Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #2 (Public review):

Summary:

In this study Amason et al employ spatial transcriptomics and intervention studies to probe the spatial and temporal dynamics of chemokines and their receptors, and their influence on cellular dynamics in C. violaceum granulomas. As a result of their spatial transcriptomic analysis, the authors narrow in on the contribution of neutrophil-and monocyte-recruiting pathways to host response. This results in the observation that monocyte recruitment is critical for granuloma formation and infection control, while neutrophil recruitment via CXCR2 may be dispensable.

Strengths:

Since C. violaceum is a self-limiting granulomatous infection, it makes an excellent case study for 'successful' granulomatous inflammation. This stands in contrast to chronic, unproductive granulomas that can occur during M. tuberculosis infection, sarcoidosis, and other granulomatous conditions, infectious or otherwise. Given the short duration of C. violaceum infection, this study specifically highlights the importance of innate immune responses in granulomas.

Another strength of this study is the temporal analysis. This proves to be important when considering the spatial distribution and timing of cellular recruitment. For example, the authors observe that the intensity and distribution of neutrophil and monocyte recruiting chemokines vary substantially across infection time and correlate well with their previous study of cellular dynamics in C. violaceum granulomas.

The intervention studies done in the last part of the paper bolster the relevance of the authors' focus on chemokines. The authors provide important negative data demonstrating the null effect of CXCR1/2 inhibition on neutrophil recruitment during C. violaceum infection. That said, the authors' difficulty with solubilizing reparixin in PBS is an important technical consideration given the negative result. On the other hand, monocyte recruitment via CCR2 proves to be indispensable for granuloma formation and infection control.

Weaknesses:

There are several shortcomings that limit the impact of this study. The first is that the cohort size is very limited. While the transcriptomic data is rich, the authors analyze just one tissue from one animal per timepoint. This assumes that the selected individual will have a representative lesion and prevents any analysis of inter-individual variability. Granulomas in other infectious diseases, such as schistosomiasis and tuberculosis, are very heterogeneous. The authors do assert that in C. violaceum infection granulomas are very consistent in their composition and kinetics, alleviating, in part, this concern.

Another caveat to these data is the limited or incompletely informative data analysis. This dataset has been previously published with more extensive and broad characterization. Here, the authors use Visium in a more targeted manner to interrogate certain chemokines and cytokines. While this is a great biological avenue, key findings rely on qualitative inspection of gene expression overlaid on to images or data that has been qualitatively binned or thresholded. Upon revision the authors did supplement their analyses with important information, such as the top expressed genes in each Visium cluster and the dynamic range of RNA counts retrieved across clusters.

Furthermore, the authors are underutilizing the spatial information provided by Visium with no spatial analysis conducted to quantify the patterning of expression patterns or spatial correlation between factors. The authors acknowledge the challenge of conducting this analysis given the variable size and geometry of the granulomas. In future studies, this can be overcome through size- or distance-based normalization or spatial clustering approaches that evaluate local neighborhood composition across different scales.

Impact:

The author's analysis helps highlight the chemokine profiles of protective, yet host protective granulomas. As that authors comment on in their discussion, these findings have important similarities and differences with other notable granulomatous conditions, such as tuberculosis. Beyond the relevance to C. violaceum infection, these data can help inform studies of other types of granulomas and hone candidate strategies for host-directed therapy strategies.

Reviewer #3 (Public review):

Summary:

This study presents a solid framework for the metabolic modeling of microbial species and resources in the rhizosphere environment. It is an ambitious effort to tackle the huge complexity of the rhizosphere and reveal the plant-microbiota interactions therein. Considering previously published data by Berihu et al., going through a series of steps, the framework then finds associations between an apple tree disease state and both microbes and metabolites. The framework is well explained and motivated. I think that further work should be done to validate the method, both using synthetic data, with a known ground truth and following up on key findings experimentally.

Strengths:

- The manuscript is well written with a good balance between detail and readability. The framework steps are well motivated and explained.

