Reviewer #2 (Public review):

Using multimodal closed-loop behavior and activity monitoring in the neocortex, Solyga and Keller show that the auditory cortex computes the deviation of current sensory input from expectations. Interestingly, in addition, mismatch responses within the auditory stream are non-linearly influenced by concurrent sensorimotor error computations in the visual pathway. These results suggest that non-hierarchical interactions (lateral relational cross-talk) must be considered when analyzing cortical models based on predictive processing. In my opinion, this is a fundamental study that addresses the question of hierarchical vs. no-hierarchical interactions across neocortical areas. Overall, I find the experiments elegantly designed, and the results robust, providing compelling evidence for non-hierarchical interactions across neocortical areas, and more specifically of exchange of sensorimotor prediction error signals across modalities. The authors thoroughly addressed the concerns raised. In my opinion, this has substantially strengthened the manuscript, enabling much clearer interpretation of the results reported.

Reviewer #3 (Public review):

Summary:

In this study, the authors have attempted to demonstrate a critical role for the cytoskeletal scaffold protein Ezrin, in the upstream regulation of EGFR/AKT/MTOR signaling. They show that in the absence of Ezrin, ligand-induced EGFR trafficking and activation at the endosomes is perturbed, with decreased endosomal recruitment of the TSC complex, and a corresponding decrease in AKT/MTOR signaling.

Strengths:

The authors have used a combination of novel imaging techniques, as well as conventional proteomic and biochemical assays to substantiate their findings. The findings expand our understanding of the upstream regulators of the EGFR/AKT MTOR signaling and lysosomal biogenesis, appear to be conserved in multiple species, and may have important implications for the pathogenesis and treatment of diseases involving endo-lysosomal function, such as diabetes and cancer, as well as neuro-degenerative diseases like macular degeneration. Furthermore, pharmacological targeting of Ezrin could potentially be utilized in diseases with defective TFEB/TFE3 functions like LSDs. While a majority of the findings appear to support the hypotheses, there are substantial gaps in the findings that could be better addressed. Since Ezrin appears to directly regulate MTOR activity, the effects of Ezrin KO on MTOR-regulated, TFEB/TFE3 -driven lysosomal function should be explored more thoroughly. Similarly, a more convincing analysis of autophagic flux should be carried out. Additionally, many immunoblots lack key controls (Control IgG in CO-Ips) and many others merit repetition to either improve upon the quality of the existing data, validate the findings using orthogonal approaches or to provide a more rigorous quantitative assessment of the findings, as highlighted in the recommendation for authors.

Comments on revisions:

The authors have satisfactorily addressed most of the concerns raised in the prior version, and have significantly improved upon the overall findings in the revised version.

Reviewer #2 (Public review):

This study investigates the cortical circuitry at the mesoscopic level of cortical columns in the human secondary visual cortex (V2) using high-resolution fMRI at ultra-high field strength (7T). The findings confirm the columnar organization of color-selective thin and disparity-selective thick stripes, a result previously demonstrated and replicated in human fMRI research. However, this study adds a novel layer of analysis by examining cortical depth, providing insights into feedforward and feedback connections to and from V2. Furthermore, examining texture selectivity in V2 showed no evidence of a columnar structure when compared to color- and disparity-selective activation clusters. Interestingly, texture selectivity in V2 was most pronounced in deeper cortical layers, with significant feedback connectivity from V4. The authors conclude that local columnar circuitry plays a crucial role in color and disparity processing within V2, while texture selectivity is driven by feedback modulation. This research underscores the potential of high-resolution human fMRI to explore the local circuitry of the cortex at the mesoscopic scale.

However, I still have a few comments that I would like to be addressed:

(1) In lines 401-403, the authors state that differential BOLD responses can significantly enhance the laminar specificity. Differential contrasts indeed have the potential to reduce macrovascular contributions that are unspecific to both experimental conditions, which was already discussed in the literature (e.g., Yacoub et al., 2008, High-field fMRI unveils orientation columns in humans). This might be especially true for the pial vasculature that drains a larger surface area of the cortex, e.g., multiple columns, which is probably the key factor that enables cortical column mapping using differential BOLD contrasts despite the relatively large spatial point spread function of the BOLD response. However, this may differ for laminar analyses, where neuronal and vascular responses from intracortical and pial veins might be harder to disentangle. It would, therefore, be advisable to tone down this statement somewhat since it could imply that laminar specificity can be readily achieved with GE-BOLD, while this remains an active area of research. This is not to say that the present results are incorrect, but the broader implications of this statement should be cautiously framed.

(2) Looking at Figure 3, one might also argue (excluding responses from V4) that statistically significant differences in selectivity are only observed where the cortical profiles generally show higher response levels. Could this be simply due to varying signal-to-noise ratios (SNR) achieved by different contrasts (color, disparity, texture)?

(3) In lines 480-484, the authors state that twenty blocks for each stimulus condition should be sufficient to investigate within-subject effects. It would be helpful if they could elaborate on the basis for this claim. High-resolution fMRI is typically limited by low temporal signal-to-noise ratio (tSNR), and extensive averaging is often required to achieve sufficient signal. Clarifying the rationale behind this assertion would strengthen the argument.

Reviewer #2 (Public review):

Summary:

Plectin is a cytolinker that associates with cytoskeletal and intercellular junction proteins and is essential for epithelial integrity and cell migration. Previous reports showed that PLEC regulates tumor growth and metastasis in different cancers. In this manuscript, the authors describe PLEC as a target in initiation and growth of HCC. They show that inhibiting PLEC reduced tumorigenesis in different in vitro and in vivo HCC models, including in a xenograft model, DEN model, oncogene-induced HCC model and a lung metastasis model. A drug PST had similar effects, a purported Plectin inhibitor, suggesting that PLEC inhibition could be a tumor prevention or treatment strategy. Mechanistically, the authors show that inhibiting PLEC results in a disorganized cytoskeleton, deficiency in cell migration, and changes in cancer-relevant signaling pathways. This study demonstrates the importance of understanding mechanobiology of HCC for the development of new treatment strategies.

Strengths:

(1) This study used a variety of in vivo models to explore the role of Plectin in HCC formation and metastasis, which extend beyond the cell line-based studies reported in prior research.<br /> (2) Blocking PLEC disrupts pathways that promote tumors and cell migration, thus preventing tumor progression.<br /> (3) Overall, the anti-cancer phenotype is promising, strengthening the important role of PLEC and related factors in tumor growth and metastasis.

Weaknesses:

(1) There is limited novel mechanistic insights as the effect of inhibiting PLEC on the cytoskeleton, cell migration and related signaling pathways have previously been reported.<br /> (2) The results associated with PST, should be interpretated with caution. Although it is reported as an inhibitor of PLECTIN, and the phenotypes and pathways affected are similar to the knock-out, additional research is needed to support whether it will be safe and specific in treating or preventing HCC.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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.

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 #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:

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #2 (Public review):

Summary:

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.

Strengths:

The manuscript is well-written, with a concise and clear writing style. The visual presentation is largely clear. The study involves multiple experiments with different participant groups. Each experiment involves specific considered changes to the experimental paradigm that both replicate the previous experiment's finding yet extend it in a relevant manner.

In the first revisions of the paper, the manuscript better relays the results and anticipates analyses, and this version adequately resolves some concerns I had about analysis details. In a further revision, it is clarified better how the results relate to the various competing hypotheses on how biological motion is processed.

Weaknesses:

Still, it is my view that the findings of the study are basic neural correlate results that offer only minimal constraint towards the question of how the brain realizes the integration of multisensory information in the service of biological motion perception, and the data do not address the causal relevance of observed neural effects towards behavior and cognition. The presence of an inversion effect suggests that the supra-additivity is related to cognition, but that leaves open whether any detected neural pattern is actually consequential for multi-sensory integration (i.e., correlation is not causation). In other words, the fact that frequency-specific neural responses to the [audio & visual] condition are stronger than those to [audio] and [visual] combined does not mean this has implications for behavioral performance. While the correlation to autism traits could suggest some relation to behavior and is interesting in its own right, this correlation is a highly indirect way of assessing behavioral relevance. It would be helpful to test the relevance of supra-additive cortical tracking on a behavioral task directly related to the processing of biological motion to justify the claim that inputs are being integrated in the service of behavior. Under either framework, cortical tracking or entrainment, the causal relevance of neural findings toward cognition is lacking.

Overall, I believe this study finds neural correlates of biological motion that offer some constraint toward mechanism, and it is possible that the effects are behaviorally relevant, but based on the current task and associated analyses this has not been shown (or could not have been, given the paradigm).

Reviewer #2 (Public review):

Summary:

This study utilized two complimentary techniques (EEG and 7T MRI/MRS) to directly test a theory of dyslexia: the neural noise hypothesis. The authors report finding no evidence to support an excitatory/inhibitory balance, as quantified by beta in EEG and Glutamate/GABA ratio in MRS. This is important work and speaks to one potential mechanism by which increased neural noise may occur in dyslexia.

Strengths:

This is a well-conceived study with in depth analyses and publicly available data for independent review. The authors provide transparency with their statistics and display the raw data points along with the averages in figures for review and interpretation. The data suggest that an E/I balance issue may not underlie deficits in dyslexia and is a meaningful and needed test of a possible mechanism for increased neural noise.

Weaknesses:

The researchers did not include a visual print task in the EEG task, which limits analysis of reading specific regions such as the visual word form area, which is a commonly hypoactivated region in dyslexia. This region is a common one of interest in dyslexia, yet the researchers measured the I/E balance in only one region of interest, specific to the language network.

Reviewer #2 (Public review):

In recent years, lots of researchers tried to explore the existence of new acetyltransferase and deacetylase by using specific antibody enrichment technologies and high resolution mass spectrometry. Here is an example for this effort. Yuqian Wang et al. studied a novel Zn2+- and NAD+-independent KDAC protein, AhCobQ, in Aeromonas hydrophila. They studied the biological function of AhCobQ by using biochemistry method and MS identification technology to confirm it. These results extended our understanding of the regulatory mechanism of bacterial lysine acetylation modifications. However, I find this conclusion is a little speculative, and unfortunately it also doesn't totally support the conclusion as the authors provided.

Major concerns:

-It is a little arbitrary to come to the title "Aeromonas hydrophila CobQ is a new type of NAD+- and Zn2+-independent protein lysine deacetylase in prokaryotes." It should be modified to delete the "in the prokaryotes" except that the authors get new more evidence in the other prokaryotes for the existence of the AhCobQ.<br /> -I was confused about the arrangement of the supplementary results. Because there are no citations for Figures S9-S19.<br /> -Same to the above, there are no data about Tables S1-S6.<br /> -All the load control is not integrated. Please provide all of the load controls with whole PAGE gel or whole membrane western blot results. Without these whole results, it is not convincing to come the conclusion as the authors mentioned in the context.<br /> -Thoroughly review the materials & methods section. It is unclear to me what exactly the authors describe in the method. All the experimental designs and protocols should be described in detail, including growth conditions, assay conditions, and purification conditions, etc.<br /> -Include relevant information about the experiments performed in the figure legends, such as experimental conditions, replicates, etc. Often it is not clear what was done based on the figure legend description.

