Reviewer #1 (Public review):

Summary:

The study examines human biases in a regime-change task, in which participants have to report the probability of a regime change in the face of noisy data. The behavioral results indicate that humans display systematic biases, in particular, overreaction in stable but noisy environments and underreaction in volatile settings with more certain signals. fMRI results suggest that a frontoparietal brain network is selectively involved in representing subjective sensitivity to noise, while the vmPFC selectively represents sensitivity to the rate of change.

Strengths:

(1) The study relies on a task that measures regime-change detection primarily based on descriptive information about the noisiness and rate of change. This distinguishes the study from prior work using reversal-learning or change-point tasks in which participants are required to learn these parameters from experiences. The authors discuss these differences comprehensively.

(2) The study uses a simple Bayes-optimal model combined with model fitting, which seems to describe the data well.

(3) The authors apply model-based fMRI analyses that provide a close link to behavioral results, offering an elegant way to examine individual biases.

Weaknesses:

My major concern is about the correlational analysis in the section "Under- and overreactions are associated with selectivity and sensitivity of neural responses to system parameters", shown in Figures 5c and d (and similarly in Figure 6). The authors argue that a frontoparietal network selectively represents sensitivity to signal diagnosticity, while the vmPFC selectively represents transition probabilities. This claim is based on separate correlational analyses for red and blue across different brain areas. The authors interpret the finding of a significant correlation in one case (blue) and an insignificant correlation (red) as evidence of a difference in correlations (between blue and red) but don't test this directly. This has been referred to as the "interaction fallacy" (Niewenhuis et al., 2011; Makin & Orban de Xivry 2019). Not directly testing the difference in correlations (but only the differences to zero for each case) can lead to wrong conclusions. For example, in Figure 5c, the correlation for red is r = 0.32 (not significantly different from zero) and r = 0.48 (different from zero). However, the difference between the two is 0.1, and it is likely that this difference itself is not significant. From a statistical perspective, this corresponds to an interaction effect that has to be tested directly. It is my understanding that analyses in Figure 6 follow the same approach.

Relevant literature on this point is:

Nieuwenhuis, S, Forstmann, B & Wagenmakers, EJ (2011). Erroneous analyses of interactions in neuroscience: a problem of significance. Nat Neurosci 14, 1105-1107. https://doi.org/10.1038/nn.2886

Makin TR, Orban de Xivry, JJ (2019). Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript. eLife 8:e48175. https://doi.org/10.7554/eLife.48175

There is also a blog post on simulation-based comparisons, which the authors could check out: https://garstats.wordpress.com/2017/03/01/comp2dcorr/

I recommend that the authors carefully consider what approach works best for their purposes. It is sometimes recommended to directly compare correlations based on Monte-Carlo simulations (cf Makin & Orban). It might also be appropriate to run a regression with the dependent variable brain activity (Y) and predictors brain area (X) and the model-based term of interest (Z). In this case, they could include an interaction term in the model:

Y = \beta_0 + \beta_1 \cdot X + \beta_2 \cdot Z + \beta_3 \cdot X \cdot Z

The interaction term reflects if the relationship between the model term Z and brain activity Y is conditional on the brain area of interest X.

Another potential concern is that some important details about the parameter estimation for the system-neglect model are missing. In the respective section in the methods, the authors mention a nonlinear regression using Matlab's "fitnlm" function, but it remains unclear how the model was parameterized exactly. In particular, what are the properties of this nonlinear function, and what are the assumptions about the subject's motor noise? I could imagine that by using the inbuild function, the assumption was that residuals are Gaussian and homoscedastic, but it is possible that the assumption of homoscedasticity is violated, and residuals are systematically larger around p=0.5 compared to p=0 and p=1.

Relatedly, in the parameter recovery analyses, the authors assume different levels of motor noise. Are these values representative of empirical values?

The main study is based on N=30 subjects, as are the two control studies. Since this work is about individual differences (in particular w.r.t. to neural representations of noise and transition probabilities in the frontoparietal network and the vmPFC), I'm wondering how robust the results are. Is it likely that the results would replicate with a larger number of subjects? Can the two control studies be leveraged to address this concern to some extent?

It seems that the authors have not counterbalanced the colors and that subjects always reported the probability of the blue regime. If so, I'm wondering why this was not counterbalanced.

Reviewer #1 (Public review):

Summary:

Insects and their relatives are commonly infected with microbes that are transmitted from mothers to their offspring. A number of these microbes have independently evolved the ability to kill the sons of infected females very early in their development; this male killing strategy has evolved because males are transmission dead-ends for the microbe. A major question in the field has been to identify the genes that cause male killing and to understand how they work. This has been especially challenging because most male-killing microbes cannot be genetically manipulated. This study focuses on a male-killing bacterium called Wolbachia. Different Wolbachia strains kill male embryos in beetles, flies, moths, and other arthropods. This is remarkable because how sex is determined differs widely in these hosts. Two Wolbachia genes have been previously implicated in male-killing by Wolbachia: oscar (in moth male-killing) and wmk (in fly male-killing). The genomes of some male-killing Wolbachia contain both of these genes, so it is a challenge to disentangle the two.

This paper provides strong evidence that oscar is responsible for male-killing in moths. Here, the authors study a strain of Wolbachia that kills males in a pest of tea, Homona magnanima. Overexpressing oscar, but not wmk, kills male moth embryos. This is because oscar interferes with masculinizer, the master gene that controls sex determination in moths and butterflies. Interfering with the masculinizer gene in this way leads the (male) embryo down a path of female development, which causes problems in regulating the expression of genes that are found on the sex chromosomes.

Strengths:

The authors use a broad number of approaches to implicate oscar, and to dissect its mechanism of male lethality. These approaches include: a) overexpressing oscar (and wmk) by injecting RNA into moth eggs, b) determining the sex of embryos by staining female sex chromosomes, c) determining the consequences of oscar expression by assaying sex-specific splice variants of doublesex, a key sex determination gene, and by quantifying gene expression and dosage of sex chromosomes, using RNASeq, and d) expressing oscar along with masculinizer from various moth and butterfly species, in a silkmoth cell line. This extends recently published studies implicating oscar in male-killing by Wolbachia in Ostrinia corn borer moths, although the Homona and Ostrinia oscar proteins are quite divergent. Combined with other studies, there is now broad support for oscar as the male-killing gene in moths and butterflies (i.e. order Lepidoptera). So an outstanding question is to understand the role of wmk. Is it the master male-killing gene in insects other than Lepidoptera and if so, how does it operate?

Weaknesses:

I found the transfection assays of oscar and masculinizer in the silkworm cell line (Figure 4) to be difficult to follow. There are also places in the text where more explanation would be helpful for non-experts.

Joint Public Review:

Solitary Fibrous Tumors (SFTs) are a rare malignancy defined by NAB2-STAT6 fusions. Because the molecular understanding of the disease is largely lacking, there are currently no targeted treatment approaches. Using primary tumor and adjacent normal tissue samples and cells inducibly expressing NAB2-STAT6, Hill et al. perform a detailed characterization of the transcriptomic and epigenomic NAB2-STAT6 SFT signatures. They identify enrichment or EGR1/NAB2 (but not STAT6) sites bound by the fusion protein and increased expression of EGR1 targets. Their studies indicate that NAB2-STAT6 fusion may direct the nuclear translocation of NAB2 and EGR1 proteins and potentially NAB1. Transcriptionally, NAB2-STAT6 SFTs most closely resemble neuroendocrine tumors.

This pioneering study provides critical insight into the molecular pathogenesis of SFTs, pivotal for the future development of mechanistically informed treatment approaches. The study is rigorously executed and well-written. This new knowledge is an important addition to the field.

Reviewer #1 (Public review):

The paper by Auer et. makes several contributions:

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

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

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

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

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

Reviewer #1 (Public review):

Nio and colleagues address an important question about how the cerebellum and ventral tegmental area (VTA) contribute to the extinction learning of conditioned fear associations. This work tackles a critical gap in the existing literature and provides new insights into this question in humans through the use of high-field neuroimaging with robust methodology. The presented results are novel and will broadly interest both the extinction learning and cerebellar research communities. As such, this is a very timely and impactful manuscript. However, there are several points that could be addressed during the review process to strengthen the claims and enhance their value for readers and the broader scientific community.

Points to Address:

(1) Reward Interpretation and Skin Conductance Responses (SCR):<br /> A central premise of the manuscript is that 'unexpected omissions of expected aversive events' are rewarding, which plays a critical role in extinction learning. The authors also suggest that the cerebellum is involved in reward processing. However, it is unclear how this conclusion can be directly drawn from their task, which does not explicitly model 'reward.' Instead, the interpretation relies on SCR, which seems more indicative of association or prediction rather than reward per se. Is SCR a valid metric of reward experienced during the extinction of feared associations? Or could these findings reflect processes tied more closely to predictive learning? Please, discuss.

(2) Reinforcement Agent and SCR Modeling:<br /> The modeling approach with the deep reinforcement agent treats SCR as a personalized expectation of shock for a given trial. However, this interpretation seems misaligned with participants' actual experience - they are aware of the shock but exhibit evolving responses to it over time. Why is this operationalization useful or valid? It would benefit the manuscript to provide a clearer justification for this approach.

(3) Clarity and Visualization of Results:<br /> The results section is challenging to follow, and the visualization and quantification of findings could be significantly improved. Terms like 'trending' appear frequently - what does this mean, and is it worth reporting? Adding clear statistical quantifications alongside additional visualizations (e.g., bar or violin plots of group means within specific subregions within the cerebellum, or grouped mean activity in VTA and DCN) would enhance clarity and allow readers to better assess the distribution and systematicity of effects. Furthermore, the figures are overly complex and difficult to read due to the heavy use of abbreviations. Consider splitting figures by either phase of the experiment or regions, and move some details to the supplemental material for improved readability.

(4) Theoretical Context for Paradigm Phases:<br /> The manuscript benefits from the comprehensive experimental paradigm, which includes multiple phases (acquisition, extinction, recall, reacquisition, re-extinction). This design has great potential for providing a more holistic view of conditioned fear learning and extinction. However, the manuscript lacks clarity on what insights can be drawn from these distinct phases. What theoretical framework underpins the different stages, and how should the results be interpreted in this context? At present, the findings seem like a display of similar patterns across phases without sufficient interpretation. Providing a stronger theoretical rationale and reorganizing the results by experimental phase could significantly improve readability and impact.

(5) Cerebellum-VTA Connectivity Analysis:<br /> The authors argue that the cerebellum modulates VTA activity, yet they perform the PPI analysis in the reverse direction. Why does this make sense? In their DCM analysis, they found a bidirectional relationship (both cerebellum - VTA and VTA-cerebellum), yet the discussion focused on connectivity from the cerebellum to VTA. A more careful interpretation of the connectivity findings would be useful - especially the strong claims in the discussion on the cerebellum providing the reward signal to the VTA should be tempered.

Reviewer #1 (Public review):

Summary:

Identifying drugs that target specific disease phenotypes remains a persistent challenge. Many current methods are only applicable to well-characterized small molecules, such as those with known structures. In contrast, methods based on transcriptional responses offer broader applicability because they do not require prior information about small molecules. Additionally, they can be rapidly applied to new small molecules. One of the most promising strategies involves the use of "drug response signatures"-specific sets of genes whose differential expression can serve as markers for the response to a small molecule. By comparing drug response signatures with expression profiles characteristic of a disease, it is possible to identify drugs that modulate the disease profile, indicating a potential therapeutic connection.

This study aims to prioritize potential drug candidates and to forecast novel drug combinations that may be effective in treating triple-negative breast cancer (TNBC). Large consortia, such as the LINCS-L1000 project, offer transcriptional signatures across various time points after exposing numerous cell lines to hundreds of compounds at different concentrations. While this data is highly valuable, its direct applicability to pathophysiological contexts is constrained by the challenges in extracting consistent drug response profiles from these extensive datasets. The authors use their method to create drug response profiles for three different TNBC cell lines from LINCS.

To create a more precise, cancer-specific disease profile, the authors highlight the use of single-cell RNA sequencing (scRNA-seq) data. They focus on TNBC epithelial cells collected from 26 diseased individuals compared to epithelial cells collected from 10 healthy volunteers. The authors are further leveraging drug response data to develop inhibitor combinations.

Strengths:

The authors of this study contribute to an ongoing effort to develop automated, robust approaches that leverage gene expression similarities across various cell lines and different treatment regimens, aiming to predict drug response signatures more accurately. The authors are trying to address the gap that remains in computational methods for inferring drug responses at the cell subpopulation level.

Weaknesses:

One weakness is that the authors do not compare their method to previous studies. The authors develop a drug response profile by summarizing the time points, concentrations, and cell lines. The computational challenge of creating a single gene list that represents the transcriptional response to a drug across different cell lines and treatment protocols has been previously addressed. The Prototype Ranked List (PRL) procedure, developed by Iorio and co-authors (PNAS, 2010, doi:10.1073/pnas.1000138107), uses a hierarchical majority-voting scheme to rank genes. This method generates a list of genes that are consistently overexpressed or downregulated across individual conditions, which then hold top positions in the PRL. The PRL methodology was used by Aissa and co-authors (Nature Comm 2021, doi:10.1038/s41467-021-21884-z) to analyze drug effects on selective cell populations using scRNA-seq datasets. They combined PRL with Gene Set Enrichment Analysis (GSEA), a method that compares a ranked list of genes like PRL against a specific set of genes of interest. GSEA calculates a Normalized Enrichment Score (NES), which indicates how well the genes of interest are represented among the top genes in the PRL. Compared to the method described in the current manuscript, the PRL method allows for the identification of both upregulated and downregulated transcriptional signatures relevant to the drug's effects. It also gives equal weight to each cell line's contribution to the drug's overall response signature.

The authors performed experimental validation of the top two identified drugs; however, the effect was modest. In addition, the effect on TNBC cell lines was cell-line specific as the identified drugs were effective against BT20, whose transcriptional signatures from LINCS were used for drug identification, but not against the other two cell lines analyzed. An incorrect choice of genes for the signature may result in capturing similarities tied to experimental conditions (e.g., the same cell line) rather than the drug's actual effects. This reflects the challenges faced by drug response signature methods in both selecting the appropriate subset of genes that make up the signature and in managing the multiple expression profiles generated by treating different cell lines with the same drug.

Reviewer #1 (Public review):

Summary:

This study investigates the role of norepinephrine (NE) signaling in the hippocampus during event transitions, positing that NE release serves as a mechanism for marking event boundaries to facilitate episodic memory segmentation. The authors use a genetically encoded fluorescent indicator (GRABNE) to measure NE release with high temporal precision, correlating these signals with changes in hippocampal firing dynamics. By integrating photometry data, behavioral analyses, and analysis of neuronal activity from publicly available datasets, the work addresses fundamental questions about the relationship between neuromodulatory signals and memory encoding.

Strengths:

The authors present a compelling framework linking NE signaling to event boundaries, offering insight into how episodic memory segmentation may occur in the brain. The writing is clear and the data are well-described. It is easy to follow. The pharmacological validation of the GRABNE sensor enhances confidence in their NE measurements, an important methodological strength given the potential limitations of fluorescence-based neuromodulatory indicators. Moreover, the authors carefully disentangle NE signals from confounding behavioral variables, providing evidence that NE release is time-locked to event boundaries rather than movement or arousal-related behaviors. This level of analytical rigor strengthens their central claims. Additionally, the observation of NE signal dynamics that decay over hundreds of seconds is interesting, as it aligns with timescales relevant to hippocampal plasticity reported in prior literature.

Weaknesses:

While the authors establish correlations between NE signaling and hippocampal activity changes, causation is not demonstrated. Future studies using perturbative approaches (e.g., optogenetic or chemogenetic manipulation of NE release) would be necessary to establish a direct causal link. Furthermore, the persistence of NE signals over long timescales (hundreds of seconds) raises questions about its role in encoding rapid event boundaries, as it is unclear how this prolonged signaling might affect memory encoding for closely spaced events. The lack of a discussion about how NE dynamics would operate in such scenarios weakens the proposed framework. Finally, while the authors acknowledge the limitations of the GRABNE sensor, a more detailed exploration of how sensor sensitivity might influence their results would enhance the interpretation of their findings.

Reviewer #1 (Public review):

Summary:

It is known that neuronal activity in several brain regions encodes interval time. However, how interval time is encoded across distributed brain regions remains unclear. By simultaneously recording neuronal activity from the hippocampal CA1, dorsal striatum, and orbitofrontal cortex during a temporal bisection task, the authors showed that elapsed time during the interval period is encoded similarly across these regions and that the neuronal activity of time cells across these regions tends to be synchronized within 100 ms. Using Bayesian decoding, they demonstrated that the interval time decoded from the firing activity of time cells in these regions correlated with the rats' decisions and that the times decoded from the neuronal activity of different brain regions were correlated. The sound experiments and analyses support most of the main conclusions of this paper.