- The authors faithfully acknowledge the limitations of their approach and do not try to "over-sell" their conclusions.

- The presented framework has potential for significant discovery if the hypotheses generated are followed up with experimental validation.

Weaknesses:

- It would be better for the framework to be validated on synthetic data.

Justification of claims and conclusions:

The claims and conclusions are sufficiently well justified since the limitations of this approach are acknowledged by the authors.

Reviewer #2 (Public review):

Summary:

The current study is presented to assess the shift in metabolism (Glycolysis and Oxidative phosphorylation) of differently primed human Alveolar macrophages and Monocyte derived macrophages in response to TLR4 activating signals (such as LPS and dead Mtb bacteria). They conducted this macrophage characterization in response to type II interferon and IL-4 priming signals, followed by different stimuli of irradiated Mycobacterium tuberculosis and LPS.

Strengths:

(1) The study employs thorough measurement of metabolic shift in metabolism by assessing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of differentially polarized primary human macrophages using the Seahorse XFe24 Analyzer.<br /> (2) The effect of differential metabolic shift on the expression of different surface markers for macrophage activation is evaluated through immunofluorescence flow cytometry and cytokine measurement via ELISA.

Weaknesses:

(1) Prior studies with human macrophages have shown a glycolytic shift with similar signals, including live Mycobacterium tuberculosis infection.<br /> (2) Results are often described with detailed methodology for each experiment, and data are replotted and presented in duplicates for cross-analyses which can be confusing.<br /> (3) The data presented shows a distinct functional profile of airway macrophages (AMs) compared to monocyte (blood)-derived macrophages (MDMs) in response to the same priming signals. However, the study does not attempt to explore the underlying mechanisms for this difference.

Appraisal:

(1) The authors have achieved their aim of preliminarily characterizing the glycolysis-dependent cytokine profile and activation marker expression of IFN-g and IL-4 primed primary human macrophages.<br /> (2) The results of the study support its conclusion of glycolysis-dependent phenotypical differences in cytokine secretion and activation marker expression of AMs and MDMs.<br /> (3) However, the study is descriptive in nature, and the results validate IFN-g-mediated glycolytic reprogramming in primary human macrophages without providing mechanistic insights.

Impact:

The study provides evidence of metabolic reprogramming in human primary macrophages and their dependence on glycolysis for downstream secretion of cytokines and expression of activation markers.

Additional comments:

The results of this study are generated from a very large experiment with different treatments and phenotypic characterization. The data is plotted and analyzed in different figures to aid the reader.

Reviewer #2 (Public review):

Summary:

The biologically realistic model of the locomotor circuits developed by this group continues to define the state of the art for understanding spinal genesis of locomotion. Here the authors have achieved a new level of analysis of this model to generate surprising and potentially transformative new insights. They show that these circuits can operate in three very distinct states and that, in the intact spinal cord, these states come into successive operation as the speed of locomotion increases. Equally important, they show that in spinal injury, the model is "stuck" in the low-speed "state machine" behavior.

Strengths:

There are many strengths for the simulations results presented here. The model itself has been closely tuned to match a huge range of experimental data and this has a high degree of plausibility. The novel insight presented here, with the three different states, constitutes a truly major advance in the understanding of neural genesis of locomotion in spinal circuits. The authors systematically consider how the states of the model relate to presently available data from animal studies. Equally important, they provide a number of intriguing and testable predictions. It is likely that these insights are the most important achieved in the past 10 years. It is highly likely proposed multi-state behavior will have a transformative effect on this field.

Weaknesses:

I have no major weaknesses. A moderate concern is that the authors should consider some basic sensitivity analyses to determine if the 3-state behavior is especially sensitive to any of the major circuit parameters-e.g., connection strengths in the oscillators.

Reviewer #2 (Public Review):

Summary:

The authors combine a clever use of historical clinical data on infection duration in immunologically naive individuals and queuing theory to infer the force of infection (FOI) from measured multiplicity of infection (MOI) in a sparsely sampled setting. They conduct extensive simulations using agent-based modeling to recapitulate realistic population dynamics and successfully apply their method to recover FOI from measured MOI. They then go on to apply their method to real-world data from Ghana before and after an indoor residual spraying campaign.

