Reviewer #3 (Public review):

Summary:

Tanaka and colleagues addressed the role of the C-C chemokine receptor 4 (CCR4) in early atherosclerotic plaque development using ApoE-deficient mice on a standard chow diet as a model. Because several CD4+ T cell subsets express CCR4, they examined whether CCR4-deficiency alters the immune response mediated by CD4+ T cells. By histological analysis of aortic lesions, they demonstrated that the absence of CCR4 promoted the development of early atherosclerosis, with heightened inflammation linked to increased macrophages and pro-inflammatory CD4+ T cells, along with reduced collagen content. Flow cytometry and mRNA expression analysis for identifying CD4+ T cell subsets showed that CCR4 deficiency promoted higher proliferation of pro-inflammatory effector CD4+ T cells in peripheral lymphoid tissues and accumulation of Th1 cells in the atherosclerotic lesions. Interestingly, the increased pro-inflammatory CD4+ T cell response occurred despite the expansion of T CD4+ Foxp3+ regulatory cells (Tregs), found in higher numbers in lymphoid tissues of CCR4-deficient mice, suggesting that CCR4 deficiency interfered with Treg's regulatory actions. In addition, CCR4 deficiency induced an augmented Th1/Treg ratio in the aortic lesions. The CCR4-mediated mechanisms underlying the control of early inflammation and atherosclerosis development were not completely elucidated. In vitro studies suggest that CCR4 expression in Tregs plays a role in controlling DC activation and, in turn, the extent of CD4+T cell activation and proliferation. Dependence on CCR4 expression for Treg migration to the atherosclerotic aorta was not proved. The findings contrast with earlier studies in a murine model of advanced atherosclerosis, where CCR4 deficiency did not alter the development of the aortic lesions. The authors included a thoughtful discussion about hypothetical mechanisms explaining these contrasting results, including putative differences in the role played by the CCL17/CCL22-CCR4 axis along the stages of atherosclerosis development in this murine model.

Major strengths:

• Demonstration of CCR4 deficiency's impact on early atherosclerosis. CCR4 deficiency effects on the early atherosclerosis development in the Apoe-/-mice model were demonstrated by a quantitative analysis of the lesion area, inflammatory cell content and the expression profile of several pro- and anti-inflammatory markers.<br /> • Analysis of the T CD4+ response in various lymphoid tissues (peripheral and para-aortic lymph nodes and spleen) and the atherosclerotic aorta during the early phase of atherosclerosis in the Apoe-/-mice model. This analysis, combining flow cytometry and mRNA expression, showed that CCR4 deficiency enhanced T CD4+ cell activation, favouring the amplification of the typical biased Th1-mediated inflammatory response observed in the lymphoid tissues of hypercholesterolemic mice.<br /> • Treg transference experiments. Transference of Treg from Apoe-/- or Ccr4-/- Apoe-/- mice to Apoe-/- mice under a standard chow diet was useful for addressing the relevance of CCR4 expression on Tregs for the atheroprotective effect of this regulatory T cell subset during early atherosclerosis.

Major weaknesses:

• The effect of CCR4 deficiency on the Th1/Th17 balance was not evaluated. Although the role of Th17 cells in atherosclerosis remains controversial, RORγt+ cells constituted, on average, more than 10% of the effector TCD45+CD3+CD4+ lymphocytes in the aorta of Apoe-/- mice (Fig 4H). Changes in the Th1/Th17 balance in lymphoid tissues and aortic lesions may influence the type and functional properties of inflammatory cells recruited to the atherosclerotic aorta.

• Lack of in vivo evidence for Treg suppressive effects on DC activation. The proposed CCR4 requirement for the Treg suppressive activity on DC activation is supported by in vitro co-culture assays, in which CCR4-deficiency partially reverted Treg regulatory actions. Higher expression of CD86, a DC activation marker, was found in spleen DCs from Ccr4-/- Apoe-/- mice compared to Apoe-/- mice (Supplementary Fig 5), which would be worth commenting on and discussing.

• Methodological limitations. Controls in flow cytometry analysis were suboptimal (no viability and doublets were checked) which may have introduced artefacts, especially when measuring less-represented cell populations within complex samples. In addition, assessing Treg migration to the aorta in atherosclerotic mice faced methodological limitations that hindered statistical comparisons between Tregs from Apoe-/- and Ccr4-/- Apoe-/- mice, leading to inconclusive results. The dependence on CCR4 expression for Treg migration to the atherosclerotic aorta was not established.

• Treg transference experiments did not allow the detection of a reduction in the aortic lesion area by transferred CCR4 expressing Tregs (comparison between saline and Apoe-/- Tregs groups). Using Apoe-/- mice as recipients, the CCR4-dependent protective effect of Tregs was mostly evidenced by analysis of aortic inflammation, which was valuable. When using Ccr4-/- Apoe-/- mice as recipients, analysis of aortic inflammation was not mentioned.

Study limitations:

This investigation has some limitations. Current tools for single-cell characterization have revealed the phenotypic heterogeneity and dynamics of aortic leukocytes, including T cells, which are among the principal aortic leukocytes found in mouse and human atherosclerotic lesions (doi:10.1161/CIRCRESAHA.117.312513). The flow cytometry analysis applied in this study cannot distinguish the generation of particular phenotypes within T CD4+ subsets, including putative phenotypes of no-suppressive T cells expressing low levels of Foxp3, as seems could occur in other chronic inflammatory disorders (doi: 10.1038/nm.3432; doi: 10.1172/JCI79014). Limitations due to the use of a complete CCR4 knockout mouse and putative differences in CCR4-mediated mechanisms along atherosclerosis stages and in human atherosclerosis were commented on by the authors in the discussion.

Global Impact:

This work opens the way for a deeper analysis of the contribution of CCR4 and its ligands to the activation and differentiation of T CD4+ lymphocytes during atherosclerosis development, with these lymphocytes being fundamental players in the generation of pro-atherogenic and anti-atherogenic immune responses. Differences in the mechanisms mediated by the CCL17/CCL22-CCR4 axis among early and advanced atherosclerosis highlight the complex landscape to examine and validate in human samples and the need to achieve a deep knowledge for identifying genuine and safe targets capable of promoting protective anti-atherogenic immune responses.

Reviewer #1 (Public review):

Summary:

In this manuscript, Liu et al have tried to dissect the neural and molecular mechanisms that C. elegans use to avoid digestion of harmful bacterial food. Liu et al show that C. elegans use the ON-OFF state of AWC olfactory neurons to regulate the digestion of harmful gram-positive bacteria S. saprophyticus (SS). The authors show that when C. elegans are fed on SS food, AWC neurons switch to OFF fate which prevents digestion of S. saprophyticus and this helps C. elegans avoid these harmful bacteria. Using genetic and transcriptional analysis as well as making use of previously published findings, Liu et al implicate the p38 MAPK pathway (in particular, NSY-1, the C. elegans homolog of MAPKKK ASK1) and insulin signaling in this process.

Strengths:

The authors have used multiple approaches to test the hypothesis that they present in this manuscript.

Weaknesses:

Overall, I am not convinced that the authors have provided sufficient evidence to support the various components of their hypothesis. While they present data that loosely align with their hypothesis, they fail to consider alternative explanations and do not use rigorous approaches to strengthen their overall hypothesis. The selective picking of genes from the RNA sequencing data and forcing the data to fit the proposed hypothesis based on previously published findings, without exploring other approaches, indicates a lack of thoroughness and rigor. These critical shortcomings significantly diminish enthusiasm for the manuscript in its totality. In my opinion, this is the biggest weakness in this manuscript.

Reviewer #2 (Public review):

Summary:

Using C. elegans as a model, the authors present an interesting story demonstrating a new regulatory connection between olfactory neurons and the digestive system. Mechanistically, they identified key factors (NSY-1, STR-130 et.al) in neurons, as well as critical 'signaling factors' (INS-23, DAF-2) that bridge different cells/tissues to execute the digestive shutdown induced by poor-quality food (Staphylococcus saprophyticus, SS).

Strengths:

The conclusions of this manuscript are mostly well supported by the experimental results shown.

Weaknesses:

Several issues could be addressed and clarified to strengthen their conclusions.

(1) The word "olfactory" should be carefully used and checked in this manuscript. Although AWCs are classic olfactory neurons in C. elegans, no data in this manuscript supports the idea that olfactory signals from SS drive the responses in the digestive system. To validate that it is truly olfaction, the authors may want to check the responses of worms (e.g. AWC, digestive shutdown, INS-23 expression) to odors from SS.

(2) In line 113, what does "once the digestive system is activated" mean? The authors need to provide a clearer statement about 'digestive activation' and 'digestive shutdown'.

(3) No control data on OP50. This would affect the conclusions generated from Figures 2A, 2B, 2D, 3B, 3C, 3G, 4D-G, 5D-E, 6B-D.

(4) Do the authors know which factors are released from AWC neurons to drive the digestive shutdown?

Reviewer #3 (Public review):

Summary:

The study explores a molecular mechanism by which C. elegans detects low-quality food through neuron-digestive crosstalk, offering new insights into food quality control systems. Liu and colleagues demonstrated that NSY-1, expressed in AWC neurons, is a key regulator for sensing Staphylococcus saprophyticus (SS), inducing avoidance behavior and shutting down the digestive system via intestinal BCF-1. They further revealed that INS-23, an insulin peptide, interacts with the DAF-2 receptor in the gut to modulate SS digestion. The study uncovers a food quality control system connecting neural and intestinal responses, enabling C. elegans to adapt to environmental challenges.

Strengths:

The study employs a genetic screening approach to identify nsy-1 as a critical regulator in detecting food quality and initiating adaptive responses in C. elegans. The use of RNA-seq analysis is particularly noteworthy, as it reveals distinct regulatory pathways involved in food sensing (Figure 4) and digestion of Staphylococcus saprophyticus (Figure 5). The strategic application of both positive and negative data mining enhances the depth of analysis. Importantly, the discovery that C. elegans halts digestion in response to harmful food and employs avoidance behavior highlights a physiological adaptation mechanism.

Weaknesses:

Major points:

(1) While NSY-1 positively regulates str-130 expression in AWC neurons and is critical for SS avoidance and survival, the authors should examine whether similar phenotypes are observed in str-130 mutants.

(2) NSY-1 promotes the AWC-OFF state through str-130, inhibiting SS digestion. The authors should investigate whether STR-130 in AWC neurons regulates bcf-1 expression levels in the intestine.

(3) The current results rely on str-2 expression levels to indicate the AWC state. Ablating AWC neurons and testing the effects on digestion would provide stronger evidence for their role in digestive regulation.

(4) The claim that NSY-1 inhibits INS-23 and that INS-23 interacts with DAF-2 to regulate bcf-1 expression (Line 339-340) requires further validation. Neuron-specific disruption of INS-23 and gut-specific rescue of DAF-2 should be tested.

(5) Figure Reference Errors: Lines 296-297 mention Figure 6E, which does not exist in the main text. This appears to refer to Figure 5E, which has not been described.

Reviewer #1 (Public review):

Summary:

In their paper, Tutunji et al aim to investigate the dynamic effects of stress on activity of different brain networks (salience network, executive network, and default mode network). Crucially they differentiate between rapid (<1 h) and late (>1) effects of stress. Lastly, they connect acute changes in brain activity with inter-individual differences in stress reactivity in real-life assessed using EMA.

They first show the expected dynamics in stress-induced brain activity with a transient increase in salience network activity and a decrease in default mode network activity although in contrast to expectations, this did not disappear in the late phase. Notably, the increase in salience network activity was associated with a 'resilience index' derived from EMA that captures whether an individual responds with more or less reduction in positive effect than expected based on the number of above average stress events.

Linking acute stress to long-term affective stress reactivity is a crucial step to better understand how adaptive or maladaptive stress responses play out in the long term and how they might be related to mental health problems.

Strengths:

The link of the acute stress response to stress reactivity in daily life is highly relevant and a major strength of the paper. Moreover, the design of the EMA component assessing a week with low stress and one with high stress (exam week) in all participants and thus including a naturalistic manipulation enables a quantification of stress reactivity that captures 'real life'.

The authors do not only quantify the magnitude of the acute stress response but take into account an early as well as late response to disentangle the dynamic nature of the stress response. In that way, it is possible to establish which parts of the stress response are relevant for the affective response.

In addition to reporting changes in network activation, the authors also report behavioral outcomes of the tasks which is crucial to evaluate the meaning and relevance of the neural outcomes.

Weaknesses:

Although the authors assess multiple physiological outcomes to the stress task, only the cortisol response is analyzed with regard to its association with the stress-induced changes in network activity. Considering that it is mainly the salience network that shows an increase and this in the early phase that is characterized by the noradrenaline and not so much the cortisol response, an association with a marker of the NA response would be interesting.

To evaluate the association of the acute stress response with stress reactivity in real life more conclusively it would be interesting to see whether and how the affective response to the acute stress is related to stress reactivity in real life.

In the introduction, the authors hypothesize that all networks show distinct activation patterns during the stress response and expect all of them to be associated with the stress reactivity during EMA. However, no correction for multiple comparisons across the many tests (each network at two phases) is reported.

All stress-induced changes in activity are assessed by using other tasks since it is not trivial to measure changes in activation of specific regions without comparing different conditions of a task. Nonetheless, with the chosen approach it is not completely clear whether stress only modulates brain responses to other tasks or changes activation within those networks independently of any other tasks. Moreover, one of the tasks did not elicit the expected activation contrast and it is unclear whether this affects stress-effects.

Some of the less central results that are discussed in the paper such as the association of the real-life stress reactivity measure with neuroticism, the sex-effect of the cortisol response or the mediation and moderation models of the stress-induced changes in network activity and performance in the tasks seem slightly overinterpreted considering that they are either not quite significant or not hypothesized and thus it is not clear why for example once a mediation and in another outcome a moderation model was chosen.

Reviewer #2 (Public review):

Summary:

This study aimed to investigate changes in neural responses over time after acute stress and their association with real-life stress. To this end, functional MRI data was collected from 3 tasks (Oddball, 2-back, Associative retrieval) early and late following stress and control conditions. Emotional ratings during a stressful week before an exam and a non-stressful week without an exam were used to index real-world stress. In total, data from 70 individuals were used for the analyses in the paper. Results showed increased oddball related activation early after stress whereas activation to the associative retrieval was reduced across early and late trials following stress compared with control. Brain activation during the oddball task after stress contrasted against control correlated with the index used to measure stress in the real-world. This is a very ambitious study and the findings that stress has opposite effects on the oddball and the associative retrieval tasks is new. However, I am not convinced that brain responses are correlated with real-world stress from the results presented in the paper. I also have several other concerns listed below.

Strengths:

The study uses a unique design based on hypothesis firmly grounded in theories of stress related brain function. Large amounts of data are collected for all of the 70 participants included in the analyses and the hypotheses tested using paired tests have strong statistical power. Data collection methods are sound aiming to reduce stress induced by being in the scanner environment for the first time and reducing variation in cortisol due to circadian rhythm.

Weaknesses:

An important argument in the paper is that neural responses associated with stress in the lab correspond to stress in real life. This conclusion is based on a single correlation analysis. This is weak evidence because the correlation is based on 70 individuals and may be driven by outliers. In fact, the correlation between the difference in stress-related SN activation (Stress-Control) and real life stress residual is likely to be driven by outliers. In fig 5b, there are 3 persons with SN values of around 2, which is twice as much as the fourth highest value. There is also 1 person with a Real life stress residual of -3 or -4, which is three to four times as much as the person with the second lowest value. These 4 outliers should be removed before calculating the correlation coefficient. Also, no power analysis is presented in the paper showing what effect size is needed for significant results given a sample size of 70.

It is not clear why the activation maps from the tasks performed in the scanner are referred to as the SN, ECN, and DMN. They are discussed as if they were resting state networks. They are however not resting state networks because they are the results of contrasting two task conditions to each other and not the results from correlating BOLD time-series data from different regions within subjects. Even though masks corresponding to SN, ECN, and DMN are used to calculate means of all voxels, I think these contrasts should be referred to as the tasks that were used to evoke them. It becomes misleading to call them networks which usually refers to nodes and edges in fMRI studies. The first scan was a resting state scan, but these data are not presented in the paper.

Introduction<br /> In the introduction it is said that there are genomically driven effects of cortisol 1 to 2 hours after stress. This is repeated in the discussion: "[the late stress phase] is thought to be dominated by genomically driven effects of glucocorticoids". (There is no reference to this statement however.) This idea, that gene expression should only be regulated by corticosteroids following stress seems unrealistic. The increase in cortisol was only around 60% from baseline in the current study which seems to be similar to other studies. This means that the baseline cortisol level is far from zero. Therefore, effects of cortisol on gene expression must occur all the time and be tightly regulated by circadian clocks. To propose that genomically driven effects of cortisol only exist 1 to 2 hours following stress is therefore too simplistic.

In the last paragraph, it says that n=83. However, the final sample consists of 70 people. Correct this number.

Methods<br /> The EMA data analysis is difficult to understand. Why are the residuals used instead of means for example? I could not understand how the residual values used in the analysis should be interpreted from the way this section was written. Therefore, I cannot judge whether the index is valid or reliable. Using mean values is more common than using residuals when investigating individual differences in stress responses. The use of residuals needs justification and clarification. The results from an analysis using mean values should also be reported.

How was AUCi calculated? What software was used to calculate AUCi?

How was the mediation analysis performed? The only information I found was: "We additionally ran separate models with an interaction term modelled for neural activity in the targeted ROI's to examine the relationship between task performance and neural responses, with random slopes and intercepts also modelled for ROI activity." This is not how mediation analyses are done conventionally. It is common to use structural equation modelling or a series of regression analyses. What is meant by separate models? Was a reduced model compared to a full model with an interaction term? In this case, this is not a mediation analysis. I think the term moderation is better to describe this analysis.

Reviewer #3 (Public review):

This is a very interesting study that aims to examine the effect of stress induction across about two hours on physiological, behavioral, and neural measures in several brain areas. This aim is of importance for the study of stress response and recovery and their neural bases. There are several strengths to the design, including a within-subject design, adequate sample size, and multiple levels of assessment (including lab-based and real-life), and the authors should really be commended for that. The results indicate an acute cortisol response following stress induction, although HR data show that the manipulation may have been effective only among those who did the stress scan first. Behaviorally, stress induction resulted in effects on one of the tasks. Neurally, temporal changes in response were observed in what is referred to as SN and DMN networks, and associations with real-life stress were evident for SN during early stress response. Together, evidence emerged for some temporal changes in stress response on neural function and its associations with behavior and real-life stress response as indicated by self-report EMA.

These findings, both positive and null, provide important insight to the field, and the authors should be praised for that. At the same time, it is important to emphasize that some aspects or findings complicate interpretation and limit the extent of inference, that many places in the manuscript could benefit from clarification, and that more discussion should be given to the null findings.

All in all, given the importance of the questions and the strengths of the design, this study could provide a major contribution to future research. But, to accurately and optimally guide research, it is important to accurately describe and interpret both what was tested and found, and what was not found. Some more specific points are noted below, where improvements could be made to facilitate extraction of insight by the reader, and thus increase the impact of the study on the field.

Reviewer #1 (Public review):

Summary:

This is an important and interesting study that uses the split-GFP approach. Localization of receptors and correlating them to function is important in understanding the circuit basis of behavior.

Strengths:

The split-GFP approach allows visualization of subcellular enrichment of dopamine receptors in the plasma membrane of GAL4-expressing neurons allowing for high level of specificity.

The authors resolve the presynaptic localization of DopR1 and Dop2R, in "giant" Drosophila neurons differentiated from cytokinesis-arrested neuroblasts in culture as its not clear in the lobes and calyx.

Starvation induced opposite responses of dopamine receptor expression in the PPL1 and PAM DANs provides key insights into models of appetitive learning.<br /> Starvation induced increase in D2R allows for increased negative feedback that the authors test in D2R knockout flies where appetitive memory is diminished.<br /> This dual autoreceptor system is an attractive model for how amplitude and kinetics of dopamine release can be fine tunes and controlled depending on the cellular function and this paper presents a good methodology to do it and a good system where dynamics of dopamine release can be tested at the level of behavior.

Weaknesses:

Key weaknesses have been resolved: 

1) Receptor expression is consistent between time of the day and the authors picked two time points. The authors mention that the states of animals could affect LI (e.g. feeding state and anesthesia for sorting, see methods) were kept constant. These data and discussion are helpful. <br /> 2) Giant fiber system is argued to be a great model and authors have added additional references. However I am not very deeply familiar with these references or the giant fiber system so I am not completely clear but the argument seems reasonable. <br /> 3) The revised manuscript, shows data in the γ KCs (Figure 4C, Figure 5 - figure supplement 1) in addition to α/β KCs, so it appears there is consistency between lobes. <br /> 4) The new data for Dop1R1 and Dop2R in MBON-γ1pedc>αβ helps with thinking about dopamine receptor co-localization and it would be a herculean talk to do this for all the regions but still keeps room open for different scenarios. 

The papers discussion has been expanded to account for different possibilities which will help the readers get a more complete picture. I appreciate the review efforts and detailed response to reviewer comments.

Reviewer #2 (Public review):

Summary:

Hiramatsu et al. investigated how cognate neurotransmitter receptors with antagonizing downstream effects localize within neurons when co-expressed. They focus on mapping the dopaminergic Dop1R1 and Dop2R receptors, corresponding to the mammalian D1- and D2-like dopamine receptors, which have opposing effects on intracellular cAMP levels, in neurons of the Drosophila mushroom body (MB). To visualize specific receptors in single neuron types within the crowded MB neuropil, the authors use existing dopamine receptor alleles tagged with 7 copies of split GFP to target the reconstitution of GFP tags specifically in the neurons of interest, providing a readout of receptor localization.

The authors demonstrate that both Dop1R1 and Dop2R are enriched, to differing degrees, in the axonal compartments of Kenyon cells cholinergic presynaptic inputs and in different dopamine neurons (DANs) that project axons to the MB. Co-localization studies of dopamine receptors with the presynaptic marker Brp suggest that Dop1R1, and to a greater extent Dop2R, localize near release sites. This pattern in DANs suggests Dop1R1 and Dop2R serve as dual-feedback autoreceptors. Finally, they provide evidence that the balance of Dop1R1 and Dop2R in the axons of two different DAN populations is differentially modulated by starvation, which plays a role in regulating appetitive behaviors.

In their revised manuscript, Hiramatsu et al. revisited the localization and functional integrity of Dop1R1 and Dop2R within the Drosophila mushroom body. This revision strengthens their claims with new high-resolution imaging data and additional behavioral assays, supporting the functional integrity of 7X split GFP-tagged receptors and their distinct localizations within neural circuits.

The revised manuscript by Hiramatsu et al. demonstrates substantial improvements in experimental design and data presentation, effectively addressing concerns raised during the initial review. The addition of advanced imaging techniques and behavioral data confirms the functionality of tagged receptors, while providing deeper insights into their spatial and functional dynamics within neural circuits modulating responses to environmental changes like starvation. This study makes an important contribution to neuroscience, enhancing our understanding of dopamine receptor distribution in circuits underlying learning and memory.

Strengths:

The authors use reconstitution of GFP fluorescence of split GFP tags integrated at the endogenous locus of dopamine receptors, providing a precise readout of receptor localization. This method preserves endogenous transcriptional and post-transcriptional regulation, a critical feature for protein localization studies.

The choice of the Drosophila mushroom body as a model system is excellent, as it is well-studied, its connectome is carefully reconstructed, and its role in behaviors and associative memory enables linking receptor localization patterns to circuit function and behavior. This approach allows the authors to demonstrate that antagonizing dopamine receptors can act as autoreceptors within the axonal compartments of MB-innervating DANs. Moreover, they show that starvation differentially modulates the balance of these receptors in distinct DANs, highlighting the role of this regulation in circuit function and behavior.

The incorporation of higher-resolution Airyscan microscopy and functional assays in the revision provide evidence that tagged receptors retain functionality and predominantly localize at presynaptic sites within Kenyon cells and DANs. These findings support the dual autoreceptor feedback model proposed.

Weaknesses:

While the revision significantly strengthens the manuscript, the absence of specific antibodies against these receptors remains a limitation. This is understandable given the challenges of generating antibodies against such proteins. However, the use of more direct validation methods, such as specific antibodies (if available), and employing higher-resolution techniques like expansion microscopy, could further validate and enhance the robustness of the findings.

Reviewer #1 (Public review):

Shen et al. conducted three experiments to study the cortical tracking of the natural rhythms involved in biological motion (BM), and whether these involve audiovisual integration (AVI). They presented participants with visual (dot) motion and/or the sound of a walking person. They found that EEG activity tracks the step rhythm, as well as the gait (2-step cycle) rhythm. The gait rhythm specifically is tracked superadditively (power for A+V condition is higher than the sum of the A-only and V-only condition, Experiments 1a/b), which is independent of the specific step frequency (Experiment 1b). Furthermore, audiovisual integration during tracking of gait was specific to BM, as it was absent (that is, the audiovisual congruency effect) when the walking dot motion was vertically inverted (Experiment 2). Finally, the study shows that an individual's autistic traits are negatively correlated with the BM-AVI congruency effect.

Reviewer #2 (Public review):

The authors evaluate spectral changes in electroencephalography (EEG) data as a function of the congruency of audio and visual information associated with biological motion (BM) or non-biological motion. The results show supra-additive power gains in the neural response to gait dynamics, with trials in which audio and visual information was presented simultaneously producing higher average amplitude than the combined average power for auditory and visual conditions alone. Further analyses suggest that such supra-additivity is specific to BM and emerges from temporoparietal areas. The authors also find that the BM-specific supra-additivity is negatively correlated with autism traits.

Reviewer #1 (Public review):

Summary:

This paper investigates the mechanism of axon growth directed by the conserved guidance cue UNC-6/Netrin. Experiments were designed to distinguish between alternative models in which UNC-6/Netrin functions as either a short range (haptotactic) cue or a diffusible (chemotactic) signal that steers axons to their final destinations. In each case, axonal growth cones execute ventrally directed outgrowth toward a proximal source of UNC-6/Netrin. This work concludes that UNC-6/Netrin functions as both a haptotactic and chemotactic cue to polarize the UNC-40/DCC receptor on the growth cone membrane facing the direction of growth. Ventrally directed axons initially contact a minor longitudinal nerve tract (vSLNC) at which UNC-6/Netrin appears to be concentrated before proceeding in the direction of the ventral nerve cord (VNC) from which UNC-6/Netrin is secreted. Time lapse imaging revealed that growth cones appear to pause at the vSLNC before actively extending ventrally directed filopodia that eventually contact the VNC. Growth cone contacts with the vSLNC were unstable in unc-6 mutants but were restored by expression of a membrane tethered UNC-6 in vSLNC neurons. In addition, expression of membrane tethered UNC-6/Netrin in the VNC was not sufficient to rescue initial ventral outgrowth in an unc-6 mutant. Finally, dual expression of membrane tethered UNC-6/Netrin in both vSLNC and VNC partially rescued the unc-6 mutant axon guidance defect, thus suggesting that diffusible UNC-6 is also required. This work is important because it potentially resolves the controversial question of how UNC-6/Netrin directs axon guidance by proposing a model in which both of the competing mechanisms, e.g., haptotaxis vs chemotaxis, are successively employed. The impact of this work is bolstered by its use of powerful imaging and genetic methods to test models of UNC-6/Netrin function in vivo thereby obviating potential artifacts arising from in vitro analysis.

Strengths:

A strength of this approach is the adoption of the model organism C. elegans to exploit its ready accessibility to live cell imaging and powerful methods for genetic analysis.

Weaknesses:

In the revised version of this manuscript, the authors have redressed the weaknesses highlighted in my review of the original paper.

Reviewer #2 (Public review):

Nichols et al studied the role of axon guidance molecules and their receptors and how these work as long-range and/or local cues, using in-vivo time-lapse imaging in C. elegans. They found that the Netrin axon guidance system, work in different modes when acting as a long-range (chemotaxis) cue vs local cue (haptotaxis). As an initial context, they take advantage of the postembryonic-born neuron, PDE, to understand how its axon grows and then is guided into its target. They found that this process occurs in various discrete steps, during which the growth cone migrates and pauses at specific structures, such as the vSLNC. The role of the UNC-6/Netrin and UNC-40/DCC axon guidance ligand-receptor pair was then looked at in terms of its requirement for (1) initial axon outgrowth direction, (2) stabilization at the intermediate target, (3) directional branching from the sublateral region or (4) ventral growth from intermediate target to the VNC. They found that each step is disrupted in the unc-6/Netrin and unc-40/DCC mutants and observed how the localization of these proteins changed during the process of axon guidance in wild type and mutant contexts. These observations were further supported by analysis of a mutant important for the regulation of Netrin signaling, the E3 ubiquitin ligase madd-2/Trim9/Trim67. Remarkably, the authors identified that this mutant affected axonal adhesion and stabilization, but not directional growth. Using membrane-tethered UNC-6 to specific localities, they then found this to be a consequence of the availability of UNC-6 at specific localities within the axon growth path. Altogether, this data and in-vivo analysis provide compelling evidence of the mechanistic foundation of Netrin-mediated axon guidance and how it works step by step.

The conclusions are well-supported, with both imaging and quantification of each step of axon guidance and localization of UNC-6 and UNC-40. Using a different type of neuron to validate their findings further supports their conclusions and strengthens their model. They also probe the role of the axon guidance ligand-receptor pair SLT-1/Slit and SAX-3/ROBO in this process and find it to work in parallel to UNC-6. This work sets up the stage for future analysis of other axon guidance molecules or regulators using time-lapse in-vivo imaging to better understand their role as long-range and/or local cues.

Reviewer #3 (Public review):

Summary:

This manuscript from Nichols, Lee, and Shen tackles an important question of how unc6/netrin promotes axon guidance: i.e. haptotaxis vs chemotaxis. This has recently been a large topic of investigation and discussion in the axon guidance field. Using live cell imaging of unc6/netrin and unc40/DCC in several neurons that extend axons ventrally during development, as well as TM localized mutants of Unc6, they suggest that unc6 promotes first haptotaxis of the emerging growth cone followed by chemotaxis of the growth cone. This is timely, as a recent preprint from the Lundquist group, using a similar strategy to make only a TM anchored unc6 similarly found that this could rescue only the haptotaxis like growth of the PDE neuron, but not the second phase of growth. However, their conclusions were quite different based on the overexpression of unc6 everywhere rescuing the second phase, and thus they conclude that a gradient is not present.

Strengths:

As this has been quite a controversy in both the invertebrate and vertebrate fields, one strength of this paper is that they use a unc6-neon green to demonstrate unc6 localization, and show localization. Further, they provide localisation of the transmembrane tether version of netrin, showing its restriction to nerve cords.

Reviewer #1 (Public review):

Summary:

This interesting and well-written article by Tuckowski et al. summarizes work connecting the flavin-containing monooxygenase FMO-4 with increased lifespan through a mechanism involving calcium signaling in the nematode Caenorhabditis elegans.

The authors have previously studied another fmo in worms, FMO-2, prompting them to look at additional members of this family of proteins. They show that fmo-4 is up in dietary restricted worms and necessary for the increased lifespan of these animals as well as of rsks-1 (s6 kinase) knockdown animals. They then show that overexpression of fmo-4 is sufficient to significantly increase lifespan, as well as healthspan and paraquat resistance. Further, they demonstrate that overexpression of fmo-4 solely in the hypodermis of the animal recapitulates the entire effect of fmo-4 OE.

In terms of interactions between fmo-2 and fmo-4 they show that fmo-4 is necessary for the previously reported effects of fmo-2 on lifespan, while the effects of fmo-4 do not depend on fmo-2.

Next the authors use RNASeq to compare fmo-4 OE animals to wild type. Their analyses suggested the possibility that FMO-4 was modulating calcium signaling, and through additional experiments specifically identified the calcium signaling genes crt-1, itr-1, and mcu-1 as important fmo-4 interactors in this context. As previously published work has shown that loss of the worm transcription factor atf-6 can extend lifespan through crt-1, itr-1 and mcu-1, the authors asked about interactions between fmo-4 and atf-6. They showed that fmo-4 is necessary for both lifespan extension and increased paraquat resistance upon RNAi knockdown of atf-6.

Overall this clearly written manuscript summarizes interesting and novel findings of great interest in the biology of aging, and suggests promising avenues for future work in this area.

Strengths:

This paper contains a large number of careful, well executed and analysed experiments in support of its existing conclusions, and which also point toward significant future directions for this work. In addition it is clear and very well written.

Weaknesses:

Within the scope of the current work there are no major weaknesses. That said, the authors themselves note pressing questions beyond the scope of this study that remain unanswered. For instance, the mechanistic nature of the interactions between FMO-4 and the other players in this story, for example in terms of direct protein-protein interactions, is not at all understood yet. Further, powerful tools such as GCaMP expressing animals will enable a much more detailed understanding of what exactly is happening to calcium levels, and where and when it is happening, in these animals.

Reviewer #2 (Public review):

Summary:

Members of a conserved family of flavin-containing monooxygenases (FMOs) play key roles in lifespan extension induced by diet restriction and hypoxia. In C. elegans, fmo-2 has received the majority of attention, but there are multiple fmo genes in both worms and mammals, and how overlapping or distinct the functional roles of these paralogs are remains unclear. Here Tuckowski et al. identify that a new family member, fmo-4, is also a positive modulator of lifespan. Based on differential requirements of fmo-2 and fmo-4 in stress resistance and lifespan extension paradigms, however, the authors conclude that fmo-4 acts through mechanisms that are distinct from fmo-2. Ultimately, the authors place fmo-2 genetically within a pathway involving atf-6, calreticulin, the IP3 receptor, and mitochondrial calcium uniporter, which was previously shown to link ER calcium homeostasis to mitochondrial homeostasis and longevity. The authors thus achieve their overarching aim to reveal that different FMO family members regulate stress resistance and lifespan through distinct mechanisms. Furthermore, because the known enzymatic activity of FMOs involves oxygenating xenobiotic and endogenous metabolites, these findings highlight a potential new link between redox/metabolic homeostasis and ER-mitochondrial calcium signaling.

Strengths:

The authors demonstrate links between multiple conserved life-extending signaling pathways and fmo-4, expanding both the significance and mechanistic diversity of FMO-family genes in aging and stress biology.

The authors use genetics to discover an interesting and unanticipated new link between FMOs and calcium pathways known to regulate lifespan.

The genetic epistasis patterns for lifespan and stress resistance phenotypes are generally clean and compelling.

Weaknesses:

The authors achieve a necessary and valuable first step with regard to linking FMO-4 to calcium homeostasis, but the mechanisms involved remain preliminary at this stage. Specifically, the genetic interactions between fmo-4 and conserved mediators of calcium transport and signaling are convincing, but a putative molecular mechanism by which the activity of FMO-4 would alter subcellular calcium transport remains unclear and potentially indirect. The authors effectively highlight this gap as a key pursuit for subsequent studies.

The authors have shown that carbachol and EDTA produce the expected effects on a cytosolic calcium reporter in neurons, supporting the utility of the chemical approach in general, but validating that carbachol, EDTA and fmo-4 itself have an impact on calcium in the tissues and subcellular compartments relevant to the lifespan phenotypes would still be valuable in supporting the overall model. Notably, however, the hypodermal-specific role of FMO-4 suggests potential cell non-autonomous regulation of lifespan, such that this pathway may ultimately involve complex inter-cellular signaling that would necessitate substantially more time and effort.

Employing mutants and more sophisticated genetic tools for modulating calcium transport or signaling (in addition to RNAi) would strengthen key conclusions and/or help to elucidate tissue- or age-specific aspects of the proposed mechanism.

Reviewer #3 (Public review):

Summary:

The authors assessed the potential involvement of fmo-4 in a diverse set of longevity interventions, showing that this gene is required for DR and S6 kinase knockdown related lifespan extension. Using comprehensive epistasis experiments they find this gene to be a required downstream player in the longevity and stress resistance provided by fmo-2 overexpression. They further showed that fmo-4 ubiquitous overexpression is sufficient to provide longevity and paraquat (mitochondrial) stress resistance, and that overexpression specifically in the hypodermis is sufficient to recapitulate most of these effects.

Interestingly, they find that fmo-4 overexpression sensitizes worms to thapsigargin during development, an effect that they link with a potential dysregulation in calcium signalling. They go on to show that fmo-4 expression is sensitive to drugs that both increase or decrease calcium levels, and these drugs differentially affect lifespan of fmo-4 mutants compared to wild-type worms. Similarly, knockdown of genes involved in calcium binding and signalling also differentially affect lifespan and paraquat resistance of fmo-4 mutants.

Finally, they suggest that atf-6 limits the expression of fmo-4, and that fmo-4 is also acting downstream of benefits produced by atf-6 knockdown.

Strengths:

• comprehensive lifespans experiments: clear placement of fmo-4 within established longevity interventions.<br /> • clear distinction in functions and epistatic interactions between fmo-2 and fmo-4 which lays a strong foundation for a longevity pathway regulated by this enzyme family.

Weaknesses:

• no obvious transcriptomic evidence supporting a link between fmo-4 and calcium signalling: either for knockout worms or fmo-4 overexpressing strains.<br /> • no direct measures of alterations in calcium flux, signalling or binding that strongly support a connection with fmo-4.<br /> • no measures of mitochondrial morphology or activity that strongly support a connection with fmo-4.<br /> • lack of a complete model that places fmo-4 function downstream of DR and mTOR signalling (first Results section), fmo-2 (second Results section) and at the same time explains connection with calcium signalling.

Comments on revisions:

The authors have addressed and fixed all the private comments we had made. In terms of the public comments, I think nothing has changed in terms of strengths and weaknesses. They have multiple independent results (drugs, RNAi and transcriptomics) that suggest a connection between fmo-4 and calcium regulation, but there is no strong evidence for what this connection is. The work still lacks direct measures of calcium, ER or mitochondrial function in relation to fmo-4 (which they acknowledge in the discussion). The first four sections strongly place fmo-4 within established longevity interventions, but their model doesn't explain how calcium regulation would fit into these.

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 of 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 #2 (Public review):

Summary:

This manuscript provides experimental evidence on circadian behavioural cycles in Antarctic krill. The krill were obtained directly from krill fishing vessels and the experiments were carried out on board using an advanced incubation device capable of recording activity levels over a number of days. A number of different experiments were carried out where krill were first exposed to simulated light:dark (L:D) regimes for some days followed by continuous darkness (DD). These were carried out on krill collected during late autumn and late summer. A further set of experiments was performed on krill across three different seasons (summer, autumn, winter), where incubations were all DD conditions. Activity was measured as the frequency by which an infrared beam close to the top of the incubation tube was broken over unit time. Results showed that patterns of increased and decreased activity that appeared synchronised to the LD cycle persisted during the DD period. This was interpreted as evidence of the operation of an internal (endogenous) clock. The amplitude of the behavioural cycles decreased with time in DD, which further suggests that this clock is relatively weak. The authors argued that the existence of a weak endogenous clock is an adaptation to life at high latitudes since allowing the clock to be modulated by external (exogenous) factors is an advantage when there is a high degree of seasonality. This hypothesis is further supported by seasonal DD experiments which showed that the periodicity of high and low activity levels differed between seasons.

Strengths

Although there has been a lot of field observations of various circadian type behaviour in Antarctic krill, relatively few experimental studies have been published considering this behaviour in terms of circadian patterns of activity. Krill are not a model organism and obtaining them and incubating them in suitable conditions are both difficult undertakings. Furthermore, there is a need to consider what their natural circadian rhythms are without the overinfluence of laboratory-induced artefacts. For this reason alone, the setup of the present study is ideal to consider this aspect of krill biology. Furthermore, the equipment developed for measuring levels of activity is well-designed and likely to minimise artefacts.

Weaknesses

I have little criticism of the rationale for carrying out this work, nor of the experimental design. Nevertheless, the manuscript would benefit from a clearer explanation of the experimental design, particularly aimed at readers not familiar with research into circadian rhythms. Furthermore, I have a more fundamental question about the relationship between levels of activity and DVM on which I will expand below. Finally, it was unclear how the observational results made here related to the molecular aspects considered in the Discussion.

(1) Explanation of experimental design - I acknowledge that the format of this particular journal insists that the Results are the first section that follows the Introduction. This nevertheless presents a problem for the reader since many of the concepts and terms that would generally be in the Methods are yet to be explained to the reader. Hence, right from the start of the Results section, the reader is thrown into the detail of what happened during the LD-DD experiments without being fully aware of why this type of experiment was carried out in the first place. Even after reading the Methods, further explanation would have been helpful. Circadian cycle type research of this sort often entrains organisms to certain light cycles and then takes the light away to see if the cycle continues in complete darkness, but this critical piece of knowledge does not come until much later (e.g. lines 369-372) leaving the reader guessing until this point why the authors took the approach they did. I would suggest the following (1) that more effort is made in the Introduction to explain the exact LD/DD protocols adopted (2) that a schematic figure is placed early on in the manuscript where the protocol is explained including some logical flow charts of e.g. if behavioural cycle continues in DD then internal clock exists versus if cycle does not continue in DD, the exogenous cues dominate - followed by - major decrease in cyclic amplitude = weak clock versus minor decrease = strong clock and so on

(2) Activity vs kinesis - in this study, we are shown data that (i) krill have a circadian cycle - incubation experiments; (ii) that krill swarms display DVM in this region - echosounder data (although see my later point). My question here is regarding the relationship between what is being measured by the incubation experiments and the in situ swarm behaviour observations. The incubation experiments are essentially measuring the propensity of krill to swim upwards since it logs the number of times an individual (or group) break a beam towards the top of the incubation tube. I argue that krill may be still highly active in the rest of the tube but just do not swim close to the surface, so this approach may not be a good measure of "activity". Otherwise, I suggest a more correct term of what is being measured is the level of "upward kinesis". As the authors themselves note, krill are negatively buoyant and must always be active to remain pelagic. What changes over the day-night cycle is whether they decide to expend that activity on swimming upwards, downwards or remaining at the same depth. Explaining the pattern as upward kinesis then also explains by swarms move upwards during the night. Just being more active at night may not necessarily result in them swimming upwards.

(3) Molecular relevance - Although I am interested in molecular clock aspects behind these circadian rhythms, it was not made clear how the results of the present study allow any further insight into this. In lines 282 to 284, the findings of the study by Biscontin et al (2017) are discussed with regard to how TIM protein is degraded by light via the clock photreceptor CRYTOCHROME 1. This element of the Discussion would be a lot more relevant if the results of the present study were considered in terms of whether they supported or refuted this or any other molecular clock model. As it stands, this paragraph is purely background knowledge and a candidate for deletion in the interest of shortening the Discussion.

