Reviewer #3 (Public review):

Summary:

The tissue regeneration enhancer elements (TREEs) identified in zebrafish have been shown to drive injury-activated temporal-spatial gene expression in mice and large animals. These findings increase the translational potential of findings in zebrafish to mammals. In this manuscript, the authors tested TREEs in combination with different adeno-associated viral (AAV) vectors using in vivo luciferase bioluminescent imaging that allows for longitudinal tracking. The TREE-driven luciferase delivered by a liver de-targeted AAV.cc84 decreased off-target transduction in the liver. They further screened an AAV library to identify capsid variants that display enhanced transduction for myocardium post-myocardial infarction. A new capsid variant, AAV.IR41, was found to show increased transduction at the infarct border zones.

Strengths:

The authors injected AAV-cargo several days after ischemia/reperfusion (I/R) injury as a clinically relevant approach. Overall, this study is significant in that it identifies new AAV vectors for potential new gene therapies in the future. The manuscript is well-written, and their data are also of high quality.

Weaknesses:

The authors might be using MI (myocardial infarction) and I/R injury interchangeably in their text and labels. For instance, "We systemically transduced mice at 4 days after permanent left coronary artery ligation with either AAV9 or IR41 harboring a 2ankrd1aEN-Hsp68::fLuc transgene. IVIS imaging revealed higher expression levels in animals transduced with IR41 compared to AAV9, in both sham and I/R groups (Fig. 5A)". They should keep it consistent. There is also no description for the MI model.

Reviewer #3 (Public review):

Summary

This short paper aims to provide an independent validation of the transgenerational inheritance of learned behaviour (avoidance) that has been published by the Murphy lab. The robustness of the phenotype has been questioned by the Hunter lab. In this paper, the authors present one figure showing that transgenerational inheritance can be replicated in their hands. Overall, it helps to shed some light on a controversial topic.

Strengths

The authors clearly outline their methods, particularly regarding the choice of assay, so that attempting to reproduce the results should be straightforward. It is nice to see these results repeated in an independent laboratory.

Weaknesses

Previous reports on this topic have provided raw data, which is helpful when assessing sample sizes. The authors provided a spreadsheet containing the choice assay results for individual assays, but not the raw data. In the methods, it is stated that F2 animals were produced from F1 animals by bleaching, but there are many more F2 assays than F1. Were multiple F2 assays performed on the offspring from one F1 plate? If so, they do not represent independent assays.

I think that the introduction somewhat overstates their findings - do they really "address potential methodological variations that might influence results"? This makes it sound as though they test different conditions, whereas they only use one assay setup throughout.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally there is little to criticise about the study.

Weaknesses:

The methods are robust, and generally there is little to criticise about the study. The extent of the testing in human participants is fairly limited, but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later), although this could simply reflect a lengthy evolutionary process.

Reviewer #3 (Public review):

In this very thorough study, the authors characterize the function of a novel Drosophila gene, which they name Sakura. They start with the observation that sakura expression is predicted to be highly enriched in the ovary and they generate an anti-sakura antibody, a line with a GFP-tagged sakura transgene, and a sakura null allele to investigate sakura localization and function directly. They confirm the prediction that it is primarily expressed in the ovary and, specifically, that it is expressed in germ cells, and find that about 2/3 of the mutants lack germ cells completely and the remaining have tumorous ovaries. Further investigation reveals that Sakura is required for piRNA-mediated repression of transposons in germ cells. They also find evidence that sakura is important for germ cell specification during development and germline stem cell maintenance during adulthood. However, despite the role of sakura in maintaining germline stem cells, they find that sakura mutant germ cells also fail to differentiate properly such that mutant germline stem cell clones have an increased number of "GSC-like" cells. They attribute this phenotype to a failure in the repression of Bam by dpp signaling. Lastly, they demonstrate that sakura physically interacts with otu and that sakura and otu mutants have similar germ cell phenotypes. Overall, this study helps to advance the field by providing a characterization of a novel gene that is required for oogenesis. The data are generally high-quality and the new lines and reagents they generated will be useful for the field.

Comments on latest version:

With these revisions, the authors have addressed my main concerns.

Reviewer #3 (Public review):

Summary:

The authors suggest a mechanism that explains the preference of<br /> viral protein 35 (VP35) homologs to bind the backbone of double stranded RNA versus blunt ends. These preferences have a biological impact in terms of the ability of different viruses to escape the immune response of the host.<br /> The proposed mechanism involves the existence of a cryptic pocket, where VP35 binds the blunt ends of dsRNA when the cryptic pocket is closed and preferentially binds the RNA double stranded backbone when the pocket is open.<br /> The authors performed MD simulation results, thiol labelling experiments, fluorescence polarization assays, as well as point mutations to support their hypothesis.

Strengths:

This is a genuinely interesting scientific questions, which is approached through multiple complementary experiments as well as extensive MD simulations. Moreover, structural biology studies focused on RNA-protein interactions are particularly rare, highlighting the importance of further research in this area.

Weaknesses:

- Sequence similarity between Ebola-Zaire (94% similarity) explains their similar behaviour in simulations and experimental assays. Marburg instead is a more distant homolog (~80% similarity relative to Ebola/Zaire). This difference is sequence and structure can explain the propensities, without the need to involve the existence of a cryptic pocket.<br /> - No real evidence for the presence of a cryptic pocket is presented, but rather a distance probability distribution between two residues obtained from extensive MD simulations. It would be interesting to characterise the modelled RNA-protein interface in more detail

Comments on revisions:

-I still think that the term cryptic pocket is misleading here, unless the cryptic pocket is more thoroughly characterised. I would find it more appropriate to use the term open/closed state.

- Mg ions are known to be crucial in stabilising RNA structure both in vitro and in MD simulations (see e.g. Draper BJ 2008 and many others). While I understand that the authors cannot repeat simulations in presence of ions, I believe that this detail should be more clearly detailed in the manuscript.

Reviewer #3 (Public review):

The manuscript by Tie et al. provides a quantitative assessment of intra-Golgi transport of diverse cargos. Quantitative approaches using fluorescence microscopy of RUSH synchronized cargos, namely GLIM and measurement of Golgi residence time, previously developed by the author's team (publications from 20216 to 2022), are being used here.

Most of the results have been already published by the same team in 2016, 2017, 2020 and 2021. In this manuscript, the authors have put together measurement of intra-Golgi transport kinetics and Golgi residence time of many cargos. The quantitative results are supported by a large number of Golgi mini-stacks/cells analyzed. They are discussed with regard to the intra-Golgi transport models being debated in the field, namely the cisternal maturation/progression model and the stable compartments model.

The authors show that different cargos have distinct intra-Golgi transport kinetics and that the Golgi residence time of glycosyltransferases is high. From this and experiment using brefeldinA, the authors suggest that the rim progression model, adapted from the stable compartments model, fits with their experimental data.

Strengths:<br /> The major strength of this manuscript is to put together many quantitative results that the authors previously obtained and to discuss them to advance our understanding of the intra-Golgi transport mechanisms.<br /> The analysis by fluorescence microscopy of intra-Golgi transport is tough and this is a tour de force of the authors even though their approach shows limitations, which are clearly stated. Their work is remarkable in regards of the numbers of Golgi markers and secretory cargos which have been analyzed.

Weaknesses:<br /> Most of the data provided here were already published and thus accessible for the community. The tubular connections between cisternae and the diffusion/biochemical properties of cargos are not taken into account to interpret the results. Indeed, tubular connections and biochemical properties of the cargos may affect their transit through the Golgi and the kinetics with which they reach the TGN for Golgi exit.

The use of nocodazole might affect cellular homeostasis but this is clearly stated by the authors and is acceptable as we need to perturb the system to conduct this analysis.

The manual selection of the Golgi mini-stack being analyzed (where the cargo and the Golgi reference markers are clearly detectable ) might introduce a bias in the analysis.

Reviewer #3 (Public review):

Summary:

In this important work, the authors use extensive MD simulations to study how the IRE1 protein can detect unfolded peptides. Their study consolidates contradicting experimental results and offers a unique view of the different sensing models that have been proposed in the literature. Overall, it is an excellent study that is quite extensive. The research is solid, meticulous, and carefully performed, leading to convincing conclusions.

Strengths:

The strength of this work is the extensive and meticulous molecular dynamics simulations. The authors use and investigate different structural models, for example, carefully comparing a model based on a PDB structure with reconstructed loops with an AlphaFold 2 Multimer model. The author also investigates a wide range of different protein structural models that probe different aspects of the peptide sensing process. These solid and meticulous MD simulations allow the authors to obtain convincing conclusions concerning the peptide sensing process of the IRE1 protein.

Weaknesses:

A potential weakness of the study is the usage of equilibrium (unbiased) molecular dynamics simulations, so that processes and conformational changes on the microsecond time scale can be probed. Furthermore, there can be inaccuracies and biases in the description of unfolded peptides and protein segments due to the protein force fields. Here, it should be noted that the authors do acknowledge these possible limitations of their study in the conclusions.

Reviewer #3 (Public review):

Summary:

The study adapts CRISPR-based detection toolkit (SHERLOCK assay) using conserved and species-specific targets for the detection of some members of the Trypanosomatidae family of veterinary importance and species-specific assays to differentiate between the six most common animal trypanosome species responsible for AAT (SHERLOCK4AAT). The assays were able to discriminate between Trypanozoon (T. b. brucei, T. evansi, and T. equiperdum), T. congolense (Savanah, Forest Kilifi, and Dzanga sangha), T. vivax, T. theileri, T. simiae, and T. suis. The design of both broad and species-specific assays was based primarily on sequences of the 18S rRNA, GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), and invariant flagellum antigen (IFX) genes for species identification. Most importantly, the authors showed varying limits of detection for the different SHERLOCK assays, which is somewhat comparable to PCR-derived molecular techniques currently used for detecting animal trypanosomes, even though some of these methodologies have used other primers that target genes such as ITS1 and 7SL sRNA.

The data presented in the study are particularly useful and of significant interest for the diagnosis of AAT in affected areas.

Strengths:

The assays convincingly allow for the analysis and detection of most trypanosomes in AAT.

Weaknesses:

Inability for the assay to distinguish T. b. brucei, T. evansi, and T. equiperdum using the 18S rRNA gene, as well as the IFX gene, not achieving the sensitivity requirements for detection of T. vivax. Both T. brucei brucei and T. vivax are the most predominant infective species in animals (in addition to T. congolense), therefore, a reliable assay should be able to convincingly detect these to allow for proper use of the diagnostic assay.

Reviewer #3 (Public review):

Summary:

The paper investigates the TMEM16 family of membrane proteins, which play roles in lipid scrambling and ion transport. A total of 27 experimental structures from five TMEM16 family members were analyzed, including mammalian and fungal homologs (e.g., TMEM16A, TMEM16F, TMEM16K, nhTMEM16, afTMEM16). The identified structures were in both Ca²⁺-bound (open) and Ca²⁺-free (closed) states to compare conformations and were preprocessed (e.g., modeling missing loops) and equilibrated. Coarse-grain simulations were performed in DOPC membranes for 10 microseconds to capture the scrambling events. These events were identified by tracking lipids transitioning between the two membrane leaflets and they analysed correlation between scrambling rates, in addition, structural properties such as groove dilation and membrane thinning were calculated. They report 700 scrambling events across structures and the figure 2 elaborates on how open structures show higher activity, also as expected. The authors also address how structures may require open groove, this and other mechanisms around scrambling is a bit controversial in the field.

Strengths:

The strength of this study emerges from comparative analysis of multiple structural starting points and understand global/local motions of the protein with respect to lipid movement. Although the protein is well-studied, both experimentally and computationally, the understanding of conformational events in different family members, especially membrane thickness less compared to fungal scramblases offers good insights.

Weaknesses:

The weakness of the work is to fully reconcile with experimental evidence of Ca²⁺-independent scrambling rates observed in prior studies, but this part is also challenging using coarse-grain molecular simulations. Previous reports have identified lipid crossing, packing defects and other associated events, so it is difficult to place this paper in that context. However, the absence of validation leaves certain claims, like alternative scrambling pathways, speculative.

Reviewer #3 (Public review):

Summary:

Nestor and colleagues identify genes escaping X chromosome inactivation (XCI) in rare individuals with non-mosaic XCI (nmXCI) whose tissue-specific RNA-seq datasets were obtained from the GTEX database. Because XCI is non-mosaic, read counts representing a second allele are tested for statistical significant escape, in this case > 2.5% of active X expression. Whereas a prior GTEX analysis found only one nmXCI female, this study finds two additional donors in GTEX, therefore expanding the number of assessed X-linked genes to 380. Although this is fewer than half of X-linked genes, the study demonstrates that although rare, nmXCI females are represented in RNA-seq databases such as GTEX. Therefore this analytical approach is worthwhile pursuing in other (larger) databases as well, to provide deeper insight into escape from XCI which is relevant to X-linked diseases and sex differences.

Strengths:

The analysis is well-documented, straight-forward and valuable. The supplementary tables are useful, and the claims in the main text well-supported.

Weaknesses:

There are very few, except that this escape catalogue is limited to 3 donors, based on a single (representative) tissue screen in 285 female donors, mostly using muscle samples. However, if only pituitary samples had been screened, nmXCI-1 would have been missed. Additional donors in the 285 representative samples cross a lower threshold of AE = 0.4. It would be worthwhile to query all tissues of the 285 donors to discover more nmXCI cases, as currently fewer than half of X-linked genes received a call using this very worthwhile approach.

Comments on revised version:

The authors incorporated some textual changes, but deferred any new analysis, or expansion from these two new skewed donors to include more individuals/tissues, or going more in depth for individual genes to future manuscripts. They appear to have that option at eLife.

Reviewer #3 (Public review):

Summary:

This study investigates the role of BICC1 in the regulation of PKD1 and PKD2 and its impact on cytogenesis in ADPKD. By utilizing co-IP and functional assays, the authors demonstrate physical, functional, and regulatory interactions between these three proteins.

Strengths:

(1) The scientific principles and methodology adopted in this study are excellent, logical, and reveal important insights into the molecular basis of cystogenesis.

(2) The functional studies in animal models provide tantalizing data that may lead to a further understanding and may consequently lead to the ultimate goal of finding a molecular therapy for this incurable condition.

(3) In describing the patients from the Arab cohort, the authors have provided excellent human data for further investigation in large ADPKD cohorts. Even though there was no patient material available, such as HUREC, the authors have studied the effects of BICC1 mutations and demonstrated its functional importance in a Xenopus model.

Weaknesses:

This is a well-conducted study and could have been even more impactful if primary patient material was available to the authors. A further study in HUREC cells investigating the critical regulatory role of BICC1 and potential interaction with mir-17 may yet lead to a modifiable therapeutic target.

Conclusion:<br /> The authors achieve their aims. The results reliably demonstrate the physical and functional interaction between BICC1 and PKD1/PKD2 genes and their products.

The impact is hopefully going to be manifold:

(1) Progressing the understanding of the regulation of the expression of PKD1/PKD2 genes.

(2) Role of BiCC1 in mir/PKD1/2 complex should be the next step in the quest for a modifiable therapeutic target.

Reviewer #3 (Public review):

Summary:

The authors seek to determine the underlying traits that support the exceptional capacity of Aspergillus oryzae to secrete enzymes and heterologous proteins. To do so, they leverage the availability of multiple domesticated isolates of A. oryzae along with other Aspergillus species to perform comparative imaging and genomic analysis.

Strengths:

The strength of this study lies in the use of multifaceted approaches to identify significant differences in hyphal morphology that correlate with enzyme secretion, which is then followed by the use of genomics to identify candidate functions that underlie these differences.

Weaknesses:

There are aspects of the methods that would benefit from the inclusion of more detail on how experiments were performed and data interpreted.

Overall, the authors have achieved their aims in that they are able to clearly document the presence of two distinct hyphal forms in A. oryzae and other Aspergillus species, and to correlate the presence of the thicker, rapidly growing form with enhanced enzyme secretion. The image analysis is convincing. The discovery that the addition of yeast extract and specific amino acids can stimulate the formation of the novel hyphal form is also notable. Although the conclusions are generally supported by the results, this is perhaps less so for the genetic analysis as it remains unclear how direct the role of RseA and the calcium transporters might be in supporting the formation of the thicker hyphae.

The results presented here will impact the field. The complexity of hyphal morphology and how it affects secretion is not well understood despite the importance of these processes for the fungal lifestyle. In addition, the description of approaches that can be used to facilitate the study of these different hyphal forms (i.e., stimulation using yeast extract or specific amino acids) will benefit future efforts to understand the molecular basis of their formation.

Reviewer #3 (Public review):

In this manuscript, Yang et al. characterize the endocytic accessory protein CCDC32, which has implications in cardio-facio-neuro-developmental syndrome (CFNDS). The authors clearly demonstrate that the protein CCDC32 has a role in the early stages of endocytosis, mainly through the interaction with the major endocytic adaptor protein AP2, and they identify regions taking part in this recognition. Through live cell fluorescence imaging and electron microscopy of endocytic pits, the authors characterize the lifetimes of endocytic sites, the formation rate of endocytic sites and pits and the invagination depth, in addition to transferrin receptor (TfnR) uptake experiments. Binding between CCDC32 and CCDC32 mutants to the AP2 alpha appendage domain is assessed by pull down experiments. While interaction between CCDC32 and the alpha appendage domain of AP2 is clearly described, a discussion of potential association with other AP2 domains would be beneficial to understand the impact of CCDC32 in endocytosis.

Together, these experiments allow deriving a phenotype of CCDC32 knock-down and CCDC32 mutants within endocytosis, which is a very robust system, in which defects are not so easily detected. A mutation of CCDC32, mimicking CFNDS mutations, is also addressed in this study and shown to have endocytic defects.

In summary, the authors present a strong combination of techniques, assessing the impact of CCDC32 in clathrin mediated endocytosis and its binding to AP2.

Reviewer #3 (Public review):

Summary:<br /> The article explores the role of mother-child interactions in the development of children's social cognition, focusing on Theory of Mind (ToM) and Social Pain Matrix (SPM) networks. Using a naturalistic fMRI paradigm involving movie viewing, the study examines relationships among children's neural development, mother-child neural synchronization, and interaction quality. The authors identified a developmental pattern in these networks, showing that they become more functionally distinct with age. Additionally, they found stronger neural synchronization between child-mother pairs compared to child-stranger pairs, with this synchronization and neural maturation of the networks associated with the mother-child relationship and parenting quality.

Strengths:<br /> This is a well-written paper, and using dyadic fMRI and naturalistic stimuli enhances its ecological validity, providing valuable insights into the dynamic interplay between brain development and social interactions.

Weaknesses:<br /> The current sample size (N = 34 dyads) is a limitation, particularly given the use of SEM, which generally requires larger samples for stable results. Although the model fit appears adequate, this does not guarantee reliability with the current sample size.

Reviewer #3 (Public review):

Summary:

Mora et al employ published ChIP-seq and RNA-seq from embryonic tissues to nominate transcription factors that work combinatorially during development. This manuscript addresses an important gap in knowledge regarding the complexities of gene regulation. However, as written, the manuscript is focused on confirming mostly known associations and does not unveil principles that can be broadly applied, given multiple technical caveats that are outlined below.

Strengths:

(1) Instead of focusing on a single transcription factor motif enriched within peaks, the authors search the flanking regions of enriched motifs to nominate additional transcription factors that may work cooperatively to provide organ specificity. This type of analysis is a crucial next step in the gene regulation field, as transcription factors rarely work independently.

(2) Figure 6 is a good demonstration of the preliminary experiments that can be done to test the activity of co-occurring motifs.

(3) This is a really nice resource of organ-specific motif associations that can be used to generate many testable hypotheses.

(4) The rationale and writing are very clear and easy to read.

Weaknesses:

(1) Much of this manuscript focuses on confirming transcription factor relationships that have been reported previously. For example, it is well known that GATA4 interacts with MEF2 in the ventricle. There are limited new or unexpected associations discussed and tested.

(2) Embryonic tissues are highly heterogeneous, limiting the utility of the bulk ChIP-seq employed in these analyses. Does the cellular heterogeneity explain the discrepancy between TEAD binding and histone acetylation? Similarly, how does conservation between species affect the TF predictions?

(3) Some of the interpretations should also be fleshed out a bit more to clarify the advantage of the analyses presented here. For example, if Gata4 and Foxa2 transcripts are expressed during different stages of development, then it's likely that (as stated by the authors) these motifs are not used during the same stage of development. But examining the flanking regions wasn't necessary to make that statement. This type of conclusion seems tangential to the benefit of this analysis, which is to understand which TFs work together in a single organ at a single time point.

(4) This manuscript hinges on luciferase assays whose results can be difficult to translate to complex gene regulation networks. Many motifs are often clustered together, which makes designing experiments at endogenous loci important in studies such as this one.

Reviewer #3 (Public review):

This manuscript is a continuation of past work by the last author where they looked at stochasticity in developmental processes leading to inter-individual behavioural differences. In that work, the focus was on a specific behaviour under specific conditions while probing the neural basis of the variability. In this work, the authors set out to describe in detail how stable individuality of animal behaviours is in the context of various external and internal influences. They identify a few behaviours to monitor (read outs of attention, exploration, and 'anxiety'); some external stimuli (temperature, contrast, nature of visual cues, and spatial environment); and two internal states (walking and flying).

They then use high-throughput behavioural arenas - most of which they have built and made plans available for others to replicate - to quantify and compare combinations of these behaviours, stimuli, and internal states. This detailed analysis reveals that:

(1) Many individualistic behaviours remain stable over the course of many days.<br /> (2) That some of these (walking speed) remain stable over changing visual cues. Others (walking speed and centrophobicity) remain stable at different temperatures.<br /> (3) All the behaviours they tested fail to remain stable over spatially varying environment (arena shape).<br /> (4) and only angular velocity (a read out of attention) remains stable across varying internal states (walking and flying)

Thus, the authors conclude that there is a hierarchy in the influence of external stimuli and internal states on the stability of individual behaviours.

The manuscript is a technical feat with the authors having built many new high-throughput assays. The number of animals are large and many variables have been tested - different types of behavioural paradigms, flying vs walking, varying visual stimuli, different temperature among others.

Comments on revisions:'

The authors have addressed my previous concerns.

Reviewer #3 (Public review):

Summary:

The study investigates the development of reinforcement learning across the lifespan with a large sample of participants recruited for an online game. It finds that children gradually develop their abilities to learn reward probability, possibly hindered by their immature spatial processing and probabilistic reasoning abilities. Motor noise and exploration after a failure all contribute to children's subpar performance.  

Strengths:

Experimental manipulations of both the continuity of movement options and the probabilistic nature of the reward function enable the inference of what cognitive factors differ between age groups. <br /> A large sample of participants is studied.<br /> The model-based analysis provides further insights into the development of reinforcement learning ability. 

Weaknesses:

The conclusion that immature spatial processing and probabilistic reasoning abilities limit reinforcement learning here still needs more direct evidence.

Reviewer #3 (Public review):

Nucleus HVC is critical both for song production as well as learning and arguably, sitting at the top of the song control system, is the most critical node in this circuit receiving a multitude of inputs and sending precisely timed commands that determine the temporal structure of song. The complexity of this structure and its underlying organization seem to become more apparent with each experimental manipulation, and yet our understanding of the underlying circuit organization remains relatively poorly understood. In this study, Trusel and Roberts use classic whole-cell patch clamp techniques in brain slices coupled with optogenetic stimulation of select inputs to provide a careful characterization and quantification of synaptic inputs into HVC. By identifying individual projections neurons using retrograde tracer injections combined with pharmacological manipulations, they classify monosynaptic inputs onto each of the three main classes of glutamatergic projection neurons in HVC (RA-, Area X- and Av-projecting neurons). This study is remarkable in the amount of information that it generates, and the tremendous labor involved for each experiment, from the expression of opsins in each of the target inputs (Uva, NIf, mMAN and Av), the retrograde labelling of each type of projection neuron, and ultimately the optical stimulation of infected axons while recording from identified projection neurons. Taken together, this study makes an important contribution to increasing our identification, and ultimately understanding, of the basic synaptic elements that make up the circuit organization of HVC, and how external inputs, which we know to be critical for song production and learning, contribute to the intrinsic computations within this critic circuit.