Strengths:

(1) The use of historical clinical data is very clever in this context.

(2) The simulations are very sophisticated with respect to trying to capture realistic population dynamics.

(3) The mathematical approach is simple and elegant, and thus easy to understand.

Weaknesses:

(1) The assumptions of the approach are quite strong and should be made more clear. While the historical clinical data is a unique resource, it would be useful to see how misspecification of the duration of infection distribution would impact the estimates.

(2 )Seeing as how the assumption of the duration of infection distribution is drawn from historical data and not informed by the data on hand, it does not substantially expand beyond MOI. The authors could address this by suggesting avenues for more refined estimates of infection duration.

(3) It is unclear in the example how their bootstrap imputation approach is accounting for measurement error due to antimalarial treatment. They supply two approaches. First, there is no effect on measurement, so the measured MOI is unaffected, which is likely false and I think the authors are in agreement. The second approach instead discards the measurement for malaria-treated individuals and imputes their MOI by drawing from the remaining distribution. This is an extremely strong assumption that the distribution of MOI of the treated is the same as the untreated, which seems unlikely simply out of treatment-seeking behavior. By imputing in this way, the authors will also deflate the variability of their estimates.

- For similar reasons, their imputation of microscopy-negative individuals is also questionable, as it also assumes the same distributions of MOI for microscopy-positive and negative individuals.

Reviewer #2 (Public Review):

This manuscript describes the impact of deleting or enhancing the expression of the neuronal-specific kinase DLK in glutamatergic hippocampal neurons using clever genetic strategies, which demonstrates that DLK deletion had minimal effects while overexpression resulted in neurodegeneration in vivo. To determine the molecular mechanisms underlying this effect, ribotag mice were used to determine changes in active translation which identified Jun and STMN4 as DLK-dependent genes that may contribute to this effect. Finally, experiments in cultured neurons were conducted to better understand the in vivo effects. These experiments demonstrated that DLK overexpression resulted in morphological and synaptic abnormalities.

Strengths:

This study provides interesting new insights into the role of DLK in the normal function of hippocampal neurons. Specifically, the study identifies:

(1) CA1 vs CA3 hippocampal neurons have differing sensitivity to increased DLK signaling.

(2) DLK-dependent signaling in these neurons is similar to but distinct from the downstream factors identified in other cell types, highlighted by the identification of STMN4 as a downstream signal.

(3) DLK overexpression in hippocampal neurons results in signaling that is similar to that induced by neuronal injury.

The study also provides confirmatory evidence that supports previously published work through orthogonal methods, which adds additional confidence to our understanding of DLK signaling in neurons. Taken together, this is a useful addition to our understanding of DLK function.

Weaknesses:

There are a few weaknesses that limit the impact of this manuscript, most of which are pointed out by the authors in the discussion. Namely:

(1) It is difficult to distinguish whether the changes in the translatome identified by the authors are DLK-dependent transcriptional changes, DLK-dependent post-transcriptional changes or secondary gene expression changes that occur as a result of the neurodegeneration that occurs in vivo. Additional expression analysis at earlier time points could be one method to address this concern.

(2) Related to the above, it is difficult to conclusively determine from the current data whether the changes in synaptic proteins observed in vivo are a secondary result of neuronal degeneration or a primary impact on synapse formation. The in vitro studies suggest this has the potential to be a primary effect, though the difference in experimental paradigm makes it impossible to determine whether the same mechanisms are present in vitro and in vivo.

(3) The phenotype of DLK cKO mice is very subtle (consistent with previous reports) and while the outcome of increased DLK levels is interesting, the relevance to physiological DLK signaling is less clear. What does seem possible is that increased DLK may phenocopy other neuronal injuries but there are no real comparisons to directly address this in the manuscript. It would be helpful for the authors to provide this analysis as well as a table with all of the translational changes along with fold changes.