Reviewer #3 (Public review):

Summary:

The authors aimed to study the activation of gliogenesis and the role of newborn astrocytes in a post-ischemic scenario. Combining immunofluorescence, BrdU-tracing and genetic cellular labelling, they tracked the migration of newborn astrocytes (expressing Thbs4) and found that Thbs4-positive astrocytes modulate the extracellular matrix at the lesion border by synthesis but also degradation of hyaluronan. Their results point to a relevant function of SVZ newborn astrocytes in the modulation of the glial scar after brain ischemia. This work's major strength is the fact that it is tackling the function of SVZ newborn astrocytes, whose role is undisclosed so far.

Strengths:

The article is innovative, of good quality, and clearly written, with properly described Materials and Methods, data analysis and presentation. In general, the methods are designed properly to answer the main question of the authors, being a major strength. Interpretation of the data is also in general well done, with results supporting the main conclusions of this article.

In this revised version, the points raised/weaknesses were clarified and discussed in the article.

Reviewer #2 (Public review):

Summary:

This work is one of the best instances of a well-controlled experiment and theoretically impactful findings within the literature on templates guiding attentional selection. I am a fan of the work that comes out of this lab and this particular manuscript is an excellent example as to why that is the case. Here, the authors use fMRI (employing MVPA) to test whether during the preparatory search period, a search template is invoked within the corresponding sensory regions, in the absence of physical stimulation. By associating faces with scenes, a strong association was created between two types of stimuli that recruit very specific neural processing regions - FFA for faces and PPA for scenes. The critical results showed that scene information that was associated with a particular cue could be decoded from PPA during the delay period. This result strongly supports the invoking of a very specific attentional template.

Strengths:

There is so much to be impressed with in this report. The writing of the manuscript is incredibly clear. The experimental design is clever and innovative. The analysis is sophisticated and also innovative. The results are solid and convincing.

Weaknesses:

I only have a few weaknesses to point out.<br /> This point is not so much of a weakness, but a further test of the hypothesis put forward by the authors. The delay period was long - 8 seconds. It would be interesting to split the delay period into the first 4seconds and the last 4seconds and run the same decoding analyses. The hypothesis here is that semantic associations take time to evolve, and it would be great to show that decoding gets stronger in the second delay period as opposed to the period right after the cue. I don't think this is necessary for publication, but I think it would be a stronger test of the template hypothesis.<br /> Type in the abstract "curing" vs "during."<br /> It is hard to know what to do with significant results in ROIs that are not motivated by specific hypotheses. However, for Figure 3, what are the explanations for ROIs that show significant differences above and beyond the direct hypotheses set out by the authors?

Reviewer #2 (Public review):

Summary of the manuscript:

Authors present MGPfactXMBD, a novel model-based manifold-learning framework designed to address the challenges of interpreting complex cellular state spaces from single-cell RNA sequences. To overcome current limitations, MGPfactXMBD factorizes complex development trajectories into independent bifurcation processes of gene sets, enabling trajectory inference based on relevant features. As a result, it is expected that the method provides a deeper understanding of the biological processes underlying cellular trajectories and their potential determinants.

MGPfactXMBD was tested across 239 datasets, and the method demonstrated similar to slightly superior performance in key quality-control metrics to state-of-the-art methods. When applied to case studies, MGPfactXMBD successfully identified critical pathways and cell types in microglia development, validating experimentally identified regulons and markers. Additionally, it uncovered evolutionary trajectories of tumor-associated CD8+ T cells, revealing new subtypes with gene expression signatures that predict responses to immune checkpoint inhibitors in independent cohorts.

Overall, MGPfactXMBD represents a relevant tool in manifold-learning for scRNA-seq data, enabling feature selection for specific biological processes and enhancing our understanding of the biological determinants of cell fate.

Summary of the outcome:

The novel method addresses core state-of-the-art questions in biology related to trajectory identification. The design and the case studies are of relevance.

Comments on revisions:

The authors have addressed all my previous comments to satisfaction.

Reviewer #2 (Public review):

The goal of HiJee Kang et al. in this study is to explore the interaction between assemblies of neurons with similar pure-tone selectivity in mouse auditory cortex. Using holographic optogenetic stimulation in a small subset of target cells selective for a given pure tone (PTsel), while optically monitoring calcium activity in surrounding non-target cells, they discovered a subtle rebalancing process: co-tuned neurons that are not optogenetically stimulated tend to reduce their activity. The cortical network reacts as if an increased response to PTsel in some tuned assemblies is immediately offset by a reduction in activity in the rest of the PTsel-tuned assemblies, leaving the overall response to PTsel unchanged. The authors show that this rebalancing process affects only the responses of neurons to PTsel, not to other pure tones. They also show that assemblies of neurons that are not selective for PTsel don't participate in the rebalancing process. They conclude that assemblies of neurons with similar pure-tone selectivity must interact in some way to organize this rebalancing process, and they suggest that mechanisms based on homeostatic signaling may play a role.

The conclusions of this paper are very interesting but some aspects of the study including methods for optogenetic stimulation, statistical analysis of the results and interpretation of the underlying mechanisms need to be clarified and extended.

(1) This study uses an all-optical approach to excite a restricted group of neurons chosen for their functional characteristics (their frequency tuning), and simultaneously record from the entire network observable in the FOV. As stated by the authors, this approach is applied for the first time to the auditory cortex, which is a tour de force. However, such an approach is complex and requires precise controls to be convincing. In the manuscript, several methodological aspects are not sufficiently described to allow a proper understanding.<br /> (i) The use of CRmine together with GCaMP8s has been reported as problematic as the 2Ph excitation of GCaMP8s also excites the opsin. Here, the authors use a red-shifted version of CRmine to prevent such cross excitation by the imaging laser. To be convincing, they should explain how they controlled for the absence of rsCRmine activation by the 940nm light. Showing the fluorescence traces immediately after the onset of the imaging session would ensure that neurons are not excited as they are imaged.<br /> (ii) Holographic patterns used to excite 5 cells simultaneously may be associated with out-of-focus laser hot spots. Cells located outside of the FOV could be activated, therefore engaging other cells than the targeted ones in the stimulation. This would be problematic in this study as their tuning may be unrelated to the tuning of the targeted cells. To control for such an effect, one could in principle decouple the imaging and the excitation planes, and check for the absence of out-of-focus unwanted excitation.<br /> (iii) The control shown in Figure 1B is intended to demonstrate the precision of the optogenetic stimulation: when the stimulation spiral is played at a distance larger or equal to 20 µm from a cell, it does not activate it. However, in the rest of the study, the stimulation is applied with a holographic approach, targeting 5 cells simultaneously instead of just one. As the holographic pattern of light could produce out-of-focus hot spots (absent in the single cell control), we don't know what is the extent of the contamination from non-targeted cells in this case. This is important because it would determine an objective criterion to exclude non-targeted but excited cells (last paragraph of the Result section: "For the stimulation condition, we excluded non-target cells that were within 15 µm distance of the target cells...")

(2) A strength of this study comes from the design of the experimental protocol used to compare the activity in non-target co-tuned cells when the optogenetic stimulation is paired with their preferred tone versus a non-preferred pure tone. The difficulty lies in the co-occurrence of the rebalancing process and the adaptation to repeated auditory stimuli, especially when these auditory stimuli correspond to a cell's preferred pure tones. To distinguish between the two effects, the authors use a comparison with a control condition similar to the optogenetic stimulation conditions, except that the laser power is kept at 0 mW. The observed effect is shown as an extra reduction of activity in the condition with the optogenetic paired with the preferred tone, compared to the control condition. The specificity of this extra reduction when stimulation is synchronized with the preferred tone, but not with a non-preferred tone, is a potentially powerful result, as it points to an underlying mechanism that links the assemblies of cells that share the same preferred pure tones.<br /> The evidence for this specificity is shown in Figure 3A and 3D. However, the universality of this specificity is challenged by the fact that it is observed for 16kHz preferring cells, but not so clearly for 54kHz preferring cells: these 54kHz preferring cells also significantly (p = 0.044) reduce their response to 54kHz in the optogenetic stimulation condition applied to 16kHz preferring target cells compared to the control condition. The proposed explanation for this is the presence of many cells with a broad frequency tuning, meaning that these cells could have been categorized as 54kHz preferring cells, while they also responded significantly to a 16kHz pure tone. To account for this, the authors divide each category of pure tone cells into three subgroups with low, medium and high frequency preferences. Following the previous reasoning, one would expect at least the "high" subgroups to show a strong and significant specificity for an additional reduction only if the optogenetic stimulation is targeted to a group of cells with the same preferred frequency. Figure 3D fails to show this. The extra reduction for the "high" subgroups is significant only when the condition of opto-stimulation synchronized with the preferred frequency is compared to the control condition, but not when it is compared to the condition of opto-stimulation synchronized with the non-preferred frequency.<br /> Therefore, the claim that "these results indicate that the effect of holographic optogenetic stimulation depends not on the specific tuning of cells, but on the co-tuning between stimulated and non-stimulated neurons" (end of paragraph "Optogenetic holographic stimulation decreases activity in non-target co-tuned ensembles") seems somewhat exaggerated. Perhaps increasing the number of sessions in the 54kHz target cell optogenetic stimulation condition (12 FOV) to the number of sessions in the 16kHz target cell optogenetic stimulation condition (18 FOV) could help to reach significance levels consistent with this claim.

(3) To interpret the results of this study, the authors suggest that mechanisms based on homeostatic signaling could be important to allow the rebalancing of the activity of assemblies of co-tuned neurons. In particular, the authors try to rule out the possibility that inhibition plays a central role. Both mechanisms could produce effects on short timescales, making them potential candidates. The authors quantify the spatial distribution of the balanced non-targeted cells and show that they are not localized in the vicinity of the targeted cells. They conclude that local inhibition is unlikely to be responsible for the observed effect. This argument raises some questions. The method used to quantify spatial distribution calculates the minimum distance of a non-target cell to any target cell. If local inhibition is activated by the closest target cell, one would expect the decrease in activity to be stronger for non-target cells with a small minimum distance and to fade away for larger minimum distances. This is not what the authors observe (Figure 4B), so they reject inhibition as a plausible explanation. However, their quantification doesn't exclude the possibility that non-target cells in the minimum distance range could also be close and connected to the other 4 target cells, thus masking any inhibitory effect mediated by the closest target cell. In addition, the authors should provide a quantitative estimate of the range of local inhibition in layers 2/3 of the mouse auditory cortex to compare with the range of distances examined in this study (< 300 µm). Finally, the possibility that some target cells could be inhibitory cells themselves is considered unlikely by the authors, given the proportions of excitatory and inhibitory neurons in the upper cortical layers. On the other hand, it should be acknowledged that inhibitory cells are more electrically compact, making them easier to be activated optogenetically with low laser power.

Reviewer #2 (Public review):

In this work, the authors comprehensively describe the transcriptional regulatory network of Pseudomonas aeruginosa through the analysis of transcription factor binding characteristics. They reveal the hierarchical structure of the network through ChIP-seq, categorizing transcription factors into top-, middle-, and bottom-level, and reveal a diverse set of relationships among the transcription factors. Additionally, the authors conduct a pangenome analysis across the Pseudomonas aeruginosa species complex as well as other species to study the evolution of transcription factors. Moreover, the authors present a database with new and existing data to enable the storage and search of transcription factor binding sites. The findings of this study broaden our knowledge on the transcriptome of P. aeruginosa.

