Reviewer #1 (Public review):

Summary:

The study investigates how neuropeptidergic signaling affects sleep regulation in Drosophila larvae. The authors first conduct a screen of CRISPR knock-out lines of genes encoding enzymes or receptors for neuropeptides and monoamines. As a result of this screen, the authors follow up on one hit, the hugin receptor, PK2-R1. They use genetic approaches, including mutants and targeted manipulations of PK2-R1 activity in insulin-producing cells (IPCs) to increase total sleep amounts in 2nd instar larvae. Similarly, dilp3 and dilp5 null mutants and genetic silencing of IPCs show increases in sleep. The authors also show that hugin mutants and thermogenetic/optogenetic activation of hugin-expressing neurons caused reductions in sleep. Furthermore, they show through imaging-based approaches that hugin-expressing neurons activate IPCs. A key finding is that wash-on of hugin peptides, Hug-γ and PK-2, in ex vivo brain preparations activates larval IPCs, as assayed by CRTC::GFP imaging. The authors then examine how the PK2-R1, hugin, and IPC manipulations affect adult sleep. Finally, the authors examine how Ca2+ responses through CRTC::GFP imaging in adult IPCs are influenced by the wash-on of hugin peptides. The conclusions of this paper are somewhat well supported by data, but some aspects of the experimental approach and sleep analysis need to be clarified and extended.

Strengths:

(1) This paper builds on previously published studies that examine Drosophila larval sleep regulation. Through the power of Drosophila genetics, this study yields additional insights into what role neuropeptides play in the regulation of Drosophila larval sleep.

(2) This study utilizes several diverse approaches to examine larval and adult sleep regulation, neural activity, and circuit connections. The impressive array of distinct analyses provides new understanding into how Drosophila sleep-wake circuitry in regulated across the lifespan.

(3) The imaging approaches used to examine IPC activation upon hugin manipulation (either thermogenetic activation or wash-on of peptides) demonstrate a powerful approach for examining how changes in neuropeptidergic signaling affect downstream neurons. These experiments involve precise manipulations as the authors use both in vivo and ex vivo conditions to observe an effect on IPC activity.

Weaknesses:

Although the paper does have some strengths in principle, these strengths are not fully supported by the experimental approaches used by the authors. In particular:

(1) The authors show total sleep amount over an 18-hour period for all the measures of 2nd instar larval sleep throughout the paper. However, published studies have shown that sleep changes over the course of 2nd instar development, so more precise time windows are necessary for the analyses in this study.

(2) Previously published reports of sleep metrics in both Drosophila larvae and adults include the average number of sleep episodes (bout number) and the average length of sleep episodes (bout length). Neither of these metrics is included in the paper for either the larval sleep or adult sleep data. Not including these metrics makes it difficult for readers to compare the findings in this study to previously published papers in the established Drosophila sleep literature.

(3) Because Drosophila adult & larval sleep is based on locomotion, the authors need to show the activity values for the experiments supporting their key conclusions. They do show travel distances in Figure 2 - Figure Supplement 1, however, it is not clear how these distances were calculated or how the distances relate to the overall activity of individual larvae during sleep experiments. It is also concerning that inactivation of the PK2-R1-expressing neurons causes a reduction in locomotion speed. This could partially explain the increase in sleep that they observe.

(4) The authors rely on homozygous mutant larvae and adult flies to support many of their conclusions. They also rely on Gal4 lines with fairly broad expression in the Drosophila brain to support their conclusions. Adding more precise tissue-specific manipulations, including thermogenetic activation and inhibition of smaller populations of neurons in the study would be needed to increase confidence in the presented results. Similarly, demonstrating that larval development and feeding are not affected by the broad manipulations would strengthen the conclusions.

(5) Many of the experiments presented in this study would benefit from genetic and temperature controls. These controls would increase confidence in the presented results.

(6) The authors claim that their findings in larvae uncover the circuit basis for larval sleep regulation. However, there is very little comparison to published studies demonstrating that neuropeptides like Dh44 regulate larval sleep. Because hugin-expressing neurons have been shown to be downstream of Dh44 neurons, the authors need to include this as part of their discussion. The authors also do not explain why other neuropeptides in the initial screen are not pursued in the study. Given the effect that these manipulations have on larval sleep in their initial screen, it seems likely that other neuropeptidergic circuits regulate larval sleep.

Reviewer #1 (Public review):

Summary:

Using highly specific antibody reagents for biological research is of prime importance. In the past few years, novel approaches have been proposed to gain easier access to such reagents. This manuscript describes an important step forward toward the rapid and widespread isolation of antibody reagents. Via the refinement and improvement of previous approaches, the Perrimon lab describes a novel phage-displayed synthetic library for nanobody isolation. They used the library to isolate nanobodies targeting Drosophila secreted proteins. They used these nanobodies in immunostainings and immunoblottings, as well as in tissue immunostainings and live cell assays (by tethering the antigens on the cell surface).

Since the library is made freely available, it will contribute to gaining access to better research reagents for non-profit use, an important step towards the democratisation of science.

Strengths:

(1) New design for a phage-displayed library of high content.

(2) Isolation of valuble novel tools.

(3) Detailed description of the methods such that they can be used by many other labs.

Weaknesses:

My comments largely concentrate on the representation of the data in the different Figures.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

A control for non-specific binding is missing from the in vitro binding assay. The figures suffer sometimes from the very small text in the labels, which obscures understanding. Ultimately, while the study provides some interesting and novel insights, we still don't understand which phosphorylation events on which proteins are important for the events occurring at the molecular level, so the advance in knowledge is somewhat incremental.

Reviewer #1 (Public review):

Summary:

Using single-unit recording in 4 regions of non-human primate brains, the authors tested whether these regions encode computational variables related to model-based and model-free reinforcement learning strategies. While some of the variables seem to be encoded by all regions, there is clear evidence for stronger encoding of model-based information in the anterior cingulate cortex and caudate.

Strengths:

The analyses are thorough, the writing is clear, and the work is well-motivated by prior theory and empirical studies.

Weaknesses:

My comments here are quite minor.

The correlation between transition and reward coefficients is interesting, but I'm a little worried that this might be an artifact. I suspect that reward probability is higher after common transitions, due to the fact that animals are choosing actions they think will lead to higher reward. This suggests that the coefficients might be inevitably correlated by virtue of the task design and the fact that all regions are sensitive to reward. Can the authors rule out this possibility (e.g., by simulation)?

The explore/exploit section seems somewhat randomly tacked on. Is this really relevant? If yes, then I think it needs to be integrated more coherently.

Reviewer #1 (Public review):

In the manuscript entitled "Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogen Cryptococcus neoformans" by Jiang et al., the authors employ a combination of molecular genetics and biochemical approaches, along with phenotypic evaluations and animal models, to identify the conserved subunit of the Paf1 complex (Paf1C), Rtf1, and functionally characterize its critical roles in mediating H2B monoubiquitination (H2Bub1) and the consequent regulation of gene expression, fungal development, and virulence traits in C. deneoformans or C. neoformans. Specially, the authors found that the histone modification domain (HMD) of Rtf1 is sufficient to promote H2B monoubiquitination (H2Bub1) and the expression of genes related to fungal mating and filamentation, and restores the fungal morphogenesis and pathogenicity defects caused by RTF1 deletion. These findings highlight the critical contribution of Rtf1's HMD to epigenetic regulation and cryptococcal virulence. This work will be of interest to fungal biologists and medical mycologists, particularly those studying fungal epigenetic regulation and fungal morphogenesis.

Comments on revisions:

The revised manuscript addresses all my previous concerns satisfactorily.

Reviewer #1 (Public review):

Summary:

The authors show for the first time that deleting GLS from rod photoreceptors results in the rapid death of these cells. The death of photoreceptor cells could result from loss of synaptic activity because of a decrease in glutamate, as has been shown in neurons, changes in redox balance, or nutrient deprivation.

Strengths:

The strength of this manuscript is that the author shows a similar phenotype in the mice when Gls was knocked out early in rod development or the adult rod. They showed that rapid cell death is through apoptosis, and there is an increase in the expression of genes responsive to oxidative stress.

Comments on revisions:

The authors addressed all of my concerns in their responses to reviewers.

Reviewer #2 (Public review):

Summary:

The study investigates the potential influence of the response criterion on neural decoding accuracy in consciousness and unconsciousness, utilizing either simulated data or reanalyzing experimental data with post-hoc sorting data.

Strengths:

When comparing the neural decoding performance of Target versus NonTarget with or without post-hoc sorting based on subject reports, it is evident that response criterion can influence the results. This was observed in simulated data as well as in two experiments that manipulated the subject response criterion to be either more liberal or more conservative. One experiment involved a two-level response (seen vs unseen), while the other included a more detailed four-level response (ranging from 0 for no experience to 3 for a clear experience). The findings consistently indicated that adopting a more conservative response criterion could enhance neural decoding performance, whether in conscious or unconscious states, depending on the sensitivity or overall response threshold.

The uneven distribution of trails for Target (75%) and NonTarget (25%) was identified as a potential weakness in the initial review of this study. Nevertheless, we support the authors' assertion that their analysis methodology validates comparing liberal and conservative approaches. Future investigations could further explore differences between liberal and conservative on different ratios of Target vs NonTarget, particularly when the proportion of Target matches or falls below that of NonTarget.

Reviewer #1 (Public review):

Guo, Hue et al., is focused on understanding the epigenetic activity and functional dependencies for two different fusions found in spindle cell rhabdomyosarcoma, VGLL2::NCOA2 and TEAD1::NCOA2. They use a variety of models and methods; specifically, ectopic expression of the fusions in human 293T cells to perform RNAseq (both fusions), CUT&RUN (VGLL2::NCOA2) and BioID mass spec (both fusions). These data identify that the VGLL2::NCOA2 fusion has peaks that are enriched for TEAD motifs. Further, CPB/p300 CUT&RUN support an enrichment of binding sites and three TEAD targets in VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells. They also functionally evaluate genetic and chemical dependencies (TEAD inhibition), and found this was only effective for the VGLL2::NCOA2 fusion, and not for TEAD1::NCOA2. Using complementary biochemical approaches, they suggest (with other supporting data) the fusions regulate TEAD transcriptional outputs via a YAP/TAZ independent mechanism. Further, they expand into a C2C12 myoblast model and show that TEAD1::NCOA2 is transforming in colony formation assays and in mouse allograft. These strategies for TEAD1-NCOA2 are consistent with previous published strategies using VGLL2::NCOA2. Importantly, they show that a CBP/p300 (a binding partner found in their BioID mass spec) small molecule inhibitor suppresses tumor formation using this mouse allograft model, and that the tumors are less proliferative, and have a reduction in transcriptional of three TEAD target genes. They complement in vivo data with biochemical approaches, and suggest this interface with p300 (for VGLL2::NCOA2) is through the NCOA2 fusion partner, as Co-IP in HEK293T with a mutant fusion that does not contain NCOA2 loses the association with endogenous p300. The data is interesting and suggests new biology for these fusion-oncogenes. However, the choice of 293T may limit the broad applicability of the findings. Strikingly, in 293T there was more transcriptional overlap with the VGLL2-NCOA2 fusion with the YAP5SA mutant than with TEAD1-NCOA2. Further, there is an additional opportunity to directly compare transcriptional profiles in 293T to the human disease and in the mouse allograft system to directly compare and discuss VGLL2-NCOA2 and TEAD1-NCOA2 histological differences or how A485 treatment may change the histology. Overall, the breadth of methods used in this study, and comparison of the two fusion-oncogene's biology is of interest to the fusion-oncogene, pediatric sarcoma, and epigenetic therapeutic targeting fields.

Reviewer #1 (Public review):

Summary:

Giménez-Orenga et al. investigate the origin and pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM). Using RNA microarrays, the authors compare the expression profiles and evaluate the biomarker potential of human endogenous retroviruses (HERV) in these two conditions. Altogether, the authors show that HERV expression is distinct between ME/CFS and FM patients, and HERV dysregulation is associated with higher symptom intensity in ME/CFS. HERV expression in ME/CFS patients is associated with impaired immune function and higher estimated levels of plasma cells and resting CD4 memory T cells. This work provides interesting insights into the pathophysiology of ME/CFS and FM, creating opportunities for several follow-up studies.

Strengths:

(1) Overall, the data is convincing and supports the authors' claims. The manuscript is clear and easy to understand, and the methods are generally well-detailed. It was quite enjoyable to read.<br /> (2) The authors combined several unbiased approaches to analyse HERV expression in ME/CFS and FM. The tools, thresholds, and statistical models used all seem appropriate to answer their biological questions.<br /> (3) The authors propose an interesting alternative to diagnosing these two conditions. Transcriptomic analysis of blood samples using an RNA microarray could allow a minimally invasive and reproducible way of diagnosing ME/CFS and FM.

Weakness:<br /> (1) While this work makes several intriguing observations, some results will need to be validated in future studies using experimental approaches.

Reviewer #2 (Public review):

Summary:

This study uses in vivo multimodal high-resolution imaging to track how microglia and neutrophils respond to light-induced retinal injury from soon after injury to 2 months post-injury. The in vivo imaging finding was subsequently verified by ex vivo study. The results suggest that despite the highly active microglia at the injury site, neutrophils were not recruited in response to acute light-induced retinal injury.

Strengths:

An extremely thorough examination of the cellular-level immune activity at the injury site. In vivo imaging observations being verified using ex vivo techniques is a strong plus.

Weaknesses:

This paper is extremely long, and in the perspective of this reviewer, needs to be better organized. Update: Modifications have been made throughout, which has made the manuscript easier to follow.

Study weakness: though the finding prompts more questions and future studies, the findings discussed in this paper is potentially important for us to understand how the immune cells respond differently to different severity level of injury. The study also demonstrated an imaging technology which may help us better understand cellular activity in living tissue during earlier time points.

Comments on revisions:

I appreciate the thorough clarification and re-organization by the authors, and the messages in the manuscript are now more apparent. I recommend also briefly discussing limitations/future improvements in the discussion or conclusion.

Reviewer #1 (Public review):

The manuscript now compares the WNet3D quantitatively against other methods on all four datasets:

Figure 1b shows results on the mouse cortex dataset, comparing StarDist, CellPose, SegResNet, SwinUNetR against self-supervised (or learning-free methods) WNet3D and Otsu thresholding.

Figure 2b shows results on an unnamed dataset (presumably the mouse cortex dataset), comparing StarDist, CellPose, SegResNet, SwinUNetR with different levels of training data against WNet3D.

Figure 3 shows results on three datasets (Platynereis-ISH-Nuclei-CBG, Platynereis-Nuclei-CBG, and Mouse-Skull-Nuclei-CBG), comparing StarDist, CellPose against WNet3D and Otsu thresholding.

It is unclear whether the Otsu thresholding baseline was given the same post-processing as the WNet3D. Figure 1b shows two versions for WNet3D ("WNet3D - No artifacts" and "WNet3D"), but only one for Otsu thresholding. Given that post-processing (or artifact removal) seems to have a substantial impact on accuracy, the authors should clarify whether the Otsu thresholding results were treated in the same way and if Otsu thresholding was not post-processed. Figure 2a would also benefit from including the thresholding results (with and without artifact removal).

Reviewer #2 (Public review):

Summary:

The goal of the paper was to trace the transitions hippocampal microglia undergo along aging. ScRNA-seq analysis allowed the authors to predict a trajectory and hypothesize about possible molecular checkpoints, which keep the pace of microglial aging. E.g. TGF1b was predicted as a molecule slowing down the microglial aging path and indeed, loss of TGF1 in microglia led to premature microglia aging, which was associated with premature loss of cognitive ability. The authors also used the parabiosis model to show how peripheral, blood-derived signals from the old organism can "push" microglia forward on the aging path.

Strengths:

A major strength and uniqueness of this work is the in-depth single-cell dataset, which may be a useful resource for the community, as well as the data showing what happens to young microglia in heterochronic parabiosis setting and upon loss of TGFb in their environment.

Weaknesses:

All weaknesses were addressed during revision.

Overall:

In general, I think the authors did a good job following the initial observations and devised clever ways to test the emerging hypotheses. The resulting data are an important addition to what we know about microglial aging and can be fruitfully used by other researchers, e.g. those working on microglia in a disease context.

Comments on revisions:

All my comments were addressed.

Reviewer #1 (Public review):

Summary:

This study addresses the question of how task-relevant sensory information affects activity in motor cortex. The authors use various approaches to address this question, looking at single units and population activity. They find that there are three subtypes of modulation by sensory information at the single unit level. Population analyses reveal that sensory information affects the neural activity orthogonally to motor output. The authors then compare both single unit and population activity to computational models to investigate how encoding of sensory information at the single-unit level is coordinated in a network. They find that an RNN that displays similar orbital dynamics and sensory modulation to motor cortex also contains nodes that are modulated similarly to the three subtypes identified by the single unit analysis.

Strengths:

The strengths of this study lie in the population analyses and the approach of comparing single-unit encoding to population dynamics. In particular, the analysis in Figure 3 is very elegant and informative about the effect of sensory information on motor cortical activity. The task is also well designed to suit the questions being asked and well controlled.

It is commendable that the authors compare single-unit to population modulation. The addition of the RNN model and perturbations strengthen the conclusion that the subtypes of individual units all contribute to the population dynamics.

Weaknesses:

The main weaknesses of the study lie in the categorization of the single units into PD shift, gain and addition types. The single units exhibit clear mixed selectivity, as the authors highlight. Therefore, the subsequent analyses looking only at the individual classes in the RNN are a little limited. Another weakness of the paper is that the choice of windows for analyses is not properly justified and the dependence of the results on the time windows chosen for single unit analyses is not assessed. This is particularly pertinent because tuning curves are known to rotate during movements (Sergio et al. 2005 Journal of Neurophysiology).

This study uses insights from single-unit analysis to inform mechanistic models of these population dynamics, which is a powerful approach, but is dependent on the validity of the single-cell analysis, which I have expanded on below.

I have clarified some of the areas that would benefit from further analysis below:

Task:

The task is well designed, although it would have benefited from perhaps one more target speed (for each direction). One monkey appears to have experienced one more target speed than the others (seen in Figure 3C). It would have been nice to have this data for all monkeys, although, of course, unfeasible given that the study has been concluded.

Single unit analyses:

The choice of the three categories (PD shift, gain addition) is not completely justified in a satisfactory way. It would be nice to see whether these three main categories are confirmed by unsupervised methods.

The decoder analyses in Figure 2 provide evidence that target speed modulation may change over the trial. Therefore, it is important to see how the window considered for the firing rate in Figure 1 (currently 100ms pre - 100ms post movement onset) affects the results. Whilst it is of course understandable that a window must be chosen and will always be slightly arbitrary, using different windows and comparing the results of two or three different sizes or timed windows would be more convincing that the results are not dependent on this particular window.

RNN:

Mixed selectivity is not analysed in the RNN, which would help to compare the model to the real data where mixed selectivity is common. The CCA and Procrustes analysis are a good start to validate the claim of similarity between RNN and neural dynamics, rather than allowing comparisons to be dominated by geometric similarities that may be features of the task. However, some of the disparity values for the Procrustes analysis are quite high, albeit below that of the shuffle. Maybe a comment about this in the text should be included. There is also an absence of alternate models to compare the perturbation model results to.

Reviewer #2 (Public review):

Summary:

The authors describe a "beads-on-a-string" (BOAS) immunogen, where they link, using a non-flexible glycine linker, up to eight distinct hemagglutinin (HA) head domains from circulating and non-circulating influenzas and assess their immunogenicity. They also display some of their immunogens on ferritin NP and compare the immunogenicity. They conclude that this new platform can be useful to elicit robust immune responses to multiple influenza subtypes using one immunogen and that it can also be used for other viral proteins.

Strengths:

The paper is clearly written. While the use of flexible linkers has been used many times, this particular approach (linking different HA subtypes in the same construct resembling adding beads on a string, as the authors describe their display platform) is novel and could be of interest.

Comments on revisions:

The authors have addressed most comments. Some mistakes/issues remain:

TI should be defined earlier on line 61 not on line 196

No legend for Figure 3E - it looks like this is where the authors did the first immunization with the "mix" to compare to the BOAs but strangely they do not mention this in the response to reviewers letter and only mention fig 6G and 7<br /> Maybe add "mix" to the title of Figure 3?

In Figure 6G they do show the response to the mix but do not mention it in the immunizations for that figure. Also weird because obviously the mix is not a NP while this figure addresses NP format.

Line 796 - pseudo viruses

The authors should add some clarification in the paper as they did in response to reviewers.

Reviewer #1 (Public review):

Summary:

Rossi et al. asked whether gait adaptation is solely a matter of slow perceptual realignment or if it also involves fast/flexible stimulus-response mapping mechanisms. To test this, they conducted a series of split-belt treadmill experiments with ramped perturbations, revealing behavior indicative of a flexible, automatic stimulus-response mapping mechanism.

Strengths:

(1) The study includes a perceptual test of leg speed, which correlates with the perceptual realignment component of motor aftereffects. This indicates that changes in motor performance are not fully accounted for by perceptual realignment.

(2) The study evaluates the possible contributions of explicit strategy using a framework (Tsay et al., 2024) and provides evidence for minimal strategy involvement in split-belt adaptation through subjective reports.

(3) The study incorporates qualitatively distinct, hypothesis-driven models of adaptation and proposes a new framework that integrates these mechanisms. Relatedly, the study considers a range of alternative models, demonstrating that perceptual recalibration and remapping uniquely explain the patterns of behavior and aftereffects, ruling out models that focus solely on a single process (e.g., PReMo, PEA, memory of errors, optimal feedback control) and others that do not incorporate remapping (dual rate state space models).

Reviewer #1 (Public Review):

Summary:

Zhang et al. demonstrate that CD4+ single positive (SP) thymocytes, CD4+ recent thymic emigrants (RTE), and CD4+ T naive (Tn) cells from Cd11c-p28-flox mice, which lack IL-27p28 selectively in Cd11c+ cells, exhibit a hyper-Th1 phenotype instead of the expected hyper Th2 phenotype. Using IL-27R-deficient mice, the authors confirm that this hyper-Th1 phenotype is due to IL-27 signaling via IL-27R, rather than the effects of monomeric IL-27p28. They also crossed Cd11c-p28-flox mice with autoimmune-prone Aire-deficient mice and showed that both T cell responses and tissue pathology are enhanced, suggesting that SP, RTE, and Tn cells from Cd11c-p28-flox mice are poised to become Th1 cells in response to self-antigens. Regarding mechanism, the authors demonstrate that SP, RTE, and Tn cells from Cd11c-p28-flox mice have reduced DNA methylation at the IFN-g and Tbx21 loci, indicating 'de-repression', along with enhanced histone tri-methylation at H3K4, indicating a 'permissive' transcriptional state. They also find evidence for enhanced STAT1 activity, which is relevant given the well-established role of STAT1 in promoting Th1 responses, and surprising given IL-27 is a potent STAT1 activator. This latter finding suggests that the Th1-inhibiting property of thymic IL-27 may not be due to direct effects on the T cells themselves.

Strengths:

Overall the data presented are high quality and the manuscript is well-reasoned and composed. The basic finding - that thymic IL-27 production limits the Th1 potential of SP, RTE, and Tn cells - is both unexpected and well described.

Weaknesses from the original round of review:

A credible mechanistic explanation, cellular or molecular, is lacking. The authors convincingly affirm the hyper-Th1 phenotype at epigenetic level but it remains unclear whether the observed changes reflect the capacity of IL-27 to directly elicit epigenetic remodeling in developing thymocytes or knock-on effects from other cell types which, in turn, elicit the epigenetic changes (presumably via cytokines). The authors propose that increased STAT1 activity is a driving force for the epigenetic changes and resultant hyper-Th1 phenotype. That conclusion is logical given the data at hand but the alternative hypothesis - that the hyper-STAT1 response is just a downstream consequence of the hyper-Th1 phenotype - remains equally likely. Thus, while the discovery of a new anti-inflammatory function for IL-27 within the thymus is compelling, further mechanistic studies are needed to advance the finding beyond phenomenology.