Other aspects<br /> (i) 'Bimodal swimming' was used in the Abstract and later in the text without the term being fully explained. I could interpret it to mean a number of things so some explanation is required before the term is introduced.<br /> (ii) Midnight sinking - I was struck by Figure 2b with regards to the dip in activity after the initial ascent, as well as the rise in activity predawn. Cushing (1951) Biol Rev 26: 158-192 describes the different phases of a DVM common to a number of marine organisms observed in situ where there is a period of midnight sinking following the initial dusk ascent and a dawn rise prior to dawn descent. Tarling et al (2002) observe midnight sinking pattern in Calanus finmarchicus and consider whether it is a response to feeding satiation or predation avoidance (i.e. exogenous factors). Evidence from the present study indicates that midnight sinking (and potential dawn rise) behaviour could alternatively be under endogenous control to a greater or lesser degree. This is something that should certainly be mentioned in the Discussion, possibly in place of the molecular discussion element mentioned above - possibly adding to the paragraph Lines 303-319.

(iii) Lines 200-207 - I struggled to follow this argument regarding Piccolin et al identifying a 12 h rhythm whereas the present study indicates a ~24 h rhythm. Is one contradicting the other - please make this clear.

(iv) Although I agree that the hydroacoustic data should be included and is generally supportive of the results, I think that two further aspects should be made clear for context (a) whether there was any groundtruthing that the acoustic marks were indeed krill and not potentially some other group know to perform DVM such as myctophids (b) how representative were these patterns - I have a sense that they were heavily selected to show only ones with prominent DVM as opposed to other parts of the dataset where such a pattern was less clear - I am aware of a lot of krill research where DVM is not such a clear pattern and it is disingenuous to provide these patterns as the definitive way in which krill behaves. I ask this be made clear to the reader (note also that there is a suggestion of midnight sinking in Fig 5b on 28/2).

Reviewer #1 (Public review):

Summary:

The authors' research group had previously demonstrated the release of large multivesicular body-like structures by human colorectal cancer cells. This manuscript expands on their findings, revealing that this phenomenon is not exclusive to colorectal cancer cells but is also observed in various other cell types, including different cultured cell lines, as well as cells in the mouse kidney and liver. Furthermore, the authors argue that these large multivesicular body-like structures originate from intracellular amphisomes, which they term "amphiectosomes." These amphiectosomes release their intraluminal vesicles (ILVs) through a "torn-bag mechanism." Finally, the authors demonstrate that the ILVs of amphiectosomes are either LC3B positive or CD63 positive. This distinction implicates that the ILVs either originate from amphisomes or multivesicular bodies, respectively.

Strengths:

The manuscript reports a potential origin of extracellular vesicle (EV) biogenesis. The reported observations are intriguing.

Weaknesses:

In their revised version, the authors have addressed the majority of my criticisms. I have no further concerns regarding this manuscript.

Reviewer #2 (Public review):

Summary:

authors had previously identified that a colorectal cancer cell line generates small extracellular vesicles (sEVs) via a mechanism where a larger intracellular compartment containing these sEVs is secreted from the surface of the cell and then tears to release its contents. Previous studies had suggested that intraluminal vesicles (ILVs) inside endosomal multivesicular bodies and amphisomes can be secreted by fusion of the compartment with the plasma membrane. The 'torn bag mechanism' considered in this manuscript is distinctly different, because it involves initial budding off of a plasma membrane-enclosed compartment (called the amphiectosome in this manuscript, or MV-lEV). The authors successfully set out to investigate whether this mechanism is common to many cell types and to determine some of the subcellular processes involved.

The strengths of the study are:

(1) The high-quality imaging approaches used, including live-cell imaging and EN, which seem to show good examples of the proposed mechanism.<br /> (2) They screen several cell lines for these structures, also search for similar structures in vivo, and show the tearing process by real-time imaging.<br /> (3) Regarding the intracellular mechanisms of ILV production, the authors also try to demonstrate the different stages of amphiectosome production and differently labelled ILVs using immuno-EM.

Several of the techniques employed are technically challenging to do well, and so these are critical strengths of the manuscript.

Overall, I think the authors have been successful in identifying amphiectosomes secreted from multiple cell lines and cells in vivo, and in demonstrating that the ILVs inside them have at least two origins (autophagosome membrane and late endosomal multivesicular body) based on the markers that they carry. Inevitably, it remains unclear how universal this mechanism is in vivo and its overall contribution to EV function.<br /> I think there could be a significant impact on the EV field and consequently on our understanding of cell-cell signalling based on these findings. It will flag the importance of investigating the release of amphiectosomes in other studies, especially as the molecular mechanisms involved in this type of 'ectosomal-style' release will be different from multivesicular compartment fusion to the plasma membrane and should be possible to be manipulated independently.<br /> In general, the EV field has struggled to link up analysis of the subcellular biology of sEV secretion and the biochemical/physical analysis of the sEVs themselves, so from that perspective, the manuscript provides a novel angle on this problem.

Reviewer #2 (Public review):

Summary:

Here Vogt et al., provide new insights into the need for sleep and the molecular and physiological response to sleep loss. The authors expand on their previously published work (Bjorness et al., 2020) and draw from recent advances in the field to propose a neuron-centric molecular model for the accumulation and resolution of sleep need and basis of restorative sleep function. While speculative, the proposed model successfully links important observations in the field and provides a framework to stimulate further research and advances on the molecular basis of sleep function. In my review, I highlight the important advances of this current work, the clear merits of the proposed model, and indicate areas of the model that can serve to stimulate further investigation.

Strengths: Reviewer comment on new data in Vogt et al., 2024<br /> Using classic slice electrophysiology, the authors conclude that wakefulness (sleep deprivation (SD)) drives a potentiation of excitatory glutamate synapses, mediated in large part by "un-silencing" of NMDAR-active synapses to AMPAR-active synapses. Using a modern single nuclear RNAseq approach the authors conclude that SD drives changes in gene expression primarily occurring in glutamatergic neurons. The two experiments combined highlight the accumulation and resolution of sleep need centered on the strength of excitatory synapses onto excitatory neurons. This view is entirely consistent with a large body of extant and emerging literature and provides important direction for future research.

Consistent with prior work, wakefulness/SD drives an LTP-type potentiation of excitatory synaptic strength on principle cortical neurons. It has been proposed that LTP associated with wake, leads to the accumulation of sleep need by increasing neuronal excitability, and by the "saturation" of LTP capacity. This saturation subsequently impairs the capacity for further ongoing learning. This new data provides a satisfying mechanism of this saturation phenomenon by introducing the concept of silent synapses. The new data show that in mice well rested, a substantial number of synapses are "silent", containing an NMDAR component but not AMPARs. Silent synapses provide a type of reservoir for learning in that activity can drive the un-silencing, increasing the number of functional synapses. SD depletes this reservoir of silent synapses to essentially zero, explaining how SD can exhaust learning capacity. Recovery sleep led to restoration of silent synapses, explaining how recovery sleep can renew learning capacity. In their prior work (Bjorness et al., 2020) this group showed that SD drives an increase in mEPSC frequency onto these same cortical neurons, but without a clear change in pre-synaptic release probability, implying a change in the number of functional synapses. This prediction is now born out in this new dataset.

The new snRNAseq dataset indicates the sleep need is primarily seen (at the transcriptional level) in excitatory neurons, consistent with a number of other studies. First, this conclusion is corroborated by two independent, contemporary snRNAseq analysis recently published in iScience 2024 doi: 10.1016/j.isci.2024.110752 and Neuroscience Research 2024 https://doi.org/10.1016/j.neures.2024.03.004. A recently published analysis on the effects of SD in drosophila imaged synapses in every brain region in a cell-type dependent manner (Weiss et al., PNAS 2024), concluding that SD drives brain wide increases in synaptic strength almost exclusively in excitatory neurons. Further, Kim et al., Nature 2022, heavily cited in this work, show that the newly described SIK3-HDAC4/5 pathway promotes sleep depth via excitatory neurons and not inhibitory neurons.

The new experiments provided in Fig1-3 are expertly conducted and presented. This reviewer has no comments of concern regarding the execution and conclusions of these experiments.

Reviewer comment on model in Vogt et al., 2024

To the view of this reviewer the new model proposed by Vogt et al., is an important contribution. The model is not definitively supported by new data, and in this regard should be viewed as a perspective, providing mechanistic links between recent molecular advances, while still leaving areas that need to be addressed in future work. New snRNAseq analysis indicates SD drives expression of synaptic shaping components (SSCs) consistent with the excitatory synapse as a major target for the restorative basis of sleep function. SD induced gene expression is also enriched for autism spectrum disorder (ASD) risk genes. As pointed out by the authors, sleep problems are commonly reported in ASD, but the emphasis has been on sleep amount. This new analysis highlights the need to understand the impact on sleep's functional output (synapses) to fully understand the role of sleep problems in ASD.

Importantly, SD induced gene expression in excitatory neurons overlap with genes regulated by the transcription factor MEF2C and HDAC4/5 (Fig. 4). In their prior work, the authors show loss of MEF2C in excitatory neurons abolished the SD transcriptional response and the functional recovery of synapses from SD by recovery sleep. Recent advances identified HDAC4/5 as major regulators of sleep depth and duration (in excitatory neurons) downstream of the recently identified sleep promoting kinase SIK3. In Zhou et al., and Kim et al., Nature 2022, both groups propose a model whereby "sleep-need" signals from the synapse activate SIK3, which phosphorylates HDAC4/5, driving cytoplasmic targeting, allowing for the de-repression and transcriptional activation of "sleep genes". Prior work shows that HDAC4/5 are repressors of MEF2C. Therefore, the "sleep genes" derepressed by HDAC4/5 may be the same genes activated in response to SD by MEF2C. The new model thereby extends the signaling of sleep need at synapses (through SIK3-HDAC4/5) to the functional output of synaptic recovery by expression of synaptic/sleep genes by MEF2C. The model thereby links aspects of expression of sleep need with the resolution of sleep need by mediating sleep function: synapse renormalization.

Weaknesses:

Areas for further investigation.<br /> In the discussion section Vogt et al., explore the links between excitatory synapse strength, arguably the major target of "sleep function", and NREM slow-wave activity (SWA), the most established marker of sleep need. SIK3-HDAC4/5 have major effects on the "depth" of sleep by regulation NREM-SWA. The effects of MEF2C loss of function on NREM SWA activity are less obvious, but clearly impact the recovery of glutamatergic synapses from SD. The authors point out how adenosine signaling is well established as a mediator of SWA, but the links with adenosine and glutamatergic strength are far from clear. The mechanistic links between SIK3/HDAC4/5, adenosine signaling, and MEF2C, are far from understood. Therefore, the molecular/mechanistic links between a synaptic basis of sleep need and resolution with NREM-SWA activity requires further investigation.

Additional work is also needed to understand the mechanistic links between SIK3-HDAC4/5 signaling and MEF2C activity. The authors point out that constitutively nuclear (cn) HDAC4/5 (acting as a repressor) will mimic MEF2C loss of function. This is reasonable, however, there are notable differences in the reported phenotypes of each. Notably, cnHDAC4/5 suppresses NREM amount and NREM SWA but had no effect on the NREM-SWA increase following SD (Zhou et al., Nature 2022). Loss of MEF2C in CaMKII neurons had no effect on NREM amount and suppressed the increase in NREM-SWA following SD (Bjorness et al., 2020). These instances indicate that cnHDAC4/5 and loss of MEF2C do not exactly match suggesting additional factors are relevant in these phenotypes. Likely HDAC4/5 have functionally important interactions with other transcription factors, and likewise for MEF2C, suggesting areas for future analysis.

One emerging theme may be that the SIK3-HDAC4/5 axis are major regulators of the sleep state, perhaps stabilizing the NREM state once the transition from wakefulness occurs. MEF2C is less involved in regulating sleep per se, and more involved in executing sleep function, by promoting the restorative synaptic modifications to resolve sleep need.

Finally, advances in the roles of the respective SIK3-HDAC4/5 and MEF2C pathways point towards transcription of "sleep genes", as clearly indicated in the model of Fig.4. Clearly more work is needed to understand how the expression of such genes ultimately lead to resolution of sleep need by functional changes at synapses. What are these sleep genes and how do they mechanistically resolve sleep need? Thus, the current work provides a mechanistic framework to stimulate further advances in understanding the molecular basis for sleep need and the restorative basis of sleep function.

Comments on revisions:

No further comments or concerns. I believe that the manuscript has been suitably revised, and the concerns raised by reviewers have been addressed. I am completely satisfied by the revisions and responses provided by the authors.

Reviewer #1 (Public review):

The study by Aguirre-Botero et al. shows the dynamics of 3D11 anti-CSP monoclonal antibody (mAb) mediated elimination of rodent malaria Plasmodium berghei (Pb) parasites in the liver. The authors show that the anti-CSP mAb could protect against intravenous (i.v.) Pb sporozoite challenge along with the cutaneous challenge, but requires higher concentration of antibody. Importantly, the study shows that the anti-CSP mAb not only affects sporozoite motility, sinusoidal extravasation, and cell invasion but also partially impairs the intracellular development inside the liver parenchyma, indicating a late effect of this antibody during liver stage development. While the study is interesting and conducted well, the only novel yet very important observation made in this manuscript is the effect of the anti-CSP mAb on liver stage development.

Comments on latest version:

No further comments.

Reviewer #1 (Public review):

The study by Aguirre-Botero et al. shows the dynamics of 3D11 anti-CSP monoclonal antibody (mAb) mediated elimination of rodent malaria Plasmodium berghei (Pb) parasites in the liver. The authors show that the anti-CSP mAb could protect against intravenous (i.v.) Pb sporozoite challenge along with the cutaneous challenge, but requires higher concentration of antibody. Importantly, the study shows that the anti-CSP mAb not only affects sporozoite motility, sinusoidal extravasation, and cell invasion but also partially impairs the intracellular development inside the liver parenchyma, indicating a late effect of this antibody during liver stage development. While the study is interesting and conducted well, the only novel yet very important observation made in this manuscript is the effect of the anti-CSP mAb on liver stage development.

Comments on latest version:

No further comments.

Reviewer #3 (Public review):

Summary:

Aguirre-Botero et al have studied the effect of a potent monoclonal antibody against the circumsporozoite protein, the major surface protein of the malaria sporozoite. This is an elegantly designed, performed, and analyzed study. They have efficiently delineated the mode of action of anti-CSP repeat mAb and confirmed previous in vitro work (not cited) that demonstrated the same intracellular effect.

Major comments from the previous round of review:

Line 51: The authors claim a correlation between high antibody levels and protection. However, they did not provide direct proof that these antibodies were responsible for protection, nor did they establish a cut-off level of anti-CSP antibodies that would distinguish between protected and unprotected individuals.

Line 326: The late intrahepatic effect of mAb against the CSP repeat has been previously reported (see Figure 2, Nudelman et al, J Immunol, 1989). The effect was shown to affect the transition from liver trophozoites to liver schizonts. This study should be cited and discussed.

Significance:

A well-done study that elucidates the mechanisms of a protective monoclonal antibody against malaria sporozoites. These data are important and will interest a large audience of researchers working in infectious diseases and immunology.

Comments on latest version:

With the addition of new experiments and proper addition of missing references and minor text correction, the manuscript has been improved.

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 the 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:

Details of experiments are missing in the text and figure legends. Additionally, the writing of the manuscript could be improved.

Reviewer #2 (Public review):

Summary:

Kaya et al uncover an intriguing relationship between hippocampal sharp wave-ripple production and peripheral hormone exposure, food intake, and lateral hypothalamic function. These findings significantly expand our understanding of hippocampal function beyond mnemonic processes and point a direction for promising future research.

Strengths:

Some of the relationships observed in this paper are highly significant. In particular, the inverse relationship between GLP1/Leptin and Insulin/Ghrelin are particularly compelling as this aligns well with opposing hormone functions on satiety.

Weaknesses: I would be curious if there were any measurable behavioral differences that occur with different hormone manipulations.

Reviewer #3 (Public review):

Summary:

The manuscript by Kaya et al. explores the effects of feeding on sharp wave-ripples (SWRs) in the hippocampus, which could reveal a better understanding of how metabolism is regulated by neural processes. Expanding on prior work that showed that SWRs trigger a decrease in peripheral glucose levels, the authors further tested the relationship between SWRs and meal consumption by recording LFPs from the dorsal CA1 region of the hippocampus before and after meal consumption. They found an increase in SWR magnitude during sleep after food intake, in both food restricted and ad libitum fed conditions. Using fiber photometry to detect GABAergic neuron activity in the lateral hypothalamus, they found increased activity locked to the onset of SWRs. They conclude that the animal's satiety state modulates the amplitude and rate of SWRs, and that SWRs modulate downstream circuits involved in regulating feeding. These experiments provide an important step forward in understanding how metabolism is regulated in the brain. However, currently, the paper lacks sufficient analyses to control for factors related to sleep quality and duration; adding these analyses would further support the claim that food intake itself, as opposed to sleep quality, is primarily responsible for changes in SWR activity. Adding this, along with some minor clarifications and edits, would lead to a compelling case for SWRs being modulated by a satiety state. The study will likely be of great interest in the field of learning and memory while carrying broader implications for understanding brain-body physiology.

Strengths:

The paper makes an innovative foray into the emerging field of brain-body research, asking how sharp wave-ripples are affected by metabolism and hunger. The authors use a variety of advanced techniques including LFP recordings and fiber photometry to answer this question. Additionally, they perform comprehensive and logical follow-up experiments to the initial food-restricted paradigm to account for deeper sleep following meal times and the difference between consumption of calories versus the experience of eating. These experiments lay the groundwork for future studies in this field, as the authors pose several follow-up questions regarding the role of metabolic hormones and downstream brain regions.

Weaknesses:

Major comments:

(1) The authors conclude that food intake regulates SWR power during sleep beyond the effect of food intake on sleep quality. Specifically, they made an attempt to control for the confounding effect of delta power on SWRs through a mediation analysis. However, a similar analysis is not presented for SWR rate. Moreover, this does not seem to be a sufficient control. One alternative way to address this confound would be to subsample the sleep data from the ad lib and food restricted conditions (or high calorie and low calorie, etc), to match the delta power in each condition. When periods of similar mean delta power (i.e. similar sleep quality) are matched between datasets, the authors can then determine if a significant effect on SWR amplitude and rate remains in the subsampled data.

(2) Relatedly, are the animals spending the same amount of time sleeping in the ad lib vs. food restricted conditions? The amount of time spent sleeping could affect the probability of entering certain stages of sleep and thus affect SWR properties. A recent paper (Giri et al., Nature, 2024) demonstrated that sleep deprivation can alter the magnitude and frequency of SWRs. Could the authors quantify sleep quantity and control for the amount of time spent sleeping by subsampling the data, similar to the suggestion above?

(3) Plot 5I only reports significance but does not clearly show the underlying quantification of LH GABAergic activity. Upon reading the methods for how this analysis was conducted, it would be informative to see a plot of the pre-SWR and post-SWR integral values used for the paired t-test whose p-values are currently shown. For example, these values could be displayed as individual points overlaid on a pair of box-and-whisker plots of the pre- and post-distribution within the session (perhaps for one example session per mouse with the p-value reported, to supplement a plot of the distribution of p-values across sessions and mice). If these data are non-normal, the authors should also use a non-parametric statistical test.

Minor comments:

(4) A brief explanation (perhaps in the discussion) of what each change in SWR property (magnitude, rate, duration) could indicate in the context of the hypothesis may be helpful in bridging the fields of metabolism and memory. For example, by describing the hypothesized mechanistic consequence of each change, could the authors speculate on why ripple rate may not increase in all the instances where ripple power increases after feeding? Why do the authors speculate that ripple duration does not increase, given that prior work (Fernandez-Ruiz et al. 2019) has shown that prolonged ripples support enhanced memory?

(5) The authors suggest that "SWRs could modulate peripheral metabolism" as a future implication of their work. However, the lack of clear effects from GLP-1, leptin and insulin complicates this interpretation. It might be informative for readers if the authors expanded their discussion of what specific role they speculate that SWRs could play in regulating metabolism, given these negative results.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors identified that<br /> (1) CDK4/6i treatment attenuates the growth of drug-resistant cells by prolongation of the G1 phase;<br /> (2) CDK4/6i treatment results in an ineffective Rb inactivation pathway and suppresses the growth of drug-resistant tumors;<br /> (3) Addition of endocrine therapy augments the efficacy of CDK4/6i maintenance;<br /> (4) Addition of CDK2i with CDK4/6 treatment as second-line treatment can suppress the growth of resistant cell;<br /> (5) The role of cyclin E as a key driver of resistance to CDK4/6 and CDK2 inhibition.

Strengths:

To prove their complicated proposal, the authors employed orchestration of several kinds of live cell markers, timed in situ hybridization, IF and Immunoblotting. The authors strongly recognize the resistance of CDK4/6 + ET therapy and demonstrated how to overcome it.

Weaknesses:

The authors need to underscore their proposed results from what is to be achieved by them and by other researchers.

Reviewer #2 (Public review):

Summary:

This study elucidated the mechanism underlying drug resistance induced by CDK4/6i as a single agent and proposed a novel and efficacious second-line therapeutic strategy. It highlighted the potential of combining CDK2i with CDK4/6i for the treatment of HR+/HER2- breast cancer.

Strengths:

The study demonstrated that CDK4/6 induces drug resistance by impairing Rb activation, which results in diminished E2F activity and a delay in G1 phase progression. It suggests that the synergistic use of CDK2i and CDK4/6i may represent a promising second-line treatment approach. Addressing critical clinical challenges, this study holds substantial practical implications.

Weaknesses:

(1) Drug-resistant cell lines: Was a drug concentration gradient treatment employed to establish drug-resistant cell lines? If affirmative, this methodology should be detailed in the materials and methods section.

(2) What rationale informed the selection of MCF-7 cells for the generation of CDK6 knockout cell lines? Supplementary Figure 3. A indicates that CDK6 expression levels in MCF-7 cells are not notably elevated.

(3) For each experiment, particularly those involving mice, the author must specify the number of individuals utilized and the number of replicates conducted, as detailed in the materials and methods section.

(4) Could this treatment approach be extended to triple-negative breast cancer?

Reviewer #3 (Public review):

Summary:

In their manuscript, Armand and colleagues investigate the potential of continuing CDK4/6 inhibitors or combining them with CDK2 inhibitors in the treatment of breast cancer that has developed resistance to initial therapy. Utilizing cellular and animal models, the research examines whether maintaining CDK4/6 inhibition or adding CDK2 inhibitors can effectively control tumor growth after resistance has set in. The key findings from the study indicate that the sustained use of CDK4/6 inhibitors can slow down the proliferation of cancer cells that have become resistant, and the combination of CDK2 inhibitors with CDK4/6 inhibitors can further enhance the suppression of tumor growth. Additionally, the study identifies that high levels of Cyclin E play a significant role in resistance to the combined therapy. These results suggest that continuing CDK4/6 inhibitors along with the strategic use of CDK2 inhibitors could be an effective strategy to overcome treatment resistance in hormone receptor-positive breast cancer.

Strengths:

(1) Continuous CDK4/6 Inhibitor Treatment Significantly Suppresses the Growth of Drug-Resistant HR+ Breast Cancer: The study demonstrates that the continued use of CDK4/6 inhibitors, even after disease progression, can significantly inhibit the growth of drug-resistant breast cancer.

(2) Potential of Combined Use of CDK2 Inhibitors with CDK4/6 Inhibitors: The research highlights the potential of combining CDK2 inhibitors with CDK4/6 inhibitors to effectively suppress CDK2 activity and overcome drug resistance.

(3) Discovery of Cyclin E Overexpression as a Key Driver: The study identifies overexpression of cyclin E as a key driver of resistance to the combination of CDK4/6 and CDK2 inhibitors, providing insights for future cancer treatments.

(4) Consistency of In Vitro and In Vivo Experimental Results: The study obtained supportive results from both in vitro cell experiments and in vivo tumor models, enhancing the reliability of the research.

(5) Validation with Multiple Cell Lines: The research utilized multiple HR+/HER2- breast cancer cell lines (such as MCF-7, T47D, CAMA-1) and triple-negative breast cancer cell lines (such as MDA-MB-231), validating the broad applicability of the results.

Weaknesses:

(1) The manuscript presents intriguing findings on the sustained use of CDK4/6 inhibitors and the potential incorporation of CDK2 inhibitors in breast cancer treatment. However, I would appreciate a more detailed discussion of how these findings could be translated into clinical practice, particularly regarding the management of patients with drug-resistant breast cancer.

(2) While the emergence of resistance is acknowledged, the manuscript could benefit from a deeper exploration of the molecular mechanisms underlying resistance development. A more thorough understanding of how CDK2 inhibitors may overcome this resistance would be valuable.

(3) The manuscript supports the continued use of CDK4/6 inhibitors, but it lacks a discussion on the long-term efficacy and safety of this approach. Additional studies or data to support the safety profile of prolonged CDK4/6 inhibitor use would strengthen the manuscript.

Reviewer #1 (Public review):

Summary:

The authors study the effect of the addition of synthetic amphiphile on the gating mechanisms of the mechano-sensitive channel MscL. They observe that the amphiphile reduces the membrane stretching and bending modulii, and increases the channel activation pressure. They then conclude that gating is sensitive to these two membrane parameters. This is explained by the effect of the amphiphile on the so-called membrane interfacial tension.

Strengths:

The major strength is that the authors found a way to tune the membrane's mechanical properties in a controlled manner, and find a progressive change of the suction pressure at which MscL gates. If analysed thoroughly, these results could give valuable information.

Weaknesses:

The weakness is the analysis and the discussion. I would like to have answers to some basic questions.

(1) The explanation of the phenomenon involves a difference between interfacial tension and tension, without the difference between these being precisely defined. In the caption of Figure 4, one can read "Under tension, the PEO groups adsorb to the bilayer, suggesting adsorption is a thermodynamically favorable process that lowers the interfacial tension." What does this mean? Under what tension is the interfacial tension lowered? The fact that the system's free energy could be lowered by putting it under mechanical tension would result in a thermodynamic unstable situation. Is this what the authors mean?

(2) From what I understand, a channel would feel the tension exerted by the membrane at its periphery, which is what I would call membrane tension. The fact that polymers may reorganise under membrane stretch to lower the system's free energy would certainly affect the membrane stretching modulus (as measured Figure 2E), but what the channel cares about is the tension (I would say). If the membrane is softer, a larger pipette pressure is required to reach the same level of tension, so it is not surprising that a given channel requires a larger activation pressure in softer membranes. To me, this doesn't mean that the channel feels the membrane stiffness, but rather that a given pressure leads to different tensions (which is what the channel feels) for different stiffnesses.

(3) In order to support the authors' claim, the micropipette suction pressure should be appropriately translated into a membrane tension. One would then see whether the gating tension is affected by the presence of amphiphiles. In the micropipette setup used here, one can derive a relationship between pressure and tension, that involves the shape of the membrane. This relationship is simple (tension=pressure difference times pipette radius divided by 2) only in the limit where the membrane tongue inside the pipette ends with a hemisphere of constant radius independent of the pressure, and the pipette radius is much smaller than the GUV radius. None of these conditions seem to hold in Figure 2C. On the other hand, the authors do report absolute values of tension in the y-axis of Figure 2D. It seems quite straightforward to plot the activation tension (rather than pressure) as a function of the amphiphile volume fraction in Figure 2B. This is what needs to be shown.

(4) The discussion needs to be improved. I could not find a convincing explanation of the role of interfacial tension in the discussion. The equation (p.14) distinguishes three contributions, which I understand to be (i) an elastic membrane deformation such as hydrophobic mismatch or other short-range effects, (ii) the protein conformation energy, and (iii) the work done by membrane tension. Apparently, the latter is where the effect is (which I agree with), but how this consideration leads to a gating energy difference (between lipid only and modified membrane) proportional to the interfacial tension is completely obscure (if not wrong).

(5) I am rather surprised at the very small values of stretching and bending modulii found under high-volume fraction. These quantities are obtained by fitting the stress-strain relationship (Figure 2D). Such a plot should be shown for all amphiphile volume fraction, so one can assess the quality of the fits.

Reviewer #2 (Public review):

Summary:

The manuscript describes how synthetic polymers, primarily poloxamers of different sizes, influence bacterial mechanosensitive channel MscL gating by modifying the interfacial tension of the membrane. The authors expressed MscL in U2OS cells and chemically blebbed the cells to derive giant plasma membrane vesicles (GPMVs) containing MscL G22S. They applied micropipette aspiration on GPMVs to obtain bending rigidity (kc) and area expansion modulus (kA) and used patch clamping to obtain activation pressure. They found a negative correlation between kc and kA with activation pressure and attributed the changes to activation pressure to the lowering of the interfacial tension in the presence of polymers. They carried out coarse-grain molecular dynamics simulations and showed that under tension the hydrophilic PEO group adsorbs to the bilayer more, thereby lowering the interfacial tension. Besides MscL, they showed similar results with TREK-1 activation. The conclusion that differences in interfacial tension are what drive the changes in activation pressure is based on using a thermodynamic model.

Strengths:

(1) Reveals that synthetic polymer that lowers bending rigidity and area expansion modulus increases activation pressure of mechanosensitive channel by lowering interfacial tension - this is an important finding.

(2) General data quality is high with detailed and thorough analysis. The use of both micropipette aspiration and patch clamp in the same study is noteworthy.

(3) Discussion on nanoplastics and their effect on membrane properties and therefore their impact on mechanosensitivity is interesting.

Weaknesses:

Interfacial tension is not experimentally measured. Given the main argument of this paper is that synthetic polymers reduce interfacial tension, which increases MS channel activation pressure, it would be prudent to show experimental measurements to bolster their analysis.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors set out to test the "force from lipids" mechanism of mechanosensitive channel gating, which posits that mechanical properties of the membrane are directly responsible for converting membrane tension into useful energy for channel gating. They employ amphiphilic polymers called poloxamers to alter membrane mechanical properties and relate those to the threshold of mechanical activation of the MscL channel of E.coli.

The authors heterologously express the channel, perform electrical recordings, and assess the mechanical properties of vesicles derived from the same membranes. This allows them to directly compare derived mechanical parameters to channel gating in the same environment.

They further repeat experiments in an eukaryotic mechano-channel and show that the same principles apply to gating in this very different protein, providing support for the force from lipids hypothesis.

Strengths:

In this work, characterization of the mechanical properties of the plasma membrane and electrical recordings of channel activity are carried out in membranes derived from the same cells. This is a nice contribution to these experiments since usually these two properties are measured in separate membranes with differing compositions. The experiments are of high quality and the data analysis and interpretation are careful.

Weaknesses:

It is not clear to this reviewer what the relationship is between the mechanical properties the authors measure, the membrane area expansion modulus, and bending rigidity, to what they call "interfacial tension".

Reviewer #1 (Public review):

Summary

In this manuscript, the authors generate an AAV-deliverable tool that generates action potentials in response to red light, but not blue light, when expressed in neurons. To do this, they screen some red light-excitatory/blue light-inhibitory opsin pairs to find ones that are spectrally and temporally matched. They first show that this works with Chrimson and GtACR2, however, they expand their search after finding that the tau-off (inactivation after light cessation) kinetics of these two opsins are not well-matched. They directly examine a small set of options based on a literature search and settle on a variant of red light-excitatory Chrimson and blue light-inhibitory ZipACR. To even more closely match the kinetics of this pair, the authors create a structure homology model of the ZipACR retinal binding pocket and use this to guide generation of a small mutant panel, leading to a more optimized ZipACR mutant. They then show that a bicistronically expressed fusion arrangement of these opsins, plus some functional peptides, can drive action potentials up to 20hz with red light and does not do so with blue light, in hippocampal cells transduced by AAV. They also show function in vivo, in a mouse, using a physiological readout. They conclude that their new tool may be useful for complex experimental designs requiring multiple optical channels for write-in/read-out.

The major advantage claimed by the authors over existing tools is the temporal time-locking of their inhibitory opsin - this is driven by the contrast between tau-off kinetics of their ZipACR variant compared to gtACR2, which is used by the leading competitor tool (BiPOLES).

Big thoughts<br /> While the authors were carefully thoughtful about the potential influence of temporal kinetics on the efficiency of a tool such as this one, there were no experiments conducted that make use of the unique properties of this molecular strategy (although the authors state that these experiments are now underway in their lab). They share some examples of how the tool could be useful in the discussion. Where do I think this could be useful?

First, experimental designs that require multiple optical channels of control. This appears to be aligned with the author's thoughts, as they state, correctly, that opsins utilizing retinal as a light-sensing chromophore are universally activated by blue light (the so-called 'blue shoulder'). Therefore, their tool may be useful for stimulating multiple populations using a blue excitatory opsin in neuron A and their tool for red excitation of neuron B - or, in the author's own words, "A potential solution to the problem of cross-talk...". In this manuscript, the authors provide state that there this is possible in theory and that there are no obvious reasons that it would not work, but do not present data that showcases their new tool for this purpose (e.g. Vierock, Johannes, et al. "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons." Nature communications 12.1 (2021): 4527. Figure 4f-I; 6). The same set-up could be imagined for green GECI (or equivalent) imaging of cells in the same volume that their tool is being used in - for instance, interleaving red stimulation light and blue imaging light, (perhaps) without the typical concern of imaging light bleed-through activating the opsin itself. I agree that it will likely work for multi-channel control, but only time will tell, at this point.

Second, for high-frequency temporal control over both excitation and inhibition in the same neuron. Red light turns the cell on, and blue light turns the cell off (see, for instance, Zhang, Feng, et al. "Multimodal fast optical interrogation of neural circuitry." Nature 446.7136 (2007): 633-639. Figure 2; Vierock as above, Figure 4a,b). Again, here the authors are long on theory ("The new system...can drive time-locked high-frequency action potentials in response to red pulses") and short on explicit data. While they do show that red light = excitation and blue light = inhibition, they neither show 1) all-optical on/off modulation of the same cell; nor 2) high-frequency inhibition or excitation (max stim rate of 20hz, which is the same as the BiPOLES paper used for their LC stimulation paradigm; Vierock, as above, Figure 7a-d). They did provide a response to this critique that data showing excitation and inhibition spread across multiple panels were largely collected from the same cells.

Despite these major shortcomings, the further development and characterization of tandem opsins, such as this one, is of interest to the community. There is on-going work by the BiPOLES team to create new iterations (e.g. Wahid, J., et al. "P-15 BiPOLES2 is a bidirectional optogenetic tool with a narrow activation spectrum and low red-light excitability." Clinical Neurophysiology 148 (2023): e16.). The authors have collected a substantial amount of additional data along the course of review and, even aside from the final tool, the overall data and approaches shown are useful.

Reviewer #1 (Public review):

Summary:

Audio et al. measured cerebral blood volume (CBV) across cortical areas and layers using high-resolution MRI with contrast agents in non-human primates. While the non-invasive CBV MRI methodology is often used to enhance fMRI sensitivity in NHPs, its application for baseline CBV measurement is rare due to the complexities of susceptibility contrast mechanisms. The authors determined the number of large vessels and the areal and laminar variations of CBV in NHP, and compared those with various other metrics.

Strengths:

Noninvasive mapping of relative cerebral blood volume is novel for non-human primates. A key finding was the observation of variations in CBV across regions; primary sensory cortices had high CBV, whereas other higher areas had low CBV. The measured CBV values correlated with previously reported neuronal and receptor densities.

Weaknesses:

A weakness of this manuscript is that the quantification of CBV with postprocessing approaches to remove susceptibility effects from pial and penetrating vessels is not fully validated, especially on a laminar scale. Further specific comments follow.

(1) Baseline CBV indices were determined using contrast agent-enhanced MRI (deltaR2*). Although this approach is suitable for areal comparisons, its application at a laminar scale poses challenges due to significant contributions from large vessels including pial vessels. The primary concern is whether large-vessel contributions can be removed from the measured deltaR2* through processing techniques.

(2) High-resolution MRI with a critical sampling frequency estimated from previous studies (Weber 2008, Zheng 1991) was performed to separate penetrating vessels. However, this approach is still insufficient to accurately identify the number of vessels due to the blooming effects of susceptibility and insufficient spatial resolution. The reported number of penetrating vessels is only applicable to the experimental and processing conditions used in this study, which cannot be generalized.

(3) Baseline R2* is sensitive to baseline R2, vascular volume, iron content, and susceptibility gradients. Additionally, it is sensitive to imaging parameters; higher spatial resolution tends to result in lower R2* values (closer to the R2 value). Thus, it is difficult to correlate baseline R2* with physiological parameters.

(4) CBV-weighted deltaR2* is correlated with various other metrics (cytoarchitectural parcellation, myelin/receptor density, cortical thickness, CO, cell-type specificity, etc.). While testing the correlation between deltaR2* and these other metrics may be acceptable as an exploratory analysis, it is challenging for readers to discern a causal relationship between them. A critical question is whether CBV-weighted deltaR2* can provide insights into other metrics in diseased or abnormal brain states.

Reviewer #2 (Public review):

Summary:

This manuscript presents a new approach for non-invasive, MRI-based, measurements of cerebral blood volume (CBV). Here, the authors use ferumoxytol, a high-contrast agent and apply specific sequences to infer CBV. The authors then move to statistically compare measured regional CBV with known distribution of different types of neurons, markers of metabolic load and others. While the presented methodology captures and estimated 30% of the vasculature, the authors corroborated previous findings regarding lack of vascular compartmentalization around functional neuronal units in the primary visual cortex.

Strengths:

Non invasive methodology geared to map vascular properties in vivo.

Implementation of a highly sensitive approach for measuring blood volume.

Ability to map vascular structural and functional vascular metrics to other types of published data.

Weaknesses:

The key issue here is the underlying assumption about the appropriate spatial sampling frequency needed to captures the architecture of the brain vasculature. Namely, ~7 penetrating vessels / mm2 as derived from Weber et al 2008 (Cer Cor). The cited work, begins by characterizing the spacing of penetrating arteries and ascending veins using vascular cast of 7 monkeys (Macaca mulatta, same as in the current paper). The ~7 penetrating vessels / mm2 is computed by dividing the total number of identified vessels by the area imaged. The problem here is that all measurements were made in a "non-volumetric" manner and only in V1. Extrapolating from here to the entire brain seems like an over-assumption, particularly given the region-dependent heterogeneity that the current paper reports.

Comments on revisions:

I appreciate the effort made to improve the manuscript. That said, the direct validation of the underlying assumption about spatial resolution sampling remains unaddressed in the final version of this manuscript. With the only intention to further strengthen the methodology presented here, I would encourage again the authors to seek a direct validation of this assumption for other brain areas.

In their reply, the authors stated "... line scanning or single-plane sequences, at least on first impression, seem inadequate for whole-brain coverage and cortical surface mapping. ". This seems to emanate for a misunderstanding as the method could be used to validate the mapping, not to map per-se.

Reviewer #1 (Public review):

Summary:

Boldt et al test several possible relationships between trandiagnostically-defined compulsivity and cognitive offloading in a large online sample. To do so, they develop a new and useful cognitive task to jointly estimate biases in confidence and reminder-setting. In doing so, they find that over-confidence is related to less utilization of reminder-setting, which partially mediates the negative relationship between compulsivity and lower reminder-setting. The paper thus establishes that, contrary to the over-use of checking behaviors in patients with OCD, greater levels of transdiagnostically-defined compulsivity predicts less deployment of cognitive offloading. The authors offer speculative reasons as to why (perhaps it's perfectionism in less clinically-severe presentations that lowers the cost of expending memory resources), and sets an agenda to understand the divergence in cognitive between clinical and nonclinical samples. Because only a partial mediation had robust evidence, multiple effects may be at play, whereby compulsivity impacts cognitive offloading via overconfidence and also by other causal pathways.

Strengths:

The study develops an easy-to-implement task to jointly measure confidence and replicates several major findings on confidence and cognitive offloading. The study uses a useful measure of cognitive offloading - the tendency to set reminders to augment accuracy in the presence of experimentally manipulated costs. Moreover, the utilizes multiple measures of presumed biases -- overall tendency to set reminders, the empirically estimated indifference point at which people engage reminders, and a bias measure that compares optimal indifference points to engage reminders relative to the empirically observed indifference points. That the study observes convergenence along all these measures strengthens the inferences made relating compulsivity to the under-use of reminder-setting. Lastly, the study does find evidence for one of several a priori hypotheses and sets a compelling agenda to try to explain why such a finding diverges from an ostensible opposing finding in clinical OCD samples and the over-use of cognitive offloading.

Weaknesses:

Although I think this design and study are very helpful for the field, I felt that a feature of the design might reduce the tasks's sensitivity to measuring dispositional tendencies to engage cognitive offloading. In particular, the design introduces prediction errors, that could induce learning and interfere with natural tendencies to deploy reminder-setting behavior. These PEs comprise whether a given selected strategy will be or not be allowed to be engaged. We know individuals with compulsivity can learn even when instructed not to learn (e.g., Sharp, Dolan and Eldar, 2021, Psychological Medicine), and that more generally, they have trouble with structure knowledge (eg Seow et al; Fradkin et al), and thus might be sensitive to these PEs. Thus, a dispositional tendency to set reminders might be differentially impacted for those with compulsivity after an NPE, where they want to set a reminder, but aren't allowed to. After such an NPE, they may avoid moreso the tendency to set reminders. Those with compulsivity likely have superstitious beliefs about how checking behaviors lead to a resolution of catastrophes, that might in part originate from inferring structure in the presence of noise or from purely irrelevant sources of information for a given decision problem.<br /> It would be good to know if such learning effects exist, if they're modulated by PE (you can imagine PEs are higher if you are more incentivized - e.g., 9 points as opposed to only 3 points - to use reminders, and you are told you cannot use them), and if this learning effect confounds the relationship between compulsivity and reminder-setting.

A more subtle point, I think this study can be more said to be an exploration than a deductive of test of a particular model -> hypothesis -> experiment. Typically, when we test a hypothesis, we contrast it with competing models. Here, the tests were two-sided because multiple models, with mutually exclusive predictions (over-use or under-use of reminders) were tested. Moreover, it's unclear exactly how to make sense of what is called the direct mechanism, which is supported by the partial (as opposed to complete) mediation.

Reviewer #2 (Public review):

Summary:

Boldt et al., investigated whether previously established relationships between transdiagnostic psychiatric symptom dimensions and confidence distortions would result in downstream influences on the confidence-related behaviour of reminder setting. 600 individuals from the general population completed a battery of psychiatric symptom questionnaires and an online reminder-setting task. In line with previous studies, individuals high in compulsivity (CIT) showed over-confidence in their task performance, whereas individuals high in anxious-depression (AD) tended to be under-confident. Crucially, the over-confidence associated with CIT partially mediated a decreased tendency to use external reminders during task performance, whereas the under-confidence associated with AD did not result in any alteration in external reminder setting. The authors suggest that metacognitive monitoring is impaired in CIT which has a knock-on effect on reminder setting behaviour, but that a direct link also exists between CIT and reduced reminder setting independently of confidence.