Strengths:

They used a temporal bisection task in which the effects of time and distance can be dissociated. The test trials successfully revealed the relationship between the interval time estimated by Bayesian decoding and the animal's judgment of long versus short interval times. Simultaneous recording of neuronal activity from the hippocampal CA1, dorsal striatum, and orbitofrontal cortex, which is technically challenging, allowed comparison of interval time encoding across brain regions and the degree of synchrony between neurons from different brain regions.

Weaknesses:

Some analyses were not explained in detail, making it difficult to assess whether their results support the authors' conclusions.

Reviewer #1 (Public review):

This study examined the effect of blood pressure variability on brain microvascular function and cognitive performance. By implementing a model of blood pressure variability using an intermittent infusion of AngII for 25 days, the authors examined different cardiovascular variables, cerebral blood flow, and cognitive function during midlife (12-15-month-old mice). Key findings from this study demonstrate that blood pressure variability impairs baroreceptor reflex and impairs myogenic tone in brain arterioles, particularly at higher blood pressure. They also provide evidence that blood pressure variability blunts functional hyperemia and impairs cognitive function and activity. Simultaneous monitoring of cardiovascular parameters, in vivo imaging recordings, and the combination of physiological and behavioral studies reflect rigor in addressing the hypothesis. The experiments are well-designed, and the data generated are clear. I list below a number of suggestions to enhance this important work:

(1) Figure 1B: It is surprising that the BP circadian rhythm is not distinguishable in either group. Figure 2, however, shows differences in circadian rhythm at different timepoints during infusion. Could the authors explain the lack of circadian effect in the 24-h traces?

(2) While saline infusion does not result in elevation of BP when compared to Ang II, there is an evident "and huge" BP variability in the saline group, at least 40mmHg within 1 hour. This is a significant physiological effect to take into consideration, and therefore it warrants discussion.

(3) The decrease in DBP in the BPV group is very interesting. It is known that chronic Ang II increases cardiac hypertrophy, are there any changes to heart morphology, mass, and/or function during BPV? Can the the decrease in DBP in BPV be attributed to preload dysfunction? This observation should be discussed.

(4) Examining the baroreceptor reflex during the early and late phases of BPV is quite compelling. Figures 3D and 3E clearly delineate the differences between the two phases. For clarity, I would recommend plotting the data as is shown in panels D and E, rather than showing the mathematical ratio. Alternatively, plotting the correlation of ∆HR to ∆SBP and analyzing the slopes might be more digestible to the reader. The impairment in baroreceptor reflex in the BPV during high BP is clear, is there any indication whether this response might be due to loss of sympathetic or gain of parasympathetic response based on the model used?

(5) Figure 3B shows a drop in HR when the pump is ON irrespective of treatment (i.e., independent of BP changes). What is the underlying mechanism?

(6) The correlation of ∆diameter vs MAP during low and high BP is compelling, and the shift in the cerebral autoregulation curve is also a good observation. I would strongly recommend that the authors include a schematic showing the working hypothesis that depicts the shift of the curve during BPV.

(7) Functional hyperemia impairment in the BPV group is clear and well-described. Pairing this response with the kinetics of the recovery phase is an interesting observation. I suggest elaborating on why BPV group exerts lower responses and how this links to the rapid decline during recovery.

(8) The experimental design for the cognitive/behavioral assessment is clear and it is a reasonable experiment based on previous results. However, the discussion associated with these results falls short. I recommend that the authors describe the rationale to assess recognition memory, short-term spatial memory, and mice activity, and explain why these outcomes are relevant in the BPV context. Are there other studies that support these findings? The authors discussed that no changes in alternation might be due to the age of the mice, which could already exhibit cognitive deficits. In this line of thought, what is the primary contributor to behavioral impairment? I think that this sentence weakens the conclusion on BPV impairing cognitive function and might even imply that age per se might be the factor that modulates the various physiological outcomes observed here. I recommend clarifying this section in the discussion.

(9) Why were only male mice used?

(10) In the results for Figure 3: "Ang II evoked significant increases in SBP in both control and BPV groups;...". Also, in the figure legend: "B. Five-minute average HR when the pump is OFF or ON (infusing Ang II) for control and BPV groups...." The authors should clarify this as the methods do not state a control group that receives Ang II.

Reviewer #1 (Public review):

Summary:

Building on previous in vitro synaptic circuit work (Yamawaki et al., eLife 10, 2021), Piña Novo et al. utilize an in vivo optogenetic-electrophysiological approach to characterize sensory-evoked spiking activity in the mouse's forelimb primary somatosensory (S1) and motor (M1) areas. Using a combination of a novel "phototactile" somatosensory stimuli to the mouse's hand and simultaneous high-density linear array recordings in both S1 and M1, the authors report in awake mice that evoked cortical responses follow a triphasic peak-suppression-rebound pattern response. They also find that M1 responses are delayed and attenuated relative to S1. Further analysis revealed a 20-fold difference in subcortical versus corticocortical propagation speeds. They also report that PV interneurons in S1 are strongly recruited by hand stimulation. Furthermore, they report that selective activation of PV cells can produce a suppression and rebound response similar to "phototactile" stimuli. Lastly, the authors demonstrate that silencing S1 through local PV cell activation reduces M1 response to hand stimulation, suggesting S1 may directly drive M1 responses.

Strengths:

The study was technically well done, with convincing results. The data presented are appropriately analyzed. The author's findings build on a growing body of both in vitro and in vivo work examining the synaptic circuits underlying the interactions between S1 and M1. The paper is well-written and illustrated. Overall, the study will be useful to those interested in forelimb S1-M1 interactions.

Weaknesses:

Although the results are clear and convincing, one weakness is that many results are consistent with previous studies in other sensorimotor systems, and thus not all that surprising. For example, the findings that sensory stimulation results in delayed and attenuated responses in M1 relative to S1 and that PV inhibitory cells in S1 are strongly recruited by sensory stimulation are not novel (e.g., Bruno et al., J Neurosci 22, 10966-10975, 2002; Swadlow, Philos Trans R Soc Lond B Biol Sci 357, 1717-1727, 2002; Gabernet et al., Neuron 48, 315-327, 2005; Cruikshank et al., Nat Neurosci 10, 462-468, 2007; Ferezou et al., Neuron 56, 907-923, 2007; Sreenivasan et al., Neuron 92, 1368-1382, 2016; Yu et al., Neuron 104, 412-427 e414, 2019). Furthermore, the observation that sensory processing in M1 depends upon activity in S1 is also not novel (e.g., Ferezou et al., Neuron 56, 907-923, 2007; Sreenivasan et al., Neuron 92, 1368-1382, 2016). The authors do a good job highlighting how their results are consistent with these previous studies.

Perhaps a more significant weakness, in my opinion, was the missing analyses given the rich dataset collected. For example, why lump all responsive units and not break them down based on their depth? Given superficial and deep layers respond at different latencies and have different response magnitudes and durations to sensory stimuli (e.g., L2/3 is much more sparse) (e.g., Constantinople et al., Science 340, 1591-1594, 2013; Manita et al., Neuron 86, 1304-1316, 2015; Petersen, Nat Rev Neurosci 20, 533-546, 2019; Yu et al., Neuron 104, 412-427 e414, 2019), their conclusions could be biased toward more active layers (e.g., L4 and L5). These additional analyses could reveal interesting similarities or important differences, increasing the manuscript's impact. Given the authors use high-density linear arrays, they should have this data.

Similarly, why not isolate and compare PV versus non-PV units in M1? They did the photostimulation experiments and presumably have the data. Recent in vitro work suggests PV neurons in the upper layers (L2/3) of M1 are strongly recruited by S1 (e.g., Okoro et al., J Neurosci 42, 8095-8112, 2022; Martinetti et al., Cerebral cortex 32, 1932-1949, 2022). Does the author's data support these in vitro observations?

It would have also been interesting to suppress M1 while stimulating the hand to determine if any part of the S1 triphasic response depends on M1 feedback. I appreciate the control experiment showing that optical hand stimulation did not evoke forelimb movement. However, this appears to be an N=1. How consistent was this result across animals, and how was this monitored in those animals? Can the authors say anything about digit movement? A light intensity of 5 mW was used to stimulate the hand, but it is unclear how or why the authors chose this intensity. Did S1 and M1 responses (e.g., amplitude and latency) change with lower or higher intensities? Was the triphasic response dependent on the intensity of the "phototactile" stimuli?

Reviewer #1 (Public review):

In this study, the authors investigate the molecular mechanisms driving the establishment of constitutive heterochromatin during embryonic development. The experiments have been meticulously conducted and effectively address the proposed hypotheses.

The methodology stands out for its robustness, utilizing:<br /> i) an efficient system for converting ESCs to 2C-like cells via Dux overexpression;<br /> ii) a global approach through IPOTD, which unveils the chromatome at distinct developmental stages; and<br /> iii) STORM technology, enabling high-resolution visualization of DNA decompaction. These tools collectively provide clear and comprehensive insights that support the study's conclusions.

The work makes a significant contribution to the field, offering valuable insights into chromatin-bound proteins at critical stages of embryonic development. These findings may also inform our understanding of processes beyond heterochromatin maintenance.

The revised manuscript shows improvement, particularly through enhanced discussion and the addition of new references addressing the cooperation of SMARCAD1 and TOPBP1. All my previous concerns have been thoroughly addressed by the authors. However, I believe that, as this reviewer suggested, the inclusion of a model that summarizes the main findings of the study and discusses the potential mechanisms involved, would enhance the clarity and understanding of the message the manuscript aims to convey.

Reviewer #1 (Public review):

Summary:

In this study, Zaho and colleagues investigate inflammasome activation by E. tarda infections. They show that E. tarda induces the activation of the NLRC4 inflammasome as well as the non-canonical pathway in human THP1 macrophages. Further dissecting NLRC4 activation, the find that T3SS translocon components eseB, eseC and eseD are necessary for NLRC4 activation, and that delivery of purified eseB is sufficient to trigger NAIP-dependnet NLRC4 activation. Sequence analysis reveals that eseB shares homology within the C-terminus with T3SS needle and rod proteins, leading the authors to test if this region is necessary for inflammasome activation. They show that the eseB CT is required and that it mediates interaction with NAIP. Finally, they that homologs of eseB in other bacteria also share the same sequence and that they can activate NLRC4 in a HEK293T cell overexpression system.

Strengths:

This is a very nice study that convincingly shows that eseB and its homologs can be recognized by the human NAIP/NLRC4 inflammasome. The experiments are well-designed, controlled and described, and the papers is convincing as a whole.

Weaknesses:

The authors need to discuss their study in the context of previous papers that have shown an important role for E. tarda flagellin in inflammasome activation and test whether flagellin and/or E. tarda T3SSs needle or rod can activate NLRC4.

The authors show that eseB and its homologs can activate NLRC4, but there are also other translocon proteins that are very different such as YopB or PopB. and share little homology with eseB. It would be nice to include a section comparing the different type 3 secretion systems. are there 2 different families of T3SSs, those that feature translocon components that are recognized by NAIP-NLRC4 and those that cannot be recognized?

Comments on revisions:

The authors have addressed my concern with additional experiments, which strengthen the authors' conclusions.

Reviewer #1 (Public review):

Satouh et al. report giant organelle complexes in oocytes and early embryos. Although these structures have often been observed in oocytes and early embryos, their exact nature has not been characterized. The authors named these structures "endosomal-lysosomal organelles form assembly structures (ELYSAs)". ELYSAs contain organelles such as endosomes, lysosomes, and probably autophagic structures. ELYSAs are initially formed in the perinuclear region and then seem to migrate to the periphery in an actin-dependent manner. When ELYSAs are disassembled after the 2-cell stage, the V-ATPase V1 subunit is recruited to make lysosomes more acidic and active. The ELYSAs are most likely the same as the "endolysosomal vesicular assemblies (ELVAs)", reported by Elvan Böke's group earlier this year (Zaffagnini et al. doi.org/10.1016/j.cell.2024.01.031). However, it is clear that Satouh et al. identified and characterized these structures independently. These two studies could be complementary. Although the nature of the present study is generally descriptive, this paper provides valuable information about these giant structures. Since the ELYSA described in this paper and ELVA proposed by Elvan Böke appear to be the same structure, it would be helpful to the field if the two groups discuss unifying the nomenclature in the future.

Comments on latest version:

In this revised manuscript, the authors have provided additional data supporting their conclusions and also revised the text to more accurately reflect the experimental results.

Reviewer #1 (Public review):

Summary:

The paper details a study of endothelial cell vessel formation during zebrafish development. The results focus on the role of aquaporins, which mediate the flow of water across the cell membrane, leading to cell movement. The authors show that actin and water flow together drive endothelial cell migration and vessel formation. If any of these two elements are perturbed, there are observed defects in vessels. Overall, the paper significantly improves our understanding of cell migration during morphogenesis in organisms.

Strengths:

The data are extensive and are of high quality. There is a good amount of quantification with convincing statistical significance. The overall conclusion is justified given the evidence.

Weaknesses:

There are two weaknesses, which if addressed, would improve the paper.

(1) The paper focuses on aquaporins, which while mediates water flow, cannot drive directional water flow. If the osmotic engine model is correct, then ion channels such as NHE1 are the driving force for water flow. Indeed this water is shown in previous studies. Moreover, NHE1 can drive water intake because the export of H+ leads to increased HCO3 due to reaction between CO2+H2O, which increases the cytoplasmic osmolarity (see Li, Zhou and Sun, Frontiers in Cell Dev. Bio. 2021). If NHE cannot be easily perturbed in zebrafish, it might be of interest to perturb Cl channels such as SWELL1, which was recently shown to work together with NHE (see Zhang, et al, Nat. Comm. 2022).

After revision, this concern has been addressed.

(2) In some places the discussion seems a little confusing where the text goes from hydrostatic pressure to osmotic gradient. It might improve the paper if some background is given. For example, mention water flow follows osmotic gradients, which will build up hydrostatic pressure. The osmotic gradients across the membrane are generated by active ion exchangers. This point is often confused in literature and somewhere in the intro, this could be made clearer.

After revision, this concern has been addressed.

Reviewer #1 (Public review):

Summary:

This paper is an elegant, mostly observational work, detailing observations that polysome accumulation appears to drive nucleoid splitting and segregation. Overall I think this is an insightful work with solid observations.

Strengths:

The strengths of this paper are the careful and rigorous observational work that leads to their hypothesis. They find the accumulation of polysomes correlates with nucleoid splitting, and that the nucleoid segregation occurring right after splitting correlates with polysome segregation. These correlations are also backed up by other observations:

(1) Faster polysome accumulation and DNA segregation at faster growth rates.<br /> (2) Polysome distribution negatively correlating with DNA positioning near asymmetric nucleoids.<br /> (3) Polysomes form in regions inaccessible to similarly sized particles.

These above points are observational, I have no comments on these observations leading to their hypothesis.

Weaknesses:

It is hard to state weaknesses in any of the observational findings, and furthermore, their two tests of causality, while not being completely definitive, are likely the best one could do to examine this interesting phenomenon.

Points to consider / address:

Notably, demonstrating causality here is very difficult (given the coupling between transcription, growth, and many other processes) but an important part of the paper. They do two experiments toward demonstrating causality that help bolster - but not prove - their hypothesis. These experiments have minor caveats, my first two points.

(1) First, "Blocking transcription (with rifampicin) should instantly reduce the rate of polysome production to zero, causing an immediate arrest of nucleoid segregation". Here they show that adding rifampicin does indeed lead to polysome loss and an immediate halting of segregation - data that does fit their model. This is not definitive proof of causation, as rifampicin also (a) stops cell growth, and (b) stops the translation of secreted proteins. Neither of these two possibilities is ruled out fully.

1a) As rifampicin also stops all translation, it also stops translational insertion of membrane proteins, which in many old models has been put forward as a possible driver of nucleoid segregation, and perhaps independent of growth. This should at last be mentioned in the discussion, or if there are past experiments that rule this out it would be great to note them.

1b) They address at great length in the discussion the possibility that growth may play a role in nucleoid segregation. However, this is testable - by stopping surface growth with antibiotics. Cells should still accumulate polysomes for some time, it would be easy to see if nucleoids are still segregated, and to what extent, thereby possibly decoupling growth and polysome production. If successful, this or similar experiments would further validate their model.