Reviewer #1 (Public review):

Summary:

The authors define the principles that, based on first principles, should be guiding the optimisation of trascription factors with intrinsically disordered regions (IDR). The first part of the study defines the following principles to optimize the binding affinities to the genome in the receiving region that is called the "antenna": (i) reduce the target to IDR-binding distance on the genome, (ii) optimise the distance betwee the DNA binding domain and the binding sites on the IDR to be as close as possible to the distance between their binding sites on the genome; (iii) keep the same number of binding sites and their targets and modulate this number with binding strength, reducing them with increased strenght; (iv) modulate the binding strenght to be above a threshold that depends on the proportion of IDR binding sites in the antenna. The second part defines the scaling of the seach time in function of key parameters such as the volume of the nucleus, and the size of the antenna, derived as a combination of 3D search of the antenna and 1D "octopusing" on the antenna. The third part focuses on validation, where the current results are compared to binding probabilith data from a single experiment, and new experiment are proposed to further validate the model as well as testing designed transcription factors.

Strengths:

The strength of this work is that it provides simple, interpretable and testable theoretical conclusions. This will allow the derived design principles to be understood, evaluated and improved in the future. The theoretical derivations are rigorous. The authors provides a comparison to experiments, and also propose new experiments to be performed in the future, this is a great value in the paper since it will set the stage and inspire new experimental techniques. Further, the field needs inspiration and motivations to develop these techniques, since they are required to benchmark the transcription factors designed with the methods presented in this paper, as well as to develop novel data based or in vivo methods that would greatly benefit the field. As such, this paper is a fundamental contribution to the field.

Weaknesses:

The model assumption that the interaction between the transcription factor and the DNA outside of the antenna region is negligible is probably too strong for many/most transcription factors, particularly in organisms with a longer genome than yeasts. The model presents many first principles to drive the design of transcription factor, but arguably, other principles and mechanisms might also play a role by being beneficial to the search and binding process. Specifically: (i) a role of the IDR in complex formation and cooperativity between multiple trascription factors, (ii) ability of the IDR to do parallel searching based on multiple DNA binding sites spaced by disordered regions, (iii) affinity of the IDR to specific compartmentalisations in the nucleus reducing the search time, etc. The paper would be improved by a discussion over alternative mechanisms.

Reviewer #1 (Public review):

Summary:

This paper uses state-of-the-art techniques to define the cellular composition and its complexity in two rodent species (mice and rats). The study is built on available datasets but extends those in a way that future research will be facilitated. The study will be of high impact for the study of metabolic control.

Strengths:

(1) The study is based on experiments that are combined with two exceptional data sets to provide compelling evidence for the cellular composition of the DVC.

(2) The use of two rodent species is very useful.

Weaknesses:<br /> There is no conceptual weakness, the performance of experiments is state-of-the-art, and the discussion of results is appropriate. One minor point that would further strengthen the data is a more distinct analysis of receptors that are characteristic of the different populations of neuronal and non-neuronal cells; this part could be improved. Currently, it is only briefly mentioned, e.g., line 585ff. See also lines 603ff; it is true that the previous studies lack some information about the neurotransmitter profile of cells, but combining all data sets should result in an analysis of the receptors as well, e.g. in the form of an easy-to-read table.

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data. In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph.

This work provides a useful resource for studying nitrogenase evolution. However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

Reviewer #1 (Public review):

Summary:

The present study addresses whether physiological signals influence aperiodic brain activity with a focus on age-related changes. The authors report age effects on aperiodic cardiac activity derived from ECG in low and high-frequency ranges in roughly 2300 participants from four different sites. Slopes of the ECGs were associated with common heart variability measures, which, according to the authors, shows that ECG, even at higher frequencies, conveys meaningful information. Using temporal response functions on concurrent ECG and M/EEG time series, the authors demonstrate that cardiac activity is instantaneously reflected in neural recordings, even after applying ICA analysis to remove cardiac activity. This was more strongly the case for EEG than MEG data. Finally, spectral parameterization was done in large-scale resting-state MEG and ECG data in individuals between 18 and 88 years, and age effects were tested. A steepening of spectral slopes with age was observed, particularly for ECG and, to a lesser extent, in cleaned MEG data in most frequency ranges and sensors investigated. The authors conclude that commonly observed age effects on neural aperiodic activity can mainly be explained by cardiac activity.

Strengths:

Compared to previous investigations, the authors demonstrate effects of aging on the spectral slope in the currently largest MEG dataset with equal age distribution available. Their efforts of replicating observed effects in another large MEG dataset and considering potential confounding by ocular activity, head movements, or preprocessing methods are commendable and highly valuable to the community. This study also employs a wide range of fitting ranges and two commonly used algorithms for spectral parameterization of neural and cardiac activity, hence providing a comprehensive overview of the impact of methodological choices. The authors discuss their findings in-depth and give recommendations for the separation of physiological and neural sources of aperiodic activity.

Weaknesses:

While the study's aim is well-motivated and analyses rigorously conducted, it remains vague what is reflected in the ECG at higher frequency ranges that contributed to the confounding of the age effects in the neural data. However, the authors address this issue in their discussion.

Reviewer #1 (Public review):

Summary:

In this paper Weber et al. investigate the role of 4 dopaminergic neurons of the Drosophila larva in mediating the association between an aversive high-salt stimulus and a neutral odor. The 4 DANs belong to the DL1 cluster and innervate non-overlapping compartments of the mushroom body, distinct from those involved in appetitive associative learning. Using specific driver lines for individual neurons, the authors show that activation of the DAN-g1 is sufficient to mimic an aversive memory and it is also necessary to form a high-salt memory of full strength, although optogenetic silencing of this neuron has only a partial phenotype. The authors use calcium imaging to show that the DAN-g1 is not the only DAN responding to salt. DAN-c1 and d1 also respond to salt, but they seem to play no role for the associative memory. DAN-f1, which does not respond to salt, is able to lead to the formation of a memory (if optogenetically activated), but it is not necessary for the salt-odor memory formation in normal conditions. However, when silenced together with DAN-g1, it enhances the memory deficit of DAN-g1. Overall, this work brings evidence of a complex interaction between DL1 DANs in both the encoding of salt signals and their teaching role in associative learning, with none of them being individually necessary and sufficient for both functions.

Strengths:

Overall, the manuscript contributes interesting results that are useful to understand the organization and function of the dopaminergic system. The behavioral role of the specific DANs is accessed using specific driver lines which allow to test their function individually and in pairs. Moreover, the authors perform calcium imaging to test whether DANs are activated by salt, a prerequisite for inducing a negative association to it. Proper genetic controls are carried across the manuscript.

Weaknesses:

The authors use two different approaches to silence dopaminergic neurons: optogenetics and induction of apoptosis. The results are not always consistent, but the authors discuss these differences appropriately. In general, the optogenetic approach is more appropriate as developmental compensations are not of major interest for the question investigated.

The physiological data would suggest the role of a certain subset of DANs in salt-odor association, but a different partially overlapping set is necessary in behavioral assays (with a partial phenotype). No manipulation completely abolishes the salt-odor association, leaving important open questions on the identity of the neural circuits involved in this behavior.

The EM data analysis reveals a non-trivial organization of sensory inputs into DANs, but it is difficult to extrapolate a link to the functional data presented in the paper.

Reviewer #2 (Public review):

Summary:

Using the well-studied oxalate-microbiome-host system, the authors propose a novel conceptual and experimental framework for developing targeted bacteriotherapies using a three-phase pre-clinical workflow. The third phase is based on a 'complex system theoretical approach' in which multi-omics technologies are combined in independent in vivo and in vitro models to successfully identify the most pertinent variables that influence specific phenotypes in diet-host-microbe systems. The innovation relies on the third phase since phase I and phase II are the dominant approaches everyone in the microbiome field uses.

Strengths:

The authors used a multidisciplinary approach which included i] fecal transplant of two distinct microbial communities into Swiss-Webster mice (SWM) to characterize the host response (hepatic response-transcriptomics) and microbial activity (untargeted metabolomics of the stool samples) to different oxalate concentrations; 2] longitudinal analysis of the N. albigulia gut microbiome composition in response to varying concentrations of oxalate by shotgun metagenomics, with deep bioinformatic analyses of the genomes assembled; and 3] development of synthetic microbial communities around oxalate metabolisms and evaluation of these communities' activity into oxalate degradation in vivo.

Weaknesses:

This study presents a valuable finding on the oxalate-microbiome-host system using a multitude of approaches. Although the multidisciplinary approach allows for a unique perspective on the system and more robust conclusions, it is challenging for any authors to present all the data clearly and systematically in a conclusive way-especially when introducing unfamiliar concepts such as a complex systems theoretical approach.

Reviewer #1 (Public Review):

In this study, the authors build upon previous research that utilized non-invasive EEG and MEG by analyzing intracranial human ECoG data with high spatial resolution. They employed a receptive field mapping task to infer the retinotopic organization of the human visual system. The results present compelling evidence that the spatial distribution of human alpha oscillations is highly specific and functionally relevant, as it provides information about the position of a stimulus within the visual field.

Using state-of-the-art modeling approaches, the authors not only strengthen the existing evidence for the spatial specificity of the human dominant rhythm but also provide new quantification of its functional utility, specifically in terms of the size of the receptive field relative to the one estimated based on broad band activity.

Reviewer #1 (Public review):

Astrocytes are known to express neuroligins 1-3. Within neurons, these cell adhesion molecules perform important roles in synapse formation and function. Within astrocytes, a significant role for neuroligin 2 in determining excitatory synapse formation and astrocyte morphology was shown in 2017. However, there has been no assessment of what happens to synapses or astrocyte morphology when all three major forms of neuroligins within astrocytes (isoforms 1-3) are deleted using a well characterized, astrocyte specific, and inducible cre line. By using such selective mouse genetic methods, the authors here show that astrocytic neuroligin 1-3 expression in astrocytes is not consequential for synapse function or for astrocyte morphology. They reach these conclusions with careful experiments employing quantitative western blot analyses, imaging and electrophysiology. They also characterize the specificity of the cre line they used. Overall, this is a very clear and strong paper that is supported by rigorous experiments. The discussion considers the findings carefully in relation to past work. This paper is of high importance, because it now raises the fundamental question of exactly what neuroligins 1-3 are actually doing in astrocytes. In addition, it enriches our understanding of the mechanisms by which astrocytes participate in synapse formation and function. The paper is very clear, well written and well illustrated with raw and average data.

Comments on revisions:

My previous comments have been addressed. I have no additional points to make and congratulate the authors.

Reviewer #1 (Public review):

Summary:

The behavioral strategies underlying decisions based on perceptual evidence are often studied in the lab with stimuli whose elements provide independent pieces of decision-related evidence that can thus be equally weighted to form a decision. In more natural scenarios, in contrast, the information provided by these pieces is often correlated, which impacts how they should be weighted. Tardiff, Kang & Gold set out to study decisions based on correlated evidence and compare observed behavior of human decision makers to normative decision strategies. To do so, they presented participants with visual sequences of pairs of localized cues whose location was either uncorrelated, or positively or negatively correlated, and whose mean location across a sequence determined the correct choice. Importantly, they adjusted this mean location such that, when correctly weighted, each pair of cues was equally informative, irrespective of how correlated it was. Thus, if participants follow the normative decision strategy, their choices and reaction times should not be impacted by these correlations. While Tardiff and colleagues found no impact of correlations on choices, they did find them to impact reaction times, suggesting that participants deviated from the normative decision strategy. To assess the degree of this deviation, Tardiff et al. adjusted drift diffusion models (DDMs) for decision-making to process correlated decision evidence. These fits, and a comparison of different model variants revealed that participants considered correlations when weighing evidence, but did so with a slight underestimation of magnitude of this correlation. This finding made Tardiff et al. conclude that participants followed a close-to normative decision strategy that adequately took into account correlated evidence.

Strength:

The authors adjust a previously used experimental design to include correlated evidence in a simple, yet powerful way. The way it does so is easy to understand and intuitive, such that participants don't need extensive training to perform the task. Limited training makes it more likely that the observed behavior is natural and reflective of every-day decision-making. Furthermore, the design allowed the authors to make the amount of decision-related evidence equal across different correlation magnitudes, which makes it easy to assess whether participants correctly take account of these correlations when weighing evidence: if they do, their behavior should not be impacted by the correlation magnitude.

The relative simplicity with which correlated evidence is introduced also allowed the authors to fall back to the well-established DDM for perceptual decisions, that has few parameters, is known to implement the normative decision strategy in certain circumstances, and enjoys a great deal of empirical support. The authors show how correlations ought to impact these parameters, and which changes in parameters one would expect to see if participants mis-estimate these correlations or ignore them altogether (i.e., estimate correlations to be zero). This allowed them to assess the degree to which participants took into account correlations on the full continuum from perfect evidence weighting to complete ignorance. More specifically, the authors showed that a consistent mis-estimation of the correlation magnitude would not impact the fraction of correct choices (as they observe), but only the reaction times. With this, they could show that participants in fact performed rational evidence weighting if one assumed that they slightly underestimated the correlation magnitude.

Weaknesses:

While the authors convincingly demonstrate that the observed decision-making behavior seems to stem from a slight underestimation of the correlation magnitudes, their experimental paradigm did not allow them to determine the origin of this bias. Through additional analyses they rule out various possibilities, like the impact of a Bayesian prior on estimated correlations. Nonetheless, the authors provide no normative explanation of the observed bias.

A further minor weakness is that the authors only focus on a single normative aspect of the observed behavior, namely on whether participants optimally accumulate decision-related evidence across time. Another question is whether participants tune their decision boundaries to maximize reward rates or some other overall performance measures. While the authors discuss that the chosen diffusion models (DDMs) have the potential of also implementing normative decisions in the latter sense, the authors' analysis does not address this question in the context of their task.

Reviewer #1 (Public review):

"Unraveling the Role of Ctla-4 in Intestinal Immune Homeostasis: Insights from a novel Zebrafish Model of Inflammatory Bowel Disease" generates a 14bp deletion/early stop codon mutation that is viable in a zebrafish homolog of ctla-4. This mutant exhibits an IBD-like phenotype, including decreased intestinal length, abnormal intestinal folds, decreased goblet cells, abnormal cell junctions between epithelial cells, increased inflammation, and alterations in microbial diversity. Bulk and single-cell RNA-seq show upregulation of immune and inflammatory response genes in this mutant (especially in neutrophils, B cells, and macrophages) and downregulation of genes involved in adhesion and tight junctions in mutant enterocytes. The work suggests that the makeup of immune cells within the intestine is altered in these mutants, potentially due to changes in lymphocyte proliferation. Introduction of recombinant soluble Ctla-4-Ig to mutant zebrafish rescued body weight, histological phenotypes, and gene expression of several pro-inflammatory genes, suggesting a potential future therapeutic route.

Strengths:

- Generation of a useful new mutant in zebrafish ctla-4<br /> - The demonstration of an IBD-like phenotype in this mutant is extremely comprehensive.<br /> - Demonstrated gene expression differences provide mechanistic insight into how this mutation leads to IBD-like symptoms.<br /> - Demonstration of rescue with a soluble protein suggests exciting future therapeutic potential<br /> - The manuscript is mostly well organized and well written.

Initial Weaknesses were addressed during review.

Reviewer #1 (Public review):

This work presents data from three species (mice, rats, and humans) performing an evidence accumulation task, that has been designed to be as similar as possible between species (and is based on a solid foundation of previous work on decision-making). The tasks are well-designed, and the analyses are solid and clearly presented - showing that there are differences in the overall parameters of the decision-making process between the species. This is valuable to neuroscientists who aim to translate behavioral and neuroscientific findings from rodents to humans and offers a word of caution for the field in readily claiming that behavioral strategies and computations are representative of all mammals. The dataset would be of great interest to the community and may be a source of further modelling of across-species behavior, but unfortunately, neither data or code are currently shared.

A few other questions remain, that make the conclusions of the paper a bit hard to assess:

(1) The main weakness is that the authors claim that all species rely on evidence accumulation as a strategy, but this is not tested against other models (see e.g. Stine et al. https://elifesciences.org/articles/55365): the fact that the DDM fits rather well does not mean that this is the strategy that each species was carrying out.

(2) In all main analyses, it is unclear what the effect is of the generative flash rate and how this has been calibrated between species. Only in Figure 6C do we see basic psychometric functions, but these should presumably also feature as a crucial variable dominating the accuracy and RTs (chronometric functions) across species. The very easy trials are useful to constrain the basic sensorimotor differences that may account for RT variability, e.g. perhaps the small body of mice requires them to move a relatively longer distance to trigger the response.

(3) The GLM-HMM results (that mice are not engaged in all trials) are very important, but they imply that mouse DDM fits may well be more similar to rats and humans if done only on engaged trials. Could it be that the main species differences are driven by different engagement state occupations?

(4) It would be very helpful if the authors could present a comprehensive overview (perhaps a table) of the factors that may be relevant for explaining the observed species differences. This may include contextual/experimental variables (age range (adolescent humans vs. mice/rats, see https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old; reward source, etc) and also outcomes (e.g. training time required to learn the task, # trials per session and in total).

Reviewer #1 (Public review):

Summary:

Sakelaris and Riecke used computational modeling to explore how neurogenesis and sequential integration of new neurons into a network support memory formation and maintenance. They focus on the integration of granule cells in the olfactory bulb, a brain area where adult neurogenesis is prominent. Experimental results published in recent years provide an excellent basis to address the question at hand by biologically constrained models. The study extends previous computational models and provides a coherent picture of how multiple processes may act in concert to enable rapid learning, high stability of memories, and high memory capacity. This computational model generates experimentally testable predictions and is likely to be valuable to understand the roles of neurogenesis and related phenomena in memory. One of the key findings is that important features of the memory system depend on transient properties of adult-born granule cells such as enhanced excitability and apoptosis during specific phases of the development of individual neurons. The model can explain many experimental observations and suggests specific functions for different processes (e.g., importance of apoptosis for continual learning). While this model is obviously a massive simplification of the biological system, it conceptualizes diverse experimental observations into a coherent picture, it generates testable predictions for experiments, and it will likely inspire further modeling and experimental studies. Nonetheless, there are issues that the authors should address.

Strengths:

(1) The model can explain diverse experimental observations.

(2) The model directly represents the biological network.

Weaknesses:

As with many other models of biological networks, this model contains major simplifications.

Reviewer #1 (Public review):

Summary:

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

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

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

Strengths:

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

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

Comments on revised version:

This updated version of the paper is improved compared to its initial version. As such, the strengths remain the same as before, with a fascinating model system and an interesting research question. The earlier weaknesses related to overinterpretation of the data have been largely fixed by shortening the paper and adding appropriate caveats throughout. The paper now also includes a significance test for its overlap between gene lists. While this turned out to be negative (i.e., there is not more overlap between lists than expected by chance), reporting this result transparently has strengthened the paper.

Reviewer #1 (Public review):

Summary

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

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

Strengths

The combination of scRNAseq, cytologic markers and gradient based hemocyte sorting offers an integrative view of the immune cell diversity.<br /> Hemocytes represent a very plastic cell population that has key roles in homeostatic and challenged conditions. Grasping the molecular features of these cells at the single-cell level will help understand their biology.<br /> This type of study may help elucidate the diversification of immune cells in comparative studies and evolutionary immunology.

Weaknesses

Several figures show inconsistency leading to erroneous conclusions and some conclusions are poorly supported. Moreover, the manuscript remains highly descriptive with limited comparison with the available literature.

Comments on revisions:

The authors replied to most comments.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors develop a novel method to infer ecologically-informative parameters across healthy and diseased states of the gut microbiota, although the method is generalizable to other datasets for species abundances. The authors leverage techniques from theoretical physics of disordered systems to infer different parameters - mean and standard deviation for the strength of bacterial interspecies interactions, a bacterial immigration rate, and the strength of demographic noise - that describe the statistics of microbiota samples from two groups-one for healthy subjects and another one for subjects with chronic inflammation syndromes. To do this, the authors simulate communities with a modified version of the Generalized Lotka-Volterra model and randomly-generated interactions, and then use a moment-matching algorithm to find sets of parameters that better reproduce the data for species abundances. They find that these parameters are different for the healthy and diseased microbiota groups. The results suggest, for example, that bacterial interaction strengths, relative to noise and immigration, are more dominant for microbiota dynamics in diseased states than in healthy states.

We think that this manuscript brings an important contribution that will be of interest in the areas of statistical physics, (microbiota) ecology, and (biological) data science. The evidence of their results is solid and the work improves the state-of-the-art in terms of methods. There are a few weaknesses that, in our opinion, the authors could address to further improve the work.

Strengths:

(1) Using a fairly generic ecological model, the method can identify the change in the relative importance of different ecological forces (distribution of interspecies interactions, demographic noise, and immigration) in different sample groups. The authors focus on the case of the human gut microbiota, showing that the data are consistent with a higher influence of species interactions (relative to demographic noise and immigration) in a disease microbiota state than in healthy ones.

(2) The method is novel, original, and it improves the state-of-the-art methodology for the inference of ecologically relevant parameters. The analysis provides solid evidence for the conclusions.

Weaknesses:

In the way it is written, this work might be mostly read by physicists. We believe that, with some rewriting, the authors could better highlight the ecological implications of the results and make the method more accessible to a broader audience.

Reviewer #1 (Public review):

The manuscript by Liao et al investigates the mechanisms that induce ephrin expression in spinal cord lateral motor column (LMC) neurons to facilitate axon guidance into the dorsal and ventral limb. The authors show that Sp1 and its co-activators p300 and CBP are required to induce ephrin expression to modulate the responsiveness of motor neurons to external ephrin cues. The study is well done and convincingly demonstrates the role of Sp1 in motor neuron axon guidance.

Further discussion and clarification of some results would further improve the study.

(1) The mechanism that the authors propose (Figure 7) and is also supported by their data is that Sp1 induces ephrinA5 in LMCm and ephrinB2 in LMCl to attenuate inappropriate responses to external ephrins in the limb. Therefore, deletion of Sp1 should result in mistargeting of LMCl and LMCm axons, as shown in the mouse data, but no overt changes in the number of axons in the ventral and dorsal limb. From the mouse backfills, it seems that an equal number of LMCm/LMCl project into the wrong side of the limb. However, the chick data show an increase of axons projecting into the ventral limb in the Sp1 knockout. Is this also true in the mouse? The authors state that medial and lateral LMC neurons differ in their reliance on Sp1 function but that is not supported by the mouse backfill data (27% vs 32% motor neurons mistargeted). Also, the model presented in Figure 7 does not explain how Sp1 overexpression leads to axon guidance defects.

(2) The authors do not directly show changes in ephrin expression in motor neurons, either in chick or mouse, after Sp1 knockout, which is the basis of their model. The experiment in Figure 4G seems to be Sp1 overexpression rather than knockdown (as mentioned in the results) and NSC-34 cells may not be relevant to motor neurons in vivo. NSC-34 experiments are also not described in the methods.

(3) There is no information about how the RNA-sequencing experiment was done (which neurons were isolated, how, at what age, how many replicates, etc) so it is hard to interpret the resulting data.

(4) It is unclear why the authors chose to use a Syn1-cre driver rather than a motor neuron restricted cre driver. Since this is a broad neuronal cre driver, the behavioral defects shown in Figure 7 may not be solely due to Sp1 deletion in motor neurons. Are there other relevant neuronal populations that express Sp1 that are targeted by this cre-mediated deletion?

Reviewer #1 (Public review):

In this study, Li et al et al. investigated the role of miR-283 in regulating cardiac aging and its potential contribution to age-related bradyarrhythmia. Using Drosophila as a model, the authors demonstrated that systemic overexpression or knockdown of miR-283 induced age-associated bradycardia. Notably, the study found that miR-283 knockdown in ventral-lateral neurons (LNvs), rather than in the heart, was sufficient to induce bradyarrhythmia, an effect the authors linked to the upregulation of miR-283 expression in both the brain and heart. The study also explored the beneficial impact of exercise on cardiac aging, showing that endurance training mitigated bradyarrhythmia, correlating with reduced miR-283 accumulation in the brain and myocardium.

The conclusions of this paper are mostly well supported by data; however, some concerns arise from the unexpected finding that bradyarrhythmia was triggered by miR-283 knockdown in LNvs rather than in the heart, suggesting a non-cell-autonomous mechanism. A more precise mechanistic explanation linking miR-283 dysregulation in LNvs to cardiac dysfunction would strengthen the study's conclusions. While the authors propose cwo as a potential target of miR-283, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Additionally, it remains unclear whether reduced miR-283 levels in LNvs lead to accelerated aging rather than a cardiac-specific effect. Likewise, the potential influence of miR-283 on the circadian clock and its broader impact on aging warrant further investigation.

Major Comments:

(1) A significant concern arises from the unexpected outcome observed in miR-283 knockdown in LNvs, which suggests a non-cell-autonomous mechanism. Elucidating the mechanisms by which miR-283 deficiency leads to the observed phenotypes would provide a more comprehensive understanding of the study's implications.