This study is impressive in its scope, rigorous in its implementation and thoughtful regarding its limitations. The manuscript is well written, and I appreciate the clarity with which the authors use our latest understanding of the evolutionary origins of this circuit to place these studies within a larger context and their relevance to the study of vocal control, including human speech. My comments are minor and primarily about legibility, clarification of certain manipulations and organization of some of the summary figures.

Comments on revisions:

The authors have done a very nice job addressing the reviewers' comments.

Reviewer #3 (Public review):

Summary:

Wang et al., examined the brain activity patterns during sleep, especially when locked to those canonical sleep rhythms such as SO, spindle, and their coupling. Analyzing data from a large sample, the authors found significant coupling between spindles and SOs, particularly during the up-state of the SO. Moreover, the authors examined the patterns of whole-brain activity locked to these sleep rhythms. The authors next investigated the functional connectivity analyses, and found enhanced connectivity between the hippocampus and the thalamus and the medial PFC. These results reinforced the theoretical model of sleep-dependent memory consolidation, such that SO-spindle coupling is conducive for systems-level memory reactivation and consolidation.

Strengths:

There are obvious strengths in this work, including the large sample size, state-of-the-art neuroimaging and neural oscillation analyses, and the richness of results. The results now inform hemodynamic neural activity that coincided with SO-spindle couplings.

Weaknesses:

My earlier comments were about the inability to make inferences on memory given the lack of memory tasks, and the weakness in using the open-ended cognitive state decoding.

The current revision has addressed these major concerns. The authors expanded discussions regarding the theoretical implications of the work in a more nuanced manner.

Reviewer #3 (Public review):

Summary:

Kim et al. present a study of the neural dynamics underlying reversal learning in monkey PFC and neural networks. Their main finding is that neural activity during fixation resembles a line attractor storing the current belief of the reversal state of the task. This is followed by richer dynamics unfolding throughout the remainder of the trial, which eventually converge to a new point on the line attractor by the start of the next trial. The idea of studying neural dynamics throughout the task (including intervening behaviour) is interesting, and the data provides some insights into the neural dynamics driving reversal learning. The modelling seems to support the analyses, but both the modelling and analyses also leave several open questions.

Strengths:

The paper addresses an interesting topic of the neural dynamics underlying reversal learning in PFC, using a combination of biological and simulated data. Reversal learning has been studied extensively in neuroscience, but this paper takes a step further by analysing neural dynamics throughout the trials instead of focusing on just the evidence integration epoch.

The authors show some close parallels between the experimental data and RNN simulations, both in terms of behaviour and neural dynamics. The analyses of how rewarded and unrewarded trials differentially affect dynamics throughout the trials in RNNs and PFC were particularly interesting. This work has the potential to provide new insights into the neural underpinnings of reversal learning.

Weaknesses:

Data analyses:

While the analyses seem mostly sound, one shortcoming is that they are all aligned to the inferred reversal trial rather than the true experimental reversal trial. For example, the analyses showing that 'x_rev' decays strongly after the reversal trial, irrespective of the reward outcome, seem like they are true essentially by design. The choice to align to the inferred reversal trial also makes this trial seem 'special' (e.g. in Fig 2 & Fig 6A), but it is unclear whether this is a real feature of the data or an artifact of effectively conditioning on a change in behaviour. It would be useful to investigate whether any of these analyses differ when aligned to the true reversal trial. It is also unsurprising that x_rev increases before the reversal and decreases after the reversal (it is hard to imagine a system where this is not the case), yet all of Fig 6 and several other analyses are devoted to this point.

Most of the analyses focus on the dynamics specifically in the x_rev subspace, but a major point of the paper is to say that biological (and artificial) networks may also have to do other things at different times in the trial. If that is the case, it would be interesting to also ask what happens in other subspaces of neural activity, which are not specifically related to evidence integration or choice - are there other subspaces that explain substantial variance? Do they relate to any meaningful features of the experiment?

This is especially important when considering analyses trying to establish the presence (or absence) of attractor dynamics in the circuit. In particular, activity in the x_rev subspace both affects and depends on other subspaces of neural activity, so it is not as meaningful to analyse the dynamics of this subspace in isolation. It would e.g. have been preferable to analyse the early-trial dynamics in the full state space and then possibly projecting onto x_rev, rather than first projecting activity onto x_rev and then fitting a linear autoregressive model.

Modelling:

There are a number of surprising and non-standard modelling choices made in this paper. For example, the choice to only use inhibitory neurons is non-conventional and it is not clear whether and how this impacts the results. The inputs are also provided without any learnable input weights, which makes it harder to interpret the input-driven dynamics during the different phases of a trial.

It is surprising that the RNN is "trained to flip its preferred choice a few trials after the inferred scheduled reversal trial", with the reversal trial inferred by an ideal Bayesian observer. A more natural approach would be to directly train the RNN to solve the task (by predicting the optimal choice) and then investigating the emergent behaviour & dynamics. If the authors prefer their imitation learning approach, it is also surprising that the network is trained to predict the reversal trial inferred using Bayesian smoothing instead of Bayesian filtering.

Finally, it was surprising that the network is trained and tested with different block lengths (24 & 36 trials, respectively), and it is not mentioned whether or how this affects behaviour.

Reviewer #3 (Public review):

Summary:

The sex determination mechanism governed by the complementary sex determination (CSD) locus is one of the mechanisms that support the haplodiploid sex determination system evolved in hymenopteran insects. While many ant species are believed to possess a CSD locus, it has only been specifically identified in two species. The authors analyzed diploid females and the rarely occurring diploid males of the clonal ant Ooceraea biroi and identified a 46 kb CSD candidate region that is consistently heterozygous in females and predominantly homozygous in males. This region was found to be homologous to the CSD locus reported in distantly related ants. In the Argentine ant, Linepithema humile, the CSD locus overlaps with an lncRNA (ANTSR) that is essential for female development and is associated with the heterozygous region (Pan et al. 2024). Similarly, an lncRNA is encoded near the heterozygous region within the CSD candidate region of O. biroi. Although this lncRNA shares low sequence similarity with ANTSR, its potential functional involvement in sex determination is suggested. Based on these findings, the authors propose that the heterozygous region and the adjacent lncRNA in O. biroi may trigger female development via a mechanism similar to that of L. humile. They further suggest that the molecular mechanisms of sex determination involving the CSD locus in ants have been highly conserved for approximately 112 million years. This study is one of the few to identify a CSD candidate region in ants and is particularly noteworthy as the first to do so in a parthenogenetic species.

Strengths:

(1) The CSD candidate region was found to be homologous to the CSD locus reported in distantly related ant species, enhancing the significance of the findings.

(2) Identifying the CSD candidate region in a parthenogenetic species like O. biroi is a notable achievement and adds novelty to the research.

Weaknesses

(1) Functional validation of the lncRNA's role is lacking, and further investigation through knockout or knockdown experiments is necessary to confirm its involvement in sex determination.

(2) The claim that the lncRNA is essential for female development appears to reiterate findings already proposed by Pan et al. (2024), which may reduce the novelty of the study.

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Weaknesses:

Relatively minor. This is a technically strong and thorough study that is broadly useful to investigators studying retinal development and retinoblastoma.

Comments on revisions:

The authors have addressed all points raised in the review and considerably strengthened the manuscript. No additional changes are required.

Reviewer #3 (Public review):

Summary:

Central pattern generator (CPG) circuits underly rhythmic motor behaviors. To date, it is thought that these CPG networks are rather local and multiple CPG circuits are serially connected to allow locomotion across the entire body. Distributed CPG networks that incorporate long-range connections have not been proposed, although such connectivity has been experimentally shown for several different spinal populations. In this manuscript, the authors use this existing literature on long-range spinal interneuron connectivity to build a new computational model that reproduces basic features of locomotion like left-right alternation, rostrocaudal propagation, and independent control of frequency and amplitude. Interestingly, the authors show that a model solely based on inhibitory neurons can recapitulate these basic locomotor features. Excitatory sources were then added that increased the dynamic range of frequencies generated. Finally, the authors were also able to reproduce experimentally observed consequences of cell-type-specific ablations, showing that local and long-range, cell-type-specific connectivity could be sufficient for generating locomotion.

Strengths:

This work is novel, providing an interesting alternative to distributed CPGs to the local networks traditionally predicted. It shows cell type cell-type-specific network connectivity is as important, if not more than intrinsic cell properties for rhythmogenesis and that inhibition plays a crucial role in shaping locomotor features. Given the importance of local CPGs in understanding motor control, this alternative concept will be of broad interest to the larger motor control field, including invertebrate and vertebrate species.

Weaknesses:

I have the following minor concerns/clarifications:

(1) The authors describe a single unit as a neuron, be it excitatory or inhibitory, and the output of the simulation is the firing rate of these neurons. Experimentally and in other modeling studies, motor neurons are incorporated in the model, and the output of the network is based on motor neuron firing rate, not the interneurons themselves. Why did the authors choose to build the model this way?

(2) In the single population model (Figure 1), the authors use ipsilateral inhibitory connections that are long-range in an ascending direction. Experimentally, these connections have been shown to be local, while long-range ipsilateral connections have been shown to be descending. What were the reasons the authors chose this connectivity? Do the authors think local ascending inhibitions contribute to rostrocaudal propagation, and how?

(3) In the two-population model, the authors show independent control of frequency and rhythm, as has been reported experimentally. However, in these previous experimental studies, frequency and amplitude are regulated by different neurons, suggesting different networks dedicated to frequency and amplitude control. However, in the current model, the same population with the same connections can contribute to frequency or amplitude depending on relative tonic drive. Can the authors please address these differences either by changes in the model or by adding to the Discussion?

(4) It would be helpful to add a paragraph in the Discussion on how these results could be applicable to other model systems beyond zebrafish. Cell intrinsic rhythmogenesis is a popular concept in the field, and these results show an interesting and novel alternative. It would help to know if there is any experimental evidence suggesting such network-based propagation in other systems, invertebrates, or vertebrates.

Reviewer #3 (Public review):

Summary:

In this study, the authors were looking at neurocorrelates of behavioural differences within the genus Macaca. To do so, they engaged in real-world dissection of dead animals (unconnected to the present study) coming from a range of different institutions. They subsequently compare different brain areas, here the amygdala and the hippocampus, across species. Crucially, these species have been sorted according to different levels of social tolerance grades (from 1 to 4). 12 species are represented across 42 individuals. The sampling process has weaknesses ("only half" of the species contained by the genus, and Macaca mulatta, the rhesus macaque, representing 13 of the total number of individuals), but also strengths (the species are decently well represented across the 4 grades) for the given purpose and for the amount of work required here. I will not judge the dissection process as I am not a neuroanatomist, and I will assume that the different interventions do not alter volume in any significant ways / or that the different conditions in which the bodies were kept led to the documented differences across species.

There are two main results of the study. First, in line with their predictions, the authors find that more tolerant macaque species have larger amygdala, compared to the hippocampus, which remains undifferentiated across species. Second, they also identify developmental effects, although with different trends: in tolerant species, the amygdala relative volume decreases across the lifespan, while in intolerant species, the contrary occurs. The results look quite strong, although the authors could bring up some more clarity in their replies regarding the data they are working with. From one figure to the other, we switch from model-calculated ratio to model-predicted volume. Note that if one was to sample a brain at age 20 in all the grades according to the model-predicted volumes, it would not seem that the difference for amygdala would differ much across grades, mostly driven with Grade 1 being smaller (in line with the main result), but then with Grade 2 bigger than Grade 3, and then Grade 4 bigger once again, but not that different from Grade 2.

Overall, despite this, I think the results are pretty strong, the correlations are not to be contested, but I also wonder about their real meaning and implications. This can be seen under 3 possible aspects:

(1) Classification of the social grade

While it may be familiar to readers of Thierry and collaborators, or to researchers of the macaque world, there is no list included of the 18 behavioral traits used to define the three main cognitive requirements (socio-cognitive demands, predictability of the environment, inhibitory control). It would be important to know which of the different traits correspond to what, whether they overlap, and crucially, how they are realized in the 12 study species, as there could be drastic differences from one species to the next. For now, we can only see from Table S1 where the species align to, but it would be a good addition to have them individually matched to, if not the 18 behavioral traits, at least the 3 different broad categories of cognitive requirements.

(2) Issue of nature vs nurture

Another way to look at the debate between nature vs nurture is to look at phylogeny. For now, there is no phylogenetic tree that shows where the different grades are realized. For example, it would be illuminating to know whether more related species, independently of grades, have similar amygdala or hippocampus sizes. Then the question will go to the details, and whether the grades are realized in particular phylogenetic subdivisions. This would go in line with the general point of the authors that there could be general species differences.

With respect to nurture, it is likely more complicated: one needs to take into account the idiosyncrasies of the life of the individual. For example, some of the cited literature in humans or macaques suggests that the bigger the social network, the bigger the brain structure considered. Right, but this finding is at the individual level with a documented life history. Do we have any of this information for any of the individuals considered (this is likely out of the scope of this paper to look at this, especially for individuals that did not originate from CdP)?

(3) Issue of the discussion of the amygdala's function

The entire discussion/goal of the paper, states that the amygdala is connected to social life. Yet, before being a "social center", the amygdala has been connected to the emotional life of humans and non-humans alike. The authors state L333/34 that "These findings challenge conventional expectations of the amygdala's primary involvement in emotional processes and highlight the complexity of the amygdala's role in social cognition". First, there is no dichotomy between social cognition and emotion. Emotion is part of social cognition (unless we and macaques are robots). Second, there is nowhere in the paper a demonstration that the differences highlighted here are connected to social cognition differences per se. For example, the authors have not tested, say, if grade 4 species are more afraid of snakes than grade 1 species. If so, one could predict they would also have a bigger amygdala, and they would probably also find it in the model. My point is not that the authors should try to correlate any kind of potential aspect that has been connected to the amygdala in the literature with their data (see for example the nice review by Domínguez-Borràs and Vuilleumier, https://doi.org/10.1016/B978-0-12-823493-8.00015-8), but they should refrain from saying they have challenged a particular aspect if they have not even tested it. I would rather engage the authors to try and discuss the amygdala as a multipurpose center, that includes social cognition and emotion.

Strengths:

Methods & breadth of species tested.

Weaknesses:

Interpretation, which can be described as 'oriented' and should rather offer additional views.

Reviewer #3 (Public review):

The manuscript by Rios-Jimenez developed a software tool, BEHAV3D Tumor Profiler, to analyze 3D intravital imaging data and identify distinctive tumor cell migratory phenotypes based on the quantified 3D image data. Moreover, the heterogeneity module in this software tool can correlate the different cell migration phenotypes with variable features of the tumor microenvironment. Overall, this is a useful tool for intravital imaging data analysis and its open-source nature makes it accessible to all interested users.

Strengths:

An open-source software tool that can quantify cell migratory dynamics from intravital imaging data and identify distinctive migratory phenotypes that correlate with variable features of the tumor microenvironment.

Weaknesses:

Motility is only one tumor cell feature and is probably not sufficient to characterize and identify the heterogeneity of the tumor cell population that impacts their behaviors in the complex tumor microenvironment (TME). For instance, there are important non-tumor cell types in the TME, and the interaction dynamics of tumor cells with other cell types, e.g., fibroblasts and distinct immune cells, play a crucial role in regulating tumor behaviors. BEHAV3D-TP focuses on only motility feature analysis, and cannot be applied to analyze other tumor cell dynamic features or cell-cell interaction dynamics.

Reviewer #3 (Public review):

Why mitochondria are finely maintained in the female germ cell (oocyte), zygotes, and preimplantation embryos? Mitochondrial fusion seems beneficial in somatic cells to compensate for unhealthy mitochondria, for example, mitochondria with mutated mtDNA that potentially defuel the respiratory activity if accumulated above a certain threshold. However, in the germ cells, it may rather increase the risk of transmitting mutated mtDNA to the next generation. Also, finely maintained mitochondria would also be beneficial for efficient removal when damaged, as authors briefly discussed. Due in part to the limited suitable model, physiological role of mitochondrial fission in embryos were obscure. In this study, authors demonstrated that mitochondrial fission prevents multiple adverse outcomes, especially including the aberrant demixing of parental genome (a clinical phenotype of human embryos) in zygotic stage. Thus, this study would be also of clinical importance that could contribute by proposing a novel mechanism.

After reading through the comments of other reviewers, what authors could potentially improve their manuscript had been largely summarized in three following points.

(1) Authors would better clarify whether a loss of Drp1 contributes to the chromosome segregation defects directly (e.g. checking SAC-like activity) or indirectly (aggregated mitochondria became physically obstacle; maybe in part getting the cytoskeleton involved).

(2) Although the level of Myo19 may not be so high (given the low level of TRAK2 in oocytes: Lee et al. PNAS 2024, PMID 38917013), authors would better further clarify the effect of Myo19-Trim with timelapse (e.g. EB3-GFP/Mt-DsRed) and EM analysis (detailed mitochondrial architecture).

(3) Authors would better clarify phenotypic heterogeneity/variety regarding the degree of alteration in mitochondrial morphology/ architecture dependent on the levels of Drp1 loss with detailed quantification of EM images to address why aggregation of mitochondria in Drp1-/- parthenote (possibly, more likely Drp1 protein-free) looks different/weaker than Trim-awayed one. Employment of the parthenotes of Trim-awayed MII oocytes might also complement the further discussion.

The revised preprinted have addressed all the points described above. Authors have also adequately indicated the limitations at each of the specific points. Revisions authors made have consolidated their conclusion, thus still, making this study an excellent one.

Reviewer #3 (Public review):

Summary:

The study by Squiers and colleagues reveals a novel, Commander-independent role for COMMD3 in endosomal recycling. Through unbiased genetic screens, the authors identified COMMD3 as a regulator of GLUT4-SPR trafficking and validated its function using knockout experiments, which demonstrated its impact on endosomal morphology and trafficking independent of the Commander complex. Importantly, they mapped the interaction between the N-terminal domain (NTD) of COMMD3 and the GTPase Arf1, and through structure-guided mutagenesis, established that this interaction is essential for COMMD3's Commander-independent activity. The manuscript provides compelling evidence supporting this newly identified function of COMMD3, and I find the authors' interpretations well-justified. This is an excellent and intriguing study.

Comments on revisions:

The authors addressed all comments. Congratulations on this exciting work.

Reviewer #3 (Public review):

Summary:

This paper develops a model to account for flexible and context-dependent behaviors, such as where the same input must generate different responses or representations depending on context. The approach is anchored in the hippocampal place cell literature. The model consists of a module X, which represents context, and a module H (hippocampus), which generates "sequences". X is a binary attractor RNN, and H appears to be a discrete binary network, which is called recurrent but seems to operate primarily in a feedforward mode. H has two types of units (those that are directly activated by context, and transition/sequence units). An input from X drives a winner-take-all activation of a single unit H_context unit, which can trigger a sequence in the H_transition units. When a new/unpredicted context arises, a new stable context in X is generated, which in turn can trigger a new sequence in H. The authors use this model to account for some experimental findings, and on a more speculative note, propose to capture key aspects of contextual processing associated with schizophrenia and autism.

Strengths:

Context-dependency is an important problem. And for this reason, there are many papers that address context-dependency - some of this work is cited. To the best of my knowledge, the approach of using an attractor network to represent and detect changes in context is novel and potentially valuable.

Weaknesses:

The paper would be stronger, however, if it were implemented in a more biologically plausible manner - e.g., in continuous rather than discrete time. Additionally, not enough information is provided to properly evaluate the paper, and most of the time, the network is treated as a black box, and we are not shown how the computations are actually being performed.

Reviewer #3 (Public review):

In this study, the authors employed the protein complex structure prediction tool AlphaFold-Multimer to obtain a predicted structure of the protein complex composed of ULK1-ATG13-FIP200 and validated the structure using mutational analysis. This complex plays a central role in the initiation of autophagy in mammals. The results obtained in this study reveal extensive binary interactions between ULK1 and ATG13, between ULK1 and FIP200, and between ATG13 and FIP200, and pinpoint the critical residues at each interaction interface. Mutating these critical residues led to the loss of binary interactions. Interestingly, the authors showed that the ATG13-ULK1 interaction and the ATG13-FIP200 interaction are partially redundant for maintaining the complex. The experimental data presented by the authors are of high quality and convincing. The revised manuscript offers enhanced details about the prediction procedure and results, along with additional experimental findings, significantly increasing the scientific value of this paper.

Reviewer #3 (Public review):

The authors have significantly improved the paper in revising to make its contributions distinct from their prior paper. They have also responded to my concerns about quantification and parameter dependency of the integration conclusion. While I think there is still more that could be done in this capacity, especially in terms of the temporal statistics and quantification of the conflict responses, they have a made a case for the conclusions as stated. The paper still stands as an important paper with solid evidence a bit limited by these concerns.

Reviewer #3 (Public review):

Summary:

In their revised manuscript, Sinha and colleagues aim to identify distinct causes of motor impairments seen when perturbing cerebellar circuits. This goal is an important one, given the diversity of movement related phenotypes in patients with cerebellar lesion or injury, which are especially difficult to dissect given the chronic nature of the circuit damage. To address this goal, the authors use high-frequency stimulation (HFS) of the superior cerebellar peduncle in monkeys performing reaching movements. HFS provides an attractive approach for transiently disrupting cerebellar function previously published by this group. First, they find a reduction in hand velocities during reaching, which was more pronounced for outward versus inward movements. By modeling inverse dynamics, they find evidence that shoulder muscle torques are especially affected. Next, the authors examine the temporal evolution of movement phenotypes over successive blocks of HFS trials. Using this analysis, they find that in addition to the acute, specific effects on torques in early HFS trials, there was an additional progressive reduction in velocity during later trials, which they interpret as an adaptive response to the inability to effectively compensate for interaction torques during cerebellar block. Finally, the authors examine movement decomposition and trajectory, finding that even when low velocity reaches are matched to controls, HFS produces abnormally decomposed movements and higher than expected variability in trajectory.

Strengths:

Overall, this work provides important insight into how perturbation of cerebellar circuits can elicit diverse effects on movement across multiple timescales.

The HFS approach provides temporal resolution and enables analysis that would be hard to perform in the context of chronic lesions or slow pharmacological interventions. Thus, this study describes an important advance over prior methods of circuit disruption in the monkey, and their approach can be used as a framework for future studies that delve deeper into how additional aspects of sensorimotor control are disrupted (e.g., response to limb perturbations).

In addition, the authors use well-designed behavioral approaches and analysis methods to distinguish immediate from longer-term adaptive effects of HFS on behavior. Moreover, inverse dynamics modeling provides important insight into how movements with different kinematics and muscle dynamics might be differentially disrupted by cerebellar perturbation.

Remaining comments:

The argument that there are acute and adaptive effects to perturbing cerebellar circuits is compelling, but there seems to be a lost opportunity to leverage the fast and reversible nature of the perturbations to further test this idea and strengthen the interpretation. Specifically, the authors could have bolstered this argument by looking at the effects of terminating HFS - one might hypothesize that the acute impacts on joint torques would quickly return to baseline in the absence of HFS, whereas the longer-term adaptive component would persist in the form of aftereffects during the 'washout' period. As is, the reversible nature of the perturbation seems underutilized in testing the authors' ideas. While this experimental design was not implemented here, it seems like a good opportunity for future work using these approaches.

The analysis showing that there is a gradual reduction in velocity during what the authors call an adaptive phase is convincing. While it is still not entirely clear why disruption of movement during the adaptive phase is not seen for inward targets, despite the fact that many of the inward movements also exhibit large interaction torques, the authors do raise potential explanations in the Discussion.