(4) For the in vivo experiments, it is unclear whether multiple sections from each animal were quantified for each condition. More information here would be helpful and it is important that any quantification takes multiple sections from each animal into account to account for natural variability.

Reviewer #2 (Public Review):

Summary:

This manuscript focuses on the apparent involvement of a proposed copper-responsive regulator in the chemotactic response of Pseudomonas putida to Cu(II), a chemorepellent. Broadly, this area is of interest because it could provide insight into how soil microbes mitigate metal stress. Additionally, copper has some historical agricultural use as an antimicrobial, thus can accumulate in soil. The manuscript bases its conclusions on an in vitro screen to identify interacting partners of CheA, an essential kinase in the P. putida chemotaxis-signaling pathway. Much of the subsequent analysis focuses on a regulator of the CsoR/RcnR family (PP_2969).

Weaknesses:

The data presented in this work does not support the model (Figure 8). In particular, PP_2969 is linked to Ni/Co resistance, not Cu resistance. Further, it is not clear how the putative new interactions with CheA would be integrated into diverse responses to various chemoattract/repellents. These two comments are justified below.

PP_2969

(1) The authors present a sequence alignment (Figure S5) that is the sole basis for their initial assignment of this ORF as a CsoR protein. There is a conservation of the primary coordinating ligands (highlighted with asterisks) known to be involved in Cu(I) binding to CsoR (ref 31). There are some key differences, though, in residues immediately adjacent to the conserved Cys (the preceding Ala, which is Tyr in the other sequences). The effect of these changes may be significant in a physiological context.

(2) The gene immediately downstream of PP_2969 is homologous to E. coli RcnA, a demonstrated Ni/Co efflux protein, suggesting that P2969 may be Ni or Co responsive. Indeed PP_2970 has previously been reported as Ni/Co responsive (J. Bact 2009 doi:10.1128/JB.00465-09). The host cytosol plays a critical role in determining metal response, in addition to the protein, which can explain the divergence from the metal response expected from the alignment.

(3) The previous JBact study also explains the lack of an effect (Figure 5b) of deleting PP_2969 on copper-efflux gene expression (copA-I, copA-II, and copB-II) as these are regulated by CueR not PP_2969 consistent with the previous report. Deletion of CsoR/RcnR family regulator will result in constitutive expression of the relevant efflux/detoxification gene, at a level generally equivalent to the de-repression observed in the presence of the signal.

(4) Further, CsoR proteins are Cu(I) responsive so measuring Cu(II) binding affinity is not physiologically relevant (Figures 5a and S5b). The affinities of demonstrated CsoR proteins are 10-18 M and these values are determined by competition assay. The MTS assay and resulting affinities are not physiologically relevant.

(5) The DNA-binding assays are carried out at protein concentrations well above physiological ranges (Figures 5c and d, and S5c, d). The weak binding will in part result from using DNA sequences upstream of the copA genes and not from from PP_2970.

CheA interactions

(1) There is no consideration given to the likely physiological relevance of the new interacting partners for CheA.

(2) How much CheA is present in the cell (copies) and how many copies of other proteins are present? How would specific responses involving individual interacting partners be possible with such a heterogenous pool of putative CheA-complexes in a cell? For PP_2969, the affinity reported (Figure 5A) may lay at the upper end of the CsoR concentration range (for example, CueR in Salmonella is present at ~40 nM).

(3) The two-hybrid system experiment uses a long growth time (60 h) before analysis. Even low LacZ activity levels will generate a blue colour, depending upon growth medium (see doi: 10.1016/0076-6879(91)04011-c). It is also not clear how Miller units can be accurately or precisely determined from a solid plate assay (the reference cited describes a protocol for liquid culture).