(2) The authors propose cwo as a potential target of miR-283; however, no functional experiments were conducted to confirm its role in mediating miR-283's effects. Similarly, direct evidence demonstrating that cwo is a bona fide target of miR-283 in LNvs should be provided.

(3) It remains unclear whether miR-283 knockdown in LNvs results in accelerated aging rather than a cardiac-specific effect. This hypothesis is supported by observations that pdf>miR-283SP animals exhibit systemic premature senescence (elevated SA-β-gal activity in both the heart and brain), cardiac dysfunction, impaired climbing ability, and reduced lifespan.

(4) The finding that reduced miR-283 levels in LNvs lead to accelerated aging raises an important, yet unexplored, question: does miR-283 influence the circadian clock, thereby broadly affecting aging?

Two aspects of this question should be addressed:<br /> (a) Is the circadian rhythm disrupted in miR-283 knockdown experiments?<br /> (b) Do circadian rhythm defects impact aging?

(5) The authors state that miR-283 knockdown in LNvs led to bradyarrhythmia, which was mainly caused by miR-283 upregulation in the whole brain and heart. However, it is unclear which experiments support this conclusion. Could the authors clarify this point?

(6) Given that miR-283 expression varies with age, could the upregulation of miR-283 in both the brain and heart be a consequence of accelerated aging rather than a specific effect of miR-283 knockdown in LNvs?

(7) While the beneficial effects of exercise on cardiac function appear clear, the claim that this effect is mediated through miR-283 function in LNvs seems premature. The data suggest that exercise-induced improvement occurs in both wild-type and miR-283-SP animals, raising the possibility that exercise acts through a miR-283-independent mechanism.

Reviewer #1 (Public review):

Summary:

In this manuscript, Rosero and Bai examined how the well-known thermosensory neuron in C. elegans, AFD, regulates context-dependent locomotory behavior based on the tactile experience. Here they show that AFD uses discrete cGMP signalling molecules and independent of its dendritic sensory endings regulates this locomotory behavior. The authors also show here that AFD's connection to one of the hub interneurons, AIB, through gap junction/electrical synapses, is necessary and sufficient for the regulation of this context-dependent locomotion modulation.

Strengths:

This is an interesting paper showcasing how a sensory neuron in C. elegans can employ a distinct set of molecular strategies and different physical parts to regulate a completely distinct set of behaviors, which were not been shown to be regulated by AFD before. The experiments were well performed and the results are clear. However, there are some questions about the mechanism of this regulation. This reviewer thinks that the authors should address these concerns before the final published version of this manuscript.

Weaknesses:

(1) The authors argued about the role of prior exposure to different physical contexts which might be responsible for the difference in their locomotory behaviour. However, the worms in the binary chamber (with both non-uniformly sized and spaced pillars) experienced both sets of pillars for one hour prior to the assay and they were also free to move between two sets of environments during the assay. So, this is not completely a switch between two different types of tactile barriers (or not completely restricted to prior experience), but rather a difference between experiencing a more complex environment vs a simple uniform environment. They should rephrase their findings. To strictly argue about the prior experience, the authors need to somehow restrict the worms from entering the uniform assay zone during the 1hr training period.

(2) The authors here argued that the sensory endings of AFD are not required for this novel role of AFD in context-dependent locomotion modulation. However, gcy-18 has been shown to be exclusively localized to the ciliated sensory endings of AFD and even misexpression of GCY-18 in other sensory neurons also leads to localizations in sensory endings (Nguyen et. al., 2014 and Takeishi et. al., 2016). They should check whether gcy-18 or tax-2 gets mislocalized in kcc-3 or tax-1 mutants.

(3) MEC-10 was shown to be required for physical space preference through its action in FLP and not the TRNs (PMID: 28349862). Since FLP is involved in harsh touch sensation while TRNs are involved in gentle touch sensation, which are the neuron types responsible for tactile sensation in the assay arena? Does mec-10 rescue in TRNs rescue the phenotype in the current paper?

(4) The authors mention that the most direct link between TRNs and AFD is through AIB, but as far as I understand, there are no reports to suggest synapses between TRNs and AIB. However, FLP and AIB are connected through both chemical and electrical synapses, which would make more sense as per their mec-10 data. (the authors mentioned about the FLP-AIB-AFD circuit in their discussion but talked about TRNs as the sensory modality). mec-10 rescue experiment in TRNs would clarify this ambiguity.

(5) Do inx-7 or inx-10 rescue in AFD and AIB using cell-specific promoters rescue the behaviour?

(6) How Guanylyl cyclase gcy-18 function is related to the electrical synapse activity between AFD and AIB? Is AFD downstream or upstream of AIB in this context?

Reviewer #1 (Public review):

Summary:

In this manuscript, Campbell et al. assess how intracranial theta-burst stimulation (TBS) applied to the basolateral amygdala in 23 epilepsy patients affects neuronal spiking in the medial temporal lobe and prefrontal cortex during a visual recognition memory task.

Strengths:

This is an incredibly rare dataset; collecting single-unit spiking data from behaving humans during active intracranial stimulation is a Herculaean task, with immense potential for translational studies of how stimulation may be applied to modulate biological mechanisms of memory. The authors utilize careful, high-quality methodology throughout (e.g. task design, spike recording and sorting, statistical analysis), providing high confidence in the validity of their findings.

Weaknesses:

(1) This is an exploratory study that doesn't explore quite enough. Critically, the authors make a point of mentioning that neuronal firing properties vary across cell types, but only use baseline firing rate as a proxy metric for cell type. This leaves several important explorations on the table, not limited to the following:<br /> a) Do waveform shape features, which can also be informative of cell type, predict the effect of stimulation?<br /> b) Is the autocorrelation of spike timing, which can be informative about temporal dynamics, altered by stimulation? This is especially interesting if theta-burst stimulation either entrains theta-rhythmic spiking or is more modulatory of endogenously theta-modulated units.<br /> c) The authors reference the relevance of spike-field synchrony (30-55 Hz) in animal work, but ignore it here. Does spike-field synchrony (comparing the image presentation to post-stimulation) change in this frequency range? This does not seem beyond the scope of investigation here.<br /> d) How does multi-unit activity respond to stimulation? At this somewhat low count of neurons (total n=156 included) it would be valuable to provide input on multi-unit responses to stimulation as well.<br /> e) Several intracranial studies have implicated proximity to white matter in determining the effects of stimulation on LFPs; do the authors see an effect of white matter proximity here?

(2) It is a little confusing to interpret stimulation-induced modulation of neuronal spiking in the absence of stimulation-induced change in behavior. How do the authors findings tell us anything about the neural mechanisms of stimulation-modulated memory if memory isn't altered? In line with point #1, I would suggest a deeper dive into behavior (e.g. reaction time? Or focus on individual sessions that do change in Figure 4A?) to make a stronger statement connecting the neural results to behavioral relevance.

(3) It is not clear to me why the assessment of firing rates after image onset and after stim offset is limited to one second - this choice should be more theoretically justified, particularly for regions that spike as sparsely as these.

(4) This work coincides with another example of human intracranial stimulation investigating the effect on firing rates (doi: https://doi.org/10.1101/2024.11.28.625915). Given how incredibly rare this type of work is, I think the authors should discuss how their work converges with this work (or doesn't).

(5) What information does the pseudo-population analysis add? It's not totally clear to me.

Reviewer #1 (Public review):

This is an interesting manuscript aimed at improving the transcriptome characterization of 52 C. elegans neuron classes. Previous single-cell RNA seq studies already uncovered transcriptomes for these, but the data are incomplete, with a bias against genes with lower expression levels. Here, the authors use cell-specific reporter combinations to FACS purify neurons and bulk RNA sequencing to obtain better sequencing depth. This reveals more rare transcripts, as well as non-coding RNAs, pseudogenes, etc. The authors develop computational approaches to combine the bulk and scRNA transcriptome results to obtain more definitive gene lists for the neurons examined.

To ultimately understand features of any cell, from morphology to function, an understanding of the full complement of the genes it expresses is a pre-requisite. This paper gets us a step closer to this goal, assembling a current "definitive list" of genes for a large proportion of C. elegans neurons. The computational approaches used to generate the list are based on reasonable assumptions, the data appear to have been treated appropriately statistically, and the conclusions are generally warranted. I have a few issues that the authors may choose to address:

(1) As part of getting rid of cross-contamination in the bulk data, the authors model the scRNA data, extrapolate it to the bulk data and subtract out "contaminant" cell types. One wonders, however, given that low expressed genes are not represented in the scRNA data, whether the assignment of a gene to one or another cell type can really be made definitive. Indeed, it's possible that a gene is expressed at low levels in one cell, and high levels in another, and would therefore be considered a contaminant. The result would be to throw out genes that actually are expressed in a given cell type. The definitive list would therefore be a conservative estimate, and not necessarily the correct estimate.

(2) It would be quite useful to have tested some genes with lower expression levels using in vivo gene-fusion reporters to assess whether the expression assignments hold up as predicted. i.e. provide another avenue of experimentation, non-computational, to confirm that the decontamination algorithm works.

(3) In many cases, each cell class would be composed of at least 2 if not more neurons. Is it possible that differences between members of a single class would be missed by applying the cleanup algorithms? Such transcripts would be represented only in a fraction of the cells isolated by scRNAseq, and might then be considered not real.

(4) I didn't quite catch whether the precise staging of animals was matched between the bulk and scRNAseq datasets. Importantly, there are many genes whose expression is highly stage-specific or age-specific so even slight temporal differences might yield different sets of gene expression.

(5) To what extent does FACS sorting affect gene expression? Can the authors provide some controls?

Reviewer #1 (Public review):

Summary:

The authors investigate the role of different specific dopaminergic neurons in the mushroom body of Drosophila larvae for learning and innate behavior. All the tested neurons are thought to be involved in punishment learning. The authors discover that artificial activation of single DANs in training leads to safety learning, but not punishment learning. Furthermore, activation of single DANs can lead to changes in locomotion behavior, which can affect light preference. The authors provide a deeper understanding of the functional diversity of single dopamine neurons; however, it is unclear how translatable these findings are to learning experiments with real punishment stimuli.

Strengths:

The authors attempt to disentangle what kind of memories are formed with the activation of different dopamine neurons - safety learning, and punishment learning, will the US be required to test for recall or not? They do indeed find differences and the results will be of interest to the learning and memory community.

Interestingly, optogenetic activation of a single DAN during training leads to safety memory, but not punishment memory. Furthermore, DAN activation also affects innate locomotion, and the authors can show that optogenetic activation of different DANs affects locomotion differently.

Weaknesses:<br /> All experiments in the manuscript use optogenetic activation of DANs, thus it is not clear what kind of memories are formed. Several stimuli can be used as punishment, such as electric shock, salt, bitter, and light - it is not clear what kind of memory the authors investigate here. The findings could be discussed in the context of what DANs respond to. Furthermore, studies in adults and larvae showed that most DANs can code for both valences - etc., aversive DANs can be activated by punishment, and inhibited by reward. Thus, safety learning might be a result of a decrease in activity in DANs during odor presentation. The authors also do not discuss possible feedback loops from MBONs to DANs across compartments. Could such connections allow for safety learning in larvae?

The authors show that artificial activation with different light intensities can form different memories and that increasing the light intensity sometimes leads to no memories. Also, using different optogenetic tools reveals different results. This again raises the question of how applicable the results will be for learning with real stimuli. Is there a natural stimulus that only induces safety learning, but no punishment learning?<br /> The authors provide a detailed behavioral analysis of locomotion behavior; however, the detailed analysis seems unnecessary for that dataset. Modulation of speed and bending rate has been described before with simpler methods (specifically for MBONs). The revealed locomotion phenotypes probably affect larval locomotion during memory recall with light activation, thus the authors should show that larvae are potentially able to move during light-on memory tests.

Reviewer #1 (Public review):

Summary:

The authors sequenced 888 individuals from the 1000 Genomes Project using the Oxford Nanopore long-read sequencing method to achieve highly sensitive, genome-wide detection of structural variants (SVs) at the population level. They conducted solid benchmarking of SV calling and systematically characterized the identified SVs. While short-read sequencing methods, including those used in the 1000 Genomes Project, have been widely applied, they exhibit high accuracy in detecting single nucleotide variants (SNVs) and small insertions and deletions but have limited sensitivity for SV detection. This study significantly enhances SV detection capabilities, establishing it as a valuable resource for human genetic research. Furthermore, the authors constructed an SV imputation panel using the generated data and imputed SVs in 488,130 individuals from the UK Biobank. They then conducted a proof-of-principle genome-wide association study (GWAS) analysis based on the imputed SVs and selected traits within the UK Biobank. Their findings demonstrate that incorporating SV-GWAS analysis provides additional insights beyond conventional GWAS frameworks focusing on SNVs, particularly in improving fine mapping.

Strengths:

The authors constructed a high-sensitivity reference panel of genome-wide SVs at the population level, addressing a critical gap in the field of human genetics. This resource is expected to significantly advance research in human genetics. They demonstrated the imputation of SVs in individuals from the UK Biobank using this panel and conducted a proof-of-concept SV-based GWAS. Their findings highlight a novel and effective strategy for integrating SVs into GWAS, which will facilitate the analysis of human genetic data from the UK Biobank and other datasets. Their conclusions are supported by comprehensive analyses.

Weaknesses:

(1) Although the authors employ state-of-the-art analytical approaches for the identification of SVs, the overall accuracy remains suboptimal, as indicated by an F1 score of 74.0%, particularly in tandem repeat regions. To enhance accuracy, it would be beneficial to explore alternative SV detection methods or develop novel approaches. Given the value of the reference panel and the fact that improved SV accuracy would lead to more precise SV imputation and GWAS results, investing effort in methodological refinement is highly encouraged.

(2) From the Methods section, it appears that the authors employed Beagle for both the "leave-one-out" imputation and the UK Biobank imputation. It would be better to explicitly clarify this in the Results section and provide a detailed description of the corresponding procedures and parameters in the Methods section for both analyses, as this represents a key aspect of the study. Additionally, Beagle is not specifically designed for SV imputation, the imputation quality of SVs is generally lower than that of SNVs. Exploring strategies to improve SV imputation, such as developing a novel method with reference panel data, may enhance performance. It is also important to assess how this reduced imputation quality may influence GWAS results. For instance, it would be useful to examine whether associated SVs exhibit higher imputation quality and whether SVs with lower quality are less likely to achieve significant association signals. In addition, the lower imputation quality observed for INV, DUP, and BND variants (Figure 3) may be due to their greater lengths (Figure 2). It is better to investigate the relationship between SV length and imputation quality.

(3) All examples presented in the manuscript focus on SVs that overlap with genes. It may also be valuable to investigate SVs that do not overlap with genes but intersect with enhancer regions. SVs can contribute to disease by altering regulatory elements, such as enhancers, which play a crucial role in gene expression. Including such analyses would further demonstrate the utility of SV-GWAS and provide deeper insights into the functional impact of SVs.

(4) The data availability link currently provides only a VCF file ("sniffles2_joint_sv_calls.vcf.gz") containing the identified SVs. It would be beneficial for the authors to make all raw sequencing data (FASTQ files) and key processed datasets (such as alignment results and merged SV and SNV files) available. Providing these resources would enable other researchers to develop improved SV detection and imputation methods or conduct further genetic analyses. Furthermore, establishing a dedicated website for data access, along with a genome browser for SV visualization, could significantly enhance the impact and accessibility of the study. Additionally, all code, particularly the SV imputation pipeline accompanied by a detailed tutorial, should be deposited in a public repository such as GitHub. This would support researchers in imputing SVs and conducting SV-GWAS on their own datasets.

Joint Public Review:

Pannexin (Panx) hemichannels are a family of heptameric membrane proteins that form pores in the plasma membrane through which ions and relatively large organic molecules can permeate. ATP release through Panx channels during the process of apoptosis is one established biological role of these proteins in the immune system, but they are widely expressed in many cells throughout the body, including the nervous system, and likely play many interesting and important roles that are yet to be defined. Although several structures have now been solved of different Panx subtypes from different species, their biophysical mechanisms remain poorly understood, including what physiological signals control their activation. Electrophysiological measurements of ionic currents flowing in response to Panx channel activation have shown that some subtypes can be activated by strong membrane depolarization or caspase cleavage of the C-terminus. Here, Henze and colleagues set out to identify endogenous activators of Panx channels, focusing on the Panx1 and Panx2 subtypes, by fractionating mouse liver extracts and screening for activation of Panx channels expressed in mammalian cells using whole-cell patch clamp recordings. The authors present a comprehensive examination with robust methodologies and supporting data that demonstrate that lysophospholipids (LPCs) directly Panx-1 and 2 channels. These methodologies include channel mutagenesis, electrophysiology, ATP release and fluorescence assays, and molecular modelling. Mouse liver extracts were initially used to identify LPC activators, but the authors go on to individually evaluate many different types of LPCs to determine those that are more specific for Panx channel activation. Importantly, the enzymes that endogenously regulate the production of these LPCs were also assessed along with other by-products that were shown not to promote pannexin channel activation. In addition, the authors used synovial fluid from canine patients, which is enriched in LPCs, to highlight the importance of the findings in pathology. Overall, we think this is likely to be an important study because it provides strong evidence that LPCs can function as activators of Panx1 and Panx2 channels, linking two established mediators of inflammatory responses and opening an entirely new area for exploring the biological roles of Panx channels. This study provides an excellent foundation for future studies and importantly provides clinical relevance.

[Editors' note: this paper has been through two rounds of review and revisions, available here: https://sciety.org/articles/activity/10.1101/2023.10.23.563601]

Reviewer #1 (Public review):

Summary:

This study aims to understand the malaria antigen-specific cTfh profile of children and adults living in malaria holoendemic area. PBMC samples from children and adults were unstimulated or stimulated with PfSEA-1A or PfGARP in vitro for 6h and analysed by a cTfh-focused panel. Unsupervised clustering and analysis on cTfh was performed. The main conclusions are: A) the children cohort has a more diverse (cTfh1/2/17) recall responses compared to adults (mainly cTfh17) and, B) Pf-GARP stimulates better cTfh17 responses in adults, thus a promising vaccine candidate.

Strengths:

This study is, in general, well-designed and with excellent data analysis. The use of unsupervised clustering is a nice attempt to understand the heterogeneity of cTfh cells.

Weaknesses:

The authors have provided additional data in Supplementary Figures 14-16. However, I remain concerned about whether cTfh cells are truly responding to antigen stimulation. In Supplementary Figure 15A-F, the IFNg responses appear as expected, SEB elicits the strongest response, as it stimulates bulk T cells, and the staining is promising, showing a clear distinction between IFNg+ and IFNg- populations. However, in Supplementary Figure 15I-N, the IL-21 secretion assay is concerning. The FACS plots make it difficult to distinguish IL-21+ from IL-21- cells, raising concerns about the validity of this analysis. Additionally, in panel J, the responses to PfSEA-1A or PfGARP appear even greater than those to SEB stimulation. In PBMCs, only a small percentage of T cells should be specific to a particular antigen. How can the positive control (SEB) produce a weaker response than stimulation with a specific antigen? This suggests that the IL-21 secretion assay may not have worked, making the authors' interpretation unreliable.

I also have similar concerns about the IL-4 secretion in Sup Figure 16. First, the FACS plot shows that appear double-positive for IL-21 and IL-4, so it suggests the staining may be due to autofluorescence rather than true cytokine signals. Also in B-C the responses of SEB stimulation is generally weaker than stimulated by one antigen, further questioning the reliability of the IL-4 assay. In summary, I am not convinced that the in vitro antigen stimulation assay worked as intended. Consequently, the manuscript's claims regarding PfSEA-1A- and PfGARP-specific cTfh responses are not sufficiently supported by the presented data.

Reviewer #1 (Public review):

This is a comprehensive study that sheds light on how Wag31 functions and localises in mycobacterial cells. A clear link to interactions with CL is shown using a combination of microscopy in combination with fusion fluorescent constructs, and lipid specific dyes. Furthermore, studies using mutant versions of Wag31 shed light on the functionalities of each domain in the protein. My concerns/suggestions for the manuscript are minor:

(1) Ln 130. A better clarification/discussion is required here. It is clear that both depletion and overexpression have an effect on levels of various lipids, but subsequent descriptions show that they affect different classes of lipids.<br /> (2) The pulldown assays results are interesting, but the links are tentative.<br /> (3) The authors may perhaps like to rephrase claims of effects lipid homeostasis, as my understanding is that lipid localisation rather than catabolism/breakdown is affected.

In response to the above reviews the authors have made the required changes in the revised manuscript.

Reviewer #1 (Public review):

Summary:

In this study, the authors re-analyzed a public dataset (Rademaker et al, 2019, Nature Neuroscience) which includes fMRI and behavioral data recorded while participants held an oriented grating in visual working memory (WM) and performed a delayed recall task at the end of an extended delay period. In that experiment, participants were pre-cued on each trial as to whether there would be a distracting visual stimulus presented during the delay period (filtered noise or randomly-oriented grating). In this manuscript, the authors focused on identifying whether the neural code in retinotopic cortex for remembered orientation was 'stable' over the delay period, such that the format of the code remained the same, or whether the code was dynamic, such that information was present, but encoded in an alternative format. They identify some timepoints - especially towards the beginning/end of the delay - where the multivariate activation pattern fails to generalize to other timepoints, and interpret this as evidence for a dynamic code. Additionally, the authors compare the representational format of remembered orientation in the presence vs absence of a distracting stimulus, averaged over the delay period. This analysis suggested a 'rotation' of the representational subspace between distracting orientations and remembered orientations, which may help preserve simultaneous representations of both remembered and viewed stimuli. Intriguingly, this rotation was a bit smaller for Expt 2, in which the orientation distractor had a greater behavioral impact on the participants' behavioral working memory recall performance, suggesting that more separation between subspaces is critical for preserving intact working memory representations.

Strengths:

(1) Direct comparisons of coding subspaces/manifolds between timepoints, task conditions, and experiments is an innovative and useful approach for understanding how neural representations are transformed to support cognition

(2) Re-use of existing dataset substantially goes beyond the authors' previous findings by comparing geometry of representational spaces between conditions and timepoints, and by looking explicitly for dynamic neural representations

(3) Simulations testing whether dynamic codes can be explained purely by changes in data SNR are an important contribution, as this rules out a category of explanations for the dynamic coding results observed

Weaknesses:

(1) Primary evidence for 'dynamic coding', especially in early visual cortex, appears to be related to the transition between encoding/maintenance and maintenance/recall, but the delay period representations seem overall stable, consistent with some previous findings. However, given the simulation results, the general result that representations may change in their format appears solid, though the contribution of different trial phases remains important for considering the overall result.

(2) Converting a continuous decoding metric (angular error) to "% decoding accuracy" serves to obfuscate the units of the actual results. Decoding precision (e.g., sd of decoding error histogram) would be more interpretable and better related to both the previous study and behavioral measures of WM performance.

Reviewer #1 (Public review):

Summary:

The authors investigate ligand and protein-binding processes in GPCRs (including dimerization) by the multiple walker supervised molecular dynamics method. The paper is interesting and it is very well written.

Strengths:

The authors' method is a powerful tool to gain insight on the structural basis for the pharmacology of G protein-coupled receptors.

Reviewer #1 (Public review):

Summary:

Activated male Plasmodium gametocytes undergo very rapid nuclear division, while keeping the nuclear envelope intact. There is interest in how events inside the nucleus are co-ordinated with events in the parasite cytoplasm, to ensure that each nucleus is packaged into a nascent male gamete.

This manuscript by Zeeshan et al describes the organisation of a nuclear membrane bridging protein, SUN1, during nuclear division. SUN1 is expected from studies in other organisms to be a component of a bridging complex (LINC) that connects the inner nuclear membrane to the outer nuclear membrane, and from there to the cytoplasmic microtubule-organising centres, the centrosome and the basal body.

The authors show that knockout of the SUN1 in gametocytes leads to severe disruption of the mitotic spindle and failure of the basal bodies to segregate. The authors show convincingly that functional SUN1 is required for male gamete formation and subsequent oocyst development.

The authors identified several SUN1-interacting proteins, thus providing information about the nuclear membrane bridging machinery.

Strengths:

The authors have used state of the art imaging, genetic manipulation and immunoprecipitation approaches.

Weaknesses:

Technical limitations of some of the methods used make it difficult to interpret some of the micrographs.