(2) In the second experiment, they express excess TagBFP2 to delocalize polysomes from midcell. Here they again see the anticorrelation of the nucleoid and the polysomes, and in some cells, it appears similar to normal (polysomes separating the nucleoid) whereas in others the nucleoid has not separated. The one concern about this data - and the differences between the "separated" and "non-separated" nuclei - is that the over-expression of TagBFP2 has a huge impact on growth, which may also have an indirect effect on DNA replication and termination in some of these cells. Could the authors demonstrate these cells contain 2 fully replicated DNA molecules that are able to segregate?

(3) What is not clearly stated and is needed in this paper is to explain how polysomes do (or could) "exert force" in this system to segregate the nucleoid: what a "compaction force" is by definition, and what mechanisms causes this to arise (what causes the "force") as the "compaction force" arises from new polysomes being added into the gaps between them caused by thermal motions.

They state, "polysomes exert an effective force", and they note their model requires "steric effects (repulsion) between DNA and polysomes" for the polysomes to segregate, which makes sense. But this makes it unclear to the reader what is giving the force. As written, it is unclear if (a) these repulsions alone are making the force, or (b) is it the accumulation of new polysomes in the center by adding more "repulsive" material, the force causes the nucleoids to move. If polysomes are concentrated more between nucleoids, and the polysome concentration does not increase, the DNA will not be driven apart (as in the first case) However, in the second case (which seems to be their model), the addition of new material (new polysomes) into a sterically crowded space is not exerting force - it is filling in the gaps between the molecules in that region, space that needs to arise somehow (like via Brownian motion). In other words, if the polysome region is crowded with polysomes, space must be made between these polysomes for new polysomes to be inserted, and this space must be made by thermal (or ATP-driven) fluctuations of the molecules. Thus, if polysome accumulation drives the DNA segregation, it is not "exerting force", but rather the addition of new polysomes is iteratively rectifying gaps being made by Brownian motion.

The authors use polysome accumulation and phase separation to describe what is driving nucleoid segregation. Both terms are accurate, but it might help the less physically inclined reader to have one term, or have what each of these means explicitly defined at the start. I say this most especially in terms of "phase separation", as the currently huge momentum toward liquid-liquid interactions in biology causes the phrase "phase separation" to often evoke a number of wider (and less defined) phenomena and ideas that may not apply here. Thus, a simple clear definition at the start might help some readers.

(4) Line 478. "Altogether, these results support the notion that ectopic polysome accumulation drives nucleoid dynamics". Is this right? Should it not read "results support the notion that ectopic polysome accumulation inhibits/redirects nucleoid dynamics"?

(5) It would be helpful to clarify what happens as the RplA-GFP signal decreases at midcell in Figure 1- is the signal then increasing in the less "dense" parts of the cell? That is, (a) are the polysomes at midcell redistributing throughout the cell? (b) is the total concentration of polysomes in the entire cell increasing over time?

(6) Line 154. "Cell constriction contributed to the apparent depletion of ribosomal signal from the mid-cell region at the end of the cell division cycle (Figure 1B-C and Movie S1)" - It would be helpful if when cell constriction began and ended was indicated in Figures 1B and C.

(7) In Figure 7 they demonstrate that radial confinement is needed for longitudinal nucleoid segregation. It should be noted (and cited) that past experiments of Bacillus l-forms in microfluidic channels showed a clear requirement role for rod shape (and a given width) in the positing and the spacing of the nucleoids.<br /> Wu et al, Nature Communications, 2020 . "Geometric principles underlying the proliferation of a model cell system" https://dx.doi.org/10.1038/s41467-020-17988-7

(8) "The correlated variability in polysome and nucleoid patterning across cells suggests that the size of the polysome-depleted spaces helps determine where the chromosomal DNA is most concentrated along the cell length. This patterning is likely reinforced through the displacement of the polysomes away from the DNA dense region"

It should be noted this likely functions not just in one direction (polysomes dictating DNA location), but also in the reverse - as the footprint of compacted DNA should also exclude (and thus affect) the location of polysomes

(9) Line 159. Rifampicin is a transcription inhibitor that causes polysome depletion over time. This indicates that all ribosomal enrichments consist of polysomes and therefore will be referred to as polysome accumulations hereafter". Here and throughout this paper they use the term polysome, but cells also have monosomes (and 2 somes, etc). Rifampicin stops the assembly of all of these, and thus the loss of localization could occur from both. Thus, is it accurate to state that all transcription events occur in polysomes? Or are they grouping all of the n-somes into one group?

Reviewer #1 (Public review):

Summary:

In this study, Wang and colleagues generate single-cell transcriptome and chromatin accessibility data from testicular tissues of two OA and three NOA cases. The authors analyze this dataset to identify novel cellular populations, marker genes, and inter-population interactions that may contribute to proper spermatogenesis. Then they propose a role of specific Sertoli cell subtypes and their interactions via Notch signaling in germ cell development. However, I remain skeptical of their central argument (also highlighted in the title) that stage-specific interactions between Sertoli and germ cells are a key component in NOA development, as my initial concerns regarding potential data misrepresentation, lack of statistical testing, and the rationale behind some of the analyses have not been sufficiently addressed.

(1) As noted in my previous comments, the analysis of Sertoli cell subtypes is potentially misleading and lacks proper statistical support. The authors claim a significant loss of Sertoli subpopulations in NOA cases, and provide the absolute number of cells in Figure 6B. However, this observation could easily be driven by the total number of cells captured during the experiment and the anatomical location of the specimens. There is no statistical basis to make the claim that this loss is significant. Furthermore, the same analysis should be performed on scATAC-seq cells and presented alongside.

(2) As pointed out in my initial concerns, some parts of the analyses require additional explanation to clarify their logical flow. For example, the logic of using between-sample correlations to assess colocalization of Sertoli and germ cells is lost on me. How can this be used to infer the important role of specific Sertoli cell populations in spermatogenesis, other than the fact that some of the genes are more co-expressed in the sub-populations? And how is this related to the claim that these cell populations are actually co-localized in the tissue? The authors then dedicate nearly a page describing the pathways enriched in Sertoli and germ cells, but the relevance is unclear, and the argument that these subtypes are functionally related is not convincing enough.

(3) The statement regarding Notch signaling as a critical component in Sertoli and germ cell interaction is not supported by actual evidence. The inference based on CellphoneDB and an epigenome snapshot that shows not much difference are insufficient to justify this claim.

(4) The manuscript is overly wordy and descriptive, making it difficult to read and understand the points. The main text needs to be more concise and on point, with unnecessary details removed to sharpen the key points. Non-essential results (e.g. Figure S10 and S11) unrelated to the main argument should be removed.

Joint Public Review:

Summary:

Hossain and coworkers investigate the mechanisms of recognition of xCas9, a variant of Cas9 with expanded targeting capability for DNA. They do so by using molecular simulations and combining different flavors of simulation techniques, ranging from long classical MD simulations, to enhanced sampling, to free energy calculations of affinity differences. Through this, the authors are able to develop a consistent model of expanded recognition based on the enhanced flexibility of the protein receptor.

Strengths:

The paper is solidly based on the ability of the authors to master molecular simulations of highly complex systems. In my opinion, this paper shows no major weaknesses. The simulations are carried out in a technically sound way. Comparative analyses of different systems provide valuable insights, even within the well-known limitations of MD. Plus, the authors further investigate why xCas9 exhibits improved recognition of the TGG PAM sequence compared to SpCas9 via well-tempered metadynamics simulations focusing on the binding of R1335 to the G3 nucleobase and the DNA backbone in both SpCas9 and xCas9. In this context, the authors provide a free-energy profiling that helps support their final model.

The implementation of FEP calculations to mimic directed evolution improvement of DNA binding is also interesting, original and well-conducted.

Overall, my assessment of this paper is that it represents a strong manuscript, competently designed and conducted, and highly valuable from a technical point of view.

Weaknesses:

To make their impact even more general, the authors may consider expanding their discussion on entropic binding to other recent cases that have been presented in the literature recently (such as e.g. the identification of small molecules for Abeta peptides, or the identification of "fuzzy" mechanisms of binding to protein HMGB1). The point on flexibility helping adaptability and expansion of functional properties is important, and should probably be given more evidence and more direct links with a wider picture.

Comments on revisions:

We have read the revised version and the response letter and I find that this manuscript is ready. There is no need for further additions/revisions.

Reviewer #1 (Public review):

This study by Wu et al. provides valuable computational insights into PROTAC-related protein complexes, focusing on linker roles, protein-protein interaction stability, and lysine residue accessibility. The findings are significant for PROTAC development in cancer treatment, particularly breast and prostate cancers.

Strengths:

(1) Comprehensive computational analysis of PROTAC-related protein complexes.<br /> (2) Focus on critical aspects: linker role, protein-protein interaction stability, and lysine accessibility.

Weaknesses:

(1) Limited examination of lysine accessibility despite its stated importance.<br /> (2) Use of RMSD as the primary metric for conformational assessment, which may overlook important local structural changes.

The authors' claims about the role of PROTAC linkers and protein-protein interaction stability are generally supported by their computational data. However, the conclusions regarding lysine accessibility could be strengthened with more in-depth analysis. The use of the term "protein functional dynamics" is not fully justified by the presented work, which focuses primarily on structural dynamics rather than functional aspects.

Comments on revisions:

The authors have addressed the questions raised substantially.

Anh - Napoleon Pháp - Wellington => cuộc chiến tranh kéo theo sự ảnh hưởng lớn về mặt trái phiếu

Reviewer #1 (Public review):

In the resubmission Simões et al. emphasize the efficacy of their novel, non-invasive imaging methodology in mapping glucose-kinetics to predict key tumor features in two commonly used syngeneic mouse models of glioblastoma. The authors highlight that DGE-DMI has the potential to capture metabolic fluxes with greater sensitivity and acknowledge that future validation of DGE-DMI in patient-derived and spontaneous GBM models, as well as in the context of genetic manipulation of metabolism, would strengthen its clinical application. To further demonstrate the ability of DGE-DMI to predict tumor features, they included an assessment of myeloid cell infiltration along with proliferation, peritumoral invasion, and distant migration. Overall, the authors offer a novel method to the scientific community that can be further tested and adapted for interrogating GBM heterogeneity.

Reviewer #1 (Public review):

Summary:

In this revised report, Yamanaka and colleagues investigate a proposed mechanism by which testosterone modulates seminal plasma metabolites in mice. The authors identify oleic acid as a particularly important metabolite, derived from seminal vesicle epithelium, that stimulates linear progressive motility in isolated cauda epidydimal sperm in vitro. The authors provide additional experimental evidence of a testosterone dependent mechanism of oleic acid production by the seminal vesicle epithelium.

Strengths:

Often, reported epidydimal sperm from mice have lower percent progressive motility compared with sperm retrieved from the uterus or by comparison with human ejaculated sperm. The findings in this report may improve in vitro conditions to overcome this problem, as well as add important physiological context to the role of reproductive tract glandular secretions in modulating sperm behaviors. The strongest observations are related to the sensitivity of seminal vesicle epithelial cells to testosterone. The revisions include addition of methodological detail, modified language to reflect the nuance of some of the measurements, as well as re-performed experiments with more appropriate control groups. The findings are likely to be of general interest to the field by providing context for follow-on studies regarding the relationship between fatty acid beta oxidation and sperm motility pattern.

Weaknesses:

Support for the proposed mechanism is stronger in this revised report than in the previous report, but there are many challenges in measuring sperm metabolism and its direct relationship with motility patterns. This study is no exception and largely relies on correlations between various experiments in lieu of direct testing. Additionally, the discussion is framed from a human pre-clinical perspective, and it should be noted that the reproductive physiology between mice and humans is very different.

Reviewer #3 (Public review):

Summary:

In this manuscript, Sur and colleagues present insights into the potential pathways and mechanisms underlying the pathogenesis of cystinosis - a prototypical lysosomal storage disorder caused by the loss of the cystine transporter cystinosin (CTNS). This deficiency results in early dysfunction of proximal tubule (PT) cells and proximal tubulopathy, which progresses to chronic kidney disease and multisystem complications later in life. The authors utilize patient-derived cell lines and knockout (KO) strategies in immortalized PT cell systems, alongside transcriptomics and pathway enrichment analyses, to demonstrate that the loss of CTNS function reduces V-ATPase subunits (specifically V-ATP6V0A1), impairing autophagy and mitochondrial homeostasis. These findings are consistent with their prior work and follow-up studies conducted in preclinical models (mouse, rat, and zebrafish) of cystinosis and CTNS deficiency.

Importantly, the authors highlight rescue strategies that involve correcting V-ATP6V0A1 expression or modulating redox dyshomeostasis through ATX treatment. These interventions restore cellular homeostasis in patient-derived cells, providing actionable therapeutic targets for patients in need of novel causal therapies.

Strengths:

The implications for health, disease, and therapeutic discovery are considerable, given the central role of autophagy and lysosome-related pathways in regulating critical cellular processes and physiological functions.

Weaknesses:

Despite these promising findings, further experimental research is required to strengthen the study's framework and conclusions. This includes characterizing the physiological properties of the PT cellular systems used, performing appropriate control or sentinel experiments in lysosome function assays, and further delineating disease phenotypes associated with cystinosis. Follow-up investigations into lysosome abnormalities and autophagy dysfunctions are also needed, along with a detailed exploration of the molecular mechanisms underlying the rescue of lysosomal, autophagic, and mitochondrial phenotypes through ATX treatment.

Reviewer #1 (Public review):

Summary:

Busch and Hansel present a morphological and histological comparison between mouse and human Purkinje cells (PCs) in the cerebellum. The study reveals species-specific differences that have not previously been reported despite numerous observations of these species. While mouse PCs show morphological heterogeneity and occasional multi-innervation by climbing fibers (CFs), human PCs exhibit a widespread, multi-dendritic structure that exceeds expectations based on allometric scaling. Specifically, human PCs are significantly larger, and exhibit increased spine density, with a unique cluster-like morphology not found in mice.

Strengths:

The manuscript provides an exceptionally detailed analysis of PC morphology across species, surpassing any prior publication. Major strengths include a systematic and thorough methodology, rigorous data analysis, and clear presentation of results. This work is likely to become the go-to resource for quantitation in this field. The authors have largely achieved their aims, with the results effectively supporting their conclusions.

Weaknesses:

There are a few concerns that need to be addressed, specifically related to details of the methodolology as well as data interpretation based on the limits of some experimental approaches. Overall, these weaknesses are minor.

Reviewer #1 (Public review):

Summary:

The authors track the motion of multiple consortia of Multicellular Magnetotactic Bacteria moving through an artificial network of pores and report a discovery of a simple strategy for such consortia to move fast through the network: an optimum drift speed is attained for consortia that swim a distance comparable to the pore size in the time it takes to align the with an external magnetic field. The authors rationalize their observations using dimensional analysis and numerical simulations. Finally, they argue that the proposed strategy could generalize to other species by demonstrating the positive correlation between the swimming speed and alignment time based on parameters derived from literature.

Strengths:

The underlying dimensional analysis and model convincingly rationalize the experimental observation of an optimal drift velocity: the optimum balances the competition between the trapping in pores at large magnetic fields and random pore exploration for weak magnetic fields.

Weaknesses:

The convex pore geometry studied here creates convex traps for cells, which I expect enhances their trapping. The more natural concave geometries, resulting from random packing of spheres, would create no such traps. In this case, whether a non-monotonic dependence of the drift velocity on the Scattering number would persist is unclear.

Reviewer #1 (Public review):

Summary:

Cruz-González and colleagues draw on DNA methylation and paired genetic data from 621 participants (n=308 controls; n=313 participants with Alzheimer's Disease). The authors generate a panel of epigenetic biomarkers of aging with a primary focus on the Horvath multi-tissue clock. The authors find weaker correlations between predicted epigenetic age and chronological age in subgroups with higher African ancestry than within a subgroup identified as White. The authors then examine genetic variation as a potential source for between-group differences in epigenetic clock performance. The authors draw on a large collection of publicly available methylation quantitative trait loci datasets and find evidence for substantial overlap between clock CpGs located within the Horvath clock and methQTLs. Going further, the authors show that methQTLs that overlap with Horvath clock CpGs show greater allelic variation in African ancestral groups pointing to a potential explanation for poorer clock performance within this group.

Strengths:

This is an interesting dataset and an important research question. The authors cite issues of portability regarding polygenic risk scores as a motivation to examine between-group differences in the performance of a panel of epigenetic clocks. The authors benefit from a diverse cohort of individuals with paired genetic data and focus on a clinical phenotype, Alzheimer's disease, of clear relevance for studies evaluating age-related biomarkers.