This study sheds light on the complex interconnections between various cellular functions that contribute to the pathogenicity of P. aeruginosa, along with the associated regulatory mechanisms. Certain findings, such as the regulatory tendencies of DNA-binding domain-types, provides valuable insights on the possible functions of uncharacterized transcription factors and new functions of those that have already been characterized. The techniques used hold great potential for discovery of transcription factor functions in understudied organisms as well.

The study would benefit from a more clear discussion on the implications of various findings, such as binding preferences, regulatory preferences, and the link between regulatory crosstalk and virulence. Additionally, the pangenome analysis would be furthered through a discussion of the divergence of the transcription factors of P. aeruginosa PAO1across species in relation to the findings on the hierarchical structure of the transcriptional regulatory network.

Reviewer #2 (Public review):

Summary:

The study tries to assess how the rise of the Qinghai-Tibet Plateau affected patterns of bird migration between their breeding and wintering sites. They do so by correlating the present distribution of the species with a set of environmental variables. The data on species distributions come from eBird. The main issue lies in the problematic assumption that species correlations between their current distribution and environment were about the same before the rise of the Plateau. There is no ground truthing and the study relies on Movebank data of only 7 species which are not even listed in the study. Similarly, the study does not outline the boundaries of breeding sites NE of the Plateau. Thus it is absolutely unclear potentially which breeding populations it covers.

Strengths:

I like the approach for how you combined various environmental datasets for the modelling part.

Weaknesses:

The major weakness of the study lies in the assumption that species correlations between their current distribution and environments found today are back-projected to the far past before the rise of the Q-T Plateau. This would mean that species responses to the environmental cues do not evolve which is clearly not true. Thus, your study is a very nice intellectual exercise of too many ifs.

The second major drawback lies in the way you estimate the migratory routes of particular birds. No matter how good the data eBird provides is, you do not know population-specific connections between wintering and breeding sites. Some might overwinter in India, some populations in Africa and you will never know the teleconnections between breeding and wintering sites of particular species. The few available tracking studies (seven!) are too coarse and with limited aspects of migratory connectivity to give answer on the target questions of your study.

Your set of species is unclear, selection criteria for the 50 species are unknown and variability in their migratory strategies is likely to affect the direction of the effects. In addition, the position of the breeding sites relative to the Q-T plate will affect the azimuths and resulting migratory flyways. So in fact, we have no idea what your estimates mean in Figure 2.

There is no way one can assess the performance of your statistical exercises, e.g. performances of the models.

Reviewer #2 (Public review):

In this manuscript Luo et al uncover that the ZNRF3/RNF43 E3 ubiquitin ligases participate in the selective endocytosis and degradation of FZD5/8 receptors in response to Wnt stimulation. Interestingly, DVL proteins have previously been shown to be important for RNF43/ZNRF3-dependent ubiquitination of Frizzled receptors but in this study the authors show that DVL proteins are only important for ligand and RNF43/ZNRF3-independent FZD endocytosis. Although it is well established that ZNRF3 and RNF43 promote the endocytosis and degradation of FZD receptors as part of a negative regulatory loop to dampened B-catenin signaling, the dependency of Wnt stimulation for this process and the specificity of this degradation for different FZD receptors remained poorly characterized.

In my opinion there are two significant findings of this study: 1) Wnt proteins are required for ZNRF3/RNF43 mediated endocytosis and degradation of FZD receptors and this constitutes an important negative regulatory loop. 2) The ZNRF3/RNF43 substrate selectivity for FZD5/8 over the other 8 Frizzleds. Of course, many questions remain, and new ones emerge as is often the case, but these findings challenge our dogmatic view on how the ZNRF3/RNF43 regulate Wnt signaling and emphasize their role in Wnt-dependent Frizzled endocytosis/degradation and beta-catenin signaling. Below I have suggestions to strengthen the manuscript.

(1) Given their results the authors conclude that upregulation of Frizzled on the plasma membrane is not sufficient to explain the stabilization of beta-catenin seen in the ZNRF3/RNF43 mutant cells. This interpretation is sound, and they suggest in the discussion that ZNRF3/RNF43-mediated ubiquitination could serve as a sorting signal to sort endocytosed FZD to lysosomes for degradation and that absence or inhibition of this process would promote FZD recycling. This should be relatively easy to test using surface biotinylation experiments and would considerably strengthen the manuscript.<br /> (2) The authors show that the FZD5 CRD domain is required for endocytosis since a mutant FZD5 protein in which the CRD is removed does not undergo endocytosis. This is perhaps not surprising since this is the site of Wnt binding, but the authors show that a chimeric FZD5CRD-FZD4 receptor can confer Wnt-dependent endocytosis to an otherwise endocytosis incompetent FZD4 protein. Since the linker region between the CRD and the first TM differs between FZD5 and FZD4 it would be interesting to understand whether the CRD specifically or the overall arrangement (such as the spacing) is the most important determinant.<br /> (3) I find it surprising that only FZD5 and FZD8 appear to undergo endocytosis or be stabilized at the cell surface upon ZNRF3/RNF43 knockout. Is this consistent with previous literature? Is that a cell-specific feature? These findings should be tested in a different cell line, with possibly different relative levels of ZNRF3 and RNF43 expression.<br /> (4) If FZD7 is not a substrate of ZNRF3/RNF43 and therefore is not ubiquitinated and degraded, how do the authors reconcile that its overexpression does not lead to elevated cytosolic beta-catenin levels in Figure 5B?<br /> (5) For Figure 5B, it would be interesting if the authors could evaluate whether overexpression of FZD5 in the ZNRF3/RNF43 double knockout lines would synergize and lead to further increase in cytosolic beta-catenin levels. As control if the substrate selectivity is clear FZD7 overexpression in that line should not do anything.<br /> (6) In Figure 6G, the authors need to show cytosolic levels of beta-catenin in the absence of Wnt in all cases.<br /> (7) Since the authors show that DVL is not involved in the Wnt and ZRNF3-dependent endocytosis they should repeat the proximity biotinylation experiment in figure 7 in the DVL triple KO cells. This is an important experiment since previous studies showed that DVL was required for the ZRNF3/RNF43-mediated ubiqtuonation of FZD.

Reviewer #2 (Public review):

Summary:

It was first reported in 2000 that Smad2/3/4 are sequestered to microtubules in resting cells and TGF-β stimulation releases Smad2/3/4 from microtubules, allowing activation of the Smad signaling pathway. Although the finding was subsequently confirmed in a few papers, the underlying mechanism has not been explored. In the present study, the authors found that Rudhira/breast carcinoma amplified sequence 3 is involved in release Smad2/3 from microtubules in response to TGF-β stimulation. Rudhira is also induced by TGF-β and probably involved in stabilization of microtubules in the delayed phase after TGF-β stimulation. Therefore, Rudhira has two important functions downstream of TGF-β in the early as well as delayed phase.

Strengths:

This work aimed to address an unsolved question on one of the earliest events after TGF-β stimulation. Based on loss-of-function experiments, the authors identified Rudhira, as a key player that triggers Smad2/3 release from microtubules after TGF-β stimulation. This is an important first step for understanding the initial phase of Smad signaling activation.

Weaknesses:

Currently, the processes how Rudhira causes the release of Smad proteins from microtubules and how Rudhira is mobilized to microtubules in response to TGF-β remain unclear. The authors are expected to address these points experimentally in the future.

This reviewer is also afraid that some of the biochemical data lack appropriate controls and are not convincing enough.

Reviewer #2 (Public review):

Summary:

In their manuscript, Ardaya et al address the impact of ischemia-induced astrogliogenesis from the adult SVZ and their effect on remodeling of the extracellular matrix (ECM) in the glial scar. The authors show that the levels of Thbs4, a marker previously identified to be expressed in astrocytes and neural stem cells (NSCs) of the SVZ, strongly increase upon ischemia. While proliferation is significantly increase shortly after ischemia, Nestin and DCX (markers for NSCs and neuroblasts, respectively) decrease and Thbs4 levels suggesting that the neurogenic program is halted and astrogenesis is enhanced. By fate-mapping, the authors show that astrocytes derive from SVZ NSCs and migrate towards the lesion. These SVZ-derived astrocytes strongly express Thbs4 and populate the border of the lesion, while local astrocytes do not express Thbs4 and localize to both scar and border. Interestingly, the Thbs4-positive astrocytes appear to represent a second wave of astrocytes accumulating at the scar, following an immediate reaction of first wave reactive gliosis by local astrocytes. Mechanistically, the study presents evidence that the degradation of hyaluronan (HA), a key component of the extracellular matrix (ECM) is downregulated in the SVZ after ischemia, potentially inducing astrogliogenesis, while HA accumulation at the lesion side represents at least one signal to recruit the newly generated astrocytes. In the aim to facilitate tissue regeneration after ischemic injury, the authors propose that the Thbs4-positive astrocytes could be a promising therapeutical target to modulate the glial scar after brain ischemia.

Strengths:

This topic is timely and important since the focus of previous studies was almost exclusively on the role of neurogenesis. The generation of adult-born astrocytes has been proven in both neurogenic niches under physiological conditions, but the implicated function in pathology has not been sufficiently addressed yet.

Weaknesses:

The study presented by Ardaya et al presents good evidence that a population of astrocytes that express Thbs4 contribute to scar formation after ischemic injury. The authors demonstrate that ischemic injury increases proliferation in the SVZ, decreases neurogenesis and increases astrogenesis. However, whether astrogenesis is a result of terminal differentiation of type B cells or their proliferation remains unclear. Here, a combination of fate mapping and thymidine analogue-tracing would have been conclusively.

Reviewer #2 (Public review):

The authors present behaviorMate, an open-source behavior control system including a central GUI and compatible treadmill and display components. Notably, the system utilize the "Intranet of things" scheme and the components communicate through local network, making the system modular, which in turn allows user to configure the setup to suit their experimental needs. Overall, behaviorMate is a useful resource for researchers performing head-fixed VR imaging studies involving 1D navigation tasks, as the commercial alternatives are often expensive and inflexible to modify.

One major utility of behaviorMate is an open-source alternative to commercial behavior apparatus for head-fixed imaging studies involving 1D navigation tasks. The documentation, BOM, CAD files, circuit design, source and compiled software, along with the manuscript, create an invaluable resource for neuroscience researcher looking to set up a budget-friendly VR and head-fixed imaging rig. Some features of behaviorMate, including the computer vision-based calibration of treadmill, and the decentralized, Android-based display devices, are very innovative approaches and can be quite useful in practical settings.

behaviorMate can also be used as a set of generic schema and communication protocols that allows the users to incorporate recording and stimulation devices during a head-fixed imaging experiment. Due to the "Intranet of things" approach taken in the design, any hardware that supports UDP communication can in theory be incorporated into the system. In terms of current capability, behaviorMate supports experimental contingencies based on animal position and time and synchronization with external recording devices using a TTL start signal. Further customization involving more complicated experimental contingencies, more accurate recording synchronization (for example with ephys recording devices), incorporation of novel behavior and high-speed neural recording hardware beyond GPIO signaling would require modification of the Java source and custom hardware implementation. Modification to the Java source of behaviorMate can be performed with basic familiarity with object-oriented programming using the Java programming language, and a JavaFX-based plugin system is under development to make such customizations more approachable for users.

