneuron innervation are a little more difficult to interpret. One would expect to only capture a small part of the local interneuron dendritic field, as compared to the smaller thalamocortical neurons, right? Doesn't that imply that finding some evidence of promiscuous connectivity means that other dendrites that were not observed probably connect to many different RGCs?

Reviewer #1 (Public review):

Summary:

In this study, the authors use ChEC-seq, an MNase based method to map yeast RNA pol II. Part of the reasoning for this study is that earlier biochemical work suggested pol II initiation and termination should involve slow steps at the UAS/promoter and termination regions that are not well visualized by formaldehyde-based ChIP methods. Here the authors find that pol II ChIP and ChEC give complementary patterns. Pol II ChIP signals are strongest in the coding region (where ChIP signal correlates well with transcription (rho = 0.62)). In contrast, pol II ChEC signals are strongest at promoters (rho = 0.52) and terminator regions. Weaker upstream ChEC signals are also observed at the STM class genes where biochemical studies have suggested a form of Pol (and maybe other general factors) is recruited to UAS sites. ChEC of TFIIA and TFIIE give promoter-specific ChEC signals as expected. Extending this work to elongation factors Ctk1 and Spt5 unexpectedly give strong signals near the PIC location and little signals over the coding region. This, and mapping CTD S2 and S5 phosphorylation by ChEC suggests to me that, for some reason, ChEC isn't optimal for detecting components of the elongation complex over coding regions.

Examples are also presented where perturbations of transcription can be measured by ChEC. Modeling studies are shown where adjustment of kinetic parameters agree well with ChEC data and that these models can be used to estimate which steps in transcription are affected by various perturbations. However, no tests were performed to see if the predictions could be validated by other means. Finally, the role of nuclear pore binding by Gcn4 is explored, although the effects are small and this proposal should be explored more completely in future studies. Overall, the authors show that pol II ChEC is a valuable and complementary method for investigating transcription mechanisms and slow steps at the initiation and termination regions.

Reviewer #1 (Public Review):

Summary:

The main goal of the paper was to identify signals that activate FLP-1 release from AIY neurons in response to H2O2, previously shown by the authors to be an important oxidative stress response in the worm.

Strengths:

This study builds upon the authors' previous work (Jia and Sieburth 2021) by further elucidating the gut-derived signaling mechanisms that coordinate the organism-wide antioxidant stress response in C. elegans.

By detailing how environmental cues like oxidative stress are transduced into gut-derived peptidergic signals, this study represents a valuable advancement in understanding the integrated physiological responses governed by the gut-brain axis.

This work provides valuable mechanistic insights into the gut-specific regulation of the FLP-2 peptide signal.

Weaknesses:

Although the authors identify intestinal FLP-2 as the endocrine signal important for regulating the secretion of the neuronal antioxidant neuropeptide, FLP-1, there is no effort made to identify how FLP-2 levels regulate FLP-1 secretion or identify whether this regulation is occurring directly through the AIY neuron or indirectly. This is brought up in the discussion, but identifying a target for FLP-2 in this pathway seems like a crucial missing piece of information in characterizing this pathway.

Comments on revised version:

In general I think the revision is improved and addresses my comments. It is unfortunate though that the authors did not address my main question (did they test the frpr-18 mutant, and if not, why?). The fact that there are other potentially relevant receptors which bind to some FLP-2 peptides with low affinity is not really a justification not to test the known high-affinity receptor (i.e. FRPR-18).

Reviewer #1 (Public review):

Summary:

The manuscript by Choi and co-authors presents "P3 editing", which leverages dual-component guide RNAs (gRNA) to induce protein-protein proximity. They explore three strategies for leveraging prime-editing gRNA (pegRNA) as a dimerization module to create a molecular proximity sensor that drives genome editing, splitting a pegRNA into two parts (sgRNA and petRNA), inserting self-splicing ribozymes within pegRNA, and dividing pegRNA at the crRNA junction. Among these, splitting at the crRNA junction proved the most promising, achieving significant editing efficiency. They further demonstrated the ability to control genome editing via protein-protein interactions and small molecule inducers by designing RNA-based systems that form active gRNA complexes. This approach was also adaptable to other genome editing methods like base editing and ADAR-based RNA editing.

Strengths:

The study demonstrates significant advancements in leveraging guide RNA (gRNA) as a dimerization module for genome editing, showcasing its high specificity and versatility. By investigating three distinct strategies-splitting pegRNA into sgRNA and petRNA, inserting self-splicing ribozymes within the pegRNA, and dividing the pegRNA at the repeat junction-the researchers present a comprehensive approach to achieving molecular proximity and reconstituting function. Among these methods, splitting the pegRNA at the repeat junction emerged as the most promising, achieving editing efficiencies up to 76% of the control, highlighting its potential for further development in CRISPR-Cas9 systems. Additionally, the study extends genome editing control by linking protein-protein interactions to RNA-mediated editing, using specific protein-RNA interaction pairs to regulate editing through engineered protein proximity. This innovative approach expands the toolkit for precision genome editing, demonstrating the feasibility of controlling genome editing with enhanced specificity and efficiency.

Weaknesses:

The initial experiments with splitting the pegRNA into sgRNA and petRNA showed low editing efficiency, less than 2%. Similarly, inserting self-splicing ribozymes within pegRNA was inefficient, achieving under 2% editing efficiency in all constructs tested, possibly hindered by the prime editing enzyme. The editing efficiency of the crRNA and petracrRNA split at the repeat junction varied, with the most promising configurations only reaching 76% of the control efficiency. The RNA-RNA duplex formation's inefficiency might be due to the lack of additional protein binding, leading to potential degradation outside the Cas9-gRNA complex. Extending the approach to control genome editing via protein-protein interactions introduced complexity, with a significant trade-off between efficiency and specificity, necessitating further optimization. The strategy combining RADARS and P3 editing to control genome editing with specific RNA expression events exhibited high background levels of non-specific editing, indicating the need for improved specificity and reduced leaky expression. Moreover, P3 editing efficiencies are exclusively quantified after transfecting DNA into HEK cells, a strategy that has resulted in past reproducibility concerns for other technologies. Overall, the various methods and combinations require further optimization to enhance efficiency and specificity, especially when integrating multiple synthetic modules.

Comments on revisions:

I think the authors have successfully addressed the initial concerns. Their adaption of the main text and discussion makes the limitations of P3 editing much clearer.

Reviewer #1 (Public review):

This study is part of an ongoing effort to clarify the effects of cochlear neural degeneration (CND) on auditory processing in listeners with normal audiograms. This effort is important because ~10% of people who seek help for hearing difficulties have normal audiograms and current hearing healthcare has nothing to offer them.

The authors identify two shortcomings in previous work that they intend to fix. The first is a lack of cross-species studies that make direct comparisons between animal models in which CND can be confirmed and humans for which CND must be inferred indirectly. The second is the low sensitivity of purely perceptual measures to subtle changes in auditory processing. To fix these shortcomings, the authors measure envelope following responses (EFRs) in gerbils and humans using the same sounds, while also performing histological analysis of the gerbil cochleae, and testing speech perception while measuring pupil size in the humans.

The study begins with a comprehensive assessment of the hearing status of the human listeners. The only differences found between the young adult (YA) and middle-aged (MA) groups are in thresholds at frequencies > 10 kHz and DPOAE amplitudes at frequencies > 5 kHz. The authors then present the EFR results, first for the humans and then for the gerbils, showing that amplitudes decrease more rapidly with increasing envelope frequency for MA than for YA in both species. The histological analysis of the gerbil cochleae shows that there were, on average, 20% fewer IHC-AN synapses at the 3 kHz place in MA relative to YA, and the number of synapses per IHC was correlated with the EFR amplitude at 1024 Hz.

The study then returns to the humans to report the results of the speech perception tests and pupillometry. The correct understanding of keywords decreased more rapidly with decreasing SNR in MA than in YA, with a noticeable difference at 0 dB, while pupillary slope (a proxy for listening effort) increased more rapidly with decreasing SNR for MA than for YA, with the largest differences at SNRs between 5 and 15 dB. Finally, the authors report that a linear combination of audiometric threshold, EFR amplitude at 1024 Hz, and a few measures of pupillary slope is predictive of speech perception at 0 dB SNR.

I only have two questions/concerns about the specific methodologies used:

(1) Synapse counts were made only at the 3 kHz place on the cochlea. However, the EFR sounds were presented at 85 dB SPL, which means that a rather large section of the cochlea will actually be excited. Do we know how much of the EFR actually reflects AN fibers coming from the 3 kHz place? And are we sure that this is the same for gerbils and humans given the differences in cochlear geometry, head size, etc.?

(2) Unless I misunderstood, the predictive power of the final model was not tested on held-out data. The standard way to fit and test such a model would be to split the data into two segments, one for training and hyperparameter optimization, and one for testing. But it seems that the only split was for training and hyperparameter optimization.

While I find the study to be generally well executed, I am left wondering what to make of it all. The purpose of the study with respect to fixing previous methodological shortcomings was clear, but exactly how fixing these shortcomings has allowed us to advance is not. I think we can be more confident than before that EFR amplitude is sensitive to CND, and we now know that measures of listening effort may also be sensitive to CND. But where is this leading us?

I think what this line of work is eventually aiming for is to develop a clinical tool that can be used to infer someone's CND profile. That seems like a worthwhile goal but getting there will require going beyond exploratory association studies. I think we're ready to start being explicit about what properties a CND inference tool would need to be practically useful. I have no idea whether the associations reported in this study are encouraging or not because I have no idea what level of inferential power is ultimately required.

That brings me to my final comment: there is an inappropriate emphasis on statistical significance. The sample size was chosen arbitrarily. What if the sample had been half the size? Then few, if any, of the observed effects would have been significant. What if the sample had been twice the size? Then many more of the observed effects would have been significant (particularly for the pupillometry). I hope that future studies will follow a more principled approach in which relevant effect sizes are pre-specified (ideally as the strength of association that would be practically useful) and sample sizes are determined accordingly.

So, in summary, I think this study is a valuable but limited advance. The results increase my confidence that non-invasive measures can be used to infer underlying CND, but I am unsure how much closer we are to anything that is practically useful.

Reviewer #1 (Public review):

Summary:

The authors test the "OHC-fluid-pump" hypothesis by assaying the rates of kainic acid dispersal both in quiet and in cochleae stimulated by sounds of different levels and spectral content. The main result is that sound (and thus, presumably, OHC contractions and expansions) result in faster transport along the duct. OHC involvement is corroborated using salicylate, which yielded results similar to silence. Especially interesting is the fact that some stimuli (e.g., tones) seem to provide better/faster pumping than others (e.g., noise), ostensibly due to the phase profile of the resulting cochlear traveling-wave response.

Strengths:

The experiments appear well controlled and the results are novel and interesting. Some elegant cochlear modeling that includes coupling between the organ of Corti and the surrounding fluid as well as advective flow supports the proposed mechanism.

The current limitations and future directions of the study, including possible experimental tests, extensions of the modeling work, and practical applications to drug delivery, are thoughtfully discussed.

Weaknesses:

Although the authors provide compelling evidence that OHC motility can usefully pump fluid, their claim (last sentence of the Abstract) that wideband OHC motility (i.e., motility in the "tail" region of the traveling wave) evolved for the purposes of circulating fluid---rather then emerging, say, as a happy by-product of OHC motility that evolved for other reasons---seems too strong.

Reviewer #1 (Public review):

Summary:

In this paper, Thomas et al. set out to study seasonal brain gene expression changes in the Eurasian common shrew. This mammalian species is unusual in that it does not hibernate or migrate but instead stays active all winter while shrinking and then regrowing its brain and other organs. The authors previously examined gene expression changes in two brain regions and the liver. Here, they added data from the hypothalamus, a brain region involved in the regulation of metabolism and homeostasis. The specific goals were to identify genes and gene groups that change expression with the seasons and to identify genes with unusual expression compared to other mammalian species. The reason for this second goal is that genes that change with the season could be due to plastic gene regulation, where the organism simply reacts to environmental change using processes available to all mammals. Such changes are not necessarily indicative of adaptation in the shrew. However, if the same genes are also expression outliers compared to other species that do not show this overwintering strategy, it is more likely that they reflect adaptive changes that contribute to the shrew's unique traits.

The authors succeeded in implementing their experimental design and identified significant genes in each of their specific goals. There was an overlap between these gene lists. The authors provide extensive discussion of the genes they found.