Reviewer #2 (Public review):

The authors of this paper have done much pioneering work to decipher and understand LRRK2 structure and function, to uncover the mechanism by which LRRK2 binds to microtubules, and to study the roles that this may play in biology. Their previous data demonstrated that LRRK2 in the active conformation (pathogenic mutation or Type I inhibitor complex) bound to microtubule filaments in an ordered helical arrangement. This they showed induced a "roadblock" in the microtubule impacting vesicular trafficking. The authors have postulated that this is a potentially serious flaw with Type 1 inhibitors and that companies should consider generating Type 2 inhibitors in which the LRRK2 is trapped in the inactive conformation. Indeed the authors have published much data that LRRK2 complexed to Type 2 inhibitors does not seem to associate with microtubules and cause roadblocks in parallel experiments to those undertaken with type 1 inhibitors published above.

In the current study, the authors have undertaken an in vitro reconstitution of microtubule-bound filaments of LRRK2 in the inactive conformation, which surprisingly revealed that inactive LRRK2 can also interact with microtubules in its auto-inhibited state. The authors' data shows that while the same interphases are seen with both the active LRRK2 and inactive microtubule bound forms of LRRK2, they identified a new interphase that involves the WD40-ARM-ANK- domains that reportedly contributes to the ability of the inactive form of LRRK2 to bind to microtubule filaments. The structures of the inactive LRRK2 complexed to microtubules are of medium resolution and do not allow visualisation of side chains.

This study is extremely well-written and the figures are incredibly clear and well-presented. The finding that LRRK2 in the inactive autoinhibited form can be associated with microtubules is an important observation that merits further investigation. This new observation makes an important contribution to the literature and builds upon the pioneering research that this team of researchers has contributed to the LRRK2 fields. However, in my opinion, there is still significant work that could be considered to further investigate this question and understand the physiological significance of this observation.

Reviewer #2 (Public Review):

Summary:

Cong et al. investigated the regulatory effects of ABHD6 on AMPARs. The authors performed adequate electrophysiology recordings to show the exact pattern of this regulation and covered major critical points.

Strengths:

The authors have performed high-quality ephys recordings and examined all potential regulatory aspects of ABHD6 on AMPARs. This is important to understand the AMPAR functions.

Weaknesses:

(1) The authors discussed CNIH-2 extensively from line 92-110 in the introduction, however, they did not perform related experiments. I suggest they move this part to the discussion where they also discussed the roles of CNIH.<br /> (2) The authors need to report the "n" for all the experiments they have presented in this manuscript. How many cells were recorded in each condition? How many batches? This information has to be in all of the figure legends, but it is missing except Fig. 4.<br /> (3) One question is what the physiological meanings of this regulatory effect are. The authors may consider adding some discussions.<br /> (4) About statistics. The authors need to add more details and make sure their statistics sound. For example, they also need to check the equality of variances. In their Table EVs, where the P values are reported, the authors need to report which statistics they have used, one-way ANOVA, K-W test, or others, and the exact post-hoc test type for each comparison. For one-way ANOVA, report the F values simultaneously with the P values in all figure legends.<br /> (5) Fig. 3J, the authors need to correct the label of the Y axis. It is shifted.

Reviewer #2 (Public review):

Summary:

This is an outstanding piece of work on the potential of FLO as a viable analgesic biologic for the treatment of postsurgical pain. The authors purified the HC-HA/PTX3 from FLO and demonstrated its potential as an effective non-opioid therapy for postsurgical pain. They further unraveled the mechanisms of action of the compound at cellular and molecular levels.

Strengths:

Prominent strengths include the incorporation of behavioral assessment, electrophysiological and imaging recordings, the use of knockout and knockdown animals, and the use of antagonist agents to verify biological effects. The integrated use of these techniques, combined with the hypothesis-driven approach and logical reasoning, provides compelling evidence and novel insight into the mechanisms of the significant findings of this work.

Weaknesses:

I did not find any significant weaknesses even with a critical mindset. The only minor suggestion is that the Results section may focus on the results from this study and minimize the discussions of background information.