Reviewer #3 (Public review):

Summary:

In "A‬‭ whole-animal‬‭ phenotypic‬‭ drug‬‭ screen‬‭ identifies‬‭ suppressors‬‭ of‬‭ atherogenic‬ lipoproteins", Kelpsch et al seek to identify new, chemically targetable pathways that regulate ApoB function and could ultimately serve as treatments for elevated lipid disorders and/or cardiovascular disease. Given the interconnected nature of lipid regulation in the whole organism with interdependent organs and secreted components (i.e. lipoproteins), they use the vertebrate model zebrafish to screen a large library of ~3000 compounds for their ability to lower the important ApoB-containing lipoproteins. They find 49 hits with 19 compounds passing a higher level of scrutiny, and focus on the role of enoxolone in modulating B-Ip levels at least partly through the HNF4alpha transcription factor and, putatively, through downstream cholesterol/lipid biosynthetic pathways.

Strengths:

The study uses a well-validated in vivo stain (LipoGlo) for measuring lipoproteins in the context of a developing whole organism with a quantitative read-out on a high-throughput platform, allowing for screening of thousands of compounds altering the complex metabolic/physiologic functions necessary for lipoprotein production.

The use of genetic mutant HNF4alpha to assign the mechanism of action to the prime candidate compound studied (enoxolone) is a powerful approach for this challenging aspect of chemical genetics studies. See caveats in weaknesses.

Weaknesses:

As shown in Figure 5A, the HNF4alpha mutant homozygous -/- already lowers lipoproteins. Is it just that the mutant level is already at a minimum in this homozygous mutant (and thus enoxolone can not induce even lower lipoprotein levels), or is it true that the enoxolone molecule is primarily acting through this TF (i.e. HNF4alpha homozygous mutant is truly epistatic to enoxolone function) as favored in the text.

While it is definitely interesting to study enoxolone effects during whole embryo development, the link to HNF4alpha had previously been described in the literature, as pointed out by the authors. The generalizability of the approach to identify truly novel pathways remains to be fully realized, but sharing this available screen data to date will invite further inquiry and be very valuable to the community.

Figure 5 - The same allele of HNF4alpha loss of function/hypomorph (rdu14) is used in both 5A and 5B, but labeled differently in each subpanel. This is explained in the figure legend, but could be updated to use the same nomenclature in both panels to clarify the Figure presentation.

Reviewer #3 (Public review):

Summary:

This manuscript describes DOX inducible RNAi KD of Lamin A, LMNA coded isoforms as a group, and the LINC component SYNE2. The authors report on differentially expressed genes, on differentially expressed isoforms, on the large numbers of differentially expressed genes that are in iLADs rather than LADs, and on telomere mobility changes induced by 2 of the 3 knockdowns.

Strengths:

Overall, the manuscript might be useful as a description for reference data sets that could be of value to the community.

Weaknesses:

The results are presented as a type of data description without formulation of models or explanations of the questions being asked and without follow-up. Thus, conceptually, the manuscript doesn't appear to break new ground.

Not discussed is the previous extensive work by others on the nucleoplasmic forms of LMNA isoforms. Also not discussed are similar experiments- for instance, gene expression changes others have seen after lamin A knockdowns or knockouts, or the effect of lamina on chromatin mobility, including telomere mobility - see, for example, a review by Roland Foisner (doi.org/10.1242/jcs.203430) on nucleoplasmic lamina. The authors need to do a thorough search of the literature and compare their results as much as possible with previous work.

The authors don't seem to make any attempt to explore the correlation of their findings with any of the previous data or correlate their observed differential gene expression with other epigenetic and chromatin features. There is no attempt to explore the direction of changes in gene expression with changes in nuclear positioning or to ask whether the genes affected are those that interact with nucleoplasmic pools of LMNA isoforms. The authors speculate that the DEG might be related to changing mechanical properties of the cells, but do not develop that further.

The technical concerns include: 1) Use of only one shRNA per target. Use of additional shRNAs would have reduced concern about possible off-target knockdown of other genes; 2) Use of only one cell clone per inducible shRNA construct. Here, the concern is that some of the observed changes with shRNA KDs might show clonal effects, particularly given that the cell line used is aneuploid. 3) Use of a single, "scrambled" control shRNA rather than a true scrambled shRNA for each target shRNA.

Reviewer #3 (Public review):

The authors have addressed all of my concerns. Congratulations!

Reviewer #3 (Public review):

Summary:

Zhang et al sought to quantify the influence of the gut microbiome on metabolite cycling in a Drosophila model with extensive metabolomic profiling in 4 time points over a 24 hour period. The authors report that the microbiome enhances metabolite cycling in a context-dependent manner. The metabolomics data presented are comprehensive and complex, and they open up may new questions. The major strength of the work is the production of a large dataset of metabolites that can be the basis for hypothesis generation for more specific experiments. There are several weaknesses that make some of the conclusions speculative.

Strengths:

The revised manuscript is significantly improved due to the inclusion of new data and expanded analyses, particularly of time-resolved food intake. The dataset is comprehensive and of high value to the community. The experimental design includes multiple metabolomic comparisons across genetic and dietary conditions, specifically, germ-free versus microbially-colonized flies, time-restricted versus ad libitum feeding, high-sugar versus high protein diets, and wildtype genotype versus the per01 clock mutant. Additionally, the cycling of individual metabolites is presented, allowing readers to examine metabolites of interest. The datasets are made publicly available, allowing this resource to benefit the community.

Weaknesses

Many of the statistically significant differences, e.g. the effects of the microbiome on lipids and biogenic amines in Fig S5A, are quite small in magnitude, and, thus, it is difficult to believe that they are of biological significance without more mechanistic studies. Key conclusions, such as those pertaining to regulation or compensation by the microbiome, are not fully supported by mechanistic experiments. The manuscript uses terms like "regulate" or "compensate," which imply causality or a purpose of the microbiome that is not yet demonstrated, but this type of study opens up many important questions for which new hypotheses can be formed.

A minor limitation is the modest temporal resolution (only four time points in 24 hours), which constrains interpretation of rhythmicity and phase. Additional experimental controls and targeted perturbation experiments are needed to support conclusions about functional impacts of metabolite oscillations. However, these types of limitations are expected from an early study in the field such as this one. Overall, the data are valuable, and the findings demonstrate the promise of the model for studying the interplay between the microbiome, metabolome, and circadian rhythm.

Assessment of Aims

The authors explore how the microbiome interacts with host circadian rhythms and diet to shape metabolite cycling. They largely succeed in characterizing broad trends and generating a valuable resource dataset. However, the conclusion that the microbiome actively regulates or compensates for cycling under specific conditions is not convincingly demonstrated with the current data.

Impact and Utility

The dataset will be a useful reference for researchers interested in microbiome-host interactions, metabolomics, and circadian biology. Its primary value lies in descriptive insight rather than mechanistic resolution. An alternative perspective is that per01 mutants serve as a useful negative control for rhythmicity detection, providing a baseline for distinguishing signal from experimental noise ---an idea that could be emphasized more in the interpretation.

Contextual Considerations

Metabolomics datasets are valuable for understanding the influence of the microbiome. Future follow-up work using higher resolution sampling and functional perturbations (e.g., more extensive genetic or microbial manipulations) will be essential to test hypotheses about the roles of specific metabolites, regulatory pathways, and microbiota members in circadian modulation. This paper lays a strong foundation for such studies.

Reviewer #3 (Public Review):

In this manuscript, the authors use the data collected and observations made on bees' scanning behaviour during visual learning to design a bio-inspired artificial neural network. The network follows the architecture of bees visual systems, where photoreceptors project into the lamina, then the medulla, medulla neurons connect to a set of spiking neurons in the lobula. Lobula neurons project to kenyon cells and then to MBON, which controls reward and punishment. The authors then test the performance of the network in comparison with real bee data, finding it to perform well in all tasks. The paper attempts to reproduce a living organism network with a practical application in mind, and it is quite impressive! I appreciate both the potential implications for the understanding of biological systems and the applications in the development of autonomous agents, making the paper absolutely worth reading.

However, I believe that the current version somewhat lacks in clarity regarding the methodology and in some of the keywords used to describe the model.

Definitions:

Throughout the manuscript, the authors use some key terminology that I believe would benefit from some clarification.

The generated model is described in the title and once in the introduction as "neuromorphic". The model is definitely bio-inspired, but at least in some layers of the neural network, the model is built very differently from actual brain connectivity. Generally, when we use the term neuromorphic we imply many advantages of neural tissue, like energy efficiency, that I am not sure the current model is achieving. I absolutely see how this work is going in that direction, and I also fundamentally agree with the choice of terminology, but this should be clearly explained to not risk over-implications

The authors describe this as a model of "active vision". This is done in the title of the article, and in the many paragraph headings (methods, results). In the introduction, however, the term active vision is reserved to the description of bees' behavior. Indeed, the developed model is not a model of active vision, as this would require for the model to control the movement of the "camera". Here instead the stimuli display is given to the model in a fixed progression. What I suspect is that the authors' aim is to describe a model that supports the bees' active vision, not a model of active vision. I believe this should be very clear from the paper, and it may be appropriate to remove the term from the title.

In the short title, it said that this network is minimal. This is then characterized in the introduction as the minimal network capable of enabling active vision in bees. The authors, however, in their experiment only vary the number of lobula neurons, without changing other parts of the architecture. Given this, we can only say that 16 lobula neurons is the minimal number required to solve the experimental task with the given model. I don't believe that this is generalizable to bees, nor that this network is minimal, as there may be different architectures (for the other layers especially) that require overall less neurons. Moreover, the tasks attempted in the minimal network experiment did not include any of the complex stimuli presented in figure 3, like faces. It may be that 16 lobula neurons are sufficient for the X vs + and clockwise vs counter-clockwise spirals, but we do not know if increasing stimuli complexity would result in a failure of the model with 16 neurons.

Methodology:

The current explanation of the model is currently a bit lacking in clarity and details. This risks impacting negatively on the relevance of the whole work which is interesting and worth reading! This issue affects also the interpretation of the results, as it is not clear to what extent each part of the network could affect the results shown. This is especially the case when the network under-performs with respect to the best performing scenario (e.g., when varying the speed and part of the pattern that is observed, such as in Fig 2C). Adding a detailed technical scheme/drawing specific to the network architecture could have been a way of significantly increasing the clarity of the Methods section and the interpretation of the results.

On a similar note, the authors make some comparisons between the model and real bees. However, it remains unclear whether these similarities are actually indicative of an optimality in the bees visual scanning strategy, or just deriving from the authors design. This is for me particularly important in the experiments aimed at finding the best scanning procedure. If the initial model training is based on natural images it is performed by presenting left to right moving frames, the highest efficiency of lower-half scanning may be due to how the weights in the initial layers are structured and a low generalizability of the model, rather than to the strategy optimality

Reviewer #3 (Public review):

Summary:

The manuscript presents a detailed study on the role of MmMYL3 in the viral entry of NNV, focusing on its function as a receptor that mediates viral internalization through the macropinocytosis pathway. The use of both in vitro assays (e.g., Co-IP, SPR, and GST pull-down) and in vivo experiments (such as infection assays in marine medaka) adds robustness to the evidence for MmMYL3 as a novel receptor for RGNNV. The findings have important implications for understanding NNV infection mechanisms, which could pave the way for new antiviral strategies in aquaculture.

Strengths:

The authors show that MmMYL3 directly binds the viral capsid protein, facilitates NNV entry via the IGF1R-Rac1/Cdc42 pathway, and can render otherwise resistant cells susceptible to infection. This multifaceted approach effectively demonstrates the central role of MmMYL3 in NNV entry.

Reviewer #3 (Public review):

Summary:

In this study, Stein and colleagues use a clever masking/attentional blink paradigm using Kanisza stimuli, coupled with EEG decoding and the NMDA antagonist memantine, to isolate putative neural markers of feedforward, lateral, and feedback processing.

In two elegant experiments, they show that memantine selective influences EEG decoding of only illusory Kanisza surfaces (but not contour continuation or raw contrast), only when unmasked, only when attention is available (not when "blinked"), and only when task-relevant.

This neatly implicates NMDA receptors in the feedback mechanisms that are believed to be involved in inferring illusory Kanisza surfaces, and builds a difficult bridge between the large body of human perceptual experiments and pharmacological and neurophysiological work in animals.

Strengths:

Three key strengths of the paper are 1) its elegant and thorough experimental design, which includes internal replication of some key findings, and 2) the clear pattern of results across the full set of experiments, and 3) its clear writing and presentation of results.

The paper effectively reports a 4-way interaction, with memantine only influencing decoding of surfaces (1) that are unmasked (2), with attention available (3) and task-relevant (4). Nevertheless, the results are very clear, with a clear separation between null effects on other conditions and quite a strong (and thus highly selective) effect on this one intersection of conditions. This makes the pattern of findings very convincing.

Weaknesses:

Overall this is an impressive and important paper. However, to my mind there are two minor weaknesses.

First, despite its clear pattern of neural effects, there is no corresponding perceptual effect. Although the manipulation fits neatly within the conceptual framework, and there are many reasons for not finding such an effect (floor and ceiling effects, narrow perceptual tasks etc), this does leave open the possibility that the observation is entirely epiphenomenal, and that the mechanisms being recorded here are not actually causally involved in perception per se.

Second, although it is clear that there is an effect on decoding in this particular condition, what that means is not entirely clear - particularly since performance improves, rather than decreases. It should be noted here that improvements in decoding performance do not necessarily need to map onto functional improvements, and we should all be careful to remain agnostic about what is driving classifier performance. Here too, the effect of memantine on decoding might be epiphenomenal - unrelated to the information carried in the neural population, but somehow changing the balance of how that is electrically aggregated on the surface of the skull. *Something* is changing, but that might be a neurochemical or electrical side-effect unrelated to actual processing (particularly since no corresponding behavioural impact is observed.)

Comments on revisions:

I think the authors responsed fairly to my comments. Even if they weren't really able to add new insight into why behaviour didn't show the same effects as decoding, they discuss this in the revised text.

Reviewer #3 (Public review):

Summary:

The authors investigated fibroblasts' communication with key cell types in developing and neonatal hearts, with focus on critical roles of fibroblast-cardiomyocyte and fibroblast-endothelial cells network in cardiac morphogenesis. They tried to map the spatial distribution of these cell types and reported the major pathways and signaling molecules driving the communication. They also used Cre-DTA system to ablate Pdgfra labeled cells and observed myocardial and endothelial cell defects at development. They screened the pathways and genes using sequencing data of ablated heart. Lastly they reported a compensatory collagen expression in long term ablated neonate heart. Overall, this study provides us with important insight on fibroblasts' roles in cardiac development and will be a powerful resource for collagens and ECM focused research.

Strengths:

The authors utilized good analyzing tools to investigate on multiple database of single cell sequencing and Multi-seq. They identified significant pathways, cellular and molecular interactions of fibroblasts. Additionally, they compared some of their analytic findings with human database, and identified several groups of ECM genes with varying roles in mice.

Weaknesses:

This study is majorly based on sequencing data analysis. At the bench, they used very strident technique to study fibroblast functions by ablating one of the major cell population of heart. Also, experimental validation of their analyzed downstream pathways will be required eventually.

Reviewer #3 (Public review):

Summary:

The authors present new observations related to the gliding motility of the multicellular filamentous cyanobacteria Fluctiforma draycotensis. The bacteria move forward by rotating their about their long axis, which causes points on the cell surface to move along helical paths. As filaments glide forward they form visible tracks. Filaments preferentially move within the tracks. The authors device a simple model in which each cell in a filament exerts a force that either pushes forwards or backwards. Mechanical interactions between cells cause neighboring cells to align the forces they exert. The model qualitatively reproduces the tendency of filaments to move in a concerted direction and reverse at the end of tracks.

The authors seek to understand how cells in a filament synchronize their motion to move in a concerted direction. This question connects to the evolution of multicellular life and so is important well beyond the specific field of cyanobacterial locomotion.

Strengths:

The biophysical model used to describe cell-cell coordination of locomotion is clear and reasonable. This model provides a useful phenomenological framework in which to consider the roles of individual cells in the coordinated motion of the group. The qualitative consistency between theory and observation suggests that this model captures some essential qualities of the true system.

The observation that filaments reverse at the ends of tracks is compelling, but difficult to clearly connect to any one microscopic model.

The observations of helical motion of the filament are compelling.

Weaknesses:

The comparison of theory and observation is mainly qualitative. While the authors have done a good job fitting the observations to the theory, it is not possible to systematically vary parameters, independently estimate parameter values, or apply external forces. Consequently, more experiments are needed before the proposed model can the accepted or rejected. This manuscript provides a promising hypothesis but not a compelling justification for it.

Reviewer #3 (Public review):

Summary:

After salamander limb amputation, the cross-section of the stump has two major axes: anterior-posterior and dorsal-ventral. Cells from all axial positions (anterior, posterior, dorsal, ventral) are necessary for regeneration, yet the molecular basis for this requirement has remained unknown. To address this gap, Yamamoto et al. took advantage of the ALM assay, in which defined positional identities can be combined on demand and their effects assessed through the outgrowth of an ectopic limb. They propose a compelling model in which dorsal and ventral cells communicate by secreting Wnt10b and Fgf2 ligands, respectively, with this interaction inducing Shh expression in posterior cells. Shh was previously shown to induce limb outgrowth in collaboration with anterior Fgf8 (PMID: 27120163). Thus, this study completes a concept in which four secreted signals from four axial positions interact for limb patterning. Notably, this work firmly places dorsal-ventral interactions upstream of anterior-posterior, which is striking for a field that has been focussed on anterior-posterior communication. The ligands identified (Wnt10b, Fgf2) are different from those implicated in dorsal-ventral patterning in the non-regenerative mouse and chick models. The results in the context of ALM/ectopic limb engineering are impressive, but the authors do not extend their experiments to assay 'normal' regeneration after amputation.

Strengths:

(1) The ALM and use of GFP grafts for lineage tracing (Figures 1-3) take full advantage of the salamander model's unique ability to outgrow patterned limbs under defined conditions. As far as I am aware, the ALM has not been combined with precise grafts that assay 2 axial positions at once, as performed in Figure 3. The number of ALMs performed in this study deserves special mention, considering the challenging surgery involved.

(2) The authors identify that posterior Shh is not expressed unless both dorsal and ventral cells are present. This echoes previous work in mouse limb development models (AER/ectoderm-mesoderm interaction), but this link between axes was not known in salamanders. The authors elegantly reconstitute dorsal-ventral communication by grafting, finding that this is sufficient to trigger Shh expression (Figure 3 - although see also the Weaknesses section.)

(3) Impressively, the authors discovered two molecules sufficient to substitute dorsal or ventral cells through electroporation into dorsal- or ventral-depleted ALMs (Figure 5). These molecules did not change the positional identity of target cells. The same group previously identified the ventral factor (Fgf2) to be a nerve-derived factor essential for regeneration. In Figure 6, the authors demonstrate that nerve-derived factors, including Fgf2, are alone sufficient to grow out ectopic limbs from a dorsal wound. Limb induction with a 3-factor cocktail without supplementing with other cells is conceptually important for regenerative engineering.

(4) The writing style and presentation of results are very clear.

Weaknesses:

(1) The expression data are the weakest part of this study.

• Despite being a central message, I found the Shh in situs unconvincing (e.g. Figure 2I, 3C, 5C), especially without sense probe controls. An additional assay would be essential to make the Shh data convincing - perhaps like in Figure 5D (qPCR?), RNA-sequencing, or a downstream target gene.

• It is not clear what the n numbers mean for the in situ data (slides analysed / number of biological samples / other?). This is crucial to understanding the reliability of the results.

• The authors do not assay where and when Wnt10b and Fgf2 are expressed beyond the bulk RNA-sequencing (which presumably contains both epidermis and mesenchyme cells). This is a shame, as understanding which cell types express these molecules, and when, would be important for understanding the mechanism.

(2) It is important to consider that the ALM is not 'regeneration', even if the authors have previously argued that ALM bumps and regenerating blastemas are equivalent (PMID: 17959163). The start- and end- points of ALM are different from regeneration, even though there are undoubtedly common principles involved. Thus, I find the word 'regeneration' in the title and last sentence of the abstract unsubstantiated unless evidence is provided that the same mechanisms (Wnt10b/Fgf2/Shh) function during normal limb regeneration.

(3) Drawing the exact boundaries of the Ant/Pos/Dor/Ven BL and grafts in the cartoon in Figure 1 (with respect to anatomical landmarks) would help to better understand the experiments in Figures 3 and 4.

(4) I find the 'positional cue' and 'positional value' terminology confusing, despite the authors' efforts. It is not clear if they refer to cell autonomous or secreted signals, and, as the authors mention, the definitions partially overlap. Lmx1b is defined as a positional value, even though it is necessary and sufficient for dorsal identity (so, isn't it positional information?). Much simpler would be to describe Wnt10b and Fgf2 as what they are: dorsally or ventrally expressed signals that substitute for dorsal or ventral tissue without inducing changes in positional information.

Overall appraisal:

This is a logical and well-executed study that creatively uses the axolotl model to advance an important framework for understanding limb patterning. The reliability of the Shh expression data is a weak point in this otherwise impressive study. The relevance of the mechanisms to normal limb regeneration is not substantiated.

Reviewer #3 (Public review):

Summary:

The neuropeptide galanin is primarily expressed in the hypothalamus and has been shown to play critical roles in homeostatic functions such as arousal, sleep, stress, and brain disorders such as epilepsy. Previous work in rodents using galanin analogs and receptor-specific knockout have provided convincing evidence for anti-convulsant effects of galanin.

In the present study, the authors sought to determine the relationship between galanin expression and whole-brain activity. The authors took advantage of the transparent nature of larval zebrafish to perform whole-brain neural activity measurements via widefield calcium imaging. Two models of seizures were used (eaat2a-/- and pentylenetetrazol; PTZ). In the eaat2a-/- model, spontaneous seizures occur and the authors found that galanin transcript levels were significantly increased and associated with reduced frequency of calcium events. Similarly, two hours after PTZ galanin transcript levels roughly doubled and the frequency and amplitude of calcium events were reduced.

The authors also used a heat shock protein line (hsp70I:gal) where galanin transcripts levels are induced by activation of heat shock protein, but this line also shows higher basal transcript levels of galanin. Due to problems with whole-brain activity in wild-type larvae, the authors used the line without heat shock. They found higher level of galanin in hsp70I:gal larval zebrafish resulted in a reduction of calcium events and a reduction in amplitude of events. In contrast, galanin knockout (gal-/-) increased calcium activity, indicated by an increased number of calcium events, but a reduction in amplitude and duration. New data in the supplementary figure 2 used antibody staining to confirm the absence of galanin expression in gal-/- knockouts. Knockout of the galanin receptor subtype galr1a via crispants also increased the frequency of calcium events. New data in the revised manuscript reports that galr1aKO did not cause an upregulation of galanin, thereby ruling out genetic compensation effects.

In subsequent experiments in eaat2a-/- mutants were crossed with hsp70I:gal or gal-/- to increase or decrease galanin expression, respectively. These experiments showed modest effects, with eaat2a-/- x gal-/- knockouts showing an increased normalized area under the curve and seizure amplitude.

Lastly, the authors attempted to study the relationship between galanin and brain activity during a PTZ challenge. The hsp70I:gal larva showed increased number of seizures and reduced seizure duration during PTZ. In contrast, gal-/- mutants showed increased normalized area under the curve and a stark reduction in number of detected seizures, a reduction in seizure amplitude, but an increase in seizure duration. The authors then ruled out the role of Galr1a in modulating this effect during PTZ, since the number of seizures was unaffected, whereas the amplitude and duration of seizures was increased.

Strengths:

(1) The gain- and loss-of function galanin manipulations provided convincing evidence that galanin influences brain activity (via calcium imaging) during interictal and/or seizure-free periods. In particular, the relationship between galanin transcript levels and brain activity in figures 1 & 2 was convincing. New antibody staining confirms the absence of galanin in gal-/- mutants. New data also shows galanin transcript levels were unchanged in galr1ako brains.

(2) The authors use two models of epilepsy (eaat2a-/- and PTZ).

(3) Focus on the galanin receptor subtype galr1a provided good evidence for an important role of this receptor in controlling brain activity during interictal and/or seizure-free periods.

(4) The authors have added supplementary video files for calcium imaging to support their observations.

Weaknesses:

(1) Although the relationship between galanin and brain activity during interictal or seizure-free periods was clear, the revised manuscript still lacks mechanistic insight in the role of galanin during seizure-like activity induced by PTZ.