The scope of this paper is quite narrow, as it adds gene expression data for only one additional tissue compared to the authors' previous work in a 2023 preprint. The two papers even use the same animals, which had been collected for that earlier work. As a consequence, the current paper is limited in the results it can present. This is somewhat compensated by an expansive interpretation of the results in the discussion section, but I felt that much of this was too speculative. More importantly, there are several limitations to the design, making it hard to draw stronger conclusions from the data. The main contribution of this work lies in the generated data and the formulation of hypotheses to be tested by future work.

Strengths:

The unique biological model system under study is fascinating. The data were collected in a technically sound manner, and the analyses were done well. The paper is overall very clear, well-written, and easy to follow. It does a thorough job of exploring patterns and enrichments in the various gene sets that are identified.

I specifically applaud the authors for doing a functional follow-up experiment on one of the differentially expressed genes (BCL2L1), even if the results did not support the hypothesis. It is important to report experiments like this and it is terrific to see it done here.

Weaknesses:

While the paper successfully identifies differentially expressed seasonal genes, the real question is (as explained by the authors) whether these are evolved adaptations in the shrews or whether they reflect plastic changes that also exist in other species. This question was the motivation for the inter-species analyses in the paper, but in my view, these cannot rigorously address this question. Presumably, the data from the other species were not collected in comparable environments as those experienced by the shrews studied here. Instead, they likely (it is not specified, and might not be knowable for the public data) reflect baseline gene expression. To see why this is problematic, consider this analogy: if we were to compare gene expression in the immune system of an individual undergoing an acute infection to other, uninfected individuals, we would see many, strong expression differences. However, it would not be appropriate to claim that the infected individual has unique features - the relevant physiological changes are simply not triggered in the other individuals. The same applies here: it is hard to draw conclusions from seasonal expression data in the shrews to non-seasonal data in the other species, as shrew outlier genes might still reflect physiological changes that weren't active in the other species.

There is no solution for this design flaw given the public data available to the authors except for creating matched data in the other species, which is of course not feasible. The authors should acknowledge and discuss this shortcoming in the paper.

Related to the point above: in the section "Evolutionary Divergence in Expression" it is not clear which of the shrew samples were used. Was it all of them, or only those from winter, fall, etc? One might expect different results depending on this. E.g., there could be fewer genes with inferred adaptive change when using only summer samples. The authors should specify which samples were included in these analyses, and, if all samples were used, conduct a robustness analysis to see which of their detected genes survive the exclusion of certain time points.

In the same section, were there also genes with lower shrew expression? None are mentioned in the text, so did the authors not test for this direction, or did they test and there were no significant hits?

The Discussion is too long and detailed, given that it can ultimately only speculate about what the various expression changes might mean. Many of the specific points made (e.g. about the blood-brain-barrier being more permissive to sensing metabolic state, about cross-organ communication, the paragraphs on single, specific genes) are a stretch based on the available data. Illustrating this point, the one follow-up experiment the authors did (on BCL2L1) did not give the expected result. I really applaud the authors for having done this experiment, which goes beyond typical studies in this space. At the same time, its result highlights the dangers of reading too much into differential expression analyses.

There is no test of whether the five genes observed in both analyses (seasonal change and inter-species) exceed the number expected by chance. When two gene sets are drawn at random, some overlap is expected randomly. The expected overlap can be computed by repeated draws of pairs of random sets of the same size as seen in real data and by noting the overlap between the random pairs. If this random distribution often includes sets of five genes, this weakens the conclusions that can be drawn from the genes observed in the real data.

for - BEing journey - example - demonstrates system 1 vs system 2 thinking - example - unconscious bias - example - symbolic incompleteness

Reviewer #1 (Public review):

Summary:

This paper reports an interesting and clever task that allows the joint measurement of both perceptual judgments and confidence (or subjective motion strength) in real/continuous time. The task is used together with a social condition to identify the (incidental, task-irrelevant) impact of another player on decision-making and confidence.

Strengths:

The innovation on the task alone is likely to be impactful for the field, extending recent continuous report (CPR) tasks to examine other aspects of perceptual decision-making and allowing more naturalistic readouts. One interesting and novel finding is the observation of dyadic convergence of confidence estimates even when the partner is incidental to the task performance, and that dyads tend to be more risk-seeking (indicating greater confidence) than when playing solo. The paper is well-written and clear.

Weaknesses:

(1) One concern with the novel task is whether confidence is disambiguated from a tracking of stimulus strength or coherence. The subjects' task is to track motion direction and use the eccentricity of the joystick to control the arc of a catcher - thus implementing a real-time sensitivity to risk (peri-decision wagering). The variable-width catcher has been used to good effect in other confidence/uncertainty tasks involving learning the spread of targets (the Nassar papers). But in the context of an RDK task, one simple strategy here is to map eccentricity directly to (subjective) motion coherence - such that the joystick position at any moment in time is a vector with motion direction and strength. This would still be an interesting task - but could be solved without invoking metacognition or the need to estimate confidence in one's motion direction decision (the analyses in Supplementary Figure 2 are nice in showing a dissociation from (objective) coherence, such that even within a coherence level, changes in eccentricity scale with direction precision - but this does not get around the potential conflation of confidence with fluctuations in motion energy).

In other words, in this deflationary framing, what the subjects might be doing is tracking two features of the world - motion strength and direction. This possibility needs to be ruled out if the authors want to claim a mapping between eccentricity and decision confidence (for instance, an ideal observer model of the task that set eccentricity proportional to instantaneous motion strength presumably would also sensibly accrue reward targets, without the need to compute confidence in the direction response). This would be straightforward to simulate and would establish a baseline model against which to compare claims about confidence (eg when evaluating additional social modulations). More generally it casts doubt on claims such as the one on line 210 that eccentricity was "chosen freely via metacognitive assessment of the current perceptual process, [and] can be treated as a proxy measure of subjective perceptual confidence."

One route to doing this would be to ask whether the eccentricity reports show statistical signatures of confidence that have been established for more classical punctate tasks. Here a key move has been to identify qualitative patterns in the frame of reference of choice accuracy - with confidence scaling positively with stimulus strength for correct decisions, and negatively with stimulus strength for incorrect decisions (the so-called X-pattern, for instance Sanders et al. 2016 Neuron https://pubmed.ncbi.nlm.nih.gov/27151640/).

(2) I was surprised not to see more analysis of the continuous report data as a function of (lagged) task variables. Some of this analysis is shown in Figure 2b relative to an (objective) direction change, and also in the cross-correlation plots in Supplementary Figure 1d. But to fully characterise the task behaviour it also seems important to ask how and whether fluctuations in motion energy (assuming that the RDK frames were recorded) during a steady state phase are affecting continuous reporting of direction and eccentricity, prior to asking how social information is incorporated into subjects' behaviour.

Minor points:

(1) Lines 295-298, isn't it guaranteed to observe these three behavioural patterns (both participants improving, both getting worse, only one improving while the other gets worse) even in random data?

(2) Lines 703-707, it wasn't clear what the AUC values referred to here (also in Figure 3) - what are the distributions that are being compared? I think part of the confusion here comes from AUC being mentioned earlier in the paper as a measure of metacognitive sensitivity (correct vs. incorrect trial distributions), whereas my impression here is that here AUC is being used to investigate differences in variables (eg confidence) between experimental conditions.

(3) Could the findings of the worse solo player benefitting more than the better solo player (Figure 4c) be partly due to a compressive ceiling effect - eg there is less room to move up the psychometric function for the higher-scoring player?

Reviewer #1 (Public review):

Summary:

This manuscript explores the transcriptional landscape of high-grade serous ovarian cancer (HGSOC) using consensus-independent component analysis (c-ICA) to identify transcriptional components (TCs) associated with patient outcomes. The study analyzes 678 HGSOC transcriptomes, supplemented with 447 transcriptomes from other ovarian cancer types and noncancerous tissues. By identifying 374 TCs, the authors aim to uncover subtle transcriptional patterns that could serve as novel drug targets. Notably, a transcriptional component linked to synaptic signaling was associated with shorter overall survival (OS) in patients, suggesting a potential role for neuronal interactions in the tumor microenvironment. Given notable weaknesses like lack of validation cohort or validation using another platform (other than the 11 samples with ST), the data is considered highly descriptive and preliminary.

Strengths:

(1) Innovative Methodology:<br /> The use of c-ICA to dissect bulk transcriptomes into independent components is a novel approach that allows for the identification of subtle transcriptional patterns that may be overshadowed in traditional analyses.

(2) Comprehensive Data Integration:<br /> The study integrates a large dataset from multiple public repositories, enhancing the robustness of the findings. The inclusion of spatially resolved transcriptomes adds a valuable dimension to the analysis.

(3) Clinical Relevance:<br /> The identification of a synaptic signaling-related TC associated with poor prognosis highlights a potential new avenue for therapeutic intervention, emphasizing the role of the tumor microenvironment in cancer progression.

Weaknesses:

(1) Mechanistic Insights:<br /> While the study identifies TCs associated with survival, it provides limited mechanistic insights into how these components influence cancer progression. Further experimental validation is necessary to elucidate the underlying biological processes.

(2) Generalizability:<br /> The findings are primarily based on transcriptomic data from HGSOC. It remains unclear how these results apply to other subtypes of ovarian cancer or different cancer types.

(3) Innovative Methodology:<br /> Requires more validation using different platforms (IHC) to validate the performance of this bulk-derived data. Also, the lack of control over data quality is a concern.

(4) Clinical Application:<br /> Although the study suggests potential drug targets, the translation of these findings into clinical practice is not addressed. Probably given the lack of some QA/QC procedures it'll be hard to translate these results. Future studies should focus on validating these targets in clinical settings.

Reviewer #1 (Public review):

Summary:

In the present study, Chen et al. investigate the role of Endophilin A1 in regulating GABAergic synapse formation and function. To this end, the authors use constitutive or conditional knockout of Endophilin A1 (EEN1) to assess the consequences on GABAergic synapse composition and function, as well as the outcome for PTZ-induced seizure susceptibility. The authors show that EEN1 KO mice show a higher susceptibility to PTZ-induced seizures, accompanied by a reduction in the GABAergic synaptic scaffolding protein gephyrin as well as specific GABAAR subunits and eIPSCs. The authors then investigate the underlying mechanisms, demonstrating that Endophilin A1 binds directly to gephyrin and GABAAR subunits, and identifying the subdomains of Endophilin A1 that contribute to this effect. Overall, the authors state that their study places Endophilin A1 as a new regulator of GABAergic synapse function.

Strengths:

Overall, the topic of this manuscript is very timely, since there has been substantial recent interest in describing the mechanisms governing inhibitory synaptic transmission at GABAergic synapses. The study will therefore be of interest to a wide audience of neuroscientists studying synaptic transmission and its role in disease. The manuscript is well-written and contains a substantial quantity of data.

Weaknesses:

A number of questions remain to be answered in order to be able to fully evaluate the quality and conclusions of the study. In particular, a key concern throughout the manuscript regards the way that the number of samples for statistical analysis is defined, which may affect the validity of the data analysed. Addressing this weakness will be essential to providing conclusive results that support the authors' claims.

Reviewer #1 (Public review):

Summary:

Dendrotweaks provides its users with a solid tool to implement, visualize, tune, validate, understand, and reduce single-neuron models that incorporate complex dendritic arbors with differential distribution of biophysical mechanisms. The visualization of dendritic segments and biophysical mechanisms therein provide users with an intuitive way to understand and appreciate dendritic physiology.

Strengths:

(1) The visualization tools are simplified, elegant, and intuitive.

(2) The ability to build single-neuron models using simple and intuitive interfaces.

(3) The ability to validate models with different measurements.

(4) The ability to systematically and progressively reduce morphologically-realistic neuronal models.

Weaknesses:

(1) Inability to account for neuron-to-neuron variability in structural, biophysical, and physiological properties in the model-building and validation processes.

(2) Inability to account for the many-to-many mapping between ion channels and physiological outcomes. Reliance on hand-tuning provides a single biased model that does not respect pronounced neuron-to-neuron variability observed in electrophysiological measurements.

(3) Lack of a demonstration on how to connect reduced models into a network within the toolbox.

(4) Lack of a set of tutorials, which is common across many "Tools and Resources" papers, that would be helpful in users getting acquainted with the toolbox.