Strengths:

The study combines the latest advances in transdiagnostic approaches to psychopathology with a cleverly designed external reminder-setting task. The approach allows for investigation of what some of the downstream consequences associated with impaired metacognition in sub-clinical psychopathology may be.

The experimental design and hypotheses were pre-registered prior to data collection.

The manuscript is well written and rigorous analysis approaches are used throughout.

Weaknesses:

Participants only performed a single task so it remains unclear if the observed effects would generalise to reminder setting in other cognitive domains.

The sample consisted of participants recruited from the general population. Future studies should investigate whether the effects observed extend to individuals with the highest levels of symptoms (including clinical samples).

Reviewer #1 (Public review):

Summary:

The authors measured glutamate transients in the DMS of rats as they performed an action selection task. They identified diverse patterns of behavior and glutamate dynamics depending on the pre-existing behavioral phenotype of the rat (sign tracker or goal tracker). Using pathway-specific DREADDs, they showed that these behavioral phenotypes and their corresponding glutamate transients were differentially dependent on input from the prelimbic cortex to the DMS.

Strengths:

Overall there are some very interesting results that make an important contribution to the field. Notably, the results seem to point to differential recruitment of the PL-DMS pathway in goal-tracking vs sign-tracking behaviors.

Weaknesses:

(1) The controls for off-target effects of CNO are not given sufficient importance both in terms of power and in reporting of their results. There is precedent to accept that CNO at the dosage given is unlikely to disrupt the behaviour, this doesn't justify the assumption that glutamate transmission won't be affected, and this possibility hasn't been sufficiently ruled out.<br /> (2) The specificity of the viral approach needs to be clarified. Figure 8 indicates a large proportion of the PL neuron population that expresses mCherry in the absence of AAV-Cre. This infers that there are a large number of neurons inhibited by CNO administration that were outside the projection pathway, drawing into question the specificity of the effects.

Reviewer #2 (Public review):

Summary:

The authors aimed to determine whether goal-directed and cue-driven attentional strategies (goal- and sign-tracking phenotypes) were associated with variation in cued motor responses and dorsomedial striatal (DMS) glutamate transmission. They used a treadmill task in which cues indicated whether rats should turn or stop to receive a reward. They collected and analyzed several behavioral measures related to task performance with a focus on turns (performance, latency, duration) for which there are more measures than for stops. First, they established that goal-trackers perform better than sign-trackers in post-criterion turn performance (cued turns completed) and turn initiation. They used glutamate sensors to measure glutamate transmission in DMS. They performed analyses on glutamate traces that suggest phasic glutamate DMS dynamics to cues were primarily associated with successful turn performance and were more characteristic of goal-trackers (ie. rats with "goal-directed" attentional strategy). Smaller and more frequent DMS glutamate peaks were associated with other task events, cued misses (missed turns), cued stops, and reward delivery and were more characteristic of sign-trackers (i.e. rats with "cue-driven" attentional strategies). Consistent with the reported glutamate findings, chemogenetic inhibition of prelimbic-DMS glutamate transmission had an effect on goal-trackers' turn performance without affecting sign-trackers' performance in the treadmill task.

Strengths:

The power of the sign- and goal-tracking model to account for neurobiological and behavioral variability is critically important to the field's understanding of heterogeneity of the brain in health and disease. The approach and methodology are sound in their contribution to this important effort.

The authors establish behavioral differences, measure a neurobiological correlate of relevance, and then manipulate that correlate in a broader circuitry and show a causal role in behavior that is consistent with neurobiological measurements and phenotypic differences.

Sophisticated analyses provide a compelling description of the authors' observations.

Limitations:

Considerable transparency was added in the revised preprint. The "n" for each analysis is now available in Tables 1 and 3, carefully cross-referenced by figure. Readers may now carefully consider the n's in drawing their own conclusions from reported data.

While more conventional trial-averaged population activity traces are not presented or analyzed, the unique nature of the peak phenotypes is likely to "wash out" potentially meaningful signals if averaged across subjects. The distribution of peaks analyses (and shifts observed with chemogenetic inhibition) are improved in the revised preprint and are informative to illustrate this likelihood. Representative traces should theoretically be consistent with population averages within phenotype, and if not, discussion of such inconsistencies may have enriched the conclusions drawn from the study. For example, population traces of the phasic cue response in GT may resemble the representative peak examples, while smaller irregular peaks of ST may "wash out" in a population average (possibly resulting in a prolonged elevation) and could have strengthened the rationale for more sophisticated analyses of peak probability that remain the focus of the revised preprint.

Reviewer #3 (Public review):

Summary:

Avila and colleagues investigate the role of glutamate signaling in the dorsomedial striatum in a treadmill-based task where rats learn to turn or stop their walking based on learning cue-associations that allow them to acquire rewards. Phenotypic variation in Pavlovian conditioned sign and goal-tracking behavior was examined, where behavioral differences in stopping and turning were observed. Glutamate signals in the DMS were recording during the treadmill task, and were related to features of cue-controlled movement, with a stronger relationship seen for goal trackers. Finally, chemogenic inhibition of prelimbic neurons projecting to the DMS (the predicted source of those glutamate signals), preferentially affected cued movement in goal trackers. The authors couch these experiments in the context of cognitive control-attentional mechanisms, movement disorders, and individual differences in cue reactivity.

Strengths:

Overall these studies are interesting and are of general relevance to a number of research questions in neurology and psychiatry. The assessment of intersection of individual differences in cue-related learning strategies with movement-related questions - in this case cued turning behavior - is interesting and understudied question. The link between this work and growing notions of corticostriatal control of action selection makes it timely.

Weaknesses:

The clarity of the manuscript could be improved in several places, including in the graphical visualization of data. It is difficult to interpret the glutamate results, as presented, in the context of specific behaviors. It is difficult to assess how many trials/subjects are represented in the data shown, and too much emphasis is placed on representative examples. Averages traces of the glutamate data and other standard analysis approaches would improve the paper and allow for easier interpretation of the data.

Reviewer #1 (Public review):

Summary:

This paper investigates the effects of the explicit recognition of statistical structure and sleep consolidation on the transfer of learned structure to novel stimuli. The results show a striking dissociation in transfer ability between explicit and implicit learning of structure, finding that only explicit learners transfer structure immediately. Implicit learners, on the other hand, show an intriguing immediate structural interference effect (better learning of novel structure) followed by successful transfer only after a period of sleep.

Strengths:

This paper is very well written and motivated, and the data are presented clearly with a logical flow. There are several replications and control experiments and analyses that make the pattern of results very compelling. The results are novel and intriguing, providing important constraints on theories of consolidation. The discussion of relevant literature is thorough. In sum, this work makes an exciting and important contribution to the literature.

Weaknesses:

There have been several recent papers which have identified issues with alternative forced choice (AFC) tests as a method of assessing statistical learning (e.g. Isbilen et al. 2020, Cognitive Science). A key argument is that while statistical learning is typically implicit, AFC involves explicit deliberation and therefore does not match the learning process well. The use of AFC in this study thus leaves open the question of whether the AFC measure benefits the explicit learners in particular, given the congruence between knowledge and testing format, and whether, more generally, the results would have been different had the method of assessing generalization been implicit. Prior work has shown that explicit and implicit measures of statistical learning do not always produce the same results (eg. Kiai & Melloni, 2021, bioRxiv; Liu et al. 2023, Cognition).

The authors argued in their response to this point that this issue could have quantitative but not qualitative impacts on the results, but we see no reason that the impact could not be qualitative. In other words, it should be acknowledged that an implicit test could potentially result in the implicit group exhibiting immediate structure transfer.

Given that the explicit/implicit classification was based on an exit survey, it is unclear when participants who are labeled "explicit" gained that explicit knowledge. This might have occurred during or after either of the sessions, which could impact the interpretation of the effects and deserves discussion.

Reviewer #2 (Public review):

Summary:

Sleep has not only been shown to support the strengthening of memory traces, but also their transformation. A special form of such transformation is the abstraction of general rules from the presentation of individual exemplars. The current work used large online experiments with hundreds of participants to shed further light on this question. In the training phase participants saw composite items (scenes) that were made up of pairs of spatially coupled (i.e., they were next to each other) abstract shapes. In the initial training, they saw scenes made up of six horizontally structured pairs and in the second training phase, which took place after a retention phase (2 min awake, 12 hour incl. sleep, 12 h only wake, 24 h incl. sleep), they saw pairs that were horizontally or vertically coupled. After the second training phase, a two-alternatives-forced-choice (2-AFC) paradigm, where participants had to identify true pairs versus randomly assembled foils, was used to measure performance on all pairs. Finally, participants were asked five questions to identify, if they had insight into the pair structure and post-hoc groups were assigned based on this. Mainly the authors find that participants in the 2 minute retention experiment without explicit knowledge of the task structure were at chance level performance for the same structure in the second training phase, but had above chance performance for the vertical structure. The opposite was true for both sleep conditions. In the 12 h wake condition these participants showed no ability to discriminate the pairs from the second training phase at all.

Strengths:

All in all, the study was performed to a high standard and the sample size in the implicit condition was large enough to draw robust conclusions. The authors make several important statistical comparisons and also report an interesting resampling approach. There is also a lot of supplemental data regarding robustness.

Weaknesses:

My main concern regards the small sample size in the explicit group and the lack of experimental control.

Reviewer #3 (Public review):

In this project, Garber and Fiser examined how the structure of incidentally learned regularities influences subsequent learning of regularities, that either have the same structure or a different one. Over a series of six online experiments, it was found that the structure (spatial arrangement) of the first set of regularities affected learning of the second set, indicating that it has indeed been abstracted away from the specific items that have been learned. The effect was found to depend on the explicitness of the original learning: Participants who noticed regularities in the stimuli were better at learning subsequent regularities of the same structure than of a different one. On the other hand, participants whose learning was only implicit had an opposite pattern: they were better in learning regularities of a novel structure than of the same one. However, when an overnight sleep separated the first and second learning phases, this opposite effect was reversed and came to match the pattern of the explicit group, suggesting that the abstraction and transfer in the implicit case were aided by memory consolidation.

In their revision the authors addressed my major comments successfully and I commend them for that.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of the melanocortin system in puberty onset. They conclude that POMC neurons within the arcuate nucleus of the hypothalamus provide important but differing input to kisspeptin neurons in the arcuate or rostral hypothalamus.

Strengths:

Innovative and novel<br /> Technically sound<br /> Well-designed<br /> Thorough

Weaknesses:

There were no major weaknesses identified.

Reviewer #2 (Public review):

Summary:

This interesting manuscript describes a study investigating the role of MC4R signalling on kisspeptin neurons. The initial question is a good one. Infertility associated with MC4 mutations in humans has typically been ascribed to the consequent obesity and impaired metabolic regulation. Whether there is a direct role for MC4 in regulating the HPG axis has not been thoroughly examined. Here, the researchers have assembled an elegant combination of targetted loss of function and gain of function in vivo experiments, specifically targetting MC4 expression in kisspeptin neurons. This excellent experimental design should provide compelling evidence for whether melanocortin signalling dirently affects arcuate kisspeptin neurons to support normal reproductive function. There were definite effects on reproductive function (irregular estrous cycle, reduced magnitude of LH surge induced by exogenous estradiol). However, the magnitude of these responses and the overall effect on fertility were relatively minor. The mice lacking MC4R in kisspeptin neurons remained fertile despite these irregularities. The second part of the manuscript describes a series of electrophysiological studies evaluating the pharmacological effects of melanocortin signalling in kisspeptin cells in ex-vivo brain slides. These studies characterised interesting differential actions of melanocortins in two different populations of kisspeptin neurons. Collectively, the study provides some novel insights into how direct actions of melanocortin signalling via the MC4 receptor in kisspeptin neurons contribute to the metabolic regulation of the reproductive system. Importantly, however, it is clear that other mechanisms are also at play.

Strengths:

The loss of function/gain of function experiments provides a conceptually simple but hugely informative experimental design. This is the key strength of the current paper - especially the knock-in study that showed improved reproductive function even in the presence of ongoing obesity. This is a very convincing result that documents that reproductive deficits in MC4R knockout animals (and humans with deleterious MC4R gene variants) can be ascribed to impaired signalling in the hypothalamic kisspeptin neurons and not necessarily caused as a consequence of obesity. As concluded by the authors: "reproductive impairments observed in MC4R deficient mice, which replicate many of the conditions described in humans, are largely mediated by the direct action of melanocortins via MC4R on Kiss1 neurons and not to their obese phenotype." This is important, as it might change how such fertility problems are treated.

I would like to see the validation experiments for the genetic manipulation studies given greater prominence in the manuscript because they are critical to interpretation. Presently, only single unquantified images are shown, and a much more comprehensive analysis should be provided.

Weaknesses:

(1) Given that mice lacking MC4R in kisspeptin neurons remained fertile despite some reproductive irregularities, this can be described as a contributing pathway, but other mechanisms must also be involved in conveying metabolic information to the reproductive system. This is now appropriately covered in the discussion.

(2) The mechanistic studies evaluating melanocortin signalling in kisspeptin neurons were all completed in ovariectomised animals (with and without exogenous hormones) that do not experience cyclical hormone changes. Such cyclical changes are fundamental to how these neurons function in vivo and may dynamically alter how they respond to hormones and neuropeptides. Eliminating this variable makes interpretation difficult, but the authors have justified this as a reductionist approach to evaluate estradiol actions specifically. However, this does not reflect the actual complexity of reproductive function.

For example, the authors focus on a reduced LH response to exogenous estradiol in ovariectomised mice as evidence that there might be a sub-optimal preovulatory LH surge. However, the preovulatory LH sure (in intact animals) was not measured.

They have not assessed why some follicles ovulated, but most did not. They have focused on the possibility that the ovulation signal (LH surge) was insufficient rather than asking why some follicles responded and others did not. This suggests some issue with follicular development, likely due to changes in gonadotropin secretion during the cycle and not simply due to an insufficient LH surge.

Reviewer #3 (Public review):

The manuscript by Talbi R et al. generated transgenic mice to assess the reproduction function of MC4R in Kiss1 neurons in vivo and used electrophysiology to test how MC4R activation regulated Kiss1 neuronal firing in ARH and AVPV/PeN. This timely study is highly significant in neuroendocrinology research for the following reasons.

(1) The authors' findings are significant in the field of reproductive research. Despite the known presence of MC4R signaling in Kiss1 neurons, the exact mechanisms of how MC4R signaling regulates different Kiss1 neuronal populations in the context of sex hormone fluctuations are not entirely understood. The authors reported that knocking out Mc4r from Kiss1 neurons replicates the reproductive impairment of MC4RKO mice, and Mc4r expression in Kiss1 neurons in the MC4R null background partially restored the reproductive impairment. MC4R activation excites Kiss1 ARH neurons and inhibits Kiss1 AVPV/PeN neurons (except for elevated estradiol).

(2) Reproduction dysfunction is one of obesity comorbidities. MC4R loss-of-function mutations cause obesity phenotype and impaired reproduction. However, it is hard to determine the causality. The authors carefully measured the body weight of the different mouse models (Figure 1C, Figure 2A, Figure 3B). For example, the Kiss1-MC4RKO females showed no body weight difference at puberty onset. This clearly demonstrated the direct function of MC4R signaling in reproduction but was not a consequence of excessive adiposity.

(3) Gene expression findings in the "KNDy" system align with the reproduction phenotype.

(4) The electrophysiology results reported in this manuscript are innovative and provide more details of MC4R activation and Kiss1 neuronal activation.

Overall, the authors have presented sufficient background in a clear, logical, and organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, and made a justified conclusion.

Comments on revisions:

The authors have addressed my comments.

Reviewer #2 (Public review):

This work describes a highly complex automated algorithm for analyzing vascular imaging data from two-photon microscopy. This tool has the potential to be extremely useful to the field and to fill gaps in knowledge of hemodynamic activity across a regional network. The biological application provided, however, has several problems that make many of the scientific claims in the paper questionable.

The authors have commented on my main concerns. They have provided some limited evidence in the literature of prolonged vascular signals - though still nothing close to the several hundred-second long vascular responses oscillating between dilation and constriction shown here. And they have added a nice experiment showing they can resolve small beads (though still quite bigger than their average capillary diameter) with their system. They have also added comparisons with other software which shows some modest but clear improvement in some aspects. All these make the paper stronger.

However, I still think the main overall problem from the biological interpretation side of the paper is still not fixed. Perhaps I am too skeptical but I have a hard time accepting the conclusions about dilators and constrictors (depth dependence, distance from nearest neuron, etc.) because the data are just too temporally sparse and too unconventional in their duration and fluctuation. Also, the differences are often very small compared to the variability.

Regarding the spatial resolution, I was more concerned that if the pixel size is about 1 micron, then detecting around 1 micron dilations (or even less) is really below the resolution of the system. While the bead imaging is good for showing they can extract these diameters very close to the real value, this is still not like a living brain with imaging and motion artifacts. Given the temporal resolution issues already mentioned, this makes me highly skeptical of the biological claims. I think the discussion should at least strongly emphasize that a major caveat in their analysis is that the diameters are only sampled every 42 seconds, and , given the fluctuation in vessel diameter above and below baseline, this makes classification of the vessel as constrictor/dilator and by how much highly dependent on what time point the vessel diameter was sampled.

Although the computational side of the paper is not my strong point, it seems there is potential for the pipeline to be useful in other applications. But given the limitations of the system they are using, I feel that it is a methods paper in its current form more than anything that should be making the biological claims included.

Reviewer #2 (Public review):

In this study, the authors measured extracellular electrical features of colliding APs travelling in different directions down an isolated earthworm axon. They then used these features to build a model of the potential ephaptic effects of AP annihilation, i.e. the electrical signals produced by colliding/annihilating APs that may influence neighbouring tissue. The model was then applied to some different hypothetical scenarios involving synaptic connections. In a revised version of the manuscript, it was also applied, with success, to published experimental data on the cerebellar basket cell-to-Purkinje cell pinceau connection. The conclusion is that an annihilating AP at a presynaptic terminal can emphatically influence the voltage of a postsynaptic cell (the 'electrical coupling between neurons' of the title), and that the nature of this influence depends on the physical configuration of the synapse.

As an experimental neuroscientist who has never used computational approaches, I am unable to comment on the rigour of the analytical approaches that form the bulk of this paper. The experimental approaches appear very well carried out, and the data showing equal conduction velocity of anti- and orthodromically propagating APs in every preparation are convincing.

The conclusions drawn from the synaptic modelling are considerably strengthened by the data in Figure 5. Here, the authors' model - including AP annihilation at a synaptic terminal - is used to predict the amplitude and direction of experimentally observed effects at the cerebellar basket cell-to-Purkinje cell synapse (Blot & Barbour 2014). One particular form of the model (RTM with tau=0.5ms and realistic non-excitability of the terminal) matches the experimental data extremely well. The authors also include a convincing demonstration (Panel A) that a propagating but not annihilating AP has almost no effect on a neighbouring neuron's activity. Given that the authors' model of ephaptic effects can quantitatively explain key features of experimental data pertaining to synaptic function, the implications for the relevance of ephaptic coupling at different synaptic contacts may be widespread and important.

Reviewer #1 (Public review):

Summary:

The authors of this study investigated the development of interoceptive sensitivity in the context of cardiac and respiratory interoception in 3-, 9-, and 18-month-old infants using a combination of both cross-sectional and longitudinal designs. They utilised the cardiac interoception paradigm developed by Maister et al (2017) and also developed a new paradigm to investigate respiratory interoception in infants. The main findings of this research are that 9-month-old infants displayed a preference for stimuli presented synchronously with their own heartbeat and respiration. The authors found less reliable effects in the 18-month-old group, and this was especially true for the respiratory interoceptive data. The authors replicated a visual preference for synchrony over asynchrony for the cardiac domain in 3-month-old infants, while they found inconclusive evidence regarding the respiratory domain. Considering the developmental nature of the study, the authors also investigated the presence of developmental trajectories and associations between the two interoceptive domains. They found evidence for a relationship between cardiac and respiratory interoceptive sensitivity at 18 months only and preliminary evidence for an increase in respiratory interoception between 9 and 18 months.

Strengths:

The conclusions of this paper are mostly well supported by data, and the data analysis procedures are rigorous and well-justified. The main strengths of the paper are:

- A first attempt to explore the association between two different interoceptive domains. How different organ-specific axes of interoception relate to each other is still open and exploring this from a developmental lens can help shed light into possible relationships. The authors have to be commended for developing a novel interoceptive tasks aimed at assessing respiratory interoceptive sensitivity in infants and toddlers, and for trying to assess the relationship between cardiac and respiratory interoception across developmental time.<br /> - A thorough justification of the developmental ages selected for the study. The authors provide a rationale behind their choice to examine interoceptive sensitivity at 3, 9, and 18-months of age. These are well justified based on the literature pertaining to self- and social development. Sometimes, I wondered whether explaining the link between these self and social processes and interoception would have been beneficial as a reader not familiar with the topics may miss the point.<br /> - An explanation of direction of looking behaviour using latent curve analysis. I found this additional analysis extremely helpful in providing a better understanding of the data based on previous research and analytical choices. As the authors explain in the manuscript, it is often difficult to interpret the direction of infant looking behaviour as novelty and familiarity preferences can also be driven by hidden confounders (e.g. task difficulty). The authors provide compelling evidence that analytical choices can explain some of these effects. Beyond the field of interoception, these findings will be relevant to development psychologists and will inform future studies using looking time as a measure of infants' ability to discriminate among stimuli.<br /> - The use of simulation analysis to account for small sample size. The authors acknowledge that some of the effects reported in their study could be explained by a small sample size (i.e. the 3-month-olds and 18-month-olds data). Using a simulation approach, the authors try to overcome some of these limitations and provide convincing evidence of interoceptive abilities in infancy and toddlerhood (but see also my next point).

Comments on revision:

The authors have clearly addressed the comments on the previous version of this manuscript. I have no further comments.

Reviewer #2 (Public review):

Summary:

This study by Tünte et al. investigated the development of interoceptive sensitivity during the first year of life, focusing specifically on cardiac and respiratory sensitivity in infants aged 3, 9, and 18 months. The research employed a previously developed experimental paradigm for the cardiac domain and adapted it for a novel paradigm in the respiratory domain. This approach assessed infants' cardiac and respiratory sensitivity based on their preferential looking behavior toward visuo-auditory stimuli displayed on a monitor, which moved either in sync or out of sync with the infants' own heartbeats or breathing. The results in the cardiac domain showed that infants across all age groups preferred stimuli moving synchronously rather than asynchronously with their heartbeat, suggesting the presence of cardiac sensitivity as early as 3 months of age. However, it is noteworthy that this preference direction contradicts a previous study, which found that 5-month-old infants looked longer at stimuli moving asynchronously with their heartbeat (Maister et al., 2017). In the respiratory domain, only the group of 9-month-old infants showed a preference for stimuli presented synchronously with their breathing. The authors conducted various statistical analyses to thoroughly examine the obtained data, providing deeper insights valuable for future research in this field.

Strengths:

Few studies have explored the early development of interoception, making the replication of the original study by Maister et al. (2017) particularly valuable. Beyond replication, this study expands the investigation into the respiratory domain, significantly enhancing our understanding of interoceptive development. The provision of longitudinal and cross-sectional data from infants at 3, 9, and 18 months of age is instrumental in understanding their developmental trajectory.

Weaknesses:

Due to a technical error, this study failed to counterbalance the conditions of the first trial in both the iBEAT and iBREATH tests. Although the authors addressed this issue as much as possible by employing alternative analyses, it should be noted that this error may have critically influenced the results and, thus, the conclusions.

Reviewer #1 (Public Review):

The authors observed a decline in autophagy and proteasome activity in the context of Milton knockdown. Through proteomic analysis, they identified an increase in the protein levels of eIF2β, subsequently pinpointing a novel interaction within eIF subunits where eIF2β contributes to the reduction of eIF2α phosphorylation levels. Furthermore, they demonstrated that overexpression of eIF2β suppresses autophagy and leads to diminished motor function. It was also shown that in a heterozygous mutant background of eIF2β, Milton knockdown could be rescued. This work represents a novel and significant contribution to the field, revealing for the first time that the loss of mitochondria from axons can lead to impaired autophagy function via eIF2β, potentially influencing the acceleration of aging.

Reviewer #2 (Public Review):

In the manuscript, the authors aimed to elucidate the molecular mechanism that explains neurodegeneration caused by the depletion of axonal mitochondria. In Drosophila, starting with siRNA depletion of Milton and Miro, the authors attempted to demonstrate that the depletion of axonal mitochondria induces the defect in autophagy. From proteome analyses, the authors hypothesized that autophagy is impacted by the abundance of eIF2β and the phosphorylation of eIF2α. The authors followed up the proteome analyses by testing the effects of eIF2β overexpression and depletion on autophagy. With the results from those experiments, the authors proposed a novel role of eIF2β in proteostasis that underlies neurodegeneration derived from the depletion of axonal mitochondria.

The manuscript has several weaknesses. The reader should take extra care while reading this manuscript and when acknowledging the findings and the model in this manuscript.

The defect in autophagy by the depletion of axonal mitochondria is one of the main claims in the paper. The authors should work more on describing their results of LC3-II/LC3-I ratio, as there are multiple ways to interpret the LC3 blotting for the autophagy assessment. Lysosomal defects result in the accumulation of LC3-II thus the LC3-II/LC3-I ratio gets higher. On the other hand, the defect in the early steps of autophagosome formation could result in a lower LC3-II/LC3-I ratio. From the results of the actual blotting, the LC3-I abundance is the source of the major difference for all conditions (Milton RNAi and eIF2β overexpression and depletion).

Another main point of the paper is the up-regulation of eIF2β by depleting the axonal mitochondria leads to the proteostasis crisis. This claim is formed by the findings from the proteome analyses. The authors should have presented their proteomic data with much thorough presentation and explanation. As in the experiment scheme shown in Figure 4A, the author did two proteome analyses: one from the 7-day-old sample and the other from the 21-day-old sample. The manuscript only shows a plot of the result from the 7-day-old sample, but that of the result from the 21-day-old sample. For the 21-day-old sample, the authors only provided data in the supplemental table, in which the abundance ratio of eIF2β from the 21-day-old sample is 0.753, meaning eIF2β is depleted in the 21-day-old sample. The authors should have explained the impact of the eIF2β depletion in the 21-day-old sample, so the reader could fully understand the authors' interpretation of the role of eIF2β on proteostasis.

Joint Public Review:

Engineered artificial gene regulatory networks ("circuits") have a wide range of applications, but their design is often hindered by unforeseen interactions between the host and circuit processes. This manuscript employs computational modeling to investigate how growth feedback influences the performance of synthetic gene circuits capable of adaptation. By analyzing 425 hypothetical circuits previously identified as achieving nearly perfect adaptation (Ma et al., 2009; Shi et al., 2017), the authors introduce growth feedback into their models using additional terms in ordinary differential equations. Their simulations reveal that growth feedback can disrupt adaptation dynamics in diverse ways but also identify core motifs that ensure robust performance under such conditions. Additionally, they establish a scaling law linking circuit robustness to the strength of growth feedback. The findings have important implications for synthetic biology, where host-circuit interactions frequently compromise desired behaviors, and for systems biology, by advancing the understanding of network motif dynamics. The authors' classification schemes will be highly valuable to the community, offering a framework for addressing growth-related challenges in circuit design.

Strengths<br /> - A detailed investigation into the reasons for adaptation failure upon the introduction of cell growth was conducted, distinguishing this work from other studies of functional screening in gene regulatory network topologies. The comprehensiveness of the analysis is particularly noteworthy.<br /> - Approaches for assessing robustness, such as the survival ratio Q, were employed, providing tools that may be applicable to a broad range of network topologies beyond adaptation. The scaling law derived from these approaches is both novel and insightful.<br /> - A thorough numerical analysis of three gene regulatory networks exhibiting adaptation was performed. For each of the 425 topologies analyzed, approximately 2e5 circuits were sampled using Latin hypercube sampling, ensuring robust coverage of the parameter space. Among these, 1.5e5 circuits were identified as showing adaptation and subsequently subjected to further analysis, yielding approximately 350 parametric designs per topology for deeper investigation.<br /> - The systematic approach and depth of the analysis position this study as a significant contribution to the understanding of gene regulatory networks and their response to growth feedback. The combination of detailed investigation, novel robustness metrics, and rigorous computational techniques enhances the impact of this work within the field.

Weaknesses<br /> - The study focuses exclusively on a preselected set of 425 topologies previously shown to achieve adaptation, limiting the exploration of whether growth feedback could enable adaptation in circuits not inherently adaptive. While the authors have discussed and justified this choice, the focus restricts the generality of the conclusions, as the potential for growth feedback to induce adaptation in non-adaptive circuits remains unaddressed. The analysis includes scenarios where higher growth feedback restores adaptation in circuits that lose it at intermediate levels, but further elaboration on the implications for circuit design would strengthen the impact. The numerical framework and parameter choices align well with established methods, and an overview of the selected topologies has been provided. However, offering detailed information in supplementary materials or a public repository would further enhance the paper's accessibility and reproducibility.

- The model fails to capture the influence of protein levels on growth. To ensure accurate modeling of protein-level effects on growth, the b(t) term should be scaled appropriately, similar to Tan et al. Nature Chemical Biology 5:842-848 (2009).

- The authors propose bistability or multistability as the primary mechanisms behind different types of adaptation failure, explaining why the failures do not occur precisely at bifurcation points. They argue that their ODE simulations provide evidence for oscillation-related bifurcations, and an included appendix explores this phenomenon further, detailing how it can be observed in their results. While the authors choose not to apply semi-analytic methods, such as numerical continuation and eigenvalue analysis, to validate the existence of bifurcations, their approach offers valuable insights into the underlying dynamics of adaptation failures.

- The analysis in this work is carried out exclusively in a deterministic regime, as the focus is on scenarios where the effects of noise are assumed to be minimal. This approach is justified, and the authors acknowledge the complexity of extending their analysis to include stochasticity, which they suggest as an avenue for future research. The discussion has been expanded to address the potential impact of noise, its handling, and the assumptions underlying its exclusion. It is important to note, however, that noise can significantly alter system behavior-for instance, stabilizing trajectories and removing oscillations, as shown in prior studies (e.g., 10.1016/j.cels.2016.01.004). Additionally, variability in experimental implementations may influence the dynamics beyond what is predicted in deterministic models. These factors should be considered when interpreting the results.

Reviewer #2 (Public review):

Summary:

This manuscript provides a comprehensive overview of potential resistance mutations within MET Receptor Tyrosine Kinase and defines how specific mutations affect different inhibitors and modes of target engagement. The goal is to identify inhibitor combinations with the lowest overlap in their sensitivity to resistant mutations and determine if certain resistance mutations/mechanisms are more prevalent for specific modes of ATP-binding site engagement. To achieve this, the authors measured the ability of ~6000 single mutants of MET's kinase domain (in the context of a cytosolic TPR fusion) to drive IL-3-independent proliferation (used as a proxy for activity) of Ba/F3 cells (deep mutational profiling) in the presence of 11 different inhibitors. The authors then used co-crystal and docked structures of inhibitor-bound MET complexes to define the mechanistic basis of resistance and applied a protein language model to develop a predictive model of inhibitor sensitivity/resistance.

Strengths:

The major strengths of this manuscript are the comprehensive nature of the study and the rigorous methods used to measure the sensitivity of ~6000 MET mutants in a pooled format. The dataset generated will be a valuable resource for researchers interested in understanding kinase inhibitor sensitivity and, more broadly, small molecule ligand/protein interactions. The structural analyses are systematic and comprehensive, providing interesting insights into resistance mechanisms. Furthermore, the use of machine learning to define inhibitor-specific fitness landscapes is a valuable addition to the narrative. Although the ESM1b protein language model is only moderately successful in identifying the underlying mechanistic basis of resistance, the authors' attempt to integrate systematic sequence/function datasets with machine learning serves as a foundation for future efforts.

Weaknesses:

The main limitation of this study is that the authors' efforts to define general mechanisms between inhibitor classes were only moderately successful due to the challenge of uncoupling inhibitor-specific interaction effects from more general mechanisms related to the mode of ATP-binding site engagement. However, this is a minor limitation that only minimally detracts from the impressive overall scope of the study.

Reviewer #3 (Public review):

Summary:

In the manuscript 'Mapping kinase domain resistance mechanisms for the MET receptor tyrosine kinase via deep mutational scanning' by Estevam et al, deep mutational scanning is used to assess the impact of ~5,764 mutants in the MET kinase domain on the binding of 11 inhibitors. Analyses were divided by individual inhibitor and kinase inhibitor subtype (I,II, I 1/2, and III). While a number of mutants were consistent with previous clinical reports, novel potential resistance mutants were also described. This study has implications for the development of combination therapies, namely which combination of inhibitors to avoid based on overlapping resistance mutant profiles. While one suggested pair of inhibitors with least overlapping resistance mutation profiles was suggested, this manuscript presents a proof of concept toward a more systematic approach for improved selection of combination therapeutics. Furthermore, in a final part of this manuscript the data was used to train a machine learning model, the ESM-1b protein language model augmented with an XG Boost Regressor framework, and found that they could improve predictions of resistance mutations above the initial ESM-1b model.

Strengths:

Overall this paper is a tour-de-force of data collection and analysis to establish a more systematic approach for the design of combination therapies, especially in targeting MET and other kinases, a family of proteins significant to therapeutic intervention for a variety of diseases. The presentation of the work is mostly concise and clear with thousands of data points presented neatly and clearly. The discovery of novel resistance mutants for individual MET inhibitors, kinase inhibitor subtypes within the context of MET, and all resistance mutants across inhibitor subtypes for MET has clinical relevance. However, probably the most promising outcome of this paper is the proposal of the inhibitor combination of Crizotinib and Cabozantib as Type I and Type II inhibitors, respectively, with the least overlapping resistance mutation profiles and therefore potentially the most successful combination therapy for MET. While this specific combination is not necessarily the point, it illustrates a compelling systematic approach for deciding how to proceed in developing combination therapy schedules for kinases. In an insightful final section of this paper, the authors approach using their data to train a machine learning model, perhaps understanding that performing these experiments for every kinase for every inhibitor could be prohibitive to applying this method in practice.

Weaknesses:

This paper presents a clear set of experiments with a compelling justification. The content of the paper is overall of high quality. Below are mostly regarding clarifications in presentation.

Two places could use more computational experiments and analysis, however. Both are presented as suggestions, but at least a discussion of these topics would improve the overall relevance of this work. In the first case it seems that while the analyses conducted on this dataset were chosen with care to be the most relevant to human health, further analyses of these results and their implications of our understanding of allosteric interactions and their effects on inhibitor binding would be a relevant addition. For example, for any given residue type found to be a resistance mutant are there consistent amino acid mutations to which a large or small or effect is found. For example is a mutation from alanine to phenylalanine always deleterious, though one can assume the exact location of a residue matters significantly. Some of this analysis is done in dividing resistance mutants by those that are near the inhibitor binding site and those that aren't, but more of these types of analyses could help the reader understand the large amount of data presented here. A mention at least of the existing literature in this area and the lack or presence of trends would be worthwhile. For example, is there any correlation with a simpler metric like the Grantham score to predict effects of mutations (in a way the ESM-1b model is a better version of this, so this is somewhat implicitly discussed).

Indeed, this discussion relates to the second point this manuscript could improve upon: the machine learning section. The main actionable item here is that this results section seems the least polished and could do a better job describing what was done. In the figure it looks like results for certain inhibitors were held out as test data - was this all mutants for a single inhibitor, or some other scheme? Overall I think the implications of this section could be fleshed out, potentially with more experiments. As mentioned in the 'Strengths' section, one of the appealing aspects of this paper is indeed its potential wide applicability across kinases -- could you use this ML model to predict resistance mutants for an entirely different kinase? This doesn't seem far-fetched, and would be an extremely compelling addition to this paper to prove the value of this approach.

Another area in which this paper could improve its clarity is in the description of caveats of the assay. The exact math used to define resistance mutants and its dependence on the DMSO control is interesting, it is worth discussing where the failure modes of this procedure might be. Could it be that the resistance mutants identified in this assay would differ significantly from those found in patients? That results here are consistent with those seen in the clinic is promising, but discrepancies could remain. Furthermore a more in depth discussion of the MetdelEx14 results is warranted. For example, why is the DMSO signature in Figure 1 - supplement 4 so different from that of Figure 1? And finally, there is a lot of emphasis put on the unexpected results of this assay for the tivantinib "type III" inhibitor - could this in fact be because the molecule "is highly selective for the inactive or unphosphorylated form of c-Met" according to Eathiraj et al JBC 2011? These points are addressed in previous work (Estevam et al 2024) or in the detailed methods section, but are not obvious in the main text of the paper.

This paper is crisply written with beautiful figures, and the complexity of the data is easy to understand from an in depth discussion of the mutants that have been previously reported.

Finally, the potential impacts and follow-ups of this excellent study could be used as a resource for the community both as a dataset and as a proof of concept. It is exciting that his approach can be altered and/or improved in the future to facilitate the general application of this approach for combination therapies and the understanding of mechanism for other targets.

Comments on revisions:

Thank you for your additions and changes - they have improved the quality of this paper.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

The manuscript by Zhu et al describes a novel role for MED26, a subunit of the Mediator complex, in erythroid development. The authors have discovered that MED26 promotes transcriptional pausing of RNA Pol II, by recruiting pausing-related factors.

Strengths:

This is a well-executed study. The authors have employed a range of cutting-edge and appropriate techniques to generate their data, including: CUT&Tag to profile chromatin changes and mediator complex distribution; nuclear run-on sequencing (PRO-seq) to study Pol II dynamics; knockout mice to determine the phenotype of MED26 perturbation in vivo; an ex vivo erythroid differentiation system to perform additional, important, biochemical and perturbation experiments; immunoprecipitation mass spectrometry (IP-MS); and the "optoDroplet" assay to study phase-separation and molecular condensates.

This is a real highlight of the study. The authors have managed to generate a comprehensive picture by employing these multiple techniques. In doing so, they have also managed to provide greater molecular insight into the workings of the MEDIATOR complex, an important multi-protein complex that plays an important role in a range of biological contexts. The insights the authors have uncovered for different subunits in erythropoiesis will very likely have ramifications in many other settings, in both healthy biology and disease contexts.

Weaknesses:

There are almost no discernible weaknesses in the techniques used, nor the interpretation of the data. The IP-MS data was generated in HEK293 cells when it could have been performed in the human CD34+ HSPC system that they employed to generate a number of the other data. This would have been a more natural setting and would have enabled a more like-for-like comparison with the other data.

Reviewer #3 (Public review):

Summary:

The authors aim to explore whether other subunits besides MED1 exert specific functions during the process of terminal erythropoiesis with global gene repression, and finally they demonstrated that MED26-enriched condensates drive erythropoiesis through modulating transcription pausing.

Strengths:

Through both in vitro and in vivo models, the authors showed that while MED1 and MED26 co-occupy a plethora of genes important for cell survival and proliferation at the HSPC stage, MED26 preferentially marks erythroid genes and recruits pausing-related factors for cell fate specification. Gradually, MED26 becomes the dominant factor in shaping the composition of transcription condensates and transforms the chromatin towards a repressive yet permissive state, achieving global transcription repression in erythropoiesis.

Weaknesses:

In the in vitro model, the author only used CD34+ cell-derived erythropoiesis as the validation, which is relatively simple, and more in vitro erythropoiesis models need to be used to strengthen the conclusion.

Reviewer #2 (Public review):

Dipasree Hajra et al demonstrated that Salmonella was able to modulate the expression of Sirtuins (Sirt1 and Sirt3) and regulate the metabolic switch in both host and Salmonella, promoting its pathogenesis. The authors found Salmonella infection induced high levels of Sirt1 and Sirt3 in macrophages, which were skewed toward the M2 phenotype allowing Salmonella to hyper-proliferate. Mechanistically, Sirt1 and Sirt3 regulated the acetylation of HIF-1alpha and PDHA1, therefore mediating Salmonella-induced host metabolic shift in the infected macrophages. Interestingly, Sirt1 and Sirt3-driven host metabolic switch also had an effect on the metabolic profile of Salmonella. Counterintuitively, inhibition of Sirt1/3 led to increased pathogen burdens in an in vivo mouse model. Overall, this is a well-designed study.

The revised manuscript has addressed all of the previous comments. The re-analysis of flow cytometry and WB data by authors makes the results and conclusion more complete and convincing.

Reviewer #3 (Public review):

Summary:

In this paper Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knock down experiments in RAW macrophage cell line to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:

The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions have not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 result in a similar outcome.

Weaknesses:

The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidences are also lacking to prove the proposed mechanisms. For instance they show correlative data that knock down of Sirt1 mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

Reviewer #1 (Public review):

Summary:

TMEM16, OSCA/TMEM63, and TMC belong to a large superfamily of ion channels where TMEM16 members are calcium activated lipid scramblases and chloride channels, whereas OSCA/TMEM63 and TMCs are mechanically activated ion channels. In the TMEM16 family, TMEM16F is a well characterized calcium activated lipid scramblase that play an important role in processes like blood coagulation, cell death signaling, and phagocytosis. In a previous study the group has demonstrated that lysine mutation in TM4 of TMEM16A can enable the calcium activated chloride channel to permeate phospholipids too. Based on this they hypothesize that the energy barrier for lipid scramblase in these ion channels is low, and that modification in the hydrophobic gate region by introducing a charged side chain between TM4/6 interface in TMEM16 and OSCA/TMEM63 family can allow lipid scramblase. In this manuscript, using scramblase activity via Annexin V binding to phosphatidylserine, and electrophysiology, the authors demonstrate that lysine mutation in TM4 of TMEM16F and TMEM16A can cause constitutive lipid scramblase activity. The authors then go on to show that analogous mutations in OSCA1.2 and TMEM63A can lead to scramblase activity. The revised version does a thorough characterization of residues that form the hydrophobic gate region in TM4/6 of this superfamily of channels. Their results indicated that disrupting the TM4/6 interaction can reduce energy barrier for this channels to scramblase lipids.

Strengths:

Overall, the authors introduce an interesting concept that this large superfamily can permeate ions and lipids.

Weaknesses:

none noted in the revised version.