(2) The revised manuscript continues to heavily rely on calcium imaging of different mutant lines. Confirmation of knockouts has been provided with immunostaining in a new supplementary figure. Additional methods could strengthen the data, translational relevance, and interpretation (e.g., acute pharmacology using galanin agonists or antagonists, brain or cell recordings, biochemistry, etc).

Reviewer #3 (Public review):

This manuscript presents a number of interesting findings that have the potential to increase our understanding of the mechanism underlying homeostatic synaptic plasticity (HSP). The data broadly support that Rab3A plays a role in HSP, although the site and mechanism of action remain uncertain.

The authors clearly demonstrate the Rab3A plays a role in HSP at excitatory synapses, with substantially less plasticity occurring in the Rab3A KO neurons. There is also no apparent HSP in the Earlybird Rab3A mutation, although baseline synaptic strength is already elevated. In this context, it is unclear if the plasticity is absent, already induced by this mutation, or just occluded by a ceiling effect due the synapses already being strengthened. Occlusion may also occur in the mixed cultures, when Rab3A is missing from neurons but not astrocytes. The authors do appropriately discuss these options. The authors have solid data showing that Rab3A is unlikely to be active in astrocytes, Finally, they attempt to study the linkage between changes in synaptic strength and AMPA receptor trafficking during HSP, and conclude that trafficking may not be solely responsible for the changes in synaptic strength during HSP.

Strengths:

This work adds another player into the mechanisms underlying an important form of synaptic plasticity. The plasticity is likely only reduced, suggesting Rab3A is only partially required and perhaps multiple mechanisms contribute. The authors speculate about some possible novel mechanisms, including whether Rab3A is active pre-synaptically to regulate quantal amplitude.

As Rab3A is primarily known as a pre-synaptic molecule, this possibility is intriguing and novel for this system. However, it is based on the partial dissociation of AMPAR trafficking and synaptic response, and lacks strong support. On average, they saw similar magnitude of change in mEPSC amplitude and GluA2 cluster area and integral, but the GluA2 data was not significant due to higher variability. It is difficult to determine if this is due to biology or methodology - the imaging method involves assessing puncta pairs (GluA2/VGlut1) clearly associated with a MAP2 labeled dendrite. This is a small subset of synapses, with usually less than 20 synapses per neuron analyzed, which would be expected to be more variable than mEPSC recordings averaged across several hundred events. However, when they reduce the mEPSC number of events to similar numbers as the imaging, the mESPC amplitudes are still less variable than the imaging data. The reason for this remains unclear. The pool of sampled synapses is still different between the methods and recent data has shown that synapses have variable responses during HSP. Further, there could be variability in the subunit composition of newly inserted AMPARs, and only assessing GluA2 could mask this (see below). It is intriguing that pre-synaptic changes might contribute to HSP, especially given the likely localization of Rab3A. But it remains difficult to distinguish if the apparent difference in imaging and electrophysiology is a methodological issue rather than a biological one. Stronger data, especially positive data on changes in release, will be necessary to conclude that pre-synaptic factors are required for HSP, beyond the established changes in post-synaptic receptor trafficking. Specific deletion of Rab3A from pre-synaptic neurons would also be highly informative.

Other questions arise from the NASPM experiments, used to justify looking at GluA2 (and not GluA1) in the immunostaining. First, there is a strong frequency effect that is unclear in origin. One would expect NASPM to merely block some fraction of the post-synaptic current, and not affect pre-synaptic release or block whole synapses. But the change in frequency seems to argue (as the authors do) that some synapses only have CP-AMPARs, while the rest of the synapses have few or none. Another possibility is that there are pre-synaptic NASPM-sensitive receptors that influence release probability. Further, the amplitude data show a strong trend towards smaller amplitude following NASPM treatment (Fig 3B). The p value for both control and TTX neurons was 0.08 - it is very difficult to argue that there is no effect. And the decrease on average is larger in the TTX neurons, and some cells show a strong effect. It is possible there is some heterogeneity between neurons on whether GluA1/A2 heteromers or GluA1 homomers are added during HSP. This would impact the conclusions about the GluA2 imaging as compared to the mEPSC amplitude data.

To understand the role of Rab3A in HSP will require addressing two main issues:

(1) Is Rab3A acting pre-synaptically, post-synaptically or both? The authors provide good evidence that Rab3A is acting within neurons and not astrocytes. But where it is acting (pre or post) would aid substantially in understanding its role. The general view in the field has been that HSP is regulated post-synaptically via regulation of AMPAR trafficking, and considerable evidence supports this view. More concrete support for the authors suggestion of a pre-synaptic site of control would be helpful.

(2) Rab3A is also found at inhibitory synapses. It would be very informative to know if HSP at inhibitory synapses is similarly affected. This is particularly relevant as at inhibitory synapses, one expects a removal of GABARs or a decrease in GABA release (ie the opposite of whatever is happening at excitatory synapses). If both processes are regulated by Rab3A, this might suggest a role for this protein more upstream in the signaling; an effect only at excitatory synapses would argue for a more specific role just at those synapses.

Comments on revisions:

The section on TNF is a bit odd. The data on the astrocyte deletion of Rab3A only argues that Rab3A is unlikely to regulate TNF release. But it could easily be downstream of the neuronal TNF receptor. Without any data addressing the TNF response, it seems quite premature to argue that Rab3A is part of a TNF-independent pathway.

The section title (line 506-7) declaring Rab3A as the first presynaptic protein involved in HSP is also premature, as they don't know it is acting pre-synaptically.

Reviewer #3 (Public review):

Summary:

In the current manuscript entitled "Population-level morphological analysis of paired CO2- and odor-sensing olfactory neurons in D. melanogaster via volume electron microscopy", Choy, Charara et al. use volume electron microscopy and neuron reconstruction to compare the dendritic morphology of ab1C and ab1D neurons of the Drosophila basiconic ab1 sensillum. They aim to investigate the degree of dendritic heterogeneity within a functional class of neurons using ab1C and ab1D, which they can identify due to the unique feature of ab1 sensilla to house four neurons and the stereotypic location on the third antennal segment. This is a great use of volumetric electron imaging and neuron reconstruction to sample a population of neurons of the same type. Their data convincingly shows that there is dendritic heterogeneity in both investigated populations, and their sample size is sufficient to strongly support this observation. This data proposes that the phenomenon of dendritic heterogenity is common in the Drosophila olfactory system and will stimulate future investigations into the developmental origin, functional implications, and potential adaptive advantage of this feature.

Moreover, the authors discovered that there is a difference between CO2- and odour-sensing neurons of which the first show a characteristic flattened and sheet-like structure not observed in other sensory neurons sampled in this and previous studies. They hypothesize that this unique dendritic organization, which increases the surface area to volume ratio, might allow more efficient Co2 sensing by housing higher numbers of Co2 receptors. This is supported by previous attempts to express Co2 sensors in olfactory sensory neurons, which lack this dendritic morphology, resulting in lower Co2 sensitivity compared to endogenous neurons.

Overall, this detailed morphological description of olfactory sensory neurons' dendrites convincingly shows heterogeneity in two neuron classes with potential functional impacts for odour sensing.

Strength:

The volumetric EM imaging and reconstruction approach offers unprecedented details in single cell morphology and compares dendrite heterogeneity across a great fraction of ab1 sensilla.<br /> The authors identify specific shapes for ab1C sensilla potentially linked to their unique function in CO2 sensing.

Weaknesses:

While the morphological description is highly detailed, no attempts are made to link this to odour sensitivity or other properties of the neurons. It would have been exciting to see how altered morphology impacts physiology in these olfactory sensory cells.

Reviewer #3 (Public review):

Summary:

This work aims to better understand the role of arginine vasopressin (AVP) in the control of islet hormone secretion. This builds on previous literature in this area reporting on the actions of AVP to stimulate islet hormones. The gap in literature being addressed by these studies is primarily focused on the glucose-dependency of AVP on both insulin and glucagon secretion. A secondary objective is to explore the role of individual receptors with the use of newly generated peptides and existing tools. The methods include the use of Ca2+ imaging in pancreas slices from mice, with additional outcomes including insulin secretion in some areas. The conclusions presented are that AVP acts through V1b receptors in both alpha- and beta-cells, that this activity occurs in the high cAMP environment, and is glucose dependent.

Strengths:

The area of research is emerging with plenty of room for new contributions. The concept of AVP stimulating islet hormone secretion is important and deserving of further insight. The use of pancreas tissue to image primary cells makes the experiments physiologically relevant. The advancement of novel tools in this area should be helpful to other groups investigating the actions of AVP.

Weaknesses:

The conclusions are only modestly supported by the data and lack experimental depth and rigor. The rationale for only conducting studies at high cAMP conditions is not entirely clear and limits the conclusions that can be made. The use of Ca2+ is helpful, but it is a surrogate for hormone secretion. Additional measurements of hormone secretion are needed to enhance the robustness of these conclusions. Consideration of paracrine effects between alpha- and beta-cells is only superficially made and is likely essential in the context of the experimental design. For instance, there is clear literature that alpha-cells secrete several factors that work in paracrine interactions on beta-cells and autocrine actions back on alpha-cells. Conducting these studies in a high cAMP context only completely overlooks these interactions, skewing the interpretations made by the investigators. Finally, the clarity of the experiments and results could be significantly enhanced.

Reviewer #3 (Public review):

Summary:

In this paper, the authors introduce the Gcoupler software, an open-source deep learning-based platform for structure-guided discovery of ligands targeting GPCR interfaces.<br /> Overall, this manuscript represents a field-advancing contribution at the intersection of AI-based ligand discovery and GPCR signaling regulation.

Strengths:

The paper presents a comprehensive and well-structured workflow combining cavity identification, de novo ligand generation, statistical validation, and graph neural network-based classification. Notably, the authors use Gcoupler to identify endogenous intracellular sterols as allosteric modulators of the GPCR-Gα interface in yeast, with experimental validations extending to mammalian systems. The ability to systematically explore intracellular metabolite modulation of GPCR signaling represents a novel and impactful contribution. This study significantly advances the field of GPCR biology and computational ligand discovery.

Reviewer #3 (Public review):

Summary:

The authors set off with an analysis of the lysosomal integrity upon knockdown of genes of the sphingolipid metabolic pathway that they identified in a previous (yet unpublished) work of an RNA screen using a new C. elegans Tau model. They then used cell culture and C. elegans experiments to study the link between lysosomal rupture and Tau propagation.

Strengths:

The authors use two complementary model systems and use probes to assess membrane rigidity that allow a quick assessment of the membrane dynamics and offer the opportunity to treat the cells with lipids, RNAi. Tau seeds, etc.

Weaknesses:

The main weakness is that this work builds on not-yet-peer-reviewed manuscript that established a new C. elegans Tau model and RNAi screen that aimed to identify genes involved in the propagation of Tau.

This reviewer misses essential information of the C. elegans Tau strain (not included in the method section): e.g., promoter used for the expression, information on the used Tau variant, expression pattern, and aggregation, etc.

Throughout the study, I missed data on:

(1) Effect of the knockdown on Tau expression, localisation (with lysosomal membrane?), aggregation, and proteotoxicity. The effect of the RNAi-mediated knockdown could also simply lead to a reduced expression of Tau that, in turn, leads to suppressed propagation.

(2) A quantification of RNAi knockdown is needed to judge the efficiency of the RNAi, in particular for the combinatorial RNAi experiments involving 2 and even 4 genes in parallel. Ideally, these analyses should be validated with mutants for these genes.

Further:

(3) Figure 4 H, I: Would Tau also aggregate in the absence of externally added Tau?

(4) How specific is the effect for Tau? It would help if the authors could assess other amyloid proteins.

(5) The connection between sphingolipids and AD is not new. See He et al, 2010, Neurobiol. Aging + numerous publications and also not between Tau seeding and lysosomal rupture: Rose et al., PNAS 2024 (that has been cited by the authors).

Reviewer #3 (Public review):

Summary:

The manuscript authored by Song et al explores the oxidative stress response of Rubrerythrin in Pyrococcus furiosus and the formation of unique tubules that also encapsulate Encapsulin VLPs. This is an excellent study employing diverse methods to comprehensively study the formation of these assemblies under oxidative stress and lays the foundation of understanding oxidative stress through the formation of tubules among redox-sensing proteins like Rubrerythrin. The authors decipher the molecular structure of the tubules and also present a high-resolution reconstruction of the rubrerythin unit that forms the OSITs.

Strengths:

The study is done thoroughly by employing methods like cryoET, single particle cryoEM, mass spectrometry, and expression analyses of knockout strains to delve into an important mechanism to counter oxidative stress. The authors perform comprehensive analyses, and this study represents a vital contribution to understanding how anaerobic organisms can respond to oxidative stress.

Weaknesses:

Not all encapsulin particles seem to be inside the OSITs. Do the authors have any insights into how the tubules sequester these viral particles? Do the VLPs have a role in nucleating the OSIT assembly, and are there interactions between VLP and OSIT surfaces? These could be points that can be discussed in greater detail by the authors.

Can the authors get a subtomogram averaging done for the encapsulin VLPs? A higher resolution reconstruction may provide potential interaction details with the OSITs, if there are any.

The role of the dense granules observed in the rubrerythrin deletion strain is not very well discussed. Is there a way these granules counter oxidative stress? The EDX scanning seems to show a Phosphate increase similar to Ca and Mg. Are these aggregates therefore likely to be calcium and Mg phosphate aggregates? This section of the paper seems incompletely analysed.

The authors should provide density and coordination distances around the diiron ions and provide a comparison with available crystal structures and highlight differences, if any, in Figure 3. Local resolution for the high-res map may be provided for Supplementary Figure 6.

Overall, this is a well-performed study with clear conclusions. The discussion points need to be improved further.

Reviewer #3 (Public review):

Summary:

This manuscript demonstrates that mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing widespread, coordinated metabolic regulation by SCoR2.

Strengths:

(1) The extensive experimental evidence.

(2) The use of the knockout model.

Weaknesses:

(1) Lack of direct evidence for underlying mechanism(s).

(2) The mouse model used is not tissue-specific.

Reviewer #3 (Public review):

The authors use high-throughput neutralisation data to explore how different summary statistics for population immune responses relate to strain success, as measured by growth rate during the 2023 season. The question of how serological measurements relate to epidemic growth is an important one, and I thought the authors present a thoughtful analysis tackling this question, with some clear figures. In particular, they found that stratifying the population based on the magnitude of their antibody titres correlates more with strain growth than using measurements derived from pooled serum data. However, there are some areas where I thought the work could be more strongly motivated and linked together. In particular, how the vaccine responses in US and Australia in Figures 6-7 relate to the earlier analysis around growth rates, and what we would expect the relationship between growth rate and population immunity to be based on epidemic theory.

Reviewer #3 (Public review):

While the AAV capsid has long been the target of protein engineering, its Rep proteins have been comparatively less studied. Since Rep plays a variety of roles for genome replication and virion packaging, gaining a deeper mechanistic understanding of their function and/or engineering versions that enable higher packaging productivity would be of interest to the field. This study generates a library of non-synonymous mutations in AAV2 rep (intended to cover all 19 aa changes at all positions, and coming close), packaged an AAV with AAV9 capsid, and sequenced the results to assess which amino acid changes resulted in an enrichment/depletion of genomes containing that variant rep. They found that proline substitutions are disruptive, well known from protein mutagenesis studies. The most significant enrichment sfound, however, were a set of synonymous mutations (unintended members of the library, as the library was designed to contain non-synonymous mutations) that lie within the p19 promoter. However, attempts to package recombinant vector using these individual rep variants in the AAV helper construct did not increase viral titer.

A previous study conducted analogous mutagenesis on Rep: Jain et al., "Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production" eLife 2024 (cited here as reference 19). It is not clear that this current study is a significant advance relative to the prior, quite comprehensive study. Both generated a library of non-synonymous mutations and observed fitness effects on Rep. Because this study sequenced the full rep, rather than barcodes associated with each rep variant, it found the enrichment in the synonymous mutations. However, these should ideally advance a basic understanding of Rep biology and/or result in better AAV production, but they did neither. It is speculated in the Discussion that the mutations generated additional GCTC motifs in p19, elements that mediate protein-DNA interactions. However, the role of GCTC motifs is speculative, and no transcriptional analysis is conducted. Furthermore, as discussed above, the mutations do not result in higher viral titers. Perhaps there's a transcriptional effect at the much lower copy numbers of vector genome present during library selection vs. rAAV packaging. They also found stop codons in Rep domains thought to be required for viral packaging, but functional studies confirming the screening findings are not conducted. As a result, the biological or technical relevance of the findings are extremely unclear, and thus the impact is relatively low.

The description of herring DNA co-transfection and cross-packaging (which is a well-known pitfall) are somewhat technical and arguably don't merit too much main manuscript attention.

Reviewer #3 (Public review):

Summary:

The authors interrogated the putative role of microglia in determining AgRP fiber maturation in offspring exposed to a maternal high-fat diet. They found that changes in specific parts of the hypothalamus (but not in others) occur in microglia and that the effect of microglia on AgRP fibers appears to be beyond synaptic pruning, a classical function of these brain-resident macrophages.

Strengths:

The work is very strong in neuroanatomy. The images are clear and nicely convey the anatomical differences. The microglia depletion study adds functional relevance to the paper; however, the pitfalls of the technology regarding functional relevance should be discussed.

Weaknesses:

There was no attempt to functionally interrogate microglia in different parts of the hypothalamus. Morphology alone does not reflect a potential for significant signaling alterations that may occur within and between these and other cell types.

Comments on revised submission: My advice is to change the title by removing "required" and state what is interrogated and found in the paper. A more accurate title would be (for example): Implication of Microglia for Developmental Specification of AgRP Innervation in the Hypothalamus of Offspring Exposed to Maternal High-Fat Diet During Lactation.

I suggest that the authors discuss the limitations of their approach and findings, and propose future directions to address them

Reviewer #3 (Public review):

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

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

(1) Since leucine uptake and PDIM synthesis are important concepts of the manuscript, experiments would benefit from exploring other BCAAs to know if the phenotypes observed are specific to leucine, and adding additional strains to the 2D TLC experiments to provide confidence in the absence of the PDIM band.

(2) The intriguing observation that wild-type H37Rv is resistant to semapimod but the leucine-auxotroph is sensitive should be further explored. If the authors are correct and semapimod does inhibit leucine uptake through a specific transporter or disrupted cell wall (PDIM synthesis), testing semapimod activity against the leucine-auxotroph in various concentrations of BCAAs could highlight the importance of intracellular leucine. H37Rv is still able to synthesize endogenous leucine and is able to circumvent the effect of semapimod.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors presented an interesting study providing an insight into the role of Type-I interferon responses in tuberculosis (TB) pathogenesis by combining transcriptome analysis of PBMCs and TST from tuberculosis patients. The zebrafish model was used to identify the changes in the innate immune cell population of macrophages and neutrophils. The findings suggested that Type-I interferon signatures inversely correlated with disease severity in the TST transcriptome data. The authors validated the observations by CRISPR-mediated disruption of stat2 (a critical transcription factor for type I interferon signaling) in zebrafish larvae, showing increased susceptibility to M. marinum infection. Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to identify and further characterize the understanding of the role of type-I interferons in TB.

Strengths:

Traditionally, type-I interferon responses have been viewed as detrimental in mycobacterial infections, with studies suggesting enhanced susceptibility in certain mouse models. The study tried to further understand the role of type-I interferons in TB pathogenesis.

Weaknesses:

Though the study showed an inverse correlation of Type-I interferon with radiological features of TB, the molecular mechanism is largely unexplored in the study, which is making it difficult to understand the basis of the results shown in the manuscript by the authors.

Reviewer #3 (Public review):

Summary:

The study by Pothast and colleagues outlines an extension of their previously described temperature-based MHC-I peptide exchange method on 4 common HLA alleles, to enable the generation of peptide/MCH-I tetramers for characterization of antigen-specific T cells by flow cytometry.

Strengths:

This work outlines a protocol for generating MHC-I tetramers on 4 common HLA allotypes, which can then be applied to monitor T cell responses by flow cytometry studies. The work provides conditional ligands for exchange on each HLA and demonstrates proof of concept studies using clonotypic T cells and CD8+ PBMCs.

The results support that the temperature-exchanged tetramers can perform similarly to conventional tetramers in some settings.

Weaknesses:

Given that there are several proposed methodologies addressing the same task (including UV-mediated, disulfide-bond based stabilization of empty MHC-I conformers, and chaperone-based methods), the relevance of the proposed temperature-mediated technology is questionable.

More specifically, important limitations of the study include:

(1) A lack of quantification of exchanged molecules relative to molecules that retain the original placeholder peptides, or completely empty molecules present in the same sample.

(2) A lack of validation that peptide exchange has occurred in the absence of a reporter T cell line appears to be a significant limitation of the methodology for antigen / T cell discovery.

(3) The sub-optimal exchange efficiency relative to conventional prepared pMHC-I molecules, shown in Figure 4, is a significant limitation of the approach.

(4) There are no data to support that exchange proceeds through the generation of empty molecules during the temperature cycle, or by peptide binding on empty molecules that are already present in the sample. Understanding the mechanism of exchange is important for the necessary improvements to the methodology.

(5) It is possible that the temperature cycle causes protein aggregation or other irreversible changes to the sample - this should be explicitly quantified and addressed in the paper, since misfolded MHC-I molecules can lead to high levels of background staining.

(6) These potential limitations should limit detection of low-affinity/low-avidity interactions between TCRs and their cognate pMHC antigens - this should be addressed explicitly in a model antigen setting.

(7) The approach appears to be limited to the HLAs showing high thermal stability, which have been explored in this study. However, a large fraction of HLAs show sub-optimal thermal stabilities. It seems that explicit validation of peptide exchange would be required for any new HLA allele introduced into this process.

(8) Whether the approach can be used to load suboptimal peptides with lower thermal stabilities that are emerging immunotherapy targets is not addressed in the present study.

Because of these limitations, the present manuscript does not conclusively support the claim that temperature-based exchange can be used as a robust methodology to generate pMHC-I tetramers with desired peptide specificities.

As a result, the scope of applications using these suboptimal exchanged pHLA tetramers is limited, and should be addressed with further improvements of the methodology, including better characterization of exchange efficiency, demonstration of functionality across a broader range of HLA allotypes with varying thermal stability profiles, and validation with clinically relevant low-affinity peptides that would strengthen the potential utility of this approach in immunotherapy development and basic T cell biology research.

Reviewer #3 (Public review):

Summary:

Deletion of the TMA-sensor TAAR5 results in circadian alterations in gene expression, particularly in the olfactory bulb, plasma hormones, and neurobehaviors.

Strengths:

Genetic background was rigorously controlled.

Comprehensive characterization.

Weaknesses:

The weaknesses identified by this reviewer are minor.

Overall, the studies are very nicely done. However, despite careful experimentation, I note that even the controls vary considerably in their gene expression, etc, across time (eg, compare control graphs for Cry 1 in IB, 4B). It makes me wonder how inherently noisy these measurements are. While I think that the overall point that the Taar5 KO shows circadian changes is robust, future studies to dissect which changes are reproducible over the noise would be helpful.

Impact:

These data add to the growing literature pointing to a role for the TMA/TMAO pathway in olfaction and neurobehavioral.

Reviewer #3 (Public review):

Summary:

The authors report converging evidence from several brain-imaging techniques that geometric figures, notably quadrilaterals, are processed differently in visual (lower activation) and spatial (greater) areas of the human brain than representative figures. Comparison of mathematical models to fit activity for geometric figures shows the best fit for abstract geometric features like parallelism and symmetry. The brain areas active for geometric figures are also active in processing mathematical concepts, even in blind mathematicians, linking geometric shapes to abstract math concepts. The effects are stronger in adults than in 6-year-old Western children. Similar phenomena do not appear in great apes, suggesting that this is uniquely human and developmental.

Strengths:

Multiple converging techniques of brain imaging and testing of mathematical models. Careful reasoning at every step of research and presentation of research, anticipating and addressing possible reservations. Connecting these findings to other findings, brain, behavior, and historical/anthropological, to suggest broad and important fundamental connections between abstract visual-spatial forms and mathematical reasoning, further suggesting visual-spatial origins of mathematical reasoning.