Reviewer #1 (Public review):

Summary:

This manuscript uses a well-validated behavioral estimation task to investigate the degree to which optimistic belief updating was attenuated during the 2020 global pandemic. Online participants recruited during and outside of the pandemic estimated how likely different negative life events were to happen to them in the future and were given statistics about these events happening. Belief updating (measured as the degree to which estimations changed after viewing the statistics) was less optimistically biased during the pandemic (compared to outside of it). This resulted from reduced updating from "good news" (better than expected information). Computational models were used to try to unpack how statistics were integrated and used to revise beliefs. Two families of models were compared - an RL set of models where "estimation errors" (analogous to prediction errors in classic RL models) predict belief change and a Bayesian set of models where an implied likelihood ratio was calculated (derived from participants estimations of their own risk and estimation of the base rate risk) and used to predict belief change. The authors found evidence that the former set of models accounted for updating better outside of the pandemic, but the latter accounted for updating during the pandemic. In addition, the RL model provides evidence that learning was asymmetrically positively biased outside of the pandemic but symmetric during it (as a result of reduced learning rates from good news estimation errors).

Strengths:

Understanding whether biases in learning are fixed modes of information processing or flexible and adapt in response to environmental shocks (like a global pandemic or economic recession) is an important area of research relevant to a wide range of fields, including cognitive psychology, behavioral economics, and computational psychiatry. The study uses a well-validated task, and the authors conduct a power analysis to show that the sample sizes are appropriate. Furthermore, the authors test that their results hold in both a between-group analysis (the focus of the main paper) and a within-group analysis (mainly in the supplemental).

The finding that optimistic biases are reduced in response to acute stress, perceived threat, and depression has been shown before using this task both in the lab (social stress manipulation), in the real world (firefighters on duty), and clinical groups (patients with depression). However, the work does extend these findings here in important ways:

(1) Examining the effect of a new real-world adverse event (the pandemic).<br /> (2) The reduction in optimistic updating here arises due to reduced updating from positive information (previously, in the case of environmental threat, this reduction mainly arose from increased sensitivity to negative information).<br /> (3) Leveraging new RL-inspired computational approaches, demonstrating that the bias - and its attenuation - can be captured using trial-by-trial computational modeling with separate learning rates for positive and negative estimation errors.

Weaknesses:

Some interpretation and analysis (the computational modeling in particular) could be improved.

On the interpretation side, while the pandemic was an adverse experience and stressful for many people (including myself), the absence of any measures of stress/threat levels limits the conclusions one can draw. Past work that has used this task to examine belief updating in response to adverse environmental events took physiological (e.g., SCR, cortisol) and/or self-report (questionnaires) measures of mood. In SI Table 1, the authors possibly had some questionnaire measures along these lines, but this might be for the participants tested during the pandemic.

On the analysis side, it was unclear what the motivation was for the different sets of models tested. Both families of models test asymmetric vs symmetric learning (which is the main question here) and have similar parameters (scaling and asymmetry parameters) to quantify these different aspects of the learning process. Conceptually, the different behavioral patterns one could expect from the two families of models needed to be clarified. Do the "winning" models produce the main behavioral patterns in Figure 1, and are they in some way uniquely able to do so, for instance? How would updating look different for an optimistic RL learner versus an optimistic Bayesian RL learner? Would the asymmetry parameter in the former be correlated with the asymmetry parameter in the latter? Moreover, crucially, would one be able to reliably distinguish the models from one another under the model estimation and selection criteria that the authors have used here (presenting robust model recovery could help to show this)?

Reviewer #1 (Public review):

First, the authors confirm the up-regulation of the main genes involved in the three branches of the Unfolded Protein Response (UPR) system in diet-induced obese mice in AT, observations that have been extensively reported before. Not surprisingly, IRE1a inhibition with STF led to an amelioration of the obesity and insulin resistance of the animals. Moreover, non-alcoholic fatty liver disease was also improved by the treatment. More novel are their results in terms of thermogenesis and energy expenditure, where IRE1a seems to act via activation of brown AT. Finally, mice treated with STF exhibited significantly fewer metabolically active and M1-like macrophages in the AT compared to those under vehicle conditions. Overall, the authors conclude that targeting IRE1a has therapeutical potential for treating obesity and insulin resistance.

The study has some strengths, such as the detailed characterization of the effect of STF in different fat depots and a thorough analysis of macrophage populations. However, the lack of novelty in the findings somewhat limits the study´s impact on the field.

Reviewer #1 (Public review):

This study examined the interaction between two key cortical regions in the mouse brain involved in goal-directed movements, the rostral forelimb area (RFA) - considered a premotor region involved in movement planning, and the caudal forelimb area (CFA) - considered a primary motor region that more directly influences movement execution. The authors ask whether there exists a hierarchical interaction between these regions, as previously hypothesized, and focus on a specific definition of hierarchy - examining whether the neural activity in the premotor region exerts a larger functional influence on the activity in the primary motor area than vice versa. They examine this question using advanced experimental and analytical methods, including localized optogenetic manipulation of neural activity in either region while measuring both the neural activity in the other region and EMG signals from several muscles involved in the reaching movement, as well as simultaneous electrophysiology recordings from both regions in a separate cohort of animals.

The findings presented show that localized optogenetic manipulation of neural activity in either RFA or CFA resulted in similarly short-latency changes in the muscle output and in firing rate changes in the other region. However, perturbation of RFA led to a larger absolute change in the neural activity of CFA neurons. The authors interpret these findings as evidence for reciprocal, but asymmetrical, influence between the regions, suggesting some degree of hierarchy in which RFA has a greater effect on the neural activity in CFA. They go on to examine whether this asymmetry can also be observed in simultaneously recorded neural activity patterns from both regions. They use multiple advanced analysis methods that either identify latent components at the population level or measure the predictability of firing rates of single neurons in one region using firing rates of single neurons in the other region. Interestingly, the main finding across these analyses seems to be that both regions share highly similar components that capture a high degree of variability of the neural activity patterns in each region. Single units' activity from either region could be predicted to a similar degree from the activity of single units in the other region, without a clear division into a leading area and a lagging area, as one might expect to find in a simple hierarchical interaction. However, the authors find some evidence showing a slight bias towards leading activity in RFA. Using a two-region neural network model that is fit to the summed neural activity recorded in the different experiments and to the summed muscle output, the authors show that a network with constrained (balanced) weights between the regions can still output the observed measured activities and the observed asymmetrical effects of the optogenetic manipulations, by having different within-region local weights. These results put into question whether previous and current findings that demonstrate asymmetry in the output of regions can be interpreted as evidence for asymmetrical (and thus hierarchical) inputs between regions, emphasizing the challenges in studying interactions between any brain regions.

Strengths:

The experiments and analyses performed in this study are comprehensive and provide a detailed examination and comparison of neural activity recorded simultaneously using dense electrophysiology probes from two main motor regions that have been the focus of studies examining goal-directed movements. The findings showing reciprocal effects from each region to the other, similar short-latency modulation of muscle output by both regions, and similarity of neural activity patterns without a clear lead/lag interaction, are convincing and add to the growing body of evidence that highlight the complexity of the interactions between multiple regions in the motor system and go against a simple feedforward-like network and dynamics. The neural network model complements these findings and adds an important demonstration that the observed asymmetry can, in theory, also arise from differences in local recurrent connections and not necessarily from different input projections from one region to the other. This sheds an important light on the multiple factors that should be considered when studying the interaction between any two brain regions, with a specific emphasis on the role of local recurrent connections, that should be of interest to the general neuroscience community.

Weaknesses:

While the similarity of the activity patterns across regions and lack of a clear leading/lagging interaction are interesting observations that are mostly supported by the findings presented (however, see comment below for lack of clarity in CCA/PLS analyses), the main question posed by the authors - whether there exists an endogenous hierarchical interaction between RFA and CFA - seems to be left largely open. The authors note that there is currently no clear evidence of asymmetrical reciprocal influence between naturally occurring neural activity patterns of the two regions, as previous attempts have used non-natural electrical stimulation, lesions, or pharmacological inactivation. The use of acute optogenetic perturbations does not seem to be vastly different in that aspect, as it is a non-natural stimulation of inhibitory interneurons that abruptly perturbs the ongoing dynamics. Furthermore, the main finding that supports a hierarchical interaction is a difference in the absolute change of firing rates as a result of the optogenetic perturbation, a finding that is based on a small number of animals (N = 3 in each experimental group), and one which may be difficult to interpret. As the authors nicely demonstrate in their neural network model, the two regions may differ in the strength of local within-region inhibitory connections. Could this theoretically also lead to a difference in the effect of the artificial light stimulation of the inhibitory inter-neurons on the local population of excitatory projection neurons, driving an asymmetrical effect on the downstream region? Moreover, the manipulation was performed upon the beginning of the reaching movement, while the premotor region is often hypothesized to exert its main control during movement preparation, and thus possibly show greater modulation during that movement epoch. It is not clear if the observed difference in absolute change is dependent on the chosen time of optogenetic stimulation and if this effect is a general effect that will hold if the stimulation is delivered during different movement epochs, such as during movement preparation.

Another finding that is not clearly interpretable is in the analysis of the population activity using CCA and PLS. The authors show that shifting the activity of one region compared to the other, in an attempt to find the optimal leading/lagging interaction, does not affect the results of these analyses. Assuming the activities of both regions are better aligned at some unknown ground-truth lead/lag time, I would expect to see a peak somewhere in the range examined, as is nicely shown when running the same analyses on a single region's activity. If the activities are indeed aligned at zero, without a clear leading/lagging interaction, but the results remain similar when shifting the activities of one region compared to the other, the interpretation of these analyses is not clear.

Reviewer #1 (Public review):

This study investigates how ant group demographics influence nest structures and group behaviors of Camponotus fellah ants, a ground-dwelling carpenter ant species (found locally in Israel) that build subterranean nest structures. Using a quasi-2D cell filled with artificial sand, the authors perform two complementary sets of experiments to try to link group behavior and nest structure: first, the authors place a mated queen and several pupae into their cell and observe the structures that emerge both before and after the pupae eclose (i.e., "colony maturation" experiments); second, the authors create small groups (of 5,10, or 15 ants, each including a queen) within a narrow age range (i.e., "fixed demographic" experiments) to explore the dependence of age on construction. Some of the fixed demographic instantiations included a manually induced catastrophic collapse event; the authors then compared emergency repair behavior to natural nest creation. Finally, the authors introduce a modified logistic growth model to describe the time-dependent nest area. The modification introduces parameters that allow for age-dependent behavior, and the authors use their fixed demographic experiments to set these parameters, and then apply the model to interpret the behavior of the colony maturation experiments. The main results of this paper are that for natural nest construction, nest areas, and morphologies depend on the age demographics of ants in the experiments: younger ants create larger nests and angled tunnels, while older ants tend to dig less and build predominantly vertical tunnels; in contrast, emergency response seems to elicit digging in ants of all ages to repair the nest.

Reviewer #1 (Public review):

Summary:

The authors introduce a novel algorithm for the automatic identification of long-range axonal projections. This is an important problem as modern high-throughput imaging techniques can produce large amounts of raw data, but identifying neuronal morphologies and connectivities requires large amounts of manual work. The algorithm works by first identifying points in three-dimensional space corresponding to parts of labelled neural projections, these are then used to identify short sections of axons using an optimisation algorithm and the prior knowledge that axonal diameters are relatively constant. Finally, a statistical model that assumes axons tend to be smooth is used to connect the sections together into complete and distinct neural trees. The authors demonstrate that their algorithm is far superior to existing techniques, especially when dense labelling of the tissue means that neighbouring neurites interfere with the reconstruction. Despite this improvement, however, the accuracy of reconstruction remains below 90%, so manual proofreading is still necessary to produce accurate reconstructions of axons.

Strengths:

The new algorithm combines local and global information to make a significant improvement on the state-of-the-art for automatic axonal reconstruction. The method could be applied more broadly and might have applications to reconstructions of electron microscopy data, where similar issues of high-throughput imaging and relatively slow or inaccurate reconstruction remain.

Weaknesses:

There are three weaknesses in the algorithm and manuscript.

(1) The best reconstruction accuracy is below 90%, which does not fully solve the problem of needing manual proofreading.