From studies in other organisms, a protein called KASH is a critical component the bridging complex (LINC). That is, KASH links SUN1 to the outer nuclear membrane. The authors undertook a gene sequence analysis that reveals that Plasmodium lacks a KASH homologue. Thus, further work is needed to identify the functional equivalent of KASH, to understand bridging machinery in Plasmodium.

Comments on revised version:

The authors have addressed the comments and suggestions that I provided as part of a Review Commons assessment.

Reviewer #1 (Public review):

Summary:

This paper contains what could be described as a "classic" approach towards evaluating a novel taste stimuli in an animal model, including standard behavioral tests (some with nerve transections), taste nerve physiology, and immunocytochemistry of taste cells of the tongue. The stimulus being tested is ornithine, from a class of stimuli called "kokumi" (in terms of human taste); these kokumi stimuli appear to enhance other canonical tastes, increasing what are essentially hedonic attributes of other stimuli. The mechanism for ornithine detection is thought to be GPRC6A receptors expressed in taste cells. The authors showed evidence for this in an earlier paper with mice; this paper evaluates ornithine taste in a rat model, and comes to a similar conclusion, albeit with some small differences between the two rodent species.

Strengths:

The data show effects of ornithine on taste/intake in laboratory rats: In two-bottle and briefer intake tests, adding ornithine results in higher intake of most, but all not all stimuli tested. Bilateral chorda tympani (CT) nerve cuts or the addition of GPRC6A antagonists decreased or eliminated these effects. Ornithine also evoked responses by itself in the CT nerve, but mainly at higher concentrations; at lower concentrations it potentiated the response to monosodium glutamate. Finally, immunocytochemistry of taste cell expression indicated that GPRC6A was expressed predominantly in the anterior tongue, and co-localized (to a small extent) with only IP3R3, indicative of expression in a subset of type II taste receptor cells.

Weaknesses:

As the authors are aware, it is difficult to assess a complex human taste with complex attributes, such as kokumi, in an animal model. In these experiments they attempt to uncover mechanistic insights about how ornithine potentiates other stimuli by using a variety of established experimental approaches in rats. They partially succeed by finding evidence that GPRC6A may mediate effects of ornithine when it is used at lower concentrations. In the revisions they have scaled back their interpretations accordingly. A supplementary experiment measuring certain aspects of the effects of ornithine added to Miso soup in human subjects is included for the express purpose of establishing that the kokumi sensation of a complex solution is enhanced by ornithine. This (supplementary) experiment was conducted with a small sample size, and though perhaps useful, these preliminary results do not align particularly well with the animal experiments. It would be helpful to further explore human taste of ornithine in a larger and better-controlled study.

Reviewer #1 (Public review):

Summary:

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

The study reveals significant findings by identifying a transcriptional component (TC121) associated with synaptic signaling, which is linked to shorter survival in patients with high-grade serous ovarian cancer, highlighting the potential role of neurons in the tumor microenvironment. However, the evidence could be strengthened by experimental validation to confirm the functional roles of key genes within TC121 and further exploration of its spatial aspects, including deeper analysis of neuronal and synaptic and other neuronal gene expression.

Strengths:

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

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

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

Weaknesses:

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

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

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

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

Reviewer #1 (Public review):

The aim of this study was a better understanding of the reproductive life history of acoels. The acoel Hofstenia miamia, an emerging model organism, is investigated; the authors nevertheless acknowledge and address the high variability in reproductive morphology and strategies within Acoela.

The morphology of male and female reproductive organs in these hermaphroditic worms is characterised through stereo microscopy, immunohistochemistry, histology, and fluorescent in situ hybridization. The findings confirm and better detail historical descriptions. A novelty in the field is the in situ hybridization experiments, which link already published single-cell sequencing data to the worms' morphology. An interesting finding, though not further discussed by the authors, is that the known germline markers cgnl1-2 and Piwi-1 are only localized in the ovaries and not in the testes.

The work also clarifies the timing and order of appearance of reproductive organs during development and regeneration, as well as the changes upon de-growth. It shows an association of reproductive organ growth to whole body size, which will be surely taken into account and further explored in future acoel studies. This is also the first instance of non-anecdotal degrowth upon starvation in H. miamia (and to my knowledge in acoels, except recorded weight upon starvation in Convolutriloba retrogemma [1]).

Egg laying through the mouth is described in H. miamia for the first time as well as the worms' behavior in egg laying, i.e. choosing the tanks' walls rather than its floor, laying eggs in clutches, and delaying egg-laying during food deprivation. Self-fertilization is also reported for the first time.

The main strength of this study is that it expands previous knowledge on the reproductive life history traits in H. miamia and it lays the foundation for future studies on how these traits are affected by various factors, as well as for comparative studies within acoels. As highlighted above, many phenomena are addressed in a rigorous and/or quantitative way for the first time. This can be considered the start of a novel approach to reproductive studies in acoels, as the authors suggest in the conclusion. It can be also interpreted as a testimony of how an established model system can benefit the study of an understudied animal group.

The main weakness of the work is the lack of convincing explanations on the dynamics of self-fertilization, sperm storage, and movement of oocytes from the ovaries to the central cavity and subsequently to the pharynx. These questions are also raised by the authors themselves in the discussion. Another weakness (or rather missing potential strength) is the limited focus on genes. Given the presence of the single-cell sequencing atlas and established methods for in situ hybridization and even transgenesis in H. miamia, this model provides a unique opportunity to investigate germline genes in acoels and their role in development, regeneration, and degrowth. It should also be noted that employing Transmission Electron Microscopy would have enabled a more detailed comparison with other acoels, since ultrastructural studies of reproductive organs have been published for other species (cfr e.g. [2],[3],[4]). This is especially true for a better understanding of the relation between sperm axoneme and flagellum (mentioned in the Results section), as well as of sexual conflict (mentioned in the Discussion).

(1) Shannon, Thomas. 2007. 'Photosmoregulation: Evidence of Host Behavioral Photoregulation of an Algal Endosymbiont by the Acoel Convolutriloba Retrogemma as a Means of Non-Metabolic Osmoregulation'. Athens, Georgia: University of Georgia [Dissertation].<br /> (2) Zabotin, Ya. I., and A. I. Golubev. 2014. 'Ultrastructure of Oocytes and Female Copulatory Organs of Acoela'. Biology Bulletin 41 (9): 722-35.<br /> (3) Achatz, Johannes Georg, Matthew Hooge, Andreas Wallberg, Ulf Jondelius, and Seth Tyler. 2010. 'Systematic Revision of Acoels with 9+0 Sperm Ultrastructure (Convolutida) and the Influence of Sexual Conflict on Morphology'.<br /> (4) Petrov, Anatoly, Matthew Hooge, and Seth Tyler. 2006. 'Comparative Morphology of the Bursal Nozzles in Acoels (Acoela, Acoelomorpha)'. Journal of Morphology 267 (5): 634-48.

Reviewer #1 (Public review):

Summary:

The authors report the role of a novel gene Aff3ir-ORF2 in flow induced atherosclerosis. They show that the gene is anti-inflammatory in nature. It inhibits the IRF5 mediated athero-progression by inhibiting the causal factor (IRF5). Furthermore, authors show a significant connection between shear stress and Aff3ir-ORF2 and its connection to IRF5 mediated athero-progression in different established mice models which further validates the ex vivo findings.

Strengths:

(1) Adequate number of replicates were used for this study.<br /> (2) Both in vitro and in vivo validation was done.<br /> (3) Figures are well presented<br /> (4) In vivo causality is checked with cleverly designed experiments

Weaknesses:

(1) Inflammatory proteins must be measured with standard methods e.g ELISA as mRNA level and protein level does not always correlate.<br /> (2) RNA seq analysis has to be done very carefully. How does the euclidean distance correlate with the differential expression of genes. Do they represent neighborhood? If they do how does this correlation affect the conclusion of the paper?<br /> (3) Volcano plot does not indicate q value of the shown genes. It is advisable to calculate q value for each of the genes which represents the FDR probability of the identified genes.<br /> (4) GO enrichment was done against Global gene set or local geneset? Authors should provide more detailed information about the analysis.<br /> (5) If the analysis was performed against global gene set. How does that connect with this specific atherosclerotic microenvironment?<br /> (6) what was the basal expression of genes and how does the DGE (differential gene expression) values differ?<br /> (7) How did IRF5 picked from GO analysis? was it within 20 most significant genes?<br /> (8) Microscopic studies should be done more carefully? There seems to be a global expression present on the vascular wall for Aff3ir-ORF2 and the expression seems to be similar like AFF3 in fig 1.

Comments on Revision:

The authors have adequately addressed my concerns.

Reviewer #1 (Public review):

Summary:

Shihabeddin et al. used bioinformatic and molecular biology tools to study the unique regeneration of rod photoreceptors in a zebrafish model. The authors identified a few transcription factors that seem to play an important role in this process.

Strengths:

This manuscript is well prepared. The topic of this study is an interesting and important one. Bioinformatics clues are interesting.

Weaknesses:

Considering the importance of the mechanism, the knockdown experiments require further validation. The authors over-emphasized this study's relevance to RP disease (i.e. patients and mammals are not capable of regeneration like zebrafish). They under-explained this regeneration's relevance or difference to normal developmental process, which is pretty much conserved in evolution.

Reviewer #1 (Public review):

Summary:

The objective of this research is to understand how the expression of key selector transcription factors, Tal1, Gata2, Gata3, involved in GABAergic vs glutamatergic neuron fate from a single anterior hindbrain progenitor domain is transcriptionally controlled. With suitable scRNAseq, scATAC-seq, CUT&TAG, and footprinting datasets, the authors use an extensive set of computational approaches to identify putative regulatory elements and upstream transcription factors that may control selector TF expression. This data-rich study will be a valuable resource for future hypothesis testing, through perturbation approaches, of the many putative regulators identified in the study. The data are displayed in some of the main and supplemental figures in a way that makes it difficult to appreciate and understand the authors' presentation and interpretation of the data in the Results narrative. Primary images used for studying the timing and coexpression of putative upstream regulators, Insm1, E2f1, Ebf1, and Tead2 with Tal1 are difficult to interpret and do not convincingly support the authors' conclusions. There appears to be little overlap in the fluorescent labeling, and it is not clear whether the signals are located in the cell soma nucleus.

Strengths:

The main strength is that it is a data-rich compilation of putative upstream regulators of selector TFs that control GABAergic vs glutamatergic neuron fates in the brainstem. This resource now enables future perturbation-based hypothesis testing of the gene regulatory networks that help to build brain circuitry.

Weaknesses:

Some of the findings could be better displayed and discussed.

Reviewer #1 (Public review):

Summary:

The authors demonstrate impairments induced by a high cholesterol diet on GLP-1R dependent glucoregulation in vivo as well as an improvement after reduction in cholesterol synthesis with simvastatin in pancreatic islets. They also map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations, and screened for key residues selected from these sites and performed detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviour, clustering, trafficking and signalling in pancreatic beta cells and primary islets, and describe an improved insulin secretion profile for the V229A mutant receptor.

These are extensive and very impressive studies indeed. I am impressed with the tireless effort exerted to understand the details of molecular mechanisms involved in the effects of cholesterol for GLP-1 activation of its receptor. In general, the study is convincing, the manuscript well written and the data well presented. Some of the changes are small and insignificant which makes one wonder how important the observations are. For instance, in Figure 2E (which is difficult to interpret anyway because the data are presented in per cent, conveniently hiding the absolute results) does not show a significant result of the cyclodextrin except for insignificant increases in basal secretion. That is not identical to impairment of GLP-1 receptor signaling!

To me the most important experiment of them all is the simvastatin experiment, but the results rest on very few numbers and there is a large variation. Apparently, in a previous study using more extensive reduction in cholesterol the opposite response was detected casting doubt on the significance of the current observation. I agree with the authors that the use of cyclodextrin may have been associated with other changes in plasma membrane structure than cholesterol depletion at the GLP-1 receptor. The entire discussion regarding the importance of cholesterol would benefit tremendously from studies of GLP-1 induced insulin secretion in people with different cholesterol levels before and after treatment with cholesterol-lowering agents. I suspect that such a study would not reveal major differences.

Comments on revisions: The authors have responded well to my criticism.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Reviewer #2 (Public review):

The manuscript "HNF4α-1 TET2-FBP1 axis contributes to gluconeogenesis and type 2 diabetes" from Zhang et al. presents significant and convincing findings that enhance our understanding of TET2's role in liver glucose metabolism. It highlights the epigenetic regulation of FBP1, a gluconeogenic gene, by TET2, linking this pathway to HNF4alpha which recruits TET2. The in vitro and in vivo experiments are now well-described and provide convincing evidence of TET2's impact on gluconeogenesis, particularly in fasting and HFD mice.

Comments on revisions:

The authors have thoroughly addressed all the concerns raised, and their responses adequately clarify the issues previously identified.

Minor changes:

(1) Could the authors provide some comments on why glucagon was not able to stimulate PEPCK and G6Pase mRNA levels in HepG2 cells (Fig. 3D)? Although it is not the focus of the research, it is well known that glucagon has this effect and could serve as a positive control for the quality of the preparation.

(2) Please include the sequences of the qPCR primers used for PEPCK and G6Pase in the Methods section (page 17).

Reviewer #1 (Public review):

Summary:

The crystal structure of the Sld3CBD-Cdc45 complex presented by Li et al. is a significant contribution that enhances our understanding of CMG formation during the rate-limiting step of DNA replication initiation. This structure provides crucial insights into the intermediate steps of CMG formation, and the particle analysis and model predictions compellingly describe the mechanism of Cdc45 loading. Building upon previously known Sld3 and Cdc45 structures, this study offers new perspectives on how Cdc45 is recruited to MCM DH through the Sld3-Sld7 complex. The most notable finding is the structural rearrangement of Sld3CBD upon Cdc45 binding, particularly the α8-helix conformation, which is essential for Cdc45 interaction and may also be relevant to its metazoan counterpart, Treslin. Additionally, the conformational shift in the DHHA1 domain of Cdc45 suggests a potential mechanism for its binding to Mcm2NTD. Furthermore, Sld3's ssDNA-binding experiments provide evidence of its novel functions in the DNA replication process in yeast, expanding our understanding of its role beyond Cdc45 recruitment.

Strengths:

The manuscript is generally well-written, with a precise structural analysis and a solid methodological section that will significantly advance future studies in the field. The predictions based on structural alignments are intriguing and provide a new direction for exploring CMG formation, potentially shaping the future of DNA replication research. This research also opens up several new opportunities to utilize structural biology to unravel the molecular details of the model presented in the paper.

Weaknesses:

The main weakness of the manuscript lies in the lack of detailed structural validation for the proposed Sld3-Sld7-Cdc45 model, and its CMG bound models, which could be done in the future using advanced structural biology techniques such as single particle cryo-electron microscopy. It would also be interesting to explore how Sld7 interacts with the MCM helicase, and this would help to build a detailed long-flexible model of Sld3-Sld7-Cdc45 binding to MCM DH and to show where Sld7 will lie on the structure. This will help us to understand how Sld7 functions in the complex. Also, future experiments would be needed to understand the molecular details of how Sld3 and Sld7 release from CMG is associated with ssARS1 binding.

Reviewer #1 (Public review):

Summary:

Sandkuhler et al. re-evaluated the biological functions of TANGO2 homologs in C. elegans, yeast, and zebrafish. Compared to the previously reported role of TANGO2 homologs in transporting heme, Sandkuhler et al. expressed a different opinion on the biological functions of TANGO2 homologs. With the support of some results from their tests, they conclude that 'there is insufficient evidence to support heme transport as the primary function of TANGO2', in addition to their claims on the role of TANGO2 in modulating metabolism. While the differences are reported in this study, more work is needed to elucidate the biological function of TANGO2.

Strengths:

(1) This work revisited a set of key experiments, including the toxic heme analog GaPP survival assay, the fluorescent ZnMP accumulation assay, and the multi-organismal investigations documented by Sun et al. in Nature 2022, which is critical for comparing the two works.

(2) This work reported additional phenotypes for the C. elegans mutant of the TANGO2 homologs, including lawn avoidance, reduced pharyngeal pumping, smaller brood size, faster exhaustion under swimming test, and a shorter lifespan. These phenotypes are important for understanding the biological function of TANGO2 homologs, while they were missing from the report by Sun et al.

(3) Investigating the 'reduced GaPP consumption' as a cause of increased resistance against the toxic GaPP for the TANGO2 homologs, hrg-9 hrg-10 double null mutant provides a valuable perspective for studying the biological function of TANGO2 homologs.

(4) This work thoroughly evaluated the role of TANGO2 homologs in supporting yeast growth using multiple yeast strains and also pointed out the mitochondrial genome instability feature of the yeast strain used by Sun et al.

Weaknesses:

(1) A detailed comparison between this work and the work of Sun et al. on experimental protocols and reagents in the main text will be beneficial for readers to assess critically.

(2) The GaPP used by Sun et al. (purchased from Frontier Scientific) is more effective in killing the worm than the one used in this study (purchased from Santa Cruz). Is the different outcome due to the differences in reagents? Moreover, Sun et al. examined the lethality after 3-4 days, while this work examined the lethality after 72 hours. Would the extra 24 hours make any difference in the result?

(3) This work reported the opposite result of Sun et al. for the fluorescent ZnMP accumulation assay. However, the experimental protocols used by the two studies are massively different. Sun et al. did the ZnMP staining by incubating the L4-stage worms in an axenic mCeHR2 medium containing 40 μM ZnMP (purchased from Frontier Scientific) and 4 μM heme at 20 ℃ for 16 h, while this work placed the L4-stage worms on the OP50 E. coli seeded NGM plates treated with 40 μM ZnMP (purchased from Santa Cruz) for 16 h. The liquid axenic mCeHR2 medium is bacteria-free, heme-free, and consistent for ZnMP uptake by worms. This work has mentioned that the hrg-9 hrg-10 double null mutant has bacterial lawn avoidance and reduced pharyngeal pumping phenotypes. Therefore, the ZnMP staining protocol used in this work faces challenges in the environmental control for the wild type vs. the mutant. The authors should adopt the ZnMP staining protocol used by Sun et al. for a proper evaluation of fluorescent ZnMP accumulation.

(4) A striking difference between the two studies is that Sun et al. emphasize the biochemical function of TANGO2 homologs in heme transporting with evidence from some biochemical tests. In contrast, this work emphasizes the physiological function of TANGO2 homologs with evidence from multiple phenotypical observations. In the discussion part, the authors should address whether these observed phenotypes in this study can be due to the loss of heme transporting activities upon eliminating TANGO2 homologs. This action can improve the merit of academic debate and collaboration.

Reviewer #1 (Public review):

Summary:

The authors aim to formalize the mathematical underpinnings of a proposed general model and discuss the relationship of this model to the ABC Score, a widely adopted heuristic for enhancer-gene predictions. While the ABC model serves as a useful binary classifier, it struggles to predict quantitative enhancer effects on gene expression. Using a graph-theoretic linear framework, the authors derive a mathematical model (the "default model") that explains how the algebraic form of the ABC Score arises under specific assumptions. They further demonstrate that the default model's predictions of enhancer additivity are inconsistent with observed non-additive enhancer effects and propose alternative assumptions to account for these discrepancies.

Strengths:

The graph-theoretic approach enables systematic exploration of enhancer interactions beyond simple additivity and enables hypothesis generation when such expectations fail. This work makes clear where assumptions are made and the consequences of those assumptions.

Weaknesses:

While the theoretical framework is elegant, I think there is always more space to demonstrate the practicality of this approach. Further guidance for how to experimentally connect this framework with typical measurements could help bolster the immediate benefits. To be clear, I do not think this is something the authors "must" do, but rather something that might help drive home the usefulness in a more accessible way.

Reviewer #1 (Public review):

This study investigates the role of site-specific DNA methylation changes during spermatogenesis and their contribution to paternal epigenetic inheritance. Using MethylCap-seq, the authors identify a transient, site-specific loss of DNA methylation at transcription start sites (TSSs) of late spermatogenesis genes during the transition from differentiating spermatogonia (KIT+) to pachytene spermatocytes (PS). This demethylation event correlates with the gain of H3K4me3, which presets nucleosome retention sites in mouse sperm. The study proposes that selective loss of DNA methylation at a subset of promoters is required for nucleosome retention and the establishment of epigenetic states that may influence embryonic gene regulation. These findings provide complementary insights to earlier work by the Peters lab, "DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos."

Overall, the study presents a valuable dataset; however, additional analyses could strengthen the conclusions and provide further mechanistic insights.

Major Comments:

(1) Prior work should be acknowledged and used for comparative analysis. A key proposal in this study is that regions undergoing DNA methylation loss retain histones, influencing the zygote's epigenetic landscape. However, previous studies (e.g., Peters et al.) have shown that regions losing methylation in DNMT3a/b knockout (KO) mice do not necessarily retain histones, suggesting additional factors are involved. Moreover, Peters et al. demonstrated that regions of low DNA methylation in sperm render paternal alleles permissive for H3K4me3 establishment in early embryos, independent of the paternal inheritance of sperm-borne H3K4me3. Comparing these findings would refine the model presented in this study.

(2) Figure 2A: The data suggest an increase in methylation peaks in PS cells. How does this align with the hypomethylation observed in Figure 1D? Reconciling these observations would improve clarity.

(3) Figure 4A: The effect size of demethylation on nucleosome retention is unclear - do all demethylated promoters retain histones or only a subset? Quantifying this would clarify whether DNA methylation loss consistently predicts nucleosome retention.

(4) Prior studies have generated bisulfite sequencing data from Tet KO sperm. Do the regions that undergo demethylation during the KIT+ to PS transition overlap with those misregulated in TET KO sperm? Integrating this comparison could provide further insight into the regulation of site-specific demethylation.

(5) The role of SCML2 enrichment in germline stem cells and its connection to H3K27me3 deposition in later germ cells is unclear. Earlier figures show that regions undergoing DNA demethylation from KIT+ to PS include genes expressed in later-stage germ cells.

Why is SCML2 enrichment occurring in germline stem cells (GSCs)? Why is H3K27me3 only acquired at later stages if SCML2 is already present? Is SCML2 preventing premature expression independent of K27ME?

Showing the dynamics of H3K27me3 and SCML2 across these stages would clarify the proposed conclusions.

Reviewer #1 (Public review):

Turner et al. present an original approach to investigate the role of Type-1 nNOS interneurons in driving neuronal network activity and in controlling vascular network dynamics in awake head-fixed mice. Selective activation or suppression of Type-1 nNOS interneurons has previously been achieved using either chemogenetic, optogenetic, or local pharmacology. Here, the authors took advantage of the fact that Type-1 nNOS interneurons are the only cortical cells that express the tachykinin receptor 1 to ablate them with a local injection of saporin conjugated to substance P (SP-SAP). SP-SAP causes cell death in 90 % of type1 nNOS interneurons without affecting microglia, astrocytes, and neurons. The authors report that the ablation has no major effects on sleep or behavior. Refining the analysis by scoring neural and hemodynamic signals with electrode recordings, calcium signal imaging, and wide-field optical imaging, the authors observe that Type-1 nNOS interneuron ablation does not change the various phases of the sleep/wake cycle. However, it does reduce low-frequency neural activity, irrespective of the classification of arousal state. Analyzing neurovascular coupling using multiple approaches, they report small changes in resting-state neural-hemodynamic correlations across arousal states, primarily mediated by changes in neural activity. Finally, they show that nNOS type 1 interneurons play a role in controlling interhemispheric coherence and vasomotion.

In conclusion, these results are interesting, use state-of-the-art methods, and are well supported by the data and their analysis. I have only a few comments on the stimulus-evoked haemodynamic responses, and these can be easily addressed.

Reviewer #1 (Public review):

Summary:

The present study aims to associate reproduction with age-related disease as support of the antagonistic pleiotropy hypothesis of ageing predominantly using Mendelian Randomization. The authors found evidence that early-life reproductive succes is associated with advanced ageing.

Strengths:

Large sample size. Many analyses

Weaknesses:

Still a number of doubts with regard to some of the results and their interpretation.