Weaknesses:

Perhaps the manuscript could emphasize that the areas recruited by geometric figures but not objects are spatial, with reduced processing in visual areas. It also seems important to say that the images of real objects are interpreted as representations of 3D objects, as they activate the same visual areas as real objects. By contrast, the images of geometric forms are not interpreted as representations of real objects but rather perhaps as 2D abstractions. The authors use the term "symbolic." That use of that term could usefully be expanded here.

Pigeons have remarkable visual systems. According to my fallible memory, Herrnstein investigated visual categories in pigeons. They can recognize individual people from fragments of photos, among other feats. I believe pigeons failed at geometric figures and also at cartoon drawings of things they could recognize in photos. This suggests they did not interpret line drawings of objects as representations of objects.

Categories are established in part by contrast categories; are quadrilaterals, triangles, and circles different categories?

It would be instructive to investigate stimuli that are on a continuum from representational to geometric, e.g., table tops or cartons under various projections, or balls or buildings that are rectangular or triangular. Building parts, inside and out. like corners. Objects differ from geometric forms in many ways: 3D rather than 2D, more complicated shapes, and internal texture. The geometric figures used are flat, 2-D, but much geometry is 3-D (e. g. cubes) with similar abstract features. The feature space of geometry is more than parallelism and symmetry; angles are important, for example. Listing and testing features would be fascinating. Similarly, looking at younger or preferably non-Western children, as Western children are exposed to shapes in play at early ages.

What in human experience but not the experience of close primates would drive the abstraction of these geometric properties? It's easy to make a case for elaborate brain processes for recognizing and distinguishing things in the world, shared by many species, but the case for brain areas sensitive to processing geometric figures is harder. The fact that these areas are active in blind mathematicians and that they are parietal areas suggests that what is important is spatial far more than visual. Could these geometric figures and their abstract properties be connected in some way to behavior, perhaps with fabrication and construction as well as use? Or with other interactions with complex objects and environments where symmetry and parallelism (and angles and curvature--and weight and size) would be important? Manual dexterity and fabrication also distinguish humans from great apes (quantitatively, not qualitatively), and action drives both visual and spatial representations of objects and spaces in the brain. I certainly wouldn't expect the authors to add research to this already packed paper, but raising some of the conceptual issues would contribute to the significance of the paper.

Reviewer #3 (Public review):

The manuscript by Sadeqi et al. studies the interactions between the mitochondrial protein Ups1 and reconstituted membranes. The authors apply synthetic liposomal vesicles to investigate the role of pH, curvature, and charge on the binding of Ups1 to membranes and its ability to extract PA from them. The manuscript is well written and structured. With minor exceptions, the authors provide all relevant information (see minor points below) and reference the appropriate literature in their introduction. The underlying question of how the energy barrier for lipid extraction from membranes is overcome by Ups1 is interesting, and the data presented by the authors could offer a valuable new perspective on this process. It is also certainly a challenging in vitro reconstitution experiment, as the authors aim to disentangle individual membrane properties (e.g., curvature, charge, and packing density) to study protein adsorption and lipid transfer. I have one major suggestion and a few minor ones that the authors might want to consider to improve their manuscript and data interpretation:

Major Comments:

The experiments are performed with reconstituted vesicles, which are incubated with recombinant protein variants and quantitatively assessed in flotation and pelleting assays. According to the Materials and Methods section, the lipid concentration in these assays is kept constant at 5 µM. However, the authors change the size of the vesicles to tune their curvature. Using the same lipid concentration but varying vesicle sizes results in different total vesicle concentrations. Moreover, larger vesicles (produced by freeze-thawing and extrusion) tend to form a higher proportion of multilamellar vesicles, thus also altering the total membrane area available for binding. Could these differences in the experimental system account for the variation in binding? To address this, the authors would need to perform the experiments either under saturation (excess protein) conditions or find an experimental approach to normalize for these differences.

Reviewer #3 (Public review):

Summary:

The authors used recurrent neural network modelling of spatial navigation tasks to investigate border and place cell behaviour during remapping phenomena.

Strengths:

The neural network training seemed for the most part (see comments later) well-performed, and the analyses used to make the points were thorough.

The paper and ideas were well-explained.

Figure 4 contained some interesting and strong evidence for map-like generalisation as environmental geometry was warped.

Figure 7 was striking and potentially very interesting.

It was impressive that the RNN path-integration error stayed low for so long (Fig A1), given that normally networks that only work with dead-reckoning have errors that compound. I would have loved to know how the network was doing this, given that borders did not provide sensory input to the network. I could not think of many other plausible explanations... It would be even more impressive if it was preserved when the network was slightly noisy.

Update:

The analysis of how the RNN remapped, using a context signal to switch between largely independent maps, and the examination of the border like tuning in the recurrent units of the RNN, were both thorough and interesting. Further, in the updated response I appreciated the additional appendix E which helped substantiate the claim that the RNN neurons were border cells.

Weaknesses:

The remapping results were also puzzling. The authors present convincing evidence that the recurrent units effectively form 6 different maps of the 6 different environments (e.g. the sparsity of the code, or fig 6a), with the place cells remapping between environments. Yet, as the authors point out, in neural data the finding is that some cells generalise their co-firing patterns across environments (e.g. grid cells, border cells), while place cells remap, making it unclear what correspondence to make between the authors network and the brain. There are existing normative models that capture both entorhinal's consistent and hippocampus' less consistent neural remapping behaviour (Whittington et al. and probably others), what have we then learnt from this exercise?

Update: see summary below

I felt that the neural data analysis was unconvincing. Most notably, the statistical effect was found in only one of seven animals. Random noise is likely to pass statistical tests 1 in 20 times (at 0.05 p value), this seems like it could have been something similar? Further, the data was compared to a null model in which place cell fields were randomly distributed. The authors claim place cell fields have two properties that the random model doesn't (1) clustering to edges (as experimentally reported) and (2) much more provocatively, a hexagonal lattice arrangement. The test seems to collude the two; I think that nearby ball radii could be overrepresented, as in figure 7f, due to either effect. I would have liked to see a computation of the statistic for a null model in which place cells were random but with a bias towards to boundaries of the environment that matches the observed changing density, to distinguish these two hypotheses.

Update: the authors acknowledge these shortcomings and have appropriately tempered their data related claims.

Some smaller weaknesses:<br /> - Had the models trained to convergence? From the loss plot it seemed like not, and when including regularisors recent work (grokking phenomena, e.g. Nanda et al. 2023) has shown the importance of letting the regularisor minimise completely to see the resulting effect. Else you are interpreting representations that are likely still being learnt, a dangerous business.<br /> Update: I understand that practical limitations make testing this thoroughly impossible, which is fair enough.

- The claim that this work provided a mathematical formalism of the intuitive idea of a cognitive map seems strange, given that upwards of 10 of the works this paper cite also mathematically formalise a cognitive map into a similar integration loss for a neural network.<br /> Update: the introduction of these ideas hasn't changed, and my concerns above remain.

Aim Achieved? Impact/Utility/Context of Work

I think this is a thorough exploration of how this network with these losses is able to path-integrate its position and remap. This is useful, it is good to know how another neural network with slightly different constraints learns to perform these behaviours.

In the updated version of the manuscript I am happy to say that I think there are few claims that are unsubstantiated (see weakness section above that has been significantly updated). The link to neuroscience remains the biggest shortcoming of this work in my view. The authors point to two main results in this direction. First, the ability for interactions only between border-type and place cells to produce many observed place-cell results, providing a new hypothesis. Second, a connection between grid cells, place cells, and border cells, in the production of hexagonal arrangements of place cells.

Regarding the first, as the authors discuss, current evidence suggests border cells are invariant across environments whereas this work finds border cells for specific environments (they use the words rate-remapping boundary-type cells). It seems likely to me that there are many ways a neural network can path-integrate across different environments. In other models where the same base map is re-used (e.g. TEM) grid cells emerge, in this work where the maps for different environments are disjoint these border-like cells that do not match an observed cell type in their tuning to environment are involved. I find this a really interesting alternative (I think what an RNN does is interesting in its own right), but I don't see why I should think it is what the brain does, given that it appears to match observations less well (existence of grid cells, consistent firing patterns of border cells across environments). The smoking gun in favour of the author's hypothesis would be finding these sparse border like cells, or some other evidence of gating like interactions between border and place cells as they discuss. Finding such evidence sounds difficult (so not reasonable to ask for in a rebuttal), and to reiterate, I applaud the authors for clearly outlining an alternative, but I remain unconvinced.

Regarding the second point, while the grid-like placement of field centres was cool, and I applaud the authors for including real neural data comparisons, as the authors say, the data is preliminary, and further evidence would be required to fully substantiate this claim.

As such, in my mind it is an interesting alternative hypothesis. I look forward to seeing experimental predictions or comparisons that can tighten the link, substantiating the claim that what this particular RNN is doing reflects the algorithms at work in the brain.

Reviewer #3 (Public review):

Summary:

The neuropeptide galanin is primarily expressed in the hypothalamus and has been shown to play critical roles in homeostatic functions such as arousal, sleep, stress, and brain disorders such as epilepsy. Previous work in rodents using galanin analogs and receptor-specific knockout have provided convincing evidence for anti-convulsant effects of galanin.

In the present study, the authors sought to determine the relationship between galanin expression and whole-brain activity. The authors took advantage of the transparent nature of larval zebrafish to perform whole-brain neural activity measurements via widefield calcium imaging. Two models of seizures were used (eaat2a-/- and pentylenetetrazol; PTZ). In the eaat2a-/- model, spontaneous seizures occur and the authors found that galanin transcript levels were significantly increased and associated with reduced frequency of calcium events. Similarly, two hours after PTZ galanin transcript levels roughly doubled and the frequency and amplitude of calcium events were reduced, while the duration increased.

The authors also used a heat shock protein line (hsp70I:gal) where galanin transcripts levels are induced by activation of heat shock protein, but this line also shows higher basal transcript levels of galanin. Due to problems with whole-brain activity in wild-type larvae, the authors used the line without heat shock. They found higher level of galanin in hsp70I:gal larval zebrafish resulted in a reduction in the number of calcium events and amplitude. In contrast, galanin knockout (gal-/-) significantly increased calcium activity, indicated by an increased number of calcium events, but a reduction in amplitude and duration. Antibody staining confirmed the absence of galanin expression in gal-/- knockouts. Knockout of the galanin receptor subtype galr1a via crispants also increased the frequency of calcium events without influencing amplitude or duration.

In subsequent experiments in eaat2a-/- mutants were crossed with hsp70I:gal or gal-/- to modify galanin expression. These experiments showed modest effects, with eaat2a-/- x gal-/- knockouts showing an increased normalized area under the curve and seizure amplitude.

Lastly, the authors attempted to study the relationship between galanin and brain activity during a PTZ challenge. The hsp70I:gal larva showed increased number of seizures and reduced seizure duration during PTZ. In contrast, gal-/- mutants showed increased normalized area under the curve and a stark reduction in number of detected seizures, a reduction in seizure amplitude, but an increase in seizure duration. The authors then ruled out the role galanin a1 receptor in modulating this effect during PTZ, since the number of seizures was unaffected, whereas the amplitude and duration of seizures was increased in galr1a knockouts.

Strengths:

(1) The gain- and loss-of function galanin manipulations provided convincing evidence that galanin influences brain activity (via calcium imaging) during interictal and/or seizure-free periods. The relationship between galanin transcript levels and brain activity in figures 1 & 2 was convincing. Antibody staining also supports the absence of galanin in gal-/- mutants. Moreover, galanin transcript levels were unchanged in galr1ako brains, suggesting the lack of compensatory effects.

(2) The authors use two models of epilepsy (eaat2a-/- and PTZ).

(3) Supplementary video files for calcium imaging support the observations.

Weaknesses:

(1) I disagree with the idea that PTZ is a 'stressor'. This was raised in previous reviews and has not been acknowledged sufficiently.

(2) Although the relationship between galanin and brain activity during interictal or seizure-free periods was clear, the mechanisms that influence excitability during PTZ remain unclear. The authors show that galr1a does not mediate this effect, since seizure amplitude and duration were more severe in galr1a KO. Therefore, it remains unclear which galanin receptor is modulating this inhibitory effect.

(3) The manuscript is heavily reliant on calcium imaging for interpretation.<br /> Additional methods could strengthen the data, translational relevance, and interpretation (e.g., acute pharmacology using selective galanin agonists or antagonists, brain or cell recordings, biochemistry, etc).

Reviewer #3 (Public review):

Summary:

Krishnan et al. present a novel contextual fear conditioning (CFC) paradigm using a virtual reality (VR) apparatus to evaluate whether conditioned context-induced freezing can be elicited in head-fixed mice. By combining this approach with two-photon imaging, the authors aim to provide high-resolution insights into the neural mechanisms underlying learning, memory, and fear. Their experiments demonstrate that head-fixed mice can discriminate between threat and non-threat contexts, exhibit fear-related behavior in VR, and show context-dependent variability during extinction. Supplemental analyses further explore alternative behaviors and the influence of experimental parameters, while hippocampal neuron remapping is tracked throughout the experiments, showcasing the paradigm's potential for studying memory formation and extinction processes.

Strengths:

Methodological Innovation: The integration of a VR-based CFC paradigm with real-time two-photon imaging offers a powerful, high-resolution tool for investigating the neural circuits underlying fear, learning, and memory.

Versatility and Utility: The paradigm provides a controlled and reproducible environment for studying contextual fear learning, addressing challenges associated with freely moving paradigms.

Potential for Broader Applications: By demonstrating hippocampal neuron remapping during fear learning and extinction, the study highlights the paradigm's utility for exploring memory dynamics, providing a strong foundation for future studies in behavioral neuroscience.

Comprehensive Data Presentation: The inclusion of supplemental figures and behavioral analyses (e.g., licking behaviors and variability in extinction) strengthens the manuscript by addressing additional dimensions of the experimental outcomes.

Weaknesses:

Optimization: many parameters remain to be tested in the VR fear conditioning paradigm.

Extended training and attrition rate: the paradigm requires weeks of training and only 40% of mice reach criteria.

Reviewer #3 (Public review):

Summary:

In their revised manuscript, Sinha and colleagues aim to identify distinct causes of motor impairments seen when perturbing cerebellar circuits. This goal is an important one, given the diversity of movement related phenotypes in patients with cerebellar lesion or injury, which are especially difficult to dissect given the chronic nature of the circuit damage. To address this goal, the authors use high-frequency stimulation (HFS) of the superior cerebellar peduncle in monkeys performing reaching movements. HFS provides an attractive approach for transiently disrupting cerebellar function previously published by this group. First, they find a reduction in hand velocities during reaching, which was more pronounced for outward versus inward movements. By modeling inverse dynamics, they find evidence that shoulder muscle torques are especially affected. Next, the authors examine the temporal evolution of movement phenotypes over successive blocks of HFS trials. Using this analysis, they find that in addition to the acute, specific effects on torques in early HFS trials, there was an additional progressive reduction in velocity during later trials, which they interpret as an adaptive response to the inability to effectively compensate for interaction torques during cerebellar block. Finally, the authors examine movement decomposition and trajectory, finding that even when low velocity reaches are matched to controls, HFS produces abnormally decomposed movements and higher than expected variability in trajectory.

Strengths:

Overall, this work provides important insight into how perturbation of cerebellar circuits can elicit diverse effects on movement across multiple timescales.

The HFS approach provides temporal resolution and enables analysis that would be hard to perform in the context of chronic lesions or slow pharmacological interventions. Thus, this study describes an important advance over prior methods of circuit disruption in the monkey, and their approach can be used as a framework for future studies that delve deeper into how additional aspects of sensorimotor control are disrupted (e.g., response to limb perturbations).

In addition, the authors use well-designed behavioral approaches and analysis methods to distinguish immediate from longer-term adaptive effects of HFS on behavior. Moreover, inverse dynamics modeling provides important insight into how movements with different kinematics and muscle dynamics might be differentially disrupted by cerebellar perturbation.

In this revised version of the manuscript, the authors have provided additional analyses and clarification that address several of the comments from the original submission.

Remaining comments:

The argument that there are acute and adaptive effects to perturbing cerebellar circuits is compelling, but there seems to be a lost opportunity to leverage the fast and reversible nature of the perturbations to further test this idea and strengthen the interpretation. Specifically, the authors could have bolstered this argument by looking at the effects of terminating HFS - one might hypothesize that the acute impacts on joint torques would quickly return to baseline in the absence of HFS, whereas the longer-term adaptive component would persist in the form of aftereffects during the 'washout' period. As is, the reversible nature of the perturbation seems underutilized in testing the authors' ideas. While this experimental design was not implemented here, it seems like a good opportunity for future work using these approaches.

The analysis showing that there is a gradual reduction in velocity during what the authors call an adaptive phase is convincing. While it is still not entirely clear why disruption of movement during the adaptive phase is not seen for inward targets, despite the fact that many of the inward movements also exhibit large interaction torques, the authors do raise potential explanations in the Discussion.

The text in the Introduction and in the prior work developing the HFS approach overstates the selectivity of the perturbations. First, there is an emphasis on signals transmitted to the neocortex. As the authors state several times in the Discussion, there are many subcortical targets of the cerebellar nuclei as well, and thus it is difficult to disentangle target-specific behavioral effects using this approach. Second, the superior cerebellar peduncle contains both cerebellar outputs and inputs (e.g., spinocerebellar). Therefore, the selectivity in perturbing cerebellar output feels overstated. Readers would benefit from a more agnostic claim that HFS affects cerebellar communication with the rest of the nervous system, which would not affect the major findings of the study. In the revised manuscript, the authors do provide additional anatomical and evolutionary context and discuss potential limitations in the selectivity of HFS in the Materials and Methods. However, I feel that at least a brief mention of these caveats in the Introduction, where it is stated, "we then reversibly blocked cerebellar output to the motor cortex", would benefit the reader.

Reviewer #3 (Public review):

Summary:

The goal of the work by Graff et al. is to model CSVD in the zebrafish using foxf2a mutants. The mutants show loss of cerebral pericyte coverage that persists through adulthood, but it seems foxf2a does not regulate the regenerative capacity of these cells. The findings are interesting and build on previous work from the group. Limitations of the work include little mechanistic insight into how foxf2a alters pericyte recruitment/differentiation/survival/proliferation in this context, and the overlap of these studies with previous work in fox2a/b double mutants. However, the data analysis is clean and compelling, and the findings will contribute to the field.

Reviewer #3 (Public review):

Summary:

In this study, Kito et al follow up on previous work that identified Drosophila GCL as a mitotic substrate recognition subunit of a CUL3-RING ubiquitin ligase (CRL3) complex.

Here they characterize mutants of the human ortholog of GCL, GMCL1, that disrupt the interaction with CUL3 (GMCL1E142K) and that lack the substrate interaction domain (GMCL1 BBO). Immunoprecipitation followed by mass spectrometry identified 9 proteins that interacted with wild-type FLAG-GMCL1 and GMCL1 EK but not GMCL1 BBO. These proteins included 53BP1, which plays a well-characterized role in double-strand break repair but also functions in a USP28-p53-53BP1 "mitotic stopwatch" complex that arrests the cell cycle after a substantially prolonged mitosis. Consistent with the IP-MS results, FLAG-GMCL1 immunoprecipitated 53BP1. Depletion of GMCL1 during mitotic arrest increased protein levels of 53BP1, and this could be rescued by wild-type GMCL1 but not the E142K mutant or a R433A mutant that failed to immunoprecipitate 53BP1.

Using a publicly available dataset, the authors identified a relatively small subset of cell lines with high levels of GMCL1 mRNA that were resistant to the taxanes paclitaxel, cabazitaxel, and docetaxel. This type of analysis is confounded by the fact that paclitaxel and other microtubule poisons accumulate to substantially different levels in various cell lines (DOI: 10.1073/pnas.90.20.9552 , DOI: 10.1091/mbc.10.4.947 ), so careful follow-up experiments are required to validate results. The correlation between increased GMCL1 mRNA and taxane resistance was not observed in lung cancer cell lines. The authors propose this was because nearly half of lung cancers harbor p53 mutations, and lung cancer cell lines with wild-type but not mutant p53 showed the correlation between increased GMCL1 mRNA and taxane resistance. However, the other cancer cell types in which they report increased GMCL1 expression correlates with taxane sensitivity also have high rates of p53 mutation. Furthermore, p53 status does not predict taxane response in patients (DOI: 10.1002/1097-0142(20000815)89:4<769::aid-cncr8>3.0.co;2-6 , DOI: 10.1002/(SICI)1097-0142(19960915)78:6<1203::AID-CNCR6>3.0.CO;2-A , PMID: 10955790).

The authors then depleted GMCL1 and reported that it increased apoptosis in two cell lines with wild-type p53 (MCF7 and U2OS) due to activation of the mitotic stopwatch. This is surprising because the mitotic stopwatch paper they cite (DOI: 10.1126/science.add9528 ) reported that U2OS cells have an inactive stopwatch and that activation of the stopwatch results in cell cycle arrest rather than apoptosis in most cell types, including MCF7. Beyond this, it has recently been shown that the level of taxanes and other microtubule poisons achieved in patient tumors is too low to induce mitotic arrest (DOI: 10.1126/scitranslmed.3007965 , DOI: 10.1126/scitranslmed.abd4811 , DOI: 10.1371/journal.pbio.3002339 ), raising concerns about the relevance of prolonged mitosis to paclitaxel response in cancer. The findings here demonstrating that GMCL1 mediates degradation of 53BP1 during mitotic arrest are solid and of interest to cell biologists, but it is unclear that these findings are relevant to paclitaxel response in patients.

Strengths:

This study identified 53BP1 as a target of CRL3GMCL1-mediated degradation during mitotic arrest. AlphaFold3 predictions of the binding interface, followed by mutational analysis, identified mutants of each protein (GMCL1 R433A and 53BP1 IEDI1422-1425AAAA) that disrupted their interaction. Knock-in of a FLAG tag into the C-terminus of GMCL1 in HCT116 cells, followed by FLAG immunoprecipitation, confirmed that endogenous GMCL1 interacts with endogenous CUL3 and 53BP1 during mitotic arrest.

Weaknesses:

The clinical relevance of the study is overinterpreted. The authors have not taken relevant data about the clinical mechanism of taxanes into account. Supraphysiologic doses of microtubule poisons cause mitotic arrest and can activate the mitotic stopwatch. However, in physiologic concentrations of clinically useful microtubule poisons, cells proceed through mitosis and divide their chromosomes on mitotic spindles that are at least transiently multipolar. Though these low concentrations may result in a brief mitotic delay, it is substantially shorter than the arrest caused by high concentrations of microtubule poisons, and the one mimicked here by 16 hours of 0.4 mg/mL nocodazole, which is not used clinically and does not induce multipolar spindles. Resistance to mitotic arrest occurs through different mechanisms than resistance to multipolar spindles. No evidence is presented in the current version of the manuscript that GMCL1 affects cellular response to clinically relevant doses of paclitaxel.

Reviewer #3 (Public review):

Summary:

Webster et al. sought to understand if phenotypic variation in the absence of genetic variation can be predicted by variation in gene expression. To this end they quantified two reproductive traits, the onset of egg laying and early brood size in cohorts of genetically identical nematodes exposed to alternative ancestral (two maternal ages) and same generation life histories (either constant 20C temperature or 8-hour temperature shift to 25C upon hatching) in a two-factor design; then they profiled genome-wide gene expression in each individual.

Using multiple statistical and machine learning approaches, they showed that, at least for early brood size, phenotypic variation can be quite well predicted by molecular variation, beyond what can be predicted by life history alone.

Moreover, they provide some evidence that expression variation in some genes might be causally linked to phenotypic variation.

Strengths:

(1) Cleverly designed and carefully performed experiments that provide high-quality datasets useful for the community.

(2) Good evidence that phenotypic variation can be predicted by molecular variation.

Weaknesses:

What drives the molecular variation that impacts phenotypic variation remains unknown. While the authors show that variation in expression of some genes might indeed be causal, it is still not clear how much of the molecular variation is a cause rather than a consequence of phenotypic variation.