In summary, the manuscript presents a well-developed and useful open-source behavior control system for head-fixed VR imaging experiments with innovative features.

Reviewer #2 (Public review):

Sensory experiences during developmental critical periods have long-lasting impacts on neural circuit function and behavior. However, the underlying molecular and cellular mechanisms that drive these enduring changes are not fully understood. In Drosophila, the antennal lobe is composed of synapses between olfactory sensory neurons (OSNs) and projection neurons (PNs), arranged into distinct glomeruli. Many of these glomeruli show structural plasticity in response to early-life odor exposure, reflecting the sensitivity of the olfactory circuitry to early sensory experiences.<br /> In their study, the authors explored the role of glia in the development of the antennal lobe in young adult flies, proposing that glial cells might also play a role in experience-dependent plasticity. They identified a critical period during which both structural and functional plasticity of OSN-PN synapses occur within the ethyl butyrate (EB)-responsive VM7 glomerulus. When flies were exposed to EB within the first two days post-eclosion, significant reductions in glomerular volume, presynaptic terminal numbers, and postsynaptic activity were observed. The study further highlights the importance of the highly conserved engulfment receptor Draper in facilitating this critical period plasticity. The authors demonstrated that, in response to EB exposure during this developmental window, ensheathing glia increase Draper expression, infiltrate the VM7 glomerulus, and actively phagocytose OSN presynaptic terminals. This synapse pruning has lasting effects on circuit function, leading to persistent decreases in both OSN-PN synapse numbers and spontaneous PN activity as analyzed by perforated patch-clamp electrophysiology to record spontaneous activity from PNs postsynaptic to Or42a OSNs .

In my view, this is an intriguing and potentially valuable set of data.

Comments on latest version:

After carefully reviewing the revised manuscript, I am satisfied with the authors' responses to my initial suggestions, particularly regarding the synaptic readouts used in their analyses. The authors have clarified their approach with appropriate changes in wording, which enhance the manuscript's clarity and address my previous concerns. Although I believe it could have been beneficial to incorporate postsynaptic markers to further substantiate the findings, I understand this may not have been feasible within the scope of the current study.

Overall, I find that the major claims of the manuscript are now sufficiently supported by the presented data. The revisions have improved the manuscript, and I am confident it meets the standards for publication. I therefore recommend the manuscript for publication in its current form.

Reviewer #2 (Public review):

Summary:

In this work, the authors investigate the role of fluid flow in shaping the colony size of a freshwater cyanobacterium Microcystis. To do so, they have created a novel assay by combining a rheometer with a bright field microscope. This allows them to exert precise shear forces on cyanobacterial cultures and field samples, and then quantify the effect of these shear forces on the colony size distribution. Shear force can affect the colony size in two ways: reducing size by fragmentation and increasing size by aggregation. They find limited aggregation at low shear rates, but high shear forces can create erosion-type fragmentation: colonies do not break in large pieces, but many small colonies are sheared off the large colonies. Overall, bacterial colonies from field samples seem to be more inert to shear than laboratory cultures, which the authors explain in terms of enhanced intercellular adhesion mediated by secreted polysaccharides.

Strengths:

-This study is timely, as cyanobacterial blooms are an increasing problem in freshwater lakes. They are expected to increase in frequency and severeness because of rising temperatures, and it is worthwhile learning how these blooms are formed. More generally, how physical aspects such as flow and shear influence colony formation is often overlooked, at least in part because of experimental challenges. Therefore, the method developed by the authors is useful and innovative, and I expect applications beyond the presented system here.<br /> -A strong feature of this paper is the highly quantitative approach, combining theory with experiments, and the combination of laboratory experiments and field samples.

Weaknesses:

-Especially the introduction seems to imply that shear force is a very important parameter controlling colony formation. However, if one looks at the results this effect is overall rather modest, especially considering the shear forces that these bacterial colonies may experience in lakes. The main conclusion seems that not shear but bacterial adhesion is the most important factor in determining colony size. As the importance of adhesion had been described elsewhere, it is not clear what this study reveals about cyanobacterial colonies that was not known before.<br /> -The agreement between model and experiments is impressive, but the role of the fit parameters in achieving this agreement needs to be further clarified.<br /> -The article may not be very accessible for readers with a biology background. Overall, the presentation of the material can be improved by better describing their new method.

Reviewer #2 (Public review):

This work investigates the possible association between language experience and morphology of the superior temporal cortex, a part of the brain responsible for the processing of auditory stimuli. Previous studies have found associations between language and music proficiency as well as language learning aptitude and cortical morphometric measures in regions in the primary and associated auditory cortex. These studies have most often, however, focused on finding neuroanatomical effects of difference between features in a few (often two) languages or from learning single phonetic/phonological features and have often been limited in terms of N. On this background, the authors use more sophisticated measures of language experience that take into account the age of onset and the differences in phonology between languages the subjects have been exposed as well as a larger number of subjects (N = 146 + 69) to relate language experience to the shape and structure of the superior temporal cortex, measured from T1-weighted MRI data. It shows solid evidence for there being a negative relationship between language experience and the right 2nd transverse temporal gyrus as well as some evidence for the relationship representing phoneme-level cross-linguistic information.

Strengths

The use of entropy measures to quantify language experience and include typological distance measures allows for a more general interpretation of the results and is an important step toward respecting and making use of linguistic diversity in neurolinguistic experiments.

A relatively large group of subjects with a range of linguistic backgrounds.

The full analysis of the structure of the superior temporal cortex including cortical volume, area, as well as the shape of the transverse gyrus/gyri. There is a growing literature on the meaning of the shape and number of the transverse gyri in relation to language proficiency and the authors explore all measures given the available data.

The authors chose to use a replication data set to verify their data, which is applaudable. However, see the relevant point under "Weaknesses".

Weaknesses

Even if the language experience and typological distance measures are a step in the right direction for correctly associating language exposure with cortical plasticity, it still is a measure that is insensitive to the intensity of the exposure.

Only the result from the multiple transverse temporal gyri (2nd TTG) is analyzed in the replicated dataset. Only the association in the right hemisphere 2nd TTG is replicated but this is not reflected in the discussion or the conclusions. The positive correlation in the right TTG is thus not attempted to be replicated.

The replication dataset differed in more ways than the more frequent combination of English and German experience, as mentioned in the discussion. Specifically, the fraction of monolinguals was higher in the replication dataset and the samples came from different scanners. It would be better if the primary and replication datasets were more equally matched.

Reviewer #2 (Public review):

Summary:

In this manuscript, Cozzolino et al. demonstrate that inhibition of the Mediator kinase CDK8 and its paralog CDK19 suppresses hyperactive interferon (IFN) signaling in Down syndrome (DS), which results from trisomy of chromosome 21 (T21). Numerous pathologies associated with DS are considered direct consequences of chronic IFN pathway activation, and thus hyperactive IFN signaling lies at the heart of pathophysiology. The collective interrogation of transcriptomics, metabolomics, and cytokine screens in sibling-matched cell lines (T21 vs D21) allows the authors to conclude that Mediator kinase inhibition could mitigate chronic, hyperactive IFN signaling in T21. To probe the functional outcomes of Mediator kinase inhibition, the authors performed cytokine screens, transcriptomic, and untargeted metabolomics. This collective approach revealed that Mediator kinases establish IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Mediator kinase inhibition suppresses cell responses during hyperactive IFN signaling through inhibition of pro-inflammatory transcription factor activity (anti-inflammatory effect) and alteration of core metabolic pathways, including upregulation of anti-inflammatory lipid mediators, which served as ligands for specific nuclear receptors and downstream phenotypic outcomes (e.g., oxygen consumption). These data provided a mechanistic link between Mediator kinase activity and nuclear receptor function. Finally, the authors also disclosed that Mediator kinase inhibition alters splicing outcomes.

Overall, this study reveals a mechanism by which Mediator kinases regulate gene expression and establish that its inhibition antagonizes chronic IFN signaling through collective transcriptional, metabolic, and cytokine responses. The data have implications for DS and other chronic inflammatory conditions, as Mediator kinase inhibition could potentially mitigate pathological immune system hyperactivation.

Comments on revisions:

In the record of version, the authors have improved readability and also incorporated experiments that provide compelling support to the main discovery of the story. Below I summarize the previous strengths and how they improved noted weaknesses.

(1) One major strength of this study is the mechanistic evidence linking Mediator kinases to hyperactive IFN signaling through transcriptional changes impacting cell signaling and metabolism.<br /> (2) Another major strength of this study is the use of sibling matched cell lines (T21 vs D21) from various donors (not just one sibling pair), and further cross-referencing with data from large cohorts, suggesting that part of the data and conclusions are generalizable.<br /> (3) Another major strength of this study is the combined experimental approach including transcriptomics, untargeted metabolomics and cytokine screens to define the mechanisms underlying suppression of hyperactive interferon signaling in DS upon Mediator kinase inhibition.<br /> (4) Another major strength of this study is the significance of the work to DS and its potential impact to other chronic inflammatory conditions.<br /> (5) The previously noted weakness regarding the roles of nuclear receptors to activation of an anti-inflammatory program upon Mediator kinase inhibition was not directly experimentally addressed because existing data from other studies (referenced in this version) have linked specific nuclear receptors to lipid biosynthesis and anti-inflammatory cascades. This is considered acceptable.<br /> (6) The presentation of the splicing data analysis is not better integrated in the overall story.<br /> (7) The authors improved the readability of the manuscript by providing specific details throughout.<br /> (8) Figures were improved and simplified when possible to facilitate readability.<br /> (9) The authors now clarified the PRO-Seq (TFEA analysis) explaining that their data is consistent with the general observation that stimulus-responsive genes is controlled by enhancer-bound TFs.

Reviewer #2 (Public review):

Summary:

In the manuscript "Decoding m6Am by simultaneous transcription-start mapping and methylation quantification" Liu and co-workers describe the development and application of CROWN-Seq, a new specialized library preparation and sequencing technique designed to detect the presence of cap-adjacent N6,2'-O-dimethyladenosine (m6Am) with single nucleotide resolution. Such a technique was a key need in the field since prior attempts to get accurate positional or quantitative measurements of m6Am positioning yielded starkly different results and failed to generate a consistent set of targets. As noted in the strengths section below the authors have developed a robust assay that moves the field forward.

Furthermore, their results show that most mRNAs whose transcription start nucleotide (TSN) is an 'A' are in fact m6Am (85%+ for most cell lines). They also show that snRNAs and snoRNAs have a substantially lower prevalence of m6Am TSNs.

Strengths:

Critically, the authors spent substantial time and effort to validate and benchmark the new technique with spike-in standards during development, cross-comparison with prior techniques, and validation of the technique's performance using a genetic PCIF1 knockout. Finally, they assayed nine different cell lines to cross-validate their results. The outcome of their work (a reliable and accurate method to catalog cap-adjacent m6Am) is a particularly notable achievement and is a needed advance for the field.