Reviewer #1 (Public review):

Summary:

Maladaptive decision-making is a trait commonly seen in gambling disorders. Salient cues can impact decision-making and drive gambling, though how cues affect decision-making isn't well understood. This manuscript describes the impact of cueing distinct outcomes of a validated rodent cost/benefit-making task based on the human Iowa Gambling Task. Comparing six task variants, the authors describe the effect of adding salient cues to wins (that scale with the size of win or the inverse), to every outcome regardless of loss or win, randomly to losses or wins, or to losses. Behavioral results reveal that cueing wins increased risky choices. By contrast, presenting the cues randomly or cueing the losses reduced risky choices. Risk-preferring animals of the uncued, randomly cued, and loss-cued tasks showed sensitivity to devaluation, whereas win-paired cued rats did not, suggesting cues blunt behavioral updating. Behavioral analyses were paired with computational modeling of initial acquisition which revealed that risky decision-making was related to reduced punishment learning. These data provide unique insight into how cues may bias behavior and drive gambling-related phenotypes.

Strengths:

The detailed analyses provide interesting insight into how cues impact complex decision-making. While there has been a great deal of work into the impact of cues on choice, few studies integrate multiple probabilistic outcomes. Complementing these data with computational parameters helps the reader to understand what may be driving these differences in behavior. The manuscript is well-written, clearly explaining the relevance of the results and potential future directions.

Weaknesses:

Two main questions arise from these results. The first - when do behavioral differences emerge between the task variants? Based on the results and discussion, the cues increase the salience of either the wins or the losses, biasing behavior in favor of either risky or optimal choice. If this is the case, one might expect the cues to expedite learning, particularly in the standard and loss condition. Providing an analysis of the acquisition of the tasks may provide insight into how the cues are "teaching" decision-making and might explain how biases are formed and cemented.

The second question is - does the learning period used for the modeling impact the interpretation of the behavioral results? The authors indicate that computational modeling was done on the first five sessions and used these data to predict preferences at baseline. Based on these results, punishment learning predicts choice preference. However, these animals are not naïve to the contingencies because of the forced choice training prior to the task, which may impact behavior in these early sessions. Though punishment learning may initially predict risk preference, other parameters later in training may also predict behavior at baseline. The authors also present simulated data from the models for sessions 18-20, but according to the statistical analysis section, sessions 35-40 were used for analysis (and presumably presented in Figure 1). If the simulation is carried out in sessions 35-40, do the models fit the data? Finally, though the n's are small, it would be interesting to see how the devaluation impacts computational metrics. These additional analyses may help to explain the nuanced effects of the cues in the task variants.

Reviewer #1 (Public review):

Summary:

Here, the authors aim to investigate the potential improvements of ANNs when used to explain brain data using top-down feedback connections found in the neocortex. To do so, they use a retinotopic and tonotopic organization to model each subregion of the ventral visual (V1, V2, V4, and IT) and ventral auditory (A1, Belt, A4) regions using Convolutional Gated Recurrent Units. The top-down feedback connections are inspired by the apical tree of pyramidal neurons, modeled either with a multiplicative effect (change of gain of the activation function) or a composite effect (change of gain and threshold of the activation function).

To assess the functional impact of the top-down connections, the authors compare three architectures: a brain-like architecture derived directly from brain data analysis, a reversed architecture where all feedforward connections become feedback connections and vice versa, and a random connectivity architecture. More specifically, in the brain-like model the visual regions provide feedforward input to all auditory areas, whereas auditory areas provide feedback to visual regions.

First, the authors found that top-down feedback influences audiovisual processing and that the brain-like model exhibits a visual bias in multimodal visual and auditory tasks. Second, they discovered that in the brain-like model, the composite integration of top-down feedback, similar to that found in the neocortex, leads to an inductive bias toward visual stimuli, which is not observed in the feedforward-only model. Furthermore, the authors found that the brain-like model learns to utilize relevant stimuli more quickly while ignoring distractors. Finally, by analyzing the activations of all hidden layers (brain regions), they found that the feedforward and feedback connectivity of a region could determine its functional specializations during the given tasks.

Strengths:

The study introduces a novel methodology for designing connectivity between regions in deep learning models. The authors also employ several tasks based on audiovisual stimuli to support their conclusions. Additionally, the model utilizes backpropagation of error as a learning algorithm, making it applicable across a range of tasks, from various supervised learning scenarios to reinforcement learning agents. Conversely, the presented framework offers a valuable tool for studying top-down feedback connections in cortical models. Thus, it is a very nice study that also can give inspiration to other fields (machine learning) to start exploring new architectures.

Weaknesses:

Although the study explores some novel ideas on how to study the feedback connections of the neocortex, the data presented here are not complete in order to propose a concrete theory of the role of top-down feedback inputs in such models of the brain.

(1) The gap in the literature that the paper tries to fill in the ability of DL algorithms to predict behavior: "However, there are still significant gaps in most deep neural networks' ability to predict behavior, particularly when presented with ambiguous, challenging stimuli." and "[...] to accurately model the brain."

It is unclear to me how the presented work addresses this gap, as the only facts provided are derived from a simple categorization task that could also be solved by the feedforward-only model (see Figures 4 and 5). In my opinion, this statement is somewhat far-fetched, and there is insufficient data throughout the manuscript to support this claim.

(2) It is not clear what the advantages are between the brain-like model and a feedforward-only model in terms of performance in solving the task. Given Figures 4 and 5, it is evident that the feedforward-only model reaches almost the same performance as the brain-like model (when the latter uses the modulatory feedback with the composite function) on almost all tasks tested. The speed of learning is nearly the same: for some tested tasks the brain-like model learns faster, while for others it learns slower. Thus, it is hard to attribute a functional implication to the feedback connections given the presented figures and therefore the strong claims in the Discussion should be rephrased or toned down.

(3) The Methods section lacks sufficient detail. There is no explanation provided for the choice of hyperparameters nor for the structure of the networks (number of trainable parameters, number of nodes per layer, etc). Clarifying the rationale behind these decisions would enhance understanding. Moreover, since the authors draw conclusions based on the performance of the networks on specific tasks, it is unclear whether the comparisons are fair, particularly concerning the number of trainable parameters. Furthermore, it is not clear if the visual bias observed in the brain-like model is an emerging property of the network or has been created because of the asymmetries in the visual vs. auditory pathway (size of the layer, number of layers, etc).

Reviewer #1 (Public review):

Summary:

Sattin, Nardin, and colleagues designed and evaluated corrective microlenses that increase the useable field of view of two long (>6mm) thin (500 um diameter) GRIN lenses used in deep-tissue two-photon imaging. This paper closely follows the thread of earlier work from the same group (esp. Antonini et al, 2020; eLife), filling out the quiver of available extended-field-of-view 2P endoscopes with these longer lenses. The lenses are made by a molding process that appears practical and easy to adopt with conventional two-photon microscopes.

Simulations are used to motivate the benefits of extended field of view, demonstrating that more cells can be recorded, with less mixing of signals in extracted traces, when recorded with higher optical resolution. In vivo tests were performed in piriform cortex, which is difficult to access, especially in chronic preparations.

The design, characterization, and simulations are clear and thorough, but they do not break new ground in optical design or biological application. However, the approach shows much promise, including for applications such as miniaturized GRIN-based microscopes. Readers will largely be interested in this work for practical reasons: to apply the authors' corrected endoscopes to their own research.

Strengths:

The text is clearly written, the ex vivo analysis is thorough and well supported, and the figures are clear. The authors achieved their aims, as evidenced by the images presented, and were able to make measurements from large numbers of cells simultaneously in vivo in a difficult preparation.

The authors did a good job of addressing issues I raised in initial review, including analyses of chromaticity and the axial field of view, descriptions of manufacturing and assembly yield, explanations in the text of differences between ex vivo and in vivo imaging conditions, and basic analysis of the in vivo recordings relative to odor presentations. They have also shortened the text, reduced repetition, and better motivated their approach in the introduction.

Reviewer #1 (Public Review):

Summary:

The study by Seo et al highlights knowledge gaps regarding the role of cerebellar complex spike (CS) activity during different phases of learning related to optokinetic reflex (OKR) in mice. The novelty of the approach is twofold: first, specifically perturbing the activity of climbing fibers (CFs) in the flocculus (as opposed to disrupting communication between the inferior olive (IO) and its cerebellar targets globally); and second, examining whether disruption of the CS activity during the putative "consolidation phase" following training affects OKR performance.

The first part of the results provides adequate evidence supporting the notion that optogenetic disruption of normal CF-Purkinje neuron (PN) signaling results in the degradation of OKR performance. As no effects are seen in OKR performance in animals subjected to optogenetic irradiation during the memory consolidation or retrieval phases, the authors conclude that CF function is not essential beyond memory acquisition. However, the manuscript does not provide a sufficiently solid demonstration that their long-term activity manipulation of CF activity is effective, thus undermining the confidence of the conclusions.

Strengths:

The main strength of the work is the aim to examine the specific involvement of the CF activity in the flocculus during distinct phases of learning. This is a challenging goal, due to the technical challenges related to the anatomical location of the flocculus as well as the IO. These obstacles are counterbalanced by the use of a well-established and easy-to-analyse behavioral model (OKR), that can lead to fundamental insights regarding the long-term cerebellar learning process.

Weaknesses:

The impact of the work is diminished by several methodological shortcomings.

Most importantly, the key finding that prolonged optogenetic inhibition of CFs (for 30 min to 6 hours after the training period) must be complemented by the demonstration that the manipulation maintains its efficacy. In its current form, the authors only show inhibition by short-term optogenetic irradiation in the context of electrical-stimulation-evoked CSs in an ex vivo preparation. As the inhibitory effect of even the eNpHR3.0 is greatly diminished during seconds-long stimulations (especially when using the yellow laser as is done in this work (see Zhang, Chuanqiang, et al. "Optimized photo-stimulation of halorhodopsin for long-term neuronal inhibition." BMC biology 17.1 (2019): 1-17. ), we remain skeptical of the extent of inhibition during the long manipulations. In short, without a demonstration of effective inhibition throughout the putative consolidation phase (for example by showing a significant decrease in CS frequency throughout the irradiation period), the main claim of the manuscript of phase-specific involvement of CF activity in OKR learning can not be considered to be based on evidence.

Second, the choice of viral targeting strategy leaves gaps in the argument for CF-specific mechanisms. CaMKII promoters are not selective for the IO neurons, and even the most precise viral injections always lead to the transfection of neurons in the surrounding brainstem, many of which project to the cerebellar cortex in the form of mossy fibers (MF). Figure 1Bii shows sparsely-labelled CFs in the flocculus, but possibly also MFs. While obtaining homogenous and strong labeling in all floccular CFs might be impossible, at the very least the authors should demonstrate that their optogenetic manipulation does not affect simple spiking in PNs.

Finally, while the paper explicitly focuses on the effects of CF-evoked complex spikes in the PNs and not, for example, on those mediated by molecular layer interneurons or via direct interaction of the CF with vestibular nuclear neurons, it would be best if these other dimensions of CF involvement in cerebellar learning were candidly discussed.

Reviewer #1 (Public review):

Summary:

This study seeks to quantify changes in vocal behavior during development in marmosets with bilateral anterior cingulate cortex (ACC) lesions. The ACC and its role in social vocal behaviors is of great interest given previous literature on its involvement in initiation of vocalizations, processing emotional content, and its connectivity to two other critical nodes in the vocal network, the amygdala and the PAG. The authors seek to test the hypothesis that the ACC contributes to the development of mature vocal behaviors during the first few weeks of life by disrupting this process with neonatal ACC lesions. Imaging and histological analyses confirm the extent of the lesion and suggest downstream effects in connected regions. Analysis of call rates and call type proportions show no or slight differences between lesioned and controlled animals. Additional analyses on the proportion of grouped 'social' calls and certain acoustic features of a particular call, the phee, reveal more distinct differences between the groups.

Strengths:

The authors have identified that ACC lesions in early life have no or little influence on certain aspects of vocal behavior (e.g. call rate, call intervals) but larger impacts on other aspects (e.g. acoustic features of phee calls). This is difficult data to collect, especially in the difficulties of that particular time period. This data is a valuable addition to the literature on the effects of the ACC on vocal production and sparks intriguing follow-up questions on the role of different acoustic features (as related to emotional content) on vocal interactions with conspecifics over the lifespan.

The histological methods and resulting quantification of neural changes in the lesioned area and in downstream areas of interest are intriguing given the large time gap between the lesion and these analyses.

The changes to the text, figures, and additional supplemental figures to my previous review requests have made it easier to determine if conclusions are supported by the data in the manuscript. Examples include the quantification of the loss of neurons and increase in glial cells, the inclusion of changes in body weight and grip strength that could also be a result from the lesions affecting vocal behavior, and additional details on analysis methods.

Weaknesses:

The article emphasizes vocal social behavior. However, marmoset infants are recorded in isolation which allows for examining the development of vocal behavior in that particular context - reaching out to conspecifics. The text now covers the relationship between 'social' information in calls and development in this particular context. However, early-life maturation of vocal behavior is strongly influenced by social interactions with conspecifics. For example, the transition of cries and subharmonic phees which are high-entropy calls to more low-entropy mature phees is affected by social reinforcement from the parents. And this effect extends cross-context, where differences in these interaction patterns extend to vocal behavior when the marmosets are alone. Together, the results are interesting and important but may not fully capture the changes resulting from direct social interactions.

Additionally, it is an intriguing finding that the infants' phee calls have acoustic differences being 'blunted of variation, less diverse and more regular'. Though the text about how the social message conveyed by these infants was 'deficient, limited, and/or indiscriminate' is now better explained with additional text from human studies, it is still an assumption that this would directly translate to marmoset communication. Thus, experiments directed at the responses of other marmosets to these calls would still be important.

Reviewer #1 (Public review):

Summary:

This study investigates what happens to the stimulus-driven responses of V4 neurons when an item is held in working memory. Monkeys are trained to perform memory guided saccades: they must remember the location of a visual cue and then, after a delay, make an eye movement to the remembered location. In addition, a background stimulus (a grating) is presented that varies in contrast and orientation across trials. This stimulus serves to probe the V4 responses, is present throughout the trial, and is task-irrelevant. Using this design, the authors report memory-driven changes in the LFP power spectrum, changes in synchronization between the V4 spikes and the ongoing LFP, and no significant changes in firing rate.

Strengths:

- The logic of the experiment is nicely laid out.

- The presentation is clear and concise.

- The analyses are thorough, careful, and yield unambiguous results.

- Together, the recording and inactivation data demonstrate quite convincingly that the signal stored in FEF is communicated to V4 and that, under the current experimental conditions, the impact from FEF manifests as variations in the timing of the stimulus-evoked V4 spikes and not in the intensity of the evoked activity (i.e., firing rate).

Weaknesses:

The weaknesses I noted in the first round of reviews were effectively addressed by the authors. In particular, the expanded discussion on the overlapping effects of attention, working memory, and motor planning does a great job putting the current findings against the wider context concerning the neural mechanisms of visuomotor guidance.

I think this is a well-designed and well-executed study that helps to better outline the relationship between perception and working memory given their respective neural substrates. A broad range of systems neuroscientists and experimental psychologists will find it illuminating.

Reviewer #1 (Public review):

Summary:

This is an interesting study on AD(H)D. The authors combine a variety of neural and physiological metrics to study attention in a VR classroom setting. The manuscript is well written and the results are interesting, ranging from an effect of group (AD(H)D vs. control) on metrics such as envelope tracking, to multivariate regression analyses considering alpha-power, gaze, TRF, ERPs, and behaviour simultaneously. I find the first part of the results clear and strong. The multivariate analyses in Tables 1 and 2 are good ideas, but I think they would benefit from additional clarifications. Overall, I think that the methodological approach is useful in itself. The rest is interesting in that it informs us on which metrics are sensitive to group-effects and correlated with each other. I think this might be one interesting way forward. Indeed, much more work is needed to clarify how these results change with different stimuli and tasks. So, I see this as an interesting first step into more naturalistic measurement of speech attention.

Strengths:

I praise the authors for this interesting attempt to tackle a challenging topic with naturalistic experiment and metrics. I think the results broadly make sense and they contribute to a complex literature that is far from being linear and cohesive.

Weaknesses:

The authors have successfully addressed most of my concerns during the review process. Some weaknesses remain in this resubmission, but they do not make the results invalid. For example:<br /> - The EEG data was filtered twice, which is not recommended as that can introduce additional filtering artifacts. So, while I definitely do not recommend doing that, I do not expect that issue to have an impact on this specific result.<br /> - The authors did not check whether participants were somewhat familiar with the topics in the speech material. The authors agreed that this point might be beneficial for future research.<br /> - The hyperparameter tuning is consistent with previous work from the authors, and it involves selecting the optimal lambda value of the regularized regression based on the group average, thus choosing a single lambda value for all participants. In my opinion, that is not the optimal way to run those models, and I do not generally recommend using this approach. The reason is that the lambda can change depending on the magnitude of the signals and the SNR, leading to different optimal lambdas for distinct participants. On the other hand, finding those optimal lambda values for individual participants can be difficult depending on the amount of data and amount of noise, so it is sometimes necessary to apply strategies that ensure an appropriate choice of lambda. Using the group average as a metric for hyperparameter tuning produces a more stable metric and lambda value selection, which might be preferrable (even though this choice should not be taken lightly). In this specific case, I think the authors had a good reason to do so.

Comments on revisions:

The authors have done a great job at addressing my comments. I don't have any further concerns. Congratulations!

Reviewer #1 (Public review):

Summary:

For many years, there has been extensive electrophysiological research investigating the relationship between local field potential patterns and individual cell spike patterns in the hippocampus. In this study, using state-of-the-art imaging techniques, they examined spike synchrony of hippocampal cells during locomotion and immobility states. In contrast to conventional understanding of the hippocampus, the authors demonstrated that hippocampal place cells exhibit prominent synchronous spikes locked to theta oscillations.

Strengths:

The voltage imaging used in this study is a highly novel method that allows recording not only suprathreshold-level spikes but also subthreshold-level activity. With its high frame rate, it offers time resolution comparable to electrophysiological recordings.

Comments on revisions: I have no further comments.

Reviewer #1 (Public review):

Summary:

The authors propose a new model of biologically realistic reinforcement learning in the direct and indirect pathway spiny projection neurons in the striatum. These pathways are widely considered to provide a neural substrate for reinforcement learning in the brain. However, we do not yet have a full understanding of mechanistic learning rules that would allow successful reinforcement learning like computations in these circuits. The authors outline some key limitations of current models and propose an interesting solution by leveraging learning with efferent inputs of selected actions. They show that the model simulations are able to recapitulate experimental findings about the activity profile in these populations in mice during spontaneous behavior. They also show how their model is able to implement off-policy reinforcement learning.

Strengths:

The manuscript has been very clearly written and the results have been presented in a readily digestible manner. The limitations of existing models, that motive the presented work, have been clearly presented and the proposed solution seems very interesting. The novel contribution in the proposed model is the idea that different patterns of activity drive current action selection and learning. Not only does this allow the model is able to implement reinforcement learning computations well, this suggestion may have interesting implications regarding why some processes selectively affect ongoing behavior and others affect learning. The model is able to recapitulate some interesting experimental findings about various activity characteristics of dSPN and iSPN pathway neuronal populations in spontaneously behaving mice. The authors also show that their proposed model can implement off-policy reinforcement learning algorithms with biologically realistic learning rules. This is interesting since off-policy learning provides some unique computational benefits and it is very likely that learning in neural circuits may, at least to some extent, implement such computations.

Weaknesses:

A weakness in this work is that it isn't clear how a key component in the model - an efferent copy of selected actions - would be accessible to these striatal populations. The authors propose several plausible candidates, but future work may clarify the feasibility of this proposal.

Reviewer #1 (Public review):

Summary:

In the article titled "Polyphosphate discriminates protein conformational ensembles more efficiently than DNA promoting diverse assembly and maturation behaviors," Goyal and colleagues investigate the role of negatively charged biopolymers, i.e., polyphosphate (polyP) and DNA, play in phase separation of cytidine repressor (CytR) and fructose repressor (FruR). The authors find that both negative polymers drive the formation of metastable protein/polymer condensates. However, polyP-driven condensates form more gel- or solid-like structures over time while DNA-driven condensates tend to dissipate over time. The authors link this disparate condensate behavior to polyP-induced structures within the enzymes. Specifically, they observe the formation of polyproline II-like structures within two tested enzyme variants in the presence of polyP. Together their results provide a unique insight into the physical and structural mechanism by which two unique negatively charged polymers can induce distinct phase transitions with the same protein. This study will be a welcomed addition to the condensate field and provide new molecular insights into how binding partner-induced structural changes within a given protein can affect the mesoscale behavior of condensates. The concerns outlined below are meant to strengthen the manuscript.

Strengths:

Throughout the article, the authors used the correct techniques to probe physical changes within proteins that can be directly linked to phase transition behaviors. Their rigorous experiments create a clear picture of what occurs at the molecular level with CytR and FruR are exposed to either DNA or polyP, which are unique, highly negatively charged biopolymers found within bacteria. This work provides a new view of mechanisms by which bacteria can regulate the cytoplasmic organization upon the induction of stress. Furthermore, this is likely applicable to mammalian and plant cells and likely to numerous proteins that undergo condensation with nucleic acids and other charged biopolymers.

Weaknesses:

The biggest weakness of this study is that compares the phase behavior of enzymes driven by negatively charged polymers that have intrinsic differences in net charge and charge density. Because these properties are extremely important for controlling phase separation, any differences may result in the observed phase transitions driven by DNA and polyP. The authors should perform an additional experiment to control for these differences as best they can. The results from these experiments will provide additional insight into the importance of charge-based properties for controlling phase transitions.

Reviewer #1 (Public review):

Summary:

This study aimed to investigate the effects of optically stimulating the A13 region in healthy mice and a unilateral 6-OHDA mouse model of Parkinson's disease (PD). The primary objectives were to assess changes in locomotion, motor behaviors, and the neural connectome. For this, the authors examined the dopaminergic loss induced by 6-OHDA lesioning. They found a significant loss of tyrosine hydroxylase (TH+) neurons in the substantia nigra pars compacta (SNc) while the dopaminergic cells in the A13 region were largely preserved. Then, they optically stimulated the A13 region using a viral vector to deliver the channelrhodopsine (CamKII promoter). In both sham and PD model mice, optogenetic stimulation of the A13 region induced pro-locomotor effects, including increased locomotion, more locomotion bouts, longer durations of locomotion, and higher movement speeds. Additionally, PD model mice exhibited increased ipsilesional turning during A13 region photoactivation. Lastly, the authors used whole-brain imaging to explore changes in the A13 region's connectome after 6-OHDA lesions. These alterations involved a complex rewiring of neural circuits, impacting both afferent and efferent projections. In summary, this study unveiled the pro-locomotor effects of A13 region photoactivation in both healthy and PD model mice. The study also indicates the preservation of A13 dopaminergic cells and the anatomical changes in neural circuitry following PD-like lesions that represent the anatomical substrate for a parallel motor pathway.

Strengths:

These findings hold significant relevance for the field of motor control, providing valuable insights into the organization of the motor system in mammals. Additionally, they offer potential avenues for addressing motor deficits in Parkinson's disease (PD). The study fills a crucial knowledge gap, underscoring its importance, and the results bolster its clinical relevance and overall strength.

The authors adeptly set the stage for their research by framing the central questions in the introduction, and they provide thoughtful interpretations of the data in the discussion section. The results section, while straightforward, effectively supports the study's primary conclusion-the pro-locomotor effects of A13 region stimulation, both in normal motor control and in the 6-OHDA model of brain damage.

Weaknesses:

(1) Anatomical investigation. I have a major concern regarding the anatomical investigation of plastic changes in the A13 connectome (Figures 4 and 5). While the methodology employed to assess the connectome is technically advanced and powerful, the results lack mechanistic insight at the cell or circuit level into the pro-locomotor effects of A13 region stimulation in both physiological and pathological conditions. This concern is exacerbated by a textual description of results that doesn't pinpoint precise brain areas or subareas but instead references large brain portions like the cortical plate, making it challenging to discern the implications for A13 stimulation. Lastly, the study is generally well-written with a smooth and straightforward style, but the connectome section presents challenges in readability and comprehension. The presentation of results, particularly the correlation matrices and correlation strength, doesn't facilitate biological understanding. It would be beneficial to explore specific pathways responsible for driving the locomotor effects of A13 stimulation, including examining the strength of connections to well-known locomotor-associated regions like the Pedunculopontine nucleus, Cuneiformis nucleus, LPGi, and others in the diencephalon, midbrain, pons, and medulla. Additionally, identifying the primary inputs to A13 associated with motor function would enhance the study's clarity and relevance.