Reviewer #2 (Public review):

Summary:

Saijilafu et al. demonstrate that MLCK/MLCP proteins promote axonal regeneration in both the central nervous system (CNS) and peripheral nervous system (PNS) using primary cultures of adult DRG neurons, hippocampal and cortical neurons, as well as in vivo experiments involving sciatic nerve injury, spinal cord injury, and optic nerve crush. The authors show that axon regrowth is possible across different contexts through genetic and pharmacological manipulation of these proteins. Additionally, they propose that MLCK/MLCP may regulate F-actin reorganization in the growth cone, which is significant as it suggests a novel strategy for promoting axonal regeneration.

Strengths:

This manuscript presents a comprehensive array of experimental models, addressing the biological question in a broad manner. Particularly noteworthy is the use of multiple in vivo models, which significantly strengthens the overall validity of the study.

Weaknesses:

The following aspects apply:

(1) The manuscript initially references prior research by the authors suggesting that NMII inhibition enhances axonal growth and that MLCK activates NMII. However, the study introduces a contradiction by demonstrating that MLCK inhibition (via ML-7 or siMLCK) inhibits axonal growth. This inconsistency is not adequately addressed or discussed in the manuscript.

(2) While the study proposes that MLCK/MLCP regulates F-actin redistribution in the growth cone, the mechanism is not explored in depth. The only figure showing how pharmacological manipulation affects the growth cone suggests that not only F-actin but also the microtubule cytoskeleton might be affected, indicating that the mechanism may not be specific. A deeper exploration of this relationship in DRG neurons, in addition to cortical neurons, as shown in the study, would be beneficial.

(3) In the sciatic nerve injury experiments, it would be crucial to include additional controls that clearly demonstrate that siMYPT1 treatment increases MLCP in the L4-L5 ganglia. Additionally, although the manuscript mentions quantifying axons expressing EGFP, the Materials and Methods section only discusses siMYPT1 electroporation, which could lead to confusion.

(4) In some panels, it is difficult to differentiate the somas from the background (Figures 3, 4, 7). In conditions where images with shorter axonal lengths are represented, it is unclear whether this is due to fewer cells or reduced axonal growth (Figures 2, 4, 6).

Reviewer #2 (Public Review):

Summary:

In this study, the authors investigate the potential role of other cleavage products of amyloid precursor protein (APP) in neurodegeneration. They combine in vitro and in vivo experiments, revealing that β-CTF, a product cleaved by BACE1, promotes synaptic loss independently of Aβ. Furthermore, they suggest that β-CTF may interact with Rab5, leading to endosomal dysfunction and contributing to the loss of synaptic proteins.

Weaknesses:

Most experiments were conducted in vitro using overexpressed β-CTF. Additionally, the study does not elucidate the mechanisms by which β-CTF disrupts endosomal function and induces synaptic degeneration.

Reviewer #2 (Public review):

I previously reviewed this important and timely manuscript at a previous journal where, after two rounds of review, I recommended publication. Because eLife practices an open reviewing format, I will recapitulate some of my previous comments here, for the scientific record.

In that previous review, I revealed my identity to help reassure the authors that I was doing my best to remain unbiased because I work in this area and some of the authors' results directly impact my prior research. I was genuinely excited to see the earlier preprint version of this paper when it first appeared. I get a lot of joy out of trying to - collectively, as a field - really understand the nature of our data, and I continue to commend the authors here for pushing at the sources of aperiodic activity!

In their manuscript, Schmidt and colleagues provide a very compelling, convincing, thorough, and measured set of analyses. Previously I recommended that the push even further, and they added the current Figure 5 analysis of event-related changes in the ECG during working memory. In my opinion this result practically warrants a separate paper its own!

The literature analysis is very clever, and expanded upon from any other prior version I've seen.

In my previous review, the broadest, most high-level comment I wanted to make was that authors are correct. We (in my lab) have tried to be measured in our approach to talking about aperiodic analyses - including adopting measuring ECG when possible now - because there are so many sources of aperiodic activity: neural, ECG, respiration, skin conductance, muscle activity, electrode impedances, room noise, electronics noise, etc. The authors discuss this all very clearly, and I commend them on that. We, as a field, should move more toward a model where we can account for all of those sources of noise together. (This was less of an action item, and more of an inclusion of a comment for the record.)