Weaknesses:

No major concerns were identified by this reviewer.

Mid-level Concerns: All previous concerns were addressed in the revised version

Reviewer #2 (Public review):

Summary:

In this work, Cheng et al use the TPP/MS-CETSA strategy to discover new components for the mitochondria arm of the Integrated Stress Response. By using short exposures of several drugs that potentially induce mitochondrial stress, they find significant CETSA shifts for the scaffold protein PEBP1 both for antimycinA and oligomycin, making PEBP1 a candidate for mitochondrial-induced ISR signaling. After extensive follow-up work, they provide good support that PEBP1 is likely involved in ISR, and possibly act through an interaction with the key ISR effector node EIF2a.

Strengths:

The work adds an important understanding of ISR signaling where PEBP1 might also constitute a druggable node to attenuate cellular stress. Although CETSA has great potential for dissecting cellular pathways, there are few studies where this has been explored, particularly with such an extensive follow-up, also giving the work methodological implications. Together I therefore think this study could have a significant impact.

Weaknesses:

The TPP/MS-CETSA experiment is quite briefly described and might have a too relaxed cut-off. The assays confirming interactions between PEBP1 and EIF2a might not be fully conclusive.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. show the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some of mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

However, the novelty of this manuscript is questionable since this group has published similar work with different mutants (Ref 11) .

Reviewer #2 (Public Review):

Kanie et al have recently characterized DAP protein CEP89 as important for the recruitment of the ciliary vesicle. Here, they describe a novel interacting partner for CEP89 that can bind membranes and therefore mediates its role in ciliary vesicle recruitment. An initial LAP tag pull-down and mass spectrometry experiment finds NCS-1 and C3ORF14 as CEP89 interactors. This interaction is mapped in the context of the ciliary vesicle formation. From the data presented, it is clear that, upon knockout, the function of these proteins might be compensated by others, as the phenotype can eventually recover over time.

In terms of the biological significance of this interaction, it would be good to examine (via co-immunoprecipitation) whether the CEP89/NCS-1/C3ORF14 interaction takes place upon serum starvation. Does the complex change?

Also, for the subdistal appendage localization of NCS-1 and C3ORF14, would this also change upon serum starvation?

For the ciliation results and the recruitment of IFT88 in CEP89 knockout cell lines, this contradicts previous work from Tanos et al (PMID: 23348840), as well as Hou et al (PMID: 36669498). A parallel comparison using siRNA, a transient knockout system, or a degron system would help understand this. A similar point goes for Figure 4, where the effect on ciliogenesis is minimal in knockout cells, but acute siRNA has been shown to have a stronger phenotype.

An elegant phenotype rescue is shown in Figure 5. An interesting question would be, how does this mutant and/or the myristoylation affect the recruitment of C3ORF14?

For the EF-hand mutants, it would be good to use control mutants, from known Ca2+ binding proteins as a control for the experiment shown.

Reviewer #2 (Public review):

This revised manuscript mostly addresses previous concerns by doubling down on the model without providing additional direct evidence of interactions between Srs2 and PCNA, and that "precise sites of Srs2 actions in the genome remain to be determined." One additional Srs2 allele has been examined, showing some effect in combination with rfa1-zm2.

Many of the conclusions are based on reasonable assumptions about the consequences of various mutations, but direct evidence of changes in Srs2 association with PNCA or other interactors is still missing. There is an assumption that a deletion of a Rad51-interacting domain or a PCNA-interacting domain have no pleiotropic effects, which may not be the case. How SLX4 might interact with Srs2 is unclear to me, again assuming that the SLX4 defect is "surgical" - removing only one of its many interactions.

One point of concern is the use of t-tests without some sort of correction for multiple comparisons - in several figures. I'm quite sceptical about some of the p < 0.05 calls surviving a Bonferroni correction. Also in 4B, which comparison is **? Also, admittedly by eye, the changes in "active" Rad53 seem much greater than 5x. (also in Fig. 3, normalizing to a non-WT sample seems odd).

What is the WT doubling time for this strain? From the FACS it seems as if in 2 h the cells have completed more than 1 complete cell cycle. Also in 5D. Seems fast...

I have one over-arching confusion. Srs2 was shown initially to remove Rad51 from ssDNA and the suppression of some of srs2's defects by deleting rad51 made a nice, compact story, though exactly how srs2's "suppression of rad6" fit in isn't so clear (since Rad6 ties into Rad18 and into PCNA ubiquitylation and into PCNA SUMOylation). Now Srs2 is invoked to remove RPA. It seems to me that any model needs to explain how Srs2 can be doing both. I assume that if RPA and Rad51 are both removed from the same ssDNA, the ssDNA will be "trashed" as suggested by Symington's RPA depletion experiments. So building a model that accounts for selective Srs2 action at only some ssDNA regions might be enhanced by also explaining how Rad51 fits into this scheme.

As a previous reviewer has pointed out, CPT creates multiple forms of damage. Foiani showed that 4NQO would activate the Mec1/Rad53 checkpoint in G1- arrested cells, presumably because there would be single-strand gaps but no DSBs. Whether this would be a way to look specifically at one type of damage is worth considering; but UV might be a simpler way to look.

As also noted, the effects on the checkpoint and on viability are quite modest. Because it isn't clear (at least to me) why rfa1 mutants are so sensitive to CPT, it's hard for me to understand how srs2-zm2 has a modest suppressive effect: is it by changing the checkpoint response or facilitating repair or both? Or how srs2-3KR or srs2-dPIM differ from Rfa1-zm2 in this respect. The authors seem to lump all these small suppressions under the rubric of "proper levels of RPA-ssDNA" but there are no assays that directly get at this. This is the biggest limitation.

Srs2 has also been implicated as a helicase in dissolving "toxic joint molecules" (Elango et al. 2017). Whether this activity is changed by any of the mutants (or by mutations in Rfa1) is unclear. In their paper, Elango writes: "Rare survivors in the absence of Srs2 rely on structure-specific endonucleases, Mus81 and Yen1, that resolve toxic joint-molecules" Given the involvement of SLX4, perhaps the authors should examine the roles of structure-specific nucleases in CPT survival?

Experiments that might clarify some of these ambiguities are proposed to be done in the future. For now, we have a number of very interesting interactions that may be understood in terms of a model that supposes discriminating among gaps and ssDNA extensions by the presence of PCNA, perhaps modified by SUMO. As noted above, it would be useful to think about the relation to Rad6.

Reviewer #2 (Public review):

While the findings might be valid, there is enough uncertainty that these results should not be considered anything other than preliminary, warranting a more thorough and rigorous investigation.

Comments on revisions:

As the author mentioned that due to the receptor internalisation of AT1 and/or LOX1 induced by AngII or Ox-LDL makes it difficult to detect receptor interaction at the membrane by Co-IP. If so, the GPCR internalisation related pathway should be activated, such as GRKs, arrestin2 could be activated and enhanced during this process, whether they could further provide the evidence for these changes in different groups by Western blot or IF images.

If the authors don't know why the results across experiments can vary so greatly nor control them, how do we know that their interpretation of the very modest intra-experimental variability they observe is correct? They explain away the difference in biosensor activity response to the likely respective insertion sites that were used. While this can be true, and even might be true, it is important to note that the publication they cite shows that the sensors in the third loop and the C-terminus respond very similarly. In fact, the authors concluded: "Our results also suggest that positioning conformational biosensors into ICL3 and the C-tail effectively reports canonical G protein-mediated signaling downstream of the AT1R." Moreover, it is unclear why the less sensitive biosensor (as least as measured by degree of DBRET) is the one that appears to show enhancement. I suppose one could argue that the activity is maximal using the C-tail and one must use a less responsive reporter to detect the effect, but this is a rationalization for an unexplained result rather than a validated mechanistic explanation. If the other results were more compelling, perhaps this would be less of an issue. Finally, they did not explain why a control, non-specific antibody wasn't used for the studies presented in panel 2d. This would have been an easy study to have done in the interim. It also would have been important to test the effect of the LOX1-ab on the effects of AngII treatment alone.

In their response to the gene expression studies, the authors attribute the lack of a robust response for some genes to the low dose of oxLDL that was used but give no justification for their choice for this low dose. More importantly, they present the data for a number of hand-picked genes rather than a global assessment of response. Their justification---cost constraints---isn't sufficient to justify this incomplete analysis. Their selective rt-PCR results are a pilot study.

There is no direct evidence in this study that shows that "partial" EMT is occurring in vivo. The rt-PCR studies presented in Fig 8 are not sufficient. Even if one accepts their incomplete analysis of transcriptomic studies using RT-PCR rather than a complete transcriptomic assessment, the study was done on bulk RNA from the entire kidney. The source material includes all cell types, not just epithelial cells, so there is no way to be sure that EMT is occurring. As noted elsewhere, they found no histologic evidence for injury and had no immunostaining results demonstrating "partial EMT" of damaged renal epithelial cells.

All of the evidence described is indirect, and the responses, while plausible, are generally excuses for lack of truly unequivocally positive results. The authors acknowledge the potential confounders of lower BP response in the Lox1-KO, unexpected weight loss in response to high fat diet, the lack of meaningful histologic evidence of injury, and they also acknowledge the absence of increased Gq signaling in the kidney, which is central to their model, but defend the entire model based on some minor changes in urinary 8-OHdG and albumin levels and a curated set of transcriptional changes. Their data could support their model---loss of Lox1 seems to reduce the levels somewhat, but the data are preliminary.

There remain serious reservations about the immunostaining results, with explanations and new data not reassuring. The authors report that they are unable to co-stain for Lox1 and AT1R because both were generated in rabbit, but this reviewer didn't ask for co-staining of the two markers. Rather, it was co-staining showing that Lox1 and ATR1 in fact stain in a specific manner to the same nephron segments. The authors have added a supplementary figure showing co-staining for LOX1/AT1R with megalin, a marker for proximal tubules. However, several aspects of this are problematic:

i. The pattern in the new Supp Fig 10 does not look like that in Fig 9. In the latter, staining is virtually everywhere, all nephron segments, and predominantly basolateral. In Supp Fig 10, they note that the pattern is primarily in the microvilli of the proximal tubule, where megalin is present. The new studies also seem to be a bit more specific, ie there are some tubules that appear to not stain with the markers.

ii. It is difficult to be certain that the megalin staining isn't simply "bleed-through" of the signal from the other antibody. The paper doesn't describe the secondary antibody used for megalin to be sure that the emission spectra completely non-overlapping and it isn't clear that the microscope that was used offers necessary precision.

iii. Their explanation for the pattern of AT1R staining is unconvincing. AT1R immunolocalization is known to be challenging, prompting Schrankl et al to do a definitive study using RNAscope to localize its expression in mice, rats and humans (Am J Physiol Renal Physiol 320: F644-F653, 2021). It argues against the pattern seen in Figure 9 (diffuse tubular expression), though it does suggest it is present in proximal tubules in mice. But perhaps more problematic for their model is that AT1R is not expressed in human tubules (or at least the RNA is undetectable).