The study raises intriguing questions about compensatory mechanisms in Parkinson's disease a new perspective with the preservation of dopaminergic cells in A13, despite the SNc degeneration, and the plastic changes to input/output matrices. To gain inspiration for a more straightforward reanalysis and discussion of the results, I recommend the authors refer to the paper titled "Specific populations of basal ganglia output neurons target distinct brain stem areas while collateralizing throughout the diencephalon from the David Kleinfeld laboratory." This could guide the authors in investigating motor pathways across different brain regions.

(2) Description of locomotor performance. Figure 3 provides valuable data on the locomotor effects of A13 region photoactivation in both control and 6-OHDA mice. However, a more detailed analysis of the changes in locomotion during stimulation would enhance our understanding of the pro-locomotor effects, especially in the context of 6-OHDA lesions. For example, it would be informative to explore whether the probability of locomotion changes during stimulation in the control and 6-OHDA groups. Investigating reaction time, speed, total distance, and even kinematic aspects during stimulation could reveal how A13 is influencing locomotion, particularly after 6-OHDA lesions. The laboratory of Whelan has a deep knowledge of locomotion and the neural circuits driving it so these features may be instructive to infer insights on the neural circuits driving movement. On the same line, examining features like the frequency or power of stimulation related to walking patterns may help elucidate whether A13 is engaging with the Mesencephalic Locomotor Region (MLR) to drive the pro-locomotor effects. These insights would provide a more comprehensive understanding of the mechanisms underlying A13-mediated locomotor changes in both healthy and pathological conditions.

(3) Figure 2 indeed presents valuable information regarding the effects of A13 region photoactivation. To enhance the comprehensiveness of this figure and gain a deeper understanding of the neurons driving the pro-locomotor effect of stimulation, it would be beneficial to include quantifications of various cell types:

• cFos-Positive Cells/TH-Positive Cells: it can help determine the impact of A13 stimulation on dopaminergic neurons and the associated pro-locomotor effect in healthy condition and especially in the context of Parkinson's disease (PD) modeling.

• cFos-Positive Cells /TH-Negative Cells: Investigating the number of TH-negative cells activated by stimulation is also important, as it may reveal non-dopaminergic neurons that play a role in locomotor responses. Identifying the location and characteristics of these TH-negative cells can provide insights into their functional significance.<br /> Incorporating these quantifications into Figure 2 would enhance the figure's informativeness and provide a more comprehensive view of the neuronal populations involved in the locomotor effects of A13 stimulation.

(4) Referred to Figure 3. In the main text (page 5) when describing the animal with 6-OHDA the wrong panels are indicated. It is indicated in Figure 2A-E but it should be replaced with 3A-E. Please do that.

Summary of the Study after revision

The revised manuscript reflects significant efforts to improve clarity, organization, and data interpretation. The refinements in anatomical descriptions, behavioral analyses, and contextual framing have strengthened the manuscript considerably. However, the study still lacks direct causal evidence linking anatomical remodeling to behavioral improvements, and the small sample size in the anatomical analyses remains a concern. The authors have addressed many points raised in the initial review, but further acknowledgement of the exploratory nature of these findings would enhance the scientific rigor of the work.

Key Improvements in the Revision

The revised manuscript demonstrates considerable progress in clarifying data presentation, refining behavioral analyses, and improving the contextualization of anatomical findings. The restructuring of the anatomical section now provides greater precision in describing motor-related pathways, integrating terminology from the Allen Brain Atlas. The addition of new figures (Figures 4 and 5) strengthens the accessibility of these findings by illustrating key connectivity patterns more effectively. Furthermore, the correlation matrices have been adjusted to improve interpretability, ensuring that the presented data contribute meaningfully to the overall narrative of the study.

The authors have also made significant improvements in their behavioral analyses, particularly in the organization and presentation of locomotor data. Figure 3 has been revised to distinctly separate results from 6-OHDA and sham animals, providing a clearer comparison of locomotor outcomes. Additional metrics, such as reaction time, locomotion bouts, and movement speed, further enhance the granularity of the analysis, making the results more informative.

The discussion surrounding anatomical connectivity has also been strengthened. The revised manuscript now places greater emphasis on motor-related pathways and refines its analysis of A13 efferents and afferents. A newly introduced figure provides a concise summary of these connections, improving the contextualization of the anatomical data within the study's broader scope. Moreover, the authors have addressed the translational relevance of their findings by acknowledging the differences between optogenetic stimulation and deep brain stimulation (DBS). Their discussion now better situates the findings within existing literature on PD-related motor circuits, providing a more balanced perspective on the potential implications of A13 stimulation.

Remaining Concerns

Despite these substantial improvements, a number of critical concerns remain. The anatomical findings, though insightful, remain largely correlative and do not establish a causal link between structural remodeling and locomotor recovery. While the authors argue that these data will serve as a reference for future investigations, their necessity for the core conclusions of the study is not entirely clear. Additionally, while the anatomical data offer an interesting perspective on A13 connectivity, their direct relevance to the study's primary goal-demonstrating the role of A13 in locomotor recovery-remains uncertain. The authors emphasize that these data will be valuable for future research, yet their integration into the study's main narrative feels somewhat supplementary. Based on this last thought of the authors it is even more relevant another key limitation lying in the small sample size used for connectivity analyses. With only two sham and three 6-OHDA animals included, the statistical confidence in the findings is inherently limited. The absence of direct statistical comparisons between ipsilesional and contralesional projections further weakens the conclusions drawn from these anatomical studies. The authors have acknowledged that obtaining the necessary samples, acquiring the data, and analyzing them is a prolonged and resource-intensive process. While this may be a valid practical limitation, it does not justify the lack of a robust statistical approach. A more rigorous statistical framework should be employed to reinforce the findings, or alternative techniques should be considered to provide additional validation. Given these constraints, it remains unclear why the authors have not opted for standard immunohistochemistry, which could provide a complementary and more statistically accessible approach to validate the anatomical findings. Employing such an approach would not only increase the robustness of the results but also strengthen the study's impact by providing an independent confirmation of the observed structural changes.

Reviewer #2 (Public review):

Summary:

In this extensive comparative study, Moreno-Borrallo and colleagues examine the relationships between plasma glucose levels, albumin glycation levels, diet and life-history traits across birds. Their results confirmed the expected positive relationship between plasma blood glucose level and albumin glycation rate but also provided findings that are somewhat surprising or contrast with findings of some previous studies (positive relationships between blood glucose and lifespan, or absent relationships between blood glucose and clutch mass or diet). This is the first extensive comparative analysis of glycation rates and their relationships to plasma glucose levels and life history traits in birds that is based on data collected in a single study, with blood glucose and glycation measured using unified analytical methods (except for blood glucose data for 13 species collected from a database).

Strengths:

This is an emerging topic gaining momentum in evolutionary physiology, which makes this study a timely, novel and important contribution. The study is based on a novel data set collected by the authors from 88 bird species (67 in captivity, 21 in the wild) of 22 orders, except for 13 species, for which data were collected from a database of veterinary and animal care records of zoo animals (ZIMS). This novel data set itself greatly contributes to the pool of available data on avian glycemia, as previous comparative studies either extracted data from various studies or a ZIMS database (therefore potentially containing much more noise due to different methodologies or other unstandardised factors), or only collected data from a single order, namely Passeriformes. The data further represents the first comparative avian data set on albumin glycation obtained using a unified methodology. The authors used LC-MS to determine glycation levels, which does not have problems with specificity and sensitivity that may occur with assays used in previous studies. The data analysis is thorough, and the conclusions are substantiated. Overall, this is an important study representing a substantial contribution to the emerging field evolutionary physiology focused on ecology and evolution of blood/plasma glucose levels and resistance to glycation.

Weaknesses:

Unfortunately, the authors did not record handling time (i.e., time elapsed between capture and blood sampling), which may be an important source of noise because handling-stress-induced increase in blood glucose has previously been reported. Moreover, the authors themselves demonstrate that handling stress increases variance in blood glucose levels. Both effects (elevated mean and variance) are evident in Figure ESM1.2. However, this likely makes their significant findings regarding glucose levels and their associations with lifespan or glycation rate more conservative, as highlighted by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthen the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail, and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

The authors have made some changes in the revised version. However, many of the changes were superficial, and some concerns still need to be addressed. Important details are still missing from the description of some experiments. Authors should carefully revise the manuscript to ascertain that all details that could affect interpretation of their results are presented clearly. For instance, authors still need to include details of how the metabolomics analyses were performed. Just stating that samples were "frozen for metabolomics analyses" is not enough. Was this mass-spec or NMR-based metabolomics. Assuming it was mass-spec, what kind? How was metabolite identity assigned, etc? These are important details, which need to be included. Even in cases where additional information was included, the authors did not discuss how the specific way in which certain experiments were performed could affect interpretation of their results. One example is the potential for compound carryover in their experiments. Another important one is the fact that CAPE affects bacterial growth and sporulation. Therefore, it is critical that authors acknowledge that they cannot discard the possibility that other factors besides compound interactions with the toxin are involved in their phenotypes. As stated previously, authors should also be careful when drawing conclusions from the analysis of microbiota composition data, and changes to the manuscript should be made to reflect this. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Again, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Reviewer #3 (Public review):

Summary:

Retroviruses have been endogenized into the genome of all vertebrate animals. The envelope protein of the virus is not well conserved and acquires many mutations hence can be used to monitor viral evolution. Since they are incorporated into the host genome, they also reflect the evolution of the hosts. In this manuscript the authors have focused their analyses to the env genes of endogenous retroviruses in primates. Important observations made include the extensive recombination events between these retroviruses that were previously unknown and the discovery of HML species in genomes prior to the splitting of old and new world monkeys.

Strengths:

They explored a number of databases and made phylogenetic trees to look at the distribution of retroviral species in primates. The authors provide a strong rationale for their study design, they provide a clear description of the techniques and the bioinformatics tools used.

Weaknesses:

The manuscript is based on bioinformatics analyses only. The reference genomes do not reflect the polymorphisms in humans or other primate species. The analyses thus likely under estimate the amount of diversity in the retroviruses. Further experimental verification will be needed to confirm the observations.

Not sure which databases were used, but if not already analyzed, ERVmap.com and repeatmesker are ones that have many ERVs that are not present in the reference genomes. Also long range sequencing of the human genome has recently become available which may also be worth studying for this purpose.

Comments on revisions:

All comments have been adequately addressed.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the drift diffusion model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options between individuals with bulimia nervosa (BN) and healthy participants.

(2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has the potential to improve the understanding of pathological food choices. The article is based on secondary research data.

Weaknesses:

I have two major concerns and a major improvement point.

The major concerns deal with the reliability of the results of the DDM (first two sections of the Results, pages 6 and 7), which are central to the manuscript, and the consistency of the results with regards to the identification of mechanisms related to binge eating in BN patients (i.e. last section of the results, page 7).

(1) Ratcliff and McKoon in 2008 used tasks involving around 1000 trials per participant. The Chen et al. experiment the authors refer to involves around 400 trials per participant. On the other hand, Shevlin and colleagues ask each participant to make two sets of 42 choices with two times fewer participants than in the Chen et al. experiment. Shevlin and colleagues also fit a DDM with additional parameters (e.g. a drift rate that varies according to subjective rating of the options) as compared to the initial version of Ratcliff and McKoon. With regards to the number of parameters estimated in the DDM within each group of participants and each emotional condition, the 5- to 10-fold ratio in the number of trials between the Shevlin and colleagues' experiment and the experiments they refer to (Ratcliff and McKoon, 2008; Chen et al. 2022) raises serious concerns about a potential overfitting of the data by the DDM. This point is not highlighted in the Discussion. Robustness and sensitivity analyses are critical in this case.

The authors compare different DDMs to show that the DDM they used to report statistical results in the main text is the best according to the WAIC criterion. This may be viewed as a robustness analysis. However, the other DDM models (i.e. M0, M1, M2 in the supplementary materials) they used to make the comparison have fewer parameters to estimate than the one they used in the main text. Fits are usually expected to follow the rule that the more there are parameters to estimate in a model, the better it fits the data. Additionally, a quick plot of the data in supplementary table S12 (i.e. WAIC as a function of the number of parameters varying by food type in the model - i.e. 0 for M0, 2 for M1, 1 for M2 and 3 for M3) suggests that models M1 and potentially M2 may be also suitable: there is a break in the improvement of WAIC between model M0 and the three other models. I would thus suggest checking how the results reported in the main text differ when using models M1 and M2 instead of M3 (for the taste and health weights when comparing M3 with M1, for τS when comparing M3 with M2). If the differences are important, the results currently reported in the main text are not very reliable.

(2) The second main concern deals with the association reported between the DDM parameters and binge eating episodes (i.e. last paragraph of the results section, page 7). The authors claim that the DDM parameters "predict" binge eating episodes (in the Abstract among other places) while the binge eating frequency does not seem to have been collected prospectively. Besides this methodological issue, the interpretation of this association is exaggerated: during the task, BN patients did not make binge-related food choices in the negative emotional state. Therefore, it is impossible to draw clear conclusions about binge eating, as other explanations seem equally plausible. For example, the results the authors report with the DDM may be a marker of a strategy of the patients to cope with food tastiness in order to make restrictive-like food choices. A comparison of the authors' results with restrictive AN patients would be of interest. Moreover, correlating results of a nearly instantaneous behavior (i.e. a couple of minutes to perform the task with the 42 food choices) with an observation made over several months (i.e. binge eating frequency collected over three months) is questionable: the negative emotional state of patients varies across the day without systematically leading patients to engage in a binge eating episode in such states.

I would suggest in such an experiment to collect the binge craving elicited by each food and the overall binge craving of patients immediately before and after the task. Correlating the DDM results with these ratings would provide more compelling results. Without these data, I would suggest removing the last paragraph of the Results.

(3) My major improvement point is to tone down as much as possible any claim of a link with binge eating across the entire manuscript and to focus more on the restrictive behavior of BN patients in between binge eating episodes (see my second major concern about the methods). Additionally, since this article is a secondary research paper and since some of the authors have already used the task with AN patients, if possible I would run the same analyses with AN patients to test whether there are differences between AN (provided they were of the restrictive subtype) and BN.

Reviewer #1 (Public review):

Summary:

Despite accumulating prior studies on the expressions of AVP and AVPR1a in the brain, a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking. Using RNAscope, a cutting-edge technology that detects single RNA transcripts, the authors created a comprehensive neuroanatomical atlas of Avp and Avpr1a in male and female brains. The findings are important, given that: (1) a detailed, gender-specific mapping of AVP/AVPR1a neuronal nodes has been lacking, and (2) the study offers valuable new insights into Avpr1a expression across the mouse brain. The findings are solid, and with improved data presentation and analysis, this work could serve as an important resource for the neuroscience community.

Strengths:

This well-executed study provides valuable new insights into gender differences in the distribution of Avp and Avpr1a. The atlas is an important resource for the neuroscience community.

Weaknesses:

A few concerns remain to be addressed. The primary weakness of this manuscript lies in the robustness of its data presentation and analysis.

Reviewer #1 (Public review):

Summary:

The authors had previously found that brief social isolation could increase the activity of these neurons, and that manipulation of these neurons could alter social behavior in a social rank-dependent fashion. This manuscript explored which of the outputs were responsible for this, identifying the central nucleus of the amygdala as the key output region. The authors identified some discrete behavior changes associated with these outputs, and found that during photostimulation of these outputs, neuronal activity appeared altered in 'social response' neurons.

Strengths:

Rigorous analysis of the anatomy. Careful examination of the heterogenous effects on cell activity due to stimulation, linking the physiology with the behavior via photostimulation during recording in vivo.

Weaknesses:

(1) There are some clear imbalances in the sample size across the different regions parsed. The CeA has a larger sample size, likely in part to the previous work suggesting differential effects depending on social rank/dominance. Given the potential variance, it may be hard to draw conclusions about the impact of stimulation across different social ranks for other groups.

(2) It is somewhat unclear why only the 'social object ratio' was used to assess the effects versus more direct measurements of social behavior.

(3) Somewhat related, while it is statistically significant, it is unclear if the change seen in face investigation of biologically significant, on average, it looks like a few-seconds difference and that was not modulated by social rank.

(4) There are several papers studying these neurons that have explored behaviors examined here, as well as the physiological connectivity that are not cited that would provide important context for this work. In particular, multiple groups have found a dopamine-mediated IPSP in the BNST, in contrast to this work. There are technical differences that may drive these differences, but not addressing them is a major weakness.

(5) The inclusion of some markers for receptors for some of these outputs is interesting, and the authors suggest that this may be important, but this is somewhat disconnected from the rest of the work performed.

Reviewer #1 (Public review):

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

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

(2) Larger breasts are associated with lower tactile spatial resolution<br /> This conclusion is based on a strong correlation between participants' JNDs and the size of their breasts. The correlation convincingly supports the conclusion. It is of interest to the field, as it aligns with the hypothesis that nerve fibers are more sparsely distributed across larger body parts.

(3) The nipple is a landmark: perceptually a unit and an attractor for tactile percepts<br /> The data do not support these conclusions. The conclusion that the nipple is perceived as a unit is based on poor performance in tactile categorization for touches on the nipple. This categorization performance may simply mirror the breast's low tactile spatial resolution with JNDs about the size of a nipple.

The conclusion that tactile percepts are drawn towards the nipple is based on tactile localization biases towards the nipple for tactile stimuli on the breast compared to localization biases for tactile stimuli on the back. Currently, the statistical analysis of the data does not match the field, psychophysics, standards. Moreover, any bias towards the nipple could simply be another instance of regression to the mean of the stimulus distribution, given that the tested locations were centered on the nipple. This confound can only be experimentally solved by shifting the distribution of the tested locations. Finally, given that participants indicated the locations on a 3D model of the body part, further experimentation would be required to determine whether there is a perceptual bias towards the nipple or whether the authors merely find a response bias.

Further comments:

- Given that later analyses require regression models, the authors might consider using them throughout.

- The stability of the JND differences between body parts across subjects is already captured in the analysis of the JNDs; the ANOVA and the post-hoc testing would not be significant if the order were not relatively stable across participants. Thus, it is unclear why this is being evaluated again with reduced power due to improper statistics.

- The null hypothesis of an ANOVA is that at least one of the mean values is different from the others; adding participants as a factor does not provide evidence for similarity.

- The pairwise correlations between body parts seem to be exploratory in nature. Like all exploratory analyses, the question arises of how much potential extra insights outweigh the risk of false positives. It would be hard to generate data with significant differences between several conditions and not find any correlations between pairs of conditions. Thus, the a priori chance of finding a significant correlation is much higher than what a correction accounts for.

- If the JND at mid breast (measured with locations centered at the nipple) is roughly the same size as the nipple, it is not surprising that participants have difficulty with the categorical localization task on the nipple but perform better than chance on the significantly larger areola.

- To justify the conclusion that the nipple is a unit, additional data would be required. 1) One could compare psychometric curves with the nipple as the center and psychometric curves with a nearby point on the areola as the center. 2) Performance in the quadrant task could be compared for the nipple and an equally sized portion of the areola. Otherwise, the task "only" provides confirmatory evidence for a low tactile resolution in the midbreast area.

- A localization bias toward the nipple in this context does not show that the nipple is the anchor of the breast's tactile coordinate system. The result might simply be an instance of regression to the mean of the stimulus distribution (also known as experimental prior). To convincingly show localization biases towards the nipple, the tested locations should be centered at another location on the breast.

- Another problem is the visual salience of the nipple, even though Blender models were uniformly grey. With this type of direct localization, it is very difficult to distinguish perceptual from response biases even if the regression to the mean problem is solved. There are two solutions to this problem: 1) Varying the uncertainty of the tactile spatial information, for example, by using a pen that exerts lighter pressure. A perceptual bias should be stronger for more uncertain sensory information; a response bias should be the same across conditions. 2) Measure bias with a 2IFC procedure by taking advantage of the fact that sensory information is noisier if the test is presented before the standard.

- Neither signed nor absolute localization error can be compared to the results of the previous experiments. The JND should be roughly proportional to the variance of the errors.

- The statistically adequate way of testing the biases is a hierarchical regression model (LMM) with a distance of the physical location from the nipple as a predictor, and a distance of the reported location from the nipple as a dependent variable. Either variable can be unsigned or signed for greater power, for example, coding the lateral breast as negative and the medial breast as positive. The bias will show in regression coefficients smaller than 1.

- It does not matter whether distances are calculated based on skin or 3D coordinates, as Euclidean distances or based on polar coordinates. However, there should only be one consistent distance in the text across both independent and dependent variables. Calculating various versions of these measures can create issues in Frequentist Statistics. For transparency, it is good practice to report the results of other methods for calculating the distance in the supplement.

- The body part could be added as a predictor to the LMM, with differences in bias between the body parts showing a significant interaction between the two predictors. The figures suggest such an effect. However, the interpretation should take into account that 1) response biases are more likely to arise at the breast and 2) it might be harder to learn the range of locations on the back given that stimulation is not restricted to an anatomically defined region as it is the case for the breast.

Reviewer #1 (Public review):

Summary:

The study explores the use of Transport-based morphometry (TBM) to predict hematoma expansion and growth 24 hours post-event, leveraging Non-Contrast Computed Tomography (NCCT) scans combined with clinical and location-based information. The research holds significant clinical potential, as it could enable early intervention for patients at high risk of hematoma expansion, thereby improving outcomes. The study is well-structured, with detailed methodological descriptions and a clear presentation of results. However, the practical utility of the predictive tool requires further validation, as the current findings are based on retrospective data. Additionally, the impact of this tool on clinical decision-making and patient outcomes needs to be further investigated.

Strengths

(1) Clinical Relevance: The study addresses a critical need in clinical practice by providing a tool that could enhance diagnostic accuracy and guide early interventions, potentially improving patient outcomes.

(2) Feature Visualization: The visualization and interpretation of features associated with hematoma expansion risk are highly valuable for clinicians, aiding in the understanding of model-derived insights and facilitating clinical application.

(3) Methodological Rigor: The study provides a thorough description of methods, results, and discussions, ensuring transparency and reproducibility.

Weaknesses:

(1) The limited sample size in this study raises concerns about potential model overfitting. While the reported AUCROC of 0.71 may be acceptable for clinical use, the robustness of the model could be further enhanced by employing techniques such as k-fold cross-validation. This approach, which aggregates predictive results across multiple folds, mimics the consensus of diagnoses from multiple clinicians and could improve the model's reliability for clinical application. Additionally, in clinical practice, the utility of the model may depend on specific conditions, such as achieving high specificity to identify patients at risk of hematoma expansion, thereby enabling timely interventions. Consequently, while AUC is a commonly used metric, it may not fully capture the model's clinical applicability. The authors should consider discussing alternative performance metrics, such as specificity and sensitivity, which are more aligned with clinical needs. Furthermore, evaluating the model's performance in real-world clinical scenarios would provide valuable insights into its practical utility and potential impact on patient outcomes.

(2) The authors compared the performance of TBM with clinical and location-based information, as well as other machine learning methods. While this comparison highlights the relative strengths of TBM, the study would benefit from providing concrete evidence on how this tool could enhance clinicians' ability to assess hematoma expansion in practice. For instance, it remains unclear whether integrating the model's output with a clinician's own assessment would lead to improved diagnostic accuracy or decision-making. Investigating this aspect-such as through studies evaluating the combined performance of clinician judgment and model predictions-could significantly enhance the tool's practical value.

Reviewer #1 (Public review):

Summary:

Govindan and Conrad use a genome-wide CRISPR screen to identify genes regulating retention of intron 4 in OGT, leveraging an intron retention reporter system previously described (PMID: 35895270). Their OGT intron 4 reporter reliably responds to O-GlcNAc levels, mirroring the endogenous splicing event. Through a genome-wide CRISPR knockout library, they uncover a range of splicing-related genes, including multiple core spliceosome components, acting as negative regulators of OGT intron 4 retention. They choose to follow up on SFSWAP, a largely understudied splicing regulator shown to undergo rapid phosphorylation in response to O-GlcNAc level changes (PMID: 32329777). RNA-sequencing reveals that SFSWAP depletion not only promotes OGT intron 4 splicing but also broadly induces exon inclusion and intron splicing, affecting decoy exon usage. While this study offers interesting insights into intron retention regulation and O-GlcNAc signaling, the RNA-Sequencing experiments lack essential controls needed to provide full confidence to the authors' conclusions.

Strengths:

(1) This study presents an elegant genetic screening approach to identify regulators of intron retention, uncovering core spliceosome genes as unexpected positive regulators of intron retention.<br /> (2) The work proposes a novel functional role for SFSWAP in splicing regulation, suggesting that it acts as a negative regulator of splicing and cassette exon inclusion, which contrasts with expected SR-related protein functions.<br /> (3) The authors suggest an intriguing model where SFSWAP, along with other spliceosome proteins, promotes intron retention by associating with decoy exons.