I also very much appreciate the authors' excellent commentary regarding the physiological effects that pharmacological challenges such as propofol and ketamine also have on non-neural (autonomic) functions such as ECG. Previously I also asked them to discuss the possibility that, while their manuscript focuses on aperiodic activity, it is possible that the wealth of literature regarding age-related changes in "oscillatory" activity might be driven partly by age-related changes in neural (or non-neural, ECG-related) changes in aperiodic activity. They have included a nice discussion on this, and I'm excited about the possibilities for cognitive neuroscience as we move more in this direction.

Finally, I previously asked for recommendations on how to proceed. The authors convinced me that we should care about how the ECG might impact our field potential measures, but how do I, as a relative novice, proceed. They now include three strong recommendations at the end of their manuscript that I find to be very helpful.

As was obvious from previous review, I consider this to be an important and impactful cautionary report, that is incredibly well supported by multiple thorough analyses. The authors have done an excellent job responding to all my previous comments and concerns and, in my estimation, those of the previous reviewers as well.

Reviewer #2 (Public Review):

Summary:

Li et al. investigated the potential anti-ageing role of 17α-Estradiol on the hypothalamus of aged rats. To achieve this, they employed a very sophisticated method for single-cell genomic analysis that allowed them to analyze effects on various groups of neurons and non-neuronal cells. They were able to sub-categorize neurons according to their capacity to produce specific neurotransmitters, receptors, or hormones. They found that 17α-Estradiol treatment led to an improvement in several factors related to metabolism and synaptic transmission by bringing the expression levels of many of the genes of these pathways closer or to the same levels as those of young rats, reversing the ageing effect. Interestingly, among all neuronal groups, the proportion of Oxytocin-expressing neurons seems to be the one most significantly changing after treatment with 17α-Estradiol, suggesting an important role of these neurons in mediating its anti-ageing effects. This was also supported by an increase in circulating levels of oxytocin. It was also found that gene expression of corticotropin-releasing hormone neurons was significantly impacted by 17α-Estradiol even though it was not different between aged and young rats, suggesting that these neurons could be responsible for side effects related to this treatment. This article revealed some potential targets that should be further investigated in future studies regarding the role of 17α-Estradiol treatment in aged males.

Strengths:

(1) Single-nucleus mRNA sequencing is a very powerful method for gene expression analysis and clustering. The supervised clustering of neurons was very helpful in revealing otherwise invisible differences between neuronal groups and helped identify specific neuronal populations as targets.

(2) There is a variety of functions used that allow the differential analysis of a very complex type of data. This led to a better comparison between the different groups on many levels.

(3) There were some physiological parameters measured such as circulating hormone levels that helped the interpretation of the effects of the changes in hypothalamic gene expression.

Weaknesses:

(1) One main control group is missing from the study, the young males treated with 17α-Estradiol.

(2) Even though the technical approach is a sophisticated one, analyzing the whole rat hypothalamus instead of specific nuclei or subregions makes the study weaker.

(3) Although the authors claim to have several findings, the data fail to support these claims.

(4) The study is about improving ageing but no physiological data from the study demonstrated such a claim with the exception of the testes histology which was not properly analyzed and was not even significantly different between the groups.

(5) Overall, the study remains descriptive with no physiological data to demonstrate that any of the effects on hypothalamic gene expression are related to metabolic, synaptic, or other functions.

Reviewer #2 (Public Review):

The tubulin subunits that make up microtubules can be posttranslationally modified and these PTMs are proposed to regulate microtubule dynamics and the proteins that can interact with microtubules in many contexts. However, most studies investigating the roles of tubulin PTMs have been conducted in vitro either with purified components or in cultured cells. Lu et al. use CRISPR/Cas9 genome editing to mutate tubulin genes in C. elegans, testing the role of specific tubulin residues on neuronal development. This study is a real tour de force, tackling multiple proposed tubulin modifications and following the resulting phenotypes with respect to neurite outgrowth in vivo. There is a ton of data that experts in the field will likely reference for years to come as this is one of the most comprehensive in vivo analyses of tubulin PTMs in vivo.