Weaknesses:

While the authors tackle an important question using a diverse cohort the current manuscript is lacking some detail that may diminish the potential impact of this paper. For example:

(1) Information on chronological ages across groups should be reported to ensure there are no systematic differences in ages or age ranges between groups (see point below).

(2) The authors compare correlations between chronological age and epigenetic age in sub-groups within to correlations reported by Horvath (2013). Attempting to draw comparisons between these two datasets is problematic. The current study has a much smaller N (particularly for sub-group analyses) and has a more restricted age range (60-90yrs versus 0-100 yrs). Thus, is an alternative explanation simply that any weaker correlations observed in this study are driven by sample size and a restricted age range? Reporting the chronological ages (and ranges) across subgroups in the current study would help in this regard. Similarly, given the lack of association between AD status and epigenetic age (and very small effect in the white group), it may be of interest to examine the correlation between chronological age and epigenetic age in each group including the AD participants: would the between-group differences in correlations between chronological age and epigenetic be altered by increasing the sample size?

(3) The correlation between chronological age and epigenetic age, while helpful is not the most informative estimate of accuracy. Median absolute error (and an analysis of MAE across subgroups) would be a helpful addition.

(4) More information should be provided about how DNAm data were generated. Were samples from each ancestral group randomized across plates/slides to ensure ancestry and batch are not associated? How were batch effects considered? Given the relatively small sample sizes, it would be important to consider the impact of technical variation on measures of epigenetic age used in the current study. The use of principal Component-based versions of these clocks (Higgins Chen et al., 2023; Nature Aging https://doi.org/10.1038/s43587-022-00248-2) may help address concerns such concerns.

(5) Marioni et al., (2015) found a very weak cross-sectional association between DNAm Age and cognitive function (r~0.07) in a cohort of >900 participants. Given these effect sizes, I would not interpret the absence of an effect in the current study to reflect issues of portability of epigenetic biomarkers.

6) The methQTL analyses presented are suggestive of potential genetic influence on DNAm at some Horvath CpGs. Do authors see differences in DNAm across ancestral groups at these potentially affected CpGs? This seems to be a missing piece together (e.g., estimating the likely impact of methQTL on clock CpG DNAm).

Reviewer #1 (Public review):

Summary:

Using high-quality genomic data (long-reads, optical maps, short-reads) and advanced bioinformatic analysis, the authors aimed to document chromosomal rearrangements across a recent radiation (Lake Malawi Cichlids). Working on 11 species, they achieved a high-resolution inversion detection and then investigated how inversions are distributed within populations (using a complementary dataset of short-reads), associated with sex, and shared or fixed among lineages. The history and ancestry of the inversions is also explored.

On one hand, I am very enthusiastic about the global finding (many inversions well-characterized in a highly diverse group!) and impressed by the amount of work put into this study. On the other hand, I have struggled so much to read the manuscript that I am unsure about how much the data supports some claims. I'm afraid most readers may feel the same and really need a deep reorganisation of the text, figures, and tables. I reckon this is difficult given the complexity brought by different inversions/different species/different datasets but it is highly needed to make this study accessible.

The methods of comparing optical maps, and looking at inversions at macro-evolutionary scales can be useful for the community. For cichlids, it is a first assessment that will allow further tests about the role of inversions in speciation and ecological specialisation. However, the current version of the manuscript is hardly accessible to non-specialists and the methods are not fully reproducible.

Strengths:

(1) Evidence for the presence of inversion is well-supported by optical mapping (very nice analysis and figure!).

(2) The link between sex determination and inversion in chr 10 in one species is very clearly demonstrated by the proportion in each sex and additional crosses. This section is also the easiest to read in the manuscript and I recommend trying to rewrite other result sections in the same way.

(3) A new high-quality reference genome is provided for Metriaclima zebra (and possibly other assemblies? - unclear).

(4) The sample size is great (31 individuals with optical maps if I understand well?).

(5) Ancestry at those inversions is explored with outgroups.

(6) Polymorphism for all inversions is quantified using a complementary dataset.

Weaknesses:

(1) Lack of clarity in the paper: As it currently reads, it is very hard to follow the different species, ecotypes, samples, inversions, etc. It would be useful to provide a phylogeny explicitly positioning the samples used for assembly and the habitat preference. Then the text would benefit from being organised either by variant or by subgroups rather than by successive steps of analysis.

(2) Lack of information for reproducibility: I couldn't find clearly the filters and parameters used for the different genomic analyses for example. This is just one example and I think the methods need to be re-worked to be reproducible. Including the codes inside the methods makes it hard to follow, so why not put the scripts in an indexed repository?

(3) Further confirmation of inversions and their breakpoints would be valuable. I don't understand why the long-reads (that were available and used for genome assembly) were not also used for SV detection and breakpoint refinement.

(4) Lack of statistical testing for the hypothesis of introgression: Although cichlids are known for high levels of hybridization, inversions can also remain balanced for a long time. what could allow us to differentiate introgression from incomplete lineage sorting?

(5) The sample size is unclear: possibly 31 for Bionano, 297 for short-reads, how many for long-reads or assemblies? How is this sample size split across species? This would deserve a table.

(6) Short read combines several datasets but batch effect is not tested.

(7) It is unclear how ancestry is determined because the synteny with outgroups is not shown.

(8) The level of polymorphism for the different inversions is difficult to interpret because it is unclear whether replicated are different species within an eco-group or different individuals from the same species. How could it be that homozygous references are so spread across the PCA? I guess the species-specific polymorphism is stronger than the ancestral order but in such a case, wouldn't it be worth re-doing the PCa on a subset?

Reviewer #1 (Public review):

Summary:

Zacharia and colleagues investigate the role of the C-terminus of IFT172 (IFT172c), a component of the IFT-B subcomplex. IFT172 is required for proper ciliary trafficking and mutations in its C-terminus are associated with skeletal ciliopathies. The authors begin by performing a pull-down to identify binding partners of His-tagged CrIFT172968-C in Chlamydomonas reinhardtii flagella. Interactions with three candidates (IFT140, IFT144, and a UBX-domain containing protein) are validated by AlphaFold Multimer with the IFT140 and IFT144 predictions in agreement with published cryo-ET structures of anterograde and retrograde IFT trains. They present a crystal structure of IFT172c and find that a part of the C-terminal domain of IFT172 resembles the fold of a non-canonical U-box domain. As U-box domains typically function to bind ubiquitin-loaded E2 enzymes, this discovery stimulates the authors to investigate the ubiquitin-binding and ubiquitination properties of IFT172c. Using in vitro ubiquitination assays with truncated IFT172c constructs, the authors demonstrate partial ubiquitination of IFT172c in the presence of the E2 enzyme UBCH5A. The authors also show a direct interaction of IFT172c with ubiquitin chains in vitro. Finally, the authors demonstrate that deletion of the U-box-like subdomain of IFT172 impairs ciliogenesis and TGFbeta signaling in RPE1 cells.

However, some of the conclusions of this paper are only partially supported by the data, and presented analyses are potentially governed by in vitro artifacts. In particular, the data supporting autoubiquitination and ubiquitin-binding are inconclusive. Without further evidence supporting a ubiquitin-binding role for the C-terminus, the title is potentially misleading.

Strengths:

(1) The pull-down with IFT172 C-terminus from C. reinhardtii cilia lysates is well performed and provides valuable insights into its potential roles.

(2) The crystal structure of the IFT172 C-terminus is of high quality.

(3) The presented AlphaFold-multimer predictions of IFT172c:IFT140 and IFT172c:IFT144 are convincing and agree with experimental cryo-ET data.

Weaknesses:

(1) The crystal structure of HsIFT172c reveals a single globular domain formed by the last three TPR repeats and C-terminal residues of IFT172. However, the authors subdivide this globular domain into TPR, linker, and U-box-like regions that they treat as separate entities throughout the manuscript. This is potentially misleading as the U-box surface that is proposed to bind ubiquitin or E2 is not surface accessible but instead interacts with the TPR motifs. They justify this approach by speculating that the presented IFT172c structure represents an autoinhibited state and that the U-box-like domain can become accessible following phosphorylation. However, additional evidence supporting the proposed autoinhibited state and the potential accessibility of the U-box surface following phosphorylation is needed, as it is not tested or supported by the current data.

(2) While in vitro ubiquitination of IFT172 has been demonstrated, in vivo evidence of this process is necessary to support its physiological relevance.

(3) The authors describe IFT172 as being autoubiquitinated. However, the identified E2 enzymes UBCH5A and UBCH5B can both function in E3-independent ubiquitination (as pointed out by the authors) and mediate ubiquitin chain formation in an E3-independent manner in vitro (see ubiquitin chain ladder formation in Figure 3A). In addition, point mutation of known E3-binding sites in UBCH5A or TPR/U-box interface residues in IFT172 has no effect on the mono-ubiquitination of IFT172c1. Together, these data suggest that IFT172 is an E3-independent substrate of UBCH5A in vitro. The authors should state this possibility more clearly and avoid terminology such as "autoubiquitination" as it implies that IFT172 is an E3 ligase, which is misleading. Similarly, statements on page 10 and elsewhere are not supported by the data (e.g. "the low in vitro ubiquitination activity exhibited by IFT172" and "ubiquitin conjugation occurring on HsIFT172C1 in the presence of UBCH5A, possibly in coordination with the IFT172 U-box domain").

(4) Related to the above point, the conclusion on page 11, that mono-ubiquitination of IFT172 is U-box-independent while polyubiquitination of IFT172 is U-box-dependent appears implausible. The authors should consider that UBCH5A is known to form free ubiquitin chains in vitro and structural rearrangements in F1715A/C1725R variants could render additional ubiquitination sites or the monoubiquitinated form of IFT172 inaccessible/unfavorable for further processing by UBCH5A.

(5) Identification of the specific ubiquitination site(s) within IFT172 would be valuable as it would allow targeted mutation to determine whether the ubiquitination of IFT172 is physiologically relevant. Ubiquitination of the C1 but not the C2 or C3 constructs suggests that the ubiquitination site is located in TPRs ranging from residues 969-1470. Could this region of TPR repeats (lacking the IFT172C3 part) suffice as a substrate for UBCH5A in ubiquitination assays?

(6) The discrepancy between the molecular weight shifts observed in anti-ubiquitin Western blots and Coomassie-stained gels is noteworthy. The authors show the appearance of a mono-ubiquitinated protein of ~108 kDa in anti-ubiquitin Western blots. However, this molecular weight shift is not observed for total IFT172 in the corresponding Coomassie-stained gels (Figures 3B, D, F). Surprisingly, this MW shift is visible in an anti-His Western blot of a ubiquitination assay (Fig 3C). Together, this raises the concern that only a small fraction of IFT172 is being modified with ubiquitin. Quantification of the percentage of ubiquitinated IFT172 in the in vitro experiments could provide helpful context.

(7) The authors propose that IFT172 binds ubiquitin and demonstrate that GST-tagged HsIFT172C2 or HsIFT172C3 can pull down tetra-ubiquitin chains. However, ubiquitin is known to be "sticky" and to have a tendency for weak, nonspecific interactions with exposed hydrophobic surfaces. Given that only a small proportion of the ubiquitin chains bind in the pull-down, specific point mutations that identify the ubiquitin-binding site are required to convincingly show the ubiquitin binding of IFT172.

(8) The authors generated structure-guided mutations based on the predicted Ub-interface and on the TPR/U-box interface and used these for the ubiquitination assays in Fig 3. These same mutations could provide valuable insights into ubiquitin binding assays as they may disrupt or enhance ubiquitin binding (by relieving "autoinhibition"), respectively. Surprisingly, two of these sites are highlighted in the predicted ubiquitin-binding interface (F1715, I1688; Figure 4E) but not analyzed in the accompanying ubiquitin-binding assays in Figure 4.

(9) If IFT172 is a ubiquitin-binding protein, it might be expected that the pull-down experiments in Figure S1 would identify ubiquitin, ubiquitinated proteins, or E2 enzymes. These were not observed, raising doubt that IFT172 is a ubiquitin-binding protein.

(10) The cell-based experiments demonstrate that the U-box-like region is important for the stability of IFT172 but does not demonstrate that the effect on the TGFb pathway is due to the loss of ubiquitin-binding or ubiquitination activity of IFT172.

(11) The challenges in experimentally validating the interaction between IFT172 and the UBX-domain-containing protein are understandable. Alternative approaches, such as using single domains from the UBX protein, implementing solubilizing tags, or disrupting the predicted binding interface in Chlamydomonas flagella pull-downs, could be considered. In this context, the conclusion on page 7 that "The uncharacterized UBX-domain-containing protein was validated by AF-M as a direct IFT172 interactor" is incorrect as a prediction of an interaction interface with AF-M does not validate a direct interaction per se.

Reviewer #1 (Public review):

Summary:

Colorectal cancer (CRC) is the third most common cancer globally and the second leading cause of cancer-related deaths. Colonoscopy and fecal immunohistochemical testing are among the early diagnostic tools that have significantly enhanced patient survival rates in CRC. Methylation dysregulation has been identified in the earliest stages of CRC, offering a promising avenue for screening, prediction, and diagnosis. The manuscript entitled "Early Diagnosis and Prognostic Prediction of Colorectal Cancer through Plasma Methylation Regions" by Zhu et al. presents that a panel of genes with methylation pattern derived from cfDNA (27 DMRs), serving as a noninvasive detection method for CRC early diagnosis and prognosis.

Strengths:

The authors provided evidence that the 27 DMRs pattern worked well in predicting CRC distant metastasis, and the methylation score remarkably increased in stage III-IV.

Weaknesses:

The major concerns are the design of DMR screening, the relatively low sensitivity of this DMR pattern in detecting early-stage CRC, the limited size of the cohorts, and the lack of comparison with the traditional diagnosis test.

Reviewer #1 (Public review):

Summary:

In this study, Xiao et al. classified retroperitoneal liposarcoma (RPLS) patients into two subgroups based on whole transcriptome sequencing of 88 patients. The G1 group was characterized by active metabolism, while the G2 group exhibited high scores in cell cycle regulation and DNA damage repair. The G2 group also displayed more aggressive molecular features and had worse clinical outcomes compared to G1. Using a machine learning model, the authors simplified the classification system, identifying LEP and PTTG1 as the key molecular markers distinguishing the two RPLS subgroups. Finally, they validated these markers in a larger cohort of 241 RPLS patients using immunohistochemistry. Overall, the manuscript is clear and well-organized, with its significance rooted in the large sample size and the development of a classification method.

Weakness:

(1) While the authors suggest that LEP and PTTG1 serve as molecular markers for the two RPLS groups, the process through which these genes were selected remains unclear. The authors should provide a detailed explanation of the selection process.

(2) To ensure the broader applicability of LEP and PTTG1 as classification markers, the authors should validate their findings in one or two external datasets.

(3) Since molecular subtyping is often used to guide personalized treatment strategies, it is recommended that the authors evaluate therapeutic responses in the two distinct groups. Additionally, they should validate these predictions using cell lines or primary cells.

Reviewer #1 (Public review):

Summary:

Flowers et al describe an improved version of qFit-ligand, an extension of qFit. qFit and qFit-ligand seek to model conformational heterogeneity of proteins and ligands, respectively, cryo-EM and X-ray (electron) density maps using multi-conformer models - essentially extensions of the traditional alternate conformer approach in which substantial parts of the protein or ligand are kept in place. By contrast, ensemble approaches represent conformational heterogeneity through a superposition of independent molecular conformations.

The authors provide a clear and systematic description of the improvements made to the code, most notably the implementation of a different conformer generator algorithm centered around RDKit. This approach yields modest improvements in the strain of the proposed conformers (meaning that more physically reasonable conformations are generated than with the "old" qFit-ligand) and real space correlation of the model with the experimental electron density maps, indicating that the generated conformers also better explain the experimental data than before. In addition, the authors expand the scope of ligands that can be treated, most notably allowing for multi-conformer modeling of macrocyclic compounds.

Strengths:

The manuscript is well written, provides a thorough analysis, and represents a needed improvement of our collective ability to model small-molecule binding to macromolecules based on cryo-EM and X-ray crystallography, and can therefore have a positive impact on both drug discovery and general biological research.

Weaknesses:

There are several points where the manuscript needs clarification in order to better understand the merits of the described work. Overall the demonstrated performance gains are modest (although the theoretical ceiling on gains in model fit and strain energy are not clear!).

In Grammar... change the form of (a word) to express a particular grammatical function or attribute, typically tense, mood, person, number, case, and gender.

2. vary the intonation or pitch of (the voice), especially to express mood or feeling.