(2) The 'minimum information flow tree' model the authors use to construct connected axonal trees has the potential to bias data collection. In particular, the assumption that axons should always be as smooth as possible is not always correct. This is a good rule-of-thumb for reconstructions, but real axons in many systems can take quite sharp turns and this is also seen in the data presented in the paper (Figure 1C). I would like to see explicit acknowledgement of this bias in the current manuscript and ideally a relaxation of this rule in any later versions of the algorithm.

(3) The writing of the manuscript is not always as clear as it could be. The manuscript would benefit from careful copy editing for language, and the Methods section in particular should be expanded to more clearly explain what each algorithm is doing. The pseudo-code of the Supplemental Information could be brought into the Methods if possible as these algorithms are so fundamental to the manuscript.

Reviewer #1 (Public review):

Summary:

Zhang et al. addressed the question of whether hyperaltruistic preference is modulated by decision context, and tested how oxytocin (OXT) may modulate this process. Using an adapted version of a previously well-established moral decision-making task, healthy human participants in this study undergo decisions that gain more (or lose less, termed as context) meanwhile inducing more painful shocks to either themselves or another person (recipient). The alternative choice is always less gain (or more loss) meanwhile less pain. Through a series of regression analyses, the authors reported that hyperaltruistic preference can only be found in the gain context but not in the loss context, however, OXT reestablished the hyperaltruistic preference in the loss context similar to that in the gain context.

Strengths:

This is a solid study that directly adapted a previously well-established task and the analytical pipeline to assess hyperaltruistic preference in separate decision contexts. Context-dependent decisions have gained more and more attention in literature in recent years, hence this study is timely. It also links individual traits (via questionnaires) with task performance, to test potential individual differences. The OXT study is done with great methodological rigor, including pre-registration. Both studies have proper power analysis to determine the sample size.

Weaknesses:

Despite the strengths, multiple analytical decisions have to be explained, justified, or clarified. Also, there is scope to enhance the clarity and coherence of the writing - as it stands, readers will have to go back and forth to search for information. Last, it would be helpful to add line numbers in the manuscript during the revision, as this will help all reviewers to locate the parts we are talking about.

(1) Introduction:<br /> The introduction is somewhat unmotivated, with key terms/concepts left unexplained until relatively late in the manuscript. One of the main focuses in this work is "hyperaltruistic", but how is this defined? It seems that the authors take the meaning of "willing to pay more to reduce other's pain than their own pain", but is this what the task is measuring? Did participants ever need to PAY something to reduce the other's pain? Note that some previous studies indeed allow participants to pay something to reduce other's pain. And what makes it "HYPER-altruistic" rather than simply "altruistic"? Plus, in the intro, the authors mentioned that the "boundary conditions" remain unexplored, but this idea is never touched again. What do boundary conditions mean here in this task? How do the results/data help with finding out the boundary conditions? Can this be discussed within wider literature in the Discussion section? Last, what motivated the authors to examine the decision context? It comes somewhat out of the blue that the opening paragraph states that "We set out to [...] decision context", but why? Are there other important factors? Why decision context is more important than studying those others?

(2) Experimental Design:<br /> (2a) The experiment per se is largely solid, as it followed a previously well-established protocol. But I am curious about how the participants got instructed? Did the experimenter ever mention the word "help" or "harm" to the participants? It would be helpful to include the exact instructions in the SI.

(2b) Relatedly, the experimental details were not quite comprehensive in the main text. Indeed, the Methods come after the main text, but to be able to guide readers to understand what was going on, it would be very helpful if the authors could include some necessary experimental details at the beginning of the Results section.

(3) Statistical Analysis<br /> (3a) One of the main analyses uses the harm aversion model (Eq1) and the results section keeps referring to one of the key parameters of it (ie, k). However, it is difficult to understand the text without going to the Methods section below. Hence it would be very helpful to repeat the equation also in the main text. A similar idea goes to the delta_m and delta_s terms - it will be very helpful to give a clear meaning of them, as nearly all analyses rely on knowing what they mean.

(3b) There is one additional parameter gamma (choice consistency) in the model. Did the authors also examine the task-related difference of gamma? This might be important as some studies have shown that the other-oriented choice consistency may differ in different prosocial contexts.

(3c) I am not fully convinced that the authors included two types of models: the harm aversion model and the logistic regression models. Indeed, the models look similar, and the authors have acknowledged that. But I wonder if there is a way to combine them? For example:<br /> Choice ~ delta_V * context * recipient (*Oxt_v._placebo)<br /> The calculation of delta_V follows Equation 1.<br /> Or the conceptual question is, if the authors were interested in the specific and independent contribution of dalta_m and dalta_s to behavior, as their logistic model did, why did the authors examine the harm aversion first, where a parameter k is controlling for the trade-off? One way to find it out is to properly run different models and run model comparisons. In the end, it would be beneficial to only focus on the "winning" model to draw inferences.

(3d) The interpretation of the main OXT results needs to be more cautious. According to the operationalization, "hyperaltruistic" is the reduction of pain of others (higher % of choosing the less painful option) relative to the self. But relative to the placebo (as baseline), OXT did not increase the % of choosing the less painful option for others, rather, it decreased the % of choosing the less painful option for themselves. In other words, the degree of reducing other's pain is the same under OXT and placebo, but the degree of benefiting self-interest is reduced under OXT. I think this needs to be unpacked, and some of the wording needs to be changed. I am not very familiar with the OXT literature, but I believe it is very important to differentiate whether OXT is doing something on self-oriented actions vs other-oriented actions. Relatedly, for results such as that in Figure 5A, it would be helpful to not only look at the difference but also the actual magnitude of the sensitivity to the shocks, for self and others, under OXT and placebo.

Reviewer #1 (Public review):

The paper by Auer et. makes several contributions:

(1) The study developed a novel approach to map the microstructural organization of the human amygdala by applying radiomics and dimensionality reduction techniques to high-resolution histological data from the BigBrain dataset.

(2) The method identified two main axes of microstructural variation in the amygdala, which could be translated to in vivo 7 Tesla MRI data in individual subjects.

(3) Functional connectivity analysis using resting-state fMRI suggests that microstructurally defined amygdala subregions had distinct patterns of functional connectivity to cortical networks, particularly the limbic, frontoparietal, and default mode networks.

(4) Meta-analytic decoding was used to suggest that the superior amygdala subregion's connectivity is associated with autobiographical memory, while the inferior subregion was linked to emotional face processing.

(5) Overall, the data-driven, multimodal approach provides an account of amygdala microstructure and possibly function that can be applied at the individual subject level, potentially advancing research on amygdala organization.

Although these are meritorious contributions there are some concerns that I will summarize below.

(1) The paper makes little-to-no contact with the monkey literature regarding the anatomy of amygdala subregions, their functionality, and their patterns of anatomical connectivity. This is surprising because such literature on non-human primates is a very important starting point for understanding the human amygdala. I recommend taking a careful look at the work by Helen Barbas, among others. There are too many papers to cite but a notable example is: Ghashghaei, H. T., Hilgetag, C. C., & Barbas, H. (2007). Sequence of information processing for emotions based on the anatomic dialogue between prefrontal cortex and amygdala. Neuroimage, 34(3), 905-923. The work of Amaral is also highly relevant. Furthermore, the authors subscribe to a model with LB, CM, and SF sectors. How does the SF sector relate to monkey anatomy?

(2) The authors use meta-analytical decoding via NeuroSynth. If the authors like those results of course they should keep them but the quality of coordinate reporting in the literature is insufficient to conclude much in the context of amygdala subregion function in my opinion. I believe the results reported are at most "somewhat suggestive".

(3) Another significant concern has to do with the results in Figure 3. The red and yellow clusters identified are quite distinct but the differences in functional connectivity are very modest. Figure 3C reveals very similar functional connectivity with the networks investigated. This is very surprising, and the authors should include a careful comparison with related findings in the literature. Overall, there is limited comparison between the observed results and those obtained via other methods. On a more pessimistic note, the results of Figure 3 seem to question the validity of the general approach.

(4) Some statements in the Discussion feel unwarranted. For example, "significant dissociation in functional connectivity to prefrontal structures that support self-referential, reward-related, and socio-affective processes." This feels way beyond what can be stated based on the analyses performed.

Reviewer #1 (Public review):

Summary:

In this study, Fakhar et al. use a game-theoretical framework to model interregional communication in the brain. They perform virtual lesioning using MSA to obtain a representation of the influence each node exerts on every other node, and then compare the optimal influence profiles of nodes across different communication models. Their results indicate that cortical regions within the brain's "rich club" are most influential.

Strengths:

Overall, the manuscript is well-written. Illustrative examples help to give the reader intuition for the approach and its implementation in this context. The analyses appear to be rigorously performed and appropriate null models are included.

Weaknesses:

The use of game theory to model brain dynamics relies on the assumption that brain regions are similar to agents optimizing their influence, and implies competition between regions. The model can be neatly formalized, but is there biological evidence that the brain optimizes signaling in this way? This could be explored further. Specifically, it would be beneficial if the authors could clarify what the agents (brain regions) are optimizing for at the level of neurobiology - is there evidence for a relationship between regional influence and metabolic demands? Identifying a neurobiological correlate at the same scale at which the authors are modeling neural dynamics would be most compelling.

It is not entirely clear what Figure 6 is meant to contribute to the paper's main findings on communication. The transition to describing this Figure in line 317 is rather abrupt. The authors could more explicitly link these results to earlier analyses to make the rationale for this figure clearer. What motivated the authors' investigation into the persistence of the signal influence across steps?

The authors used resting-state fMRI data to generate functional connectivity matrices, which they used to inform their model of neural dynamics. If I understand correctly, their functional connectivity matrices represent correlations in neural activity across an entire fMRI scan computed for each individual and then averaged across individuals. This approach seems limited in its ability to capture neural dynamics across time. Modeling time series data or using a sliding window FC approach to capture changes across time might make more sense as a means of informing neural dynamics.

The authors evaluated their model using three different structural connectomes: one inferred from diffusion spectrum imaging in humans, one inferred from anterograde tract tracing in mice, and one inferred from retrograde tract-tracing in macaque. While the human connectome is presumably an undirected network, the mouse and macaque connectomes are directed. What bearing does experimentally inferred knowledge of directionality have on the derivation of optimal influence and its interpretation?

It would be useful if the authors could assess the performance of the model for other datasets. Does the model reflect changes during task engagement or in disease states in which relative nodal influence would be expected to change? The model assumes optimality, but this assumption might be violated in disease states.

The MSA approach is highly computationally intensive, which the authors touch on in the Discussion section. Would it be feasible to extend this approach to task or disease conditions, which might necessitate modeling multiple states or time points, or could adaptations be made that would make this possible?

Reviewer #1 (Public review):

Summary:

Winkler et al. present brain activity patterns related to complex motor behaviour by combining whole-head magnetoencephalography (MEG) with subthalamic local field potential (LFP) recordings from people with Parkinson's disease. The motor task involved repetitive circular movements with stops or reversals associated with either predictable or unpredictable cues. Beta and gamma frequency oscillations are described, and the authors found complex interactions between recording sites and task conditions. For example, they observed stronger modulation of connectivity in unpredictable conditions. Moreover, STN power varied across patients during reversals, which differed from stopping movements. The authors conclude that cortex-STN beta modulation is sensitive to movement context, with potential relevance for movement redirection.

Strengths:

This study employs a unique methodology, leveraging the rare opportunity to simultaneously record both invasive and non-invasive brain activity to explore oscillatory networks.

Weaknesses:

It is difficult to interpret the role of the STN in the context of reversals because no consistent activity pattern emerged.

Reviewer #1 (Public review):

Summary:

In this manuscript, Mendoza-Romero et al. investigate the effects of maternal high-fat diet (MHFD) on microglia and AgRP synaptic terminals in the hypothalamus of postnatal mice during lactation. The study employs 3D microglial morphology reconstruction and genetically targeted axonal labeling, offering a detailed examination of microglial changes and their implications for AgRP terminal density and body weight regulation, focusing on the PVN and ARC nuclei. The authors also use pharmacological (e.g., PLX5622) elimination of microglia to test the sufficiency of microglia to shape PVN AgRP+ synapses.

Strengths:

This is a well-written paper with a thorough introduction and discussion.

The impact of microglia on hypothalamic synaptic pruning is poorly characterized, so the findings herein are especially interesting.