Reviewer #2 (Public review):

This focused study by Lowry and colleagues that identifies a key molecular motif that controls ion permeation vs combined ion permeation and lipid transport in three families of channel/scramblase proteins, in TMEM16 channels, in the plant-expressed and stress-gated cation channel OSCA, and in the mammalian homolog and mechanosensitive cation channel, TMEM63. Between them, these three channels share low sequence similarity and have seemingly differing functions, as anion (TMEM16 channels), or stress-activated cation channels (OSCA/TMEM63). The study finds that in all three families, mutating a single hydrophobic residue in the ion permeation pathway of the channels confers lipid transport through the pores of the channels, indicating that TMEM16 and related OSCA and TMEM63 channels have a conserved potential for both ion and lipid permeation. The authors interpret the findings as revealing that these channel/scramblase proteins have a relatively low "energetic barrier for scramblase" activity. The experiments are done with a high level of rigor and the revised paper is very well written and addresses the previous concerns.

Reviewer #3 (Public review):

This study was focused on the conserved mechanisms across the Transmembrane Channel/Scramblase superfamily, which includes members of the TMEM16, TMEM63/OSCA, and TMC families. In previous work, the authors have studied the role of the inner activation gate of these proteins. Here, the authors show that the introduction of mutations at the TM4-TM6 interface, which are close to the inactivation gate, can disrupt gating and confer scramblase activity to non-scramblases proteins.

Overall, the confocal imaging experiments, patch clamping experiments, and data analysis are performed well and in line with standard methods. The molecular dynamics simulation work is focused but adds supportive evidence to their findings. Although there could have been more extensive molecular analysis to bolster the authors' arguments on the role of the TM4-TM6 interface (e.g. evaluate effects of size/hydrophobicity, double mutants, cross-linking, more in-depth simulation data), there is adequate evidence to conclude that certain residues at this interface is critical to ion conduction and phospholipid scramblase activity. The data presented only adds incremental depth of knowledge for each individual channel, but together, they show this to be true for conserved TM4 residues across TMEM16F, TMEM16A, OSCA1.2, and TMEM63A proteins. This breadth of data is a major strength of this paper, and provides strong evidence for a coupled pathway for ion conduction and phospholipid transport, though the underlying biophysical mechanism is still speculative and remains to be elucidated.

Reviewer #1 (Public review):

Summary:

The authors addressed the influence of DKK2 on colorectal cancer (CRC) metastasis to the liver using an orthotopic model transferring AKP-mutant organoids into the spleens of wild-type animals. They found that DKK2 expression in tumor cells led to enhanced liver metastasis and poor survival in mice. Mechanistically, they associate Dkk2-deficiency in donor AKP tumor organoids with reduced Paneth-like cell properties, particularly Lz1 and Lyz2, and defects in glycolysis. Quantitative gene expression analysis showed no significant changes in Hnf4a1 expression upon Dkk2 deletion. Ingenuity Pathway Analysis of RNA-Seq data and ATAC-seq data point to a Hnf4a1 motif as a potential target. They also show that HNF4a binds to the promoter region of Sox9, which leads to LYZ expression and upregulation of Paneth-like properties. By analyzing available scRNA data from human CRC data, the authors found higher expression of LYZ in metastatic and primary tumor samples compared to normal colonic tissue; reinforcing their proposed link, HNF4a was highly expressed in LYZ+ cancer cells compared to LYZ- cancer cells.

Strengths:

Overall, this study contributes a novel mechanistic pathway that may be related to metastatic progression in CRC.

Weaknesses:

The main concerns are related to incremental gains, missing in vivo support for several of their conclusions in murine models, and missing human data analyses.

Main comments

Novelty:<br /> The authors previously described the role of DKK2 in primary CRC, correlating increased DKK2 levels to higher Src phosphorylation and HNF4a1 degradation, which in turn enhances LGR5 expression and "stemness" of cancer cells, resulting in tumor progression (PMID: 33997693). A role for DKK2 in metastasis has also been previously described (sarcoma, PMID: 23204234)

Mouse data:<br /> (a) The authors analyzed liver mets, but the main differences between AKT and AKP/Dkk2 KO organoids could arise during the initial tumor cell egress from the intestinal tissue (which cannot be addressed in their splenic injection model), or during pre-liver stages, such as endothelial attachment. While the analysis of liver mets is interesting, given that Paneth cells play a role in the intestinal stem cell niche, it is questionable whether a study that does not involve the intestine can appropriately address this pathway in CRC metastasis.<br /> (b) The overall number of Paneth cells found in the scRNA-seq analysis of liver mets was low (17 cells, Fig.3), and assuming that these cells are driving the differences seems somewhat far-fetched.<br /> (c) Fig. 6 suggests a signaling cascade in which the absence of DKK2 leads to enhanced HNF4A expression, which in turn results in reduced Sox9 expression and hence reduced expression of Paneth cell properties. It is therefore crucial that the authors perform in vivo (splenic organoid injection) loss-of-function experiments, knockdown of Sox9 expression in AKP organoids, and Sox9 overexpression experiments in AKP/Dkk2 KO organoids to demonstrate Sox9 as the central downstream transcription factor regulating liver CRC metastasis.<br /> (d) Given the previous description of the role of DKK2 in primary CRC, it is important to define the step of liver metastasis affected by Dkk2 deficiency in the metastasis model. Does it affect extravasation, liver survival, etc.?

Human data:<br /> Can the authors address whether the expression of Dkk2 changes in human CRC and whether mutations in Dkk2 as correlated with metastatic disease or CRC stage?

Bioinformatic analysis<br /> GEO repositories remain not open (at the time of the re-review) and SRA links for raw data are still unavailable. Without access to raw data, it is not possible to verify the analyses or fully assess the results. A part of the article was made by re-analyzing public data so the authors should make even the raw available and not just the count tables

Reviewer #2 (Public review):

Summary:

The authors propose that DKK2 is necessary for the metastasis of colon cancer organoids. They then claim that DKK2 mediates this effect by permitting the generation of lysozyme-positive Paneth-like cells within the tumor microenvironmental niche. They argue that these lysozyme-positive cells have Paneth-like properties in both mouse and human contexts. They then implicate HNF4A as the causal factor responsive to DKK2 to generate lysozyme-positive cells through Sox9.

Strengths:

The use of a genetically defined organoid line is state-of-the-art. The data in Figure 1 and the dependence of DKK2 for splenic injection and liver engraftment, as well as the long-term effect on animal survival, are interesting and convincing. The rescue using DKK2 administration for some of their phenotype in vitro is good. The inclusion and analysis of human data sets help explore the role of DKK2 in human cancer and help ground the overall work in a clinical context.

Remaining Weaknesses after revision:

(1) The authors have effectively explained the regulation of HNF4A at both mRNA and protein levels. To further strengthen their findings, I recommend using CRISPR technology to generate DKK2 and HNF4A double knockout organoids. This approach would allow the authors to investigate whether the AKP liver metastasis is restored in the double knockout condition. Such an experiment would provide more direct evidence that HNF4A protein stabilization is the crucial mechanism for liver metastasis suppression following DKK2 knockout.

Reviewer #1 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Comments on revised version:

The authors well addressed all my concerns. It is a wonderful design to view the 3D cell spheroids.

Reviewer #2 (Public review):

Summary:

The author developed a new device to overcome current limitations in the imaging process of 3D spheroidal structures. In particular, they created a system to follow in real-time tumour spheroid formation, fusion and cell migration without disrupting their integrity. The system has also been exploited to test the effects of a therapeutic agent (chemotherapy) and immune cells.

Strengths:

The system allows the in situ observation of the 3D structures along the 3 axes (x,y and z) without disrupting the integrity of the spheroids; in a time-lapse manner it is possible to follow the formation of the 3D structure and the spheroids fusion from multiple angles, allowing a better understanding of the cell aggregation/growth and kinetic of the cells.

Interestingly the system allows the analysis of cell migration/ escape from the 3D structure analysing not only the morphological changes in the periphery of the spheroids but also from the inner region demonstrating that the proliferating cells in the periphery of the structure are more involved in the migration and dissemination process. The application of the system in the study of the effects of doxorubicin and NK cells would give new insights in the description of the response of tumor 3D structure to killing agents.

Reviewer #2 (Public review):

This study presents valuable insight on how neurons within the central amygdala may broadly encode the valence of emotional stimuli. The evidence supporting most of the authors' conclusion is solid, although some of the claims should be treated with caution due to potential alternative interpretation of the data.

In this revised manuscript the authors have addressed the reviewers' critiques in a way that acknowledges the feedback but does not fully embrace or rigorously address the reviewers' core concerns. Here are the main observations that support this impression:

(1) The authors repeatedly acknowledge the ambiguity in defining "valence" and "salience" in the literature, but their responses don't clarify how they address these terms more rigorously. They seem to justify their operational definitions by citing previous studies but do not address how their definitions impact the clarity and robustness of their findings.

(2) The reviewers highlighted that using stimuli from different sensory modalities without scaling them or including neutral cues limits the ability to distinguish between valence and salience. The authors acknowledge this but argue that using same-modality stimuli would not produce distinct responses. This response doesn't address the reviewers' point about how these design limitations could weaken the conclusions. They seem to rely on citations of similar experimental designs instead of addressing the core critique or proposing additional experiments.

(3) In response to the low number of cue-responsive units and the call for more rigorous behavioral measures (like licking or orienting), the authors provide some data but emphasize statistical rigor over behavioral insights, which was questioned during the initial review. They don't propose any methodological adjustments or consider alternative explanations.

(4) The reviewers suggested clustering or other population-level analyses to understand functional diversity within the central amygdala. The authors argue that their statistical approach was sufficient and don't believe additional clustering analyses would add value. This response seems dismissive, as they don't consider whether population-level insights might reveal patterns that single-cell responses overlook.

Overall, while the authors have responded to each concern, their rebuttals often reference other studies to justify their choices rather than addressing the specific limitations highlighted by the reviewers.

Reviewer #3 (Public review):

Summary:

The authors have performed endoscopic calcium recordings of individual CeA neuron responses to food and shock, as well as to cues predicting food and shock. They claim that a majority of neurons encode valence, with a substantial minority encoding salience.

Strengths:

The use of endoscopic imaging is valuable, as it provides the ability to resolve signals from single cells, while also being able to track these cells across time (though the latter capability was not extensively utilized). Another strength is the use of a sophisticated circular shifting analysis to avoid statistical errors caused by correlations between neighboring image pixels.

Weaknesses:

In the first version of this manuscript, my main critique was that the authors didn't fully test whether neurons encode valence. In their rebuttal, the authors justify their use of the terms valence and salience by citing prior works from different labs:

(1) Li et al., 2019, doi: 10.7554/eLife.41223<br /> (2) Yang et al., 2023, doi: 10.1038/s41586-023-05910-2<br /> (3) Huang et al., 2024, doi: 10.1038/s41586-024-07819<br /> (4) Lin and Nicolelis, 2008, doi: 10.1016/j.neuron.2008.04.031<br /> (5) Stephenson-Jones et al., 2020, doi: 10.1016/j.neuron.2019.12.006<br /> (6) Zhu et al., 2018, doi: 10.1126/science.aat0481<br /> (7) Comoli et al., 2003, doi: 10.1038/nn1113P

Among these, items #1 and #3 primarily discuss valence, while #2, #4, #6, and #7 discuss salience, and #5 discusses both.

Upon reviewing these references, the authors' identification of valence encoding patterns is still problematic, and indeed studies cited above show several lines of evidence for valence encoding that are absent here. For example, item #3 ranked behavioral responses to five different odors in drosophila, from most attractive to most repulsive, and saw neuronal responses correlated with the degree of attraction versus repulsion across all five odors. This is robust evidence for valence encoding that is absent here. Items #1 and #5 above are the other two valence-addressing studies cited, and although those only used one rewarding and one aversive stimulus (in rodents), both also added a neutral cue, and most critically, identified substantial subsets of neurons showing a rank-order response, e.g. either aversion > neutral > reward or aversion < neutral < reward. Again, that level of demonstration of valence encoding is not shown in the current study.

Finally, two of the valence studies above tested responses to omission of reward/punishment, providing yet more evidence of valence encoding that is absent in the current study.

While there is much to like about the current study, the claims of valence encoding appear hard to justify, and should be toned down.

Reviewer #1 (Public review):

The authors present an important work where they model some of the complex interactions between immune cells, fibroblasts and cancer cells. The model takes into account the increased ECM production of cancer-associated fibroblasts. These fibres trap the cancer but also protect it from immune system cells. In this way, these fibroblasts' actions both promote and hinder cancer growth. By exploring different scenarios, the authors can model different cancer fates depending on the parameters regulating cancer cells, immune system cells and fibroblasts. In this way, the model explores non-trivial scenarios. An important weakness of this study is that, though it is inspired by NSCLC tumors, it is still far from modelling tumor lesions with morphologies similar to NSCLC tumors and does not explore the formation of ramified tumors. In this way, is a general model and it is challenging how it can be adapted to simulate more realistic tumor morphologies.

Comments on revisions:

The authors have improved the manuscript and addressed my concerns.

Reviewer #2 (Public review):

Summary:

The authors develop a computational model (and a simplified version thereof) to treat an extremely important issue regarding tumor growth. Specifically, it has been argued that fibroblasts have the ability to support tumor growth by creating physical conditions in the tumor microenvironment that prevent the relevant immune cells from entering into contact with, and ultimately killing, the cancer cells. This inhibition is referred to as immune exclusion. The computational approach follows standard procedures in the formulation of models for mixtures of different material species, adapted to the problem at hand by making a variety of assumptions as to the activity of different types of fibroblasts, namely "normal" versus "cancer-associated". The model itself is relatively complex, but the authors do a convincing job of analyzing possible behaviors and attempting to relate these to experimental observations.

Strengths:

As mentioned, the authors do an excellent job of analyzing the behavior of their model both in its full form (which includes spatial variation of the concentrations of the different cellular species) and in its simplified mean field form. The model itself is formulated based on established physical principles, although the extent to which some of these principles apply to active biological systems is perhaps debatable (see Weaknesses). The results of the model do indeed offer some significant insights into the critical factors which determine how fibroblasts might affect tumor growth; these insights could lead to new experimental ways of unraveling these complex sets of issues and enhancing immunotherapy. In this revised version, the authors have properly placed this work within the general context of other research on modeling the tumor-immune ecology.

Weaknesses:

Models of the form being studied here rely on a large number of assumptions regarding cellular behavior. One major issue is the degree to which close-to-equilibrium assumptions (such as the dynamics being driven by free energy minimization) can be taken as reliable predictors of the obviously active dynamics of biological cells. The authors have recognized this conceptual issue and have argued that these assumptions provide a reasonable first step for understanding the full complexity of dynamics in the tumor microenvironment.

The problem of T cell infiltration as well as the patterning of the extracellular matrix (ECM) by fibroblasts necessarily involve understanding cell proliferation, cell motion and cell interactions due e.g. to cell signaling. There is evidence that inherently non-equilibrium interactions between the fibroblasts and the extracellular matrix can lead to patterning of the fiber network and trapping of potentially infiltrating T-cells. it is not clear the extent to which this type of interaction can be captured by the approach being used here, although the authors propose that they can be mimicked by proper terms in their formulation. This to me is the primary concern that I had with this paper.

The authors have now addressed what used to be a separate weakness concerning the assumption that fibroblasts affect T cell behavior primarily by just making a more dense ECM. Instead, the organization of the ECM (for example, its anisotropy) could be playing a much more essential role than is given credit for here. This possibility is now discussed in some detail and the authors have suggested that the introduction of a nematic order parameter field would be a useful way to treat this effect.

Reviewer #1 (Public review):

Summary:

The authors created a transgenic mouse line to read out integrated stress responses with single-cell resolution.

Strengths:

ISR plays an important role in the development, maintenance, and degeneration of the nervous system. This mouse line represents a potentially important tool to understand ISR in situ.

Weaknesses:

The current manuscript is clearly written. However, more validation experiments should be performed to understand the exact meaning of the fluorescence intensity of GFP and RFP channels. This is important because these results will define how this tool will be used in the future and in the field.

Reviewer #2 (Public review):

Summary:

In this paper, the authors create transgenic animals with a CMV promoter driving expression of their DIO-SPOTlight construct in which uORF2 and the authentic ORF of Atf4 are replaced by GFP and tdTomato respectively, such that ISR activation is predicted to diminish GFP expression and enhance RFP expression. The major experimental finding of the paper is that cholinergic neurons have the most robust activation of the reporter, consistent with and extending upon their previous work.

Strengths:

It is very likely that the reporter does indeed read out on ISR activation at some level. It is mostly likely to be useful for screening and hypothesis testing than for gaining mechanistic insight, because, as the authors note in the present version, ATF4 itself is but one component of ISR activation. Cells might have robust eIF2a phosphorylation but have suppressed translational regulation (for instance by regulating the expression of eIF2B). The mRNA and protein half-lives of the GFP and Tomato are likely quite different from that of the equivalent components in ATF4, which means that the reporter is likely to behave differently from ATF4 itself over time.

Weaknesses:

The major element that the current manuscript lacks is a detailed comparison between how the reporter behaves and how it tracks with eIF2a phosphorylation, ATF4, and the initiation of the gene expression program downstream of ATF4. While this would be difficult to do in vivo, it would seem much more feasible to isolate primary cells (neurons, fibroblasts, hepatocytes, etc.) from the animals and thoroughly characterize the kinetics of reporter-versus-ISR activation. In that way, the reader can have a better idea of how to interpret the behavior of the reporter. As it is, the authors' attempt to account for the reporter's behavior in Figure 3F is purely speculative and not backed by experiment or modeling.

Reviewer #3 (Public review):

Summary:

The previously described reporter SPOTlight is a fluorescence-based reporter of the integrated stress response, specifically, protein synthesis initiation dynamics. In the current study from the same lab, the authors describe the creation and characterization of a transgenic mouse that expresses SPOTlight.

Strengths:

The previously described reporter has now been made into a Cre-dependent transgene in mice. The authors replicate previous findings from their lab that were acquired using viral vector-mediated delivery of their reporter.

Weaknesses:

There is not a clear advantage to having the Cre-dependent SPOTlight reporter in a transgenic mouse over using a viral vector to deliver the same Cre-dependent SPOTlight based on the experiments presented. There are potential general advantages and disadvantages to virus vs transgenic mouse but no side-by-side comparisons are performed here.

It is not clear whether overexpressing the reporter alters basal ISR/UPR function and gene expression. The CAG is a strong promoter and overexpression of fluorescent proteins (or any protein) can potentially stress protein synthesis and processing mechanisms. The use of the animal as a reporter may be misleading if the presence of the reporter is already altering ISR/UPR.

Reviewer #1 (Public review):

Summary:

In organisms with an open mitosis, nuclear envelope breakdown at mitotic entry and re-assembly of the nuclear envelope at the end of mitosis are important, highly regulated processes. One key regulator of nuclear envelope re-assembly is the BAF (Barrier-to-Autointegration) protein, which contributes to cross-linking of chromosomes to the nuclear envelope. Crucially, BAF has to be in a dephosphorylated form to carry out this function, and PP2A has been shown to be the phosphatase which dephosphorylates BAF. The Ankle2/LEM4 protein has previously been identified as an important regulator of PP2A in the dephosphorylation of BAF but its precise function is not fully understood, and Li and colleagues set out to investigate the function of Ankle2/LEM4 in both Drosophila flies and Drosophila cell lines.

Strengths:

The authors use a combination of biochemical and imaging techniques to understand the biology of Ankle2/LEM4. On the whole the experiments are well conducted and the results look convincing. A particular strength of this manuscript is that the authors are able to study both cellular phenotypes and organismal effects of their mutants by studying both Drosophila D-mel cells and whole flies.<br /> The work presented in this manuscript significantly enhances our understanding of how Ankle2/LEM4 supports BAF dephosphorylation at the end of mitosis. Particularly interesting is finding that Ankle2/LEM4 appears to be a bona fide PP2A regulatory protein in Drosophila, as well as the localisation of Ankle2/LEM4 and how this is influenced by the interaction between Ankle2 and the ER protein Vap33. It would be interesting to see, though, whether these insights are conserved in mammalian cells, e.g. does mammalian Vap33 also interact with LEM4? Is LEM4 also a part of the PP2A holoenzyme complex in mammalian cells?

Weaknesses:

This work is certainly impactful but more discussion and comparison of the Drosophila versus mammalian cell system would be helpful. Also, to attract the largest possible readership, the Ankle2 protein should be referred to as Ankle2/LEM4 throughout the paper to make it clear that this is the same molecule.

A schematic model at the end of the final figure would be very useful to summarise the findings.

Comments on revisions:

The authors have carefully revised the manuscripts and have satisfactorily addressed the issues that were raised by the reviewers.

Reviewer #2 (Public review):

The authors first identify Ankle2 as a regulatory subunit and direct interactor of PP2A, showing they interact both in vitro and in vivo to promote BAF dephosphorylation. The Ankyrin domain of Ankle2 is important for the interaction with PP2A. They then show Ankle2 also interacts with the ER protein Vap33 through FFAT motifs and they particularly co-localize during mitosis. The recruitment of Ankle2 to Vap33 is essential to ER and nuclear envelop membrane in telophase while earlier in mitosis, it relies on the C terminus but not the FFAT motifs for recruitments to the nuclear membrane and spindle envelop in early mitosis. The molecular determinants and receptors are currently not known. The authors check the function of the PP2A recruitment to Ankle2/Vap33 in the context of embryos and show this recruitment pathway is functionally important. While the Ankle2/Vap33 interaction is dispensable in adult flies -looking at wing development, the PP2A/Ankle2 interaction is essential for correct wing and fly development. Overall, this is a very complete paper that reveals the molecular mechanism of PP2A recruitment to Ankle2 and studies both the cellular and the physiological effect of this interaction in the context of fly development.

The paper is well-written and the narrative is well developed. The figures are of high quality, well-controlled, clearly labelled and easy to understand. They support the claims made by the authors.

Comments on revisions:

There are still issues with the statistics. On graphs where multiple conditions are shown, you cannot perform a T-test. You have to use other tests such as ANOVA if the data is normal, and other tests such as KS test if the data is not normally distributed.

Reviewer #3 (Public review):

The authors were interested in how Ankle2 regulates nuclear envelope reformation after cell division. They show that Ankle2 can bind in a PP2A complex without other known regulatory subunits of PP2A. The authors also identity a novel interaction with ER protein Vap33 that could be important for localization. This manuscript is a useful finding linking Ankle2 function during nuclear envelope reformation to the PP2A complex. The authors present solid data showing that Ankle2 can form a complex with PP2A-29B and Mts and generate a phosphoproteomic resource that is fundamentally important to understand Ankle2 biology. The caveat should be remembered that most experiments, including subcellular localization, are based on overexpression data. Keeping this in mind, the manuscript is a valuable resource.

Reviewer #1 (Public review):

The hypothesis is based on the idea that inversions capture genetic variants that have antagonistic effects on male sexual success (via some display traits) and survival of females (or both sexes) until reproduction. Furthermore, a sufficiently skewed distribution of male sexual success will tend to generate synergistic epistasis for male fitness even if the individual loci contribute to sexually selected traits in an additive way. This should favor inversions that keep these male-beneficial alleles at different loci together at a cis-LD. A series of simulations are presented and show that the scenario works at least under some conditions. While a polymorphism at a single locus with large antagonistic effects can be maintained for a certain range of parameters, a second such variant with somewhat smaller effects tends to be lost unless closely linked. It becomes much more likely for genomically distant variants that add to the antagonism to spread if they get trapped in an inversion; the model predicts this should drive accumulation of sexually antagonistic variants on the inversion versus standard haplotype, leading to the evolution of haplotypes with very strong cumulative antagonistic pleiotropic effects. This idea has some analogies with one of predominant hypotheses for the evolution of sex chromosomes, and the authors discuss these similarities. The model is quite specific, but the basic idea is intuitive and thus should be robust to the details of model assumption. It makes perfect sense in the context of the geographic pattern of inversion frequencies. One prediction of the models (notably that leads to the evolution of nearly homozygously lethal haplotypes) does not seem to reflect the reality of chromosomal inversions in Drosophila, as the authors carefully discuss, but it is the case of some other "supergenes", notably in ants. So the theoretical part is a strong novel contribution,

To provide empirical support for this idea, the authors study the dynamics of inversions in population cages over one generation, tracking their frequencies through amplicon sequencing at three time points: (young adults), embryos and very old adult offspring of either sex (>2 months from adult emergence). Out of four inversions included in the experiment, two show patterns consistent with antagonistic effects on male sexual success (competitive paternity) and the survival of offspring, especially females, until an old age, which the authors interpret as consistent with their theory.

As I have argued in my comments on previous versions, the experiment only addresses one of the elements of the theoretical hypothesis, namely antagonistic effects of inversions on male reproductive success and other fitness components, in particular of females. Furthermore, the design of this experiment is not ideal from the viewpoint of the biological hypothesis it is aiming to test. This is in part because, rather than testing for the effects of inversion on male reproductive success versus the key fitness components of survival to maturity and female reproductive output, it looks at the effects on male reproductive success versus survival to a rather old age of 2 months. The relevance of survival until old age to fitness under natural conditions is unclear, as the authors now acknowledge. Furthermore, up to 15% of males that may have contributed to the next generation did not survive until genotyping, and thus the difference between these males' inversion frequency and that in their offspring may be confounded by this potential survival-based sampling bias. The experiment does not test for two other key elements of the proposed theory: the assumption of frequency-dependence of selection on male sexual success, and the prediction of synergistic epistasis for male fitness among genetic variants in the inversion. To be fair, particularly testing for synergistic epistasis would be exceedingly difficult, and the authors have now included a discussion of the above caveats and limitations, making their conclusions more tentative. This is good but of course does not make these limitations of the experiment go away. These limitations mean that the paper is stronger as a theoretical than as an empirical contribution.

Reviewer #2 (Public review):

Summary:

In their manuscript the authors address the question whether the inversion polymorphism in D. melanogaster can be explained by sexually antagonistic selection. They designed a new simulation tool to perform computer simulations, which confirmed their hypothesis. They also show a tradeoff between male reproduction and survival. Furthermore, some inversions display sex-specific survival.

Strengths:

It is an interesting idea on how chromosomal inversions may be maintained

Weaknesses:

The authors motivate their study by the observation that inversions are maintained in D. melanogaster and because inversions are more frequent closer to the equator, the authors conclude that it is unlikely that the inversion contributes to adaptation in more stressful environments. Rather the inversion seems to be more common in habitats that are closer to the native environment of ancestral Drosophila populations.<br /> While I do agree with the authors that this observation is interesting, I do not think that it rules out a role in local adaptation. After all, the inversion is common in Africa, so it is perfectly conceivable that the non-inverted chromosome may have acquired a mutation contributing to the novel environment.

Based on their hypothesis, the authors propose an alternative strategy, which could maintain the inversion in a population. They perform some computer simulations, which are in line with the predicted behavior. Finally, the authors perform experiments and interpret the results as empirical evidence for their hypothesis. While the reviewer is not fully convinced about the empirical support, the key problem is that the proposed model does not explain the patterns of clinal variation observed for inversions in D. melanogaster. According to the proposed model, the inversions should have a similar frequency along latitudinal clines. So in essence, the authors develop a complicated theory because they felt that the current models do not explain the patterns of clinal variation, but this model also fails to explain the pattern of clinal variation.

Reviewer #3 (Public review):

Summary:

In this study, McAllester and Pool develop a new model to explain the maintenance of balanced inversion polymorphism, based on (sexually) antagonistic alleles and a trade-off between male reproduction and survival (in females or both sexes). Simulations of this model support the plausibility of this mechanism. In addition, the authors use experiments on four naturally occurring inversion polymorphisms in D. melanogaster and find tentative evidence for one aspect of their theoretical model, namely the existence of the above-mentioned trade-off in two out of the four inversions.

Strengths:

(1) The study develops and analyzes a new (Drosophila melanogaster-inspired) model for the maintenance of balanced inversion polymorphism, combining elements of (sexually) antagonistically (pleiotropic) alleles, negative frequency-dependent selection and synergistic epistasis. Simulations of the model suggest that the hypothesized mechanism might be plausible.

(2) The above-mentioned model assumes, as a specific example, a trade-off between male reproductive display and survival; in the second part of their study, the authors perform laboratory experiments on four common D. melanogaster inversions to study whether these polymorphisms may be subject to such a trade-off. The authors observe that two of the four inversions show suggestive evidence that is consistent with a trade-off between male reproduction and survival.

Open issues:

(1) A gap in the current modeling is that, while a diploid situation is being studied, the model does not investigate the effects of varying degrees of dominance. It would thus be important and interesting, as the authors mention, to fill this gap in future work,

(2) It will also be important to further explore and corroborate the potential importance and generality of trade-offs between different fitness components in maintaining inversion polymorphisms in future work.

Reviewer #3 (Public review):

The manuscript by Goyal et al report substrate-bound and substrate-free structures of a tripartite ATP independent periplasmic (TRAP) transporter from a previously uncharacterized homolog, F. nucleatum. This is one of most mechanistically fascinating transporter families, by means of its QM domain (the domain reported in his manuscript) operating as a monomeric 'elevator', and its P domain functioning as a substrate-binding 'operator' that is required to deliver the substrate to the QM domain; together, this is termed an 'elevator with an operator' mechanism. Remarkably, previous structures had not demonstrated the substrate Neu5Ac bound. In addition, they confirm the previously reported Na+ binding sites, and report a new metal binding site in the transporter, which seems to be mechanistically relevant. Finally, they mutate the substrate binding site and use proteoliposomal uptake assays to show the mechanistic relevance of the proposed substrate binding residues.

Strengths:

The structures are of good quality, the presentation of the structural data has improved, the functional data is robust, the text is well-written, and the authors are appropriately careful with their interpretations. Determination of a substrate bound structure is an important achievement and fills an important gap in the 'elevator with an operator' mechanism.

Weaknesses:

Although the possibility of the third metal site is compelling, I do not feel it is appropriate to model in a publicly deposited PDB structure without directly confirming experimentally. The authors do not extensively test the binding sites due to technical limitations of producing relevant mutants; however, their model is consistent with genetic assays of previously characterized orthologs, which will be of benefit to the field.

Reviewer #1 (Public review):

Summary:

In this paper, the authors present an interesting strategy to interfere with the HBV life cycle: the preparation of geranyl and peptides' dimers that could impede the correct assembly of hepatitis B core protein HBc into viable capsids. These dimers are of different nature, depending on the HBc site the authors plan to target. A preliminary study with geranyl dimers (targeting a hydrophobic site of HBc) was first investigated. The second series deals with peptide-PEG linker-peptide dimers, targeting the tips of HBc dimer spikes.

Strengths:

This work is very well conducted, combining ITC experiments (for determination of dimers' KD), cellular effects (thanks to the grafting of previously developed dimers with polyarginine-based cell penetrating peptide) HBV infected HEK293 cells and Cryo-EM studies.<br /> The findings of these research teams unambiguously demonstrated the interest of such dimeric structures in impeding the correct HBV life cycle and thus, could bring solutions in the control of its development. Ultimately, a new class of HBV Capside Assembly Modulators could arise from this study.<br /> There is no doubt that this work could bring very interesting information for people working on VHB.

Comments on revisions:

Minor corrections have been made in this revised version of this work, according to the remarks of the reviewers.

Reviewer #2 (Public review):

Summary:

Vladimir Khayenko et al. discovered two novel binding pockets on HBc with in vitro binding and electron microscopy experiments. While the geranyl dimer targeting a central hydrophobic pocket displayed a micromolar affinity, the P1-dimer binding to the spike tip of HBc has a nanomolar affinity. In the turbidity assay and at the cellular level, an HBc aggregation from peptide crosslinking was demonstrated.

Strengths:

The study identifies two previously unexplored binding pockets on HBc capsids and develops novel binders targeting these sites with promising affinities.

Weaknesses:

While the in vitro and cellular HBc aggregation effects are demonstrated, the antiviral potential against HBV infection is not directly evaluated in this study.

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Comments on revisions:

I have read the author's responses and the revised manuscript. The authors did not sufficiently address my comments, nor the fundamental issue with the manuscript.

By the authors' own admission, many of the results presented in the current version of the manuscript cannot be reproduced without relying on locally saved MSAs. In other words, there is almost no evidence presented that this pipeline will predict antibody-antigen interactions using currently publicly available software. This manuscript is reduced to essentially a case study (N=1) in how one might go about making such predictions coupled with pretty good experimental evidence backing up this singular prediction.

Reviewer #2 (Public Review):

Summary:

The authors showed the applicability and usefulness of a new AlphaFold2 pipeline called PabFold, which can predict linear antibody epitopes (B-cell epitopes) that can be helpful for the selection of reagents to be applied in competitive ELISA assay.

Strengths:

The authors showed the accuracy of the pipeline to identify correctly the binding epitope for three different antibody-antigen systems (Myc, HA, and Sars-Cov2 nucleocapsid protein). The design of scFvs from Fab of the three antibodies to speed up the analysis time is extremely interesting.

Weaknesses:

The article justifies correctly the findings and no great weaknesses are present. However, it could be useful for a broader audience to show in detail how pLDDT was calculated for both Simple-Max approach (per residue-pLDDT) and Consensus analysis ( average pLDDT for each peptide), with associated equations.

Comments on revisions:

I have read the author's responses to my comments and the revised paper. They addressed the minor comments and concerns. However, I agree with Reviewer #1 that these findings cannot be reproduced without local MSAs and this is a major issue.

Reviewer #1 (Public review):

Summary:

Rigor in the design and application of scientific experiments is an ongoing concern in preclinical (animal) research. Because findings from these studies are often used in the design of clinical (human) studies, it is critical that the results of the preclinical studies are valid and replicable. However, several recent peer-reviewed published papers have shown that some of the research results in cardiovascular research literature may not be valid because their use of key design elements is unacceptably low. The current study is designed to expand on and replicate previous preclinical studies in nine leading scientific research journals. Cardiovascular research articles that were used for examination were obtained from a PubMed Search. These articles were carefully examined for four elements that are important in the design of animal experiments: use of both biological sexes, randomization of subjects for experimental groups, blinding of the experimenters, and estimating the proper size of samples for the experimental groups. The findings of the current study indicate that the use of these four design elements in the reported research in preclinical research is unacceptably low. Therefore, the results replicate previous studies and demonstrate once again that there is an ongoing problem in the experimental design of preclinical cardiovascular research.

Strengths:

This study selected four important design elements for study. The descriptions in the text and figures of this paper clearly demonstrate that the rate of use of all four design elements in the examined research articles was unacceptably low. The current study is important because it replicates previous studies and continues to call attention once again to serious problems in the design of preclinical studies, and the problem does not seem to lessen over time.

Weaknesses:

Weaknesses from the first review were adequately addressed.

Reviewer #1 (Public review):

Summary:

Authors of this article have previously shown the involvement of the transcription factor Zinc finger homeobox-3 (ZFHX3) in the function of the circadian clock and the development/differentiation of the central circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. Here, they show that ZFHX3 plays a critical role in the transcriptional regulation of numerous genes in the SCN. Using inducible knockout mice, they further demonstrate that the deletion Of Zfhx3 induces a phase advance of the circadian clock, both at the molecular and behavioral levels.

Strengths:

- Inducible deletion of Zfhx3 in adults<br /> - Behavioral analysis<br /> - Properly designed and analyzed ChIP-Seq and RNA-Seq supporting the conclusion of the behavioral analysis

Weaknesses:

- Further characterization of the disruption of the activity of the SCN is required.<br /> - The description of the controls needs some clarification.

Reviewer #2 (Public review):

Summary:

ZFHX3 is a transcription factor expressed in discrete populations of adult SCN and was shown by the authors previously to control circadian behavioral rhythms using either a dominant missense mutation in Zfhx3 or conditional null Zfhx3 mutation using the Ubc-Cre line (Wilcox et al., 2017). In the current manuscript, the authors assess the function of ZFHX3 by using a multi-omics approach including ChIPSeq in wildtype SCNs and RNAseq of SCN tissues from both wildtype and conditional null mice. RNAseq analysis showed a loss of oscillation in Bmal1 and changes in expression levels of other clock output genes. Moreover, a phase advance gene transcriptional profile using the TimeTeller algorithm suggests the presence of a regulatory network that could underlie the observed pattern of advanced activity onset in locomotor behavior in knockout mice.

In figure1, the authors identified tthe ZFHX3 bound sites using ChIPseq and compared the loci with other histone marks that occur at promoters, TSS, enhancers and intergenic regions. And the analysis broadly points to a role for ZFHX3 in transcriptional regulation. The vast majority of nearly 40000 peaks overlapped H3K4me3 and K27ac marks, active promoters which also included genes falling under the GO category circadian rhythms. However, no significant differential ZFHX3 bound peaks were detected between ZT3 and ZT15. In these experiments, it is not clear if and how the different ChIP samples (ZFHX3 and histone PTM ChIPs) were normalized/downsampled for analysis. Moreover, it seems that ZFHX3 binding or recruitment has little to do with whether the promoters are active.

Based on a enrichment of ARNT domains next to K4Me3 and K27ac PTMs, the authors propose a model where the core-clock TFs and ZFHX3 interact. If the authors develop other assays beyond just predictions to test their hypothesis, it would strengthen the argument for role in circadian transcription in the SCN. It would be important in this context to perform a ChIP-seq experiment for ZFHX3 in the knockout animal (described from Figure 2 onwards) to eliminate the possibility of non-specific enrichment of signal from "open chromatin'. Alternatively, a ChIPseq analysis for BMAL1 or CLOCK could also strengthen this argument to identify the sites co-occupied by ZFHX3 and core-clock TFs.

Next, they compared locomotor activity rhythms in floxed mice with or without tamoxifen treatment. As reported before in Wilcox et al 2017, the loss of ZFHX3 led to a shorter free running period and reduced amplitude and earlier onset of activity. Overall, the behavioral data in Figure 2 and supplementary figure 2 has been reported before and are not novel.

Next, the authors performed RNAseq at 4hr intervals on wildtype and knockout animals maintained in light/dark cycles to determine the impact of loss of ZFHX3. Overall transcriptomic analysis indicated changes in gene expression in nearly 36% of expressed genes, with nearly half being upregulated while an equal fraction was downregulated. Pathways affected included mostly neureopeptide neurotransmitter pathways. Surprisingly, there was no correlation between the direction in change in expression and TF binding since nearly all the sites were bound by ZFHX3 and the active histone PTMs. The ChIP-seq experiment for ZFHX3 in the UBC-Cre+Tam mice again could help resolve the real targets of ZFHX3 and the transcriptional state in knockout animals.

To determine the fraction of rhythmic transcripts, Using dryR, the authors categorise the rhythmic transcriptome into modules that include genes that lose rhythmicity in the KO, gain rhythmicity in the KO or remain unaffected or partially affected. The analysis indicates that a large fraction of the rhythmic transcriptome is affected in the KO model. However, among core-clock genes only Bmal1 expression is affected showing a complete loss of rhythm. The authors state a decrease in Clock mRNA expression (line 294) but the panel figure 4A does not show this data. Instead it depicts the loss in Avp expression - {{ misstated in line 321 ( we noted severe loss in 24-h rhythm for crucial SCN neuropeptides such as Avp (Fig. 3a).}}

However, core-clock genes such as Pers and Crys show minor or no change in expression patterns while Per2 and Per3 show a ~2hr phase advance. While these could only weakly account for the behavioral phase advance, the authors used TimeTeller to assess circadian phase in wildtype and ZFHX3 deficient mice. This approach clearly indicated that while the clock is not disrupted in the knockout animals, the phase advance can be correctly predicted from a network of gene expression patterns.

Strengths:

The authors use a multiomic strategy in order to reveal the role of the ZFHX3 transcription factor with a combination of TF and histone PTM ChIPseq, time-resolved RNAseq from wildtype and knockout mice and modeling the transcriptomic data using TimeTeller. The RNAseq experiments are nicely controlled and the analysis of the data indicates a clear impact on gene-expression levels in the knockout mice and the presence of a regulatory network that could underlie the advanced activity onset behavior.

Weaknesses:

It is not clear whether ZFHX3 has a direct role in any of the processes and seems to be a general factor that marks H3K4me3 and K27ac marked chromatin. Why it would specifically impact the core-clock TTFL clock gene expression or indeed daily gene expression rhythms is not clear either. Details for treatment of different ChIP samples (ZFHX3 and histone PTM ChIPs) on data normalization for analysis are needed. The loss of complete rhythmicity of Avp and other neuropeptides or indeed other TFs could instead account for the transcriptional deregulation noted in the knockout mice.

Reviewer #1 (Public review):

Summary:

Migration of the primordial germ cells (PGCs) in mice is asynchronous, such that leading and lagging populations of migrating PGCs emerge. Prior studies found that interactions between the cells the PGCs encounter along their migration routes regulates their proliferation. In this study, the authors used single cell RNAseq to investigate PGC heterogeneity and to characterize their niches during their migration along the AP axis. Unlike prior scRNAseq studies of mammalian PGCs, the authors conducted a time course covering 3 distinct stages of PGC migration (pre, mid, and post migration) and isolated PGCs from defined somite positions along the AP axis. In doing so, this allowed the authors to uncover differences in gene expression between leading and lagging PGCs and their niches and to investigate how their transcript profiles change over time. Among the pathways with the biggest differences were regulators of actin polymerization and epigenetic programming factors and Nodal response genes. In addition, the authors report changes in somatic niches, specifically greater non-canonical WNT in posterior PGCs compared to anterior PGCs. This relationship between the hindgut epithelium and migrating PGCs was also detected in reanalysis of a previously published dataset of human PGCs. Using whole mount immunofluorescence, the authors confirmed elevated Nodal signaling based on detection of the LEFTY antagonists and targets of Nodal during late stage PGC migration. Taken together, the authors have assembled a temporal and spatial atlas of mouse PGCs and their niches. This resource and the data herein provide support for the model that interactions of migrating mouse PGCs with their niches influences their proliferation, cytoskeletal regulation, epigenetic state and pluripotent state.

Overall, the findings provide new insights into heterogeneity among leading and lagging PGC populations and their niches along the AP axis, as well as comparisons between mouse and human migrating PGCs. The data are clearly presented, and the text is clear and well-written. This atlas resource will be valuable to reproductive and developmental biologists as a tool for generating hypotheses and for comparisons of PGCs across species.

Strengths:

(1) High quality atlas of individual PGCs prior to, during and post migration and their niches at defined positions along the AP axis.<br /> (2) Comparisons to available datasets, including human embryos, provide insight into potentially conserved relationships among PGCs and the identified pathways and gene expression changes.<br /> (3) Detailed picture of PGC heterogeneity.<br /> (4) Valuable resource for the field.<br /> (5) Some validation of Nodal results and further support for models in the literature based on less comprehensive expression analysis.