Reviewer #1 (Public review):

Summary:

The authors tested whether learning to suppress (ignore) salient distractors (e.g., a lone colored nontarget item) via statistical regularities (e.g., the distractor is more likely to appear in one location than any other) was proactive (prior to paying attention to the distractor) or reactive (only after first attending the distractor) in nature. To test between proactive and reactive suppression the authors relied on a recently developed and novel technique designed to "ping" the brain's hidden priority map using EEG inverted encoding models. Essentially, a neutral stimulus is presented to stimulate the brain, resulting in activity on a priority map which can be decoded and used to argue when this stimulation occurred (prior to or after attending a distracting item). The authors found evidence that despite learning to suppress the high probability distractor location, the suppression was reactive, not proactive in nature.

Overall, the manuscript was well-written, tests a timely question, and provides novel insight into a long-standing debate concerning distractor suppression.

The authors provided a thorough rebuttal and addressed the previous critiques and concerns.

Strengths (in no particular order):<br /> (1) The manuscript is well-written, clear, and concise (especially given the complexities of the method and analyses).<br /> (2) The presentation of the logic and results is clear and relatively easy to digest.<br /> (3) This question concerning whether location-based distractor suppression is proactive or reactive in nature is a timely question.<br /> (4) The use of the novel "pinging" technique is interesting and provides new insight into this particularly thorny debate over the mechanisms of distractor suppression.

Weaknesses (in no particular order):

After revision, the prior weaknesses have been largely addressed.

Reviewer #1 (Public review):

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

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

The revised manuscript is also more transparent about the model's limitations, such as the lack of excitatory-excitatory connectivity.

Reviewer #1 (Public review):

Summary:

This very short paper shows a greater likelihood of C->U substitutions at sites predicted to be unpaired in the SARS-CoV-2 RNA genome, using previously published observational data on mutation frequencies in SARS-CoV-2 (Bloom and Neher, 2023).

General comments:

A preference for unpaired bases as target for APOBEC-induced mutations has been demonstrated previously in functional studies so the finding is not entirely surprising. This of course assumes that A3A or other APOBEC is actually the cause of the majority of C->U changes observed in SARS-CoV-2 sequences.

I'm not sure why the authors did not use the published mutation frequency data to investigate other potential influences on editing frequencies, such as 5' and 3' base contexts. The analysis did not contribute any insights into the potential mechanisms underlying the greater frequency of C->U (or G->U) substitutions in the SARS-CoV-2 genome.

Comments on revisions:

The revisions have addressed my main comments in my review.

Reviewer #1 (Public review):

Summary:

This study focuses on metabolic changes in the paraventricular hypothalamic (PVH) region of the brain during acute periods of cold exposure. The authors point out that in comparison to the extensive literature on the effects of cold exposure in peripheral tissues, we know relatively little about its effects on the brain. They specifically focus on the hypothalamus, and identify the PVH as having changes in Atgl and Hsl gene expression changes during cold exposure. They then go on to show accumulation of lipid droplets, increased Fos expression, and increased lipid peroxidation during cold exposure. Further, they show that neuronal activation is required for the formation of lipid droplets and lipid peroxidation.

Strengths:

A strength of the study is trying to better understand how metabolism in the brain is a dynamic process, much like how it has been viewed in other organs. The authors also use a creative approach to measuring in vivo lipid peroxidation via delivery of BD-C11 sensor through a cannula to the region in conjunction with fiber photometry to measure fluorescence changes deep in the brain.

Comments on revised version:

The authors have attempted to address concerns brought to their attention in the initial review. They have performed one or two additional experiments to address concerns (e.g. adding fiber photometry of PVH neurons and trying to manipulate lipid peroxidation) though many of the concerns from the original review stand. The authors have also revised the text to limit the extent of their claims and to improve clarity, which is appreciated.

Reviewer #1 (Public review):

Summary:

IPF is a disease lacking regressive therapies which has a poor prognosis, and so new therapies are needed. This ambitious phase 1 study builds on the authors 2024 experience in Sci Tran Med with positive results with autologous transplantation of P63 progenitor cells in patients with COPD. The current study suggests P63+ progenitor cell therapy is safe in patients with ILD. The authors attribute this to acquisition of cells from a healthy upper lobe site, removed from the lung fibrosis. There are currently no cell based therapies for ILD and in this regard the study is novel with important potential for clinical impact if validated in Phase 2 and 3 clinical trials.

Strengths:

This study addresses the need for an effective therapy for interstitial lung disease. It offers good evidence the cell used for therapy are safe. In so doing it addresses a concern that some P63+ progenitor cells may be proinflammatory and harmful, as has been raised in the literature (articles which suggested some P63+ cells can promote honeycombing fibrosis; ref 26 &35). The authors attribute the safety they observed (without proof) to the high HOPX expression of administered cells (a marker found in normal Type 1 AECs. The totality of the RNASeq suggests the cloned cells are not fibrogenic. They also offer exploratory data suggesting a relationship between clone roundness and PFT parameters (and a negative association of patient age and clone roundness).

Weaknesses:

The authors can conclude they can isolate, clone, expand and administer P63+ progenitor cells safely; but with the small sample size and lack of placebo group no efficacy should be implied.

Comments on revisions:

The paper is meritorious as I noted initially

However, the authors did not directly address several of my concerns-i.e. their responses to the initial review were polite but did not translate into much change in the manuscript.

(1) Do these progenitor cells exert their beneficial effects by a paracrine mechanism vs transforming into lung AECs? Based on work in the field of bronchopulmonary dysplasia I suspect the benefits are mediated by a paracrine mecahnism and arguably media from these cells should be tested as an alternative to administering the cells themselves. In any case, for the revision a Discussion of the possibility of differentiation vs paracrine mechanisms, citing relevant literature, would be expected. I suggest that you add such a paragraph to a limitation section.

(2) Please address that potential implications of the fact that 5 patients had essentially normal DLCO/VA values. Saying that the "criterion for entry was DLCO" does not take away from the fact that DLCO/VA is a valid measure of lung diffusion capacity. In the absence of placebo an enrollment of mildly diseased patients would favor positive results (including stability in study endpoint parameters even without treatment). Thus, I suggest again that the limitations section should be more forthright in this regard.

(3) The authors acknowledge the lack of a placebo group but in a study of mild IPF, I worry that without a placebo group the only robust findings are those related to technique of transplantation and the safety of cell therapy. The paper still reads as if there is a clinical benefit...I would advise you further soften this (while understanding the desire to emphasize a hopeful observation). The price for not having a placebo group must be avoidance of claims of efficacy. The improvements in DLCO and CT in several cases speaks for the need for the planned phase 2 trial, which if positive will be the time to claim efficacy signals.

Reviewer #1 (Public review):

Summary:

In this manuscript, authors have investigated the effects of JNK inhibition on sucrose-induced metabolic dysfunction in rats. They used multi-tissue network analysis to study the effects of the JNK inhibitor JNK-IN-5A on metabolic dysfunction associated with excessive sucrose consumption. Their results show that JNK inhibition reduces triglyceride accumulation and inflammation in the liver and adipose tissues while promoting metabolic adaptations in skeletal muscle. The study provides new insights into how JNK inhibition can potentially treat metabolic dysfunction-associated fatty liver disease (MAFLD) by modulating inter-tissue communication and metabolic processes.

Strengths:

The study has several notable strengths:

Comprehensive Multi-Tissue Analysis: The research provides a thorough multi-tissue evaluation, examining the effects of JNK inhibition across key metabolically active tissues, including the liver, visceral white adipose tissue, skeletal muscle, and brain. This comprehensive approach offers valuable insights into the systemic effects of JNK inhibition and its potential in treating MAFLD.

Robust Use of Systems Biology: The study employs advanced systems biology techniques, including transcriptomic analysis and genome-scale metabolic modeling, to uncover the molecular mechanisms underlying JNK inhibition. This integrative approach strengthens the evidence supporting the role of JNK inhibitors in modulating metabolic pathways linked to MAFLD.

Potential Therapeutic Insights: By demonstrating the effects of JNK inhibition on both hepatic and extrahepatic tissues, the study offers promising therapeutic insights into how JNK inhibitors could be used to mitigate metabolic dysfunction associated with excessive sucrose consumption, a key contributor to MAFLD.

Behavioral and Metabolic Correlation: The inclusion of behavioral tests alongside metabolic assessments provides a more holistic view of the treatment's effects, allowing for a better understanding of the broader physiological implications of JNK inhibition.

Weaknesses:

The authors have adequately addressed all my concerns, and the revisions have significantly improved the manuscript's clarity and impact.

Reviewer #1 (Public review):

Summary:

This paper describes a new in vitro model for DRG neurons that recapitulates several key differences between the peripheral and central branches of DRG axons in vivo. These differences include morphology (with one branch being thinner than the other), and regenerative capacity (with the peripheral branch displaying higher regenerative capacity). The authors analyze the abundance of various microtubule associated protein (MAPs) in each branch, as well as the microtubule dynamics in each branch and find significant differences between branches. Importantly, they found that a well-known conditioning paradigm (prior lesion of the peripheral branch improves the regenerative capacity of the central branch) is not only reproduced in this system, but also leads to loss of the asymmetry of MAPs between branches. Zooming in on one MAP that shows differential abundance between the axons, they find that the severing enzyme Spastin is required for the asymmetry in microtubule dynamics and in regenerative capacity following a conditioning lesion

Strengths:

The establishment of an experimental system that recapitulates DRG axon asymmetry in vitro is an important step that is likely to be useful for other studies. In addition, identifying key molecular signatures that differ between central and peripheral branches, and determining how they are lost following a conditioning lesion adds to our understanding of why peripheral axons have a better regenerative capacity. Last, the authors use of an in vivo model system to support some of their in vitro findings is a strength of this work.

Weaknesses:

One weakness of the manuscript is that to a large degree, one of its main conclusions (MAP symmetry underlies differences in regenerative capacity) relies mainly on a correlation, without firmly establishing a causal link. However, this is weakness is relatively minor because (1) it is partially addressed with the Spastin KO and (2) there isn't a trivial way to show a causal relationship in this case. (3) It is addressed in the Discussion section.

Reviewer #1 (Public Review):

The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.

The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

Comments on latest version:

The authors have sufficiently addressed all concerns raised, which further enhanced data presentation. No additional concerns were raised.

Reviewer #1 (Public review):

Summary:

This study shows that the pro-inflammatory S1P signaling regulates the responses of muller glial cells to damage. The authors describe the expression of S1P signaling components. Using agonist and antagonist of the pathways they also investigate their effect on the de-differentiation and proliferation of Muller glial cells in damaged retina of postnatal chicks. They show that S1PR1 is highly expressed in resting MG and non-neurogenic MGPCs. This receptor suppresses the proliferation and neuronal activity promotes MGPC cell cycle re-entry and enhanced the number of regenerated amacrine-like cells after retinal damage. The formation of MGPCs in damaged retinas is impaired in the absence of microglial cells. This study further shows that ablation of microglial cells from the retina increases the expression of S1P-related genes in MG, whereas inhibition of S1PR1 and SPHK1 partially rescues the formation of MGPCs in damaged retinas depleted of microglia. The studies also show that expression of S1P-related genes is conserved in fish and human retinas.

Strengths:

This is well-conducted study, with convincing images and statistically relevant data

Weaknesses:

In a previous study, the authors have shown that S1P is upstream of NF-κB signaling (Palazzo et al. 2020; 2022, 2023). Although S1P and NF-κB signaling have overlapping effects, the authors here provide evidence for S1P specific effects, adding some new information to the field.

Reviewer #1 (Public review):

Summary:

The authors employed a combinatorial CRISPR-Cas9 knockout screen to uncover synthetically lethal kinase genes that could play a role in drug resistance to kinase inhibitors in triple-negative breast cancer. The study successfully reveals FYN as a mediator of resistance to depletion and inhibition of various tyrosine kinases, notably EGFR, IGF-1R, and ABL, in triple-negative breast cancer cells and xenografts. Mechanistically, they demonstrate that KDM4 contributes to the upregulation of FYN and thereby is an important mediator of the drug resistance. All together, these findings suggest FYN and KDM4A as potential targets for combination therapy with kinase inhibitors in triple-negative breast cancer. Moreover, the study may also have important implications for other cancer types and other inhibitors, as the authors suggest that FYN could be a general feature of drug-tolerant persister cells.

Strengths:

(1) The authors used a large combination matrix of druggable tyrosine kinase gene knockouts, enabling studying of co-dependence of kinase genes. This approach mitigates off-target effects typically associated with kinase inhibitors, enhancing the precision of the findings.

(2) The authors demonstrate the importance of FYN in drug resistance in multiple ways. They demonstrate synergistic interactions using both knockouts and inhibitors, while also revealing its transcriptional upregulation upon treatment, strengthening the conclusion that FYN plays a role in the resistance.

(3) The study extends its impact by demonstrating the potent in vivo efficacy of certain combination treatments, underscoring the clinical relevance of the identified strategies.

Weaknesses:

(1) The combination of FYN knockout with other gene knockouts exhibits only very modest synergy. The high standard deviation observed for FYN knockout in Figure S2A weakens the robustness of these findings. As combination treatments involving inhibitors did demonstrate stronger synergistic effects, the data still support the role of FYN in regulating sensitivity to the described drugs.

(2) While the study identifies KDM4A as a key contributor to FYN upregulation, it does not fully explore the upstream mechanisms regulating KDM4A or the downstream pathways through which FYN upregulation confers drug resistance. These unaddressed questions limit the mechanistic understanding that can be obtained from this study.

(3) FYN has been implicated in drug resistance in previous studies, and other mechanisms for its upregulation and downstream effects have already been described. While this study adds value to the existing literature in the context of breast cancer, it does not present entirely novel findings regarding FYN's role in drug resistance.

Reviewer #1 (Public review):

Summary:

The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

(3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

The RNA seq data add on to this worry and should at least be discussed.

Reviewer #1 (Public review):

Summary:

This work provides a new potential tool to manipulate Tregs function for therapeutic use. It focuses on the role of PGAM in Tregs differentiation and function. The authors, interrogating publicly available transcriptomic and proteomic data of human regulatory T cells and CD4 T cells, state that Tregs express higher levels of PGAM (at both message and protein levels) compared to CD4 T cells. They then inhibit PGAM by using a known inhibitor ECGC and show that this inhibition affects Tregs differentiation. This result was also observed when they used antisense oligonucleotides (ASOs) to knockdown PGAM1.

PGAM1 catalyzes the conversion of 3PG to 2PG in the glycolysis cascade. However, the authors focused their attention on the additional role of 3PG: acting as starting material for the de novo synthesis of serine.

They hypothesized that PGAM1 regulates Tregs differentiation by regulating the levels of 3PG that are available for de novo synthesis of serine, which has a negative impact on Tregs differentiation. Indeed, they tested whether the effect on Tregs differentiation observed by reducing PGAM1 levels was reverted by inhibiting the enzyme that catalyzes the synthesis of serine from 3PG.

The authors continued by testing whether both synthesized and exogenous serine affect Tregs differentiation and continued with in vivo experiments to examine the effects of dietary serine restriction on Tregs function.

In order to understand the mechanism by which serine impacts Tregs function, the authors assessed whether this depends on the contribution of serine to one-carbon metabolism and to DNA methylation.

The authors therefore propose that extracellular serine and serine whose synthesis is regulated by PGAM1 induce methylation of genes Tregs associated, downregulating their expression and overall impacting Tregs differentiation and suppressive functions.

Strengths:

The strength of this paper is the number of approaches taken by the authors to verify their hypothesis. Indeed, by using both pharmacological and genetic tools in in vitro and in vivo systems they identified a potential new metabolic regulation of Tregs differentiation and function.

Weaknesses:

Using publicly available transcriptomic and proteomic data of human T cells, the authors claim that both ex vivo and in vitro polarized Tregs express higher levels of PGAM1 protein compared to CD4 T cells (naïve or cultured under Th0 polarizing conditions). The experiments shown in this paper have all been carried out in murine Tregs. Publicly available resources for murine data (ImmGen -RNAseq and ImmPRes - Proteomics) however show that Tregs do not express higher PGAM1 (mRNA and protein) compared to CD4 T cells. It would be good to verify this in the system/condition used in the paper.

It would also be good to assess the levels of both PGAM1 mRNA and protein in Tregs PGAM1 knockdown compared to scramble using different methods e.g. qPCR and western blot. However, due to the high levels of cell death and differentiation variability, that would require cells to be sorted.

It is not specified anywhere in the paper whether cells were sorted for bulk experiments. Based on the variability of cell differentiation, it would be good if this was mentioned in the paper as it could help to interpret the data with a different perspective.