Reviewer #3 (Public review):

Summary:

Built on their previous pioneer expertise in studying RAD51 biology, in this paper, the authors aim to capture and investigate the structural mechanism of human RAD51 filament bound with a displacement loop (D-loop), which occurs during the dynamic synaptic state of the homologous recombination (HR) strand-exchange step. As the structures of both pre- and post-synaptic RAD51 filaments were previously determined, a complex structure of RAD51 filaments during strand exchange is one of the key missing pieces of information for a complete understanding of how RAD51 functions in the HR pathway. This paper aims to determine the high-resolution cryo-EM structure of RAD51 filament bound with the D-loop. Combined with mutagenesis analysis and biophysical assays, the authors aim to investigate the D-loop DNA structure, RAD51-mediated strand separation and polarity, and a working model of RAD51 during HR strand invasion in comparison with RecA.

Strengths:

(1) The structural work and associated biophysical assays in this paper are solid, elegantly designed, and interpreted.  These results provide novel insights into RAD51's function in HR.

(2) The DNA substrate used was well designed, taking into consideration the nucleotide number requirement of RAD51 for stable capture of donor DNA. This DNA substrate choice lays the foundation for successfully determining the structure of the RAD51 filament on D-loop DNA using single-particle cryo-EM.

(3) The authors utilised their previous expertise in capping DNA ends using monomeric streptavidin and combined their careful data collection and processing to determine the cryo-EM structure of full-length human RAD51 bound at the D-loop in high resolution. This interesting structure forms the core part of this work and allows detailed mapping of DNA-DNA and DNA-protein interaction among RAD51, invading strands, and donor DNA arms (Figures 1, 2, 3, 4). The geometric analysis of D-loop DNA bound with RAD51 and EM density for homologous DNA pairing is also impressive (Figure S5). The previously disordered RAD51's L2-loop is now ordered and traceable in the density map and functions as a physical spacer when bound with D-loop DNA. Interestingly, the authors identified that the side chain position of F279 in the L2_loop of RAD51_H differs from other F279 residues in L2-loops of E, F, and G protomers. This asymmetric binding of L2 loops and RAD51_NTD binding with donor DNA arms forms the basis of the proposed working model about the polarity of csDNA during RAD51-mediated strand exchange.

(4) This work also includes mutagenesis analysis and biophysical experiments, especially EMSA, single-molecule fluorescence imaging using an optical tweezer, and DNA strand exchange assay, which are all suitable methods to study the key residues of RAD51 for strand exchange and D-loop formation (Figure 5).

Weaknesses:

(1) The proposed model for the 3'-5' polarity of RAD51-mediated strand invasion is based on the structural observations in the cryo-EM structure. This study lacks follow-up biochemical/biophysical experiments to validate the proposed model compared to RecA or developing methods to capture structures of any intermediate states with different polarity models.

(2) The functional impact of key mutants designed based on structure has not been tested in cells to evaluate how these mutants impact the HR pathway.

The significance of the work for the DNA repair field and beyond:

Homologous recombination (HR) is a key pathway for repairing DNA double-strand breaks and involves multiple steps. RAD51 forms nucleoprotein filaments first with 3' overhang single-strand DNA (ssDNA), followed by a search and exchange with a homologous strand. This function serves as the basis of an accurate template-based DNA repair during HR. This research addressed a long-standing challenge of capturing RAD51 bound with the dynamic synaptic DNA and provided the first structural insight into how RAD51 performs this function. The significance of this work extends beyond the discovery of biology for the DNA repair field, into its medical relevance. RAD51 is a potential drug target for inhibiting DNA repair in cancer cells to overcome drug resistance. This work offers a structural understanding of RAD51's function with the D-loop and provides new strategies for targeting RAD51 to improve cancer therapies.

Reviewer #3 (Public review):

Summary:

This important study shows that stationary phase bacteria survive antimicrobial peptide treatment by switching on efflux pumps, generating low accumulating subpopulations that evade killing-a finding with clear implications for the design of peptide based antibiotics and for researchers studying antimicrobial resistance. The evidence is solid and frequently convincing, as diverse single cell assays, genetics and chemical inhibition coherently link reduced intracellular peptide to survival, even though a few mechanistic details warrant further exploration.

Strengths:

The authors investigate how Escherichia coli (and, to a lesser extent, Pseudomonas aeruginosa) survive exposure to the antimicrobial peptide (AMP) tachyplesin. Because resistance to AMPs is thought to rely heavily on non genetic adaptations rather than on classical mutation based mechanisms, the study focuses on phenotypic heterogeneity and seeks to pinpoint the cellular processes that protect a subset of cells. Using fluorescently labelled tachyplesin, single cell imaging, flow cytometry, transcriptomics, targeted genetics, and chemical perturbations, the authors report that stationary phase cultures harbor two phenotypic states: high accumulating cells that die and low accumulating cells that survive. They further propose and show that inducible efflux activity is the primary driver of survival and show that either efflux inhibition (sertraline, verapamil) or nutrient supplementation prevents the emergence of low accumulators and boosts killing.<br /> The experiments unambiguously reveal that the cells respond to stress heterogeneously, with two distinct subpopulations - one with better survival than the other. This primary phenotype is convincingly shown across various E. coli strains, including clinical isolates. The authors probed the underlying mechanism from several angles, with important additional experiments in the revised version that strengthens the original conclusions in several ways. Newly added efflux assays with ethidium bromide, together with proteinase treatment experiments and ΔacrAΔtolC and ΔqseB/qseC mutant data, illustrate that the low accumulating subpopulation can actively export intracellular compounds. The authors took great care to temper their language to acknowledge other potential alternatives that could explain some of the data such as altered influx, vesicle release or proteolysis, metabolic activity of the cells, indirect effects of sertraline treatment, etc. Additional metabolic dye measurements confirm that low accumulators are less metabolically active, and a new data on nutrient supplementation shows that forcing growth increases peptide uptake and lethality. The authors clarify the crucial point of where antimicrobial peptides actually bind on the cell within the broader survival mechanism and present their conclusions, along with potential caveats, with commendable clarity.

Weaknesses:

Despite these advances, the contribution of efflux may require more direct evidence to further dissect whether efflux is necessary, sufficient, or contributory. The facts that the key low-efflux mutant still retains a small fraction of survivors and that the inhibitors used may cause other physiological changes leading to higher efflux are still unaccounted for. The lipidomic and vesicle findings, while intriguing, remain descriptive, and direct tests of their functional relevance would further solidify the mechanistic models.

Conclusion:

Even with these limitations, the study provides valuable insight into non genetic resistance mechanisms to AMPs and highlights inducible heterogeneity as a critical obstacle to peptide therapeutics. In a much broader context, this study also underscores the importance of efflux physiology even for those antimicrobials that seemingly would not have intracellular targets.

Reviewer #3 (Public review):

Summary:

The manuscript by Guo and colleagues features the documentation and interpretation of three successions of continental to marginal marine deposits spanning the P/T transition and their respective ichnofaunas. Based on these new data inferences concerning end-Permian mass extinction and Triassic recovery in the tropical realm are discussed.

Strengths:

The manuscript is well written and organized and includes a large amount of new lithological and ichnological data that illuminate ecosystem evolution in a time of large scale transition. The lithological documentations, facies interpretations and ichnotaxonomic assignments look alright (with few exceptions).

Reviewer #3 (Public review):

The authors investigated differences in iridescence wing colouration of allopatric (geographically separated) and sympatric (coexisting) Morpho butterfly (sub)species. Their aim was to assess if iridescence wing colouration of Morpho (sub)species converged or diverged depending on coexistence and if iridescence wing colouration was involved in mating behaviour and reproductive isolation. The authors hypothesize that iridescence wing colouration of different (sub)species should converge in sympatry and diverge in allopatry. In sympatry, iridescence wing colouration can act as an effective antipredator defence with shared benefits if multiple (sub)species share the same colouration. However, shared wing colouration can have potential costs in terms of reproductive interference since wing colouration is often involved in mate recognition. If the benefits of a shared antipredator defence outweigh the costs of reproductive interference, iridescence wing colouration will show convergence and alternative mate recognition strategies might evolve, such as chemical mate recognition. In allopatry, iridescence wing colouration is expected to diverge due to adaptation to different local conditions and no alternative mate recognition is expected.

Strengths:

(1) Using allopatric and sympatric (sub)species that are closely related is a powerful way to test evolutionary hypotheses.

(2) By clearly defining iridescence and measuring colour spectra from a variety of angles, applying different methods, a very comprehensive dataset of iridescence wing colouration is achieved.

(3) By experimentally manipulating wing coloration patterns, the authors show visual mate recognition for M. h. bristowi and could, in theory, separate different visual aspects of colouration (patterns VS iridescence strength).

(4) Measurements of chemical profiles to investigate alternative mate recognition strategies in case of convergence of visual signals.

Weaknesses:

In my opinion, studies should be judged on the methods and data included, and not on additional measurements that could have been taken or additional treatments/species that should be included, since in most ecological and evolutionary studies, more measurements or treatments/species can always be included. However, studies do need to ensure appropriate replication and appropriate measurements to test their hypothesis AND support their conclusions. The current study failed to ensure appropriate replication, and in various cases, the results do not support the conclusions.

First, when using allopatric and sympatric (sub)species pairs to test evolutionary hypotheses, replication is important. Ideally, multiple allopatric and sympatric (sub)species pairs are compared to avoid outlier (sub)species or pairs that lead to biased conclusions. Unfortunately, the current study compares 1 allopatric and 1 sympatric (sub)species pair, hence having poor (no) replication on the level of allopatric and sympatric (sub)species pairs.

Second, chemical profiles were only measured for sympatric species and not for allopatric (sub)species, which limits the interpretation of this data. The allopatric (sub)species could have been measured as non-coexistence "control". If coexistence and convergence in wing colouration drives the evolution of alternative mate recognition signals, such alternative signals should not evolve/diverge for allopatric (sub)species where wing colouration is still a reliable mate recognition cue. More importantly, no details are provided on the quantification of butterfly chemical profiles, which is essential to understand such data. It is unclear how the chemical profiles were quantified and what data (concentrations, ratios, proportions) were used to perform NDMS and generate Figure 5 and the associated statistical tests.

Third, throughout the discussion, the authors mention that their results support natural selection by predators on iridescent wing colouration, without measuring natural selection by predators or any other measure related to predation. It is unclear by what predators any of the butterfly species are predated on at this point.

To continue on the interpretation of the data related to selection on specific traits by specific selection agents: This study did not measure any form of selection or any selection agent. Hence, it is not known if iridescent wing colouration is actually under selection by predators and/or mates, if maybe other selection agents are involved or if these traits converge due to genetic correlations with other traits under selection. For example, Iridescent colouration in ground beetles has functions as antipredator defence but also thermo- and water regulation. None of these issues are recognized or discussed.

Finally, some of the results are weakly supported by statistics or questionable methodology.

Most notably, the perception of the iridescence coloration of allopatric subspecies by bird visual systems. Although for females, means and errors (not indicated what exactly, SD, SE or CI) are clearly above the 1 JND line, for males, means are only slightly above this line and errors or CIs clearly overlap with the 1 JND line. Since there is no additional statistical support, higher means but overlap of SD, SE or CI with the baseline provides weak statistical support for differences.

Regarding the assortative mating experiment, the results are clearly driven by M. bristowi. For M. theodorus, females mate equally often with conspecifics (6 times) as with M. bristowi (5 times). For males, the ratio is slightly better (6 vs 3), but with such low numbers, I doubt this is statistically testable. Overall low mating for M. bristowi could indicate suboptimal experimental conditions, and hence results should be interpreted with care.

Regarding the wing manipulation experiment, M. theodorus does not show a preference when dummies with non-modified wings are presented and prefers non-modified dummies over modified dummies. This is acknowledged by the authors but not further discussed. Certainly, some control treatment for wing modification could have been added.

Overall, the fact that certain measurements only provide evidence for 1 of the 2 (sub)species (assortative mating, wing manipulation) or one sex of one of the species (bird visual systems) means overall interpretation and overgeneralization of the results to both allopatric or sympatric species should be done with care, and such nuances should ideally be discussed.

The aim of the authors, "to investigate the antagonistic effects of selective pressures generated by mate recognition and shared predation" has not been achieved, and the conclusions regarding this aim are not supported by the results. Nevertheless, the iridescence colour measurements are solid, and some of the behavioural experiments and chemical profile measurements seem to yield interesting results. The study would benefit from less overinterpretation of the results in the framework of predation and more careful consideration of methodological difficulties, statistical insecurities, and nuances in the results.

Reviewer #3 (Public review):

Summary:

This is an exciting, comprehensive paper that demonstrates the role of GATA4 on OA-like changes in chondrocytes. The authors present elegant reverse translational experiments that justify this mechanism and demonstrate the sufficiency of GATA4 in a mouse model of osteoarthritis (DMM), where GATA4 drove cartilage degeneration and pain in a manner that was significantly worse than DMM alone. This could pave the way for new therapies for OA that account for both structural changes and pain.

Strengths:

(1) GATA4 was identified in human chondrocytes.

(2) IHC and sequencing confirmed GATA4 presence.

(3) Activation of SMADs is clearly shown in vitro with GATA4 overexpression.

(4) The role of GATA4 was functionally assessed in vivo using the mouse DMM model, where the authors uncovered that GATA4 worsens OA structure and hyperalgesia in male mice.

(5) It is interesting that GATA4 is largely known to be found in cardiac cells and to have a role in cardiac repair, metabolism, and inflammation, among other things listed by the authors in the discussion (in liver, lung, pancreas). What could this new knowledge of GATA4 mean for OA as a potentially systemically mediated disease, where cardiac disease and metabolic syndrome are often co-morbid?

Weaknesses:

(1) It would be useful to explain why GATA4 was chosen over HIF1a, which was the most differentially expressed.

(2) In Figure 5, it would be useful to demonstrate the non-surgical or naive limbs to help contextualize OARSI scores and knee hyperalgesia changes.

(3) While there appear to be GATA4 small-molecule inhibitors in various stages of development that could be used to assess the effects in age-related OA, those experiments are out of scope for the current study.

Reviewer #3 (Public review):

Summary:

The manuscript by Schoeberlet et al. aims to elucidate the relationship between somatic transcription and nascent transcription. Using PRO-seq data across V regions and 275 non-immunoglobulin targets, the authors show that there is no statistically significant correlation with SHM hotspots and localized Pol II enrichment within V regions. They further confirm this conclusion by comparing SHM levels with reduced transcription and reduced activating epigenetic marks. They have revised the model for SHM regulation to emphasize transcription-independent targeting.

Comments:

(1) The sum of the mutation class percentages in Figure 3G should be one hundred percent.

(2) A quantitative bar of transcription and mutation levels could be added to make it clear across these V regions.

(3) The authors propose that transcriptional termination may contribute to the boundaries of the SHM (e.g., the ~2 kb from the V promoters). If this is the case, the slowing of Pol II velocity prior to termination would theoretically provide more opportunities for AID to access ssDNA, which should lead to higher mutation rates in regions upstream of termination sites (3-4 kb from TSSs). However, the observed SHM peaks in the V(D)J region, and declines exponentially within 1-2 kb downstream, which seems contradictory. The related statement could be revised.

(4) Recent ELOF1 stories published by the Schatz and Meng labs should be discussed. ELOF1 could be listed in the model in Figure 7.

Reviewer #3 (Public review):

Summary:

Ecker et al. utilized a biologically realistic, large-scale cortical model of the rat's non-barrel somatosensory cortex, incorporating a calcium-dependent plasticity rule to examine how various factors influence synaptic plasticity under in vivo-like conditions. Their analysis characterized the resulting plastic changes and revealed that key factors, including the co-firing of stimulus-evoked neuronal ensembles, the spatial organization of synaptic clusters, and the overall network topology, play an important role in affecting the extent of synaptic plasticity.

Strengths:

The detailed, large-scale model employed in this study enables the evaluation of diverse factors across various levels that influence the extent of plastic changes. Specifically, it facilitates the assessment of synaptic organization at the subcellular level, network topology at the macroscopic level, and the co-activation of neuronal ensembles at the activity level. Moreover, modeling plasticity under in vivo-like conditions enhances the model's relevance to experiments.

Weaknesses:

The paper lacks mechanistic insights into the observed phenomena, particularly regarding aspects that are typically inaccessible in traditional simplified models, such as layer-specific and layer-to-layer pathway-specific plasticity changes.

Reviewer #3 (Public review):

Summary:

The authors investigate the kinase activity of IKK2, a crucial regulator of inflammatory cell signaling. They describe a novel tyrosine kinase activity of this well-studied enzyme and a highly unusual phosphotransfer from phosphorylated IKK2 onto substrate proteins in the absence of ATP as a substrate.

Strengths:

The authors provide an extensive biochemical characterization of the processes with recombinant protein, western blot, autoradiography, protein engineering and provide MS data now.

Weaknesses:

The identity and purity of the used proteins has improved in the revised work. Since the findings are so unexpected and potentially of wide-reaching interest - this is important. Similar specific detection of phospho-Ser/Thr vs phospho-Tyr relies largely on antibodies which can have varying degrees of specificity. Using multiple antibodies and MS improves the quality of the data.

Reviewer #3 (Public review):

Brickwedde et al. attempt to clarify the role of alpha in sensory gain modulation by exploring the relationship between attention-related changes in alpha and attention-related changes in sensory-evoked responses, which surprisingly few studies have explicitly examined. The authors find evidence against the alpha-inhibition account, at least in early sensory processing, adding valuable data to the field to support our understanding of the alpha-inhibition hypothesis.

Due to task and measurement considerations, the EEG task is not sufficiently compelling to support the authors' claims that alpha inhibition does not occur in early sensory processing. However, the findings are bolstered by the additional MEG study which included changes in task design and a source-localization analysis. Importantly, the MEG results are aligned with the EEG study's key findings and support the authors' initial results, making a stronger case for their claims.

It is important to note that task designs can have great implications for the assessment of alpha inhibition, particularly with the use of stimuli that evoke a steady-state response, and the authors review these considerations during their discussion and interpretation of the theory. Overall, this paper is an excellent contribution to the alpha-inhibition literature and will hopefully motivate additional research on the specific relationship between these attention-related changes using both frequency-tagged and non-frequency-tagged stimuli in different task contexts.

Reviewer #3 (Public review):

In the present study, the authors aimed to achieve a better understanding of the mechanisms underlying the attentional blink, that is, a deficit in processing the second of two target stimuli when they appear in rapid succession. Specifically, they used a concurrent detection and identification task in- and outside of the attentional blink and decoupled effects of perceptual sensitivity and response bias using a novel signal detection model. They conclude that the attentional blink selectively impairs perceptual sensitivity but not response bias, and link established EEG markers of the attentional blink to deficits in stimulus detection (N2p, P3) and discrimination (fronto-parietal high-beta coherence), respectively. Taken together, their study suggests distinct mechanisms mediating detection and discrimination deficits in the attentional blink.

This innovative study appears to have been carefully conducted and the overall conclusions seem warranted given the results. In my opinion, the manuscript is a valuable contribution to the current literature on the attentional blink. Moreover, the novel paradigm and signal detection model are likely to stimulate future research.

Major strengths of the present study include its innovative approach to investigating the mechanisms underlying the attentional blink, an elegant, carefully calibrated experimental paradigm, a novel signal detection model, multifaceted data analyses using state-of-the-art model comparisons and robust statistical tests, and an interesting discussion on the neural mechanisms underlying detection versus identification.

Weaknesses concern a lack of clarity regarding specific statistical hypotheses and correction for multiple comparisons (e.g., across or within the multiple classes of tests) in the Methods, relatively low statistical power (N = 24/18 for behavioral/ERP data, respectively), unusual and heavy EEG filtering (0.5-18 Hz bandpass and 9-11 Hz bandstop), data-driven analyses (e.g., pooling of lag 1 and 3 trials a posteriori), and the absence of a discussion of limitations.

Reviewer #3 (Public review):

Summary:

Donofrio et al. report a new observation that in normal aging mice, anti-calbindin wholemount staining and coronal immunohistochemistry in the cerebellum often show a sagittally patterned loss of Purkinje cells with age. The authors address a central concern that calbindin antibody staining alone is not sufficient to definitively assess Purkinje cell loss, and corroborate their antibody staining data with transgenic Pcp2-CRE x flox-GFP reporter mice and Neutral Red staining. The authors then investigate whether this patterned Purkinje loss correlates with the known parasagittal expression of zebrin-II, finding a strong but imperfect correlation with zebrin-II antibody staining. They next draw a connection between this age-related Purkinje loss to the age-related decline in motor function in mice, with a trending but non-significant statistical association between the severity/patterning of Purkinje loss and motor phenotypes within cohorts of aged mice. Finally, the authors look at post-mortem human cerebellar tissues from deceased healthy donors between 21 and 74 years of age, finding a positive correlation between Purkinje degeneration and age, but with unknown spatial patterning.

Strengths:

The conclusions drawn from this study are well supported by the data provided. The authors highlight several examples of parasagittal patterning of Purkinje cell degeneration in disease, and show that proper methodologies must be used to account for these patterns to avoid highly variable data in the sagittal plane. The authors aptly point out that additional work is needed to investigate the spatial patterns of Purkinje cell loss in the human cerebellum.

Weaknesses:

(1) In Figure 3, the authors use Pcp2-CRE mice to drive GFP expression in Purkinje cells in order to avoid the confounding variable of loss of calbindin expression in aging Purkinje cells. The authors go on to say, "we argue that calbindin expression alone is not a reliable, sufficient indicator of Purkinje cell loss". However, in Figure 4, the authors return to calbindin staining alone to assess the correlation of Purkinje cell loss with zebrin-II expression. Could the authors comment on why zebrin-II co-staining experiments were not performed in GFP reporter mice to avoid potential confounds of calbindin expression? Without this experiment, should readers accept the data presented in Figure 4 as a "reliable, sufficient indicator of Purkinje cell loss", given the author's prior claim?

(2) Throughout the manuscript, there is a considerable reliance on the authors' interpretation of imaging data with no accompanying quantification (categorization of "striped" or "non-striped" PC loss, correlation of GFP/calbindin/zebrin-II staining, etc.). While this may be difficult to obtain, the results would be much stronger with a quantitative approach to support the stated categorizations/observations.

Reviewer #3 (Public review):

Summary:

Nigro et al examine how the locus coeruleus (LC) influences the medial prefrontal cortex (mPFC) during attentional shifts required for behavioral flexibility. Specifically, they propose that LC-mPFC inputs enable mice to shift attention effectively from texture to odor cues to optimize behavior. The LC and its noradrenergic projections to the mPFC have previously been implicated in this behavior. The authors further establish this by using chemogenetics to inhibit LC terminals in mPFC and show a selective deficit in extradimensional set-shifting behavior. However, the study's primary innovation is the simultaneous inhibition of LC while recording multineuron patterns of activity in mPFC. Analysis at the single neuron and population levels revealed broadened tuning properties, less distinct population dynamics, and disrupted predictive encoding when LC is inhibited. These findings add to our understanding of how neuromodulatory inputs shape attentional encoding in mPFC. However, several issues somewhat limit the overall impact and interpretation of the results.

Strengths:

The more naturalistic set-shifting task used in the study is a major strength, and its implementation in freely-moving animals is very useful. The inclusion of localized suppression of LC-mPFC terminals is also a strength that builds confidence in the specificity of their behavioral effect. Moreover, the combination of chemogenetic inhibition of LC while simultaneously recording neural activity in mPFC with miniscopes is state-of-the-art. The authors apply analyses to population dynamics, in particular, that can advance our understanding of how the LC modifies patterns of mPFC neural activity. The authors show that neural encoding at both the single-cell level and the population level is disrupted when LC is inhibited. They also show that activity is less able to predict key aspects of the behavior when the influence of LC is disrupted. This is quite interesting and adds to a growing understanding of how neuromodulatory systems sharpen the tuning of mPFC activity.