Why isn't there more colocalization apparent for the AT1R and LOX1 if they form a co-receptor complex? They say that the complexes may be very dynamic, yet their movie in Suppl Fig 1 does not really support that. Not only are there few overlapping puncta in the static image, there is very little change over the duration of the movie. We don't see complexes form and then disappear and we see few new complexes form.

The explanation for why the number of replicates is variable is not reassuring. The authors note that it was because of the higher variability of the results, necessitating a higher "N" to achieve significance, but this has the appearance of P-chasing.

Reviewer #2 (Public Review):

Summary:

Complementary to mammalian models, zebrafish has emerged as a powerful system to study vertebrate development and to serve as a go-to model for many human disorders. All vertebrates share the ancestral capacity to form a skeleton. Teleost fish models have been a key model to understand the foundations of skeletal development and plasticity, pairing with more classical work in amniotes such as the chicken and mouse. However, the genetic foundation of the diversity of skeletal programs in teleosts has been hampered by mapping similarities from amniotes back and not objectively establishing more ancestral states. This is most obvious in systematic, objective analysis of transcriptional regulation and tissue specification in differentiated skeletal tissues. Thus, the molecular events regulating bone-producing cells in teleosts have remained largely elusive. In this study, Petratou et al. leverage spatial experimental delineation of specific skeletal tissues -- that they term 'classical' vs 'non-classical' osteoblasts -- with associated cartilage of the endo/peri-chondrial skeleton and inter-segmental regions of the forming spine during development of the zebrafish, to delineate molecular specification of these cells by current chromatin and transcriptome analysis. The authors further show functional evidence of the utility of these datasets to identify functional enhancer regions delineating entp5 expression in 'classical' or 'non-classical' osteoblast populations. By integration with paired RNA-seq, they delineate broad patterns of transcriptional regulation of these populations as well as specific details of regional regulation via predictive binding sites within ATACseq profiles. Overall the paper was very well written and provides an essential contribution to the field that will provide a foundation to promote modeling of skeletal development and disease in an evolutionary and developmentally informed manner.

Strengths:

Taken together, this study provides a comprehensive resource of ATAC-seq and RNA-seq data that will be very useful for a wide variety of researchers studying skeletal development and bone pathologies. The authors show specificity in the different skeletal lineages and show the utility of the broad datasets for defining regulatory control of gene regulation in these different lineages, providing a foundation for hypothesis testing of not only agents of skeletal change in evolution but also function of genes and variations of unknown significance as it pertains to disease modeling in zebrafish. The paper is excellently written, integrating a complex history and experimental analysis into a useful and coherent whole. The terminology of 'classical' and 'non-classical' will be useful for the community in discussing the biology of skeletal lineages and their regulation.

Weaknesses:

Two items arose that were not critical weaknesses but areas for extending the description of methods and integration into the existing data on the role of non-classical osteoblasts and establishment/canalization of this lineage of skeletal cells.

(1) In reading the text it was unclear how specific the authors' experimental dissection of the head/trunk was in isolating different entp5a osteoblast populations. Obviously, this was successful given the specificity in DEG of results, however, analysis of contaminating cells/lineages in each population would be useful - e.g. using specific marker genes to assess. The text uses terms such as 'specific to' and 'enriched in' without seemingly grounded meaning of the accuracy of these comments. Is it really specific - e.g. not seen in one or other dataset - or is there some experimental variation in this?

(2) Further, it would be valuable to discuss NSC-specific genes such as calymmin (Peskin 2020) which has species and lineage-specific regulation of non-classical osteoblasts likely being a key mechanistic node for ratcheting centra-specific patterning of the spine in teleost fishes. What are dynamics observed in this gene in datasets between the different populations, especially when compared with paralogues - are there obvious cis-regulatory changes that correlate with the co-option of this gene in the early regulation of non-classical osteoblasts? The addition of this analysis/discussion would anchor discussions of the differential between different osteoblasts lineages in the paper.

Reviewer #3 (Public review):

Summary:

The work by Graca et al. describes a GMC flavoprotein dehydrogenase (MftG) in the ethanol metabolism of mycobacteria and provides evidence that it shuttles electrons from the mycofactocin redox cofactor to the electron transport chain.

Strengths:

Overall, this study is compelling, exceptionally well-designed and thoroughly conducted. An impressively diverse set of different experimental approaches is combined to pin down the role of this enzyme and scrutinize the effects of its presence or absence in mycobacteria cells growing on ethanol and other substrates. Other strengths of this work are the clear writing style and stellar data presentation in the figures, which makes it easy also for non-experts to follow the logic of the paper. Overall, this work therefore closes an important gap in our understanding of ethanol oxidation in mycobacteria, with possible implications for the future treatment of bacterial infections.

Weaknesses:

I see no major weaknesses in this work, which in my opinion leaves no doubt about the role of MftG.

Reviewer #2 (Public review):

Summary

Roiuk et al describe a work in which they have investigated the role of eIF2A in translation initiation in mammals without much success. Thus, the manuscript focuses on negative results. Further, the results, while original, are generally not novel, but confirmatory, since related claims have been made before independently in different systems with Haikwad et al study recently published in eLife being the most relevant.

Despite this, we find this work highly important. This is because of a massive wealth of unreliable information and speculations regarding eIF2A role in translation arising from series of artifacts that began at the moment of eIF2A discovery. This, in combination with its misfortunate naming (eIF2A is often mixed up with alpha subunit of eIF2, eIF2S1) has generated a widespread confusion among researchers who are not experts in eukaryotic translation initiation. Given this, it is not only justifiable but critical to make independent efforts to clear up this confusion and I very much appreciate the authors' efforts in this regard.

Strengths

The experimental investigation described in this manuscript is thorough, appropriate and convincing.

Weaknesses

However, we are not entirely satisfied with the presentation of this work which we think should be improved.

Reviewer #3 (Public review):

Summary:

The overall goal of this manuscript is to understand how Notch signaling is activated in specific regions of the endocardium, including the OFT and AVC, that undergo EMT to form the endocardial cushions. Using dofetilide to transiently block circulation in E9.5 mice, the authors show that Notch receptor cleavage still occurs in the valve-forming regions due to mechanical sheer stress as Notch ligand expression and oxygen levels are unaffected. The authors go on to show that changes in lipid membrane structure activate mTOR signaling, which causes phosphorylation of PKC and Notch receptor cleavage. The data are largely convincing and support their hypothesis. The conclusions are also novel and significantly add to the field of endocardial cushion biology.

The strengths of the manuscript include the dual pharmacological and genetic approaches to block blood flow in the mouse, the inclusion of many controls including those for hypoxia, the quality of the imaging, and the clarity of the text. In the revision, the authors put forth a good faith effort to address experimentally or textually the concerns of the reviewers. Most weaknesses that were identified in the first submission were addressed and the main claims are convincing. In general, the authors achieved their aims and the results support their conclusions.

Reviewer #2 (Public review):

Summary:

The authors tried to test the hypothesis that Cdk8 and Cdk19 stabilize the cytoplasmic CcNC protein, the partner protein of Mediator complex including CDK8/19 and Mediator protein via a kinase-independent function by generating induced double knockout of Cdk8/19. However the evidence presented suffer from a lack of focus and rigor and does not support their claims.

Strengths:

This is the first comprehensive report on the effect of a double knockout of CDK8 and CDK19 in mice on male fertility, hormones and single cell testicular cellular expression. The inducible knockout mice led to male sterility with severe spermatogenic defects, and the authors attempted to use this animal model to test the kinase-independent function of CDK8/19, previously reported for human. Single cell RNA-seq of knockout testis presented a high resolution of molecular defects of all the major cell types in the testes of the inducible double knockout mice. The authors also have several interesting findings such as reentry into cell cycles by Sertoli cells, loss of Testosterone in induced dko that could be investigated further.

Weaknesses:

The claim of reproductive defects in the induced double knockout of CDK8/19 resulted from the loss of CCNC via a kinase-independent mechanism is interesting but was not supported by the data presented. While the construction and analysis of the systemic induced knockout model of Cdk8 in Cdk19KO mice is not trivial, the analysis and data is weakened by systemic effect of Cdk8 loss, making it difficult to separate the systemic effect from the local testis effect.

The analysis of male sterile phenotype is also inadequate with poor image quality, especially testis HE sections. Male reproductive tract picture is also small and difficult to evaluate. The mice crossing scheme is unusual as you have three mice to cross to produce genotypes, while we could understand that it is possible to produce pups of desired genotypes with different mating schemes, such vague crossing scheme is not desirable and of poor genetics practice. Also using TAM treated wild type as control is ok, but a better control will be TAM treated ERT2-cre; CDK8f/f or TAM treated ERT2 Cre CDK19/19 KO, so as to minimize the impact from well-recognized effect of TAM.

While the authors proposed that the inducible loss of CDK8 in the CDK19 knockout background is responsible for spermatogenic defects, it was not clear in which cells CDK8/19 genes are interested and which cell types might have a major role in spermatogenesis. The authors also put forward the evidence that reduction/loss of Testosterone might be the main cause of spermatogenic defects, which is consistent with the expression change in genes involved in steroigenesis pathway in Leydig cells of inducible double knockout. But it is not clear how the loss of Testosterone contributed to the loss of CcnC protein.

The authors should clarify or present the data on where CDK8 and CDK19 as well as CcnC are expressed so as to help the readers to understand which tissues that both CDK might be functioning and cause the loss of CcnC. It should be easier to test the hypothesis of CDK8/19 stabilize CcnC protein using double knock out primary cells, instead of the whole testis.

Since CDK8KO and CDK19KO both have significantly reduced fertility in comparison with wildtype, it might be important to measure the sperm quantity and motility among CDK8 KO, CDK19KO and induced DKO to evaluate spermatogenesis based on their sperm production.

Some data for the inducible knockout efficiency of Cdk8 were presented in Supplemental figure 1, but there is no legend for the supplemental figures, it was not clear which band represented deletion band, which tissues were examined? Tail or testis? It seems that two months after the injection of Tam, all the Cdk8 were completely deleted, indicating extremely efficient deletion of Tam induction by two-month post administration. Were the complete deletion of Cdk8 happening even earlier ? an examination of timepoints of induced loss would be useful and instructional as to when is the best time to examine phenotypes.

The authors found that Sertoli cells re-entered cell cycle in the inducible double knockout but stop short of careful characterization other than increased expression of cell cycle genes.

Overall this work suffered from a lack of focus and rigor in the analysis and lack of sufficient evidence to support their main conclusions.

Comments on revisions:

This reviewer appreciated the authors' effort in improving the quality of this manuscript during their revision. While some concerns remain, the revision is a much improved work and the authors addressed most of my major concerns.<br /> Figure 2E CDK8 and CDK19 immunofluorescent staining images seem to show CDK8 and CDK19 location are completely distinct and in different cells, the authors need to elaborate on this results and discuss what such a distinct location means in line of their double knockout data.

Minor comments:

Supplemental figure 1(C) legend typo : (C) Periodic acid-Schiff stained sections of ilea of tamoxifen treated R26/Cre/ERI2 and DKO mice.

While the effort to identify and generate new antibodies is appreciated, the specificity of the antibodies used should be examined and presented if available.

Reviewer #2 (Public review):

Summary.