Reviewer #1 (Public review):

This paper measures the positioning and diffusivity of RNaseE-mEos3.2 proteins in E. coli as a function of rifampicin treatment, compares RNaseE to other E. coli proteins, and measures the effect of changes in domain composition on this localization and motion. The straightforward study is thoroughly presented, including very good descriptions of the imaging parameters and the image analysis/modeling involved, which is good because the key impact of the work lies in presenting this clear methodology for determining the position and mobility of a series of proteins in living bacteria cells.

My key notes and concerns are listed below; the most important concerns are indicated with asterisks.

(1) The very start of the abstract mentions that the domain composition of RNase E varies among species, which leads the reader to believe that the modifications made to E. coli RNase E would be to swap in the domains from other species, but the experiment is actually to swap in domains from other E. coli proteins. The impact of this work would be increased by examining, for instance, RNase E domains from B. subtilis and C. crescentus as mentioned in the introduction.

(2) Furthermore, the introduction ends by suggesting that this work will modulate the localization, diffusion, and activity of RNase E for "various applications", but no applications are discussed in the discussion or conclusion. The impact of this work would be increased by actually indicating potential reasons why one would want to modulate the activity of RNase E.

(3) Lines 114 - 115: "The xNorm histogram of RNase E shows two peaks corresponding to each side edge of the membrane": "side edge" is not a helpful term. I suggest instead: "...corresponding to the membrane at each side of the cell"

(4) ***A key concern of this reviewer is that, since membrane-bound proteins diffuse more slowly than cytoplasmic proteins, some significant undercounting of the % of cytoplasmic proteins is expected due to decreased detectability of the faster-moving proteins. This would not be a problem for the LacZ imaging where essentially all proteins are cytoplasmic, but would significantly affect the reported MB% for the intermediate protein constructs. How is this undercounting considered and taken into account? One could, for instance, compare LacZ vs. LacY (or RNase E) copy numbers detected in fixed cells to those detected in living cells to estimate it.

(5) ***The rifampicin treatment study is not presented well. Firstly, it is found that LacY diffuses more rapidly upon rifampicin treatment. This change is attributed to changes in crowding at the membrane due to mRNA. Several other things change in cells after adding rif, including ATP levels, and these factors should be considered. More importantly, since the change in the diffusivity of RNaseE is similar to the change in diffusivity of LacY, then it seems that most of the change in RNaseE diffusion is NOT due to RNaseE-mRNA-ribosome binding, but rather due to whatever crowding/viscosity effects are experienced by LacY (along these lines: the error reported for D is SEM, but really should be a confidence interval, as in Figure 1, to give the reader a better sense of how different (or similar) 1.47 and 1.25 are).

(6) Lines 185-189: it is surprising to me that the CTD mutants both have the same change in D (5.5x and 5.3x) relative to their full-length counterparts since D for the membrane-bound WT protein should be much less sensitive to protein size than D for the cytoplasmic MTS mutant. Can the authors comment?

(7) Lines 190-194. Again, the confidence intervals and experimental uncertainties should be considered before drawing biological conclusions. It would seem that there is "no significant change" in the rhlB and pnp mutants, and I would avoid saying "especially for ∆pnp" when the same conclusion is true for both (one shouldn't say 1.04 is "very minute" and 1.08 is just kind of small - they are pretty much the same within experiments like this).

(8) ***Lines 221-223 " This is remarkable because their molecular masses (and thus size) are expected to be larger than that of MTS" should be reconsidered: diffusion in a membrane does not follow the Einstein law (indeed lines 223-225 agree with me and disagree with lines 221-223). (Also the discussion paragraph starting at line 375). Rather, it is generally limited by the interactions with the transmembrane segments with the membrane. So Figure 3D does not contain the right data for a comparison, and what is surprising to me is that MTS doesn't diffuse considerably faster than LacY2.

(9) ***The logical connection between the membrane-association discussion (which seems to ignore associations with other proteins in the cell) and the preceding +/- rifampicin discussion (which seeks to attribute very small changes to mRNA association) is confusing.

(10) Separately, the manuscript should be read through again for grammar and usage. For instance, the title should be: "Single-molecule imaging reveals the *roles* of *the* membrane-binding motif and *the* C-terminal domain of RNase E in its localization and diffusion in Escherichia coli". Also, some writing is unwieldy, for instance, "RNase E's D" would be easier to read if written as D_{RNaseE}. (underscore = subscript), and there is a lot of repetition in the sentence structures.

Disease for patient 1: Von Willebrand Disease Type1, transmitter VWD-type 3

Disease for patient 2: Von Willebrand Disease Type 3

Patient1: 90 YO female (Afro-Caribbean)

Patient2: 40 YO female (Afro-Caribbean)

Notes multiple variants in the VWF gene but have focused on variants in the D4 domain. However cannot discount the impact of some other variants.

Variant 1: VWF NM_000552.5: c.6647del p.(Cys2216Phefs*9), results in VWF protein missing D4 domain and C-terminal end of molecule

Phenotype patient 1: Reduced VWF levels in VWF:Ag, VWF:ristocetin cofactor, FVIII:C, FVIII(VWF:FVIIIB). Bleeding score 0, required Helixate treatment before and after receiving surgery.

Variant 2: VWF NM_000552.5: c.6432dup p.(Pro2145Thrfs*5)

Three sequence variations in family study showed other variants highlighted p.(Cys1149Arg) and p.(Pro2145Thrfs*5) are not on the same allele.

Does have other variants in VWF but they are stated by authors to not be detrimental. p.(Val510=) is noted to be potentially deleterious.

Phenotype patient 2: severely reduced VWF levels, absence of multimers, bleeding score 32, epistaxis, bruising, oral cavity bleeding, prolonged bleeding from minor wounds, menorrhagia, hemarthrosis, ankle arthropathy.

Suggests premature termination codons in these variants may lead to NMD but that this mechanism was found to be PTC position-dependent. Degradation was not 100% and need to perform cellular experiments.

https://www.nytimes.com/2025/01/28/business/energy-environment/chevron-power-plant-ai.html

Reviewer #1 (Public review):

Summary:

This study uses single nucleus multi-omics to profile the transcriptome and chromatin accessibility of mouse XX and XY primordial germ cells (PGCs) at three time points spanning PGC sexual differentiation and entry of XX PGCs into meiosis (embryonic days 11.5-13.5). They find that PGCs can be clustered into sub-populations at each time point, with higher heterogeneity among XX PGCs and more switch-like developmental transitions evident in XY PGCs. In addition, they identify several transcription factors that appear to regulate sex-specific pathways as well as cell-cell communication pathways that may be involved in regulating XX vs XY PGC fate transitions. The findings are important and overall rigorous. The study could be further improved by better connection to the biological system, including putting the transcriptional heterogeneity of XX PGCs in the context of findings that meiotic entry is spatially asynchronous in the fetal ovary and further addressing the role of retinoic acid signaling. Overall, this study represents and advance in germ cell regulatory biology and will be a highly used resource in the field of germ cell development.

Strengths:

(1) The multi-omics data is mostly rigorously collected and carefully interpreted.

(2) The dataset is extremely valuable and helps to answer many long-standing questions in the field.

(3) In general, the conclusions are well anchored in the biology of the germ line in mammals.

Comments on revised version:

Most of my concerns have been addressed in the revised manuscript. I have one remaining concern but I believe this is important in order for the paper to be fully appreciated:

In Figures 2a, 2e, 3a, and 3e, the visualization scheme is very difficult to follow, and has not been updated or improved in the revised manuscript. It's very hard to see the colors corresponding to average expression for many genes because the circles are so small. The yellow color is hard to see and makes it hard to estimate the size of the circle. This issue is particularly egregious in Figure 2a for the data relating to ZKSCAN5, which is specifically highlighted in the text in lines 421-426. This data must be shown in a more convincing way in order to make the claims. An update to the visualization, including color scheme, is very strongly recommended; it is not difficult and would substantially improve the ability of these panels to communicate their message.

Reviewer #1 (Public review):

Summary:

Authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.

Strengths and weaknesses:

(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); this is acknowledged by the authors that no full mechanistic explanation can be given at this moment.

(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? A RNA seq analysis is required to show the effect on cellular genes.

A RNA seq analysis was done in the revised manuscript comparing the effect of HSV-1 and deleted vector in J-LAT cells (Fig S5). More than 2000 genes are upregulated after transduction with the modified vector in comparison with the WT vector. Hence, the specificity of upregulation of SIV genes is questioned. Authors do NOT comment on these findings. In my view it questions the utility of this approach.

(3) The primate groups are too small and the results to variable to make averages. In Fig 5, the group with ART and saline has two slow rebounders. It is not correct to average those with the single quick rebounder. Here the interpretation is NOT supported by the data.

Although authors provided some promising SIV DNA data, no additional animals were added. Groups of 3 animals are too small to make any conclusion, especially since the huge variability in response. The average numbers out of 3 are still presented in the paper, which is not proper science.

No data are given of the effect of the deletion in primates. Now the deleted construct is compared with an empty vector containing no SIV genes. Authors provide new data in Fig S2 on the comparison of WT and modified vector in cells from PLWH, but data are not that convincing. A significant difference in reactivation is seen for LTR in only 2/4 donors and in Gag in 3/4 donors. (Additional question what is meaning of LTR mRNA, do authors relate to genomic RNA??)

Discussion

HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.

The RNA seq data add on to this worry and should at least be discussed.

Comments on revisions:

The authors accept the limitations of the primate study (too small for strong conclusions). The new way of presenting the data clearly shows these limitations.

Reviewer #1 (Public review):

Summary:

By using the biophysical chromosome stretching, the authors measured the stiffness of chromosomes of mouse oocytes in meiosis I (MI) and meiosis II (MII). This study was the follow-up of previous studies in spermatocytes (and oocytes) by the authors (Biggs et al. Commun. Biol. 2020: Hornick et al. J. Assist. Rep. and Genet. 2015). They showed that MI chromosomes are much stiffer (~10 fold) than mitotic chromosomes of mouse embryonic fibroblast (MEF) cells. MII chromosomes are also stiffer than the mitotic chromosomes. The authors also found that oocyte aging increases the stiffness of the chromosomes. Surprisingly, the stiffness of meiotic chromosomes is independent of meiotic chromosome components, Rec8, Stag3, and Rad21L. and aging increases the stiffness.

Strengths

This provides a new insight into the biophysical property of meiotic chromosomes, that is chromosome stiffness. The stiffness of chromosomes in meiosis prophase I is ~10-fold higher than that of mitotic chromosomes, which is independent of meiotic cohesin. The increased stiffness during oocyte aging is a novel finding.

Weaknesses:

A major weakness of this paper is that it does not provide any molecular mechanism underlying the difference between MI and MII chromosomes (and/or prophase I and mitotic chromosomes).

Comments on revisions:

The main text lacks the first page with the authors' names and their affiliations (and corresponding authors etc).

Reviewer #2 (Public review):

Summary:

The authors used a yeast model for analyzing Parkinson's disease-associated synphilin-1 inclusion bodies (SY1 IBs). In this model system, large SY1 IBs are efficiently formed from smaller potentially more toxic SY1 aggregates. Using a genome-wide approach (synthetic genetic array, SGA, combined with a high content imaging approach), the authors identified the sphingolipid metabolic pathway as pivotal for SY1 IBs formation. Disturbances of this pathway increased SY1-triggered growth deficits, loss of mitochondrial membrane potential, increased production of reactive oxygen species (ROS), and decreased cellular ATP levels pointing to an increased energy crisis within affected cells. Notably, SY1 IBs were found to be surrounded by mitochondrial membranes using state-of-the-art super-resolution microscopy. Finally, the effects observed in the yeast for SY1 IBs turned out to be evolutionary conserved in mammalian cells. Thus, sphingolipid metabolism might play an important role in the detoxification of misfolded proteins by large IBs formation at the mitochondrial outer membrane.

Strengths:

• The SY1 IB yeast model is very suitable for the analysis of genes involved in IB formation.<br /> • The genome-wide approach combining a synthetic genetic array (SGA) with a high content imaging approach is a compelling approach and enabled the reliable identification of novel genes. The authors tightly checked the output of the screen.<br /> • The authors clearly showed, including a couple of control experiments, that the sphingolipid metabolic pathway is crucial for SY1 IB formation and cytotoxicity.<br /> • The localization of SY1 IBs at mitochondrial membranes has been clearly demonstrated with state-of-the-art super-resolution microscopy and biochemical methods.<br /> • Pharmacological manipulation of the sphingolipid pathway influenced mitochondrial function and cell survival.<br /> • The authors have carefully redone critical experiments to avoid any misleading interpretation of data.

Weaknesses:

• It remains unclear how sphingolipids are involved in SY1 IB formation.

Comments on revisions: No further comments

Reviewer #1 (Public review):

Summary:

This paper introduces a new approach for modeling human behavioral responses using image-computable models. They create a model (VAM) that is a combination of a standard CNN coupled with a standard evidence accumulation model (EAM). The combined model is then trained directly on image-level data using human behavioral responses. This approach is original and can have wide applicability. However, many of the specific findings reported are less compelling.

Strengths:

(1) The manuscript presents an original approach of fitting an image-computable model to human behavioral data. This type of approach is sorely needed in the field.<br /> (2) The analyses are very technically sophisticated.<br /> (3) The behavioral data are large both in terms of sample size (N=75) and in terms of trials per subject.

Weaknesses:

(1) The main advance here thus appears to be methodological rather than conceptual. It's really cool that VAMs are image computable and are also fit to human data. But what we learn about the mind or brain is perhaps more modest.<br /> (2) In the approach here, a given stimulus is always processed in the same way through the core CNN to produce activations v_k. These v_k's are then corrupted by Gaussian noise to produce drift rates d_k, which can differ from trial to trial even for the same stimulus. In other words, the assumption built into VAM appears to be that the drift rate variability stems entirely from post-sensory (decisional) noise. In contrast, the typical interpretation of EAMs is that the variability in drift rates is sensory. In response to this concern, the authors responded that one can imagine an additional (unmodeled) sensory process that adds variability to the drift rates. However, this process remains unmodeled. The authors motivate their paper by saying "EAMs do not explain how the visual system extracts these representations in the first place" (second sentence of the Abstract). VAM is definitely a step in this direction but there's still a gap between the current VAM implementation and sensory systems.

Reviewer #1 (Public review):

Summary:

The authors aimed to modify the characteristics of the extracellular matrix (ECM) produced by immortalized mesenchymal stem cells (MSCs) by employing the CRISPR/Cas9 system to knock out specific genes. Initially, they established VEGF-KO cell lines, demonstrating that these cells retained chondrogenic and angiogenic properties. Additionally, lyophilized carriage tissues produced by these cells exhibited retained osteogenic properties.

Subsequently, the authors established RUNX2-KO cell lines, which exhibited reduced COLX expression during chondrogenic differentiation and notably diminished osteogenic properties in vitro. Transplantation of lyophilized carriage tissues produced by RUNX2-KO cell lines into osteochondral defects in rat knee joints resulted in the regeneration of articular cartilage tissues as well as bone tissues, a phenomenon not observed with tissues derived from parental cells. This suggests that gene-edited MSCs represent a valuable cell source for producing ECM with enhanced quality.

Strengths:

The enhanced cartilage regeneration observed with ECM derived from RUNX2-KO cells supports the authors' strategy of creating gene-edited MSCs capable of producing ECM with superior quality. Immortalized cell lines offer a limitless source of off-the-shelf material for tissue regeneration.

Weaknesses:

Most of the data align with anticipated outcomes, offering limited novelty to advance scientific understanding. Methodologically, the chondrogenic differentiation properties of immortalized MSCs appeared deficient, evidenced by Safranin-O staining of 3D tissues and histological findings lacking robust evidence for endochondral differentiation. This presents a critical limitation, particularly as authors propose the implantation of cartilage tissues for in vivo experiments. Instead, the bulk of data stemmed from type I collagen scaffold with factors produced by MSCs stimulated by TGFβ.

In the revised version, the authors presented Safranin-O staining results of pellets prior to lyophilization. The inset of figures showing entire pellets revealed that Safranin-O-positive areas were limited, suggesting that cells in the negative regions had not differentiated into chondrocytes. In Figure 3F, DAPI staining showed devitalized cells in the outer layer but was negative in the central part, indicating the absence of cells in these areas and incomplete differentiation induction.

The rationale for establishing VEGF-KO cell lines remains unclear, and the authors' explanation in the revised manuscript is still equivocal. While they mention that VEGF is a late marker for endochondral ossification, the data in Figures 1D and 1E clearly show that VEGF-KO affects the early phase of endochondral ossification.

Insufficient depth was given to elucidate the disparity in osteogenic properties between those observed in ectopic bone formation and those observed in transplantation into osteochondral defects.