Weaknesses:

(1) A cartoon paradigm of the HFD treatment window would be a helpful addition to Figure 1. Relatedly, the authors might consider qualifying MHFD as 'lactational MHFD.' Readers might miss the fact that the exposure window starts at birth.

(2) More details on the modeling pipeline are needed either in Figure 1 or text. Of the ~50 microglia that were counted (based on Figure 1J), were all 50 quantified for the morphological assessments? Were equal numbers used for the control and MHFD groups? Were the 3D models adjusted manually for accuracy? How much background was detected by IMARIS that was discarded? Was the user blind to the treatment group while using the pipeline? Were the microglia clustered or equally spread across the PVN?

(3) Suggest toning back some of the language. For example: "...consistent with enhanced activity and surveillance of their immediate microenvironment" (Line 195) could be "...perhaps consistent with...". Likewise, "profound" (Lines 194, 377) might be an overstatement.

(4) Representative images for AgRP+ cells (quantified in Figure 2J) are missing. Why not a co-label of Iba1+/AgRP+ as per Figure 1, 3? Also, what was quantified in Figure 2J - soma? Total immunoreactivity?

(5) For the PLX experiment:<br /> a) "...we depleted microglia during the lactation period" (Line 234). This statement suggests microglia decreased from the first injection at P4 and throughout lactation, which is inaccurate. PLX5622 effects take time, upwards of a week. Thus, if PLX5622 injections started at P4, it could be P11 before the decrease in microglia numbers is stable. Moreover, by the time microglia are entirely knocked down, the pups might be supplementing some chow for milk, making it unclear how much PLX5622 they were receiving from the dam, which could also impact the rate at which microglia repopulation commences in the fetal brain. Quantifying microglia across the P4-P21 treatment window would be helpful, especially at P16, since the PVN AgRP microglia phenotypes were demonstrated and roughly when pups might start eating some chow.

b) I am surprised that ~70% of the microglia are present at P21. Does this number reflect that microglia are returning as the pups no longer receive PLX5622 from milk from the dam? Does it reflect the poor elimination of microglia in the first place?

(6) Was microglia morphology examined for all microglia across the PVN? It is possible that a focus on PVNmpd microglia would reveal a stronger phenotype? In Figure 4H, J, AgRP+ terminals are counted in PVN subregions - PVNmpd and PVNpml, with PVNmpd showing a decrease of ~300 AgRP+ terminals in MHFD/Veh (rescued in MHFD/PLX5622). In Figure 1K, AgRP+ terminals across what appears to be the entire PVN decrease by ~300, suggesting that PVNmpd is driving this phenotype. If true, then do microglia within the PVNmpd display this morphology phenotype?

(7) What chow did the pups receive as they started to consume solid food? Is this only a MHFD challenge, or could the pups be consuming HFD chow that fell into the cage?

(8) Figure 5: Does internalized AgRP+ co-localize with CD68+ lysosomes? How was 'internalized' determined?

(9) Different sample sizes are used across experiments (e.g., Figure 4 NCD n=5, MHFD n=4). Does this impact statistical significance?

Reviewer #1 (Public review):

Summary:

The study by Hu et al. investigated the role of olfactory ErbB4 in regulating olfactory information processing. The authors demonstrated that ErbB4 deletion impairs odor discrimination, sensitivity, habituation, and dishabituation by using an impressive combination of techniques from morphological to electrophysiology (both slice and in vivo) and from viral injection to cell-type-specific mutation to behavioral analysis. The findings underscore the crucial role of ErbB4 in olfactory PV neurons in modulating mitral cell function and odor perception.

Strengths:

This study contains a pretty comprehensive set of experiments.

Major concerns:

(1) Line 151 page 7, "PV-Erbb4+/+ mice (generated by crossing PV-Cre mice (Wen et al., 2010) with loxP flanked Erbb4 mice". Does this mean mice carrying PV-Cre and ErbB4 floxed allele? Or with the WT allele? This is confusing. Figures 2B and 2C, ErbB4 expression was evident in many cells that were not positive for PV. What are the identities of those cells? Are they important?

(2) In Figure 4, the authors performed tetrode recordings in awake head-fixed animals. Although individual neuron spikes could be obtained by spike-sorting, this is not a "single-unit" experiment due to the nature of this approach.

What is the odor used in Figure 4? How did the authors clean up the odor to limit the stimulation within 2 seconds? In what layer were the tetrodes placed? What is the putative cell type presented in Figure 4C? If Figure 4C is a representative neuron recorded, the odor-induced suppression of spike activity seems to be impaired in PV-ErbB4-/- animals. However, Figure 4D shows that suppressed neurons were similar between the two types of animals. Such comparisons among individual mice are difficult for in vivo electrophysiological experiments because the recorded cell type and placement of electrodes would be different. The authors should apply ErbB4 inhibitors to the same animals and compare the effects before and after. This would ensure the recoding of the same population of neurons.

(3) At a glance in the heatmap in Figure 4D, excited neurons were reduced in PV-ErbB4-/- mice, but not inhibited neurons. This was different from Figure 4L. The authors need to have a criteria or threshold to show how they categorized each population.

(4) Figure 4D, 4F and 4J seemed to be inconsistent. In Figure 4D before odor, there was no clear increase in the spontaneous activity in PV-ErbB4-/- mice; in Figure 4F-4G and 4J-4K, clearly, there was a high spontaneous activity in PV-ErbB4-/- mice.

(5) What are the neurons recorded in Figure 6E-6F? If they were MCs, loss of ErbB4 in PV neurons should not alter their intrinsic electrical properties. Rather GABAergic inputs could be altered. Indeed, the authors presented a reduction of GABAergic inputs from PV neurons to MCs.

(6) Figure 8E-8H, a better experiment would be specifically expressing ErbB4 or PV neurons. In Figure 8F and Figure 8I, was it the excitability after the current injection? Why not perform the spontaneous activity recording?

Reviewer #1 (Public review):

Summary:

In this study from Zhu and colleagues, a clear role for MED26 in mouse and human erythropoiesis is demonstrated that is also mapped to amino acids 88-480 of the human protein. The authors also show the unique expression of MED26 in later-stage erythropoiesis and propose transcriptional pausing and condensate formation mechanisms for MED26's role in promoting erythropoiesis. Despite the author's introductory claim that many questions regarding Pol II pausing in mammalian development remain unanswered, the importance of transcriptional pausing in erythropoiesis has actually already been demonstrated (Martell-Smart, et al. 2023, PMID: 37586368, which the authors notably did not cite in this manuscript). Here, the novelty and strength of this study is MED26 and its unique expression kinetics during erythroid development.

Strengths:

The widespread characterization of kinetics of mediator complex component expression throughout the erythropoietic timeline is excellent and shows the interesting divergence of MED26 expression pattern from many other mediator complex components. The genetic evidence in conditional knockout mice for erythropoiesis requiring MED26 is outstanding. These are completely new models from the investigators and are an impressive amount of work to have both EpoR-driven deletion and inducible deletion. The effect on red cell number is strong in both. The genetic over-expression experiments are also quite impressive, especially the investigators' structure-function mapping in primary cells. Overall the data is quite convincing regarding the genetic requirement for MED26. The authors should be commended for demonstrating this in multiple rigorous ways.

Weaknesses:

(1) The authors state that MED26 was nominated for study based on RNA-seq analysis of a prior published dataset. They do not however display any of that RNA-seq analysis with regards to Mediator complex subunits. While they do a good job showing protein-level analysis during erythropoiesis for several subunits, the RNA-seq analysis would allow them to show the developmental expression dynamics of all subunit members.

(2) The authors use an EpoR Cre for red cell-specific MED26 deletion. However, other studies have now shown that the EpoR Cre can also lead to recombination in the macrophage lineage, which clouds some of the in vivo conclusions for erythroid specificity. That being said, the in vitro erythropoiesis experiments here are convincing that there is a major erythroid-intrinsic effect.

(3) The donor chimerism assessment of mice transplanted with MED26 knockout cells is a bit troubling. First, there are no staining controls shown and the full gating strategy is not shown. Furthermore, the authors use the CD45.1/CD45.2 system to differentiate between donor and recipient cells in erythroblasts. However, CD45 is not expressed from the CD235a+ stage of erythropoiesis onwards, so it is unclear how the authors are detecting essentially zero CD45-negative cells in the erythroblast compartment. This is quite odd and raises questions about the results. That being said, the red cell indices in the mice are the much more convincing data.

(4) The authors make heavy use of defining "erythroid gene" sets and "non-erythroid gene" sets, but it is unclear what those lists of genes actually are. This makes it hard to assess any claims made about erythroid and non-erythroid genes.

(5) Overall the data regarding condensate formation is difficult to interpret and is the weakest part of this paper. It is also unclear how studies of in vitro condensate formation or studies in 293T or K562 cells can truly relate to highly specialized erythroid biology. This does not detract from the major findings regarding genetic requirements of MED26 in erythropoiesis.

(6) For many figures, there are some panels where conclusions are drawn, but no statistical quantification of whether a difference is significant or not.

Reviewer #1 (Public Review):

The authors investigate whether during free exploration of an environment with an internal structure of corridors and occasionally fluid-rewarded alleys, rat CA1 place cells generate multiple firing fields in repeating patterns, allowing the investigators to analyze whether firing field positional properties like alley orientation, and non-positional properties like heading, field-rate modulation and other properties are similar or different within and across single place cell place fields. They adopt a standard cognitive map analysis framework, conceiving each cell as an individual map element and characterizing each cell's individual activity independently of the activity of other cells, such that the main unit of analysis is a place field averaged across recording times of many minutes. Despite framing the work as an investigation of a fundamentally-subjective episodic memory system sensitive to hidden cognitive and attentional variables, the experiment and analyses are conceived as if the cells respond to positional and non-positional features of experience as static "inputs" that the investigators infer. These "inputs" are conceptualized as effectively stationary and steady, and they are not manipulated. The authors find that there are many "repeated" firing fields, that they tend to have similar orientation more than expected by chance, and that each field's rate is modulated distinctly by heading direction and other factors, leading them to conclude that each field's nonpositional inputs are "individually addressable." The authors do not consider alternative possibilities for which there are strong indications in the contemporary literature like 1) CA1 activity could be internally generated; 2) that there could be hidden cognitive variables that influence CA1 activity episodically and in non-stationary ways rather than consistently; 3) that CA1 cells exhibit mixed tuning to a variety of environmental and navigational variables; 4) that CA1 activity is better interpreted from the point-of-view of a neural ensemble or a neural manifold of conjoint neural activity that represents multiple information variables, or 5) that stable neural representations of information need not depend on stable stimulus-response properties of individual cells. In fact, the analyses provide evidence consistent with each of these alternatives, but they are not considered. There is a case to be made that the authors are allowed to ignore these alternatives because they properly engage the dogmatic point of view, in which case there is little to adjust in the manuscript, which is both well-conceived and well-executed in the classic (but not contemporary) norms of place cell investigations.

My comments are focused on improving the manuscript without insisting that the authors adopt alternative (contemporary) points of view, but requiring them to clarify their point of view and explain that there are alternatives.

(1) The authors define what they mean by "positional" and "non-positional" "inputs" later in the manuscript. Since the experimental apparatus and task have been designed to isolate these "inputs" the authors should in the initial description of the environment and task explain what the task does and does not allow them to analyze. Instead, they have repeatedly asserted that the environment is a hybrid of an open-field and a linear track environment. This may be the case, but so what? The authors need to better explain, up front, why that matters and what they will be able to investigate as a result. As written, this all seems to me rather vague and post hoc.

(2) The abstract states "Previous work implies a distinction between positional inputs to the hippocampus that provide information about an animal's location and non-positional inputs which provide information about the content of experience." While I understand what the authors mean, I want to point out that it is not straightforward to identify the "positional inputs" and the "non-positional inputs." What are they, how can they be measured? Is it not also possible that hippocampus generates "positional" information rather than receiving it, that is in fact the longstanding view of the cognitive map framework that the authors have adopted, and yet they frame the essential issue as one of differential receipt of positional and non-positional inputs. This seems to me imprecise and hard to defend but demonstrates the authors' opinion in framing this work. In my view a more objective and accurate statement might be "Previous work implies a distinction between hippocampal (positional) activity representing information about an animal's location and (non-positional) activity which represents information about the content of experience." This opinion about "inputs" is found throughout the manuscript over 50 times, starting with the title. While in my view this is not an objective treatment of the experimental design or data (positional and non-positional inputs are never identified or manipulated, they are merely inferred), I accept that the authors can say whatever they want so long as they make it clear to the reader that theirs is an opinion or assumption rather than a measurement. The manuscript is written as if the different inputs are identified and valid, rather than inferred.