Weaknesses:

(1) No indication of which sex(es) were used for the mouse data and whether or not sex-related differences exist or can excluded at the stages examined. This should be clarified.

Reviewer #2 (Public review):

Summary:

This work addresses the question of how 'leading' and 'lagging' PGCs differ, molecularly, during their migration to the mouse genital ridges/gonads during fetal life (E9.5, E10.5, E11.5), and how this is regulated by different somatic environments encountered during the process of migration. E9.5 and E10.5 cells differed in expression of genes involved in canonical WNT signaling and focal adhesions. Differences in cell adhesion, actin cytoskeletal dynamics were identified between leading and lagging cells, at E9.5, before migration into the gonads. At E10.5, when some PGCs have reached the genital ridges, differences in Nodal signaling response genes and reprogramming factors were identified. This last point was verified by whole mount IF for proteins downstream of Nodal signaling, Lefty1/2. At E11.5, there was upregulation of genes associated with chromatin remodeling and oxidative phosphorylation. Some aspects of the findings were also found to be likely true in human development, established via analysis of a dataset previously published by others.

Strengths:

The work is strong in that a large number of PGCs were isolated and sequenced, along with associated somatic cells. The authors dealt with problem of very small number of migrating mouse PGCs by pooling cells from embryos (after ascertaining age matching using somite counting). 'Leading' and 'lagging' populations were separated by anterior and posterior embryo halves and the well-established Oct4-deltaPE-eGFP reporter mouse line was used.

Weaknesses:

The work seems to have been carefully done, but I do not feel the manuscript is very accessible, and I do not consider it well written. The novel findings are not easy to find. The addition of at least one figure to show the locations of putative signaling etc. would be welcome.

(1) The initial discussion of CellRank analysis (under 'Transcriptomic shifts over developmental time...' heading) is somewhat confusing - e.g. If CellRank's 'pseudotime analysis' produces a result that seems surprising (some E9.5 cells remain in a terminal state with other E9.5 cells) and 'realtime analysis' produces something that makes more sense, is there any point including the pseudotime analysis (since you have cells from known timepoints)? Perhaps the 'batch effects' possible explanation (in Discussion) should be introduced here. Do we learn anything novel from this CellRank analysis? The 'genetic drivers' identified seem to be genes already known to be key to cell transitions during this period of development.

(2) In Discussion - with respect to Y-chromosome correlation, it is not clear why this analysis would be done at E10.5, when E11.5 data is available (because some testis-specific effect might be more apparent at the later stage).

(3) Figure 2A - it seems surprising that there are two clusters of E9.5 anterior cells

(4) Figure 5F - there does seem to be more LEFTY1/2 staining in the anterior region, but also more germ cells as highlighted by GFP

Reviewer #3 (Public review):

Summary:

The migration of primordial germ cells (PGCs) to the developing gonad is a poorly understood, yet essential step in reproductive development. Here, the authors examine whether there are differences in leading and lagging migratory PGCs using single-cell RNA sequencing of mouse embryos. Cleverly, the authors dissected embryonic trunks along the anterior-to-posterior axis prior to scRNAseq in order to distinguish leading and lagging migratory PGCs. After batch corrections, their analyses revealed several known and novel differences in gene expression within and around leading and lagging PGCs, intercellular signaling networks, as well as number of genes upregulated upon gonad colonization. The authors then compared their datasets with publicly available human datasets to identify common biological themes. Altogether, this rigorous study reveals several differences between leading and lagging migratory PGCs, hints at signatures for different fates among the population of migratory PGCs, and provides new potential markers for post-migratory PGCs in both humans and mice. While many of the interesting hypotheses that arise from this work are not extensively tested, these data provide a rich platform for future investigations.

Strengths:

-The authors have successfully navigated significant technical challenges to obtain a substantial number of mouse migratory primordial germ cells for robust transcriptomic analysis. Here the authors were able to collect quality data on ~13,000 PGCs and ~7,800 surrounding somatic cells, which is ten times more PGCs than previous studies.

- The decision to physically separate leading and lagging primordial germ cells was clever and well-validated based on expected anterior-to-posterior transcriptional signatures.

- Within the PGCs and surrounding tissues, the authors found many gene expression dynamics they would expect to see both along the PGC migratory path as well as across developmental time, increasing confidence in the new differentially expressed genes they found.

- The comparison of their mouse-based migratory PGC datasets with existing human migratory PGC datasets is appreciated.

- The quality control, ambient RNA contamination elimination, batch correction, cell identification and analysis of scRNAseq data were thorough and well-done such that the new hypotheses and markers found through this study are dependable.

- The subsetting of cells in their trajectory analysis is appreciated, further strengthening their cell terminal state predictions.

Weaknesses:

- Although it is useful to compare their mouse-based dataset with human datasets, the authors used two different analysis pipelines for each dataset. While this may have been due to the small number of cells in the human dataset as mentioned, it does make it difficult to compare them.

- There were few validation experiments within this study. For one such experiment, whether there is a difference in pSMAD2/3 along the AP axis is unclear and not quantified as was nicely done for Lefty1/2.

Reviewer #1 (Public review):

This manuscript introduces a useful curation pipeline of antibody-antigen structures downloaded from the PDB database. The antibody-antigen structures are presented in a new database called AACDB, alongside annotations that were either corrected from those present in the PDB database or added de-novo with a solid methodology. Sequences, structures, and annotations can be very easily downloaded from the AACDB website, speeding up the development of structure-based algorithms and analysis pipelines to characterize antibody-antigen interactions. However, AACDB is missing some key annotations that would greatly enhance its usefulness.

Here are detailed comments regarding the three strengths above:

(1) I think potentially the most significant contribution of this database is the manual data curation to fix errors present in the PDB entries, by cross-referencing with the literature. However, as a reviewer, validating the extent and the impact of these corrections is hard, since the authors only provided a few anecdotal examples in their manuscript.

I have personally verified some of the examples presented by the authors and found that SAbDab appears to fix the mistakes related to the misidentification of antibody chains, but not other annotations.

(a) "the species of the antibody in 7WRL was incorrectly labeled as "SARS coronavirus B012" in both PDB and SabDab" → I have verified the mistake and fix, and that SAbDab does not fix is, just uses the pdb annotation.<br /> (b) "1NSN, the resolution should be 2.9 , but it was incorrectly labeled as 2.8" → I have verified the mistake and fix, and that saabdab does not fix it, just uses the PDB annotation.<br /> (c) "mislabeling of antibody chains as other proteins (e.g. in 3KS0, the light chain of B2B4 antibody was misnamed as heme domain of flavocytochrome b2)" → SAbDab fixes this as well in this case.<br /> (d) "misidentification of heavy chains as light chains (e.g. both two chains of antibody were labeled as light chain in 5EBW)" → SAbDab fixes this as well in this case.

I personally believe the authors should make public the corrections made, and describe the procedures - if systematic - to identify and correct the mistakes. For example, what was the exact procedure (e.g. where were sequences found, how were the sequences aligned, etc.) to find mutations? Was the procedure run on every entry?

(2) I believe the splitting of the pdb files is a valuable contribution as it standardizes the distribution of antibody-antigen complexes. Indeed, there is great heterogeneity in how many copies of the same structure are present in the structure uploaded to the PDB, generating potential artifacts for machine learning applications to pick up on. That being said, I have two thoughts both for the authors and the broader community. First, in the case of multiple antibodies binding to different epitopes on the same antigen, one should not ignore the potentially stabilizing effect that the binding of one antibody has on the complex, thereby enabling the binding of the second antibody. In general, I urge the community to think about what is the most appropriate spatial context to consider when modeling the stability of interactions from crystal structure data. Second, and in a similar vein, some antigens occur naturally as homomultimers - e.g. influenza hemagglutinin is a homotrimer. Therefore, to analyze the stability of a full-antigen-antibody structure, I believe it would be necessary to consider the full homo-trimer, whereas, in the current curation of AACDB with the proposed data splitting, only the monomers are present.

(3) I think the annotation of interface residues is a useful addition to structural datasets, but their current presentation is lacking on several fronts.

I think the manuscript is lacking in justification about the numbers used as cutoffs (1A^2 for change in SASA and 5A for maximum distance for contact) The authors just cite other papers applying these two types of cutoffs, but the underlying physico-chemical reasons are not explicit even in these papers. I think that, if the authors want AACDB to be used globally for benchmarks, they should provide direct sources of explanations of the cutoffs used, or provide multiple cutoffs. Indeed, different cutoffs are often used (e.g. ATOM3D uses 6A instead of 5A to determine contact between a protein and a small molecule https://datasets-benchmarks-proceedings.neurips.cc/paper/2021/hash/c45147dee729311ef5b5c3003946c48f-Abstract-round1.html)

I think the authors should provide a figure with statistics pertaining to the interface atoms. I think showing any distribution differences between interface atoms determined according to either strategy (number of atoms, correlation between change in SASA and distance...) would be fundamental to understanding the two strategies. I think other statistics would constitute an enhancement as well (e.g. proportion of heavy vs. light chain residues).

Some obvious limitations of AACDB in its current form include:

AACDB only contains entries with protein-based antigens of at most 50 amino acids in length. This excludes non-protein-based antigens, such as carbohydrate- and nucleotide-based, as well as short peptide antigens.

AACDB does not include annotations of binding affinity, which are present in SAbDab and have been proven useful both for characterizing drivers of antibody-antigen interactions (cite https://www.sciencedirect.com/science/article/pii/S0969212624004362?via%3Dihub) and for benchmarking antigen-specific antibody-design algorithms (cite https://www.biorxiv.org/content/10.1101/2023.12.10.570461v1)).

In conclusion, I believe AACDB has the potential to be a more standardized and error-light database for antibody-antigen complex structures. It is, however, hard to evaluate the extent to which errors have been corrected since the authors do not provide a list of the errors or a step-by-step procedure for fixing the errors. Unfortunately, AACDB is currently missing binding affinity annotations, which hinders its usefulness.

Reviewer #2 (Public review):

Summary:

Antibodies, thanks to their high binding affinity and specificity to cognate protein targets, are increasingly used as research and therapeutic tools. In this work, Zhou et al. have created, curated, and made publicly available a new database of antibody-antigen complexes to support research in the field of antibody modelling, development, and engineering.

Strengths:

The authors have performed a manual curation of antibody-antigen complexes from the Protein Data Bank, rectifying annotation errors; they have added two methods to estimate paratope-epitope interfaces; they have produced a web interface that is capable of both effective visualisation and of summarising the key useful information in one page. The database is also cross-linked to other databases that contain information relevant to antibody developability and therapeutic applications.

Weaknesses:

The database does not import all the experimental information from PDB and contains only complexes with large protein targets.

Reviewer #1 (Public review):

Summary:

Mackie and colleagues compare chemosensory preferences between C. elegans and P. pacificus, and the cellular and molecular mechanisms underlying them. The nematodes have overlapping and distinct preferences for different salts. Although P. pacificus lacks the lsy-6 miRNA important for establishing asymmetry of the left/right ASE salt-sensing neurons in C. elegans, the authors find that P. pacificus ASE homologs achieve molecular (receptor expression) and functional (calcium response) asymmetry by alternative means. This work contributes an important comparison of how these two nematodes sense salts and highlights that evolution can find different ways to establish asymmetry in small nervous systems to optimize the processing of chemosensory cues in the environment.

Strengths:

The authors use clear and established methods to record the response of neurons to chemosensory cues. They were able to show clearly that ASEL/R are functionally asymmetric in P. pacificus, and combined with genetic perturbation establish a role for che-1-dependent gcy-22.3 in in the asymmetric response to NH4Cl.

Weaknesses:

The mechanism of lsy-6-independent establishment of ASEL/R asymmetry in P. pacificus remains uncharacterized.

Reviewer #2 (Public review):

Summary:

In this manuscript, Mackie et al. investigate gustatory behavior and the neural basis of gustation in the predatory nematode Pristionchus pacificus. First, they show that the behavioral preferences of P. pacificus for gustatory cues differ from those reported for C. elegans. Next, they investigate the molecular mechanisms of salt sensing in P. pacificus. They show that although the C. elegans transcription factor gene che-1 is expressed specifically in the ASE neurons, the P. pacificus che-1 gene is expressed in the Ppa-ASE and Ppa-AFD neurons. Moreover, che-1 plays a less critical role in salt chemotaxis in P. pacificus than C. elegans. Chemogenetic silencing of Ppa-ASE and Ppa-AFD neurons results in more severe chemotaxis defects. The authors then use calcium imaging to show that both Ppa-ASE and Ppa-AFD neurons respond to salt stimuli. Calcium imaging experiments also reveal that the left and right Ppa-ASE neurons respond differently to salts, despite the fact that P. pacificus lacks lsy-6, a microRNA that is important for ASE left/right asymmetry in C. elegans. Finally, the authors show that the receptor guanylate cyclase gene Ppa-gcy-23.3 is expressed in the right Ppa-ASE neuron (Ppa-ASER) but not the left Ppa-ASE neuron (Ppa-ASEL) and is required for some of the gustatory responses of Ppa-ASER, further confirming that the Ppa-ASE neurons are asymmetric and suggesting that Ppa-GCY-23.3 is a gustatory receptor. Overall, this work provides insight into the evolution of gustation across nematode species. It illustrates how sensory neuron response properties and molecular mechanisms of cell fate determination can evolve to mediate species-specific behaviors. However, the paper would be greatly strengthened by a direct comparison of calcium responses to gustatory cues in C. elegans and P. pacificus, since the comparison currently relies entirely on published data for C. elegans, where the imaging parameters likely differ. In addition, the conclusions regarding Ppa-AFD neuron function would benefit from additional confirmation of AFD neuron identity. Finally, how prior salt exposure influences gustatory behavior and neural activity in P. pacificus is not discussed.

Strengths:

(1) This study provides exciting new insights into how gustatory behaviors and mechanisms differ in nematode species with different lifestyles and ecological niches. The results from salt chemotaxis experiments suggest that P. pacificus shows distinct gustatory preferences from C. elegans. Calcium imaging from Ppa-ASE neurons suggests that the response properties of the ASE neurons differ between the two species. In addition, an analysis of the expression and function of the transcription factor Ppa-che-1 reveals that mechanisms of ASE cell fate determination differ in C. elegans and P. pacificus, although the ASE neurons play a critical role in salt sensing in both species. Thus, the authors identify several differences in gustatory system development and function across nematode species.

(2) This is the first calcium imaging study of P. pacificus, and it offers some of the first insights into the evolution of gustatory neuron function across nematode species.

(3) This study addresses the mechanisms that lead to left/right asymmetry in nematodes. It reveals that the ASER and ASEL neurons differ in their response properties, but this asymmetry is achieved by molecular mechanisms that are at least partly distinct from those that operate in C. elegans. Notably, ASEL/R asymmetry in P. pacificus is achieved despite the lack of a P. pacificus lsy-6 homolog.

Weaknesses:

(1) The authors observe only weak attraction of C. elegans to NaCl. These results raise the question of whether the weak attraction observed is the result of the prior salt environment experienced by the worms. More generally, this study does not address how prior exposure to gustatory cues shapes gustatory responses in P. pacificus. Is salt sensing in P. pacificus subject to the same type of experience-dependent modulation as salt sensing in C. elegans?

(2) A key finding of this paper is that the Ppa-CHE-1 transcription factor is expressed in the Ppa-AFD neurons as well as the Ppa-ASE neurons, despite the fact that Ce-CHE-1 is expressed specifically in Ce-ASE. However, additional verification of Ppa-AFD neuron identity is required. Based on the image shown in the manuscript, it is difficult to unequivocally identify the second pair of CHE-1-positive head neurons as the Ppa-AFD neurons. Ppa-AFD neuron identity could be verified by confocal imaging of the CHE-1-positive neurons, co-expression of Ppa-che-1p::GFP with a likely AFD reporter, thermotaxis assays with Ppa-che-1 mutants, and/or calcium imaging from the putative Ppa-AFD neurons.

(3) Loss of Ppa-che-1 causes a less severe phenotype than loss of Ce-che-1. However, the loss of Ppa-che-1::RFP expression in ASE but not AFD raises the question of whether there might be additional start sites in the Ppa-che-1 gene downstream of the mutation sites. It would be helpful to know whether there are multiple isoforms of Ppa-che-1, and if so, whether the exon with the introduced frameshift is present in all isoforms and results in complete loss of Ppa-CHE-1 protein.

(4) The authors show that silencing Ppa-ASE has a dramatic effect on salt chemotaxis behavior. However, these data lack control with histamine-treated wild-type animals, with the result that the phenotype of Ppa-ASE-silenced animals could result from exposure to histamine dihydrochloride. This is an especially important control in the context of salt sensing, where histamine dihydrochloride could alter behavioral responses to other salts.

(5) The calcium imaging data in the paper suggest that the Ppa-ASE and Ce-ASE neurons respond differently to salt solutions. However, to make this point, a direct comparison of calcium responses in C. elegans and P. pacificus using the same calcium indicator is required. By relying on previously published C. elegans data, it is difficult to know how differences in growth conditions or imaging conditions affect ASE responses. In addition, the paper would be strengthened by additional quantitative analysis of the calcium imaging data. For example, the paper states that 25 mM NH4Cl evokes a greater response in ASEL than 250 mM NH4Cl, but a quantitative comparison of the maximum responses to the two stimuli is not shown.

(6) It would be helpful to examine, or at least discuss, the other P. pacificus paralogs of Ce-gcy-22. Are they expressed in Ppa-ASER? How similar are the different paralogs? Additional discussion of the Ppa-gcy-22 gene expansion in P. pacificus would be especially helpful with respect to understanding the relatively minor phenotype of the Ppa-gcy-22.3 mutants.

(7) The calcium imaging data from Ppa-ASE is quite variable. It would be helpful to discuss this variability. It would also be helpful to clarify how the ASEL and ASER neurons are being conclusively identified during calcium imaging.

(8) More information about how the animals were treated prior to calcium imaging would be helpful. In particular, were they exposed to salt solutions prior to imaging? In addition, the animals are in an M9 buffer during imaging - does this affect calcium responses in Ppa-ASE and Ppa-AFD? More information about salt exposure, and how this affects neuron responses, would be very helpful.

(9) In Figure 6, the authors say that Ppa-gcy-22.3::GFP expression is absent in the Ppa-che-1(ot5012) mutant. However, based on the figure, it looks like there is some expression remaining. Is there a residual expression of Ppa-gcy-22.3::GFP in ASE or possibly ectopic expression in AFD? Does Ppa-che-1 regulate rGC expression in AFD? It would be helpful to address the role of Ppa-che-1 in AFD neuron differentiation.

Reviewer #1 (Public review):

Summary:

In this paper Kawasaki et al describe a regulatory role for the PIWI/piRNA pathway in rRNA regulation in Zebrafish. This regulatory role was uncovered through a screen for gonadogenesis defective mutants, which identified a mutation in the meioc gene, a coiled-coil germ granule protein. Loss of this gene leads to redistribution of Piwil1 from germ granules to the nucleolus, resulting in silencing of rRNA transcription.

Strengths:

Most of the experimental data provided in this paper is compelling. It is clear that in the absence of meioc, PiwiL1 translocates in to the nucleolus and results in down regulation of rRNA transcription. the genetic compensation of meioc mutant phenotypes (both organismal and molecular) through reduction in PiwiL1 levels are evidence for a direct role for PiwiL1 in mediating the phenotypes of meioc mutant.

Weaknesses:

Questions remain on the mechanistic details by which PiwiL1 mediated rRNA down regulation, and whether this is a function of Piwi in an unperturbed/wildtype setting. There is certainly some evidence provided in support of the natural function for piwi in regulating rRNA transcription (figure 5A+5B). However, the de-enrichment of H3K9me3 in the heterozygous (Figure 6F) is very modest and in my opinion not convincingly different relative to the control provided. It is certainly possible that PiwiL1 is regulating levels through cleavage of nascent transcripts. Another aspect I found confounding here is the reduction in rRNA small RNAs in the meioc mutant; I would have assumed that the interaction of PiwiL1 with the rRNA is mediated through small RNAs but the reduction in numbers do not support this model. But perhaps it is simply a redistribution of small RNAs that is occurring. Finally, the ability to reduce PiwiL1 in the nucleolus through polI inhibition with actD and BMH-21 is surprising. What drives the accumulation of PiwiL1 in the nucleolus then if in the meioc mutant there is less transcription anyway?

Despite the weaknesses outlined, overall I find this paper to be solid and valuable, providing evidence for a consistent link between PIWI systems and ribosomal biogenesis. Their results are likely to be of interest to people in the community, and provide tools for further elucidating the reasons for this link.

Reviewer #2 (Public review):

Summary:

In this study, the authors report that Meioc is required to upregulate rRNA transcription and promote differentiation of spermatogonial stem cells in zebrafish. The authors show that upregulated protein synthesis is required to support spermatogonial stem cells' differentiation into multi-celled cysts of spermatogonia. Coiled coil protein Meioc is required for this upregulated protein synthesis and for increasing rRNA transcription, such that the Meioc knockout accumulates 1-2 cell spermatogonia and fails to produce cysts with more than 8 spermatogonia. The Meioc knockout exhibits continued transcriptional repression of rDNA. Meioc interacts with and sequesters Piwil1 to the cytoplasm. Loss of Meioc increases Piwil1 localization to the nucleolus, where Piwil1 interacts with transcriptional silencers that repress rRNA transcription.

Strengths:

This is a fundamental study that expands our understanding of how ribosome biogenesis contributes to differentiation and demonstrates that zebrafish Meioc plays a role in this process during spermatogenesis. This work also expands our evolutionary understanding of Meioc and Ythdc2's molecular roles in germline differentiation. In mouse, the Meioc knockout phenocopies the Ythdc2 knockout, and studies thus far have indicated that Meioc and Ythdc2 act together to regulate germline differentiation. Here, in zebrafish, Meioc has acquired a Ythdc2-independent function. This study also identifies a new role for Piwil1 in directing transcriptional silencing of rDNA.

Weaknesses:<br /> There are limited details on the stem cell-enriched hyperplastic testes used as a tool for mass spec experiments, and additional information is needed to fully evaluate the mass spec results. What mutation do these testes carry? Does this protein interact with Meioc in the wildtype testes? How could this mutation affect the results from the Meioc immunoprecipitation?

Reviewer #3 (Public review):

Summary:

The paper describes the molecular pathway to regulate germ cell differentiation in zebrafish through ribosomal RNA biogenesis. Meioc sequesters Piwil1, a Piwi homolog, which suppresses the transcription of the 45S pre-rDNA by the formation of heterochromatin, to the perinuclear bodies. The key results are solid and useful to researchers in the field of germ cell/meiosis as well as RNA biosynthesis and chromatin.

Strengths:

The authors nicely provided the molecular evidence on the antagonism of Meioc to Piwil1 in the rRNA synthesis, which supported by the genetic evidence that the inability of the meioc mutant to enter meiosis is suppressed by the piwil1 heterozygosity.

Weaknesses:

(1) Although the paper provides very convincing evidence for the authors' claim, the scientific contents are poorly written and incorrectly described. As a result, it is hard to read the text. Checking by scientific experts would be highly recommended. For example, on line 38, "the global translation activity is generally [inhibited]", is incorrect and, rather, a sentence like "the activity is lowered relative to other cells" is more appropriate here. See minor points for more examples.<br /> (2) In some figures, it is hard for readers outside of zebrafish meiosis to evaluate the results without more explanation and drawing.<br /> (3) Figure 1E, F, cycloheximide experiments: Please mention the toxicity of the concentration of the drug in cell proliferation and viability.

Reviewer #1 (Public review):

Summary:

By way of background, the Jiang lab has previously shown that loss of the type II BMP receptor Punt (Put) from intestinal progenitors (ISCs and EBs) caused them to differentiate into EBs, with a concomitant loss of ISCs (Tian and Jiang, eLife 2014). The mechanism by which this occurs was activation of Notch in Put-deficient progenitors. How Notch was upregulated in Put-deficient ISCs was not established in this prior work. In the current study, the authors test whether a very low level of Dl was responsible. But co-depletion of Dl and Put led to a similar phenotype as depletion of Put alone. This result suggested that Dl was not the mechanism. They next investigate genetic interactions between BMP signaling and Numb, an inhibitor of Notch signaling. Prior work from Bardin, Schweisguth and other labs has shown that Numb is not required for ISC self-renewal. However the authors wanted to know whether loss of both the BMP signal transducer Mad and Numb would cause ISC loss. This result was observed for RNAi depletion from progenitors and for mad, numb double mutant clones. Of note, ISC loss was observed in 40% of mad, numb double mutant clones, whereas 60% of these clones had an ISC. They then employed a two-color tracing system called RGT to look at the outcome of ISC divisions (asymmetric (ISC/EB) or symmetric (ISC/ISC or EB/EB)). Control clones had 69%, 15% and 16%, respectively, whereas mad, numb double mutant clones had much lower ISC/ISC (11%) and much higher EB/EB (37%). They conclude that loss of Numb in moderate BMP loss of function mutants increased symmetric differentiation which lead caused ISC loss. They also reported that numb15 and numb4 clones had a moderate but significant increase in ISC-lacking clones compared to control clones, supporting the model that Numb plays a role in ISC maintenance. Finally, they investigated the relevance of these observation during regeneration. After bleomycin treatment, there was a significant increase in ISC-lacking clones and a significant decrease in clone size in numb4 and numb15 clones compared to control clones. Because bleomycin treatment has been shown to cause variation in BMP ligand production, the authors interpret the numb clone under bleomycin results as demonstrating an essential role of Numb in ISC maintenance during regeneration.

Strengths:

(i) Most data is quantified with statistical analysis<br /> (ii) Experiments have appropriate controls and large numbers of samples<br /> (iii) Results demonstrate an important role of Numb in maintaining ISC number during regeneration and a genetic interaction between Mad and Numb during homeostasis.

Weaknesses:

(i) No quantification for Fig. 1<br /> (ii) The premise is a bit unclear. Under homeostasis, strong loss of BMP (Put) leads to loss of ISCs, presumably regardless of Numb level (which was not tested). But moderate loss of BMP (Mad) does not show ISC loss unless Numb is also reduced. I am confused as to why numb does not play a role in Put mutants. Did the authors test whether concomitant loss of Put and Numb leads to even more ISC loss than Put-mutation alone.<br /> (iii) I think that the use of the word "essential" is a bit strong here. Numb plays an important role but in either during homeostasis or regeneration, most numb clones or mad, numb double mutant clones still have ISCs. Therefore, I think that the authors should temper their language about the role of Numb in ISC maintenance.

Reviewer #2 (Public review):

Summary:

This work assesses the genetic interaction between the Bmp signaling pathway and the factor Numb, which can inhibit Notch signalling. It follows up on the previous studies of the group (Tian, Elife, 2014; Tian, PNAS, 2014) regarding BMP signaling in controlling stem cell fate decision as well as on the work of another group (Sallé, EMBO, 2017) that investigated the function of Numb on enteroendocrine fate in the midgut. This is an important study providing evidence of a Numb-mediated back up mechanism for stem cell maintenance.

Strengths:

(1) Experiments are consistent with these previous publications while also extending our understanding of how Numb functions in the ISC.<br /> (2) Provides an interesting model of a "back up" protection mechanism for ISC maintenance.

Weaknesses:<br /> (1) Aspects of the experiments could be better controlled or annotated:<br /> (a) As they "randomly chose" the regions analyzed, it would be better to have all from a defined region (R4 or R2, for example) or to at least note the region as there are important regional differences for some aspects of midgut biology.<br /> (b) It is not clear to me why MARCM clones were induced and then flies grown at 18{degree sign}C? It would help to explain why they used this unconventional protocol.

(2) There are technical limitations with trying to conclude from double-knockdown experiments in the ISC lineage, such as those in Figure 1 where Dl and put are both being knocked down: depending on how fast both proteins are depleted, it may be that only one of them (put, for example) is inactivated and affects the fate decision prior to the other one (Dl) being depleted. Therefore, it is difficult to definitively conclude that the decision is independent of Dl ligand.

(3) Additional quantification of many phenotypes would be desired.<br /> (a) It would be useful to see esg-GFP cells/total cells and not just field as the density might change (2E for example).<br /> (b) Similarly, for 2F and 2G, it would be nice to see the % of ISC/ total cell and EB/total cell and not only per esgGFP+ cell.<br /> (c) Fig1: There is no quantification - specifically it would be interesting to know how many esg+ are su(H)lacZ positive in Put- Dl- condition compared to WT or Put- alone. What is the n?<br /> (d) Fig2: Pros + cells are not seen in the image? Are they all DllacZ+?<br /> (e) Fig3: it would be nice to have the size clone quantification instead of the distribution between groups of 2 cell 3 cells 4 cell clones.<br /> (f) How many times were experiments performed?

(4) The authors do not comment on the reduction of clone size in DSS treatment in Figure 6K. How do they interpret this? Does it conflict with their model of Bleo vs DSS?

(5) There is probably a mistake on sentence line 314 -316 "Indeed, previous studies indicate that endogenous Numb was not undetectable by Numb antibodies that could detect Numb expression in the nervous system".

Reviewer #3 (Public review):

Summary:

The authors provide an in-depth analysis of the function of Numb in adult Drosophila midgut. Based on RNAi combinations and double mutant clonal analyses, they propose that Numb has a function in inhibiting Notch pathway to maintain intestinal stem cells, and is a backup mechanism with BMP pathway in maintaining midgut stem cell mediated homeostasis.

Strengths:

Overall, this is a carefully constructed series of experiments, and the results and statistical analyses provides believable evidence that Numb has a role, albeit weak compared to other pathways, in sustaining ISC and in promoting regeneration especially after damage by bleomycin, which may damage enterocytes and therefore disrupt BMP pathway more. The results overall support their claim.

The data are highly coherent, and support a genetic function of Numb, in collaborating with BMP signaling, to maintain the number and proliferative function of ISCs in adult midguts. The authors used appropriate and sophisticated genetic tools of double RNAi, mutant clonal analysis and dual marker stem cell tracing approaches to ensure the results are reproducible and consistent. The statistical analyses provide confidence that the phenotypic changes are reliable albeit weaker than many other mutants previously studied.

Weaknesses:<br /> In the absence of Numb itself, the midgut has a weak reduction of ISC number (Fig. 3 and 5), as well as weak albeit not statistically significant reduction of ISC clone size/proliferation. I think the authors published similar experiments with BMP pathway mutants. The mad1-2 allele used here as stated below may not be very representative of other BMP pathway mutants. Therefore, it could be beneficial to compare the number of ISC number and clone sizes between other BMP experiments to provide the readers with a clearer picture of how these two pathways individually contribute (stronger/weaker effects) to the ISC number and gut homeostasis.

The main weakness of this manuscript is the analysis of the BMP pathway components, especially the mad1-2 allele. The mad RNAi and mad1-2 alleles (P insertion) are supposed to be weak alleles and that might be suitable for genetic enhancement assays here together with numb RNAi. However, the mad1-2 allele, and sometimes the mad RNAi, showed weakly increased ISC clone size. This is kind of counter-intuitive that they should have a similar ISC loss and ISC clone size reduction.

A much stronger phenotype was observed when numb mutants were subject to treatment of tissue damaging agents Bleomycin, which causes damage in different ways than DSS. Bleomycin as previously shown to be causing mainly enterocyte damage, and therefore disrupt BMP signaling from ECs more likely. Therefore, this treatment together with loss of numb led to a highly significant reduction of ISC in clones and reduction of clone size/proliferation. One improvement is that it is not clear whether the authors discussed the nature of the two numb mutant alleles used in this study and the comparison to the strength of the RNAi allele. Because the phenotypes are weak and more variable, the use of specific reagents is important.

Furthermore, the use of possible activating alleles of either or both pathways to test genetic enhancement or synergistic activation will provide strong support for the claims.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors set out to determine how a DNA demethylation enzyme TET2 regulates beta cell senescence in the context of Type 2 Diabetes and aging. They analyze public RNA-seq data and found upregulation of TET2 coincident with downregulation of MOF and PTEN, genes involved in chromatin regulation and cell cycle. TET2 is upregulated during aging, high-fat diet feeding, high glucose on rat beta cell line INS1E, and in leptin receptor deficient (db/db) mice islets. This was not found for TET1 and TET3. TET2 global KO mice show improved glucose tolerance during aging, but not TET1 or TET3. The authors show improved beta cell identity genes in TET2 KO islets. They they performed DNA methyalation/hydroxymethylation analyses of TET2 KO transformed rat beta cell line INS1E followed by ChIP-seq of Histone H4K16 acetylation to find this mark relies on TET2 expression. Finally they demonstrate in the cell lines that overexpressing TET2 leads to loss of MOF and increased PTEN and p16, linking TET2 to a regulatory mechanism with these factors that may influence senescence.

Strengths:

The study uses a number of orthogonal approaches and evidence from cell lines and the genetic TET2 KO as well as primary islets. The concept is interesting and potentially useful to the field. Efforts were made to examine TET1 and TET3 paralogues to rule out their compensation.

Weaknesses:

The study has several major weaknesses that mean the data presented did not fully support the main conclusions. These include the following:

(1) From the beginning of the manuscript the authors first sentence does not seem to indicate which datasets were analysed, the rationale behind why public datasets were used and what the main conclusions are being drawn from the plots shown throughout Fig. 1. This section of the manuscript was very hard to follow, and lacked rationale and explanation as to what these data show.

(2) All of the metabolic phenotypic data come from global TET2 KO mice, where TET2 is lost from all cells. The authors need to use a beta cell-specific KO of TET2 to ensure that metabolic changes are not due to cross-talk with other tissues (e.g. liver, adipose, even effects on central control of metabolism). No insulin tolerance tests were done to ascertain phenotypes in other metabolic tissues. This was a major weakness of the study. The authors should also provide clear validation of their global TET2 KO mice demonstrating a total lack of protein in islets and metabolic tissues.

(3) TET2 localization and expression pattern in islets was not clearly demonstrated and the data shown are not convincing from Fig 3 and Fig 4. In Fig 3e the staining for TET2 in green looks ubiquitous in acinar tissue (not nuclear) and not in the islet. In Fig 4d there is an increase in nuclear stain shown during aging, but no INS stain is used to show specificity to beta cells. Thus there is not sufficient data to support the expression pattern and localization of TET2 and specificity of the antibody.

(4) In Fig. 5: The effect sizes for the beta cell identity gene expression differences by qRT-PCR between WT and TET2 KO islets shown in Fig 5 are extremely modest so as to be questionable whether they are biologically meaningful. The same is true of the senescence markers quantified from isolated islets by qRT-PCR in Fig 5f. The immunostains for Pdx1 are hard to see and signal should be quantified. The SA-Bgal staining is quantified but no representative image is shown. The p16 immunostaining is not clear and should be quantified. Given that a lack of truly specific p16 antibodies in mouse immunostainings have been a major issue for the field, the authors would be advised to demonstrate specificity of the antibody if possible on mouse KO tissue, or to at least validate the predicted increase in p16 staining comparing young versus old pancreas as has been shown in other studies.

(5) Throughout the manuscript the figures colors are difficult to see and text difficult to read. Text in the p-values above the bars on most Figures is not legible (particularly Figs 4, 5, and 9). The legends simply do not contain sufficient information to interpret the data panels. This is true from Figures 1 through 9. P-value and specific statistical tests are missing from legends as well. For instance, in Fig 6c, what is being shown in LV-Ctrl vs LV-TET2 and why are these sample labels the same for two sets of images with two different outcomes of the staining? How many cells were quantified here?

(6) There is an over-reliance on cell lines throughout the manuscript. INS1E and BTC6 are not truly representative of mature adult mouse or rat beta cells, and hence the connections between H4K16ac/MOF/PTEN and TET2 must be assessed in primary mouse or rat islets to confirm these phenotypes.

(7) In the in vitro studies of senescence markers, it is not convincingly shown that the cells are actually senescent. Even though there changes found in expression of p16 and SA-Bgal in the cultures, the authors did not evaluate key senescence phenotypes such as the actual cell cycle arrest, SASP proteins or apoptosis resistance. Are the cells actually senescent or are these markers simply increasing? Hence much of the changes driven by TET2 overexpression in the in vitro cell lines could likely changes in p16 protein but not actually a senescence phenotype. BTC6, INS1E, and MIN6 are cell lines that are transformed, and while they can undergo some senescence-like changes in response to specific stressors like lipotoxicity, DNA damage, or oxidative stress, the authors did not evaluate these, only senescence genes/proteins in otherwise unstressed cells. Thus the claim that TET2 modifies senescence of beta cells remains unsubstantiated from the in vitro studies. It was not clear how any of these studies related to beta cell senescence in T2DM where there is metabolic and/or gluco-lipotoxic stress. Although it is claimed from Fig 9 that TET2 regulates PTEN/MOF axis to regulate beta cell function, no functional data (e.g. GSIS) are shown.

(8) There were issues and difficulties with the writing in the introduction and discussion in that they did not clearly or adequately describe, discuss or interpret the main conclusions and their significance. The work is not positioned within the current state of the field and it is very difficult to follow the rationales for the study and the advances in knowledge provided.

Reviewer #2 (Public review):

Summary:<br /> Epigenetic regulation is critical for maintaining cellular function, and its dysregulation contributes to senescence and disease. This manuscript investigates the role of TET2 in β cell aging, proposing that TET2-mediated PTEN DNA methylation promotes H4K16 acetylation (H4K16ac) through MOF, driving β cell senescence. Using TET2 inhibitors, RNA interference, lentiviral overexpression, and knockout mouse models, the authors aim to establish TET2 as a key player in β cell aging and a potential therapeutic target in type 2 diabetes mellitus (T2DM).<br /> However, significant limitations reduce the manuscript's impact. Figures are poorly presented, with illegible fonts and unquantified staining panels, while key analyses, such as β cell specificity and senescence inducers, are missing. The rationale for focusing on H4K16ac and MOF is unclear, and the authors fail to address whether β cell identity gene changes reflect altered gene expression or mass. Additionally, critical controls, such as low-fat diet cohorts, are absent, and the writing lacks clarity and coherence. Together, these weaknesses undermine the validity of the findings.

Main Comments<br /> Figures 1 and 2:<br /> The fonts in Figures 1 and 2 are barely visible and should be improved for readability. Additionally, do TET2 protein levels change in mouse and human β cells with aging? Is there evidence from regression analyses using single-cell RNA sequencing on human islets that TET2 expression correlates with age-associated gene signatures in β cells? Are these correlations specific to β cells, or do they extend to other islet cell types? It would also be informative to assess whether TET2 levels increase with senescence inducers such as DNA damage agents (e.g., bleomycin, doxorubicin) or reactive oxygen species (e.g., H₂O₂).<br /> Figure 3:<br /> Why do TET2 protein levels appear stronger in acinar cells? Additionally, the predominant cellular localization of TET2 seems to be cytoplasmic. Can the authors clarify or expand on this observation?<br /> Figure 4:<br /> The data on the impact of TET2 insufficiency in vivo is compelling. There are several quality control experiments to validate their model and main hypothesis (That T2t2 expression increases with aging in beta-cells). Here, authors have the right system to validate their initial Tet2 protein dynamics in the mouse, since they have a KO mouse model. Here, it would be useful to co-stain Tet2 with insulin and glucagon, to infer the dynamics of Tet2 in the two most abundant islet cell types.<br /> Figure 5:<br /> The upregulation of β-cell identity genes in the KO mouse model raises an important question: Is this effect due to an actual increase in gene expression or simply a higher proportion of β cells? Quantifying β-cell mass and performing gene expression analyses on FACS-sorted β cells would help address this. Additionally, the staining panels lack quantification. For instance, GLUT2 staining appears cytoplasmic when it should be membranous. The authors focus on cellular senescence, but does apoptosis increase in wild-type mice under a high-fat diet (HFD)? Including animals on a low-fat diet (LFD) for comparison would add valuable context.<br /> Figure 6:<br /> The data suggest an increase in cell numbers in TET2-overexpressing cells. Does this indicate an effect on β-cell proliferation? Quantification would provide clarity.<br /> Figure 8:<br /> The rationale for focusing on H4K16ac is insufficiently discussed. What is the mechanism linking TET2-induced changes to decreased H4K16ac levels? Including a more thorough explanation in the introduction and discussion would enhance the manuscript.<br /> Figure 9:<br /> The introduction lacks any discussion of H4K16ac or MOF. The discussion paragraph (lines 530-540) that elaborates on these points should instead be moved to the introduction to improve the manuscript's flow. Furthermore, the authors should cite their 2022 paper on H4K16ac as part of the rationale for focusing on this histone modification.

Minor Comments:<br /> The manuscript would benefit from language refinement. Examples include:<br /> Line 183: Replace "the blood included" with a more precise description.<br /> Line 315: "treated with RNA seq" should be rephrased to clarify methodology (e.g., "analyzed via RNA sequencing").<br /> Line 456: Replace "expression of H4K16ac" with "levels of H4K16ac."<br /> Line 496: The phrase "can solve scientific problems from multiple dimensions" sounds vague and overly broad; consider rephrasing to be more specific.

Reviewer #3 (Public review):

Summary:<br /> This study advances the field of β cell dysfunction by unveiling an epigenetic mechanism of β cell senescence. By connecting TET2-mediated DNA methylation to histone acetylation and cellular aging, it opens promising new avenues for therapeutic intervention. In particular, the authors aimed at identifying the mechanisms of pancreatic β cell senescence by epigenetic regulation. They conclude that increased TET2 expression in β cells is associated with ageing and metabolic dysfunction in type 2 diabetes by inducing β cell senescence. The authors further propose that TET2-mediated PTEN promoter methylation promotes β cell senescence by regulating H4K16ac. Last, the authors suggest that this could represent new molecular mechanism and therapeutic target against β cell senescence during type 2 diabetes.

Strengths:<br /> The major strengths of the study are the use of both biased and unbiased experimental tools to approach the topic. The authors also provide in vivo and in vitro mechanistic approaches to answer their questions. All of these approaches are valuable and provides robustness to their study. The authors provide solid evidence that TET2 is associated with ageing and that its absence improves glucose metabolism in ageing and β cell senescence. In addition, the mechanistic studies showing that TET2 regulates the PTEN/MOF/H4K16ac signaling pathway in β cell lines is convincing.

Weaknesses:<br /> Although the use of such a variety of tools is a strength, the outcome of each individual tool is somehow superficial. For instance, the authors focus on very specific targets emanating from their omics studies without a clear or logical justification. In addition, the metabolic studies are inaccurate and the authors do not follow an understandable and rational examination of the ageing versus their obesity cohorts. Last, the mechanistic studies using model cell lines are not validated in the available mouse models.