Weaknesses:

There are some concerns about tying the results to noradrenergic circuit activity. The authors use a DBH-Cre mouse line, but the histology images provided are low resolution, and surprisingly, there appears to be little overlap between HM4Di expression and TH immunostaining. It is unclear what explains this, but without further confirmation, it is hard to be sure whether the manipulation selectively impacts a specific LC population. While the authors are generally conservative in relating their findings to norepinephrine (NE) signaling, it is still implied that this is likely. But even if HM4Di is expressed specifically in DBH+ LC neurons, there is no confirmation that NE release is suppressed, and these neurons may release other neurotransmitters, including glutamate and dopamine. In the absence of careful controls, it is important to recognize that effects may or may not be due to LC-mPFC NE.

Another weakness is that the behavior of miniscope mice is not shown. These experiments make up the bulk of the study, including the most significant results (Figures 2-4). Interpreting the chemogenetics + imaging results without this data is more challenging and relies on the assumption that they were affected similarly to an animal from Figure 1. More fundamentally, the imaging analyses are entirely from the extradimensional shift session. Showing similar analyses from the intradimensional shift (IDS) session would confirm that test group mice do not exhibit broadened tuning prior to injecting CNO and would help to establish whether the observed changes are to some feature of activity that is specific to extradimensional shifts. The ideal experiment would also include a separate group of animals with LC suppression during the IDS, which would show whether the observed effects are specific to an extradimensional shift and might explain behavioral effects.

There are also some weaknesses in how the single neuron encoding data is analyzed and presented. First, the corresponding methods section is insufficient to fully understand how selectively tuned neurons were classified. The authors perform ROC analysis for the period 0 - 5s before choice to reveal choice-tuned neurons. It would be useful to know what proportion of the total neurons this represents, and whether this includes neurons with activity that is significantly increased, decreased, or both. Further, insufficient detail is provided to be able to understand how neurons are further classified into 'choice', 'history', and 'switch' categories, or what percentage of ROC-identified neurons fall into each category (only % of total neurons is provided).

Finally, there are some concerns about lumping all the identified neurons together (as in Figure 2F). The miniscope experiments include very few mice (n=4 controls, n=5 test), and effects may be driven by only 1 or 2 subjects. Also, plotting the data on a per-animal basis would help to better understand the effects in greater detail. Overall, the results are interesting, but these weaknesses limit the strength and specificity of the claims that can be made.

Reviewer #3 (Public review):

This study investigated the in vitro amplification of donor fecal virus using chemostat culturing technology, aiming to reduce eukaryotic virus load while preserving bacteriophage community diversity, thereby optimizing the safety and efficacy of FVT. The research employed a preterm pig model to evaluate the effects of chemostat-propagated viromes (CVT) in preventing necrotizing enterocolitis (NEC) and mitigating adverse effects such as diarrhea.

Strengths:

(1) Enhanced Safety Profile:<br /> Chemostat cultivation effectively reduced eukaryotic virus load, thereby minimizing the potential infection risks associated with virome transplantation and offering a safer virome preparation method for clinical applications.

(2) Process Reproducibility:<br /> The chemostat system achieved stable amplification of bacteriophage communities (Bray-Curtis similarity >70%), mitigating the impact of donor fecal variability on therapeutic efficacy.

Weaknesses:

(1) Loss of Phage Functionality:<br /> The chemostat cultivation resulted in a reduction in phage diversity (e.g., the loss of Lactobacillaceae phages), which may compromise their protective effects against NEC (potentially linked to the immunomodulatory functions of Lactobacilli). The authors should explicitly address this limitation in the discussion section, particularly if additional experiments cannot be conducted to resolve it within the current study.

(2) Limitations in Experimental Design:<br /> The low incidence of NEC lesions in the control group reduced the statistical power of the study. This limitation undermines the ability to conclusively evaluate the efficacy and safety of the chemostat-propagated virome as a novel intervention for NEC. Future studies should optimize experimental conditions (e.g., using a more NEC-susceptible model or diet) to ensure adequate disease incidence for robust statistical comparisons.

Reviewer #3 (Public review):

Summary:

Predicting how two different drugs act together by looking at their specific gene targets and pathways is crucial for understanding the biological significance of drug combinations. This study incorporates drug-specific pathway activation scores (PASs) to estimate synergy scores as one of the key advancements for synergy prediction. The new algorithm, Drug synergy Interaction Prediction (DIPx), developed in this study, uses gene expression, mutation profiles, and drug synergy data to train the model and predict synergy between two drugs. Comprehensive comparisons with another best-performing algorithm, TAIJI-M, highlight the potential of its capabilities.

Strengths:

DIPx uses target and driver genes to elucidate pathway activation scores (PASs) to predict drug synergy. Its performance was tested using the AstraZeneca-Sanger (AZS) DREAM Challenge dataset, especially in Test Set 1, where the Spearman correlation coefficient between predicted and observed drug synergy was 0.50 (95% CI: 0.47-0.53). DIPx's ability to handle novel combinations, as evidenced by its performance in test set 2, indicates the potential for predicting new and untested drug combinations, even though it's lower than that of the test set 1.

Weaknesses:

While the DIPx algorithm shows promise in predicting drug synergy based on pathway activation scores, it's essential to consider its limitations. One limitation is that the availability of training data for specific drug combinations may influence its predictive capability. Further testing and experimental validation of the predictions in future studies would be necessary to assess the algorithm's generalizability and robustness.

Reviewer #3 (Public review):

Summary:

This manuscript utilized zebrafish bcas2 mutants to study the role of bcas2 in primitive hematopoiesis, and further confirms that it has a similar function in mice. Moreover, they showed that bcas2 regulates the transition of hematopoietic differentiation from angioblasts via activating Wnt signaling. By performing a series of biochemical experiments, they also showed that bcas2 accomplishes this by sequestering b-catenin within the nucleus, rather than through its known function in pre-mRNA splicing.

Strengths:

The work is well-performed, and the manuscript is well-written.

Comments on revisions:

The revised manuscript is substantially improved, and all my previous questions are now well addressed.

Reviewer #3 (Public review):

This paper describes a new mechanism for the clearance of protein aggregates associated to endoplasmic reticulum re-organization that occurs during mitosis.

Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting. The authors made several new experiments included in the revised version to address the concerns raised by reviewers. A new proteomic analysis, co-localization of the aggregates with the ER membrane Sec61beta protein, expression of the aggregate-prone protein in the nucleus does not result in accumulation of aggregates, detection of protein aggregates in the insoluble faction after cell disruption and mostly importantly knockdown of ATL proteins involved in the organization of ER shape and structure impaired the clearance mechanism. This last observation addresses one of the weakest points of the original version which was the lack of experimental correlation between ER structure capability to re-shape and the clearance mechanism.

In conclusion, this new mechanism of protein aggregate clearance from the ER was not completely understood in this work but the manuscript presented, particularly in the revised version, an ensemble of solid observations and mechanistic information to scaffold future studies that clarify more details of this mechanism. As stated by the authors: "How protein aggregates are targeted and assembled into the intranuclear membranous structure waits for future investigation". This new mechanism of aggregate clearance from the ER is not expected to be fully understood in a single work but this paper may constitute one step to better comprehend the cell capability to resolve protein aggregates in different cell compartments.

[Editors' note: The authors have appropriately addressed the previous reviewers' concerns.]

Reviewer #3 (Public review):

Summary:

This manuscript by Luo et al. applied SHAPE-Map to analyze the secondary structure of the Porcine Epidemic Diarrhoea Virus (PEDV) RNA genome in infected cells. By combining SHAPE reactivity and Shannon entropy, the study indicated that the folding of the PEDV genomic RNA was nonuniform, with the 5' and 3' untranslated regions being more compactly structured, which revealed potentially antiviral targetable RNA regions. Interestingly, the study also suggested that compounds bound to well-folded RNA structures in vitro did not necessarily exhibit antiviral activity in cells, because the binding of these compounds did not necessarily alter the functions of the well-folded RNA regions. Later in the manuscript, the authors focus on guanine-rich regions, which may form G-quadruplexes and be potential targets for small interfering RNA (siRNA). The manuscript shows the binding effect of Braco-19 (a G-quadruplex-binding ligand) to a predicted G4 region in vitro, along with the inhibition of PEDV proliferation in cells. This suggests that targeting high SHAPE-high Shannon G4 regions could be a promising approach against RNA viruses. Lastly, the manuscript identifies 73 single-stranded regions with high SHAPE and low Shannon entropy, which demonstrated high success in antiviral siRNA targeting.

Strengths:

The paper presents valuable data for the community. Additionally, the experimental design and data analysis are well documented.

Weaknesses:

I have no further comments after the authors validated their concept by adding the ThT fluorescence assay in the revised version.

Reviewer #3 (Public review):

This paper investigates invariance to natural background noise in the auditory cortex of ferrets and humans. The authors first replicate, in ferrets, a finding from human neuroimaging showing that invariance to background noise increases along the cortical hierarchy (i.e., from primary to non-primary auditory cortex). Next, the authors ask whether this pattern of invariance could be explained by differences in tuning to low-level acoustic features across primary and non-primary regions. The authors conclude that this tuning can explain the spatial organization of background invariance in ferrets, but not in humans. The conclusions of the paper are generally well supported by the data, but additional control analyses are needed to fully substantiate the paper's claims. Finally, additional discussion and potentially analysis, are needed to reconcile these findings with similar work in the literature (particularly that of Hamersky et al. 2025 J. Neurosci.).

The paper is very straightforwardly written, with a generally clear presentation including well-designed and visually appealing figures. Not only does this paper provide an important replication in a non-human animal model commonly used in auditory neuroscience, but it also extends the original findings in three ways. First, the authors reveal a more fine-grained gradient of background invariance by showing that background invariance increases across primary, secondary, and tertiary cortical regions. Second, the authors address a potential mechanism that might underlie this pattern of invariance by considering whether differences in tuning to frequency and spectrotemporal modulations across regions could account for the observed pattern of invariance. The spectrotemporal modulation encoding model used here is a well-established approach in auditory neuroscience and seems appropriate for exploring potential mechanisms underlying invariance in auditory cortex, particularly in ferrets. However, as discussed below, the analyses based on this simple encoding model are only informative to the extent that the model accurately captures neural responses. Thus, its limitations in modeling non-primary human auditory cortex should be considered when interpreting cross-species comparisons. Third, the authors provide a more complete picture of invariance by additionally analyzing foreground invariance, a complementary measure not explored in the original study. While this analysis feels like a natural extension and its inclusion is appreciated, the interpretation of these foreground invariance findings remains somewhat unclear, as the authors offer limited discussion of their significance or relation to existing literature.

As mentioned above, interpretation of the invariance analyses using predictions from the spectrotemporal modulation encoding model hinges on the model's ability to accurately predict neural responses. Although Figure S5 suggests the encoding model was generally able to predict voxel responses accurately, the authors note in the introduction that, in human auditory cortex, this kind of tuning can explain responses in primary areas but not in non-primary areas (Norman-Haignere & McDermott, PLOS Biol. 2018). Indeed, the prediction accuracy histograms in Figure S5C suggest a slight difference in the model's ability to predict responses in primary versus non-primary voxels. Additional analyses should be done to a) determine whether the prediction accuracies are meaningfully different across regions and b) examine whether controlling for prediction accuracy across regions (i.e., sub-selecting voxels across regions with matched prediction accuracy) affects the outcomes of the invariance analyses.

A related concern is the procedure used to train the encoding model. From the methods, it appears that the model may have been fit using responses to both isolated and mixture sounds. If so, this raises questions about the interpretability of the invariance analyses. In particular, fitting the model to all stimuli, including mixtures, may inflate the apparent ability of the model to "explain" invariance, since it is effectively trained on the phenomenon it is later evaluated on. Put another way, if a voxel exhibits invariance, and the model is trained to predict the voxel's responses to all types of stimuli (both isolated sounds and mixtures), then the model must also show invariance to the extent it can accurately predict voxel responses, making the result somewhat circular. A more informative approach would be to train the encoding model only on responses to isolated sounds (or even better, a completely independent set of sounds), as this would help clarify whether any observed invariance is emergent from the model (i.e., truly a result of low-level tuning to spectrotemporal features) or simply reflects what it was trained to reproduce.

Finally, the interpretation of the foreground invariance results remains somewhat unclear. In ferrets (Figure 2I), the authors report relatively little foreground invariance, whereas in humans (Figure 5G), most participants appear to show relatively high levels of foreground invariance in primary auditory cortex (around 0.6 or greater). However, the paper does not explicitly address these apparent cross-species differences. Moreover, the findings in ferrets seem at odds with other recent work in ferrets (Hamersky et al. 2025 J. Neurosci.), which shows that background sounds tend to dominate responses to mixtures, suggesting a prevalence of foreground invariance at the neuronal level. Although this comparison comes with the caveat that the methods differ substantially from those used in the current study, given the contrast with the findings of this paper, further discussion would nonetheless be valuable to help contextualize the current findings and clarify how they relate to prior work.

Reviewer #3 (Public review):

Summary:

Soffers et al. developed a comprehensive genetic toolkit that enables researchers to access neuronal hemilineages during developmental and adult time points using scRNA-seq analysis to guide gene cassette exchange-based or CRISPR-based tool building. Currently, research groups studying neural circuit development are challenged with tying together findings in the development and mature circuit function of hemilineage related neurons. Here, authors leverage publicly available scRNA-seq datasets to inform the development of a split-Gal4 library that targets 32 of 34 hemilineages in development and adult stages. The authors demonstrated that the split-Gal4 library, or genetic toolkit, can be used to assess the functional roles, neurotransmitter identity, and morphological changes in targeted cells. The tools presented in this study should prove to be incredibly useful to Drosophila neurobiologists seeking to link neural developmental changes to circuit assembly and mature circuit function. Additionally, some hemilineages have more than one split-Gal4 combination that will be advantageous for studies seeking to disrupt associated upstream genes.

Strengths:

Informing genetic tool development with publicly available scRNA-seq datasets is a powerful approach to creating specific driver lines. Additionally, this approach can be easily replicated by other researchers looking to generate similar driver lines for more specific subpopulations of cells, as mentioned in the Discussion.

The unification of optogenetic stimulation data of 8B neurons and connectomic analysis of the Giant-Fiber-induced take-off circuit was an excellent example of the utility of this study. The link between hemilineage-specific functional assays and circuit assembly has been limited by insufficient genetic tools. The tools and data present in this study will help better understand how collections of hemilineages develop in a genetically constrained manner to form circuits amongst each other selectively.

Reviewer #3 (Public review):

Summary:

The manuscript by Guo and colleagues features the documentation and interpretation of three successions of continental to marginal marine deposits spanning the P/T transition and their respective ichnofaunas. Based on these new data inferences concerning end-Permian mass extinction and Triassic recovery in the tropical realm are discussed.

Strengths:

The manuscript is well written and organized and includes a large amount of new lithological and ichnological data that illuminate ecosystem evolution in a time of large scale transition. The lithological documentations, facies interpretations and ichnotaxonomic assignments look alright (with few exceptions).

Weaknesses: [all eliminated in revision]

Reviewer #3 (Public review):

Summary:

Papagiannakis et al. present a detailed study exploring the relationship between DNA/polysome phase separation and nucleoid segregation in Escherichia coli. Using a combination of experiments and modelling, the authors aim to link physical principles with biological processes to better understand nucleoid organisation and segregation during cell growth.

Strengths:

The authors have a conducted a large number of experiments under different growth conditions and physiological perturbations (using antibiotics) to analyse the biophysical factors underlying the spatial organisation of nucleoids within growing E. coli cells. A simple model of ribosome-nucleoid segregation has been developed to explain the observations and tested with cleverly designed perturbation experiments.

The model and explanation presented in the original version have been strengthened with additional results and consideration of new factors. In particular, the radial attachment of the nucleoid, supported by previous studies and the A22 treatment data in this study, provides a plausible mechanism that prevents ribosomes from diffusing between and around the nucleoid lobes through the radial shells surrounding the nucleoid. The revised version of the paper incorporates this effect, resulting in model predictions that align well with the drug treatment outcomes and the observed mid-cell accumulation and confinement of ribosomes.

Furthermore, experiments involving plasmid-based gene expression, designed to redirect transcription away from chromosomal loci, offer compelling validation of the model's predictions. Overall, this is a robust and insightful study that will be of significant value to the quantitative microbiology community.

Reviewer #3 (Public review):

Summary:

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying them to simulated results from a previously published agent based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

Strengths:

It would be great to be able to infer FOI from cross sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross sectional studies. They attempt to validate this process using a previously published agent based model which helps us understand the complexity of parasite population genetics.

Weaknesses:

(1) I fear that the work could be easily over-interpreted as no true validation was done as no field estimates of FOI (I think considered true validation) were measured. You have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions acts as a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

(2) Another aspect of the paper is adding greater realism to the previous agent based model, by including assumptions on missing data and under sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the later before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

(5) The text is a little difficult to follow at times, and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

Comments on revisions:

I think the authors gave a robust but thorough response to our reviews and made some important changes to the manuscript which certainly clarify things for me.

Reviewer #3 (Public review):

Summary:

The data and experiments presented in that study convincingly show that a subpopulation of endothelial cells undergo transformation into pericyte-like cells after stroke in mice. These so-called "E-pericytes" are protective and might present a new target for stroke recovery. The authors used a huge battery of different techniques and modified signaling pathways and cellular interactions using several genetic and pharmacological tools to show that TGFbeta and EndoMT are causes of this transformation.

Strengths:

The amount of different genetic and pharmacological approaches in combination with sophisticated techniques such as single-cell RNAseq is impressive and convincing. The results support their conclusions and the authors achieved their aims. The findings will strongly impact the field of cerebrovascular recovery after stroke and might open up new therapeutic targets.

Weaknesses:

In addition to improving the written and graphical presentation of the results, there is only one point I would like to see clarified: the inclusion of additional experiments, even if they have already been performed but are not applicable due to methodological difficulties regarding the role of Procr+ cells. Negative results also help the scientific community avoid unnecessary experiments and advance understanding.

Reviewer #3 (Public review):

The authors provide convincing data to support an elegant model in which ribosome stalling by ToiL promotes downstream topAI translation and prevents premature Rho-dependent transcription termination. However, the physiological consequences of activating topAI-yjhQP expression upon exposure to various ribosome-targeting antibiotics remain unresolved. The authors have satisfactorily addressed all major concerns raised by the reviewers, particularly regarding the SHAPE-seq data. Overall, this study underscores the diversity of regulatory ribosome-stalling peptides in nature, highlighting ToiL's uniqueness in sensing multiple antibiotics and offering significant insights into bacterial gene regulation coordinated by transcription and translation.

[Editors' note: The earlier public reviews are included. No additional reviews were requested.]

Reviewer #3 (Public review):

Summary:

The authors have extended their previous research to develop TOPBP1 as a potential drug target for colorectal cancer by inhibiting its condensation. Utilizing an optogenetic approach, they identified the small molecule AZD2858, which inhibits TOPBP1 condensation and works synergistically with first-line chemotherapy to suppress colorectal cancer cell growth. The authors investigated the mechanism and discovered that disrupting TOPBP1 assembly inhibits the ATR/Chk1 signaling pathway, leading to increased DNA damage and apoptosis, even in drug-resistant colorectal cancer cell lines.

Comments on latest version:

The authors have addressed most of the concerns that I raised in the first round of revision and I have no further questions. I appreciate the authors's efforts in carrying out an preliminary in vivo work, although as the authors pointed out the compound seems to be not effective in vivo. Future work is desired to address this to clarify the significance of the work.

Reviewer #3 (Public review):

Summary:

The heat shock response (HSR) is an inducible transcriptional program that has provided paradigmatic insight into how stress cues feed information into the control of gene expression. The recent elucidation that the chaperone Hsp70 controls the DNA binding activity of the central HSR transcription factor Hsf1 by direct binding has spurred the question of how such a general chaperone obtains specificity. This study has addressed the next logical question: how J-domain proteins execute this task in budding yeast, the leading cell model for studying the HSR. While an involvement and in part overlapping function of general class A and B J-domain proteins, Ydj1 and Sis1 are indicated by the genetic analysis, a highly specific role for the class A Apj1 in displacing Hsf1 from the promoters is found, unveiling specificity in the system.

Strengths

The central strong point of the paper is the identification of class A J-domain protein Apj1 as a specific regulator of the attenuation of the HSR by removing Hsf1 from HSEs at the promoters. The genetic evidence and the ChIP data strongly support this claim. This identification of a specific role for a lowly expressed nuclear J-domain protein changes how the wiring of the HSR should be viewed. It also raises important questions regarding the model of chaperone titration, the concept that a chaperone with limited availability is involved in a tug of war involving competing interactions with misfolded protein substrates and regulatory interactions with Hsf1. Perhaps Apj1, with its low levels and interactions with misfolded and aggregated proteins in the nucleus, is the titrated Hsp70 (co)chaperone that determines the extent of the HSR? This would mean that Apj1 is at the nexus of the chaperone titration mechanism. Although Apj1 is not a highly conserved J domain protein among eukaryotes the strength of the study is that is provides a conceptual framework for what may be required for chaperone titration in other eukaryotes: One or more nuclear J-domain proteins with low nuclear levels that has an affinity for Hsf1 and that can become limiting due to interactions with misfolded Hsp70 proteins. The provides a pathway for how these may be identified using, for example, ChIP-seq.

Weaknesses

A built-in challenge when studying the mechanism of the HSR is the general role of the Hsp70 chaperone system and its J domain proteins. Indeed, a weakness of the study is that it is unclear which of the phenotypic effects have to do with directly recruiting Hsp70 to Hsf1 dependent on a J domain protein and what instead is an indirect effect of protein misfolding caused by the mutation. This interpretation problem is clearly and appropriately dealt with in the manuscript text and in experiments, but is of such fundamental nature that it cannot easily be fully ruled out. One way forward is a reconstituted biochemical system that monitors how Hsf1 DNA binding is affected by the Hsp70 system, misfolded proteins, and the various J domain proteins. Yet this approach is clearly beyond the scope of this study.

Reviewer #3 (Public review):

Summary

Kerlin et.al combined single-molecule RNA FISH with oligonucleotide-based DNA FISH to directly examine the transcriptional activities of three adjacent genes at individual alleles in MCF7 cells. Importantly, they provided quantitative methods to resolve allele-specific (cis) and cell-to-cell (trans) variation and quantified the contribution of burst co-occurrence and burst size, which may help to more accurately analyze transcription coregulation. They found that transcriptional variability is largely gene-autonomous, and by disentangling burst co-occurrence and burst size after E2 induction, they proposed two distinct mechanisms of local gene regulation.

Strengths:

(1) Innovative Research Methods: Successfully integrates single-molecule RNA FISH with oligonucleotide-based DNA FISH to directly image the transcriptional activities of three adjacent genes at individual alleles. This enables the observation of transcriptional dynamics more precisely and provides a powerful tool for studying gene regulation.

(2) Novel Data Analysis Approaches: Develops two new analysis methods to dissect the sources of gene activity (co)variation. One approach separates allele-extrinsic, allele-intrinsic, and gene-autonomous components, and the other quantifies the contributions of burst co-occurrence and burst size correlations. These methods help to more accurately analyze transcriptional correlations between genes and reveal potential regulatory mechanisms.

Weaknesses:

Biological Insights: The findings challenge the traditional view of contact insulation sites as strict regulators of gene coregulation and suggest two distinct coregulatory mechanisms influenced by local chromosome folding. However, expression activity of multiple genes is differentially correlated at the population-level or cell-level versus single-allele-level. More in-depth analysis is needed for further biological insights.

Reviewer #3 (Public review):

Summary:

The authors present a technically impressive data set showing that repeated excitation or restraint stress internalises somato dendritic α2A adrenergic autoreceptors (α2A ARs) in locus coeruleus (LC) neurons. Loss of these receptors weakens GIRK-dependent autoinhibition, raises neuronal excitability, and is accompanied by higher MAO-A, DOPEGAL, AEP, and tau N368 levels. The work combines rigorous whole-cell electrophysiology with barbadin-based trafficking assays, qPCR, Western blotting, and immunohistochemistry. The final schematic is appealing and could, in principle, explain early LC hyperactivity followed by degeneration in ageing and Alzheimer's disease.