(3) The abstract states "even though the animal's behavior was not constrained to 1-D trajectories" whereas page 13 states "but their trajectories were constrained to orthogonal directions by the city-maze architecture" and page 23 states "but their trajectories were constrained to a rectilinear grid." While I understand what the authors mean, the first statement appears to contradict the others. There are additional examples that I do not identify here. In any case, I would like to have seen examples of the animals' trajectories through the maze. A figure showing the raw trajectories and another after the unwanted behaviors have been filtered out should be given, allowing the reader to understand how much the animals tended to travel through the alleys, how much they turned and lingered within them, etc.

(4) The abstract ends with "These results demonstrate that the positional inputs that drive a cell to fire in similar locations across the maze can be behaviorally and temporally dissociated from the nonpositional inputs that alter the firing rates of the cell within its place fields, thereby increasing the flexibility of the system to encode episodic variables within a spatiotemporal framework provided by place cells." I don't see the evidence for the "thereby ..." claim. The authors are free to speculate and discuss but they should say they are speculating and/or discussing a possibility, rather than assert as if they have demonstrated a fact.

(5) The Introduction begins with "All behavior is embedded within a spatial and temporal framework." By this statement, I believe the authors mean to assert, or at least they cause a reader to understand that there is a spatial and temporal framework that is separate from the behaving subject. They will use this point of view to design their experiment around the utility of a city- maze. Since the authors appeal to cognitive map theory so much, I point out that O'Keefe and Nadel write in The Hippocampus as a Cognitive Map that "Space was a way of perceiving, not a thing to be perceived." Sentence number 2 of the book states "We shall argue that the hippocampus is the core of a neural memory system providing an objective spatial framework within which the items and events of an organism's experience are located and interrelated." Consistent with Kant and O'Keefe and Nadel, the present authors might more accurately state "All behavior is embedded within a subjective spatial and temporal framework." but then they will have to explain why they conceive of there being "positional inputs" to which they are measuring CA1 responses. This framing seems to me problematic and not logically self-consistent.

(6) On page 2 the authors assert "Neurons within the hippocampus respond to a wide array of sensory and otherwise nonspatial cues..." then they go on to list sensory features and "non-positional" features of experience to which CA1 cells respond. It seems to me they leave out a class of features of experience that might be considered "subjective spatial frames" that have been investigated by Gothard and Redish when they were in the McNaughton and Barnes lab, as well the Fenton and Muller labs, amongst others. All of these papers describe non-stationary, multi-stable place cell phenomena that are tied to subjective variables, which have the potential to undermine the premise of the present work's analyses and so they should be considered. I list a sample but certainly not all the work that might be considered.

Gothard KM, Skaggs WE, Moore KM, McNaughton BL (1996) Binding of hippocampal CA1 neural activity to multiple reference frames in a landmark-based navigation task. J Neurosci 16:823-835.

Gothard KM, Skaggs WE, McNaughton BL (1996) Dynamics of mismatch correction in the hippocampal ensemble code for space: interaction between path integration and environmental cues. J Neurosci 16:8027-8040.

Gothard KM, Hoffman KL, Battaglia FP, McNaughton BL (2001) Dentate gyrus and ca1 ensemble activity during spatial reference frame shifts in the presence and absence of visual input. J Neurosci 21:7284-7292.

Redish AD, Rosenzweig ES, Bohanick JD, McNaughton BL, Barnes CA (2000) Dynamics of hippocampal ensemble activity realignment: time versus space. J Neurosci 20:9298-9309.

Rosenzweig ES, Redish AD, McNaughton BL, Barnes CA (2003) Hippocampal map realignment and spatial learning. Nat Neurosci 6:609-615.

Jackson J, Redish AD (2007) Network dynamics of hippocampal cell-assemblies resemble multiple spatial maps within single tasks. Hippocampus 17:1209-1229

Lenck-Santini PP, Fenton AA, Muller RU (2008) Discharge properties of hippocampal neurons during performance of a jump avoidance task. J Neurosci 28:6773-6786.

Fenton AA, Lytton WW, Barry JM, Lenck-Santini PP, Zinyuk LE, Kubik S, Bures J, Poucet B, Muller RU, Olypher AV (2010) Attention-like modulation of hippocampus place cell discharge. J Neurosci 30:4613-4625.

Kelemen E, Fenton AA (2013) Key features of human episodic recollection in the cross-episode retrieval of rat hippocampus representations of space. PLoS Biol 11:e1001607.

(7) The Introduction asserts that "rate remapping" is a hypothesis. Rate remapping is a phenomenon, something that is observed. The interpretation of the observation as being the substrate of episodic memory is certainly a hypothesis that in my opinion has not been tested and is not being tested in the present work. After making the above statement, the authors go on to describe that firing rates differ across "repeated" firing fields, which seems to be a form of rate remapping, and predicted by the relevant hypothesis that different episodes of experience at the same locations are represented by different firing rates. This is very speculative and there are many other explanations.

(8) The Introduction ends with the statement "Here, we show that repeating fields of the same neuron do not always display the same nonpositional rate modulation, demonstrating that nonpositional cues are dissociable from, and more flexible than, the positional inputs onto place cells in a given environment." Apart from my concern about using the "input" terminology I which to point out that there is very little novel in this statement. It has been described many times before that on linear tracks CA1 firing fields are directionally modulated such that the field rates for traversals in one direction are different compared to field traversals in the opposite direction. Jackson and Redish (2007) cited above show this to be due to reference frame or map switching. That and other work allow one to state that "Others show that repeating fields of the same neuron do not always display the same nonpositional rate modulation, demonstrating that nonpositional cues are dissociable from, and more flexible than, the positional inputs onto place cells in a given environment." Either the present authors should acknowledge that they are demonstrating what others have already demonstrated, or they should more precisely describe what about their contribution is unique.

(9) Page 6 Methods - Data Filtering and Pre-processing. How did the authors handle theta cells and others that fired more or less everywhere but with spatial modulation?

(10) Page 9 Methods - Why was the session-wide activity used to normalize the firing rates for the activity vector input to the random forest classifier? The authors state "The normalized firing rate was computed as discussed above with the change that the session-wide activity in the alley was used." It seems to me better to have used the session-averaged firing rate map because the activity would be normalized by the expected positional firing. I imagine "The classifier used the population vector of firing rates as the input." is incorrect and the authors mean to state "The classifier used the population vector of normalized firing rates as the input."

(11) What does "spatially-gated" mean? The use of such jargon should be explained, or better avoided.

(12) Page 12: Since fields tend to have similar orientations, but not repeat at all geometrically similar locations, did they tend to be clustered? Was there a proximity feature to their distribution?

(13) Page 18 states "Thus, although there was a slight trend for repeating field ..." The authors are reporting a significant effect not a "slight trend." They do something similar in reporting Figure 5's result. Despite significant effects, they seem to think the findings are not large enough so state that repeating-field directionality is not conserved. It is fine to explain that a significant effect was small (for example give the effect size, which would have been welcome throughout) but as in these cases and others, the authors should be more objective in their reporting of the outcomes. Either a statistical test was or was not significant. It is not "a little" or "a lot" significant.

(14) Page 18: What do the authors mean by "topology?" Might they mean "topography?"

(15) Figure 6 shows field instability and multi-stability (termed temporal dynamics) as described on page 22. The recording sessions were 60 min. Is this impression simply due to long recording sessions? If 10 or 15 minutes of data were analyzed (which is more the norm), would similar instability be observed/detectable?

(16) I found the Discussion very confusing. On the one hand, there is an assertion that because the location of firing fields is stable there is a "positional code." How would that actually work? Any neural system has to signal by firing rates or firing coincidences across groups of cells (that are affected by changes in rate) so if there is firing field firing rate instability the authors should explain how position can be accurately decoded on a behaviorally-meaningful time scale. In fact, they should demonstrate such decoding explicitly. Just because there is modulation and instability, it is a rather long leap to assert that this is how episodic experience/memory is encoded (as stated at the end of the abstract and elsewhere for example on page 24: "The present data utilize repeating fields to suggest that, within an environment, the positional inputs are relatively rigid, whereas the nonpositional inputs are more flexible, allowing different repeating fields to show different directional preferences. In other words, fields are individually addressable with respect to the nonpositional inputs they receive; they do not inherit their nonpositional tuning as a global property of the cell." What does it mean that a field is "individually addressable?" How is that achieved by neurons? If the authors want to make such assertions they should explain and demonstrate how their assertions can be valid, given the data and findings. At least they should explain what they are assuming.<br /> The main findings seem related to the published finding that in large environments place cells have multiple firing fields, with distinct rates in each field, quite similar to what is here described in the city maze. In my opinion, positional representations can only plausibly work in such cases by using the conjoint population activity moment to moment, which necessarily marginalizes the value of individual firing fields, yet the present work focuses the discussion (and analyses) on interpretations of single firing fields (which they assert are individually addressable multiple times). I don't know what that means exactly and the authors should explain why maintaining the standard single-field perspective is appropriate and how position can be represented in such a system, given the data. In fact, I would have thought that the present findings would cause the authors to reject as invalid the framework they have adopted.

(17) This is a further example, on page 25 which asserts that "Directionality is affected by an animal's experience through the field (Navratilova et al., 2012), so it is possible the difference in experience between sampling fields on the same versus different corridors affects the directional tuning properties between them." I do not understand how "the difference in experience between sampling fields on the same versus different corridors affects the directional tuning properties between them." If I follow the logic then the so-called directionality would depend on experience and so only emerge after a certain time for experience, or else the firing during one traversal would need to be modulated by information about future traversals, which I suppose the authors would agree does not make sense.

(18) I found it at times confusing to follow the arguments because the terms "route" and "trajectory" and also "direction" and "heading" were used sometimes interchangeably and sometimes in ways that appear distinct.

(19) Page 25 states "One explanation for these data is that fields sampled along contiguous routes, without interruptions from heading change or reward delivery, are more likely to share their directionality." The authors should consider alternative explanations like reference frame shifts as mentioned in comment 6 above. These alternatives can be rejected based on data, but they should be considered because they seem to offer more parsimonious explanations for the observations than what the authors have offered. For example, what can explain the bimodality reported in Fig. 5G?

(20) The authors assert on page 15 that "In the present study, turns at the ends of corridors, along with reward deliveries, may be salient task boundaries at which point theta sequences are terminated. Fields active within the same theta sequence (typically same corridor fields) may be functionally coupled, while fields active on opposite sides of a theta sequence termination (different corridor fields) may be uncoupled and their tuning uncorrelated." The authors should check this. They recorded the LFPs. Why speculate when they can evaluate the speculation?

(21) The authors assert on page 26 "It is important to note that because a Pearson correlation was used, it is possible the fields are related in time with a phase shift, and we did not have the statistical power to test this possibility adequately." I either do not understand this statement or it is untrue. Please clarify.

(22) The authors continue on page 26, asserting "Thus, although it is clear that the place fields of repeating cells do not change their firing rates in synchrony, as if the cell had a global excitability change that made all its fields wax and wane together, it nonetheless remains an open question as to whether the subfields of repeating cells engage in certain types of competitive interactions or other network dynamics that couple changes in their firing rates in more complex ways." This statement implies that it might even be possible for firing fields in distinct and distant locations to be modulated together. Could the authors please explain how that is possible? A firing field is an observation that requires averaging over minutes and behavioral sampling across minutes. How might one cell be modulated to fire at a low rate during one minute and then at another minute later be modulated to fire at a high rate everywhere in the environment? Perhaps I am again not understanding the assertion - please clarify.

Reviewer #1 (Public review):

Summary:

This paper describes the covalent interactions of small molecule inhibitors of carbonic anhydrase IX, utilizing a pre-cursor molecule capable of undergoing beta-elimination to form the vinyl sulfone and covalent warhead.

Strengths:

The use of a novel covalent pre-cursor molecule that undergoes beta-elimination to form the vinyl sulfone in situ. Sufficient structure-activity relationships across a number of leaving groups, as well as binding moieties that impact binding and dissociation constants.

Weaknesses:

No major weaknesses noted. Suggested corrections were addressed.