In my opinion, the evidence that TET2 regulates β cell senescence during obesity is not very strong. This is because the effect of deletion of TET2 in senescence markers is the same under 24weeks of age or 52 weeks of age (16 weeks HFD). Both ageing and HFD promoted the same extent of reduction of senescent markers and increase in β cell markers in the absence of TET2. There is no comparison between young glucose tolerant mice and old glucose intolerant mice. There is also no direct comparison of aged matched lean or obese mice. It may seem as if the mechanism by which TET2 regulates senescence in β cells is independent of the diabetic status but it is more related to ageing. Given that there is evidence that TET2 expression in β cells coordinates inflammatory responses in autoimmune diabetes, it would have been interested to check whether this is also the case for T2DM. Also, considering that expression of TET2 in Figure 3 does not seem to be in β cells in db/db mice but rather in the exocrine pancreas. In addition, senescent marker p16 in Figure 5 in the presence of TET2, seems to be localized in alpha cells or immune cells but not in β cells.<br /> Regarding the mechanistic studies, the authors convincingly show that TET2 regulates the PTEN/MOF/H4K16ac signaling pathway in β cell lines and that this is important for β cell senescence. However, there is no validation of whether this holds true in aged, or prediabetic, mice. Given the availability of mice and model samples, this should be possible and meaningful. Last, in the genome-wide bisulfite sequencing (Figure 7), it seems that the authors are cherry picking for PTEN and in the RNAseq, the same applies for MOF. Thus, although the mechanism seems valid, the lack of in vivo validation, and a proper rational for the selected targets in the omics studies, renders the mechanistic studies rather correlative.

In sum, I believe that the study in its current version, unfortunately, does not bear the conceptual advance or the robustness that is required to offer a strong impact on the field. The methods, on the other hand, mainly the omics analyses provided here, could be of potential benefit for the field of epigenetics in β cell biology. However, in the benefit of the current study, the relevance of this data could be more rigorously assessed experimentally. I believe that the study has the potential to provide the required impact, should the authors work on it further to provide more solid functional and mechanistic validation.

Reviewer #1 (Public review):

Summary

This very interesting article describes extensive work by the authors connecting topoisomerase 2 to aging across multiple model systems. The authors began by analyzing published transcriptomes for genes previously reported to be connected to increased lifespan in S. cerevisiae, focusing on genes whose downregulation is highly correlated with increased lifespan. One of these candidates was topoisomerase 2, which had previously been shown to be connected to lifespan in yeast.

The authors here show that reduction in topoisomerase 2 levels can significantly extend lifespan in yeast (by damp), C. elegans (by RNAi), and mice (by CRISPR CasRx).

Next, the authors demonstrate in both C. elegans and mice that in addition to increased survival times, animals with decreased top2 levels also show increased healthspan, as measured by using rates of body bends and of pharyngeal pumping in C. elegans, and using the Frailty Index (FI) for mice. Further, they report that lowered top2 levels result in less aged tissue phenotypes in multiple tissues in mice as assayed by histology, and positively affect multiple hallmarks of aging in both mouse tissues and human IMR-90 cells.

The authors go on to perform thorough transcriptomic analysis of reduced top2 animals in both C. elegans and mice. Many interesting GO terms are highly overrepresented among both up- and down-regulated transcripts from these experiments, and the authors conclude that in the case of mice there is significant tissue specific biology based on differing results in the tissues they examined.

Given the previously known biological roles of top2, the authors looked at changes in the epigenetic landscape of reduced top2 organisms as evidenced by changes in H3K4me3, H3K9me3 and H3K27me3. Overall, the authors conclude from these data that reduction of top2 "differentially down-regulates genes with active promoters/high abundance".

Overall this well-written manuscript summarizes a great deal of new data that will be of great interest to aging researchers broadly.

The figures and tables are all very clear and well-designed, and all add greatly to the manuscript overall including the use of color which is in all cases justified.

Reviewer #2 (Public review):

Summary:

Previous studies have shown that Topoisomerase 2 (Top2) depletion in yeast can extend the lifespan of the organism, but no known mechanisms have been reported. In the current study, Zhu et al. reported that reduction of Top2 enhances longevity and mitigates aging phenotypes across multiple model organisms, including not yeast, but also C. elegans and mouse. The evidence of reduction of aging phenotypes is particularly strong, which include markers of cellular senescence, nutrient sensing, epigenetic markers, and lysosome biogenesis. They propose that Top2b reduction confers longevity through a conserved mechanism, and may be used a novel therapeutic strategy for countering aging. Overall, their findings should be of broad interest to the fields of Aging and Topoisomerase research. The technical quality of the work is in general solid but can be improved.

Strengths:

Top2 is an essential type II topoisomerase that resolves DNA topological stress generated during transcription, replication, chromosome segregation, and other DNA metabolic processes by introducing transient double-strand breaks (DSBs), passing the DNA strands, and re-ligating them. Top2 is a target for anticancer therapies, but its connection to aging and longevity remains largely unexplored. The authors' findings are notable, as Top2 has been deemed indispensable for normal development. Yet, this study suggests that its reduction confers benefits in the context of healthy aging. Their results convincingly show extended lifespan and improvements in physiological and molecular aging phenotypes, supported by behavioral assays and tissue morphology analyses.

Weaknesses:

Despite these strengths, the manuscript is weak on the proposed "conserved mechanism". The authors proposed in Discussion that Top2/Top2b knockdown may be similar to the classical insulin/IGF1 and the mTORC pathway, but did not provide any genetic evidence to support this.

The authors also mentioned in the Discussion that the potential mechanism could be selective down-regulation of transcription of genes of active promoter and high abundance, such as ribosomal genes, which could be relevant to yeast aging. But there is no evidence in worms or mouse that Top2b directly binds and promotes transcription of certain high abundance genes critical for aging.

I understand that this mechanism issue may be difficult to address, and I do not expect that the authors can fully address this issue. However, as both yeast and worms have been widely-used in aging studies with many tools available, I suggest that the authors can improve their studies by performing the following experiments.

Reviewer #1 (Public review):

Koren et al. derive and analyse a spiking network model optimised to represent external signals using the minimum number of spikes. Unlike most prior work using a similar setup, the network includes separate populations of excitatory and inhibitory neurons. The authors show that the optimised connectivity has a like-to-like structure, which leads to the experimentally observed phenomenon of feature competition. The authors also examine how various (hyper)parameters-such as adaptation timescale, the excitatory-to-inhibitory cell ratio, regularization strength, and background current-affect the model. These findings add biological realism to a specific implementation of efficient coding. They show that efficient coding explains, or at least is consistent with, multiple experimentally observed properties of excitatory and inhibitory neurons.

As discussed in the first round of reviews, the model's ability to replicate biological observations such as the 4:1 ratio of excitatory vs. inhibitory neurons hinges on somewhat arbitrary hyperparameter choices. Although this may limit the model's explanatory power, the authors have made significant efforts to explore how these parameters influence their model. It is an empirical question whether the uncovered relationships between, e.g., metabolic cost and the fraction of excitatory neurons are biologically relevant.

The revised manuscript is also more transparent about the model's limitations, such as the lack of excitatory-excitatory connectivity. Further improvements could come from explicitly acknowledging additional discrepancies with biological data, such as the widely reported weak stimulus tuning of inhibitory neurons in the primary sensory cortex of untrained animals.

Reviewer #2 (Public review):

Summary:

In this work, the authors present a biologically plausible, efficient E-I spiking network model and study various aspects of the model and its relation to experimental observations. This includes a derivation of the network into two (E-I) populations, the study of single-neuron perturbations and lateral-inhibition, the study of the effects of adaptation and metabolic cost, and considerations of optimal parameters. From this, they conclude that their work puts forth a plausible implementation of efficient coding that matches several experimental findings, including feature-specific inhibition, tight instantaneous balance, a 4 to 1 ratio of excitatory to inhibitory neurons, and a 3 to 1 ratio of I-I to E-I connectivity strength.

Strengths:

While many network implementations of efficient coding have been developed, such normative models are often abstract and lacking sufficient detail to compare directly to experiments. The intention of this work to produce a more plausible and efficient spiking model and compare it with experimental data is important and necessary in order to test these models. In rigorously deriving the model with real physical units, this work maps efficient spiking networks onto other more classical biophysical spiking neuron models. It also attempts to compare the model to recent single-neuron perturbation experiments, as well as some long-standing puzzles about neural circuits, such as the presence of separate excitatory and inhibitory neurons, the ratio of excitatory to inhibitory neurons, and E/I balance. One of the primary goals of this paper, to determine if these are merely biological constraints or come from some normative efficient coding objective, is also important. Lastly, though several of the observations have been reported and studied before, this work arguably studies them in more depth, which could be useful for comparing more directly to experiments.

Weaknesses:

This work is the latest among a line of research papers studying the properties of efficient spiking networks. Many of the characteristics and findings here have been discussed before, thereby limiting the new insights that this work can provide. Thus, the conclusions of this work should be considered and understood in the context of those previous works, as the authors state. Furthermore, the number of assumptions and free parameters in the model, though necessary to bring the model closer to biophysical reality, make it more difficult to understand and to draw clear conclusions from. As the authors state, many of the optimality claims depend on these free parameters, such as the dimensionality of the input signal (M=3), the relative weighting of encoding error and metabolic cost, and several others. This raises the possibility that it is not the case that the set of biophysical properties measured in the brain are accounted for by efficient coding, but rather that theories of efficient coding are flexible enough to be consistent with this regime. With this in mind, some of the conclusions made in the text may be overstated and should be considered in this light.

Conclusions, Impact, and additional context:

Notions of optimality are important for normative theories, but they are often studied in simple models with as few free parameters as possible. Biophysically detailed and mechanistic models, on the other hand, will often have many free parameters by their very nature, thereby muddying the connection to optimality. This tradeoff is an important concern in neuroscientific models. Previous efficient spiking models have often been criticized for their lack of biophysically-plausible characteristics, such as large synaptic weights, dense connectivity, and instantaneous communication. This work is an important contribution in showing that such networks can be modified to be much closer to biophysical reality without losing their essential properties. Though the model presented does suffer from complexity issues which raise questions about its connections to "optimal" efficient coding, the extensive study of various parameter dependencies offers a good characterization of the model and puts its conclusions in context.

Reviewer #3 (Public review):

Summary:

In their paper the authors tackle three things at once in a theoretical model: how can spiking neural networks perform efficient coding, how can such networks limit the energy use at the same time, and how can this be done in a more biologically realistic way than previous work.

They start by working from a long-running theory on how networks operating in a precisely balanced state can perform efficient coding. First, they assume split networks of excitatory (E) and inhibitory (I) neurons. The E neurons have the task to represent some lower dimensional input signal, and the I neurons have the task to represent the signal represented by the E neurons. Additionally, the E and I populations should minimize an energy cost represented by the sum of all spikes. All this results in two loss functions for the E and I populations, and the networks are then derived by assuming E and I neurons should only spike if this improves their respective loss. This results in networks of spiking neurons that live in a balanced state, and can accurately represent the network inputs.

They then investigate in depth different aspects of the resulting networks, such as responses to perturbations, the effect of following Dale's law, spiking statistics, the excitation (E)/inhibition (I) balance, optimal E/I cell ratios, and others. Overall, they expand on previous work by taking a more biological angle on the theory and show the networks can operate in a biologically realistic regime.

Strengths:

* The authors take a much more biological angle on the efficient spiking networks theory than previous work, which is an essential contribution to the field<br /> * They make a very extensive investigation of many aspects of the network in this context, and do so thoroughly<br /> * They put sensible constraints on their networks, while still maintaining the good properties these networks should have

Weaknesses:

* One of the core goals of the paper is to make a more biophysically realistic network than previous work using similar optimization principles. One of the important things they consider is a split into E and I neurons. While this works fine, and they consider the coding consequences of this, it is not clear from an optimization perspective why the split into E and I neurons and following Dale's law would be beneficial. This would be out of scope for the current paper however.<br /> * The theoretical advances in the paper are not all novel by themselves, as most of them (in particular the split into E and I neurons and the use of biophysical constants) had been achieved in previous models. However, the authors discuss these links thoroughly and do more in-depth follow-up experiments with the resulting model.

Assessment and context:

Overall, although much of the underlying theory is not necessarily new, the work provides an important addition to the field. The authors succeeded well in their goal of making the networks more biologically realistic, and incorporate aspects of energy efficiency. For computational neuroscientists this paper is a good example of how to build models that link well to experimental knowledge and constraints, while still being computationally and mathematically tractable. For experimental readers the model provides a clearer link of efficient coding spiking networks to known experimental constraints and provides a few predictions.

Reviewer #1 (Public review):

Summary:

The Authors investigated the anatomical features of the excitatory synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of the synapse, the macular or the perforated appearance and the size of the synaptic active zone, the number and volume of the mitochondria, the number of the synaptic and the dense core vesicles, also differentiating between the readily releasable, the recycling and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The Authors conclude that the subcellular morphology of the layer 1 synapses is suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow the glutamate spillover from the synapses enhancing synpatic crosstalk within this cortical layer.

Strengths:

The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable, since this is a highly time- and energy consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the Authors are all solid, strengthen this manuscript, and mainly support the conclusions drawn in the discussion.

Comments on latest version:

The corrected version of the article titled „Ultrastructural sublaminar specific diversity of excitatory synaptic boutons in layer 1 of the adult human temporal lobe neocortex" has been improved thanks to the comments and suggestions of the reviewers. The Authors implemented several of my comments and suggestions. However, many of them were not completed. It is understandable that the Authors did not start a whole new series of experiment investigating inhibitory synapses (as it was a misunderstanding affecting 2 reviewers from the three). But the English text is still very hard to understand and has many mistakes, although I suggested to extensively review the use of English. Furthermore, my suggestion about avoiding many abbreviations in the abstract, analyse and discuss more the perforated synapses, the figure presentation (Figure 3) and including data about the astrocytic coverage in the Results section were not implemented. My questions about the number of docked vesicles and p10 vesicles, as well as about the different categories of the vesicle pools have not been answered neither. Many other minor comments and suggestions were answered, corrected and implemented, but I think it could have been improved more if the Authors take into account all of the reviewers' suggestions, not only some of them. I still have several main and minor concerns, with a few new ones as well I did not realized earlier, but still think it is important.

Main concerns:

(1) Epileptic patients:<br /> As all patients were epileptic, it is not correct to state in the abstract that non-epileptic tissue was investigated. Even if the seizure onset zone was not in the region investigated, seizures usually invade the temporal lobe in TLE. If you can prove that no spiking activity occured in the sample you investigated and the seizures did not invade that region, then you can write that it is presumably non-epileptic. I would suggest to write „L1 of the human temporal lobe neocortical biopsy tissue". See also Methods lines 608-612. Write only „non-epileptic" or „non-affected" if you verified it with EcoG. If this was the case, please write a few sentences about it in the Methods.

(2) About the inhibitory/excitatory synapses.<br /> Since our focus was on excitatory synaptic boutons as already stated in the title we have not analyzed inhibitory SBs.<br /> Now, I do understand that only excitatory synapses were investigated. Although it was written in the title, I did not realized, since all over the manuscript the Authors were writing synapses, and were distinguishing between inhibitory and excitatory syanpses in the text and showing numerous excitatory and inhibitory synapses on Figure 2 and discussing inhibitory interneurons in the Discussion as well. Maybe this was the reason why two reviewers out of the three (including myself) thought you investigated both types of synapses but did not differentiated between them. So, please, emphasize in the Abstract (line 40), Introduction (for ex. line 92-97) and the Discussion (line 369) that only excitatory synaptic boutons were investigated.<br /> As this paper investigated only excitatory synaptic boutons, I think it is irrelevant to write such a long section in the Discussion about inhibitory interneurons and their functions in the L1 of the human temporal lobe neocortex. Same applies to the schematic drawing of the possible wiring of L1 (Figure 7). As no inhibitory interneurons were examined, neither the connection of the different excitatory cells, only the morphology of single synaptic boutons without any reference on their origin, I think this figure does not illustrate the work done in this paper. This could be a figure of a review paper about the human L1, but is is inappropriate in this study.

(3) Perforated synapses<br /> "the findings of the Geinismann group suggesting that perforated synapses are more efficient than non-perforated ones is nowadays very controversially discussed"<br /> I did not ask the Authors to say that perforated synapses are more efficient. However, based on the literature (for ex. Harris et al, 1992; Carlin and Siekievitz, 1982; Nieto-Sampedro et al., 1982) the presence of perforated synapses is indeed a good sign of synapse division/formation - which in turn might be coupled to synaptic plasticity (Geinisman et al, 1993), increased synaptic activity (Vrensen and Cardozo, 1981), LTP (Geinisman et al, 1991, Harris et al, 2003), pathological axonal sprouting (Frotscher et al, 2006), etc. I think it is worth mentioning this at least in the Discussion.

(4) Question about the vesicle pools<br /> Results, Line 271: Still not understandable, why the RRP was defined as {less than or equal to}10 nm and {less than or equal to}20nm. Why did you use two categories? One would be sufficient (for example {less than or equal to}20nm). Or the vesicles between 10 and 20nm were considered to be part of RRP? In this case there is a typo, it should be {greater than or equal to}10 nm and {less than or equal to}20nm.<br /> The answer of the Authors was to my question raised: We decided that also those very close within 10 and 20 nm away from the PreAZ, which is less than a SV diameter may also contribute to the RRP since it was shown that SVs are quite mobile.<br /> This does not clarify why did you use two categories. Furthermore, I did not receive answer (such as Referee #2) for my question on how could you have 3x as many docked vesicles than vesicles {less than or equal to}10nm. The category {less than or equal to}10nm should also contain the docked vesicles. Or if this is not the case, please, clarify better what were your categories.

(5) Astrocytic coverage<br /> On Fig. 6 data are presented on the astrocytic coverage derived from L1 and L4. In my previous review I asked to include this in the text of the Results as well, but I still do not see it. It is also lacking from the Results how many samples from which layer were investigated in this analysis. Only percentages are given, and only for L1 (but how many patients, L1a and/or L1b and/or L4 is not provided). In contrast, Figure 6 and Supplementary Table 2 (patient table) contains the information that this analysis has been made in L4 as well. Please, include this information in the text as well (around lines 348-360).<br /> About how to determine glial elements. I cannot agree with the Authors that glial elements can be determined with high certainty based only on the anatomical features of the profiles seen in the EM. „With 25 years of experience in (serial) EM work" I would say, that glial elements can be very similar to spine necks and axonal profiles.<br /> All in all, if similar methods were used to determine the glial coverage in the different layers of the human neocortex, than it can be compared (I guess this is the case). However, I would say in the text that proper determination would need immunostaining and a new analysis. This only gives an estimatation with the possibility of a certain degree of error.

(6) Large interindividual differences in the synapse density should be discussed in the Discussion.

Reviewer #2 (Public review):

Summary:

The study of Rollenhagen et al examines the ultrastructural features of Layer 1 of human temporal cortex. The tissue was derived from drug-resistant epileptic patients undergoing surgery, and was selected as further from the epilepsy focus, and as such considered to be non-epileptic. The analyses has included 4 patients with different age, sex, medication and onset of epilepsy. The MS is a follow-on study with 3 previous publications from the same authors on different layers of the temporal cortex:

Layer 4 - Yakoubi et al 2019 eLife<br /> Layer 5 - Yakoubi et al 2019 Cerebral Cortex,<br /> Layer 6 - Schmuhl-Giesen et al 2022 Cerebral Cortex

They find, the L1 synaptic boutons mainly have single active zone a very large pool of synaptic vesicles and are mostly devoid of astrocytic coverage.

Strengths:

The MS is well written easy to read. Result section gives a detailed set of figures showing many morphological parameters of synaptic boutons and surrounding glial elements. The authors provide comparative data of all the layers examined by them so far in the Discussion. Given that anatomical data in human brain are still very limited, the current MS has substantial relevance.<br /> The work appears to be generally well done, the EM and EM tomography images are of very good quality. The analyses is clear and precise.

Weaknesses:

The authors made all the corrections required, answered most of my concerns, included additional data sets, and clarified statements where needed.

My remaining points are:

Synaptic vesicle diameter (that has been established to be ~40nm independent of species) can properly be measured with EM tomography only, as it provides the possibility to find the largest diameter of every given vesicle. Measuring it in 50 nm thick sections result in underestimation (just like here the values are ~25 nm) as the measured diameter will be smaller than the true diameter if the vesicle is not cut in the middle, (which is the least probable scenario). The authors have the EM tomography data set for measuring the vesicle diameter properly.

It is a bit misleading to call vesicle populations at certain arbitrary distances from the presynaptic active zone as readily releasable pool, recycling pool and resting pool, as these are functional categories, and cannot directly be translated to vesicles at certain distances. Even it is debated whether the morphologically docked vesicles are the ones, that are readily releasable, as further molecular steps, such as proper priming is also a prerequisite for release.<br /> It would help to call these pools as "putative" correlates of the morphological categories.

Reviewer #3 (Public review):

Summary:

Rollenhagen at al. offer a detailed description of layer 1 of the human neocortex. They use electron microscopy to assess the morphological parameters of presynaptic terminals, active zones, vesicle density/distribution, mitochondrial morphology and astrocytic coverage. The data is collected from tissue from four patients undergoing epilepsy surgery. As the epileptic focus was localized in all patients to the hippocampus, the tissue examined in this manuscript is considered non-epileptic (access) tissue.

Strengths:

The quality of the electron microscopic images is very high, and the data is analyzed carefully. Data from human tissue is always precious and the authors here provide a detailed analysis using adequate approaches, and the data is clearly presented.

Weaknesses:

The text connects functional and morphological characteristics in a very direct way. For example, connecting plasticity to any measurement the authors present would be rather difficult without any additional functional experiments. References to various vesicle pools based on the location of the vesicles is also more complex than it is suggested in the manuscript. The text should better reflect the limitations of the conclusions that can be drawn from the authors' data.

Reviewer #1 (Public review):

Summary:

Trutti and colleagues used 7T fMRI to identify brain regions involved in subprocesses of updating the content of working memory. Contrary to past theoretical and empirical claims that the striatum serves a gating function when new information is to be entered into working memory, the relevant contrast during a reference-back task did not reveal significant subcortical activation. Instead, the experiment provided support for a role of subcortical (and cortical) regions in other subprocesses.

Strengths

The use of high-field imaging optimized for subcortical regions in conjunction with the theory-driven experimental design mapped well to the focus on a hypothetical striatal gating mechanism.

Consideration of multiple subprocesses and the transparent way of identifying these, summarized in a table, will make it easy for future studies to replicate and extend the present experiment.

Weaknesses:

The reference-back paradigm seems to only require holding a single letter in working memory (X or O; Fig 1). It remains unclear how such low demand on working memory influences associated fMRI updating responses. It is also not clear whether reference-switch trials with 'same' response truly taxes working-memory updating (and gate opening), as the working-memory content/representation does not need to be updated in this case. These potential design issues, together with the rather low number of experimental trials, raise concerns about the demonstrated absence of evidence for striatal gate opening. Adding an experiment with higher working-memory demand and additional trials could strengthen the evidence for the authors present claim

The authors provide a motivation for their multi-step approach to fMRI analyses. Still, the three subsections of fMRI results (3.2.1; 3.2.2; 3.3.3) for 4 subprocesses each (gate opening, gate closing, substitution, updating mode) made the Results section complex and it was not always easy to understand why some but not other approaches revealed significant effects (as the midbrain in gate opening).<br /> It could be helpful to readers to further revise the Results section and/or more clearly convey the analytic strategy.

The many references to the role of dopamine are interesting, but the discussion of dopaminergic pathways and signals remains speculative and must be confirmed in future studies (e.g., with PET imaging).

Several relevant studies were not cited (e.g., Dahlin et al., 2008, Science; Bäckman et al., 2011, Science).

Reviewer #2 (Public review):

Summary:

The study reported by Trutti et al. uses high-field fMRI to test the hypothesized involvement of subcortical structure, particularly striatum, in WM updating. Specifically, participants were scanned while performing the Reference Back task (e.g., Rac-Lubashevsky and Kessler, 2016), which tests constructs like working memory gate opening and closing and substitution. While striatal activation was involved in substitution, it was not observed in gate opening.

While there have been prior fMRI studies of the reference back task (Nir-Cohen et al., 2020), the present study overcomes limitations in prior work, particularly with regard to subcortical structures, by applying high-field imaging with more precise definition of ROIs. And, the fMRI methods are careful and rigorous, overall. Thus, the empirical observations here are useful and will be of interest to specialists interested in working memory gating or the reference back task specifically. I do not have additional concerns about this contribution.

Reviewer #1 (Public review):

This study uses a variety of approaches to explore the role of cerebellum, and in particular Purkinje cells (PCs), in the development of postural control in larval zebrafish. A chemogenetic approach is used to either ablate PCs or disrupt their normal activity and a powerful, high-throughput behavioural tracking system then enables quantitative assessment of swim kinematics. Using this strategy, convincing evidence is presented that PCs are required for normal postural control in the pitch axis. Calcium imaging further shows that PCs encode tilt direction. Evidence is also presented that suggests the role of the cerebellum changes over the course of early development, although this claim is less robust. Finally, the authors build on their prior work showing that both axial muscles and pectoral fins contribute to "climbs" and show convincing evidence that PCs are required for speed-dependent engagement of the fins during this behavior. Overall, establishing a role for cerebellum in postural control is not very surprising. However, a clear motivation of this study was to establish a robust experimental platform to investigate the changing role of cerebellar circuits in the development of postural control in the highly experimentally accessible zebrafish larvae and in this regard the authors have certainly succeeded.

This revised version of the manuscript incorporates several improvements. In particular, additional analysis and methodological detail is provided regarding the chemogenetic manipulation, there is expanded analysis of the speed-dependency of pectoral fin engagement, and aspects of the decoding analysis are clearer. However, it is still not certain that the emergence of a dive phenotype over development (from 7 to 14 day post fertilisation) really represents changing role for the cerebellum as opposed to changing sensitivity of Purkinje cells to the chemogenetic treatment.

Reviewer #2 (Public review):

Franziska Auer et al. successfully applied the TRPV1/capsaicin tool to study the contribution of Purkinje cells to postural control. They leveraged the ability of this tool to both activate and ablate neurons within the same construct and tested its effects using their smart, high-throughput behavioral setup for postural control monitoring. With Purkinje cells ablated, balance did not appear to be disrupted; however, postural control was clearly modified along the pitch axis, with larval zebrafish maintaining, on average, a more nose-down posture compared to controls. While this effect is subtle, it is statistically robust and consistent with the group's previous findings using KillerRed-mediated ablation of Purkinje cells, where the observed postural angle change was explained by a disruption in cerebellar-mediated fin-trunk coordination. Here, the authors present a novel insight, demonstrating that this coordination is swim-speed dependent.

Furthermore, the authors convincingly activated Purkinje cells at 7 dpf, and reported modifications in posture pitch angle comparable to those observed when ablating Purkinje cells. The authors suggest a potential desynchronization of Purkinje cells to explain this observation. Future characterization and application of this activation method to other developmental time points could be of major interest. The authors successfully validated the transfer of the TRPV1/capsaicin method for targeted cell ablation and activation to the study of cerebellar functions and reinforced our current understanding of the role of Purkinje cells in postural control.

This study also explores the developmental evolution of cerebellar function in postural control by comparing the effects of Purkinje cell ablation at 7 dpf and 14 dpf. Interestingly, only dive bout posture showed differential effects across these time points, with no significant impact at 7 dpf but a significant change in postural pitch angle at 14 dpf. In contrast, the effect of Purkinje cell ablation on the climbing bout postural angle remained comparable at both ages. Including additional developmental time points would further strengthen this critical characterization of cerebellar maturation in the context of postural control.

To examine whether Purkinje cell activity encodes postural tilt angle, the authors performed calcium imaging on 31 cells from 8 fish using their Tilt In Place Microscope (TIPM). They found that tilt-angle could be decoded from individual neurons with highly tuned responses, as well as from neurons that were not obviously tuned when pooling their data. The authors refer to this effect as pseudo-population coding because recordings were performed non-simultaneously across animals.

This study successfully integrates cutting-edge genetic tools, high-throughput behavioral assays, and advanced optical microscopy to investigate the role of populations of Purkinje cells in postural control. The authors have not only validated these powerful tools but have also provided novel insights into the cerebellar involvement in postural control, including the swim-speed dependence of fin-trunk coordination.

This work represents an important step toward a detailed understanding of cerebellar contributions to postural control and highlights the potential of combining genetically targeted perturbation with quantitative behavioral analysis.

The authors have addressed my previous concerns, and I congratulate them for their excellent work.

Reviewer #3 (Public review):

Summary:

This paper uses a new chemogenetic tool to investigate the role of cerebellar Purkinje cells in postural control. Using a high-throughput behavioral assay, they show that activation or ablation of Purkinje cells affects various aspects of postural control in zebrafish larvae during spontaneous swimming, and that the effects are more pronounced at later developmental time points, where the Purkinje cell number is much greater. Using a sophisticated imaging assay, they record Purkinje cell activity in response to tilt of the fish, and show that some Purkinje cells are tuned to tilt direction, and that the direction can even be decoded from untuned neurons.

Strengths:

Overall the study is nice, using a variety of genetic tools and behavioral analysis to address a fundamental question about the role of the cerebellum in postural control in fish

Reviewer #1 (Public review):

Summary:

In this paper, Bose et al. investigated the role of Foxg1 transcription factor in the progenitors at late stages of cerebral cortex development.

They discover that Foxg1 is a repressor of gliogenesis and has a dual function, first as a repressor of Fgfr3 receptor in progenitors, and second as a suppressor of the Fgf ligands in young neurons.

They found that the inactivation of Foxg1 in cortical progenitors causes premature astrogliogenesis at the expense of neurogenesis. They identify Fgfr3 as a novel FOXG1 target. They show that suppression of Fgfr3 by FOXG1 in progenitors is required to maintain neurogenesis. On the other hand, they also show that FOXG1 negatively regulates the expression of Fgf gliogenic secreted factors in young neurons suppressing gliogenesis cells extrinsically.

Strengths:

The authors used time-consuming in vivo experiments utilizing several mouse strains including Foxg1-MADM in combination with RNA-Seq and ChIP to convincingly show that Foxg1 acts upstream of FGF signalling in the control of gliogenesis onset. The conclusions of this paper are mostly well supported by data.

Reviewer #2 (Public review):

Summary:

We have known for some time that neural progenitors in the cerebral cortex switch their output from cortical neurons to glia at late embryonic stages, however little is known about how this switch is regulated at the molecular level. Bose et al present a convincing set of findings, demonstrating that the transcription factor Foxg1 plays a key role in this process, mediated through FGF signalling. Foxg1 cell-autonomously inhibits gliogenesis in progenitor cells (thereby promoting neuronal identity), and lower Foxg1 expression in postnatal neurons leads to increased expression of FGF ligand, promoting glial development from nearby progenitors.

Strengths:

The study is very well designed, having a systematic, thorough, and logical approach. The data is convincing. The authors make full use of a range of existing transgenic strains, published 'omics data, and elegant genetic approaches such as MADM. This combination of approaches is particularly rigorous, lending significant weight to the study. The manuscript is well-written, clear, and easy to follow.

Impact

This manuscript identifies a previously unknown role for Foxg1 in forebrain development and a mechanism underlying the neurogenic-to-gliogenic switch that occurs at late embryonic stages of cortex development. These findings will stimulate further research to uncover more details of how this important switch is controlled and may provide useful insight into some of the symptoms experienced by children with FOXG1 Syndrome.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #1 (Public review):

Summary:

The authors investigate the effects of aging on auditory system performance in understanding temporal fine structure (TFS), using both behavioral assessments and physiological recordings from the auditory periphery, specifically at the level of the auditory nerve. This dual approach aims to enhance understanding of the mechanisms underlying observed behavioral outcomes. The results indicate that aged animals exhibit deficits in behavioral tasks for distinguishing between harmonic and inharmonic sounds, which is a standard test for TFS coding. However, neural responses at the auditory nerve level do not show significant differences when compared to those in young, normal-hearing animals. The authors suggest that these behavioral deficits in aged animals are likely attributable to dysfunctions in the central auditory system, potentially as a consequence of aging. To further investigate this hypothesis, the study includes an animal group with selective synaptic loss between inner hair cells and auditory nerve fibers, a condition known as cochlear synaptopathy (CS). CS is a pathology associated with aging and is thought to be an early indicator of hearing impairment. Interestingly, animals with selective CS showed physiological and behavioral TFS coding similar to that of the young normal-hearing group, contrasting with the aged group's deficits. Despite histological evidence of significant synaptic loss in the CS group, the study concludes that CS does not appear to affect TFS coding, either behaviorally or physiologically.

Strengths:

This study addresses a critical health concern, enhancing our understanding of mechanisms underlying age-related difficulties in speech intelligibility, even when audiometric thresholds are within normal limits. A major strength of this work is the comprehensive approach, integrating behavioral assessments, auditory nerve (AN) physiology, and histology within the same animal subjects. This approach enhances understanding of the mechanisms underlying the behavioral outcomes and provides confidence in the actual occurrence of synapse loss and its effects. The study carefully manages controlled conditions by including five distinct groups: young normal-hearing animals, aged animals, animals with CS induced through low and high doses, and a sham surgery group. This careful setup strengthens the study's reliability and allows for meaningful comparisons across conditions. Overall, the manuscript is well-structured, with clear and accessible writing that facilitates comprehension of complex concepts.

Weaknesses:

The stimulus and task employed in this study are very helpful for behavioral research, and using the same stimulus setup for physiology is advantageous for mechanistic comparisons. However, I have some concerns about the limitations in auditory nerve (AN) physiology. Due to practical constraints, it is not feasible to record from a large enough population of fibers that covers a full range of best frequencies (BFs) and spontaneous rates (SRs) within each animal. This raises questions about how representative the physiological data are for understanding the mechanism in behavioral data. I am curious about the authors' interpretation of how this stimulus setup might influence results compared to methods used by Kale and Heinz (2010), who adjusted harmonic frequencies based on the characteristic frequency (CF) of recorded units. While, the harmonic frequencies in this study are fixed across all CFs, meaning that many AN fibers may not be tuned closely to the stimulus frequencies. If units are not responsive to the stimulus further clarification on detecting mistuning and phase locking to TFS effects within this setup would be valuable. Given the limited number of units per condition-sometimes as few as three for certain conditions - I wonder if CF-dependent variability might impact the results of the AN data in this study and discussing this factor can help with better understanding the results. While the use of the same stimuli for both behavioral and physiological recordings is understandable, a discussion on how this choice affects interpretation would be beneficial. In addition a 60 dB stimulus could saturate high spontaneous rate (HSR) AN fibers, influencing neural coding and phase-locking to TFS. Potentially separating SR groups, could help address these issues and improve interpretive clarity.

A deeper discussion on the role of fiber spontaneous rate could also enhance the study. How might considering SR groups affect AN results related to TFS coding? While some statistical measures are included in the supplement, a more detailed discussion in the main text could help in interpretation.

Although Figure S2 indicates no change in median SR, the high-dose treatment group lacks LSR fibers, suggesting a different distribution based on SR for different animal groups, as seen in similar studies on other species. A histogram of these results would be informative, as LSR fiber loss with CS-whether induced by ouabain in gerbils or noise in other animals-is well documented (e.g., Furman et al., 2013).

Although ouabain effects on gerbils have been explored in previous studies, since these data already seems to be recorded for the animal in this study, a brief description of changes in auditory brainstem response (ABR) thresholds, wave 1 amplitudes, and tuning curves for animals with cochlear synaptopathy (CS) in this study would be beneficial. This would confirm that ouabain selectively affects synapses without impacting outer hair cells (OHCs). For aged animals, since ABR measurements were taken, comparing hearing differences between normal and aged groups could provide insights into the pathologies besides CS in aged animals. Additionally, examining subject variability in treatment effects on hearing and how this correlates with behavior and physiology would yield valuable insights. If limited space maybe a brief clarification or inclusion in supplementary could be good enough.

Another suggestion is to discuss the potential role of MOC efferent system and effect of anesthesia in reducing efferent effects in AN recordings. This is particularly relevant for aged animals, as CS might affect LSR fibers, potentially disrupting the medial olivocochlear (MOC) efferent pathway. Anesthesia could lessen MOC activity in both young and aged animals, potentially masking efferent effects that might be present in behavioral tasks. Young gerbils with functional efferent systems might perform better behaviorally, while aged gerbils with impaired MOC function due to CS might lack this advantage. A brief discussion on this aspect could potentially enhance mechanistic insights.

Lastly, although synapse counts did not differ between the low-dose treatment and NH I sham groups, separating these groups rather than combining them with the sham might reveal differences in behavior or AN results, particularly regarding the significance of differences between aged/treatment groups and the young normal-hearing group.

Reviewer #2 (Public review):

Summary:

Using a gerbil model, the authors tested the hypothesis that loss of synapses between sensory hair cells and auditory nerve fibers (which may occur due to noise exposure or aging) affects behavioral discrimination of the rapid temporal fluctuations of sounds. In contrast to previous suggestions in the literature, their results do not support this hypothesis; young animals treated with a compound that reduces the number of synapses did not show impaired discrimination compared to controls. Additionally, their results from older animals showing impaired discrimination suggest that age-related changes aside from synaptopathy are responsible for the age-related decline in discrimination.

Strengths:

(1) The rationale and hypothesis are well-motivated and clearly presented.

(2) The study was well conducted with strong methodology for the most part, and good experimental control. The combination of physiological and behavioral techniques is powerful and informative. Reducing synapse counts fairly directly using ouabain is a cleaner design than using noise exposure or age (as in other studies), since these latter modifiers have additional effects on auditory function.

(3) The study may have a considerable impact on the field. The findings could have important implications for our understanding of cochlear synaptopathy, one of the most highly researched and potentially impactful developments in hearing science in the past fifteen years.

Weaknesses:

(1) My main concern is that the stimuli may not have been appropriate for assessing neural temporal coding behaviorally. Human studies using the same task employed a filter center frequency that was (at least) 11 times the fundamental frequency (Marmel et al., 2015; Moore and Sek, 2009). Moore and Sek wrote: "the default (recommended) value of the centre frequency is 11F0." Here, the center frequency was only 4 or 8 times the fundamental frequency (4F0 or 8F0). Hence, relative to harmonic frequency, the harmonic spacing was considerably greater in the present study. By my calculations, the masking noise used in the present study was also considerably lower in level relative to the harmonic complex than that used in the human studies. These factors may have allowed the animals to perform the task using cues based on the pattern of activity across the neural array (excitation pattern cues), rather than cues related to temporal neural coding. The authors show that mean neural driven rate did not change with frequency shift, but I don't understand the relevance of this. It is the change in response of individual fibers with characteristic frequencies near the lowest audible harmonic that is important here.

The case against excitation pattern cues needs to be better made in the Discussion. It could be that gerbil frequency selectivity is broad enough for this not to be an issue, but more detail needs to be provided to make this argument. The authors should consider what is the lowest audible harmonic in each case for their stimuli, given the level of each harmonic and the level of the pink noise. Even for the 8F0 center frequency, the lowest audible harmonic may be as low as the 4th (possibly even the 3rd). In human, harmonics are thought to be resolvable by the cochlea up to at least the 8th.

(2) The synapse reductions in the high ouabain and old groups were relatively small (mean of 19 synapses per hair cell compared to 23 in the young untreated group). In contrast, in some mouse models of the effects of noise exposure or age, a 50% reduction in synapses is observed, and in the human temporal bone study of Wu et al. (2021, https://doi.org/10.1523/JNEUROSCI.3238-20.2021) the age-related reduction in auditory nerve fibres was ~50% or greater for the highest age group across cochlear location. It could be simply that the synapse loss in the present study was too small to produce significant behavioral effects. Hence, although the authors provide evidence that in the gerbil model the age-related behavioral effects are not due to synaptopathy, this may not translate to other species (including human). This should be discussed in the manuscript.

It would be informative to provide synapse counts separately for the animals who were tested behaviorally, to confirm that the pattern of loss across the group was the same as for the larger sample.

(3) The study was not pre-registered, and there was no a priori power calculation, so there is less confidence in replicability than could have been the case. Only three old animals were used in the behavioral study, which raises concerns about the reliability of comparisons involving this group.

Reviewer #3 (Public review):

This study is a part of the ongoing series of rigorous work from this group exploring neural coding deficits in the auditory nerve, and dissociating the effects of cochlear synaptopathy from other age-related deficits. They have previously shown no evidence of phase-locking deficits in the remaining auditory nerve fibers in quiet-aged gerbils. Here, they study the effects of aging on the perception and neural coding of temporal fine structure cues in the same Mongolian gerbil model.

They measure TFS coding in the auditory nerve using the TFS1 task which uses a combination of harmonic and tone-shifted inharmonic tones which differ primarily in their TFS cues (and not the envelope). They then follow this up with a behavioral paradigm using the TFS1 task in these gerbils. They test young normal hearing gerbils, aged gerbils, and young gerbils with cochlear synaptopathy induced using the neurotoxin ouabain to mimic synapse losses seen with age.

In the behavioral paradigm, they find that aging is associated with decreased performance compared to the young gerbils, whereas young gerbils with similar levels of synapse loss do not show these deficits. When looking at the auditory nerve responses, they find no differences in neural coding of TFS cues across any of the groups. However, aged gerbils show an increase in the representation of periodicity envelope cues (around f0) compared to young gerbils or those with induced synapse loss. The authors hence conclude that synapse loss by itself doesn't seem to be important for distinguishing TFS cues, and rather the behavioral deficits with age are likely having to do with the misrepresented envelope cues instead.

The manuscript is well written, and the data presented are robust. Some of the points below will need to be considered while interpreting the results of the study, in its current form. These considerations are addressable if deemed necessary, with some additional analysis in future versions of the manuscript.

Spontaneous rates - Figure S2 shows no differences in median spontaneous rates across groups. But taking the median glosses over some of the nuances there. Ouabain (in the Bourien study) famously affects low spont rates first, and at a higher degree than median or high spont rates. It seems to be the case (qualitatively) in Figure S2 as well, with almost no units in the low spont region in the ouabain group, compared to the other groups. Looking at distributions within each spont rate category and comparing differences across the groups might reveal some of the underlying causes for these changes. Given that overall, the study reports that low-SR fibers had a higher ENV/TFS log-z-ratio, the distribution of these fibers across groups may reveal specific effects of TFS coding by group.

Threshold shifts - It is unclear from the current version if the older gerbils have changes in hearing thresholds, and whether those changes may be affecting behavioral thresholds. The behavioral stimuli appear to have been presented at a fixed sound level for both young and aged gerbils, similar to the single unit recordings. Hence, age-related differences in behavior may have been due to changes in relative sensation level. Approaches such as using hearing thresholds as covariates in the analysis will help explore if older gerbils still show behavioral deficits.