Weaknesses:

(1) The conclusions regarding SFSWAP's impact on alternative splicing rely on cells treated with a single pool of two siRNAs for five days. The absence of independent siRNA treatments raises concerns about potential off-target effects, which may reduce confidence in the observed SFSWAP-dependent splicing changes. Rescue experiments or using additional independent siRNA treatments would strengthen the conclusions.<br /> (2) The mechanistic role of SFSWAP in splicing would benefit from further exploration, though this may be more appropriate for future studies.

Comments on revisions:

The authors have addressed all my previous recommendations.

Reviewer #1 (Public review):

Summary:

This study has preliminarily revealed the role of ACVR2A in trophoblast cell function, including its effects on migration, invasion, proliferation, and clonal formation, as well as its downstream signaling pathways.

Strengths:

The use of multiple experimental techniques, such as CRISPR/Cas9-mediated gene knockout, RNA-seq, and functional assays (e.g., Transwell, colony formation, and scratch assays), is commendable and demonstrates the authors' effort to elucidate the molecular mechanisms underlying ACVR2A's regulation of trophoblast function. The RNA-seq analysis and subsequent GSEA findings offer valuable insights into the pathways affected by ACVR2A knockout, particularly the Wnt and TCF7/c-JUN signaling pathways.

Weaknesses:

The current findings provide valuable insights into the role of ACVR2A in trophoblast cell function and its involvement in the regulation of migration, invasion, and proliferation, further validation in both in vitro and in vivo models would strengthen the conclusions. Additional techniques, such as animal models and more advanced clinical sample analyses, would help strengthen the conclusions and provide a more comprehensive understanding of the molecular pathways involved.

Reviewer #1 (Public review):

Lu et. al. proposed here a direct role of LPS in inducing hepatic fat accumulation and that metabolism of LPS therefore can mitigate fatty liver injury. With an Acyloxyacyl hydrolase whole-body KO mice, they demonstrated that Acyloxyacyl hydrolase deletion resulted in higher hepatic fat accumulation over 7 months of high glucose/high fructose diet. Previous literature has found that hepatocyte TLR4 (which is a main receptor for binding LPS) KO reduced fatty liver in MAFLD model, and this paper complement this by showing that degradation/metabolism of LPS can also reduce fatty liver. Using clodronate-liposomes to deplete KC, the authors went on to show that AOAH level decreased significantly with increased SREBP1 level, suggesting that KCs were the major source of AOAH in the liver. To explain the mechanism of LPS induced lipogenesis, the authors demostrated in vitro that LPS alone without KC can induce SREBP1 level in primary hepatocytes via mTOR activation. This result proposed a very interesting mechanism, and the translational implications of utilizing Acyloxyacyl hydrolase to decrease LPS exposure is intriguing.

The strengths of the present study include that they raised a very simplistic mechanism with LPS that is of interest in many diseases. The phenotype shown in the study is strong. The mechanism proposed by the findings are generally well supported. Manuscript significantly improved with revision. Overall, this work adds to the current understanding of the gut-liver axis and development of MAFLD, and will be of interest to many readers.

Reviewer #3 (Public review):

Summary:

Chen et al. present a thorough statistical analysis of social interactions, more precisely, co-occupying the same chamber in the Eco-HAB measurement system. They also test the effect of manipulating the prelimbic cortex by using TIMP-1 that inhibits the MMP-9 matrix metalloproteinase. They conclude that altering neural plasticity in the prelimbic cortex does not eliminate social interactions, but it strongly impacts social information transmission.

Strengths:

The quantitative approach to analyzing social interactions is laudable and the study is interesting. It demonstrates that the Eco-HAB can be used for high throughput, standardized and automated tests of the effects of brain manipulations on social structure in large groups of mice.

Weaknesses:

A demonstration of TIMP-1 impairing neural plasticity specifically in the prelimbic cortex of the treated animals would greatly strengthen the biological conclusions. The Eco-HAB provides coarser spatial information compared to some other approaches, which may influence the conclusions.

Reviewer #1 (Public review):

Summary:

In this report, the authors made use of a murine cell line derived from a MYC-driven liver cancer to investigate the gene expression changes that accompany the switch from normoxic to hypoxia conditions during 2D growth and the switch from 2D monolayer to 3D organoid growth under normoxic conditions. They find a significant (ca. 40-50%) overlap among the genes that are dysregulated in response to hypoxia in 2D cultures and in response to spheroid formation. Unsurprisingly, hypoxia-related genes were among the most prominently deregulated under both sets of conditions. Many other pathways pertaining to metabolism, splicing, mitochondrial electron transport chain structure and function, DNA damage recognition/repair and lipid biosynthesis were also identified.

Comments on the revised manuscript:

In my original review of this manuscript, I raised 11 points that I thought needed to be addressed and/or clarified by the authors. In response, they have provided an adequate answer to only one of these (point 6), which is little more than a more thorough description of how spheroids were generated. The remaining points that I raised, which would have provided more mechanistic insight into their study were addressed by the authors with the following such comments:

- It is not the focus of this study (Points 1 and 4)

- It is worthy of further validation (Point 2)

- We apologize for not being able to validate everything (Point 3)

- This reviewer has raised an interesting question. We are investigating this hypothesis and hopefully we can give a clear answer in the future (Point 5)

- This is an excellent idea that we certainly will do it in our future experiments (Point 7)

As to responses that the authors made to the other two reviewers' comments: Most pertained to cosmetic alterations involving clarification of methods, inclusion of a new figure or rearrangement of old figures. These were generally answered. However, in response to the last point raised by Rev. 3 to compare "sgRNA abundances at the earliest harvesting time with the distribution in the library...to see whether and to what extent selection has already taken place before the three culture conditions were established", the authors responded with the comment: "This is great point. Unfortunately, we did not perform such an analysis."

I understand that it is often impossible to address all points raised by the reviewers. This can be for a variety of reasons and many times the omissions can be overlooked and accepted if the reviewer can be convinced that a good faith attempt has otherwise been made to address the other deficiencies. However, no such effort has been made here and the study remains deficient and largely descriptive as I pointed out in my original review.

Reviewer #1 (Public review):

Summary:

The authors address the role of the centromere histone core in force transduction by the kinetochore

Strengths:

They use a hybrid DNA sequence that combines CDEII and CDEIII as well as Widom 601 so they can make stable histones for biophysical studies (provided by the Widom sequence) and maintain features of the centromere (CDE II and III).

Weaknesses:

The main results are shown in one figure (Fig 2). Indeed the Centromere core of Widom and CDE II and III contribute to strengthening the binding force for the OA-beads. The data are very nicely done and convincingly demonstrate the point. The weakness is that this is the entire paper. It is certainly of interest to investigators in kinetochore biology, but beyond that the impact is fairly limited in scope.

Comments on revisions:

The additional information provided by the authors will help the reader understand and interpret the manuscript.

Reviewer #1 (Public review):

The authors set out to analyse the roles of the teichoic acids of Streptococcus pneumoniae in supporting the maintenance of the periplasmic region. Previous work has proposed the periplasm to be present in Gram positive bacteria and here advanced electron microscopy approach was used. This also showed a likely role for both wall and lipo-teichoic acids in maintaining the periplasm. Next, the authors use a metabolic labelling approach to analyse the teichoic acids. This is a clear strength as this method cannot be used for most other well studied organisms. The labelling was coupled with super-resolution microscopy to be able to map the teichoic acids at the subcellular level and a series of gel separation experiments to unravel the nature of the teichoic acids and the contribution of genes previously proposed to be required for their display. The manuscript could be an important addition to the field but there are a number of technical issues which somewhat undermine the conclusions drawn at the moment. These are shown below and should be addressed. More minor points are covered in the private

Recommendations for Authors.

Weaknesses to be addressed:

(1) l. 144 Was there really only one sample that gave this resolution? Biological repeats of all experiments are required.

(2) Fig. 4A. Is the pellet recovered at "low" speeds not just some of the membrane that would sediment at this speed with or without LTA? Can a control be done using an integral membrane protein and Western Blot? Using the tacL mutant would show the behaviour of membranes alone.

(3) Fig. 4A. Using enzymatic digestion of the cell wall and then sedimentation will allow cell wall associated proteins (and other material) to become bound to the membranes and potentially effect sedimentation properties. This is what is in fact suggested by the authors (l. 1000, Fig. S6). In order to determine if the sedimentation properties observed are due to an artefact of the lysis conditions a physical breakage of the cells, using a French Press, should be carried out and then membranes purified by differential centrifugation. This is a standard, and well-established method (low-speed to remove debris and high-speed to sediment membranes) that has been used for S. pneumoniae over many years but would seem counter to the results in the current manuscript (for instance Hakenbeck, R. and Kohiyama, M. (1982), Purification of Penicillin-Binding Protein 3 from Streptococcus pneumoniae. European Journal of Biochemistry, 127: 231-236).

(4) l. 303-305. The authors suggest that the observed LTA-like bands disappear in a pulse chase experiment (Fig. 6B). What is the difference between this and Fig. 5B, where the bands do not disappear? Fig. 5C is the WT and was only pulse labelled for 5 min and so would one not expect the LTA-like bands to disappear as in 6B?

(5) Fig. 6B, l. 243-269 and l. 398-410. If, as stated, most of the LTA-like bands are actually precursor then how can the quantification of LTA stand as stated in the text? The "Titration of Cellular TA" section should be re-evaluated or removed? If you compare Fig. 6C WT extract incubated at RT and 110oC it seems like a large decrease in amount of material at the higher temperature. Thus, the WT has a lot of precursors in the membrane? This needs to be quantified.

(6) L. 339-351, Fig. 6A. A single lane on a gel is not very convincing as to the role of LytR. Here, and throughout the manuscript, wherever statements concerning levels of material are made, quantification needs to be done over appropriate numbers of repeats and with densitometry data shown in SI.

(7) 14. l. 385-391. Contrary to the statement in the text, the zwitterionic TA will have associated counterions that results in net neutrality. It will just have both -ve and +ve counterions in equal amounts (dependent on their valency), which doesn't matter if it is doing the job of balancing osmolarity (rather than charge).

Comments on revisions:

The resubmitted manuscript now contains new data and changes to the text.

The authors have largely covered my previous points in both sets of reviews (Public/Recommendations).

Public Review Points:

1 & 6: I still do not see a reproducibility statement as such, with details of the number of biological repeats etc.

2 & 3. Fig S7 seems to be quite telling. As predicted after physical breakage the membrane proteins sediment at high speed (rather than low speed). This presumably also means that the LTA comes down at high and not low speed. LTA was not measured due to cost of reagents. The Microfluidizer breaks the cells using a shear force and thus is unlikely to create very small membrane fragments. Thus, the sedimentation properties of membranes containing LTA are likely dependent on the way in which the cells are lysed. It is therefore worthwhile qualifying the statements on l. 35-36, 46-47 and 212 (as Ref 8 used mechanical breakage). This will give better direction to those in the field following up the findings.

It is also a little alarming that the mutanolysin is contaminated by protease and one hopes this does not affect any of the properties of the materials being analysed.

Joint Public Review:

Summary:

The behavioral switch between foraging and mating is important for resource allocation in insects. This study characterizes the role of sulfakinin and the sulfakinin receptor 1 in changes in olfactory responses associated with foraging versus mating behavior in the oriental fruit fly (Bactrocera dorsalis), a significant agricultural pest. This pathway regulates food consumption and mating receptivity in other species; here the authors use genetic disruption of sulfakinin and sulfakinin receptor 1 to provide strong evidence that changes in sulfakinin signaling modulate antennal responses to food versus pheromonal cues and alter the expression of ORs that detect relevant stimuli.

Strengths:

The authors utilize multiple complementary approaches including CRISPR/Cas9 mutagenesis, behavioral characterization, electroantennograms, RNA sequencing and heterologous expression to convincingly demonstrate the involvement of the sulfakinin pathway in the switch between foraging and mating behaviors. The use of both sulfakinin peptide and receptor mutants is a strength of the study and implicates specific signaling actors.

Weaknesses:

The authors demonstrate that SKR is expressed in olfactory neurons, however there are additional potential sites of action that may contribute to these results.

Reviewer #1 (Public review):

Summary:

This research article by Nath et al. from the Lee Lab addresses how lipolysis under starvation is achieved by a transient receptor potential channel, TRPγ, in the neuroendocrine neurons to help animals survive prolonged starvation. Through a series of genetic analyses, the authors identify that trpγ mutations specifically lead to a failure in lipolytic processes under starvation, thereby reducing animals' starvation resistance. The conclusion was confirmed through total triacylglycerol levels in the animals and lipid droplet staining in the fat bodies. This study highlights the importance of transient receptor potential (TRP) channels in the fly brain to modulate energy homeostasis and combat metabolic stress. However, the co-expression of trpγ and Dh44-R2 in the gut is not convincing, especially in the picture of the arrows pointing at the autofluorescence signals in the gut (Figure 7P). Therefore, the authors should either confirm the co-expression or acknowledge that trpγ and Dh44-R2 are not co-expressed in the gut and modify their model in Figure 8 accordingly, although clarifying their co-expression may not change the main conclusions of this study. Overall, the revised version includes the required clarifications on their important results that strengthen the interpretations of the research as well as the visibility of this study.

Strengths:

This study identifies the biological meaning of TRPγ in promoting lipolysis during starvation, advancing our knowledge about the TRPγ channel and the neural mechanisms to combat metabolic stress. Furthermore, this study demonstrates the potential of the TRPγ channel as a target to develop new therapeutic strategies for human metabolic disorders by showing that metformin and AMPK pathways are involved in its function in lipid metabolisms during starvation in Drosophila.

Reviewer #1 (Public review):

This manuscript presents SAVEMONEY, a computational tool designed to enhance the utilization of Oxford Nanopore Technologies (ONT) long-read sequencing for the design and analysis of plasmid sequencing experiments. In the past few years, with the improvement in both sequencing length and accuracy, ONT sequencing is being rapidly extended to almost all omics analyses which are dominated by short-read sequencing (e.g., Illumina). However, relatively higher sequencing errors of long-read sequencing techniques including PacBio and ONT is still a major obstacle for plasmid/clone-based sequencing service that aims to achieve single base/nucleotide accuracy. This work provides a guideline for sequencing multiple plasmids together using the same ONT run without molecular barcoding, followed by data deconvolution. The whole algorithm framework is well-designed, and some real data and simulation data are utilized to support the conclusions. The tool SAVEMONEY is proposed to target users who have their own ONT sequencers and perform library preparation and sequencing by themselves, rather than relying on commercial services. As we know and discussed by the authors, in the real world, to ensure accuracy, the researchers will routinely pick up multiple colonies in the same plasmid construction and submit for Sanger sequencing. However, SAVEMONEY is not able to support the simultaneous analysis of multiple colonies in the same run, as compared to the barcoding-based approaches. This is a major limitation in the significance of this work. Encouraging computational efforts in ONT data debarcoding for mixed-plasmid or even single-cell sequencing would be more valuable in the field.

Comments on revisions:

My previous concerns have been addressed, and the revised manuscript has been significantly approved.

Reviewer #2 (Public review):

Summary:

This study investigates cold induced states in C. elegans, using polysome profiling and RNA seq to identify genes that are differentially regulated and concluding that cold-specific gene regulation occurs at the transcriptional level. This study also includes analysis of one gene from the differentially regulated set, lips-11 (a lipase), and finds that it is regulated in response to a specific set of ER stress factors.

Strengths:

(1) Understanding how environmental conditions are linked to stress pathways is generally interesting.<br /> (2) The study used well-established genetic tools to analyze ER stress pathways.

Weaknesses:

(1) The conclusions regarding a general transcriptional response are based on a few genes, with much of the emphasis on lips-11, which does not affect survival in response to cold.

(2) Definitive conclusions regarding transcription vs translational effects would require the use of blockers such as alpha-amanitin or cyclohexamide. Although this may be beyond the scope of the study, it does affect the breadth of the conclusions that can be made.

(3) Conclusions regarding the role of lipids are based on supplementation with oleic acid or choline, yet there is no lipid analysis of the cold animals, or after lips-1 knockdown. Although choline is important for PC production, adding choline in normal PC could have many other metabolic impacts and doesn't necessarily implicate PC without lipidomic or genetic evidence. Although they note the caveats, their evidence falls short of proving a role in PC production.

Reviewer #1 (Public Review):

The mechanisms that regulate establishment of the germline stem cells and germline progenitors during zebrafish reproductive development are not understood. Prior single cell analysis characterized the cell types of the early zebrafish ovary during and at stages after sexual differentiation. In this work Hsu et al. took a single approach to analyze the cell types present in the early gonad during early sex determination. As expected, they identified germline stem cells (GSCs) that express canonical GSC markers and distinct populations of progenitors. Unexpectedly, they found multiple populations of transcriptionally distinct progenitor populations that the authors termed early (those lacking the differentiation marker foxl2l), committed (those expressing fox2l2 and S-phase genes) and late (those expressing fox2l2 and meiotic genes) progenitors. Comparisons of their dataset to the published zebrafish ovary datasets confirmed the presence of these distinct progenitor populations in the ovary. Further, they convincingly validated the presence of these progenitor subtypes using fluorescent in situ hybridization. To investigate the relationship between progenitor subsets and known regulators of ovary differentiation, the authors conducted single cell analysis of gonads lacking the transcription factor, Foxl2l. As previously reported, Foxl2l absence blocks ovary differentiation and all foxl2l mutants develop testes. The single cell analysis here indicates that foxl2l is inappropriately expressed in GSCs and early progenitors and that germ cell differentiation is blocked at the committed progenitor stage since few committed progenitors and no late progenitors or meiotic transcripts were detected in the single cell analysis of foxl2l mutants. Based on the coexpression of genes that are not typically expressed together in normally developing germ cells, specifically nanos2 and foxl2l, and dmrt1 and foxl2l, the authors conclude that Foxl2l is required for the committed progenitor program and that it prevents committed progenitors from returning to the GSC state.

Overall, the data provide new insights into the cell populations of the early differentiating gonad, define distinct progenitor states, pinpoint a requirement for the ovary differentiation factor Foxl2l at a specific stage of progenitor differentiation, and generate new hypotheses to be tested. Many but not all of the conclusions are supported by compelling data, and some findings and conclusions need to be clarified in the context of the published literature.

(1) The authors conclude that the committed progenitors revert to GSCs based on the coexpression of nanos2 and foxl2l nanos2 and based on expression of id1 in mutants but not in WT. Without functional data demonstrating that the progenitors revert to an earlier state, alternative interpretations should be considered. For example, it is possible that the cells initiate the committed progenitor program but continue to express the GSC program and that the coexpression of both programs blocks differentiation. Consistent with this possibility, some Fox family members, FoxL2 and FoxPs for example, are known to be both activators and repressors of transcription or act primarily as repressors. Potentially relevant to this work, repressive activity of FoxL2 has been previously reported in the mammalian ovary (Pisarska et al Endocrinology 2004, Pisarska Am J. Phys Endo. Metabolism 2010, Kuo Reproduction 2012, Kuo Endocrinology 2011, as well as more recent publications). In that context interfering with FoxL2 was proposed to cause upregulated expression of genes normally repressed by FoxL2, accelerated follicle recruitment, and premature ovarian failure.

(2) The authors conclude that the committed progenitor stage is "the gate toward female determination" and that the cells "stay at S-Phase temporarily before differentiation". This conclusion seems to be based solely on single cell RNAseq expression. In several species, including zebrafish, meiotic entry occurs earlier in females and has been correlated with ovary development. The possibility that the late progenitor stage, the stage when meiotic genes are detected in this study and a stage missing in foxl2l mutants, is actually the key stage for female determination cannot be excluded by the data provided.

(3) The authors discuss prior working showing that loss of germ cells leads to male development and that germ cells are required for female development and claim to extend that work by showing here that some progenitors are already sexually differentiated. First, the stages compared are completely different. The earlier work looks at the primordial germ cells and their loss in the first few days of development before a gonad forms. In contrast, this work examines stages well after the gonad has formed and during sex determination. The second concern is that the conclusion that the progenitors are differentiated is based solely on the expression of foxl2l, which is initially expressed in the juvenile ovary state that lab strains have been shown to develop through (Wilson et al Front Cell Dev Bio 2024). While it is fair to state that some cells express ovary markers at this stage, it is unclear that this is sufficient evidence that the cells are differentiated. For example, in the context of the foxl2l mutant, the authors observe that GSCs and early progenitors inappropriately express foxl2l, but the mutants develop as males. Thus, expression of foxl2l transcripts alone is insufficient evidence to claim that the cells are already differentiated as female.

(4) The comparison between medaka and zebrafish foxl2l mutants seems to suggest that Foxl2l is required for meiosis in medaka but has a different role in zebrafish. However, if foxl2l represses the earlier developmental programs of GSCs and early progenitors, it is possible that continued expression of these early programs interferes with activation of meiotic genes. This could account for the absence of the late progenitor stage in foxl2l mutants since the late progenitor stage is defined by and distinguished from the earlier stages by expression of foxl2l and meiotic genes. If so, foxl2l may be similarly required in both systems.

(5) The authors state that "Foxl2l may ensure female differentiation by preventing stemness and antagonizing male development." It is unclear why suppressing stemness would be necessary for female differentiation since female zebrafish have stem cells as do male zebrafish. It seems likely that turning off the GSC and early differentiation programs is important for allowing expression of meiosis and oocyte differentiation genes, and that a gene other than Foxl2l is required for differentiation from GSCs to spermatocytes.

(6) Based on its expression in mutant progenitors, p53 is proposed to assist with alternative differentiation of mutant germ cells. Although p53 transcripts are expressed, no evidence is provided that p53 is involved in differentiation of germ cells, and sex bias has not been associated with the published p53 mutants in zebrafish. Furthermore, while p53 has been shown to be important for ovary to testis transformation in mutant contexts in adults, it appears dispensable for testis development in mutants that disrupt ovary differentiation in earlier stages (Rodriguez-Mari et al PLoS Gen 2010, Shive PNAS 2010, Hartung et al Mol. Reprod. Dev 2014, Miao Development 2017, Kaufman et al PLoSGen 2018, Bertho et al Development 2021. It is possible that p53 eliminates foxl2l mutant germ cells that are simultaneously expressing multiple developmental programs, but this possibility would need to be tested.

Reviewer #1 (Public review):

Summary:

Pradhan et al investigated the potential gustatory mechanisms that allow flies to detect cholesterol. They found that flies are indifferent to low cholesterol and avoid high cholesterol. They further showed that the ionotropic receptors Ir7g, Ir51b, and Ir56d are important for the cholesterol sensitivity in bitter neurons. The figures are clear and the behavior result is interesting. However, I have several major comments, especially on the discrepancy of the expression of these Irs with other lab published results, and the confusing finding that the same receptors (Ir7g, Ir51b) have been implicated in the detection of various seemingly unrelated compounds.

Strengths:

The results are very well presented, the figures are clear and well-made, text is easy to follow.

Weaknesses:

(1) Regarding the expression of Ir56d. The reported Ir56d expression pattern contradicts multiple previous studies (Brown et al., 2021 eLife, Figure 6a-c; Sanchez-Alcaniz et al., 2017 Nature Communications, Figure 4e-h; Koh et al., 2014 Neuron, Figure 3b). These studies, using three different driver lines, consistently showed Ir56d expression in sweet-sensing neurons and taste peg neurons. Importantly, Sanchez-Alcaniz et al. demonstrated that Ir56d is not expressed in Gr66a-expressing (bitter) neurons. This discrepancy is critical since Ir56d is identified as the key subunit for cholesterol detection in bitter neurons, and misexpression of Ir7g and Ir51b together is insufficient to confer cholesterol sensitivity (Fig.4b,d). Which Ir56d-GAL4 (and Gr66a-I-GFP) line was used in this study? Is there additional evidence (scRNA sequencing, in-situ hybridization, or immunostaining) supporting Ir56d expression in bitter neurons?