Mitochondrial dysfunction is associated with a wide spectrum of genetic and age-related diseases. Healthy mitochondria form a dynamic reticular network and constantly fuse, divide, and move. In contrast, dysfunctional mitochondria have altered dynamic properties resulting in fragmentation of the network and more static mitochondria. It has recently been reported that different types of mitochondrial stress or dysfunction activate kinases that control the integrated stress response, including HRI, PERK and GCN2. Kinase activity results in decreased global translation and increased transcription of stress response genes via ATF4, including genes that encode mitochondrial protein chaperones and proteases (HSP70 and LON). In addition, the ISR kinases regulate other mitochondrial functions including mitochondrial morphology, phospholipid composition, inner membrane organization, and respiratory chain activity. Increased mitochondrial connectivity may be a protective mechanism that could be initiated by pharmacological activation of ISR kinases, as was recently demonstrated for GCN2.

A small molecule screening platform was used to identify nucleoside mimetic compounds that activate HRI. These compounds promote mitochondrial elongation and protect against acute mitochondrial fragmentation induced by a calcium ionophore. Mitochondrial connectivity is also increased in patient cells with a dominant mutation in MFN2 by treatment with the compounds.

Strengths:

(1) The screen leverages a well-characterized reporter of the ISR: translation of ATF4-FLuc is activated in response to ER stress or mitochondrial stress. Nucleoside mimetic compounds were screened for activation of the reporter, which resulted in the identification of nine hits. The two most efficacious in dose response tests were chosen for further analysis (0357 and 3610). The authors clearly state that the compounds have low potency. These compounds were specific to the ISR and did not activate the unfolded protein response or the heat shock response. Kinases activated in the ISR were systematically depleted by CRISPRi revealing that the compounds activate HRI.<br /> (2) The status of the mitochondrial network was assessed with an Imaris analysis pipeline and attributes such as length, sphericity, and ellipsoid principal axis length were quantified. The characteristics of the mitochondrial network in cells treated with the compounds were consistent with increased connectivity. Rigorous controls were included. These changes were attenuated with pharmacological inhibition of the ISR.<br /> (3) Treatment of cells with the calcium ionophore results in rapid mitochondrial fragmentation. This was diminished by pre-treatment with 0357 or 3610 and control treatment with thapsigargin and halofuginone.<br /> (4) Pathogenic mutations in MFN2 result in the neurodegenerative disease Charcot-Marie-Tooth Syndrome Type 2A (CMT2A). Patient cells that express Mfn2-D414V possess fragmented mitochondrial networks and treatment with 0357 or 3610 increased mitochondrial connectivity in these cells.

Weaknesses:

The weakness is the limited analysis of cellular changes following treatment with the compounds.<br /> (1) Unclear how 0357 or 3610 alter other aspects of cellular physiology. While this would be satisfying to know, it may be that the authors determined that broad, unbiased experiments such as RNAseq or proteomic analysis are not justified due to the limited translational potential of these specific compounds.<br /> (2) There are many changes in Mfn2-D414V patient cells including reduced respiratory capacity, reduced mtDNA copy number, and fewer mitochondrial-ER contact sites. These experiments are relatively narrow in scope and quantifying more than mitochondrial structure would reveal if the compounds improve mitochondrial function, as is predicted by their model.

Comments on revisions:

Many reviewer concerns have been addressed or will be addressed in forthcoming manuscripts.

Reviewer #2 (Public review):

Summary:

Complementary to mammalian models, zebrafish has emerged as a powerful system to study vertebrate development and serve as a go-to model for many human disorders. All vertebrates share the ancestral capacity to form a skeleton. Teleost fish models have been a key model to understand the foundations of skeletal development and plasticity, pairing with more classical work in amniotes such as the chicken and mouse. However, the genetic foundation of the diversity of skeletal programs in teleosts have been hampered by mapping similarities from amniotes back and not objectively establishing more ancestral states. This is most obvious in systematic, objective analysis of transcriptional regulation and tissue specification in differentiated skeletal tissues. Thus, the molecular events regulating bone-producing cells in teleosts have remained largely elusive. In this study, Petratou et al. leverage spatial experimental delineation of specific skeletal tissues -- that they term 'classical' vs 'non-classical' osteoblasts -- with associated cartilage of the endo/peri-chondrial skeleton and inter-segmental regions of the forming spine during development of the zebrafish, to delineate molecular specification of these cells by current chromatin and transcriptome analysis. The authors further show functional evidence of the utility of these datasets to identify functional enhancer regions delineating entp5 expression delineated in 'classical' or 'non-classical' osteoblast populations. By integration with paired RNA-seq, they delineate broad patterns of transcriptional regulation of these populations as well as specific detail of regional regulation via predictive binding sites within ATACseq profiles. Overall the paper was very well written and provides an essential contribution to the field that will provide a foundation to promote modeling of skeletal development and disease in an evolutionary and developmentally informed manner.

Strengths:

Taken together, this study provides a comprehensive resource of ATAC-seq and RNA-seq data that will be very useful for a wide variety of researchers studying skeletal development and bone pathologies. The authors show specificity in the different skeletal lineages and show utility of the broad datasets for defining regulatory control of gene regulation in these different lineages, providing the foundation for hypothesis testing of not only agents of skeletal change in evolution but also function of genes and variations of unknown significance as it pertains to disease modeling in zebrafish. The paper is excellently written, integrating a complex history and experimental analysis into a useful and coherent whole. The terminology of 'classical' and 'non-classical' will be useful for the community in discussing biology of skeletal lineages and their regulation.

Weaknesses:

Two items arose that proposed areas for extending the description to integrate the data into the existing data on role of non-classical osteobasts and establishment/canalization of this lineage of skeletal cells.

(1) It was unclear how specific the authors' experimental dissection of head/trunk was in isolating different entp5a osteoblast populations. Obviously, this was successful given the specificity in DEG of results, however an analysis of contaminating cells/lineages in each population would be useful - e.g. maybe use specific marker genes to assess. The text uses terms such as 'specific to' and 'enriched in' without seemingly grounded meaning of the accuracy of these comments. Is it really specific e.g. not seen in one or other dataset, or is there some experimental variation in this?

(2) Further, it would be valuable to discuss NSC-specific genes such as calymmin (Peskin 2020) which has species and lineage specific regulation of non-classical osteoblasts likely being a key mechanistic node for ratcheting centra-specific patterning of the spine in teleost fishes. What are dynamics observed in this gene in datasets between the different populations, especially when compared with paralogues - is there obvious cis-regulatory changes that correlate with the co-option of this gene in early regulation of non-classical osteoblasts? The addition of this analysis/discussion would anchor discussions of a differential between different osteoblasts lineages in the paper.

Comments on revisions: All issues have been addressed.

Reviewer #2 (Public Review):

Summary:

The authors report results of QM/MM simulations and kinetic measurements for the phosphoryl-transfer step in adenylate kinase. The main assertion of the paper is that a wide transition state ensemble is a key concept in enzyme catalysis as a strategy to circumvent entropic barriers. This assertion is based on observation of a "structurally wide" set of energetically equivalent configurations that lie along the reaction coordinate in QM/MM simulations, together with kinetic measurements that suggest a decrease of the entropy of activation.

Strengths:

The study combines theoretical calculations and supporting experiments.

Weaknesses:

The current paper hypothesizes a "wide" transition state ensemble as a catalytic strategy and key concept in enzyme catalysis. Overall, it is not clear the degree to which this hypothesis is fully supported by the data. The reasons are as follows:

(1) Enzyme catalysis reflects a rate enhancement with respect to a baseline reaction in solution. In order to assert that something is part of a catalytic strategy of an enzyme, it would be necessary to demonstrate from simulations that the activation entropy for the baseline reaction is indeed greater and the transition state ensemble less "wide". Alternatively stated, when indicating there is a "wide transition state ensemble" for the enzyme system - one needs to indicate that is with respect to the non-enzymatic reaction. However, these simulations were not performed and the comparisons not demonstrated. The authors state "This chemical step would take about 7000 years without the enzyme" making it impossible to measure; nonetheless, the simulations of the nonenzymatic reaction would be fairly straightforward to perform in order to demonstrate this key concept that is central to the paper. Rather, the authors examine the reaction in the absence of a catalytically important Mg ion.

(2) The observation of a "wide conformational ensemble" is not a quantitative measure of entropy. In order to make a meaningful computational prediction of the entropic contribution to the activation free energy, one would need to perform free energy simulations over a range of temperatures (for the enzymatic and non-enzymatic systems). Such simulations were not performed, and the entropy of activation was thus not quantified by the computational predictions. The authors instead use a wider TS ensemble as a proxy for larger entropy, and miss an opportunity to compare directly to the experimental measurements.

Comments on revisions:

Overall, I do not think the authors have been able to quantitatively support their conclusion, and the qualitative support is somewhat weak. This makes the interpretation of the computational results somewhat speculative. Nonetheless, comparison was made for models with and without divalent ions, and the experimental data is valuable.

Reviewer #2 (Public review):

In this paper, Banerjee & Banerjee argue that a solely autocatalytic assembly model of the centrosome leads to size inequality. The authors instead propose a catalytic growth model with a shared enzyme pool. Using this model, the authors predict that size control is enzyme-mediate and are able to reproduce various experimental results such as centrosome size scaling with cell size and centrosome growth curves in C. elegans.

The paper contains interesting results and is well-written and easy to follow/understand.

Comments on revised version:

The authors made a number of revisions that significantly improved the manuscript, including analyzing the impact of finite diffusion, more thorough stability analysis, and enhanced comparison to experimental results.

Reviewer #2 (Public review):

Summary:

This manuscript addresses an important impediment in the field of Alzheimer's disease (AD) and tauapathy research by showing that 12 specific phosphomimetic mutations in full-length tau allow the protein to aggregate into fibrils with the AD fold and the fold of chronic traumatic encephalopathy fibrils in vitro. The paper presents comprehensive structural and cell based seeding data indicating the improvement of their approach over previous in vitro attempts on non-full-length tau constructs. The main weaknesses of this work results from the fact that only up to 70% of the tau fibrils form the desired fibril polymorphs. In addition, some of the figures are of low quality and confusing.

Strengths:

This study provides significant progress towards a very important and timely topic in the amyloid community, namely the in vitro production of tau fibrils found in patients.

The 12 specific phosphomimetic mutations presented in this work will have an immediate impact in the field since they can be easily reproduced.

Multiple high-resolution structures support the success of the phosphomimetic mutation approach.

Additional data show the seeding efficiency of the resulting fibrils, their reduced tendency to bundle, and their ability to be labeled without affecting core structure or seeding capability.

Weaknesses:

Despite the success of making full-length AD tau fibrils, still ~30% of the fibrils are either not PHF, or not accounted for. A small fraction of the fibrils are single filaments and another ~20% are not accounted for. The authors mention that ~20% of these fibrils were not picked by the automated algorithm. However, it would be important to get additional clarity about these fibrils. Therefore, it would improve the impact of the paper if the authors could manually analyze passed-over particles to see if they are compatible with PHF or fall into a different class of fibrils. In addition, it would be helpful if the authors could comment on what can be done/tried to get the PHF yield closer to 90-100%

Reviewer #2 (Public review):

Summary:

The work in this paper discusses the use of CG-MD simulations and nMS to describe cardiolipin binding sites in a synthetically designed that can be extrapolated to a naturally occurring membrane protein. While the authors acknowledge their work illuminates the challenges in engineering lipid binding they are able to describe some features that highlight residues within GlpG that may be involved in lipid regulation of protease activity, although further study of this site is required to confirm it's role in protein activity.