Reviewer #1 (Public review):

Previous studies have used a randomly induced label to estimate the number of hematopoietic precursors that contribute to hematopoiesis. In particular, the McKinney-Freeman lab established a measurable range of precursors of 50-2500 cells using random induction of one of the 4 fluorescent proteins (FPs) of a Confetti reporter in the fetal liver to show that hundreds of precursors establish lifelong hematopoiesis. In the presented work, Liu and colleagues aim to extend the measurable range of precursor numbers previously established and enable measurement in a variety of contexts beyond embryonic development. To this end, the authors investigated whether the random induction of a given Confetti FP follows the principles of binomial distribution such that the variance inversely correlates with the precursor number. The authors validated their hypothesis and identified sampling conditions to minimize experimental error using a simplified in vitro system. They use tamoxifen-inducible Scl-CreER, active in hematopoietic stem and progenitor cells (HSPCs), to induce Confetti labeling and investigate whether they could extend their model to cell numbers below 50 with in vivo transplantation of high versus low numbers of Confetti total bone marrow (BM) cells. The data generated are generally robust. While the lower and upper limits of the model may show some small error or have not yet been completely validated experimentally, it extends the measurable range of precursor from 15 - 10^5 cells. The authors then apply their model to estimate the number of hematopoietic precursors that contribute to hematopoiesis in a variety of contexts including adult steady state, fetal liver, following myeloablation, and a genetic model of Fanconi anemia.

Their data highlight the importance of estimating precursor numbers and not just donor frequency in transplantation settings and show that native hematopoiesis is highly polyclonal. Their data also confirm previous findings from Ganuza et al, 2022 that demonstrate no major expansion of precursors between E11.5 - E14.5. Finally, their work reveals intact Fancc-/-precursor numbers following transplantation, suggesting that the observed reduced chimerism is due to defects in cell proliferation.

The conclusions are generally sound and based on high-quality data. As the authors note, future studies should validate the model using alternative Cre-drivers to exclude any potential functional difference between labelled and non-labelled cells. Although this system does not permit tracing of individual clones, the modeling presented allows measurements of clonal activity covering nearly the entire HSPC population (as recently estimated by Cosgrove et al, 2021) and can be applied to a wide range of in vivo contexts with relative ease.

Reviewer #1 (Public review):

Summary:

This study serves as a proof of concept for KMO inhibition as a new non-hormonal treatment for endometriosis. The authors investigated KMO expression in human endometrial and endometriosis lesion tissues, confirmed that KNS898 effectively inhibits KMO and alleviates manifestations of endometriosis in mice - reduced endometriosis lesions and improved hyperalgesia and cage behaviour.

Strengths:

(1) Inhibition of KMO may present as a promising first-in-class non-hormonal therapeutic agent for patients suffering from endometriosis and the side-effects of hormonal treatments.<br /> (2) The expression of KMO in endometrial tissues was demonstrated in both human (multiple patients per AFS stage of disease) and mice tissues.<br /> (3) Measurement of multiple substrates/analytes of the KMO regulatory pathway was performed and demonstrated strong correlation to each other in response to KMO inhibition.<br /> (4) The aims of study (as proof-of-concept) were achieved in the study and the results support their conclusions.

Weaknesses:

If any dysregulation in the KMO/tryptophan metabolic activity, expression and/or pathway in endometriosis can be shown, this will strengthen the rationale for the use of KMO inhibitor in the disease.

Reviewer #1 (Public review):

Summary:

Orlovski and his colleagues revealed an interesting phenomenon that SAP54-overexpressing leaf exposure to leafhopper males is required for the attraction of followed females. By transcriptomic analysis, they demonstrated that SAP54 effectively suppresses biotic stress response pathways in leaves exposed to the males. Furthermore, they clarified how SAP54, by targeting SVP, heightens leaf vulnerability to leafhopper males, thus facilitating female attraction and subsequent plant colonization by the insects.

Strengths:

The phenomenon of this study is interesting and exciting.

Reviewer #1 (Public review):

Summary:

By employing human primary microvascular endothelial cells, along with live confocal imaging, proteomics, and chemical validation studies, the authors reveal a novel cellular mechanism underlying mycolactone's effects in Buruli ulcer lesions. This finding provides important insights into the specific mechanisms of skin pathogenesis.

Strengths:

The techniques employed are state-of-the-art.

Weaknesses:

The study lacks genetic validation of the findings.

Reviewer #1 (Public review):

Summary:

The investigators undertook detailed characterization of a previously proposed membrane targeting sequence (MTS), a short N-terminal peptide, of the bactofilin BacA in Caulobacter crescentus. Using light microscopy, single molecule tracking, liposome binding assays, and molecular dynamics simulations, they provide data to suggest that this sequence indeed does function in membrane targeting and further conclude that membrane targeting is required for polymerization. While the membrane association data are reasonably convincing, there are no direct assays to assess polymerization and some assays used lack proper controls as detailed below. Since the MTS isn't required for bactofilin polymerization in other bacterial homologues, showing that membrane binding facilitates polymerization would be a significant advance for the field.

Major concerns

(1) This work claims that the N-termina MTS domain of BacA is required for polymerization, but they do not provide sufficient evidence that the ∆2-8 mutant or any of the other MTS variants actually do not polymerize (or form higher order structures). Bactofilins are known to form filaments, bundles of filaments, and lattice sheets in vitro and bundles of filaments have been observed in cells. Whether puncta or diffuse labeling represents different polymerized states or filaments vs. monomers has not been established. Microscopy shows mis-localization away from the stalk, but resolution is limited. Further experiments using higher resolution microscopy and TEM of purified protein would prove that the MTS is required for polymerization.<br /> (2) Liposome binding data would be strengthened with TEM images to show BacA binding to liposomes. From this experiment, gross polymerization structures of MTS variants could also be characterized.<br /> (3) The use of the BacA F130R mutant throughout the study to probe the effect of polymerization on membrane binding is concerning as there is no evidence showing that this variant cannot polymerize. Looking through the papers the authors referenced, there was no evidence of an identical mutation in BacA that was shown to be depolymerized or any discussion in this study of how the F130R mutation might to analogous to polymerization-deficient variants in other bactofilins mentioned in these references.<br /> (4) Microscopy shows that a BacA variant lacking the native MTS regains the ability to form puncta, albeit mis-localized, in the cell when fused to a heterologous MTS from MreB. While this swap suggests a link between puncta formation and membrane binding the relationship between puncta and polymerization has not been established (see comment 1).<br /> (5) The authors provide no primary data for single molecule tracking. There is no tracking mapped onto microscopy images to show membrane localization or lack of localization in MTS deletion/variants. A known soluble protein (e.g. unfused mVenus) and a known membrane bound protein would serve as valuable controls to interpret the data presented. It also is unclear why the authors chose to report molecular dynamics as mean squared displacement rather than mean squared displacement per unit time, and the number of localizations is not indicated. Extrapolating from the graph in figure 4 D for example, it looks like WT BacA-mVenus would have a mobility of 0.5 (0.02/0.04) micrometers squared per second which is approaching diffusive behavior. Further justification/details of their analysis method is needed. It's also not clear how one should interpret the finding that several of the double point mutants show higher displacement than deleting the entire MTS. These experiments as they stand don't account for any other cause of molecular behavior change and assume that a decrease in movement is synonymous with membrane binding.<br /> (6) The experiments that map the interaction surface between the N-terminal unstructured region of PbpC and a specific part of the BacA bactofilin domain seem distinct from the main focus of the paper and the data somewhat preliminary. While the PbpC side has been probed by orthogonal approaches (mutation with localization in cells and affinity in vitro), the BacA region side has only been suggested by the deuterium exchange experiment and needs some kind of validation.

Reviewer #1 (Public review):

Summary:

This paper tests the hypothesis that neuronal adaptation to spatial frequency affects the estimation of spatial population receptive field sizes as commonly measured using the pRF paradigm in fMRI. To this end, the authors modify a standard pRF setup by presenting either low or high SF (near full field) adaptation stimuli prior to the start of each run and interleaved between each pRF bar stimulus. The hypothesis states that adaptation to a specific spatial frequency (SF) should affect only a specific subset of neurons in a population (measured with an fMRI voxel), leaving the other neurons in the population intact, resulting in a shift in the tuning of the voxel in the opposite direction of the adapted stimulus (so high SF adaptation > larger pRF size and vice versa). The paper shows that this 'repelling' effect is robustly detectable psychophysically and is evident in pRF size estimates after adaptation in line with the hypothesized direction, thereby demonstrating a link between SF tuning and pRF size measurements in the human visual cortex.

Strengths:

The paper introduces a new experimental design to study the effect of adaptation on spatial tuning in the cortex, nicely combining the neuroimaging analysis with a separate psychophysical assessment.

The paper includes careful analyses and transparent reporting of single-subject effects, and several important control analyses that exclude alternative explanations based on perceived contrast or signal-to-noise differences in fMRI.

The paper contains very clear explanations and visualizations, and a carefully worded Discussion that helpfully contextualizes the results, elucidating prior findings on the effect of spatial frequency adaptation on size illusion perception.

Weaknesses:

The fMRI experiments consist of a relatively small sample size (n=8), of which not all consistently show the predicted pattern in all ROIs. For example, one subject shows a strong effect in the pRF size estimates in the opposite direction in V1. It's not clear if this subject is also in the psychophysical experiment and if there is perhaps a behavioral correlate of this deviant pattern. The addition of a behavioral task in the scanner testing the effect of adaptation could perhaps have helped clarify this (although arguably it's difficult to do psychophysics in the scanner). Although the effects are clearly robust at the group level here, a larger sample size could clarify how common such deviant patterns are, and potentially allow for the assessment of individual differences in adaption effects on spatial tuning as measured with fMRI, and their perceptual implications.

The psychophysical experiment in which the perceptual effects are shown included a neutral condition, which allowed for establishing a baseline for each subject and the discovery of an asymmetry in the effects with stronger perceptual effects after high SF adaptation compared to low SF. This neutral condition was lacking in fMRI, and thus - as acknowledged - this asymmetry could not be tested at the neural level, also precluding the possibility of comparing the obtained pRF estimates to the typical ranges found using standard pRF mapping procedures (without adaptation), or to compare the SNR using in the adaptation pRF paradigm with that of a regular paradigm, etc.

The results indicate quite some variability in the magnitude of the shift in pRF size across eccentricities and ROIs (Figure 5B). It would be interesting to know more about the sources of this variability, and if there are other effects of adaptation on the estimated retinotopic maps other than on pRF size (there is one short supplementary section on the effects on eccentricity tuning, but not polar angle).

Reviewer #1 (Public review):

Summary:

This study addresses the issue of rapid skill learning and whether individual sequence elements (here: finger presses) are differentially represented in human MEG data. The authors use a decoding approach to classify individual finger elements, and accomplish an accuracy of around 94%. A relevant finding is that the neural representations of individual finger elements dynamically change over the course of learning. This would be highly relevant for any attempts to develop better brain machine interfaces - one now can decode individual elements within a sequence with high precision, but these representations are not static but develop over the course of learning.

Strengths:

The work follows a large body of work from the same group on the behavioural and neural foundations of sequence learning. The behavioural task is well established and neatly designed to allow for tracking learning and how individual sequence elements contribute. The inclusion of short offline rest periods between learning epochs has been influential because it has revealed that a lot, if not most of the gains in behaviour (ie speed of finger movements) occur in these so-called micro-offline rest periods.

The authors use a range of new decoding techniques, and exhaustively interrogate their data in different ways, using different decoding approaches. Regardless of the approach, impressively high decoding accuracies are observed, but when using a hybrid approach that combines the MEG data in different ways, the authors observe decoding accuracies of individual sequence elements from the MEG data of up to 94%.

Weaknesses:

There are a few concerns which the authors may well be able to resolve. These are not weaknesses as such, but factors that would be helpful to address as these concern potential contributions to the results that one would like to rule out.

Regarding the decoding results shown in Figure 2 etc, a concern is that within individual frequency bands, the highest accuracy seems to be within frequencies that match the rate of keypresses. This is a general concern when relating movement to brain activity, so is not specific to decoding as done here. As far as reported, there was no specific restraint to the arm or shoulder, and even then it is conceivable that small head movements would correlate highly with the vigor of individual finger movements. This concern is supported by the highest contribution in decoding accuracy being in middle frontal regions - midline structures that would be specifically sensitive to movement artefacts and don't seem to come to mind as key structures for very simple sequential keypress tasks such as this - and the overall pattern is remarkably symmetrical (despite being a unimanual finger task) and spatially broad. This issue may well be matching the time course of learning, as the vigor and speed of finger presses will also influence the degree to which the arm/shoulder and head move.