(2) Ir51b has previously been implicated in detecting nitrogenous waste (Dhakal 2021), lactic acid (Pradhan 2024), and amino acids (Aryal 2022), all by the same lab. Additionally, both Ir7g and Ir51b have been implicated in detecting cantharidin, an insect-secreted compound that flies may or may not encounter in the wild, by the same lab. Is Ir51b proposed to be a specific receptor for these chemically distinct compounds or a general multimodal receptor for aversive stimuli? Unlike other multimodal bitter receptors, the expression level of Ir51b is rather low and it's unclear which subset of GRNs express this receptor. The chemical diversity among nitrogenous waste, amino acids, lactic acid, cantharidin, and cholesterol raises questions about the specificity of these receptors and warrants further investigation and at a minimum discussion in this paper. Given the wide and seemingly unrelated sensitivity of Ir51b and Ir7g to these compounds I'm leaning towards the hypothesis that at least some of these is non-specific and ecologically irrelevant without further supporting evidence from the authors.

(3) The Benton lab Ir7g-GAL4 reporter shows no expression in adults. Additionally, two independent labellar RNA sequencing studies (Dweck, 2021 eLife; Bontonou et al., 2024 Nature Communications) failed to detect Ir7g expression in the labellum. This contradicts the authors' previous RT-PCR results (Pradhan 2024 Fig. S4, Journal of Hazardous Materials) showing Ir7g expression in the labellum. Additionally the Benton and Carlson lab Ir51b-GAL4 reporters show no expression in adults as well. Please address these inconsistencies.

(4) The premise that high cholesterol intake is harmful to flies, which makes sensory mechanisms for cholesterol avoidance necessary, is interesting but underdeveloped. Animal sensory systems typically evolve to detect ecologically relevant stimuli with dynamic ranges matching environmental conditions. Given that Drosophila primarily consume fruits and plant matter (which contain minimal cholesterol) rather than animal-derived foods (which contain higher cholesterol), the ecological relevance of cholesterol detection requires more thorough discussion. Furthermore, at high concentrations, chemicals often activate multiple receptors beyond those specifically evolved for their detection. If the cholesterol concentrations used in this study substantially exceed those encountered in the fly's natural diet, the observed responses may represent an epiphenomenon rather than an ecologically and ethologically relevant sensory mechanism. What is the cholesterol content in flies' diet and how does that compare to the concentrations used in this paper?

Reviewer #1 (Public review):

The aim of this study is to test the overarching hypothesis that plasticity in BNST CRF neurons drives distinct behavioral responses to unpredictable threat in males and females. The manuscript provides solid evidence for a sex-specific role for CRF-expressing neurons in the BNST in unpredictable aversive conditioning and subsequent hypervigilance across sexes. As the authors note, this is an important question given the high prevalence of sex differences in stress-related disorders, like PTSD, and the role of hypervigilance and avoidance behaviors in these conditions. The study includes in vivo manipulation, bulk calcium imaging, and cellular resolution calcium imaging, which yield important insights into cell-type specific activity patterns. A major strength of this manuscript is the inclusion of both males and females and attention to possible behavioral and neurobiological differences between them throughout.

Reviewer #1 (Public review):

In this study, Osiurak and colleagues investigate the neurocognitive basis of technical reasoning. They use multiple tasks from two neuroimaging studies to show that the area PF is central to technical reasoning and plays an essential role in tool-use and non-tool-use physical problem-solving, as well as both conditions of mentalizing tasks. They also demonstrate the specificity of technical reasoning, finding that area PF is not involved in the fluid-cognition task or the mentalizing network (INT+PHYS vs. PHYS-only). This work enhances our understanding of the neurocognitive basis of technical reasoning that supports advanced technologies.

Strengths:

- The topic this study focuses on is intriguing and can help us understand the neurocognitive processes involved in technical reasoning and advanced technologies.<br /> - The researchers collected fMRI data from multiple tasks. The data is rich and encompasses the mechanical problem-solving task, psychotechnical task, fluid-cognition task, and mentalizing tasks.<br /> - The article is well written.

The authors have addressed many of the reviewers' concerns in their response. They utilized both correlation analysis and coordinate analysis to tackle alternative hypotheses, namely the same-region-but-different-function interpretation and the adjacency interpretation. Additionally, ROI analysis was conducted to validate the negative results. These additional analyses have enhanced the reliability of the findings. This study offers valuable insights into the neurocognitive mechanisms underlying technical reasoning.

Weaknesses:

While the authors attempted to address the limitations of overlap analysis by correlating activation across different tasks within subjects, this issue could not be entirely resolved due to the constraints of the current experimental design. The mechanical problem-solving task was not included since the sample of subjects differed from that of other tasks. Furthermore, the fluid-cognition task was not scanned in the same run as the psychotechnical and mentalizing tasks, which may have contributed to a lack of correlation between them, thereby affecting result interpretation. Moreover, the core cognitive focus of this study, technical reasoning, may be influenced by assumptions about motion-related information. While this issue has been discussed in the discussion section, further evidence is needed to substantiate this interpretation.

Reviewer #1 (Public review):

Hüppe and colleagues had already developed an apparatus and an analytical approach to capture swimming activity rhythms in krill. In a previous manuscript they explained the system, and here they employ it to show a circadian clock, supplemented by exogenous light, produces an activity pattern consistent with "twilight" diel vertical migration (DVM; a peak at sunset, a midnight sink, and a peak in the latter half of the night).

They used light:dark (LD) followed by dark:dark (DD) photoperiods at two times of the year to confirm the circadian clock, coupled with DD experiments at four times a year to show rhythmicity occurs throughout the year along with DVM in the wild population. The individual activity data show variability in the rhythmic response, which is expected. However, their results showed rhythmicity was sustained in DD throughout the year, although the amplitude decayed quickly. The interpretation of a weak clock is reasonable, and they provide a convincing justification for the adaptive nature of such a clock in a species that has a wide distributional range and experiences various photic environments. These data also show that exogenous light increases the activity response and can explain the morning activity bouts, with the circadian clock explaining the evening and late-night bouts. This acknowledgement that vertical migration can be driven by multiple proximate mechanisms is important.

The work is rigorously done, and the interpretations are sound. I see no major weaknesses in the manuscript. Because a considerable amount of processing is required to extract and interpret the rhythmic signals (see Methods and previous AMAZE paper), it is informative to have the individual activity plots of krill as a gut check on the group data.

The manuscript will be useful to the field as it provides an elegant example of looking for biological rhythms in a marine planktonic organism and disentangling the exogenous response from the endogenous one. Furthermore, as high-latitude environments change, understanding how important organisms like krill have the potential to respond will become increasingly important. This work provides a solid behavioral dataset to complement the earlier molecular data suggestive of a circadian clock in this species.

Reviewer #1 (Public review):

Summary:

This manuscript by Kaya et al. studies the effect of food consumption on hippocampal sharp wave ripples (SWRs) in mice. The authors use multiple foods and forms of food delivery to show that the frequency and power of SWRs increase following food intake, and that this effect depends on the caloric content of food. The authors also studied the effects of administration of various food-intake-related hormones on SWRs during sleep, demonstrating that ghrelin negatively affects SWR rate and power, but not GLP-1, insulin, or leptin. Finally, the authors use fiber photometry to show that GABAergic neurons in the lateral hypothalamus, increase activity during a SWR event.

Strengths:

The experiments in this study seem to be well performed, and the data are well presented, visually. The data support the main conclusions of the manuscript that food intake enhances hippocampal SWRs. Taken together, this study is likely to be impactful to the study of the impact of feeding on sleep behavior, as well as the phenomena of hippocampal SWRs in metabolism.

Weaknesses:

None

Reviewer #2 (Public review):

This manuscript addresses an important question which has not yet been solved in the field, what is the contribution of different gamma oscillatory inputs to the development of "theta sequences" in the hippocampal CA1 region. Theta sequences have received much attention due to their proposed roles in encoding short-term behavioral predictions, mediating synaptic plasticity, and guiding flexible decision-making. Gamma oscillations in CA1 offer a readout of different inputs to this region and have been proposed to synchronize neuronal assemblies and modulate spike timing and temporal coding. However, the interactions between these two important phenomena have not been sufficiently investigated. The authors conducted place cell and local field potential (LFP) recordings in the CA1 region of rats running on a circular track. They then analyzed the phase locking of place cell spikes to slow and fast gamma rhythms, the evolution of theta sequences during behavior and the interaction between these two phenomena. They found that place cell with the strongest modulation by fast gamma oscillations were the most important contributors to the early development of theta sequences and that they also displayed a faster form of phase precession within slow gamma cycles nested with theta.

Comments on revisions:

Several important shortcomings were noted in the original manuscript. These have been addressed in this revised version with the addition of multiple new analysis, controls and clarifications. The revised manuscript has been significantly improved and its conclusions are adequately supported by the results presented.

Reviewer #1 (Public review):

Summary:

This very interesting manuscript proposes a general mechanism for how activating signaling proteins respond to species specific signals arising from a variety of stresses. In brief, the authors propose that the activating signal alters the structure by a universal allosteric mechanism.

Strengths:

The unitary mechanism proposed is appealing and testable. The propose that the allosteric module consists of crossed alpha-helical linkers with similar architecture and that their attached regulatory domains connect to phosphatases or other molecules through coiled-coli domains, such that the signal is transduced via rigidifying the alpha helices, permitting downstream enzymatic activity. The authors present genetic and structural prediction data in favor of the model for the system they are studying, and stronger structural data in other systems.

Weaknesses:

I thank the authors for making significant revisions that addressed almost all of my concerns. I hope that the authors will consider addressing my last concern, which is that the title is inappropriate. However, I do not believe that this should hold up the publication of the ms.

"A General Mechanism for Initiating the General Stress Response in Bacteria" is misleading because it suggests a broadly applicable, universal mechanism across all bacterial species, whereas the study primarily focuses on Bacillus subtilis and its RsbU phosphatase activation. While the authors propose that the mechanism may extend to other bacteria, the evidence is largely based on structural modeling rather than direct experimental validation across multiple phyla. Additionally, the phrase "General Stress Response" might imply that the paper broadly explains stress response regulation, but it specifically examines the activation of RsbU by RsbT, which is just one really small part of the broader GSR network. The redundancy in "A General Mechanism for the General Stress Response" could also create an impression of an oversimplified, universal model when stress responses are often species- and context-specific. Furthermore, the study builds upon existing knowledge of partner-switching mechanisms rather than introducing an entirely new concept, making the claim of a general mechanism overstated and misleading for the field.

Title options could be "A Conserved Activation Mechanism for the General Stress Response Phosphatase in Bacteria", "Coiled-Coil Linker-Mediated Activation of a General Stress Response Phosphatase", all of which more accurately reflect the study's scope and findings.

Reviewer #1 (Public review):

Summary:

Kimura et al performed a saturation mutagenesis study of CDKN2A to assess functionality of all possible missense variants and compare them to previously identified pathogenic variants. They also compared their assay result with those from in silico predictors.

Strengths:

CDKN2A is an important gene that modulate cell cycle and apoptosis, therefore it is critical to accurately assess functionality of missense variants. Overall, the paper reads well and touches upon major discoveries in a logical manner.

Weaknesses:

The paper lacks proper details for experiments and basic data, leaving the results less convincing. Analyses are superficial and does not provide variant-level resolution.

Comments on revisions

The manuscript was improved during the revision process.

Reviewer #3 (Public review):

Summary:

The authors investigate the effect of high concentrations of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in a yeast deletion strain lacking the detoxification enzyme. Transcriptomic analyses as global read out reveals that a large range of cellular functions across all compartments are affected (transcriptomic changes affect 1/3 of all genes). The authors provide additional analyses, which indicate that mitochondrial protein import is affected.

Strengths:

Global analyses (transcriptomic and functional genomics approach) to obtain an overview of changes upon yeast treatment with high doses of t-2-hex.

Weaknesses:

The use of high concentrations of t-2-hex in combination with a deletion of the detoxifying enzyme Hfd1 limits the possibility to identify physiological relevant changes. For the follow-up analysis, the authors focus on mitochondrial proteins and describe an impairment of mitochondrial protein biogenesis, but the underlying molecular modification resulting in the observed impairment is not yet known.

Reviewer #1 (Public review):

Summary:

The authors have developed self-amplifying RNAs (saRNAs) encoding additional genes to suppress dsRNA-related inflammatory responses and cytokine release. Their results demonstrate that saRNA constructs encoding anti-inflammatory genes effectively reduce cytotoxicity and cytokine production, enhancing the potential of saRNAs. This work is significant for advancing saRNA therapeutics by mitigating unintended immune activation.

Strengths:

This study successfully demonstrates the concept of enhancing saRNA applications by encoding immune-suppressive genes. A key challenge for saRNA-based therapeutics, particularly for non-vaccine applications, is the innate immune response triggered by dsRNA recognition. By leveraging viral protein properties to suppress immunity, the authors provide a novel strategy to overcome this limitation. The study presents a well-designed approach with potential implications for improving saRNA stability and minimizing inflammatory side effects.

Weaknesses:

(1) Impact on Cellular Translation:

The authors demonstrate that modified saRNAs with additional components enhance transgene expression by inhibiting dsRNA-sensing pathways. However, it is unclear whether these modifications influence global cellular translation beyond the expression of GFP and mScarlet-3 (which are encoded by the saRNA itself). Conducting a polysome profiling analysis or a puromycin labeling assay would clarify whether the modified saRNAs alter overall translation efficiency. This additional data would strengthen the conclusions regarding the specificity of dsRNA-sensing inhibition.

(2) Stability and Replication Efficiency of Long saRNA Constructs:

The saRNA constructs used in this study exceed 16 kb, making them more fragile and challenging to handle. Assessing their mRNA integrity and quality would be crucial to ensure their robustness.<br /> Furthermore, the replicative capacity of the designed saRNAs should be confirmed. Since Figure 4 shows lower inflammatory cytokine production when encoding srIkBα and srIkBα-Smad7-SOCS1, it is important to determine whether this effect is due to reduced immune activation or impaired replication. Providing data on replication efficiency and expression levels of the encoded anti-inflammatory proteins would help rule out the possibility that reduced cytokine production is a consequence of lower replication.

(3) Comparative Data with Native saRNA:

Including native saRNA controls in Figures 5-7 would allow for a clearer assessment of the impact of additional genes on cytokine production. This comparison would help distinguish the effect of the encoded suppressor proteins from other potential factors.

(4) In vivo Validation and Safety Considerations:

Have the authors considered evaluating the in vivo potential of these saRNA constructs? Conducting animal studies would provide stronger evidence for their therapeutic applicability. If in vivo experiments have not been performed, discussing potential challenges - such as saRNA persistence, biodistribution, and possible secondary effects-would be valuable.

(5) Immune Response to Viral Proteins:

Since the inhibitors of dsRNA-sensing proteins (E3, NSs, and L*) are viral proteins, they would be expected to induce an immune response. Analyzing these effects in vivo would add insight into the applicability of this approach.

(6) Streamlining the Discussion Section:

The discussion is quite lengthy. To improve readability, some content - such as the rationale for gene selection-could be moved to the Results section. Additionally, the descriptions of Figure 3 should be consolidated into a single section under a broader heading for improved coherence.

Reviewer #1 (Public review):

Summary:

The authors present a novel usage of fluorescence lifetime imaging microscopy (FLIM) to measure NAD(P)H autofluorescence in the Drosophila brain, as a proxy for cellular metabolic/redox states. This new method relies on the fact that both NADH and NADPH are autofluorescent, with a different excitation lifetime depending on whether they are free (indicating glycolysis) or protein-bound (indicating oxidative phosphorylation). The authors successfully use this method in Drosophila to measure changes in metabolic activity across different areas of the fly brain, with a particular focus on the main center for associative memory: the mushroom body.

Strengths:

The authors have made a commendable effort to explain the technical aspects of the method in accessible language. This clarity will benefit both non-experts seeking to understand the methodology and researchers interested in applying FLIM to Drosophila in other contexts.

Weaknesses:

(1) Despite being statistically significant, the learning-induced change in f-free in α/β Kenyon cells is minimal (a decrease from 0.76 to 0.73, with a high variability). The authors should provide justification for why they believe this small effect represents a meaningful shift in neuronal metabolic state.

(2) The lack of experiments examining the effects of long-term memory (after spaced or massed conditioning) seems like a missed opportunity. Such experiments could likely reveal more drastic changes in the metabolic profiles of KCs, as a consequence of memory consolidation processes.

(3) The discussion is mostly just a summary of the findings. It would be useful if the authors could discuss potential future applications of their method and new research questions that it could help address.

Joint Public Review:

The manuscript describes the role of mmp21, a metallopeptidase, in left-right patterning. MMP21 has been implicated in genetic studies of patients with heterotaxy and the authors add an additional case. However, a molecular mechanism for Htx/LR patterning defects is not clear although one previous study implicated Notch signaling. The authors find that mmp21 does indeed cause LR patterning defects in Xenopus consistent with work in mice and zebrafish without affecting cilia motility. Importantly, the authors extend this work to place mmp21 in the LR pathway between dand5 (in the nodal cascade) and the cilia-driven sensation of flow. With RNA overexpression studies, the authors show MMP21 can induce Nodal signaling bilaterally suggesting it is an activator of the pathway, potentially through regulation of dand5 asymmetry. The authors also show that the role of MMP21 is upstream of another matrix metalloprotease CIROP which is tethered to the plasma membrane and possibly the cilium. They propose that mmp21, which is secreted, may represent a morphogen that is asymmetrically distributed along the LR axis due to cilia-driven flow and sensed by sensory cilia in the LRO.

The authors attempt to address a highly controversial subject in the LR patterning field, that is, the debate between Nodal Vesicular Particles (NVP, ie morphogens) being driven by cilia to activate signaling on the left and the Two Cilia model which posits that mechanosensation of fluid flow and not morphogens drive asymmetric organogenesis.

The model they propose is that mmp21 is secreted in the center of the LRO. LRO cilia generate leftward flow driving mmp21 to the left where sensory cilia at the LRO margin detect the mmp21 via cirop and suppress dand5, leading to activation of Nodal and Pitx2 expression.

First and foremost, the authors need to consider alternative models in the discussion and acknowledge the strengths and weaknesses of their work. All three reviewers felt that their conclusion that mmp21 is a morphogen is premature and that other models could also fit their data which needs to be discussed. The authors need to soften the conclusion that other models have been excluded.

Reviewer #1 (Public review):

Summary:

The authors demonstrate that two human preproprotein human mutations in the BMP4 gene cause a defect in proprotein cleavage and BMP4 mature ligand formation, leading to hypomorphic phenotypes in mouse knock-in alleles and in Xenopus embryo assays.

Strengths:

They provide compelling biochemical and in vivo analyses supporting their conclusions, showing the reduced processing of the proprotein and concomitant reduced mature BMP4 ligand protein from impressively mouse embryonic lysates. They perform excellent analysis of the embryo and post-natal phenotypes demonstrating the hypomorphic nature of these alleles. Interesting phenotypic differences between the S91C and E93G mutants are shown with excellent hypotheses for the differences. Their results support that BMP4 heterodimers act predominantly throughout embryogenesis whereas BMP4 homodimers play essential roles at later developmental stages.

Weaknesses:

In the revision the authors have appropriately addressed the previous minor weaknesses.

Reviewer #2 (Public review):

Summary:

In this paper, the authors first examined lens phenotypes in mice with Le-Cre-mediated knockdown (KD) of all the four FGFR (FGFR1-4), and found that pERK signals, Jag1 and foxe3 expression are absent or drastically reduced, indicating that FGF signaling is essential for lens induction. Next, the authors examined lens phenotypes of FGFR1/2-KD mice and found that lens fiber differentiation is compromised, and that proliferative activity and cell survival are also compromised in lens epithelium. Interestingly, Kras activation rescues defects in lens growth and lens fiber differentiation in FGFR1/2-KD mice, indicating that Ras activation is a key step for lens development, downstream of FGF signaling. Next, the authors examined the role of Frs2, Shp2 and Grb2 in FGF signaling for lens development. They confirmed that lens fiber differentiation is compromised in FGFR1/3-KD mice combined with Frs2-dysfunctional FGFR2 mutants, which is similar to lens phenotypes of Grb2-KD mice. However, lens defects are milder in mice with Shp2YF/YF and Shp2CS mutant alleles, indicating that involvement of Shp2 is limited for the Grb2 recruitment for lens fiber differentiation. Lastly, the authors showed new evidence on the possibility that another adapter protein, Shc1, promotes Grb2 recruitment independent of Frs2/Shp2-mediated Grb2 recruitment.

Strengths:

Overall, the manuscript provides valuable data on how FGFR activation leads to Ras activation through the adapter platform of Frs2/Shp2/Grb2, which advances our understanding on complex modification of FGF signaling pathway. The authors applied a genetic approach using mice, whose methods and results are valid to support the conclusion. The discussion also well summarizes the significance of their findings.

Weaknesses:

The authors found that the new adaptor protein Shc1 is involved in Grb2 recruitment in response to FGF receptor activation. However, the main data on Shc1 are only histological sections and statistical evaluation of lens size. Cellular-level evidence on Shc1 makes the authors' conclusion more convincing.

Comments on latest version:

In the 2nd revised version of the manuscript, the authors responded to my recommendation to show the number of biological replicates for Prox1 and αA-crystallin (Fig. 1F) and conductedstatistical analysis for pmTOR, and pS6 (Supplementary figure 1B).

The authors also explained why the animals are no longer available for the additional experiments that I requested. I may understand the situation, but hope that the authors will investigate the cellular-level evidence on Shc1 in more detail and report it maybe as another paper in future.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

The main concern (noted by all reviewers) is the interpretation of the evoked activity in visual trials. In the revised manuscript, the authors have not provided much data to disentangle movement related activity from sensory related activity. The only new data is on the visual response dynamics in supplementary figure 2, which is unconvincing both in terms of visual response latency and response dynamics. Therefore, the response of the authors has been insufficient to prove the visual nature of the evoked responses.

In this supplemental figure 2 the same example neuron as in the original manuscript is shown again as well as the average z-scored visual response. First, the visual response latency is inconsistent with literature. The first evoked activity in mouse V1 (!) is routinely reported around 50 ms (for example, 45 ms in Niell Stryker 2008, 52 ms, Schnabel et al. 2018, 54 ms in Oude Lohuis et al. 2024). According to the authors the potential route of crossmodal modulation of AC can occur through either corticocortical connections (which will impose further polysynaptic delays - monosynaptic projection from dLGN or V1 incredibly sparse), or through pulvinar (but pulvinar visual responses are much later (they find 170 vs 80 ms in dLGN, Roth et al. 2019) as expected from a higher order thalamic nucleus). One can also critique the estimation of the response latency which depends on the signal strength (visual response is smaller) and thus choice of threshold. With a different arbitrary threshold one would come to different conclusions.

Second, the temporal response dynamics to visual input are the same as the auditory response. It can be observed that if the data were normalized by the max response the dynamics are very similar, with the response back to near baseline levels at 100 ms post stimulus. I am not aware of publications that have observed response dynamics that are similar between A and V stimuli, nor such short-lasting visual response. In the visual system, mean activity typically drops again around 150-200ms.<br /> With the nature of the observed activity unclear, careful interpretation is warranted about audiovisual interactions in auditory cortex.

Reviewer #2 (Public review):

Summary:

In this work, the authors manage to optimize a simple and rapid protocol using SEC followed by DGCU to isolate sEVs with adequate purity and yield from small volumes of plasma. Isolated fractions containing sEVs using SEC, DGCU, SEC-DGCU and DGCU-SEC are compared in terms of their yield, purity surface protein profile and RNA content. Although the combined use of these methodologies has already been evaluated in previous works, the authors manage to adapt them for the use of small volumes of plasma, which allows working in 1.5 mL tubes and reducing the centrifugation time to 2 hours.<br /> The authors finally find that although both the SEC-DGCU and DGCU-SEC combinations achieve isolates with high purity, the SEC-DGCU combination results in higher yields.<br /> This work provides an interesting tool for the rapid obtention of sEVs with sufficient yield and purity for detailed characterization which could be very useful in research and clinical therapy.

Strengths:

The work is well written and organized.<br /> The authors clearly state the problem they want to address, that is, optimizing a method that allows sEV to be isolated from small volumes of plasma.<br /> Although these methodologies have been tested in previous works, the authors manage to isolate sEVs of high purity and good performance through a simple and fast methodology.<br /> The characteristics of all isolated fractions are exhaustively analyzed through various state-of-the-art methodologies.<br /> They present a good interpretation of the results obtained through the methodologies used.

Weaknesses:

Although this work focuses on comparing different techniques and their combinations to find an optimal option, the authors could strengthen their analysis by using statistical methods that reliably show the differences between the explored techniques.

Comments on revisions:

Although superiority of the proposed method was demonstrated by other techniques, it is always advisable to calculate the differences between different methodologies through different statistical methods, whenever possible, to strengthen the obtained results.