In the ectopic bone formation study, most of the collagenous matrix observed at 2 weeks was resorbed by 6 weeks, with only a small amount contributing to bone formation in MSOD-B cells (Figs. 2I and 4C). This finding does not align with the micro-CT data presented in Figures 2H and 4B. For the micro-CT experiments, it would be more appropriate to use a standard window for bone and present the data accordingly.

While the regeneration of articular cartilage in RUNX2-KO ECM presents intriguing results, the study lacked an exploration into underlying mechanisms, such as histological analyses at earlier time points.

Reviewer #1 (Public review):

Summary:

In their previous publication (Dong et al. Cell Reports 2024), the authors showed that citalopram treatment resulted in reduced tumor size by binding to the E380 site of GLUT1 and inhibiting the glycolytic metabolism of HCC cells, instead of the classical citalopram receptor. Given that C5aR1 was also identified as the potential receptor of citalopram in the previous report, the authors focused on exploring the potential of the immune-dependent anti-tumor effect of citalopram via C5aR1. C5aR1 was found to be expressed on tumor-associated macrophages (TAMs) and citalopram administration showed potential to improve the stability of C5aR1 in vitro. Through macrophage depletion and adoptive transfer approaches in HCC mouse models, the data demonstrated the potential importance of C5aR1-expressing macrophage in the anti-tumor effect of citalopram in vivo. Mechanistically, their in vitro data suggested that citalopram may regulate the phagocytosis potential and polarization of macrophages through C5aR1. Next, they tried to investigate the direct link between citalopram and CD8+T cells by including an additional MASH-associated HCC mouse model. Their data suggest that citalopram may upregulate the glycolytic metabolism of CD8+T cells, probability via GLUT3 but not GLUT1-mediated glucose uptake. Lastly, as the systemic 5-HT level is down-regulated by citalopram, the authors analyzed the association between a low 5-HT and a superior CD8+T cell function against a tumor. Although the data is informative, the rationale for working on additional mechanisms and logical links among different parts is not clear. In addition, some of the conclusion is also not fully supported by the current data.

Strengths:

The idea of repurposing clinical-in-used drugs showed great potential for immediate clinical translation. The data here suggested that the anti-depression drug, citalopram displayed an immune regulatory role on TAM via a new target C5aR1 in HCC.

Weaknesses:

(1) The authors concluded that citalopram had a 'potential immune-dependent effect' based on the tumor weight difference between Rag-/- and C57 mice in Figure 1. However, tumor weight differences may also be attributed to a non-immune regulatory pathway. In addition, how do the authors calculate relative tumor weight? What is the rationale for using relative one but not absolute tumor weight to reflect the anti-tumor effect?

(2) The authors used shSlc6a4 tumor cell lines to demonstrate that citalopram's effects are independent of the conventional SERT receptor (Figure 1C-F). However, this does not entirely exclude the possibility that SERT may still play a role in this context, as it can be expressed in other cells within the tumor microenvironment. What is the expression profiling of Slc6a4 in the HCC tumor microenvironment? In addition, in Figure 1F, the tumor growth of shSlc6a4 in C57 mice displayed a decreased trend, suggesting a possible role of Slc6a4.

(3) Why did the authors choose to study phagocytosis in Figures 3G-H? As an important player, TAM regulates tumor growth via various mechanisms.

(4) The information on unchanged deposition of C5a has been mentioned in this manuscript (Figures 3D and 3F), the authors should explain further in the manuscript, for example, C5a could bind to receptors other than C5aR1 and/or C5a bind to C5aR1 by different docking anchors compared with citalopram.

(5) Figure 3I-M - the flow cytometry data suggested that citalopram treatment altered the proportions of total TAM, M1 and M2 subsets, CD4+ and CD8+T cells, DCs, and B cells. Why does the author conclude that the enhanced phagocytosis of TAM was one of the major mechanisms of citalopram? As the overall TAM number was regulated, the contribution of phagocytosis to tumor growth may be limited.

(6) Figure 4 - what is the rationale for using the MASH-associated HCC mouse model to study metabolic regulation in CD8+T cells? The tumor microenvironment and tumor growth would be quite different. In addition, how does this part link up with the mechanisms related to C5aR1 and TAM? The authors also brought GLUT1 back in the last part and focused on CD8+T cell metabolism, which was totally separated from previous data.

(7) Figure 5, the authors illustrated their mechanism that citalopram regulates CD8+T cell anti-tumor immunity through proinflammatory TAM with no experimental evidence. Using only CD206 and MHCII to represent TAM subsets obviously is not sufficient.

Reviewer #1 (Public review):

Summary:

In a previous work, Prut and colleagues had shown that during reaching, high-frequency stimulation of the cerebellar outputs resulted in reduced reach velocity. Moreover, they showed that the stimulation produced reaches that deviated from a straight line, with the shoulder and elbow movements becoming less coordinated. In this report, they extend their previous work by the addition of modeling results that investigate the relationship between the kinematic changes and torques produced at the joints. The results show that the slowing is not due to reductions in interaction torques alone, as the reductions in velocity occur even for movements that are single joints. More interestingly, the experiment revealed evidence for the decomposition of the reaching movement, as well as an increase in the variance of the trajectory.

Strengths:

This is a rare experiment in a non-human primate that assessed the importance of cerebellar input to the motor cortex during reaching.

Weaknesses:

My major concerns are described below.

If I understand the task design correctly, the monkeys did not need to stop their hand at the target. I think this design may be suboptimal for investigating the role of the cerebellum in control of reaching because a number of earlier works have found that the cerebellum's contributions are particularly significant as the movement ends, i.e., stopping at the target. For example, in mice, interposed nucleus neurons tend to be most active near the end of the reach that requires extension, and their activation produces flexion forces during the reach (Becker and Person 2019). Indeed, the inactivation of interposed neurons that project to the thalamus results in overshooting of reaching movements (Low et al. 2018). Recent work has also found that many Purkinje cells show a burst-pause pattern as the reach nears its endpoint, and stimulation of the mossy fibers tends to disrupt endpoint control (Calame et al. 2023). Thus, the fact that the current paper has no data regarding endpoint control of the reach is puzzling to me.

Because stimulation continued after the cursor had crossed the target, it is interesting to ask whether this disruption had any effects on the movements that were task-irrelevant. The reason for asking this is because we have found that whereas during task-relevant eye or tongue movements the Purkinje cells are strongly modulated, the modulations are much more muted when similar movements are performed but are task-irrelevant (Pi et al., PNAS 2024; Hage et al. Biorxiv 2024). Thus, it is interesting to ask whether the effects of stimulation were global and affected all movements, or were the effects primarily concerned with the task-relevant movements.

If the schematic in Figure 1 is accurate, it is difficult for me to see how any of the reaching movements can be termed single joint. In the paper, T1 is labeled as a single joint, and T2-T4 are labeled as dual-joint. The authors should provide data to justify this.

Because at least part of this work was previously analyzed and published, information should be provided regarding which data are new.

Reviewer #1 (Public review):

Summary:

Measurement of BOLD MR imaging has regularly found regions of the brain that show reliable suppression of BOLD responses during specific experimental testing conditions. These observations are to some degree unexplained, in comparison with more usual association between activation of the BOLD response and excitatory activation of the neurons (most tightly linked to synaptic activity) in the same brain location. This paper finds two patients whose brains were tested with both non-invasive functional MRI and with invasive insertion of electrodes, which allowed the direct recording of neuronal activity. The electrode insertions were made within the fusiform gyrus, which is known to process information about faces, in a clinical search for the sites of intractable epilepsy in each patient. The simple observation is that the electrode location in one patient showed activation of the BOLD response and activation of neuronal firing in response to face stimuli. This is the classical association. The other patient showed an informative and different pattern of responses. In this person, the electrode location showed a suppression of the BOLD response to face stimuli and, most interestingly, an associated suppression of neuronal activity at the electrode site.

Strengths:

Whilst these results are not by themselves definitive, they add an important piece of evidence to a long-standing discussion about the origins of the BOLD response. The observation of decreased neuronal activation associated with negative BOLD is interesting because, at various times, exactly the opposite association has been predicted. It has been previously argued that if synaptic mechanisms of neuronal inhibition are responsible for the suppression of neuronal firing, then it would be reasonable

Weaknesses:

The chief weakness of the paper is that the results may be unique in a slightly awkward way. The observation of positive BOLD and neuronal activation is made at one brain site in one patient, while the complementary observation of negative BOLD and neuronal suppression actually derives from the other patient. Showing both effects in both patients would make a much stronger paper.

Reviewer #1 (Public review):

Summary:

This study demonstrates that strip cropping enhances the taxonomic diversity of ground beetles across organically-managed crop systems in the Netherlands. In particular, strip cropping supported 15% more ground beetle species and 30% more individuals compared to monocultures.

Strengths:

A well-written study with well-analyzed data of a complex design. The data could have been analyzed differently e.g. by not pooling samples, but there are pros and cons for each type of analysis and I am convinced this will not affect the main findings. A strong point is that data were collected for 4 years. This is especially strong as most data on biodiversity in cropping systems are only collected for one or two seasons. Another strong point is that several crops were included.

Weaknesses:

This study focused on the biodiversity of ground beetles and did not examine crop productivity. Therefore, I disagree with the claim that this study demonstrates biodiversity enhancement without compromising yield. The authors should present results on yield or, at the very least, provide a stronger justification for this statement.

Reviewer #1 (Public review):

Summary:

Goal: Find downstream targets of cmk-1 phosphorylation, identify one that also seems to act in thermosensory habituation, test for genetic interactions between cmk-1 and this gene, and assess where these genes are acting in the thermosensory circuit during thermosensory habituation.

Methods: Two in vitro analyses of cmk-1 phosphorylation of C. elegans proteins. Thermosensory habituation of cmk-1 and tax-6 mutants and double mutants was assessed by measuring the rate of heat-evoked reversals (reversal probability) of C. elegans before and after 20s ISI repeated heat pulses over 60 minutes.

Conclusions: cmk-1 and tax-6 act in separate habituation processes, primarily in AFD, that interact complexly, but both serve to habituate the thermosensory reversal response. They found that cmk-1 primarily acts in AFD and tax-6 primarily acts in RIM (and FLP for naïve responses). They also identified hundreds of potential cmk-1 phosphorylation substrates in vitro.

Strengths:

The effect size in the genetic data is quite strong and a large number of genetic interaction experiments between cmk-1 and tax-1 demonstrate a complex interaction.

Weaknesses:

The major concern about this manuscript is the assumption that the process they are observing is habituation. The two previously cited papers using this (or a very similar) protocol, Lia and Glauser 2020 and Jordan and Glauser 2023, both use the word 'adaptation' to describe the observed behavioral decrement. Jordan and Glauser 2023 use the words 'habituation' or 'habituation-like' 10 times, however, they use 'adaptation' over 100 times. It is critical to distinguish habituation from sensory adaptation (or fatigue) in this thermal reversal protocol. These processes are often confused/conflated, however, they are very different; sensory adaptation is a process that decreases how much the nervous system is activated by a repeated stimulus, therefore it can even occur outside of the nervous system. Habituation is a learning process where the nervous system responds less to a repeated stimulus, despite (at least part of the nervous system) the nervous system still being similarly activated by the stimulus. Habituation is considered an attentional process, while adaptation is due to the fatigue of sensory transduction machinery. Control experiments such as tests for dishabituation (where the application of a different stimulus causes recovery of the decremented response) or rate of spontaneous recovery (more rapid recovery after short inter-stimulus intervals) are required to determine if habituation or sensory adaptation are occurring. These experiments will allow the results to be interpreted with clarity, without them, it isn't actually clear what biological process is actually being studied.

While the discrepancy between the in vitro phosphorylation experiments and the in silico predictions was discussed, the substantial discrepancy (over 85% of the substrates in the smaller in vitro dataset were not identified in the larger dataset) between the two different in vitro datasets was not discussed. This is surprising, as these approaches were quite similar, and it may indicate a measure of unreliability in the in vitro datasets (or high false negative rates). Additionally, the rationale for, and distinction between, the two separate in vitro experiments is not made clear.

Line 207: After reporting that both tax-6 and cnb-1 mutants have high spontaneous reversals, it is not made clear why cnb-1 is not further explored in the paper. Additionally, this spontaneous reversal data should be in a supplementary figure.

Figure 3 -S1: This model doesn't explain why the cmk-1(gf) group and the cmk-1(gf) +cyclo A group cause enhanced response decrement (presumably by reducing the inhibition by tax-6) but the +cyclo A group (inhibited tax-6) showed weaker response decrement, as here there is even further weakened inhibition of tax-6 on this process. Also, the cmk-1(lf) +cyclo A group is labeled as constitutive habituation, however, this doesn't appear to be the case in Figure 3 (seems like a similar initial level and response decrement phenotype to wildtype).

More discussion of the significance of the sites of cmk-1 and tax-6 function in the neural circuit should take place. Additionally, incorporating the suspected loci of cmk-1 and tax-6 in the neural circuit into the model would be interesting (using proper hypothetical language). For example, as it seems like AFD is not required for the naïve reversal response but just its reduction, cmk-1 activity in AFD might be generating inhibition of the reversal response by AFD. It certainly would be understandable if this isn't workable, given extrasynaptic signaling and other unknowns, but it potentially could also be helpful in generating a working model for these complex interactions. For example, cmk-1 induces AIZ inhibition of AVA (AIZ is electrically coupled to AFD), and tax-6 reduces RIM activation of AVA (these neurons are also electrically coupled according to the diagram). RIM is also a neuropeptide-rich neuron, so this could allow it to interact with the cmk-1-related process(es) in AFD. Some discussion of possibilities like this could be informative.

Provide an explanation for why some of the experiments in Figure 4 have such a high N, compared to other experiments.

Because the loss of function and gain of function mutations in cmk-1 have a similar effect, it is likely that this thermosensory plasticity phenotype is sensitive to levels of cmk-1 activity. Therefore, it is not surprising that the cmk-1 promoter failed to rescue very well as these plasmid-driven rescues often result in overexpression. Given this and that the cmk-1p rescue itself was so modest, these rescue experiments are not entirely convincing (and very hard to interpret; for example, is the AFD rescue or the ASER rescue more complete? The ASER one is actually closer to the cmk-1p rescue). Given the sensitivity to cmk-1 activity levels, a degradation strategy would be more likely to deliver clear results (or perhaps even the overactivation approach used for tax-6).

Reviewer #1 (Public review):

Summary:

This study presents convincing findings that oligodendrocytes play a regulatory role in spontaneous neural activity synchronisation during early postnatal development, with implications for adult brain function. Utilising targeted genetic approaches, the authors demonstrate how oligodendrocyte depletion impacts Purkinje cell activity and behaviours dependent on cerebellar function. Delayed myelination during critical developmental windows is linked to persistent alterations in neural circuit function, underscoring the lasting impact of oligodendrocyte activity.

Strengths:

(1) The research leverages the anatomically distinct olivocerebellar circuit, a well-characterized system with known developmental timelines and inputs, strengthening the link between oligodendrocyte function and neural synchronization.

(2) Functional assessments, supported by behavioral tests, validate the findings of in vivo calcium imaging, enhancing the study's credibility.

(3) Extending the study to assess the long-term effects of early-life myelination disruptions adds depth to the implications for both circuit function and behavior.

Weaknesses:

(1) The study would benefit from a closer analysis of myelination during the periods when synchrony is recorded. Direct correlations between myelination and synchronized activity would substantiate the mechanistic link and clarify if observed behavioral deficits stem from altered myelination timing.

(2) Although the study focuses on Purkinje cells in the cerebellum, neural synchrony typically involves cross-regional interactions. Expanding the discussion on how localized Purkinje synchrony affects broader behaviors - such as anxiety, motor function, and sociality - would enhance the findings' functional significance.

(3) The authors discuss the possibility of oligodendrocyte-mediated synapse elimination as a possible mechanism behind their findings, drawing from relevant recent literature on oligodendrocyte precursor cells. However, there are no data presented supporting this assumption. The authors should explain why they think the mechanism behind their observation extends beyond the contribution of myelination or remove this point from the discussion entirely.

(4) It would be valuable to investigate the secondary effects of oligodendrocyte depletion on other glial cells, particularly astrocytes or microglia, which could influence long-term behavioral outcomes. Identifying whether the lasting effects stem from developmental oligodendrocyte function alone or also involve myelination could deepen the study's insights.

(5) The authors should explore the use of different methods to disturb myelin production for a longer time, in order to further determine if the observed effects are transient or if they could have longer-lasting effects.