Comments<br /> Discrepancy between total CDL binding in CG simulations (Fig 1d) and nMS (Fig 2b,c) should be further discussed. Limitations in nMS methodology selecting for tightest bound lipids?<br /> Mutation of helical residues to alanine not only results in loss of lipid binding residues but may also impact overall helix flexibility, is this observed by the authors in CG-MD simulations? Change in helix overall RMSD throughout simulation? The figures shown in Fig.1H show what appear to be quite significant differences in APO protein arrangement between ROCKET and ROCKET AAXWA.<br /> CG-MD force experiments could be corroborated experimentally with magnetic tweezer unfolding assays as has been performed for the unfolding of artificial protein TMHC2. Alternatively this work could benefit to referencing Wang et al 2019 "On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations" to support MD vs experimental values.<br /> Did the authors investigate if ROCKET or ROCKETAAXWA copurifies with endogenous lipids? Membrane proteins with stabilising CDL often copurify in detergent and can be detected by MS without the addition of CDL to the detergent solution. Differences in retention of endogenous lipid may also indicate differences in stability between the proteins and is worth investigation.<br /> Do the AAXWA and ROCKET have significantly similar intensities from nMS? The AAXWA appears to show slight lower intensities than the ROCKET.<br /> Can the authors extend their comments on why densities are observed only around site 2 in the cryo-em structures when site 1 is the apparent preferential site for ROCKET.<br /> The authors state that nMS is consistent with CDL binding preferentially to Site 1 in ROCKET and preferentially to Site 2 in the ROCKET AAXWA variant, yet it unclear from the text exactly how these experiments demonstrate this.<br /> As carried out for ROCKET AAXWA the total CDL binding to A61P and R66A would add to supporting information of characterisation of lipid stabilising mutations.<br /> Did the authors investigate a double mutation to Site 2 (e.g. R66A + M16A)?<br /> Was the stability of R66A ever compared to the WT or only to AAXWA?<br /> How many CDL sites in the database used are structurally verified?<br /> The work on GlpG could benefit from mutagenesis or discussion of mutagenesis to this site. The Y160F mutation has already been shown to have little impact on stability or activity (Baker and Urban Nat Chem Biol. 2012).

Reviewer #2 (Public review):

The Gram-positive cell wall contains for a large part of TAs, and is essential for most bacteria. However, TA biosynthesis and regulation is highly understudied because of the difficulties in working with these molecules. This study closes some of our important knowledge gaps related to this and provides new and improved methods to study TAs. It also shows an interesting role for TAs in maintaining a 'periplasmic space' in Gram positives. Overall, this is an important piece of work. It would have been more satisfying if the possible causal link between TAs and periplasmic space would have been more deeply investigated with complemented mutants and CEMOVIS. For the moment, there is clearly something happening but it is not clear if this only happens in TA mutants or also in strains with capsules/without capsules and in PG mutants, or in lafB (essential for production of another glycolipid) mutants. Finally, some very strong statements are made suggesting several papers in the literature are incorrect, without actually providing any substantiation/evidence supporting these claims. This work pioneers some new methods that will definitively move the field forward.

Reviewer #2 (Public review):

Summary:

Jia and colleagues developed a fluorescence resonance energy transfer (FRET)-based biosensor to study programmed cell death in the zebrafish spinal cord. They applied this tool to study death of zebrafish spinal motor neurons.

Strengths:

Their analysis shows that the tool is a useful biosensor of motor neuron apoptosis in living zebrafish and can reveal which part of the neuron undergoes caspase activation first, achieving two of their aims.

Weaknesses:

The third aim, to provide novel insights into the spatiotemporal properties and occurrence rates of motor neuron death requires additional context and investigation, especially to understand the significance of the differences they report between zebrafish motor neuron programmed cell death and what has been previously described in chicks and rodents. For example, mnx1 expresses not only in motor neurons, but also in interneurons. However, the way the authors counted living and dead cells does not take this into consideration, potentially underestimating the percentage of motor neurons that died. Previous studies of chicks and rodents showed widespread differences in the timing of motor neuron programmed cell death and the number of cells that died depending on the spinal cord region examined. The authors have not described which spinal cord segments they examined or whether they examined motor neurons in limb-bearing segments which have been best studied in other species. Previous literature investigated the death of an identified zebrafish motor neuron and provided experimental evidence that it is independent of limitations in muscle innervation area, suggesting it is not coupled to muscle-derived neurotrophic factors. Thus, the authors need to acknowledge that even previous to their study, there was literature suggesting that programmed cell death of at least one motor neuron in zebrafish does not easily fit into the "neurotrophic hypothesis" as it is generally formulated. Finally, the authors need to be mindful that showing that something does not happen in an observational study cannot reveal the capabilities of the cells involved without an experimental test.

Reviewer #2 (Public review):

Summary:

This manuscript by Yu and coworkers investigates the potential role of Secretory leukocyte protease inhibitor (SLPI) in Lyme arthritis. They show that, after needle inoculation of the Lyme disease (LD) agent, B. burgdorferi, compared to wild type mice, a SLPI-deficient mouse suffers elevated bacterial burden, joint swelling and inflammation, pro-inflammatory cytokines in the joint, and levels of serum neutrophil elastase (NE). They suggest that SLPI levels of Lyme disease patients are diminished relative to healthy controls. Finally, they find that SLPI may interact directly the B. burgdorferi.

Strengths:

Many of these observations are interesting and the use of SLPI-deficient mice is useful (and has not previously been done).

Weaknesses:

(a) The known role of SLPI in dampening inflammation and inflammatory damage by inhibition of NE makes the enhanced inflammation in the joint of B. burgdorferi-infected mice a predicted result; (b) The potential contribution of the greater bacterial burden to the enhanced inflammation is not addressed; (c) The relationship of SLPI binding by B. burgdorferi to the enhanced disease of SLPI-deficient mice is not clear; and (d) Several methodological aspects of the study are unclear.

Reviewer #2 (Public review):

Summary:

The Crenarchaeal Cdv division system represents a reduced form of the universal and ubiquitous ESCRT membrane reverse-topology scission machinery, and therefore a prime candidate for synthetic and reconstitution studies. The work here represents a solid extension of previous work in the field, clarifying the order of recruitment of Cdv proteins to curved membranes.

Strengths:

The use of a recently developed approach to produce dumbbell-shaped liposomes (De Franceschi et al. 2022), which allowed the authors to assess recruitment of various Cdv assemblies to curved membranes or membrane necks; reconstitution of a quaternary Cdv complex at a membrane neck.

Weaknesses:

The manuscript is a bit light on quantitative detail, across the various figures, and several key controls are missing (CdvA, B alone to better interpret the co-polymerisation phenotypes and establish the true order of recruitment, for example) - addressing this would make the paper much stronger. The authors could also include in the discussion a short paragraph on implications for our understanding of ESCRT function in other contexts and/or in archaeal evolution, as well as a brief exploration of the possible reasons for the discrepancy between the foci observed in their liposome assays and the large rings observed in cells - to better serve the interests of a broad audience.

Reviewer #2 (Public review):

Summary:

This manuscript investigates the role of IκBα in regulating mouse embryonic stem cell (ESC) pluripotency and differentiation. The authors demonstrate that IκBα knockout impairs the exit from the naïve pluripotent state during embryoid body differentiation. Through mechanistic studies using various mutants, they show that IκBα regulates ESC differentiation through chromatin-related functions, independent of the canonical NF-κB pathway.

Strengths:

The authors nicely investigate the role of IκBα in pluripotency exit, using embryoid body formation and complementing the phenotypic analysis with a number of genome-wide approaches, including transcriptomic, histone marks deposition, and DNA methylation analyses. Moreover, they generate a first-of-its-kind mutant set that allows them to uncouple IκBα's function in chromatin regulation versus its NF-κB-related functions. This work contributes to our understanding of cellular plasticity and development, potentially interesting a broad audience including developmental biologists, chromatin biology researchers, and cell signaling experts.

Weaknesses:<br /> - The study's main limitation is the lack of crucial controls using bona fide naïve cells across key experiments, including DNA methylation analysis, gene expression profiling in embryoid bodies, and histone mark deposition. This omission makes it difficult to evaluate whether the observed changes in IκBα-KO cells truly reflect naïve pluripotency characteristics.<br /> - Several conclusions in the manuscript require a more measured interpretation. The authors should revise their statements regarding the strength of the pluripotency exit block, the extent of hypomethylation, and the global nature of chromatin changes.<br /> - From a methodological perspective, the manuscript would benefit from additional orthogonal approaches to strengthen the knockout findings, which may be influenced by clonal expansion of ES cells.

Overall, this study makes an important contribution to the field. However, the concerns raised regarding controls, data interpretation, and methodology should be addressed to strengthen the manuscript and support the authors' conclusions.

Reviewer #2 (Public review):

Summary:<br /> Authors introduced a computational framework, DyNoPy, that integrates residue coevolution analysis with molecular dynamics (MD) simulations to identify functionally important residues in proteins. DyNoPy identifies key residues and residue-residue coupling to generate an interaction graph and attempts to validate using two clinically relevant β-lactamases (SHV-1 and PDC-3).

Strengths:<br /> DyNoPy could not only show clinically relevance of mutations but also predict new potential evolutionary mutations. Authors have provided biologically relevant insights into protein dynamics which can have potential applications in drug discovery and understanding molecular evolution.

Weaknesses:<br /> Although DyNoPy could show the relevance of key residues in active and non-active site residues, no experiments have been performed to validate their predictions. In addition, they should compare their method with conventional techniques and show how their method could be different.

An explanation of "communities" divided in the work and how these communities are relevant to the article should be provided. In addition, choice of collective variables and their relevance in residue coupling movement is also not very well explained. Dynamics cross correlation map can also be a good method for understanding the residue movements and can explain the residue-residue coupling, it is not explained how DyNoPy is different from the conventional methods or can perform better.

In the sentence "DyNoPy identified eight significant communities of strongly coupled residues within SHV-1 (Supporting Fig. S4A)" I could not find a clear description of eight significant communities.

Again the description of communities is not clear to me in the following sentence "Detailed description of the other three communities is provided in the supporting information (Fig. S6)."

In the sentence "N170 acts as an intermediary between N136 and E166". Kindly cite the reference figure to show N179 as intermediate residue.

Please be careful with the numbers. In the sentence "These residues not only interact with each other directly but are also indirectly coupled via 21 other residues." I could count 22 other residues and not 21.

In the sentence "Unlike other substitution sites that are adjacent to the active site, R205 is situated more than 16 Å away from catalytic serine S70". Please add this label somewhere in the figure.

Please cite a reference in the sentence "This indicates that mutations on G238 would result in an alteration on protein catalytic function, as well as an increased flexibility of the protein, which strongly aligns with previous finding."