This is not to say that useful information is contained within either of the frequencies or broadband data. But it raises the question of whether a lot is dominated by movement "artefacts" and one may get a more specific answer if removing any such contributions.

A somewhat related point is this: when combining voxel and parcel space, a concern is whether a degree of circularity may have contributed to the improved accuracy of the combined data, because it seems to use the same MEG signals twice - the voxels most contributing are also those contributing most to a parcel being identified as relevant, as parcels reflect the average of voxels within a boundary. In this context, I struggled to understand the explanation given, ie that the improved accuracy of the hybrid model may be due to "lower spatially resolved whole-brain and higher spatially resolved regional activity patterns". Firstly, there will be a relatively high degree of spatial contiguity among voxels because of the nature of the signal measured, ie nearby individual voxels are unlikely to be independent. Secondly, the voxel data gives a somewhat misleading sense of precision; the inversion can be set up to give an estimate for each voxel, but there will not just be dependence among adjacent voxels, but also substantial variation in the sensitivity and confidence with which activity can be projected to different parts of the brain. Midline and deeper structures come to mind, where the inversion will be more problematic than for regions along the dorsal convexity of the brain, and a concern is that in those midline structures, the highest decoding accuracy is seen.

Some of these concerns could be addressed by recording head movement (with enough precision) to regress out these contributions. The authors state that head movement was monitored with 3 fiducials, and their timecourses ought to provide a way to deal with this issue. The ICA procedure may not have sufficiently dealt with removing movement-related problems, but one could eg relate individual components that were identified to the keypresses as another means for checking. An alternative could be to focus on frequency ranges above the movement frequencies. The accuracy for those still seems impressive, and may provide a slightly more biologically plausible assessment.

One question concerns the interpretation of the results shown in Figure 4. They imply that during the course of learning, entirely different brain networks underpin the behaviour. Not only that, but they also include regions that would seem rather unexpected to be key nodes for learning and expressing relatively simple finger sequences, such as here. What then is the biological plausibility of these results? The authors seem to circumnavigate this issue by moving into a distance metric that captures the (neural network) changes over the course of learning, but the discussion seems detached from which regions are actually involved; or they offer a rather broad discussion of the anatomical regions identified here, eg in the context of LFOs, where they merely refer to "frontoparietal regions".

If I understand correctly, the offline neural representation analysis is in essence the comparison of the last keypress vs the first keypress of the next sequence. In that sense, the activity during offline rest periods is actually not considered. This makes the nomenclature somewhat confusing. While it matches the behavioural analysis, having only key presses one can't do it in any other way, but here the authors actually do have recordings of brain activity during offline rest. So at the very least calling it offline neural representation is misleading to this reviewer because what is compared is activity during the last and during the next keypress, not activity during offline periods. But it also seems a missed opportunity - the authors argue that most of the relevant learning occurs during offline rest periods, yet there is no attempt to actually test whether activity during this period can be useful for the questions at hand here.

Reviewer #1 (Public review):

Summary:

Mollá-Albaladejo et al. investigate the neurons downstream of GR64f and Gr66a, called G2Ns. They identify downstream neurons using trans-Tango labeling with RFP and then perform bulk RNA-seq on the RFP-sorted cells. Gene expression is up- or downregulated between the cell populations and between fed and starved states. They specifically identify Leukocinin as a neuropeptide that is upregulated in starved Gr66a cells. Leucokinin cells, identified by a GAL4 line indeed show higher expression when starved, especially in the SEZ. Furthermore, Leucokinin cells colocalize with the trans-Tango signal from downstream neurons of both GRs. This connection is confirmed with GRASP. According to EM data, Leucokinin cells in the SEZ receive a lot of input and connect to many downstream neurons. In behavior experiments performed with flies lacking Leucokinin neurons, flies show reduced responsiveness to sugar and bitter mixtures when starved. The authors suggest that Leucokinin neurons integrate bitter and sugar tastes and that their output is modified by a hunger state.

Strengths:

The authors use a multitude of tools to identify SELK neurons downstream of taste sensory neurons and as starvation-sensitive cells. This study provides an example of how combining genetic labeling, RNA-seq, and EM analysis can be combined to investigate neural circuits.

Weaknesses:

The authors do not show a functional connection between sensory neurons and SELK neurons. Additionally, data from RNA seq, anatomical studies, and EM analysis are sometimes contradictory in terms of connectivity. GRASP signal is not foolproof that cells are synaptically connected.

The authors describe a behavioral phenotype when flies are starved, however, they do not use a specific driver for the described cell type, thus they should also tone down their claims.

Generally, the authors do not provide a big advancement to the field and some of the results are contradictory with previous publications.

Reviewer #1 (Public review):

This paper describes "Ais", a new software tool for machine-learning based segmentation and particle picking of electron tomograms. The software can visualise tomograms as slices and allows manual annotation for the training of a provided set of various types of neural networks. New networks can be added, provided they adhere to a python file with an (undescribed) format. Once networks have been trained on manually annotated tomograms, they can be used to segment new tomograms within the same software. The authors also set up an online repository to which users can upload their models, so they might be re-used by others with similar needs. By logically combining the results from different types of segmentations, they further improve the detection of distinct features. The authors demonstrate the usefulness of their software on various data sets. Thus, the software appears to be a valuable tool for the cryo-ET community that will lower the boundaries of using a variety of machine-learning methods to help interpret tomograms.

Reviewer #4 (Public review):

Summary:

This study describes an understudied migration pattern of dynamic non-breeding range using data from an Arctic raptor. Using data from GPS tags, the study describes the known pattern of fast migration during autumn and spring, and an undescribed pattern of slow migration, at much slower pace, throughout the over-wintering season.

Strengths:

The study presents a comprehensive analysis of the annual cycle of an interesting and undescribed migration system. The conceptual advancement is original and the data is rich and persuading. The Discussion part of the manuscript is well written.

Weaknesses:

Other sections of the manuscript need some more polish, both in terms of the terminology, the language and the logic of the presentation of the subject. The title is not good. During most of the text, the authors do not properly follow a certain terminology regarding migration, over-wintering, non-breeding range, and this is very confusing. So, consistency of the text is warranted. A bigger issue is the selection of latitudes (or the actual reason for movement) during the over-wintering period. The study claims that this relates to snow cover but fails to properly demonstrate it. It is likely that the birds move because of changes in snow cover rather than because of the level of snow cover. This is a testable prediction. A possible explanation is that there is a cost for moving further south and thus the birds are reluctant of moving unless they are forced to do it by the high snow cover. Another, similar and testable prediction is that the birds aim at selecting latitudes where snow cover is partial and move slowly during the winter to areas that are only partially covered by the snow with the progression of the winter. A modified, non-linear, snow cover analysis using GAMM could uncover such patterns.

Reviewer #1 (Public review):

Summary:

In an era of increasing antibiotic resistance, there is a pressing need for the development of novel sustainable therapies to tackle problematic pathogens. In this study, the authors hypothesize that pyoverdines - metal-chelating compounds produced by fluorescent pseudomonads - can act as antibacterials by locking away iron, thereby arresting pathogen growth. Using biochemical, growth and virulence assays on 12 opportunistic pathogens strains, the authors demonstrate that pyoverdines induce iron starvation, but this affect was highly context dependent. This same effect has been demonstrated for plant pathogens, but not for human opportunistic pathogens exposed to natural siderophores. Only those pathogens lacking (1) a matching receptor to take up pyoverdine-bound iron and/or (2) the ability to produce strong iron chelators themselves experienced strong growth arrest. This would suggest that pyoverdines might not be effective against all pathogens, thereby potentially limiting the utility of pyoverdines as global antibacterials.

Strengths:

The work addresses an important and timely question - can pyoverdines be used as an alternative strategy to deal with opportunistic pathogens? In general, the work is well conducted with rigorous biochemical, growth and virulence assays. In line, the work is clearly written, and the findings are supported by high-quality figures.

Weaknesses:

I do not think there are any 'weaknesses' as such. The authors have taken all suggestions on board and this has greatly improved the quality and robustness of the work

Joint Public Review:

Summary:

This study presents a strategy to efficiently isolate PcrV-specific BCRs from human donors with cystic fibrosis who have/had Pseudomonas aeruginosa (PA) infection. Isolation of mAbs that provide protection against PA may be a key to developing a new strategy to treat PA infection as the PA has intrinsic and acquired resistance to most antibiotic drug classes. Hale et al. developed fluorescently labeled antigen-hook and isolated mAbs with anti-PA activity. Overall, the authors' conclusion is supported by solid data analysis presented in the paper. Four of five recombinantly expressed PcrV-specific mAbs exhibited anti-PA activity in a murine pneumonia challenge model as potent as the V2L2MD mAb (equivalent to gremubamab). However, therapeutic potency for these isolated mAbs is uncertain as the gremubamab has failed in Phase 2 trials. Clarification of this point would greatly benefit this paper.

Strengths:

(1) High efficiency of isolating antigen-specific BCRs using an antigenic hook.

(2) The authors' conclusion is supported by data.

Weaknesses:

Although the authors state that the goal of this study was to generate novel protective mAbs for therapeutic use (P12; Para. 2), it is unclear whether PcrV-specific mAbs isolated in this study have therapeutic potential better than the gremubamab, which has failed in Phase 2 trials. Four of five PcrV-specific mAbs isolated in this study reduced bacterial burdens in mice as potent as, but not superior to, gremubamab-equivalent mAb. Clarification of this concern by revising the text or providing experimental results that show better potential than gremubamab would greatly benefit this paper.

Reviewer #1 (Public review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.

The authors observed that NMIIA is required for durotaxis and, buiding on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.

The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

Reviewer #1 (Public review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.

The authors observed that NMIIA is required for durotaxis and, buiding on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.

The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

Joint Public review:

Summary

This manuscript offers significant insights into the impact of maternal obesity on oocyte methylation and its transgenerational effects. Chao and colleagues demonstrated the potential mechanisms behind the DNA methylation changes. The major observations of the work include transgenerational DNA methylation changes in offspring of maternal obesity and metabolites such as methionine and melatonin which correlated with the epigenetic changes. Exogenous melatonin treatment could reverse the effects of obesity. The authors further hypothesized that the linkage may be mediated by the cAMP/PKA/CREB pathway to regulate the expression of DNMTs. This work has done lots of breeding and DNA Methylation analysis across multiple generations, which provides solid data for future research. The results of this work may benefit from deeper data analysis to make more causal analyses and conclusions more concrete.

Strengths

The study employs comprehensive methodologies, including transgenerational breeding experiments, whole genome bisulfite sequencing, and metabolomics analysis, and provides the convincing data.

Weaknesses

The results of this work are correlational, which may require further analysis to establish more concrete conclusions on causal relationships.

Reviewer #1 (Public review):

Summary:

In this work by Wang et al., the authors use single-molecule super-resolution microscopy together with biochemical assays to quantify the organization of Nipah virus fusion protein F (NiV-F) on cell and viral membranes. They find that these proteins form nanoscale clusters which favors membrane fusion activation, and that the physical parameters of these clusters are unaffected by protein expression level and endosomal cleavage. Furthermore, they find that the cluster organization is affected by mutations in the trimer interface on the NiV-F ectodomain and the putative oligomerization motif on the transmembrane domain, and that the clusters are stabilized by interactions among NiV-F, the AP2-complex, and the clathrin coat assembly. This work improves our understanding of the NiV fusion machinery, which may also have implications for our understanding of the function of other viruses.

Strengths:

The conclusions of this paper are well-supported by the presented data. This study sheds light on the activation mechanisms underlying the NiV fusion machinery.

Reviewer #1 (Public review):

Summary:

The authors make a new contribution with careful computational validation/exploration of their method on synthetic and real-world datasets. Overall, I find their results significant and their presentation compelling.

Strengths:

The authors provide extensive computational validation of their approach to synthetic and real-world datasets of increasing complexity.

Weaknesses:

The authors should provide a comparison of their approach to other state-of-the-art neural network-based methods. Without this, it is difficult to tell which aspects of their approach (novel coupling metric, or network architecture) are most important for their results.