(6) Throughout the paper, there are concerns about statistical analyses, particularly on the use of the Mann-Whitney test or using fields of view as biological replicates.

Reviewer #1 (Public review):

Summary:

The paper by Lee and Ouellette explores the role of cyclic-d-AMP in chlamydial developmental progression. The manuscript uses a collection of different recombinant plasmids to up- and down-regulate cdAMP production, and then uses classical molecular and microbiological approaches to examine the effects of expression induction in each of the transformed strains.

Strengths:

This laboratory is a leader in the use of molecular genetic manipulation in Chlamydia trachomatis and their efforts to make such efforts mainstream is commendable. Overall, the model described and defended by these investigators is thorough and significant.

Weaknesses:

The biggest weakness in the document is their reliance on quantitative data that is statistically not significant, in the interpretation of results. These challenges can be addressed in a revision by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors Eapen et al. investigated the peptide inhibitors of Cdc20. They applied a rational design approach, substituting residues found in the D-box consensus sequences to better align the peptides with the Cdc20-degron interface. In the process, the authors designed and tested a series of more potent binders, including ones that contain unnatural amino acids, and verified binding modes by elucidating the Cdc-20-peptide structures. The authors further showed that these peptides can engage with Cdc20 in the cellular context, and can inhibit APC/CCdc20 ubiquitination activity. Finally, the authors demonstrated that these peptides could be used as portable degron motifs that drive the degradation of a fused fluorescent protein.

Strengths:

This manuscript is clear and straightforward to follow. The investigation of different peptide variations was comprehensive and well-executed. This work provided the groundwork for the development of peptide drug modalities to inhibit degradation or apply peptides as portable motifs to achieve targeted degradation. Both of which are impactful.

Weaknesses:

A few minor comments:

(1) In my opinion, more attention to the solubility issue needs to be discussed and/or tested. On page 10, what is the solubility of D2 before a modification was made? The authors mentioned that position 2 is likely solvent exposed, it is not immediately clear to me why the mutation made was from one hydrophobic residue to another. What was the level of improvement in solubility? Are there any affinity data associated with the peptide that differ with D2 only at position 2?

(2) I'm not entirely convinced that the D19 density not observed in the crystal structure was due to crystal packing. This peptide is peculiar as it also did not induce any thermal stabilization of Cdc20 in the cellular thermal shift assay. Perhaps the binding of this peptide could be investigated in more detail (i.e., NMR?) Or at least more explanation could be provided.

Reviewer #1 (Public review):

This study investigates alterations in the autophagic-lysosomal pathway in the Q175 HD knock-in model crossed with the TRGL autophagy reporter mouse. The findings provide valuable insights into autophagy dynamics in HD and the potential therapeutic benefits of modulating this pathway. The study suggests that autophagy stimulation may offer therapeutic benefits in the early stages of HD progression, with mTOR inhibition showing promise in ameliorating lysosomal pathology and reducing mutant huntingtin accumulation.

However, the data raises concerns regarding the strength of the evidence. The observed changes in autophagic markers, such as autolysosome and lysosome numbers, are relatively modest, and the Western blot results do not fully match the quantitative results. These discrepancies highlight the need for further validation and more pronounced effects to strengthen the conclusions. While the study suggests the potential of autophagy regulation as a long-term therapeutic strategy, additional experiments and more reliable data are necessary to confirm the broader applicability of the TRGL/Q175 mouse model.

Furthermore, the 2004 publication by Ravikumar et al. demonstrated that inhibition of mTOR by rapamycin or the rapamycin ester CCI-779 induces autophagy and reduces the toxicity of polyglutamine expansions in fly and mouse models of Huntington's disease. mTOR is a key regulator of autophagy, and its inhibition has been explored as a therapeutic strategy for various neurodegenerative diseases, including HD. Studies suggest that inhibiting mTOR enhances autophagy, leading to the clearance of mHTT aggregates. Given that dysfunction of the autophagic-lysosomal pathway and lysosomal function in HD is already well-established, and that mTOR inhibition as a therapeutic approach for HD is also known, this study does not present entirely novel findings.

Major Concerns:

(1) In Figure 3A1 and A2, delayed and/or deficient acidification of AL causes deficits in the reformation of LY to replenish the LY pool. However, in Figure S2D, there is no difference in AL formation or substrate degradation, as shown by the Western blotting results for CTSD and CTSB. How can these discrepancies be explained?

(2) The results demonstrate that in the brain sections of 17-month-old TRGL/Q175 mice, there was an increase in the number of acidic autolysosomes (AL), including poorly acidified autolysosomes (pa-AL), alongside a decrease in lysosome (LY) numbers. These AL/pa-AL changes were not significant in 2-month-old or 7-month-old TRGL/Q175 mice, where only a reduction in lysosome numbers was observed. This indicates that these changes, representing damage to the autophagy-lysosome pathway (ALP), manifest only at later stages of the disease. Considering that the ALP is affected predominantly in the advanced stages of the disease (e.g., at 17 months), why were 6-month-old TRGL/Q175 mice selected for oral mTORi INK treatment, and why was the treatment duration restricted to just 3 weeks?

(3) Is the extent of motor dysfunction in TRGL/Q175 mice comparable to that in Q175 mice? Does the administration of mTORi INK improve these symptoms?

(4) Why is eGFP expression not visible in Fig. 6A in TRGL-Veh mice? Additionally, why do normal (non-poly-Q) mice have fewer lysosomes (LY) than TRGL/Q175-INK mice? IHC results also show that CTSD levels are lower in TRGL mice compared to TRGL/Q175-INK mice. Does this suggest lysosome dysfunction in TRGL-Veh mice?

(5) In Figure 5A, the phosphorylation of ATG14 (S29) shows minimal differences in Western blotting, which appears inconsistent with the quantitative results. A similar issue is observed in the quantification of Endo-LC3.

(6) In Figure S2A and Figure S2B, 17-month-old TRGL/Q175 mice show a decrease in p-p70S6K and the p-ULK1/ULK1 ratio, but no changes are observed in autophagy-related markers. Do these results indicate only a slight change in autophagy at this stage in TRGL/Q175 mice? Since the mTOR pathway regulates multiple cellular mechanisms, could mTOR also influence other processes? Is it possible that additional mechanisms are involved?

Reviewer #2 (Public review):

Summary:

In their manuscript, Quian and colleagues identified a novel mechanisms by which Pseudomonas control inflammatory responses upon inflammasome activation. They identified a caspase-11 substrates (VgrG2b) which, upon cleavage, binds and inhibit the NLRP3 to reduce the production of pro-inflammatory cytokines. This is a unique mechanism that allow for the tailoring of the innate immune response upon bacterial recognition.

Strengths:

The authors are presenting here a novel conceptual framework in host-pathogen interactions. Their work is supported by a range of approaches (biochemical, cellular immunology, microbiology, animal models) and their conclusions are supported by multiple independent evidences. The work is likely to have an important impact in the innate immunity field and host-pathogen interactions field and may guide the development of novel inhibitors.

Weaknesses:

Although quite exhaustive, a few of the authors conclusions are not fully supported (e.g, caspase-11 directly cleaving VgrG2b, the unique affinity of VgrG2b-C for NLRP3) and would require complementary approaches to validate their findings fully. This is minimal.

Comments on revisions:

I command the authors's effort to address my comments. They have addressed all my concerns.

Reviewer #1 (Public review):

Summary:

The authors aim to explore the effects of the electrogenic sodium-potassium pump (Na+/K+-ATPase) on the computational properties of highly active spiking neurons, using the weakly-electric fish electrocyte as a model system. Their work highlights how the pump's electrogenicity, while essential for maintaining ionic gradients, introduces challenges in neuronal firing stability and signal processing, especially in cells that fire at high rates. The study identifies compensatory mechanisms that cells might use to counteract these effects, and speculates on the role of voltage dependence in the pump's behavior, suggesting that Na+/K+-ATPase could be a factor in neuronal dysfunctions and diseases

Strengths:

(1) The study explores a less-examined aspect of neural dynamics-the effects of Na+/K+-ATPase electrogenicity. It offers a new perspective by highlighting the pump's role not only in ion homeostasis but also in its potential influence on neural computation.<br /> (2) The mathematical modeling used is a significant strength, providing a clear and controlled framework to explore the effects of the Na+/K+-ATPase on spiking cells. This approach allows for the systematic testing of different conditions and behaviors that might be difficult to observe directly in biological experiments.<br /> (3) The study proposes several interesting compensatory mechanisms, such as sodium leak channels and extracellular potassium buffering, which provide useful theoretical frameworks for understanding how neurons maintain firing rate control despite the pump's effects.

Weaknesses:

(1) While the modeling approach provides valuable insights, the lack of experimental data to validate the model's predictions weakens the overall conclusions.<br /> (2) The proposed compensatory mechanisms are discussed primarily in theoretical terms without providing quantitative estimates of their impact on the neuron's metabolic cost or other physiological parameters.

Reviewer #1 (Public review):

Summary:

The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on visual cortex. By correlating mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in he visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.<br /> While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

Strengths:

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

Weaknesses:

(1) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.<br /> (2) Ambiguity regarding the identity of the [AM+VM] MM neurons.

Reviewer #2 (Public review):

Summary:

The authors begin with the stated goal of gaining insight into the known repression of autophagy by Ezrin, a major membrane-actin linker that assembles signaling complexes on membranes. RNA and protein expression analysis is consistent with upregulation of lysosomal proteins in Ezrin-deficient MEFs, which the authors confirm by immunostaining and western blotting for lysosomal markers. Expression analysis also implicates EGF signaling as being altered downstream of Ezrin loss, and the authors demonstrate that Ezrin promotes relocalization of EGFR from the plasma membrane to endosomes. Ezrin loss reduces downstream MAPK and Akt signaling, and represses mTORC1 signaling by promoting lysosomal localization of the TSC complex. An Ezrin mutant Medaka fish line is then generated to test its role in retinal cells, which are known to be sensitive to changes in autophagy regulation. Phenotypes in this model appear generally consistent with observations made in cultured cells, though milder overall.

Strengths:

Data on the impact of Ezrin-loss on relocalization of EGFR from the plasma membrane are extensive, and thoroughly demonstrate that Ezrin is required for EGFR internalization in response to EGF.

A new Ezrin-deficient in vivo model (Medaka fish) is generated.

Strong data demonstrating that Ezrin loss suppresses Akt signaling and mTORC1 signaling by promoting TSC complex localization to the lysosome.

Weaknesses:

The authors have addressed all concerns

Reviewer #1 (Public review):

Summary:

This study examines the cortical modular functional organization of visual texture in comparison with that of color and disparity. While color, disparity, and orientation have been shown to exhibit clear functional organizations within the thin, thick, and thick/pale stripes of V2, whether the feature of texture is also organized within V2 is unknown. Using ultrahigh field 7T fMRI in humans viewing color-, disparity-, and texture-specific visual stimuli, the authors find that, unlike color and disparity, texture does not exhibit stripe-specific organization in V2. Moreover, using laminar imaging methods and calculations of informational connectivity, they find V2 color and disparity stripes exhibit the expected feedforward and feedback relationships with V1 & V4, and with V1 & V3ab, respectively. In contrast, texture activation, found predominantly in the deep layers of V2, is driven preferentially by feedback from V4. Based on these findings, the authors suggest that texture is a visual feature computed in higher-order areas and not generated by local intra-V2 computation.

Strengths:

This study poses an interesting and fundamental question regarding the relationship between functional modularity and hierarchical origin of computed properties. This question is thus highly significant and deserves study. The methodology is appropriate for the question and the areal and laminar resolution achieved across 10 subjects is commendable. The combination of high-resolution functional imaging and informational connectivity analysis introduces a useful way for examining feedforward and feedback relationships in mesoscale imaging data.

Comments on latest version:

The authors have responded adequately to my comments. The lack of texture organization in V2 is now strengthened by the apparently more clustered texture response in V4 (Fig. S9). The paired results in V2 and V4 make the study stronger. The authors may suggest that texture response, while present at the neural level, may not emerge as a primary organizational cue in V2, based on this texture stimulus paradigm. The negative results should still be presented cautiously. The connectivity inferences are interesting but should also be stated cautiously, as there are multiple assumptions. Overall, this study makes a contribution to emerging views about texture processing in the early visual pathways.

Reviewer #1 (Public review):

Summary:

This study investigated the role of PLECTIN, a cytoskeletal crosslinker protein, in liver cancer formation and progression. Using the liver-specific Plectin knockout mouse model, the authors convincingly showed that PLECTIN is critical for hepatocarcinogenesis, as functional inhibition of PLECTIN suppressed tumor formation in several models. They also provided evidence to show that inhibition of PLECTIN inhibited HCC cell invasion and reduced metastatic outgrowth in the lung. Mechanistically, they suggested that PLECTIN inhibition attenuated FAK, MAPK/ERK, and PI3K/AKT signaling.

Strengths:

The authors generated a liver-specific Plectin knockout mouse model. By using DEN and sgP53/MYC models, the authors convincingly demonstrated an oncogenic role of PLECTIN in HCC development. plecstatin-1 (PST), as a plectin inhibitor, showed promising efficacy in inhibiting HCC growth, which provides a basis for potentially treating HCC using PST.

The MIR images for tracking tumor growth in animal models were compelling. The high-quality confocal images and related qualifications convincingly showed the impact of plectin functional inhibition on contractility and adhesions in HCC cells.

Comments on latest version:

My concerns have been largely addressed. The authors did a good job in addressing the questions and clarifying the inconsistent results. I have two comments:

(1) The current data still cannot support the conclusion that plectin inactivation attenuates HCC oncogenic potential through FAK, Erk1/2, and PI3K/Akt axis, unless they can reactivate these signaling to restore the HCC congenic potential in plectin inactivated cells. It might be more appropriate to claim that plectin inactivation suppresses FAK, Erk1/2, and PI3K/Akt oncogenic signaling.

(2) I think it would be beneficial to include the H&E and HNF4α staining from lung tissue of mice inoculated with WT Huh7 cells indicated in the rebuttal letter.

Reviewer #2 (Public review):

Summary:

Tanaka et al. investigated the role of CCR4 in early atherosclerosis, focusing on the immune modulation elicited by this chemokine receptor under hypercholesterolemia. The study found that Ccr4 deficiency led to qualitative changes in atherosclerotic plaques, characterized by an increased inflammatory phenotype. The authors further analyzed the CD4 T cell immune response in para-aortic lymph nodes and atherosclerotic aorta, showing an increase mainly in Th1 cells and the Th1/Treg ratio in Ccr4-/-Apoe-/- mice compared to Apoe-/- mice. They then focused on Tregs, demonstrating that Ccr4 deficiency impaired their immunosuppressive function in in vitro assays. Authors also states that Ccr4-deficient Tregs had, as expected, impaired migration to the atherosclerotic aorta. Adoptive cell transfer of Ccr4-/- Tregs to Apoe-/- mice mimicked early atherosclerosis development in Ccr4-/-Apoe-/- mice. Therefore, this work shows that CCR4 plays an important role in early atherosclerosis but not in advanced stages.

Strengths:

Several in vivo and in vitro approaches were used to address the role of CCR4 in early atherosclerosis. Particularly, through the adoptive cell transfer of CCR4+ or CCR4- Tregs, the authors aimed to directly demonstrate the role of CCR4 in Tregs' protection against early atherosclerosis.

Weaknesses:

Flow cytometry experiments are not well controlled. Dead cells and doublets were not excluded from analysis.

Clinical relevance is unclear.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

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

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

Strengths:

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

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

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

Weaknesses:

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

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

Weaknesses:

Key weaknesses have been resolved: 

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

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

Reviewer #1 (Public review):

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

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

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

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

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

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

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

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

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

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

Reviewer comment on model in Vogt et al., 2024

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

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

Weaknesses:

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

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

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

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

Comments on latest version:

No further comments.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary

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

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

There were no major weaknesses identified.

Reviewer #2 (Public review):

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

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

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

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

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

Reviewer #2 (Public review):

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

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

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

Comments on revision:

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

Reviewer #1 (Public Review):

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

Joint Public Review:

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

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

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

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

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Reviewer #2 (Public review):

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

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

none noted in the revised version.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Main comments

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

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

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

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

Reviewer #1 (Public review):

Summary:

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

Comments on revised version:

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

Reviewer #2 (Public review):

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

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

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

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

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

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

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

Reviewer #1 (Public review):

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

Comments on revisions:

The authors have improved the manuscript and addressed my concerns.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Comments on revisions:

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

Reviewer #1 (Public review):

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

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

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

Reviewer #3 (Public review):

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

Strengths:

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

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Comments on revisions:

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

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Major Point-by-Point Comments:

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