Task learning in aged gerbils - It is unclear if the aged gerbils really learn the task well in two of the three TFS1 test conditions. The d' of 1 which is usually used as the criterion for learning was not reached in even the easiest condition for aged gerbils in all but one condition for the aged gerbils (Fig. 5H) and in that condition, there doesn't seem to be any age-related deficits in behavioral performance (Fig. 6B). Hence dissociating the inability to learn the task from the inability to perceive TFS 1 cues in those animals becomes challenging.

Increased representation of periodicity envelope in the AN - the mechanisms for increased representation of periodicity envelope cues is unclear. The authors point to some potential central mechanisms but given that these are recordings from the auditory nerve what central mechanisms these may be is unclear. If the authors are suggesting some form of efferent modulation only at the f0 frequency, no evidence for this is presented. It appears more likely that the enhancement may be due to outer hair cell dysfunction (widened tuning, distorted tonotopy). Given this increased envelope coding, the potential change in sensation level for the behavior (from the comment above), and no change in neural coding of TFS cues across any of the groups, a simpler interpretation may be -TFS coding is not affected in remaining auditory nerve fibers after age-related or ouabain induced synapse loss, but behavioral performance is affected by altered outer hair cell dysfunction with age.

Emerging evidence seems to suggest that cochlear synaptopathy and/or TFS encoding abilities might be reflected in listening effort rather than behavioral performance. Measuring some proxy of listening effort in these gerbils (like reaction time) to see if that has changed with synapse loss, especially in the young animals with induced synaptopathy, would make an interesting addition to explore perceptual deficits of TFS coding with synapse loss.

Reviewer #1 (Public review):

Turi, Teng and the team used state-of-the-art techniques to provide convincing evidence on the infraslow oscillation of DG cells during NREM sleep, and how serotonergic innervation modulates hippocampal activity pattern during sleep and memory. First, they showed that the glutamatergic DG cells become activated following an infraslow rhythm during NREM sleep. In addition, the infraslow oscillation in the DG is correlated with rhythmic serotonin release during sleep. Finally, they found that specific knockdown of 5-HT receptors in the DG impairs the infraslow rhythm and memory, suggesting that serotonergic signaling is crucial for regulating DG activity during sleep. Given that the functional role of infraslow rhythm still remains to be studied, their findings deepen our understanding on the role of DG cells and serotonergic signaling in regulating infraslow rhythm, sleep microarchitecture and memory.

Reviewer #2 (Public review):

Summary:

The authors investigated DG neuronal activity at the population and single cell level across sleep/wake periods. They found an infraslow oscillation (0.01-0.03 Hz) in both granule cells (GC) and mossy cells (MC) during NREM sleep. The important findings are 1) the antiparallel temporal dynamics of DG neuron activities and serotonin neuron activities/extracellular serotonin levels during NREM sleep, and 2) the GC Htr1a-mediated GC infraslow oscillation.

Strengths:

(1) The combination of polysomnography, Ca-fiber photometry, two-photon microscopy and gene depletion is technically sound. The coincidence of microarousals and dips in DG population activity is convincing. The dip in activity in upregulated cells is responsible for the dip at the population level.<br /> (2) DG GCs express excitatory Htr4 and Htr7 in addition to inhibitory Htr1a, but deletion of Htr1a is sufficient to disrupt DG GC infraslow oscillation, supporting the importance of Htr1a in DG activity during NREM sleep.

Weaknesses:

(1) The current data set and analysis are insufficient to interpret the observation correctly.<br /> a. In Fig 1A, during NREM, the peaks and troughs of GC population activities seem to gradually decrease over time. Please address this point.<br /> b. In Fig 1F, about 30% of Ca dips coincided with MA (EMG increase) and 60% of Ca dips did not coincide with EMG increase. If this is true, the readers can find 8 Ca dips which are not associated with MAs from Fig 1E. If MAs were clustered, please describe this properly.<br /> c. In Fig 1F, the legend stated the percentage during NREM. If the authors want to include the percentage of wake and REM, please show the traces with Ca dips during wake and REM. This concern applies to all pie charts provided by the authors.<br /> d. In Fig 1C, please provide line plots connecting the same session. This request applies to all related figures.<br /> e. In Fig 2C, the significant increase during REM and the same level during NREM are not convincing. In Fig 2A, the several EMG increasing bouts do not appear to be MA, but rather wakefulness, because the duration of the EMG increase is greater than 15 seconds. Therefore, it is possible that the wake bouts were mixed with NREM bouts, leading to the decrease of Ca activity during NREM. In fact, In Fig 2E, the 4th MA bout seems to be the wake bout because the EMG increase lasts more than 15 seconds.<br /> f. Fig 5D REM data are interesting because the DRN activity is stably silenced during REM. The varied correlation means the varied DG activity during REM. The authors need to address it.<br /> g. In Fig 6, the authors should show the impact of DG Htr1a knockdown on sleep/wake structure including the frequency of MAs. I agree with the impact of Htr1a on DG ISO, but possible changes in sleep bout may induce the DG ISO disturbance.

(2) It is acceptable that DG Htr1a KO induces the reduced freezing in the CFC test (Fig. 6E, F), but it is too much of a stretch that the disruption of DG ISO causes impaired fear memory. There should be a correlation.

(3) It is necessary to describe the extent of AAV-Cre infection. The authors injected AAV into the dorsal DG (AP -1.9 mm), but the histology shows the ventral DG (Supplementary Fig. 4), which reduces the reliability of this study.

Comments on revisions:

In the first revision, I pointed out the inappropriate analysis of the EEG/EMG/photometry data and gave examples. The authors responded only to the points raised and did not seem to see the need to improve the overall analysis and description. In this second revision, I would like to ask the authors to improve them. The biggest problem is that the detection criteria and the quantification of the specific event are not described at all in Methods and it is extremely difficult to follow the statement. All interpretations are made by the inappropriate data analysis; therefore, I have to say that the statement is not supported by the data.

Please read my following concerns carefully and improve them.

(1) The definition of the event is critical to the detection of the event and the subsequent analysis. In particular, the authors explicitly describe the definition of MA (microarousal), the trough and peak of the population level of intracellular Ca concentrations, or the onset of the decline and surge of Ca levels.

(1-1) The authors categorized wake bouts of <15 seconds with high EMG activity as MA (in Methods). What degree of high EMG is relevant to MA and what is the lower limit of high EMG? In Fig 1E, there are some EMG spikes, but it was unclear which spike/wave (amplitude/duration) was detected as MA-relevant spike and which spike was not detected. In Fig 2E, the 3rd MA coincides with the EMG spike, but other EMG spikes have comparable amplitude to the 3rd MA-relevant EMG spike. Correct counting of MA events is critical in Fig 1F, 2F, 4C.

(1-2) Please describe the definition of Ca trough in your experiments. In Fig 1G, the averaged trough time is clear (~2.5 s), so I can acknowledge that MA is followed by Ca trough. However, the authors state on page 4 that "30% of the calcium troughs during NREM sleep were followed by an MA epoch". This discrepancy should be corrected.

(1-3) Relating comment 1-2, I agree that the latency is between MA and Ca through in page 4, as the authors explain in the methods, but, in Fig 1G, t (latency) is labeled at incorrect position. Please correct this.

(1-4) The authors may want to determine the onset of the decline in population Ca activity and the latency between onset and trough (Fig 1G, latency t). If so, please describe how the onset of the decline is determined. In Fig 1G, 2G, S6, I can find the horizontal dashed line and infer that the intersection of the horizontal line and the Ca curve is considered the onset. However, I have to say that the placement of this horizontal line is super arbitrary. The results (t and Drop) are highly dependent on the position of horizontal line, so the authors need to describe how to set the horizontal line.

(1-5) In order to follow Fig 1F correctly, the authors need to indicate the detection criteria of "Ca dip (in legend)". Please indicate "each Ca dip" in Fig 1E. As a reader, I would like to agree with the Ca dip detection of this Ca curve based on the criteria. Please also indicate "each Ca dip" in Fig 2E and 2F. In the case of the 2nd and 3rd MAs, do they follow a single Ca dip or does each MA follow each Ca dip? This chart is highly dependent on the detection criteria of Ca dip.

As I mentioned above, most of the quantifications are not based on the clear detection criteria. The authors need to re-analyze the data and fix the quantification. Please interpret data and discuss the cellular mechanism of ISO based on the re-analyzed quantification.

Reviewer #3 (Public review):

Summary:

The authors employ a series of well-conceived and well-executed experiments involving photometric imaging of the dentate gyrus and raphe nucleus, as well as cell-type specific genetic manipulations of serotonergic receptors that together serve to directly implicate serotonergic regulation of dentate gyrus (DG) granule (GC) and mossy cell (MC) activity in association with an infra slow oscillation (ISO) of neural activity has been previously linked to general cortical regulation during NREM sleep and microarousals.

Strengths:

There are a number of novel and important results, including the modulation of dentage granule cell activity by the infraslow oscillation during NREM sleep, the selective association of different subpopulations of granule cells to microarousals (MA), the anticorrelation of raphe activity with infraslow dentate activity.

The discussion includes a general survey of ISOs and recent work relating to their expression in other brain areas and other potential neuromodulatory system involvement, as well as possible connections with infraslow oscillations, micro arousals, and sensory sensitivity.

Weaknesses:

- The behavioral results showing contextual memory impairment resulting from 5-HT1a knockdown are fine, but are over-interpreted. The term memory consolidation is used several times, as well as references to sleep-dependence. This is not what was tested. The receptor was knocked down, and then 2 weeks later animals were found to have fear conditioning deficits. They can certainly describe this result as indicating a connection between 5-HT1a receptor function and memory performance, but the connection to sleep and consolidation would just be speculation. The fact that 5-HT1a knockdown also impacted DG ISOs does not establish dependency. Some examples of this are:<br /> o The final conclusion asserts "Together, our study highlights the role of neuromodulation in organizing neuronal activity during sleep and sleep-dependent brain functions, such as memory.", but the reported memory effects (impairment of fear conditioning) were not shown to be explicitly sleep-dependent.<br /> o Earlier in the discussion it mentions "Finally, we showed that local genetic ablation of 5-HT1a receptors in GCs impaired the ISO and memory consolidation". The effect shown was on general memory performance - consolidation was not specifically implicated.

- The assertion on page 9 that the results demonstrate "that the 5-HT is directly acting in the DG to gate the oscillations" is a bit strong given the magnitude of effect shown in Fig. 6D, and the absence of demonstration of negative effect on cortical areas that also show ISO activity and could impact DG activity (see requested cortical sigma power analysis).

- Recent work has shown that abnormal DG GC activity can result from the use of the specific Ca indicator being used (GCaMP6s). (Teng, S., Wang, W., Wen, J.J.J. et al. Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures. Sci Rep 14, 8104 (2024). https://doi.org/10.1038/s41598-024-58819-9). The authors of that study found that the effect seemed to be specific to GCaMP6s and that GCaMP6f did not lead to abnormal excitability. Note this is of particular concern given similar infraslow variation of cortical excitability in epilepsy (cf Vanhatalo et al. PNAS 2004). While I don't think that the experiments need to be repeated with a different indicator to address this concern, you should be able to use the 2p GCaMP7 experiments that have already been done to provide additional validation by repeating the analyses done for the GCaMP6s photometry experiments. This should be done anyway to allow appropriate comparison of the 2p and photometry results.

- While the discussion mentions previous work that has linked ISOs during sleep with regulation of cortical oscillations in the sigma band, oddly no such analysis is performed in the current work even though it is presumably available and would be highly relevant to the interpretation of a number of primary results including the relationship between the ISOs and MAs observed in the DG and similar results reported in other areas, as well as the selective impact of DG 5-HT1a knockdown on DG ISOs. For example, in the initial results describing the cross correlation of calcium activity and EMG/EEG with MA episodes (paragraph 1, page 4), similar results relating brief arousals to the infraslow fluctuation in sleep spindles (sigma band) have been reported also at .02 Hz associated with variation in sensory arousability (cf. Cardis et al., "Cortico-autonomic local arousals and heightened somatosensory arousability during NREMS of mice in neuropathic pain", eLife 2021). It would be important to know whether the current results show similar cortical sigma band correlations. Also, in the results on ISO attenuation following 5-HT1 knockdown on page 7 (fig. 6), how is cortical EEG affected? is ISO still seen in EEG but attenuated in DG?

- The illustrations of the effect of 5-HT1a knockdown shown in Figure 6 are somewhat misleading. The examples in panels B and C show an effect that is much more dramatic than the overall effect shown in panel D. Panels B and C do not appear to be representative examples. Which of the sample points in panel D are illustrated in panels B, C? it is not appropriate to arbitrarily select two points from different animals for comparison, or worse, to take points from the extremes of the distributions. If the intent is to illustrate what the effect shown in D looks like in the raw data, then you need to select examples that reflect the means shown in panel D. It is also important to show the effect on cortical EEG, particularly in sigma band to see if the effects are restricted to the DG ISOs. It would also be helpful to show that MAs and their correlations as shown in Fig 1 or G as well as broader sleep architecture are not affected.

- On page 9 of the results it states that GCs and MCs are upregulated during NREM and their activity is abruptly terminated by MAs through a 5-HT mediated mechanism. I didn't see anything showing the 5-HT dependence of the MA activity correlation. The results indicate a reduction in ISO modulation of GC activity but not the MA correlated activity. I would like to see the equivalent of Fig 1,2 G panels with the 5-HT1a manipulation.

Reviewer #2 (Public review):

Summary:

This computational modeling study addresses the observation that variable observations are interpreted differently depending on how much uncertainty an agent expects from its environment. That is, the same mismatch between a stimulus and an expected stimulus would be less significant, and specifically would represent a smaller prediction error, in an environment with a high degree of variability than in one where observations have historically been similar to each other. The authors show that if two different classes of inhibitory interneurons, the PV and SST cells, (1) encode different aspects of a stimulus distribution and (2) act in different (divisive vs. subtractive) ways, and if (3) synaptic weights evolve in a way that causes the impact of certain inputs to balance the firing rates of the targets of those inputs, then pyramidal neurons in layer 2/3 of canonical cortical circuits can indeed encode uncertainty-modulated prediction errors. To achieve this result, SST neurons learn to represent the mean of a stimulus distribution and PV neurons its variance.

The impact of uncertainty on prediction errors in an understudied topic, and this study provides an intriguing and elegant new framework for how this impact could be achieved and what effects it could produce. The ideas here differ from past proposals about how neuronal firing represents uncertainty. The developed theory is accompanied by several predictions for future experimental testing, including the existence of different forms of coding by different subclasses of PV interneurons, which target different sets of SST interneurons (as well as pyramidal cells). The authors are able to point to some experimental observations that are at least consistent with their computational results. The simulations shown demonstrate that if we accept its assumptions, then the authors' theory works very well: SSTs learn to represent the mean of a stimulus distribution, PVs learn to estimate its variance, firing rates of other model neurons scale as they should, and the level of uncertainty automatically tunes the learning rate, so that variable observations are less impactful in a high uncertainty setting.

Strengths:

The ideas in this work are novel and elegant, and they are instantiated in a progression of simulations that demonstrate the behavior of the circuit. The framework used by the authors is biologically plausible and matches some known biological data. The results attained, as well as the assumptions that go into the theory, provide several predictions for future experimental testing. The authors have taken into account earlier review comments to revise their paper in ways that enhance its clarity.

Weaknesses:

One weakness could be that the proposed theory does rely on a fairly large number of assumptions. However, there is at least some biological support for these. Importantly, the authors do lay out and discuss their key assumptions in the Discussion section, so readers can assess their validity and implications for themselves.

Comments on revisions:

I have no further suggestions for the authors.

Reviewer #4 (Public review):

Summary:

Wilmes and colleagues develop a model for the computation of uncertainty modulated prediction errors based on an experimentally inspired cortical circuit model for predictive processing. Predictive processing is a promising theory of cortical function. An essential aspect of the model is the idea of precision weighting of prediction errors. There is ample experimental evidence for prediction error responses in cortex. However, a central prediction of the theory is that these prediction error responses are regulated by the uncertainty of the input. Testing this idea experimentally has been difficult due to a lack of concrete models. This work provides one such model and makes experimentally testable predictions.

Strengths:

The model proposed is novel and well-implemented. It has sufficient biological accuracy to make useful and testable predictions.

Weaknesses:

One key idea the model hinges on is that stimulus uncertainty is encoded in the firing rate of parvalbumin positive interneurons. While this assumption is rather speculative, the model also here makes experimentally testable predictions.

Comments on revisions:

Congratulations on a very nice paper.

Reviewer #1 (Public review):

The focus of this manuscript was to investigate the role of Cldn9 in the development of the mammalian cochlea. The main rationale of the study is the fact that cochlear hair cells do not regenerate, so when damaged they are lost forever, causing irreparable hearing loss. The authors have attempted to address this problem by inducing the ectopic production of additional hair cells and test whether they acquire the morphological and functional characteristics of native hair cells. They show that downregulation of Cldn9 using a well-established genetic manipulation of transgenic mice led to the production of extra numerary inner hair cells, which were able to survive for several months. By performing a large battery of experiments, the authors were able to determine that the native and ectopic inner hair cells have comparable morphological and physiological characteristics. There are several conclusions highlighted by the authors in different parts of the manuscript, including the key role of Cldn9 in coordinating embryonic and postnatal development, the differentiation of supporting cells into inner hair cells, and the possible use of Cldn9 to induce inner hair cell differentiation following deafness induced by hair cell loss.

Comments on revised version:

The authors have addressed the following points raised during the first submission: statistical analysis and wave 1 analysis. However, very little was done to address the other key aspects of my report, which are essential for the interpretation of the results. As mentioned in my previous report, some aspects of the work are not justified by the current data and will require either a tone-down of the claims or further experiments.

For example, one puzzling finding that is not addressed in the manuscript is the lack of functional benefit from these additional inner hair cells. In fact, it appears to be detrimental based on the increased ABR thresholds and EP. So, it is not clear to this reviewer the advantage of this approach.

It is not clear what direct evidence there is, apart from some immunostaining, indicating that the ectopic inner hair cells derive from the supporting cells. This part would benefit from a more careful consideration and maybe an attempt at a more direct experimental approach. Alternatively, the text should be modified accordingly.

One point that should be made clear throughout the manuscript is that the ectopic inner hair cells are generated in a cochlea that is undergoing normal maturation. Thus, there is no guarantee that modulating the expression levels of Cldn9 in a deaf mouse lacking hair cells would produce the same result as that shown in this study. This point should be at least discussed.

Reviewer #3 (Public review):

The study by Chen et al reports an interesting and previously unknown phenomenon of generation of supernumerary inner hair cells (IHCs) in response to downregulation of Cldn9 during embryonic or postnatal development. The authors developed an inducible doxycycline (dox)-tet-OFF-Cldn9 transgenic mice to regulate expression levels of Cldn9 and show that downregulation of Cldn9 resulted in additional, although incomplete row of IHCs immediately adjacent to the original IHC row. These induced extra IHCs had similar well-developed hair bundles, able to mechanotransduce and were innervated by auditory neurons, resembling wild-type IHCs. In addition, the authors knock down Cldn9 postnatally using shRNA injections in P1-7 mice with similar induction of extranumerary IHCs next to the original row of IHCs. The conclusions of this paper are mostly well supported by the data. However, some data analyses are limited, and some important controls are not shown.<br /> The data from this study are important and promising for future gene therapy applications. The generation of extra IHCs postnatally using downregulation of Cldn9 by shRNA could potentially be used as a replacement of IHCs lost after noise-induced trauma, ototoxic agents, or other environmental trauma. However, it is not clear if downregulation of CLDN9 in adult mice would lead to extranumerary IHCs. On the other hand, the replacement of lost inner hair cells due to various genetic mutations by inducing supernumerary mutant IHCs with the same abnormalities would not be reasonable.

The authors show that postnatally generated ectopic IHCs are viable and mechanotransducive, but the hearing function of the mice with ectopic hair cells is not improved. However, the ectopic hair cells seems to be generated from supporting cell trans-differentiation, and the intricate mosaic of the organ of Corti is altered (the extra row of IHCs seems to be positioned immediately adjacent to the original IHC row), which could by itself lead to hearing issues. It is not clear if the newly formed unusual junctions between the ectopic and original IHCs are sufficiently tight to prevent leakage of the endolymph to the basolateral surface of IHCs. Also, it is not clear if the other organ of Corti tight junctions could lose their tightness due to the downregulation of Cldn9, which could over time affect the endocochlear potential and hearing abilities as shown by this study.

Overall, the manuscript could be of interest to scientists working in the inner ear development and regeneration field, and to the hearing researchers in general and perhaps developmental biologists and cell biologists interested in tight junction proteins and their function.

Strength

The methodologies used are solid and convincing. There is a great potential for practical use of these valuable findings and new knowledge on IHC developmental regulation by Cldn9 expression.

Weakness

Some of the data in this study would benefit from showing corresponding negative controls and higher-resolution images of CLDN9 localization, which the authors chose not to show in the revised manuscript. Importantly, CLDN9 immunofluorescence staining data look different from previously published observations and show cytoplasmic staining of supporting cells only and did not show the staining of tight junctions between the OHCs and supporting cells as well as between the IHCs and supporting cells as reported previously (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021). The organ of Corti schematics showing CLDN9 expression reflects the authors' immunostaining data but is unusual considering that CLDN9 localizes to the tight junctions of the reticular lamina as was shown by immuno-EM in this study and described in previous publications (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021). However, the authors did not provide an explanation for these discrepancies in the Discussion of the manuscript.

Also, more detailed investigations would in some instances clarify the data. For example, it is not clear if the downregulation of Cldn9 affects the other genes known to participate in cell fate determination, and why downregulation of Cldn9 expression resulted in production of extranumerary inner hair cells only and not the other cell types, like OHCs, for example.

Reviewer #4 (Public review):

The work by Yingying Chen, Jeong Han Lee, and co-authors summarizes the morphological and functional outcomes of Cldn9 loss in the inner ear, particularly in the organ of Corti. While the study does not provide mechanistic insights into how the developmental loss of Cldn9 leads to ectopic hair cell formation, the phenomenon itself is curious. The work primarily focuses on a detailed characterization of the ectopic hair cells, which is well done. Despite the lack of mechanistic insights, the study will be of interest to the inner ear field if several major issues with the manuscript are addressed.

(1) The title, "Genetic and pharmacologic alterations of claudin9 levels suffice to induce functional and mature inner hair cells," is misleading. First, both manipulations (knockout and knockdown) are genetic, and no pharmacology is involved. Second, both manipulations are carried out during the embryonic and neonatal periods, and there is no evidence of mature hair cell regeneration in this study. The title should be revised to reflect this. A more accurate title could be: "Developmental loss of Cldn9 results in functional ectopic inner hair cells that persist through adulthood."<br /> (2) Contact-mediated lateral inhibition in hair cell fate determination is one of the most well-studied phenomena in the inner ear field, and numerous groups have shown that it is mediated by Notch signaling. This must be added to the introduction.<br /> (3) A large body of literature has demonstrated that Notch inhibition alone is not sufficient to regenerate hair cells in adult mice. Therefore, if the loss of claudins disrupts Notch signaling-the proposed mechanisms in the discussion - it is unlikely to be a viable therapeutic strategy for hair cell regeneration in the adult ear. Furthermore, no hair cell ablation experiments were conducted to demonstrate what could be considered true regeneration. These speculative statements should be removed or revised accordingly.<br /> (4) Cldn9 is a tight junction protein and should localize to the membrane. Yet, the data presented show what appears to be diffuse cytoplasmic staining, which is concerning.

Joint Public Review:

Summary:

The authors aimed to identify the neural sources of behavioral variation in fruit flies deciding between odor and air, or between two odors.

Strengths:

- The question is of fundamental importance.<br /> - The behavioral studies are automated, and high-throughput.<br /> - The data analyses are sophisticated and appropriate.<br /> - The paper is clear and well-written aside from some initially strong wording.<br /> - The figures beautifully illustrate their results.<br /> - The modeling efforts mechanistically ground observed data correlations.

Weaknesses:

-The correlations between behavioral variations and neural activity/synapse morphology are statistically significant but relatively weak.

Reviewer #1 (Public review):

Summary:

This manuscript details the results of a small pilot study of neoadjuvant radiotherapy followed by combination treatment with hormone therapy and dalpiciclib for early-stage HR+/HER2-negative breast cancer.

Strengths:

The strengths of the manuscript include the scientific rationale behind the approach and the inclusion of some simple translational studies.

Weaknesses:

The main weakness of the manuscript is that overly strong conclusions are made by the authors based on a very small study of twelve patients. A study this small is not powered to fully characterize the efficacy or safety of a treatment approach, and can, at best, demonstrate feasibility. These data need validation in a larger cohort before they can have any implications for clinical practice, and the treatment approach outlined should not yet be considered a true alternative to standard evidence-based approaches.

I would urge the authors and readers to exercise caution when comparing results of this 12-patient pilot study to historical studies, many of which were much larger, and had different treatment protocols and baseline patient characteristics. Cross-trial comparisons like this are prone to mislead, even when comparing well powered studies. With such a small sample size, the risk of statistical error is very high, and comparisons like this have little meaning.

Reviewer #2 (Public review):

The author and his team explored a novel neoadjuvant strategy of radiotherapy followed by CDK4/6 inhibitor and exemestane for HR+/HER2- breast cancer. This strategy interestingly reached an ORR of 91.7% and RCB 0-I of 16.7%, with satisfying tolerance.

There are several questions for your further consideration.

Firstly, as this is a single-arm preliminary study, we are curious about the order of radiotherapy and the endocrine therapy. Besides, considering the radiotherapy, we also concern about the recovery of the wound after the surgery and whether related data were collected.

Secondly, in the methodology, please describe the sample size estimation of this study and follow up details.

Thirdly, in Table 1, the item HER2 expression, it's better to categorise HER2 into 0, 1+, 2+ and FISH-.

Reviewer #1 (Public review):

Summary:

The authors set out to evaluate the regulation of interferon (IFN) gene expression in fish, using mainly zebrafish as a model system. Similar to more widely characterized mammalian systems, fish IFN is induced during viral infection through the action of the transcription factor IRF3 which is activated by phosphorylation by the kinase TBK1. It has been previously shown in many systems that TBK1 is subjected to both positive and negative regulation to control IFN production. In this work, the authors find that the cell cycle kinase CDK2 functions as a TBK1 inhibitor by decreasing its abundance through recruitment of the ubiquitinylation ligase, Dtx4, which has been similarly implicated in the regulation of mammalian TBK1. Experimental data are presented showing that CDK2 interacts with both TBK1 and Dtx4, leading to TBK1 K48 ubiqutinylation on K567 and its subsequent degradation by the proteasome.

Strengths:

The strengths of this manuscript are its novel demonstration of the involvement of CDK2 in a process in fish that is controlled by different factors in other vertebrates and its clear and supportive experimental data.

Weaknesses:

The weaknesses of the study include the following. 1) It remains unclear how CDK is regulated during viral infection and how it specifically recruits E3 ligase to TBK1. 2) The implications and mechanisms for a relationship between the cell cycle and IFN production will be a fascinating topic for future studies.

Reviewer #1 (Public review):

Summary:

The authors set out to evaluate the regulation of interferon (IFN) gene expression in fish, using mainly zebrafish as a model system. Similar to more widely characterized mammalian systems, fish IFN is induced during viral infection through the action of the transcription factor IRF3 which is activated by phosphorylation by the kinase TBK1. It has been previously shown in many systems that TBK1 is subjected to both positive and negative regulation to control IFN production. In this work, the authors find that the cell cycle kinase CDK2 functions as a TBK1 inhibitor by decreasing its abundance through recruitment of the ubiquitinylation ligase, Dtx4, which has been similarly implicated in the regulation of mammalian TBK1. Experimental data are presented showing that CDK2 interacts with both TBK1 and Dtx4, leading to TBK1 K48 ubiqutinylation on K567 and its subsequent degradation by the proteasome.

Strengths:

The strengths of this manuscript are its novel demonstration of the involvement of CDK2 in a process in fish that is controlled by different factors in other vertebrates and its clear and supportive experimental data.

Weaknesses:

The weaknesses of the study include the following. 1) It remains unclear how CDK is regulated during viral infection and how it specifically recruits E3 ligase to TBK1. 2) The implications and mechanisms for a relationship between the cell cycle and IFN production will be a fascinating topic for future studies.

Reviewer #1 (Public review):

Summary:

Lodhiya et al. demonstrate that antibiotics with distinct mechanisms of action, norfloxacin and streptomycin, cause similar metabolic dysfunction in the model organism Mycobacterium smegmatis. This includes enhanced flux through the TCA cycle and respiration as well as a build-up of reactive oxygen species (ROS) and ATP. Genetic and/or pharmacologic depression of ROS or ATP levels protect M. smegmatis from norfloxacin and streptomycin killing. Because ATP depression is protective, but in some cases does not depress ROS, the authors surmise that excessive ATP is the primary mechanism by which norfloxacin and streptomycin kill M. smegmatis. In general, the experiments are carefully executed; alternative hypotheses are discussed and considered; the data are contextualized within the existing literature.

Strengths:

The authors tackle a problem that is both biologically interesting and medically impactful, namely, the mechanism of antibiotic-induced cell death.

Experiments are carefully executed, for example, numerous dose- and time-dependency studies; multiple, orthogonal readouts for ROS; and several methods for pharmacological and genetic depletion of ATP.

There has been a lot of excitement and controversy in the field, and the authors do a nice job of situating their work in this larger context.

Inherent limitations to some of their approaches are acknowledged and discussed e.g., normalizing ATP levels to viable counts of bacteria.

Weaknesses:

All of the experiments performed here were in the model organism M. smegmatis. As the authors point out, the extent to which these findings apply to other organisms (most notably, slow-growing pathogens like M. tuberculosis) is to be determined.

At first glance, some of the results in the manuscript seem to conflict with what has been previously reported in the (referenced) literature. In their response to reviewers, the authors addressed these concerns. Ideally they would have addressed them in the main manuscript too.

Figs. 9 and 10A-B and associated text make the manuscript significantly longer and more descriptive. They are more appropriate to the beginning of a new story rather than the end of the current one.

Reviewer #2 (Public review):

Summary:

The authors are trying to test the hypothesis that ATP bursts are the predominant driver of antibiotic lethality of Mycobacteria

Strengths:

No significant strengths in the current state as it is written.

Weaknesses:

A major weakness is that M. smegmatis has a doubling time of three hours and the authors are trying to conclude that their data would reflect the physiology of M. tuberculossi which has a doubling time of 24 hours. Moreover, the authors try to compare OD measurements with CFU counts and thus observe great variabilities.

Comments on revisions:

The authors confirm they are using CFU counts, but then Figure 1 has 0 as the first data point on the Y-axis. This should be somewhere between 10e5 or 10e6. CFU would not start at 0, your initial inoculum has to be more than 0 to have something to challenge.

Reviewer #1 (Public review):

Summary:

This comprehensive study employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure regulation in a mouse model of TGFβ2 -induced ocular hypertension. TGFβ2 upregulated expression of TRPV4 and PIEZO1 transcripts and time-dependently augmented functional TRPV4 activation. TRPV4 activation induced TM contractility whereas pharmacological inhibition suppressed TGFβ2-induced hypercontractility and abrogated ocular hypertension in eyes overexpressing TGFβ2. Trpv4-/- mice resisted TGFβ2-driven increases in IOP. These data establish a fundamental role of TGFβ as a modulator of mechanosensing and identifies TRPV4 channel as a common mechanism for TM contractility and pathological ocular hypertension.

Strengths:

The manuscript is very well written and details the important function of TRPV4 in TM cell function. These data provide novel therapeutic targets and potential for disease-altering therapeutics.

Weaknesses:

The experimental rigor and design of the noctural IOP experiments was weak with low n values and differing methods of IOP measurement (conscious versus anesthetized). The same method of IOP measurement needs to be used for all measurements to make any conclusions on the circadian patterns of IOP in each condition.

Reviewer #2 (Public review):

The manuscript by Christopher N. Rudzitis et al. describes the role of TGFβ2 in the transcription and functional expression of mechanosensitive channel isoforms, alongside studies on TM contractility in biomimetic hydrogels and intraocular pressure. Overall, it is a very interesting study, nicely designed, and will contribute to the available literature on TRPV4 sensitivity to mechanical forces.

I have the following comment for the authors to address.

Figure 1A-C.<br /> Often there is a difference between the massage and transcript data. I recommend the authors to confirm with qPCR data with another mode of protein measurements.<br /> Does direct TRPV4 activation also induce the expression of these markers? Does inhibition of TRPV4, after TGF-β treatment, prevent the expression of these markers? Is TRPV4 acting downstream of this response?

Figure 1D. Beta tubulin is not a membrane marker. Having staining of b tubulin in membrane fraction shows contamination from the cytoplasm.<br /> Does the overall expression also increase?

Figure 4A: it is not very clear. I recommend including a zoom image or better resolution image.

Figure 5B and 6B.<br /> Why there is a difference between groups in pre-injection panel. As Figure 5A, in pre-injection, there is no difference between LV-TGFβ and LV-control while in 5B there is a significant difference between these groups.<br /> Discussion section.

Line 279, . "TRPV4 channels in cells treated with TGFβ2 are likely to be constitutively active" ... needs to be discussed further.

Line 280: "The residual contractility in HC-06-treated cells may reflect TGFβ2-mediated contributions from Piezo1."<br /> Piezo1 has a low threshold for mechanosensitivity. How do the authors discuss the observation that, in the presence of Piezo1, TRPV4 has a more prominent mechanosensory function? Is this tied to TGFβ signalling?

Reviewer #1 (Public review):

Summary:

The authors use a sophisticated task design and Bayesian computational modeling to test their hypothesis that information generalization (operationalized as a combination of self-insertion and social contagion) in social situations is disrupted in Borderline Personality Disorder. Their main finding relates to the observation that two different models best fit the two tested groups: While the model assuming both self-insertion and social contagion to be present when estimating others' social value preferences fit the control group best, a model assuming neither of these processes provided the best fit to BPD participants.

Strengths:

The strengths of the presented work lie in the sophisticated task design and the thorough investigation of their theory by use of mechanistic computational models to elucidate social decision-making and learning processes in BPD.

Weaknesses:

The manuscript's primary weakness relates to the number of comparisons conducted and a lack of clarity in how those comparisons relate to the authors' hypotheses. The authors specify a primary prediction about disruption to information generalization in social decision making & learning processes, and it is clear from the text how their 4 main models are supposed to test this hypothesis. With regards to any further analyses however (such as the correlations between multiple clinical scales and eight different model parameters, but also individual parameter comparisons between groups), this is less clear. I recommend the authors clearly link each test to a hypothesis by specifying, for each analysis, what their specific expectations for conducted comparisons are, so a reader can assess whether the results are/aren't in line with predictions. The number of conducted tests relating to a specific hypothesis also determines whether multiple comparison corrections are warranted or not. If comparisons are exploratory in nature, this should be explicitly stated.

Furthermore, the authors present some measures for external validation of the models, including comparison between reaction times and belief shifts, and correlations between model predicted accuracy and behavioural accuracy/total scores. However it would be great to see some more formal external validation of how the model parameters relate to participant behaviour, e.g., the correlation between the number of pro-social choices and ß-values, or the correlation between the change in absolute number of pro-social choices and the change in ß. From comparing the behavioural and computational results it looks like they would correlate highly, but it would be nice to see this formally confirmed.

The statement in the abstract that 'Overall, the findings provide a clear explanation of how self-other generalisation constrains and assists learning, how childhood adversity disrupts this through separation of internalised beliefs' makes an unjustified claim of causality between childhood adversity and separation of self - and other beliefs, although the authors only present correlations. I recommend this should be rephrased to reflect the correlational nature of the results.

Currently, from the discussion the findings seem relevant in explaining certain aberrant social learning and -decision making processes in BPD. However, I would like to see a more thorough discussion about the practical relevance of their findings in light of their observation of comparable prediction accuracy between the two groups.

Relatedly, the authors mention that a primary focus of mentalization based therapy for BPD is 'restoring a stable sense of self' and 'differentiating the self from the other'. These goals are very reminiscent of the findings of the current study that individuals with BPD show lower uncertainty over their own and relative reward preferences, and that they are less susceptible to social contagion. Could the observed group differences therefore be a result of therapy rather than adverse early life experiences?

Regarding partner similarity: It was unclear to me why the authors chose partners that were 50% similar when it would be at least equally interesting to investigate self-insertion and social contagion with those that are more than 50% different to ourselves? Do the authors have any assumptions or even data that shows the results still hold for situations with lower than 50% similarity?

Reviewer #2 (Public review):

Summary:

The paper investigates social-decision making, and how this changes after observing the behaviour of other people, in borderline personality disorder. The paper employs a task including three phases, the first where participants make decision on how to allocate rewards to oneself and to a virtual partner, the second where they observe the same task performed by someone else, and a third phase equivalent to phase one, but with a new partner. Using sophisticated computational modelling to analyse choice data, the study reports that borderline participants (versus controls) are more certain about their preferences in phase one, used more neutral priors and are less flexible during phase two, and are less influenced by partners in phase three.

Strengths:

The topic is interesting and important, and the findings are potentially intriguing. The computational methods employed is clever and sophisticated, at the cutting edge of research in the field.

Weaknesses:

There are two major weaknesses. First, the paper lacks focus and clarity. The introduction is rather vague and, after reading it, I remained confused about the paper's aims. Rather than relying on specific predictions, the analysis is exploratory. This implies that it is hard to keep track, and to understand the significance, of the many findings that are reported. Second, although the computational approach employed is clever and sophisticated, there is important information missing about model comparison which ultimately makes some of the results hard to assess from the perspective of the reader.

Reviewer #3 (Public review):

In this paper, the authors use a three-phase economic game to examine the tendency to engage in prosocial versus competitive exchanges with three anonymous partners. In particular, they consider individual differences in the tendency to infer about others' tendencies based on one's preferences and to update one's preferences based on observations of others' behavior. The study includes a sample of individuals diagnosed with borderline personality disorder and a matched sample of psychiatrically healthy control participants.

On the whole, the experimental design is well-suited to the questions and the computational model analyses are thorough, including modern model-fitting procedures. I particularly appreciated the clear exposition regarding model parameterization and the descriptive Table 2 for qualitative model comparison. My broad question about the experiment (in terms of its clinical and cognitive process relevance): Does the task encourage competition or give participants a reason to take advantage of others? I don't think it does, so it would be useful to clarify the normative account for prosociality in the introduction (e.g., some of Robin Dunbar's work).

The finding that individuals with BPD do not engage in self-other generalization on this task of social intentions is novel and potentially clinically relevant. The authors find that BPD participants' tendency to be prosocial when splitting points with a partner does not transfer into their expectations of how a partner will treat them in a task where they are the passive recipient of points chosen by the partner. In the discussion, the authors reasonably focus on model differences between groups (Bayesian model comparison), yet I thought this finding -- BPD participants not assuming prosocial tendencies in phase 2 while CON participant did -- merited greater attention. Although the BPD group was close to 0 on the \beta prior in Phase 2, their difference from CON is still in the direction of being more mistrustful (or at least not assuming prosociality). This may line up with broader clinical literature on mistrustfulness and attributions of malevolence in the BPD literature (e.g., a 1992 paper by Nigg et al. in Journal of Abnormal Psychology). My broad point is to consider further the Phase 2 findings in terms of the clinical interpretation of the shift in \beta relative to controls.

On the conceptual level, I had two additional concerns. First, the authors note that they have "proposed a theory with testable predictions" (p. 4 but also elsewhere) but they do not state any clear predictions in the introduction, nor do they consider what sort of patterns will be observed in the BPD group in view of extant clinical and computational literature. Rather, the paper seems to be somewhat exploratory, largely looking at group differences (BPD vs. CON) on all of the shared computational parameters and additional indices such as belief updating and reaction times. Given this, I would suggest that the authors make stronger connections between extant research on intention representation in BPD and their framework (model and paradigm). In particular, the authors do not address related findings from Ereira (2020) and Story (2024) finding that in a false belief task that BPD participants *overgeneralize* from self to other. A critical comparison of this work to the present study, including an examination of the two tasks differ in the processes they measure, is important.

In addition, perhaps it is fairer to note more explicitly the exploratory nature of this work. Although the analyses are thorough, many of them are not argued for a priori (e.g., rate of belief updating in Figure 2C) and the reader amasses many individual findings that need to by synthesized.

Second, in the discussion, the authors are too quick to generalize to broad clinical phenomena in BPD that are not directly connected to the task at hand. For example, on p. 22: "Those with a diagnosis of BPD also show reduced permeability in generalising from other to self. While prior research has predominantly focused on how those with BPD use information to form impressions, it has not typically examined whether these impressions affect the self." Here, it's not self-representation per se (typically, identity or one's view of oneself), but instead cooperation and prosocial tendencies in an economic context. It is important to clarify what clinical phenomena may be closely related to the task and which are more distal and perhaps should not be approached here.

On a more technical level, I had two primary concerns. First, although the authors consider alternative models within a hierarchical Bayesian framework, some challenges arise when one analyzes parameter estimates fit separately to two groups, particularly when the best-fitting model is not shared. In particular, although the authors conduct a model confusion analysis, they do not as far I could tell (and apologies if I missed it) demonstrate that the dynamics of one model are nested within the other. Given that M4 has free parameters governing the expectations on the absolute and relative reward preferences in Phase 2, is it necessarily the case that the shared parameters between M1 and M4 can be interpreted on the same scale? Relatedly, group-specific model fitting has virtues when believes there to be two distinct populations, but there is also a risk of overfitting potentially irrelevant sample characteristics when parameters are fit group by group.

To resolve these issues, I saw one straightforward solution (though in modeling, my experience is that what seems straightforward on first glance may not be so upon further investigation). M1 assumes that participants' own preferences (posterior central tendency) in Phase 1 directly transfer to priors in Phase 2, but presumably the degree of transfer could vary somewhat without meriting an entirely new model (i.e., the authors currently place this question in terms of model selection, not within-model parameter variation). I would suggest that the authors consider a model parameterization fit to the full dataset (both groups) that contains free parameters capturing the *deviations* in the priors relative to the preceding phase's posterior. That is, the free parameters $\bar{\alpha}_{par}^m$ and $\bar{\beta}_{par}^m$ govern the central tendency of the Phase 2 prior parameter distributions directly, but could be reparametrized as deviations from Phase 1 $\theta^m_{ppt}$ parameters in an additive form. This allows for a single model to be fit all participants that encompasses the dynamics of interest such that between-group parameter comparisons are not biased by the strong assumptions imposed by M1 (that phase 1 preferences and phase 2 observations directly transfer to priors). In the case of controls, we would expect these deviation parameters to be centred on 0 insofar as the current M1 fit them best, whereas for BPD participants should have significant deviations from earlier-phase posteriors (e.g., the shift in \beta toward prior neutrality in phase 2 compared to one's own prosociality in phase 1). I think it's still valid for the authors to argue for stronger model constraints for Bayesian model comparison, as they do now, but inferences regarding parameter estimates should ideally be based on a model that can encompass the full dynamics of the entire sample, with simpler dynamics (like posterior -> prior transfer) being captured by near-zero parameter estimates.