Strengths:

(1) Multi-level approach - The study integrates electrophysiology, pharmacology, mRNA quantification, and protein-level analysis.

(2) The use of barbadin to block β-arrestin/AP-2-dependent internalisation is both technically precise and mechanistically informative.

(3) Well-executed electrophysiology.

(4) Translation relevance - converges to a model that can be discussed by peers (scientists can only discuss models - not data!).

Weaknesses:

Nevertheless, the manuscript currently reads as a sequence of discrete experiments rather than a single causal chain. Below, I outline the key points that should be addressed to make the model convincing.

Reviewer #3 (Public review):

Summary:

A subset of cancer cells attain replicative immortality by activating the ALT mechanism of telomere maintenance, which is currently the subject of intense research due to its potential for novel targeted therapies. Key questions remain in the field, such as whether ALT telomeres adhere to the same end-protection rules as telomeres in telomerase-expressing cells, or if ALT telomeres possess unique properties that could be targeted with new, less toxic cancer therapies. Both questions, along with the approaches developed by the authors to address them, are highly relevant.

Strengths:

Since chromosome ends resemble one-ended DSBs, the authors hypothesized that the previously described END-SEQ protocol could be used to accurately sequence the 5' end of telomeres on the C-rich strand. As expected, most reads corresponded to the C-rich strand and, confirming a previous observation by de Lange's group, most chromosomes end with the ATC-5' sequence, a feature that was found to be dependent on POT1 and to be conserved in both human ALT cells and mouse cells. Through a complementary method, S1-END-SEQ, the authors further explored ssDNA regions at telomeres, providing new insights into the characteristics of ALT telomeres. The study is original, the experiments were well-controlled and excellently executed.

Weaknesses:

Overall, the discussion section is lacking depth and should be expanded and a few additional experiments should be performed to clarify the results.

(1) The finding that the abundance of variant telomeric repeats (VTRs) within the final 30 nucleotides of the telomeric 5' ends is similar in both telomerase-expressing and ALT cells is intriguing, but the authors do not address this result. Could the authors provide more insight into this observation and suggest potential explanations? As the frequency of VTRs does not seem to be upregulated in POT1-depleted cells, what then drives the appearance of VTRs on the C-strand at the very end of telomeres? Is CST-Pola complex responsible?

(2) The authors also note that, in ALT cells, the frequency of VTRs in the first 30 nucleotides of the S1-END-SEQ reads is higher compared to END-SEQ, but this finding is not discussed either. Do the authors think that the presence of ssDNA regions is associated with the VTRs? Along this line, what is the frequency of VTRs in the END-SEQ analysis of TRF1-FokI-expressing ALT cells? Is it also increased? Has TRF1-FokI been applied to telomerase-expressing cells to compare VTR frequencies at internal sites between ALT and telomerase-expressing cells?

Finally, in these experiments (S1-END-SEQ or END-SEQ in TRF1-Fok1), is the frequency of VTRs the same on both the C- and the G-rich strands? It is possible that the sequences are not fully complementary in regions where G4 structures form.

(3) Based on the ratio of C-rich to G-rich reads in the S1-END-SEQ experiment, the authors estimate that ALT cells contain at least 3-5 ssDNA regions per chromosome end. While the calculation is understandable, this number could be discussed further to consider the possibility that the observed ratios (of roughly 0.5) might result from the presence of extrachromosomal DNA species, such as C-circles. The observed increase in the ratio of C-rich to G-rich reads in BLM-depleted cells supports this hypothesis, as BLM depletion suppresses C-circle formation in U2OS cells. To test this, the authors should examine the impact of POLD3 depletion on the C-rich/G-rich read ratio. Alternatively, they could separate high-molecular-weight (HMW) DNA from low-molecular-weight DNA in ALT cells and repeat the S1-END-SEQ in the HMW fraction.

(4) What is the authors' perspective on the presence of ssDNA at ALT telomeres? Do they attribute this to replication stress? It would be helpful for the authors to repeat the S1-END-SEQ in telomerase-expressing cells with very long telomeres, such as HeLa1.3 cells, to determine if ssDNA is a specific feature of ALT cells or a result of replication stress. The increased abundance of G4 structures at telomeres in HeLa1.3 cells (as shown in J. Wong's lab) may indicate that replication stress is a factor. Similar to Wong's work, it would be valuable to compare the C-rich/G-rich read ratios in HeLa1.3 cells to those in ALT cells with similar telomeric DNA content.

Minor Points:

(1) The Y-axes of Figure 4 should be relabeled to account for the G-strand reads. Additionally, statistical analyses are absent in Figure 4 and Figure S3.

(2) A careful proofreading of the manuscript is necessary.

Reviewer #3 (Public review):

This study investigates how two cortical regions which are central to the study of rodent motor control (rostral forelimb area, RFA, and caudal forelimb area, CFA) interact during directional forelimb reaching in mice. The authors investigate this interaction using (1) optogenetic manipulations in one area while recording extracellularly from the other, (2) statistical analyses of simultaneous CFA/RFA extracellular recordings, and (3) network modeling. The authors provide solid evidence that asymmetry between RFA and CFA can be observed, although such asymmetry is only observed in certain experimental and analytical contexts.

The authors find asymmetry when applying optogenetic perturbations, reporting a greater impact of RFA inactivation on CFA activity than vice-versa. The authors then investigate asymmetry in endogenous activity during forelimb movements and find asymmetry with some analytical methods but not others. Asymmetry was observed in the onset timing of movement-related deviations of local latent components with RFA leading CFA (computed with PCA) and in a relatively higher proportion and importance of cross-area latent components with RFA leading than CFA leading (computed with DLAG). However, no asymmetry was observed using several other methods that compute cross-area latent dynamics, nor with methods computed on individual neuron pairs across regions. The authors follow up this experimental work by developing a two-area model with asymmetric dependence on cross-area input. This model is used to show that differences in local connectivity can drive asymmetry between two areas with equal amounts of across-region input.

Overall, this work provides a useful demonstration that different cross-area analysis methods result in different conclusions regarding asymmetric interactions between brain areas and suggests careful consideration of methods when analyzing such networks is critical. A deeper examination of why different analytical methods result in observed asymmetry or no asymmetry, analyses that specifically examine neural dynamics informative about details of the movement, or a biological investigation of the hypothesis provided by the model would provide greater clarity regarding the interaction between RFA and CFA.

Strengths:

The authors are rigorous in their experimental and analytical methods, carefully monitoring the impact of their perturbations with simultaneous recordings and providing valid controls for their analytical methods. They cite relevant previous literature that largely agrees with the current work, highlighting the continued ambiguity regarding the extent to which there exists an asymmetry in endogenous activity between RFA and CFA.

A strength of the paper is the evidence for asymmetry provided by optogenetic manipulation. They show that RFA inactivation causes a greater absolute difference in muscle activity than CFA interaction (deviations begin 25-50 ms after laser onset, Figure 1) and that RFA inactivation causes a relatively larger decrease in CFA firing rate than CFA inactivation causes in RFA (deviations begin <25ms after laser onset, Figure 3). The timescales of these changes provide solid evidence for an asymmetry in impact of inactivating RFA/CFA on the other region that could not be driven by differences in feedback from disrupted movement (which would appear with a ~50ms delay).

The authors also utilize a range of different analytical methods, showing an interesting difference between some population-based methods (PCA, DLAG) that observe asymmetry, and single neuron pair methods (granger causality, transfer entropy, and convergent cross mapping) that do not. Moreover, the modeling work presents an interesting potential cause of "hierarchy" or "asymmetry" between brain areas: local connectivity that impacts dependence on across-region input, rather than the amount of across-region input actually present.

Weaknesses:

There is no attempt to examine neural dynamics that are specifically relevant/informative about the details of the ongoing forelimb movement (e.g., kinematics, reach direction). Thus, it may be preemptive to claim that firing patterns alone do not reflect functional influence between RFA/CFA. For example, given evidence that the largest component of motor cortical activity doesn't reflect details of ongoing movement (reach direction or path; Kaufman, et al. PMID: 27761519) and that the analytical tools the authors use likely include this component (PCA, CCA), it may not be surprising that CFA and RFA do not show asymmetry if such asymmetry is related to control of movement details. An asymmetry may still exist in the components of neural activity that encode information about movement details, and thus it may be necessary to isolate and examine the interaction of behaviorally-relevant dynamics (e.g., Sani, et al. PMID: 33169030).

The idea that local circuit dynamics play a central role in determining the asymmetry between RFA and CFA is not supported by experimental data in this paper. The plausibility of this hypothesis is supported by the model but is not explored in any analyses of the experimental data collected. Further experimental investigation is needed to separate this hypothesis from other possibilities.

Comments on revisions:

The authors have improved the manuscript by reviewing several aspects of the text and the addition of supplemental materials. I believe these revisions have clarified some important aspects of the results.

Reviewer #3 (Public review):

Summary:

The authors demonstrated MK2i could enhance the therapeutic efficacy of MTAs. With the tumour xenograft and migration assay, the author suggested that the p38-MK2 pathway may serve as a promising therapeutic target in combination with MTAs in cancer treatment.

Strengths:

The authors provided a potential treatment for breast cancer.

Comments on revisions:

A xenograft experiment should be included to evaluate the synergistic effect of MK2i and vinblastine.

Reviewer #3 (Public review):

Hawes et al. combined behavioral, optical imaging, and activity manipulation techniques to investigate the role of striatal patch SPNs in locomotion regulation. Using Sepw1-Cre transgenic mice, they found that patch SPNs encode locomotion deceleration in a light-dark box procedure through optical imaging techniques. Moreover, genetic ablation of patch SPNs increased locomotion speed, while chemogenetic activation of these neurons decreased it. The authors concluded that a subtype of patch striatonigral neurons modulates locomotion speed based on external environmental cues. Below are some major concerns:

The study concludes that patch striatonigral neurons regulate locomotion speed. However, unless I missed something, very little evidence is presented to support the idea that it is specifically striatonigral neurons, rather than striatopallidal neurons, that mediate these effects. In fact, the optogenetic experiments shown in Fig. 6 suggest otherwise. What about the behavioral effects of optogenetic stimulation of striatonigral versus striatopallidal neuron somas in Sepw1-Cre mice?

In the abstract, the authors state that patch SPNs control speed without affecting valence. This claim seems to lack sufficient data to support it. Additionally, speed, velocity, and acceleration are very distinct qualities. It is necessary to clarify precisely what patch neurons encode and control in the current study.

One of the major results relies on chemogenetic manipulation (Figure 5). It would be helpful to demonstrate through slice electrophysiology that hM3Dq and hM4Di indeed cause changes in the activity of dorsal striatal SPNs, as intended by the DREADD system. This would support both the positive (Gq) and negative (Gi) findings, where no effects on behavior were observed.

Finally, could the behavioral effects observed in the current study, resulting from various manipulations of patch SPNs, be due to alterations in nigrostriatal dopamine release within the dorsal striatum?

Reviewer #3 (Public review):

Summary:

Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since it's reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice, are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mouse, these two neuropeptides are not coexpressed in medaka neurons and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mouse due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

Strengths:

This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

The manuscript has been further strengthened by the inclusion of additional information according to my advice: The analysis of ovariectomized female fish and juvenille fish has now been reported in terms of their feeding behaviour and so provide a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the discussion section has been expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function. Modifications made in order to address technical concerns of the other 2 reviewers have also significantly strengthened the presentation of this work.

Weaknesses:

These have now been addressed in the revised version.

Reviewer #3 (Public review):

Summary:

This study presents a valuable finding on the mechanism used by WTAP to modulate the IFN-β stimulation. It describes the phase transition of WTAP driven by IFN-β-induced dephosphorylation. The evidence supporting the claims of the authors is solid.

Strength:

The key finding is the revelation that WTAP undergoes phase separation during virus infection or IFN-β treatment. The authors conducted a series of precise experiments to uncover the mechanism behind WTAP phase separation and identified the regulatory role of 5 phosphorylation sites. They also succeeded in pinpointing the phosphatase involved.

Reviewer #3 (Public review):

I thank the authors for their extensive revision of this paper, and I found some elements greatly improved.<br /> In particular, the authors do embrace a somewhat more speculative tone in the current version, which I think is fitting for this work, as the data seem (to me) to be not fully conclusive. The data set collected here is clearly valuable and unique (and I would encourage the authors to make it publicly available!), however, my overall impression is that the specific analyses reported here might not fully

Despite the revised description of methods, results and figures, I still have trouble understanding many of the results and the authors conclusive interpretation of them. These are my main reservations:

(1) Regarding "individual prediction tendency" - thank you for adding clarifying methodological details and showing the data in a new Figure (#2). Honestly, however, I still can't say that I fully understand the result. For example, why is there also a significant response in the random condition as well? And how do you interpret the interesting time-course (with a peak ~200ms prior to the stimulus, and a reduction overtime from there?<br /> Also (I may have missed this, but..) what neural data was used to train the classifier and derive the "prediction tendency" index? Was it just the broadband neural response? Is there a way to know which sensors contributed to this metric (e.g., are they predominantly auditory? Frontal?)? And is there a way to establish the statistical significance of this metric (e.g., how good the decoder actually was in predicting behavioral sensitivity?). I don't see any statistics in the results section describing the individual prediction tendency.

(2) Regarding the TRF analysis - Thanks for clarifying the approach used to obtain 2-second long "segments" of speech tracking. This is an interesting approach, however I think quite new(?) , and for me it raises a whole new set of questions, as well as additional controls and data that I would have liked to see, to be convinced that results are significant. I will elaborate:

- Do I understand correctly that you segment the real and predicted neural response into 2-second long segments and then calculate the Pearsons' correlation between them to assess the goodness of the model? This is very unclear, since in the methods section you state only that "the same" analysis was performed as for the full data - but what exactly? Clearly, values will be very different when using such short segments. I feel that additional details are still required (and perhaps data shown) to fully understand the "semantic violation" analysis of TRFs.

- I would like to reiterate my previous comment regarding the use of permutation tests to verify the validity of TRF-based measures derived. This would be especially important when using new approaches (such as the segmentation used here). The authors argue that this is not needed since this was not done in their previously published study. However, this sounds a bit like "two wrongs make a right" argument... why not just do it, and let us know that this 2-second segmentation approach allows estimating reliable speech tracking?

- Following up on my previous comment that defining "clusters" as at least two neighboring channels (Figure 3) - the fact that this is a default in Fieldtrip is by no means sufficient justification!. This seems quite liberal to me, especially given the many comparisons performed. Here too, permutations can help to determine the necessary data-driven threshold for corrections. This is of course critical for interpreting the result shown in Figures 3E&G that are critical "take home messages" of the paper - i.e., that the prediction-index from the first part of the experiment is related to speech tracking in the second part of the experiment. To my eyes, this does not look extremely convincing, but perhaps the authors can show more conclusive data to support this (e.g., scatter plots of the betas across participant?).<br /> - A similar point can be made for the effect of semantic violations (though here the scalp-level result is somewhat more clustered). The authors point out that the semantic effect is a "replication" of their result reported in Schubert et al. 2023, but if I am not mistaken the results there were somewhat different (as was the manipulation). It would be nice to explicitly discuss the similarity/difference between these effects.

(3) Regarding the ocular-TRFs -

- Maybe this is just me, but I believe that effects that are robust should be clearly visible in the data, without the need for fancy "black-box" statistical models. In the case of the ocular TRFs, it is hard for me to see how these time-courses are not just noise (and, again, a permutation test would have helped to convince me..). The inconsistent results for horizontal and vertical eye-movements vis a vis the experimental conditions (single vs. multi-speaker conditions) don't help either, despite the authors argument that these are "independent" - but why should this be the case, especially if there is nothing really to look at in this task?<br /> - I remain with this scepticism for the mediation-portion of the analysis as well... But perhaps replications from other groups or making the data public will help shed further light on this in the future.

Minor<br /> - Thanks for adding information about the creation of semantic-violation stimuli. Since the violations and lexical-controls were taken from different audio recordings, it would have been nice to verify that differences between neural responses cannot be attributed to differences in articulations (e.g., by comparing their spectro-temporal properties).

Reviewer #3 (Public review):

Kobayashi et al identify MER21C as a common promoter of GPR1-AS/Liz in Euarchontoglires, which establishes a somatic DMR that controls ZFDB2 imprinting. In mice, MER21C appears to have diverged significantly from its primate counterparts and is no longer annotated as such.

The authors used high-quality cross-species RNA-seq data to characterise GPR1-AS-like transcripts, which included generating new data in five different species. The association between MER21C/B elements and the promoter of GPR1-AS in most species is clear and convincing. The expression pattern of MER21C/B elements overall further supports their role in enabling correct temporal expression of GPR1-AS during embryonic development.

In the revised version of the manuscript the authors provided additional support for the common evolutionary origin of the GPR1-AS/Liz promoter between primates and rodents. They also showed a more extensive concordance between the presence of GPR1-AS-like transcripts and ZDBF2 imprinting.

Altogether, these findings robustly support the conclusions of the paper, shedding light into the events underlying the evolution of imprinting at the ZDBF2 locus.

Reviewer #3 (Public review):

The study of Weber et al. provides a thorough investigation of the roles of four individual dopamine neurons for aversive associative learning in the Drosophila larva. They focus on the neurons of the DL-1 cluster which already have been shown to signal aversive teaching signals. But the authors go beyond the previous publications and test whether each of these dopamine neurons responds to salt or sugar, is necessary for learning about salt, bitter, or sugar, and is sufficient to induce a memory when optogenetically activated. In addition, previously published connectomic data is used to analyze the synaptic input to each of these dopamine neurons. The authors conclude that the aversive teaching signal induced by salt is distributed across the four DL-1 dopamine neurons, with two of them, DAN-f1 and DAN-g1, being particularly important. Overall, the experiments are well designed and performed, support the authors' conclusions, and deepen our understanding of the dopaminergic punishment system.

Strengths:

(1) This study provides, at least to my knowledge, the first in vivo imaging of larval dopamine neurons in response to tastants. Although the selection of tastants is limited, the results close an important gap in our understanding of the function of these neurons.

(2) The authors performed a large number of experiments to probe for the necessity of each individual dopamine neuron, as well as combinations of neurons, for associative learning. This includes two different training regimen (1 or 3 trials), three different tastants (salt, quinine and fructose) and two different effectors, one ablating the neuron, the other one acutely silencing it. This thorough work is highly commendable, and the results prove that it was worth it. The authors find that only one neuron, DAN-g1, is partially necessary for salt learning when acutely silenced, whereas a combination of two neurons, DAN-f1 and DAN-g1, are necessary for salt learning when either being ablated or silenced.

(3) In addition, the authors probe whether any of the DL-1 neurons is sufficient for inducing an aversive memory. They found this to be the case for two of the neurons, largely confirming previous results obtained by a different learning paradigm, parameters and effector.

(4) This study also takes into account connectomic data to analyze the sensory input that each of the dopamine neurons receives. This analysis provides a welcome addition to previous studies and helps to gain a more complete understanding. The authors find large differences in inputs that each neuron receives, and little overlap in input that the dopamine neurons of the "aversive" DL-1 cluster and the "appetitive" pPAM cluster seem to receive.

(5) Finally, the authors try to link all the gathered information in order to describe an updated working model of how aversive teaching signals are carried by dopamine neurons to the larva's memory center. This includes important comparisons both between two different aversive stimuli (salt and nociception) and between the larval and adult stages.

Reviewer #3 (Public review):

Summary:

In this work, Yamada, Brandani, and Takada have developed a mesoscopic model of the interacting proteins in the postsynaptic density. They have performed simulations, based on this model and using the software ReaDDy, to study the phase separation in this system in 2D (on the membrane) and 3D (in the bulk). They have carefully investigated the reasons behind different morphologies observed in each case, and have looked at differences in valency, specific/non-specific interactions, and interfacial tension.

Strengths:

The simulation model is developed very carefully, with strong reliance on binding valency and geometry, experimentally measured affinities, and physical considerations like the hydrodynamic radii. The presented analyses are also thorough, and great effort has been put into investigating different scenarios that might explain the observed effects.

Weaknesses:

The biggest weakness of the study, in my opinion, has to do with a lack of more in-depth physical insight about phase separation. For example, the authors express surprise about similar interactions between components resulting in different phase separation in 2D and 3D. This is not surprising at all, as in 3D, higher coordination numbers and more available volume translate to lower free energy, which easily explains phase separation. The role of entropy is also significantly missing from the analyses. When interaction strengths are small, entropic effects play major roles.

In the introduction, the authors present an oversimplified view of associative and segregative phase transitions based on the attractive and repulsive interactions, and I'm afraid that this view, in which all the observed morphologies should have clear pairwise enthalpic explanations, diffuses throughout the analysis. Meanwhile, I believe the authors correctly identify some relevant effects, where they consider specific/non-specific interactions, or when they investigate the reduced valency of CaMKII in the 2D system.

Also, I sense some haste in comparing the findings with experimental observations. For example, the authors mention that "For the current four component PSD system, the product of concentrations of each molecule in the dilute phase is in good agreement with that of the experimental concentrations (Table S2)." But the data used here is the dilute phase, which is the remnant of a system prepared at very high concentrations and allowed to phase separate. The errors reported in Table S2 already cast doubt on this comparison. Or while the 2D system is prepared via confining the particles to the vicinity of the membrane, the different diffusive behavior in the membrane, in contrast to the bulk (i.e., the Saffman-Delbrück model), is not considered. This would thus make it difficult to interpret the results of a coupled 2D/3D system and compare them to the actual system.

Reviewer #3 (Public review):

Summary:

A very thorough technical report of a new standalone, open-source software for microscopy image processing and analysis (MorphoNet 2.0), with a particular emphasis on automated segmentation and its curation to obtain accurate results even with very complex 3D stacks, including timelapse experiments.

Strengths:

The authors did a good job of explaining the advantages of MorphoNet 2.0, as compared to its previous web-based version and to other software with similar capabilities. What I particularly found more useful to actually envisage these claimed advantages is the five examples used to illustrate the power of the software (based on a combination of Python scripting and the 3D game engine Unity). These examples, from published research, are very varied in both types of information and image quality, and all have their complexities, making them inherently difficult to segment. I strongly recommend the readers to carefully watch the accompanying videos, which show (although not thoroughly) how the software is actually used in these examples.

Weaknesses:

Being a technical article, the only possible comments are on how methods are presented, which is generally adequate, as mentioned above. In this regard, and in spite of the presented examples (chosen by the authors, who clearly gave them a deep thought before showing them), the only way in which the presented software will prove valuable is through its use by as many researchers as possible. This is not a weakness per se, of course, but just what is usual in this sort of report. Hence, I encourage readers to download the software and give it time to test it on their own data (which I will also do myself).

In conclusion, I believe that this report is fundamental because it will be the major way of initially promoting the use of MorphoNet 2.0 by the objective public. The software itself holds the promise of being very impactful for the microscopists' community.

Reviewer #3 (Public review):

Summary:

The authors aimed to improve cryo-TEM workflows for plant cells. The authors present details on high-pressure-freezing protocols to vitrify, ion-mill, and image certain plant cell types.

Strengths:

Clear step-by-step outline on how to preserve and image cryo samples derived from plants.

Weaknesses:

A general current weakness of cryo-TEM is the problem of vitrifying cells that are embedded in tissues. The vast majority of cells in the plant body are currently not accessible to this technology. This is not a weakness of this specific manuscript but a general problem.

The manuscript is well organized and well written, and the discussion covers practically all questions I had while reading the results section. I only have a few comments, all of which I consider minor.

Reviewer #3 (Public review):

Summary

This paper investigates how disinformation affects reward learning processes in the context of a two-armed bandit task, where feedback is provided by agents with varying reliability (with lying probability explicitly instructed). They find that people learn more from credible sources, but also deviate systematically from optimal Bayesian learning: They learned from uninformative random feedback, learned more from positive feedback, and updated too quickly from fully credible feedback (especially following low-credibility feedback). Overall, this study highlights how misinformation could distort basic reward learning processes, without appeal to higher-order social constructs like identity.

Strengths

(1) The experimental design is simple and well-controlled; in particular, it isolates basic learning processes by abstracting away from social context.

(2) Modeling and statistics meet or exceed the standards of rigor.