My second concern pertains to the psychometric individual difference analyses. These were not clearly justified in the introduction, though I agree that they could offer potentially meaningful insight into which scales may be most related to model parameters of interest. So, perhaps these should be earmarked as exploratory and/or more clearly argued for. Crucially, however, these analyses appear to have been conducted on the full sample without considering the group structure. Indeed, many of the scales on which there are sizable group differences are also those that show correlations with psychometric scales. So, in essence, it is unclear whether most of these analyses are simply recapitulating the between-group tests reported earlier in the paper or offer additional insights. I think it's hard to have one's cake and eat it, too, in this regard and would suggest the authors review Preacher et al. 2005, Psychological Methods for additional detail. One solution might be to always include group as a binary covariate in the symptom dimension-parameter analyses, essentially partialing the correlations for group status. I remain skeptical regarding whether there is additional signal in these analyses, but such controls could convince the reader. Nevertheless, without such adjustments, I would caution against any transdiagnostic interpretations such as this one in the Highlights: "Higher reported childhood trauma, paranoia, and poorer trait mentalizing all diminish other-to-self information transfer irrespective of diagnosis." Since many of these analyses relate to scales on which the groups differ, the transdiagnostic relevance remains to be demonstrated.

Reviewer #1 (Public review):

Summary:

This manuscript reports a very interesting, novel and important research angle to add to the now enormous interest in how pesticides can be toxic to beneficial insects like the honey bee. Many studies have reported on how pesticides in standard use formulations show both lethality as well as sublethal negative effects on behavior and reproduction. The authors propose to use machine learning algorithms to identify new volatile compounds that can be tested for repellency. They use as input chemical structures that are derived from chemicals that have known repellent effects as identified in their initial behavioral assays.

Strengths:

The conclusion is that such chemicals specific to repelling bees and not pest insects (using the fruit fly as a model for the latter) can be identified using the ML approach. Have a list of such chemicals that can be rotated among in any field application would be a benefit because of the honey bees' ability to learn its way around any kind of stimulus designed to keep it from nectar and pollen, even when they may be tainted by pesticide.

Weaknesses:

The use of machine learning seems well-executed and legitimate. But this is beyond my expertise. So other reviewers can maybe comment more on that.

The behavioral data report on the use of a two-choice assay for bees in small Petrie plates. Bess can feed from two small wells place of filter paper impregnated with control or the control containing a chemical. The primary behavior, for ex in Fig 2C, is the first choice by one of the five bees in the plate of which well to feed from. For some chemical compound, there seems to be a 50:50 choice, indicating no repellent effects. In other cases the first bee making the choice chose the control, indicating possible repellent effects of the test chemical. Choices in this assay were validated in a free flying assay.

Concerns with the choice assay:<br /> - 50-70 microliters amounts to what one hungry bee will drink. Did the first bee drink most of it, such that measures of bait consumed reflect a single bee or multiple bees?<br /> - How many bees were repelled to the control side? Was it just the one bee? Were other measures considered? E.g. time to first approach; the number of bees feeding at different time points; the total number of bees observed feeding per unit time.

Reviewer #2 (Public review):

Summary:

The search for new repellent odors for honey bees has significant practical implications. The authors developed an iterative pipeline through machine learning to predict honey bee-repellent odors based on molecular structures. By screening a large number of candidate compounds, they identified a series of novel repellents. Behavioral tests were then conducted to validate the effectiveness of these repellents. Both the discovery and the methodological approach hold value for related fields.

Strengths:

* The study demonstrates that using molecular structures and a relatively small training dataset, the model could predict repellents with a reasonably high success rate. If the iterative approach works as described, it could benefit a wide range of olfaction-related fields.<br /> * The effectiveness of the predicted repellents was validated through both laboratory and field behavioral tests.

Weaknesses:

The small size of the training dataset poses a common challenge for machine learning applications. However, the authors did not clearly explain how their iterative approach addresses this limitation in this study. Quantitative evidence demonstrating improvements achieved in the second round of training would strengthen their claims. For instance, details on whether the success rate of predictions or the identification of higher-affinity components would be helpful. Furthermore, given that only 15 new components were added for the second round of training, it is surprising that such a small dataset could result in significant improvements.

Reviewer #3 (Public review):

The manuscript of Kowalewski et al. titled "Machine learning of honey bee olfactory behavior identifies repellent odorants in free flying bees in the field" did machine learning to predict potential candidates for honeybee repellents, which may keep foraging bees from pesticides. This is a pilot research with strong significance in the research of olfactory behavior and in pest control. However, some major issues need to be addressed to enhance the manuscript's clarity, strength, and overall coherence.

(1) Drosophila melanogaster is not considered as a true agricultural pest. The manuscript would be more compelling if using true pests, for example, Drosophila suzukii or others.<br /> (2) For repellency test, the result relies on dosage. An attractant may become a repellent at high concentration. Test a range of concentrations for each chemicals and compare responses between honeybees and pests.<br /> (3) Be more clear about bee behavior data and their scores (as in Page 4 Results "184 training chemicals and later for 203 chemicals" and Page 10 Methods). I suggest that authors add a supplemental table with each chemical and its behavioral score, feature and reference - which ones were used for training, and which ones for testing. Also add your own behavioral test data (second input) to this table.<br /> (4) The AUC in the first validation was 0.88 (Page 4), and in Page 5, "As expected, the computational validation results based on the AUC values, show an improvement." However, there were no other AUC values to show improvement.<br /> (5) Show plots of ROC AUC curves from Round 1 and Round 2.<br /> (6) In the Discussion, the authors mentioned olfactory receptors in honeybees. It would be useful to provide a general review of the current understanding of these receptors and their (potential) functions.<br /> (7) I suggest combining Fig. 1 and Fig. 3A as one pipeline for this work.<br /> (8) Figure 2C, some sample sizes are very small, such as 2-piperidone: 1 first-choice control vs 0 first-choice repellent? Increase sample size and do statistical analysis.<br /> (9) In general, to assist reviewers, include line numbers to the manuscript.

Reviewer #1 (Public review):

Summary:

This work is meant to help create a foundation for future studies of the Central Complex, which is a critical integrative center in the fly brain. The authors present a systematic description of cellular elements, cell type classifications, behavioral evaluations and genetic resources available to the Drosophila neuroscience community.

Strengths:

The work contributes new, useful and systematic technical information in compelling fashion to support future studies of the fly brain. It also continues to set a high and transparent standard by which large-scale resources can be defined and shared.

Weaknesses:

manuscript p. 1<br /> "The central complex (CX) of the adult Drosophila melanogaster brain consists of approximately 2,800 cells that have been divided into 257 cell types based on morphology and connectivity (Scheer et al., 2020; Hulse et al. 2021; Wolff et al., 2015)."<br /> The 257 accumulated cell types have informational names (e.g., PBG2‐9.s‐FBl2.b‐NO3A.b) in addition to their associations with specific Gal4 lines and specific EM Body IDs. All this is very useful. I have one suggestion to help a reader trying to get a "bird's eye view" of such a large amount of detailed and multi-layered information. Give each of the 257 CX cell types an arbitrary number: 1 to 257. In fact, Supplemental File 2 lists ~277 cell types each with a number in sequence, so perhaps in principle, it is there. This could expedite the search function when a reader is trying to cross-reference CX cell type information from the text, to the Figures and/or to the Supplemental Figures. Also, the use of (arbitrary) cell type numbers could expedite the explanation of which cell types are included in any compilation of information (e.g., which ones were tested for specific NT expression).

manuscript p 2<br /> "Figure 2 and Figure 2-figure supplements 1-4 show the expression of 52 new split-GAL4 lines with strong GAL4 expression that is largely limited to the cell type of interest. .... We also generated lines of lesser quality for other cell types that in total bring overall coverage to more than three quarters of CX cell types."<br /> This section describes the generation and identification of specific split Gal4 lines, and the presentation is generally excellent. It represents an outstanding compendium of information. My reading of the text suggests ~200 cell types have Gal4 lines that are of immediate use (having high specificity or v close-to-high). Use of an arbitrary number system (mentioned above) could augment that description for the reasons stated. For example, which of the 257 cell types are represented by split Gal4 lines that constitute the ~1/3 representing "high-quality lines "? A second comment relates to this study 's functional analysis of the contributions of CX cell types to sleep physiology. The recent literature contains renewed interest in the specific expression patterns of Gal4 lines that can promote sleep-like behaviors. In particular Gal4 line expression outside the brain (in the VNC and outside the CNS) have been raised as important elements that need be included for interpretation interpretation of sleep regulation. This present study offers useful information about a large number of expression patterns, as well as a basis with which to seek additional information., including mention of VNC expression in many cases However, perhaps I missed it, but I could not find a short description of the over-all strategy used to describe the expression patterns and feel that could be helpful. Were all Gal4 lines studied for expression in the VNC? and in the peripheral NS? It is probably published elsewhere, but even a short reprise would still be useful.

manuscript p 9<br /> Neurotransmitter expression in CX cell types<br /> "To determine what neurotransmitters are used by the CX cell types, we carried out fluorescent in situ hybridization using EASI-FISH (Eddison and Irkhe, 2022; Close et al., 2024) on brains that also expressed GFP driven from a cell-type-specific split GAL4 line. In this way, we could determine what neurotransmitters were expressed in over 100 different CX cell types based on ...."<br /> Reading this description, I was uncertain whether the >100 cell types mentioned were tested with all the NT markers by EASI-FISH? Also, assigning arbitrary numbers to the cell types (same suggestion as above) could help the reader more readily ascertain which were the ~100 cell types classified in this context.

manuscript p 10<br /> "Our full results are summarized below, together with our analysis of neuropeptide expression in the same cell types."<br /> I recommend specifying which Figures and Tables contain the "full results" indicated.

NP expression in CX cell types<br /> Similar to the comments regarding studies of NT expression: were all ~100 cell types tested with each of the 17 selected NPs? Arbitrary numerical identifies could be useful for the reader to determine which cell types/ lines were tested and which were not yet tested

manuscript p. 11<br /> "The neuropeptide expression patterns we observed fell into two broad categories."<br /> This section presents information that is extensive and extremely useful. It supports consideration of peptidergic cell signaling at a circuits level and in a systematic fashion that will promote future progress in this field. I have two comments. First, regarding the categorization of two NP expression patterns, discernible by differences in cell number: this idea mirrors one present in prior literature. Recently the classification of the transcription factor DIMM summarizes this same two-way categorization (e.g., doi: 10.1371/journal.pone.0001896). That included the fact that a single NP can be utilized by cell of either category.

Second, regarding this comment:<br /> "In contrast, neuropeptides like those shown in Figure 6 appear to be expressed in dozens to hundreds of cells and appear poised to function by local volume transmission in multiple distinct circuits."<br /> Signaling by NPs in this second category (many small cells) suggests more local diffusion, a smaller geographic expanse compared to "volume" signaling by the sparser larger peptidergic cells. Given this, I suggest re-consideration in using the term "volume" in this instance, perhaps in favor of "local" or "paracrine". This is only a suggestion and in fact rests almost entirely on speculation/ interpretation, as the field lacks a strong empirical basis to say how far NPs diffuse and act. A recent study in the fly brain of peptide co-transmitters (doi: 10.1016/j.cub.2020.04.025) provides an instructive example in which differences between the spatial extents of long-range (peptide 1) versus short-range (peptide 2) NP signaling may be inferred in vivo.

Spab was mentioned (Figure 6 legend) but discarded as a candidate NP to include based on a personal communication, as was Nplp1. The manuscript did not include reasons to do so, nor include a reference to spab peptide. I suggest including explicit reasons to discard candidate NPs.

In Fig 9-supplement 1, neurotransmitter biosynthetic enzymes were measured by RNA-seq for given CX cell types to augment the cell type classification. The same methods could be used to support cell type classification regarding putative peptidergic character (in Figure 9 supplement 2) by measuring expression levels of critical, canonical neuropeptide biosynthetic enzymes. These include the proprotein convertase dPC2 (amon); the carboxypeptidase dCPD/E (silver); and the amidating enzymes dPHM; dPal1; dPal2. PHM is most related to DBM (dopamine beta monooxygenase), the rate limiting enzyme for DA production, and greater than 90% of Drosophila neuropeptides are amidated. If the authors are correct in surmising widespread use of NPs by CX cell types (and I expect they are), there could be diagnostic value to report expression levels of this enzyme set across many/most CX cell types.

Comment #6<br /> Screen of effects on Sleep behavior<br /> This work is large in scope and as suggested likely presents excellent starting points for many follow-up studies. I again suggest assigning stable number identities to the elements described. In this case, not cell types, but split Gal4 lines. This would expedite the cross-referencing of results across the four Supplemental Files 3-6. For example, line SS00273 is entry line #27 in S Files 3 and 4, but line entry #18 in S Files 5 and 6.

manuscript p 26<br /> Clock to CX<br /> "Not surprisingly, the connectome reveals that many of the intrinsic CX cell types with sleep phenotypes are connected by wired pathways (Figure 12 and Figure 12-figure supplement 1)."<br /> Do intrinsic CX cells with sleep phenotypes also connect by wired pathways to CX cells that do not have sleep phenotypes?

"The connectome also suggested pathways from the circadian clock to the CX. Links between clock output DN1 neurons to the ExR1 have been described in Lamaze et al. (2018) and Guo et al. (2018), and Liang et al. (2019) described a connection from the clock to ExR2 (PPM3) dopaminergic neurons."<br /> The introduction to this section indicates a focus on connectome-defined synaptic contacts. Whereas the first two studies cited featured both physiological and anatomic evidence to support connectivity from clock cells to CX, the third did not describe any anatomical connections, and that connection may in fact be due to diffuse not synaptic signaling

I could not easily discern the difference between Figs 12 and 12-S1? These appear to be highly-related circuit models, wherein the second features more elements. Perhaps spell out the basis for the differences between the two models to avoid ambiguity.

"...the cellular targets of Dh31 released from ER5 are unknown, however previous work (Goda et al., 2017; Mertens et al., 2005) has shown that Dh31 can activate the PDF receptor raising the possibility of autocrine signaling."<br /> Regarding pharmacological evidence for Dh31 activation of Pdfr: strong in vivo evidence was developed in doi: 10.1016/j.neuron.2008.02.018: a strong pdfr mutation greatly reduces response to synthetic dh31 in neurons that normally express Pdfr

manuscript p 30<br /> "Unexpectedly, we found that all neuropeptide-expressing cell types also expressed a small neurotransmitter."<br /> Did this conclusion apply only to CX cell types? - or was it also true for large peptidergic neurons? Prior evidence suggests the latter may not express small transmitters (doi: 10.1016/j.cub.2009.11.065). The question pertains to the broader biology of peptidergic neurons, and is therefore outside the strict scope of the main focus area - the CX. However, the text did initially consider peptidergic neurons outside the CX, so the information may be pertinent to many readers.

Reviewer #2 (Public review):

Summary:

In this paper, Wolff et al. describe an impressive collection of newly created split-GAL4 lines targeting specific cell types within the central complex (CX) of Drosophila. The CX is an important area in the brain that has been involved in the regulation of many behaviors including navigation and sleep/wake. The authors advocate that to fully understand how the CX functions, cell-specific driver lines need to be created. In that respect, this manuscript will be of very important value to all neuroscientists trying to elucidate complex behaviors using the fly model. In addition, and providing a further very important finding, the authors went on to assess neurotransmitter/neuropeptides and their receptors expression in different cells of the CX. These findings will also be of great interest to many and will help further studies aimed at understanding the CX circuitries. The authors then investigated how different CX cell types influence sleep and wake. While the description of the new lines and their neurochemical identity is excellent, the behavioral screen seems to be limited.

Strengths:

(1) The description of dozens of cell-specific split-GAL4 lines is extremely valuable to the fly community. The strength of the fly system relies on the ability to manipulate specific neurons to investigate their involvement in a specific behavior. Recently, the need to use extremely specific tools has been highlighted by the identification of sleep-promoting neurons located in the VNC of the fly as part of the expression pattern of the most widely used dorsal-Fan Shaped Body (dFB) GAL4 driver. These findings should serve as a warning to every neurobiologist, make sure that your tool is clean. In that respect, the novel lines described in this manuscript are fantastic tools that will help the fly community.<br /> (2) The description of neurotransmitter/neuropeptides expression pattern in the CX is of remarkable importance and will help design experiments aimed at understanding how the CX functions.

Weaknesses:

(1) I find the behavioral (sleep) screen of this manuscript to be limited. It appears to me that this part of the paper is not as developed as it could be. The authors have performed neuronal activation using thermogenetic and/or optogenetic approaches. For some cell types, only thermogenetic activation is shown. There is no silencing data and/or assessment of sleep homeostasis or arousal threshold. The authors find that many CX cell types modulate sleep and wake but it's difficult to understand how these findings fit one with the other. It seems that each CX cell type is worthy of its own independent study and paper. I am fully aware that a thorough investigation of every CX neuronal type in sleep and wake regulation is a herculean task. So, altogether I think that this manuscript will pave the way for further studies on the role of CX neurons in sleep regulation.<br /> (2) Linked to point 1, it is possible that the activation protocols used in this study are insufficient for some neuronal types. The authors have used 29{degree sign} for thermogenetic activation (instead of the most widely used 31{degree sign}) and a 2Hz optogenetic activation protocol. The authors should comment on the fact that they may have missed some phenotypes by using these mild activation protocols.<br /> (3) There are multiple spelling errors in the manuscript that need to be addressed.

Reviewer #3 (Public review):

Summary:

The authors created and characterized genetic tools that allow for precise manipulation of individual or small subsets of central complex (CX) cell types in the Drosophila brain. They developed split-GAL4 driver lines and integrated this with a detailed survey of neurotransmitter and neuropeptide expression and receptor localization in the central brain. The manuscript also explores the functional relevance of CX cell types by evaluating their roles in sleep regulation and linking circadian clock signals to the CX. This work represents an ambitious and comprehensive effort to provide both molecular and functional insights into the CX, offering tools and data that will serve as a critical resource for researchers.

Strengths:

(1) The extensive collection of split-GAL4 lines targeting specific CX cell types fills a critical gap in the genetic toolkit for the Drosophila neuroscience community.<br /> (2) By combining anatomical, molecular, and functional analyses, the authors provide a holistic view of CX cell types that is both informative and immediately useful for researchers across diverse disciplines.<br /> (3) The identification of CX cell types involved in sleep regulation and their connection to circadian clock mechanisms highlights the functional importance of the CX and its integrative role in regulating behavior and physiological states.<br /> (4) The authors' decision to present this work as a single, comprehensive manuscript rather than fragmenting it into smaller publications each focusing on separate central complex components is commendable. This decision prioritizes accessibility and utility for the broader neuroscience community, which will enable researchers to approach CX-related questions with a ready-made toolkit.

Weaknesses:

While the manuscript is an outstanding resource, it leaves room for more detailed mechanistic exploration in some areas. Nonetheless, this does not diminish the immediate value of the tools and data provided.

Appraisal:

The authors have succeeded in achieving their aims of creating well-characterized genetic tools and providing a detailed survey of neurochemical and functional properties in the CX. The results strongly support their conclusions and open numerous avenues for future research. The work effectively bridges the gap between genetic manipulation, molecular characterization, and functional assessment, enabling a deeper understanding of the CX's diverse roles.

Impact and Utility

This manuscript will have a significant and lasting impact on the field, providing tools and data that facilitate new discoveries in the study of the CX, sleep regulation, circadian biology, and beyond. The genetic tools developed here are likely to become a standard resource for Drosophila researchers, and the comprehensive dataset on neurotransmitter and neuropeptide expression will inspire investigations into the interplay between neuromodulation and classical neurotransmission.

Additional Context

By delivering an integrated dataset that spans anatomy, molecular properties, and functional relevance, the authors have created a resource that will serve the neuroscience community for years to come.

Reviewer #1 (Public review):

Summary:

Recent work has demonstrated that the hummingbird hawkmoth, Macroglossum stellatarum, like many other flying insects, use ventrolateral optic flow cues for flight control. However, unlike other flying insects, the same stimulus presented in the dorsal visual field elicits a directional response. Bigge et al., use behavioral flight experiments to set these two pathways in conflict in order to understand whether these two pathways (ventrolateral and dorsal) work together to direct flight and if so, how. The authors characterize the visual environment (the amount of contrast and translational optic flow) of the hawkmoth and find that different regions of the visual field are matched to relevant visual cues in their natural environment and that the integration of the two pathways reflects a priortiziation for generating behavior that supports hawkmoth safety rather than than the prevalence for a particular visual cue that is more prevalent in the environment.

Strengths:

This study creatively utilizes previous findings that the hawkmoth partitions their visual field as a way to examine parallel processing. The behavioral assay is well-established and the authors take the extra steps to characterize the visual ecology of the hawkmoth habitat to draw exciting conclusions about the hierarchy of each pathway as it contributes to flight control.

Weaknesses:

The work would be further clarified and strengthened by additional explanation included in the main text, figure legends, and methods that would permit the reader to draw their own conclusions more feasibly. It would be helpful to have all figure panels referenced in the text and referenced in order, as they are currently not. In addition, it seems that sometimes the incorrect figure panel is referenced in the text, Figure S2 is mislabeled with D-E instead of A-C and Table S1 is not referenced in the main text at all. Table S1 is extremely important for understanding the figures in the main text and eliminating acronyms here would support reader comprehension, especially as there is no legend provided for Table S1. For example, a reader that does not specialize in vision may not know that OF stands for optic flow. Further detail in figure legends would also support the reader in drawing their own conclusions. For example, dashed red lines in Figures 3 and 4 A and B are not described and the letters representing statistical significance could be further explained either in the figure legend or materials to help the reader draw their own conclusions.

Reviewer #2 (Public review):

Summary:

Bigge and colleagues use a sophisticated free-flight setup to study visuo-motor responses elicited in different parts of the visual field in the hummingbird hawkmoth. Hawkmoths have been previously shown to rely on translational optic flow information for flight control exclusively in the ventral and lateral parts of their visual field. Dorsally presented patterns, elicit a formerly completely unknown response - instead of using dorsal patterns to maintain straight flight paths, hawkmoths fly, more often, in a direction aligned with the main axis of the pattern presented (Bigge et al, 2021). Here, the authors go further and put ventral/lateral and dorsal visual cues into conflict. They found that the different visuomotor pathways act in parallel, and they identified a 'hierarchy': the avoidance of dorsal patterns had the strongest weight and optic flow-based speed regulation the lowest weight.

Strengths:

The data are very interesting, unique, and compelling. The manuscript provides a thorough analysis of free-flight behavior in a non-model organism that is extremely interesting for comparative reasons (and on its own). These data are both difficult to obtain and very valuable to the field.

Weaknesses:

While the present manuscript clearly goes beyond Bigge et al, 2021, the advance could have perhaps been even stronger with a more fine-grained investigation of the visual responses in the dorsal visual field. Do hawkmoths, for example, show optomotor responses to rotational optic flow in the dorsal visual field?

Reviewer #3 (Public review):

The central goal of this paper as I understand it is to extract the "integration hierarchy" of stimulus in the dorsal and ventrolateral visual fields. The segregation of these responses is different from what is thought to occur in bees and flies and was established in the authors' prior work. Showing how the stimuli combine and are prioritized goes beyond the authors' prior conclusions that separated the response into two visual regions. The data presented do indeed support the hierarchy reported in Figure 5 and that is a nice summary of the authors' work. The moths respond to combinations of dorsal and lateral cues in a mixed way but also seem to strongly prioritize avoiding dorsal optic flow which the authors interpret as a closed and potentially dangerous ecological context for these animals. The authors use clever combinations of stimuli to put cues into conflict to reveal the response hierarchy.

My most significant concern is that this hierarchy of stimulus responses might be limited to the specific parameters chosen in this study. Presumably, there are parameters of these stimuli that modulate the response (spatial frequency, different amounts of optic flow, contrast, color, etc). While I agree that the hierarchy in Figure 5 is consistent for the particular stimuli given, this may not extend to other parameter combinations of the same cues. For example, as the contrast of the dorsal stimuli is reduced, the inequality may shift. This does not preclude the authors' conclusions but it does mean that they may not generalize, even within this species. For example, other cue conflict studies have quantified the responses to ranges of the parameters (e.g. frequency) and shown that one cue might be prioritized or up-weighted in one frequency band but not in others. I could imagine ecological signatures of dorsal clutter and translational positioning cues could depend on the dynamic range of the optic flow, or even having spatial-temporal frequency-dependent integration independent of net optic flow.

The second part of this concern is that there seems to be a missed opportunity to quantify the integration, especially when the optic flow magnitude is already calculated. The discussion even highlights that an advantage of the conflict paradigm is that the weights of the integration hierarchy can be compared. But these weights, which I would interpret as stimulus-responses gains, are not reported. What is the ratio of moth response to optic flow in the different regions? When the moth balances responses in the dorsal and ventrolateral region, is it a simple weighted average of the two? When it prioritizes one over the other is the response gain unchanged? This plays into the first concern because such gain responses could strongly depend on the specific stimulus parameters rather than being constant.

The authors do explain the choice of specific stimuli in the context of their very nice natural scene analysis in Fig. 1 and there is an excellent discussion of the ecological context for the behaviors. However, I struggled to directly map the results from the natural scenes to the conclusions of the paper. How do they directly inform the methods and conclusions for the laboratory experiments? Most important is the discussion in the middle paragraph of page 12, which suggests a relationship with Figure 1B, but seems provocative but lacking a quantification with respect to the laboratory stimuli.

The central conclusion of the first section of the results is that there are likely two different pathways mediating the dorsal and the ventrolateral response. This seems reasonable given the data, however, this was also the message that I got from the authors' prior paper (ref 11). There are certainly more comparisons being done here than in that paper and it is perfectly reasonable to reinforce the conclusion from that study but I think what is new about these results needs to be highlighted in this section and differentiated from prior results. Perhaps one way to help would be to be more explicit with the open hypotheses that remain from that prior paper.

Reviewer #1 (Public review):

Summary:

The authors investigate the neuroprotective effect of reserpine in a retinitis pigmentosa (P23H-1) model, characterized by a mutation in the rhodopsin gene. Their results reveal that female rats show better preservation of both rod and cone photoreceptors following reserpine treatment compared to males.

Strengths:

This study effectively highlights the neuroprotective potential of reserpine and underscores the value of drug repositioning as a strategy for accelerating the development of effective treatments. The findings are significant for their clinical implications, particularly in demonstrating sex-specific differences in therapeutic response.

Weaknesses:

The main limitation is the lack of precise identification of the specific pathway through which reserpine prevents photoreceptor death.

Reviewer #2 (Public review):

Summary:

In the manuscript entitled "Sex-specific attenuation of photoreceptor degeneration by reserpine in a rhodopsin P23H rat model of autosomal dominant retinitis pigmentosa" by Beom Song et al., the authors explore the transcriptomic differences between male and female wild-type (WT) and P23H retinas, highlighting significant gene expression variations and sex-specific trends. The study emphasizes the importance of considering biological sex in understanding inherited retinal degeneration and the impact of drug treatments on mutant retinas.

Strengths:

(1) Relevance to Clinical Challenges: The study addresses a critical limitation in inherited retinal degeneration (IRD) therapies by exploring a gene-agnostic approach. It emphasizes sex-specific responses, which aligns with recent NIH mandates on sex as a biological variable.<br /> (2) Multi-dimensional Methodology: Combining electroretinography (ERG), optical coherence tomography (OCT), histology, and transcriptomics strengthens the study's findings.<br /> (3) Novel Insights: The transcriptomic analysis uncovers sex-specific pathways impacted by reserpine, laying the foundation for personalized approaches to retinal disease therapy.

Weaknesses:

Dose Optimization<br /> The study uses a fixed dose (40 µM), but no dose-response analysis is provided. Sex-specific differences in efficacy might be influenced by suboptimal dosing, particularly considering potential differences in metabolism or drug distribution.

Statistical Analysis

In my opinion, there is room for improvement. How were the animals injected? Was the contralateral eye used as control? (no information in the manuscript about it!, line 390 just mentions the volume and concentration of injections). If so, why not use parametric paired analysis? Why use a non-parametric test, as it is the Mann-Whitney U? The Mann-Whitney U test is usually employed for discontinuous count data; is that the case here?<br /> Therefore, please specify whether contralateral eyes or independent groups served as controls. If contralateral controls were used, paired parametric tests (e.g., paired t-tests) would be statistically appropriate. Alternatively, if independent cohorts were used, non-parametric Mann-Whitney U tests may suffice but require clear justification.

Sex-Specific Pathways

The authors do identify pathways enriched in female vs. male retinas but fail to explicitly connect these to the changes in phenotype analysed by ERG and OCT. The lack of mechanistic validation weakens the argument.

The study does not explore why female rats respond better to reserpine. Potential factors such as hormonal differences, retinal size, or differential drug uptake are not discussed.<br /> It remains open, whether observed transcriptomic trends (e.g., proteostasis network genes) correlate with sex-specific functional outcomes.

Reviewer #1 (Public review):

Summary:

The study addresses the growing threat of multi-drug-resistant (MDR) pathogens, focusing on the efficacy of colistin (COL), a last-resort antibiotic, and its enhanced activity when combined with artesunate (AS) and ethylenediaminetetraacetic acid (EDTA) against colistin-resistant Salmonella strains. The researchers aim to explore whether these combinations can restore the effectiveness of colistin and understand the underlying mechanisms. The study used a combination of microbiological and molecular techniques to evaluate the antibacterial activity and mechanisms of action of COL, AS, and EDTA. Key methods included: (i) Antimicrobial Susceptibility Testing: Determining minimum inhibitory concentrations (MICs) of COL, AS, and EDTA, both alone and in combination, against various Salmonella strains; (ii) Time-Kill Assays: Measuring bacterial growth inhibition over time with different drug combinations; (iii) Fluorescent Probe-Permeability Assays: Assessing cell membrane integrity using fluorescent dyes; (iv) Proton Motive Force Assay: Evaluating the impact on the electrochemical proton gradient (PMF); (v) Reactive Oxygen Species (ROS) Measurement: Quantifying intracellular ROS levels; (vi) Scanning Electron Microscopy (SEM): Observing morphological changes in bacterial cells; and (vii) Omics Analysis: Transcriptome and metabolome profiling to identify differentially expressed genes (DEGs) and significant differential metabolites (SDMs). The combination of COL, AS, and EDTA (AEC) showed significant antibacterial activity against colistin-resistant Salmonella strains, reducing the MICs and enhancing bacterial killing compared to individual treatments. The AEC treatment caused extensive damage to both the outer and inner bacterial membranes, as evidenced by increased fluorescence of membrane-impermeant dyes and SEM images showing deformed cell membranes. AEC treatment selectively collapsed the Δψ component of PMF, indicating disruption of vital cellular processes. The combination therapy increased intracellular ROS levels, contributing to bacterial killing. Transcriptome data revealed changes in genes related to two-component systems, flagellar assembly, and ABC transporters. Metabolome analysis highlighted disruptions in pathways such as arachidonic acid metabolism. The findings suggest that AS and EDTA can potentiate the antibacterial effects of colistin by disrupting bacterial membranes, collapsing PMF, and increasing ROS levels. This combination therapy could serve as a promising approach to combat colistin-resistant Salmonella infections.

Strengths:

- The study employs a wide range of techniques to thoroughly investigate the antibacterial mechanisms and efficacy of the drug combinations.<br /> - The results are consistent across multiple assays and supported by both in vitro and in vivo data.<br /> - Combining AS and EDTA with COL represents a novel strategy to tackle antibiotic resistance.

Weaknesses:

- The methodology used for interpreting and reporting time-kill assay results.

Comments on revised version:

Overall, the authors have adequately addressed the suggestions provided.

Reviewer #2 (Public review):

The study by Zhai et al describes repurposing of artesunate, to be used in combination with EDTA to resensitize Salmonella spp. to colistin. The observed effect applied both to strains with and without mobile colistin resistance determinants (MCR). It is known since earlier that EDTA in combination with colistin has an inhibitory effect on MCR-enzymes, but at the same time both colistin and EDTA can contribute to nephrotoxicity, something which is also true for artesunate. Thus, the triple combination of three nephrotoxic agents has significant challenges in vivo, which is not particularly discussed in this paper.

The study is sound from a methodological point of view and has many interesting angles to address mechanistically how the three compounds can synergize.

Comments on revised version:

After having read the revised version, I have the following comments:

(1) The antimicrobials tested in Figure 9 are not really very relevant. I would want to see carbapenems and novel beta-lactam/beta-lactamase inhibitors rather than many old drugs with a debatable role in the treatment of Gram-negative infections. At least the authors should be able to test carbapenem resistance<br /> (2) The genomics analysis of the strains should be fairly quick - both in terms of characterizing the mobile resistome and the sequence types. There are publicly available databases for this purpose

The rest of my comments have been addressed in the revised version. There are still some remaining valid points from other reviewers that could be debatable whether they should be address. The authors refer to plans of studying these aspects in subsequent studies, but it could be discussed whether some of the data could be expected already in this study.

Reviewer #1 (Public review):

Summary:

SARS-CoV-2 infection induces syncytia formation, which promotes viral transmission. In this paper, the authors aimed to understand how host-derived inflammatory cytokines IL-1α/β combat SARS-CoV-2 infection.

Strengths:

First, they used a cell-cell fusion assay developed previously to identify IL-1α/β as the cytokines that inhibit syncytia formation. They co-cultured cells expressing the spike protein and cells expressing ACE2 and found that IL-1β treatment decreased syncytia formation and S2 cleavage.

Second, they investigated the IL-1 signaling pathway in detail, using knockouts or pharmacological perturbation to understand the signaling proteins responsible for blocking cell fusion. They found that IL-1 prevents cell-cell fusion through MyD88/IRAK/TRAF6 but not TAK1/IKK/NF-κB, as only knocking out MyD88/IRAK/TRAF6 eliminates the inhibitory effect on cell-cell fusion in response to IL-1β. This revealed that the inhibition of cell fusion did not require a transcriptional response and was mediated by IL-1R proximal signaling effectors.

Third, the authors identified RhoA/ROCK activation by IL-1 as the basis for this inhibition of cell fusion. By visualizing a RhoA biosensor and actin, they found a redistribution of RhoA to the cell periphery and cell-cell junctions after IL-1 stimulation. This triggered the formation of actin bundles at cell-cell junctions, preventing fusion and syncytia formation. The authors confirmed this molecular mechanism by using constitutively active RhoA and an inhibitor of ROCK.<br /> Diverse Cell types and in vivo models were used, and consistent results were shown across diverse models. These results were convincing and well-presented.

In summary, the authors have provided compelling evidence regarding how IL-1 signaling induces a prophylactic response to viral infection. While the mechanistic details of how IL-1R and MyD88 induce RhoA/Rock pathway to mediate actin remodeling remain unclear, this manuscript serves as the basis for future studies.

Reviewer #2 (Public review):

Summary:

In this study, Zheng et al investigated the role of inflammatory cytokines in protecting cells against SARS-CoV-2 infection. They demonstrate that soluble factors in the supernatants of TLR stimulated THP1 cells reduce fusion events between HEK293 cells expressing SARS-CoV-2 S protein and the ACE2 receptor. Using qRT-PCR and ELISA, they demonstrate that IL-1 cytokines are (not surprisingly) upregulated by TLR treatment in THP1 cells. Further, they convincingly demonstrate that recombinant IL-1 cytokines are sufficient to reduce cell-to-cell fusion mediated by the S protein. Using chemical inhibitors and CRISPR knock-out of key IL-1 receptor signaling components in HEK293 cells, they demonstrate that components of the myddosome (MYD88, IRAK1/4, and TRAF6) are required for fusion inhibition, but that downstream canonical signaling (i.e., TAK1 and NFKB activation) is not required. Instead, they provide evidence that IL-1-dependent non-canonical activation of RhoA/Rock is important for this phenotype. Importantly, the authors demonstrate that expression of a constitutively active RhoA alone is sufficient to inhibit fusion and that chemical inhibition of Rock could reverse this inhibition. The authors followed up these in vitro experiments by examining the effects of IL-1 on SARS-COV-2 infection in vivo and they demonstrate that recombinant IL-1 can reduce viral burden and lung pathogenesis in a mouse model of infection. Use of a ROCK inhibitor in IL-1 treated mice restored the ability of SARS-CoV-2 to spread in the lung, suggesting that this inhibitory process functions in vivo.

Strengths:

(1) The bioluminescence cell-cell fusion assay provides a robust quantitative method to examine cytokine effects on viral glycoprotein-mediated fusion.

(2) The study identifies a new mechanism by which IL-1 cytokines can limit virus infection.

(3) The authors tested IL-1 mediated inhibition of fusion induced by many different coronavirus S proteins and several SARS-CoV-2 strains.

(4) The authors demonstrate that recombinant IL-1 mediated inhibition of SARS-CoV-2 infection in mice is dependent on the RhoA/Rock pathway.

Reviewer #1 (Public review):

Summary:

In this study, Bu et al examined the dynamics of TRPV4 channel in cell overcrowding in carcinoma conditions. They investigated how cell crowding (or high cell confluence) triggers a mechano-transduction pathway involving TRPV4 channels in high-grade ductal carcinoma in situ (DCIS) cells that leads to large cell volume reduction (or cell volume plasticity) and pro-invasive phenotype.

In vitro, this pathway is highly selective for highly malignant invasive cell lines derived from a normal breast epithelial cell line (MCF10CA) compared to the parent cell line, but not present in another triple-negative invasive breast epithelial cell line (MDA-MB-231). The authors convincingly showed that enhanced TRPV4 plasmamembrane localization correlates with high-grade DCIS cells in patient tissue samples. Specifically in invasive MCF10DCIS.com cells they showed that overcrowding or over-confluence leads to a decrease in cell volume and intracellular calcium levels. This condition also triggers the trafficking of TRPV4 channels from intracellular stores (nucleus and potentially endosomes), to the plasma membrane (PM). When these over-confluent cells are incubated with a TRPV4 activator, there is an acute and substantial influx of calcium, attesting the fact that there are high number of TRPV4 channels present on the PM. Long-term incubation of these over-confluent cells with the TRPV4 activator results in the internalization of the PM-localized TRPV4 channels.

In contrast, cells plated at lower confluence primarily have TRPV4 channels localized in the nucleus and cytosol. Long-term incubation of these cells at lower confluence with a TRPV4 inhibitor leads to the relocation of TRPV4 channels to the plasma membrane from intracellular stores and a subsequent reduction in cell volume. Similarly, incubation of these cells at low confluence with PEG 3000 (a hyperosmotic agent) promotes the trafficking of TRPV4 channels from intracellular stores to the plasma membrane.

Strengths:

The study is elegantly designed and the findings are novel. Their findings on this mechano-transduction pathway involving TRPV4 channels, calcium homeostasis, cell volume plasticity, motility and invasiveness will have a great impact in the cancer field and potentially applicable to other fields as well. Experiments are well-planned and executed, and the data is convincing. Authors investigated TRVP4 dynamics using multiple different strategies- overcrowding, hyperosmotic stress, pharmacological and genetic means, and showed a good correlation between different phenomena.

All of my previous concerns have been addressed. The quality of the manuscript has improved significantly.

Reviewer #2 (Public review):

Summary:

The metastasis poses a significant challenge in cancer treatment. During the transition from non-invasive cells to invasive metastasis cells, cancer cells usually experience mechanical stress due to a crowded cellular environment. The molecular mechanisms underlying mechanical signaling during this transition remain largely elusive. In this work, the authors utilize an in vitro cell culture system and advanced imaging techniques to investigate how non-invasive and invasive cells respond to cell crowding, respectively.

The results clearly show that pre-malignant cells exhibit a more pronounced reduction in cell volume and are more prone to spreading compared to non-invasive cells. Furthermore, the study identifies that TRPV4, a calcium channel, relocates to the plasma membrane both in vitro and in vivo (patient's samples). Activation and inhibition of TRPV4 channel can modulate the cell volume and cell mobility. These results unveil a novel mechanism of mechanical sensing in cancer cells, potentially offering new avenues for therapeutic intervention targeting cancer metastasis by modulating TRPV4 activity. This is a very comprehensive study, and the data presented in the paper are clear and convincing. The study represents a very important advance in our understanding of the mechanical biology of cancer.

Reviewer #1 (Public review):

Summary:

The authors report a study aimed at understanding the brain's representations of viewed actions, with a particular aim to distinguish regions that encode observed body movements, from those that encode the effects of actions on objects. They adopt a cross-decoding multivariate fMRI approach, scanning adult observers who viewed full-cue actions, pantomimes of those actions, minimal skeletal depictions of those actions, and abstract animations that captured analogous effects to those actions. Decoding across different pairs of these action conditions allowed the authors to pull out the contributions of different action features in a given region's representation. The main hypothesis, which was largely confirmed, was that the superior parietal lobe (SPL) more strongly encodes movements of the body, whereas the anterior inferior parietal lobe (aIPL) codes for action effects of outcomes. Specifically, region of interest analyses showed dissociations in the successful cross-decoding of action category across full-cue and skeletal or abstract depictions. Their analyses also highlight the importance of the lateral occipito-temporal cortex (LOTC) in coding action effects. They also find some preliminary evidence about the organisation of action kinds in the regions examined, and take some steps to distinguishing the differences and similarities of action-evoked patterns in primary visual cortex and the other examined regions.

Strengths:

The paper is well-written, and it addresses a topic of emerging interest where social vision and intuitive physics intersect. The use of cross-decoding to examine actions and their effects across four different stimulus formats is a strength of the study. Likewise the a priori identification of regions of interest (supplemented by additional full-brain analyses) is a strength. Finally, the authors successfully deployed a representational-similarity approach that provides more detailed evidence about the different kinds of action features that seem to be captured in each of the regions that were examined.

Weaknesses:

Globally, the findings provide support for the predicted anatomical distinctions, and for the distinction between body-focused representations of actions and more abstract "action effect structures". Viewed more narrowly, the picture is rather complex, and the patterns of (dis)similarity in the activity evoked by different action kinds do not always divide neatly. Probably, examining many more kinds of actions with the multi-format decoding approach developed here will be needed to more effectively disentangle the various contributions of movement, posture, low-level visual properties, and action outcomes/effects.