This paper will be very important to the field, however would be strengthened if: 1) the authors demonstrated that the mutations they introduced had the intended consequences on microtubule PTMs, 2) the authors explored how the various tubulin mutations directly affect microtubules, and 3) the findings are made generally more accessible to non C. elegans neurobiologists.

(1) The authors introduce several mutations to perturb tubulin PTMs, However, it is unclear to what extent the engineered mutations affect tubulin in the intended way i.e. are the authors sure that the PTMs they want to perturb are actually present in C. elegans. Many of the antibodies used did not appear to be specific and antibody staining was not always impacted in the mutant cases as expected. For example, is there any evidence that S172 is phosphorylated in C. elegans, e.g. from available phosphor-proteomic data? Given the significant amount of staining left in the S172A mutant, the antibody seems non-specific in this context and therefore not a reliable readout of whether MTs are actually phosphorylated at this residue. As another example, there is no evidence presented that K252 is acetylated in C. elegans. At the very least, the authors should consider demonstrating the conservation of these residues and the surrounding residues with other organisms where studies have demonstrated PTMs exist.

(2) Given that the authors have the mutants in hand, it would be incredibly valuable to assess the impact of these mutations on microtubules directly in all cases. MT phenotypes are inferred from neurite outgrowth phenotypes in several cases, the authors should look directly at microtubules and/or microtubule dynamics via EBP-2 when possible OR show evidence that the only way to derive the neurite phenotypes shown is through the inferred microtubule phenotypes. For example, the effect of the acetylation or detyrosination mutants on MTs was not assessed.

(3) There is a ton of data here that will be important for experts working in this field to dig into, however, for the more general cell biologist, some of the data are quite inaccessible. More cartoons and better labeling will be helpful as will consistent comparisons to control worms in each experiment.

(4) In addition, I am left unconvinced of the negative data demonstrating that MBK does not phosphorylate tubulin. First, the data described in lines 207-211 does not appear to be presented anywhere. Second, RNAi is notoriously finicky in neurons, thus necessitating tissue-specific degradation using either the ZF/ZIF-1 or AID/TIR1 systems which both work extremely well in C. elegans. Third, there appears to be increasing S172 phosphorylation in Figure 3 Supplement 2 with added MBK-2, but there is no anti-tubulin blot to show equal loading, so this experiment is hard to interpret.

Reviewer #2 (Public Review):

Summary:

The authors show that a spiking network model with clustered neurons produces intrinsic spike sequences when driven with a ramping input, which are recapitulated in the absence of input. This behavior is only seen for some network parameters (neuron cluster participation and number of clusters in the network), which correspond to those that produce a small world network. By changing the strength of ramping input to each network cluster, the network can show different sequences.

Strengths:

A strength of the paper is the direct comparison between the properties of the model and neural data.

Weaknesses:

My main critiques of the paper relate to the form of the input to the network.

First, because the input is the same across trials (i.e. all traversals are the same duration/velocity), there is no ability to distinguish a representation of space from a representation of time elapsed since the beginning of the trial. The authors should test what happens e.g. with traversals in which the animal travels at different speeds, and in which the animal's speed is not constant across the entire track, and then confirm that the resulting tuning curves are a better representation of position or duration.

Second, it's unclear how much the results depend on the choice of a one-dimensional environment with ramping input. While this is an elegant idealization that allows the authors to explore the representation and replay properties of their model, it is a strong and highly non-physiological constraint. The authors should verify that their results do not depend on this idealization. Specifically, I would suggest the authors also test the spatial coding properties of their network in 2-dimensional environments, and with different kinds of input that have a range of degrees of spatial tuning and physiological plausibility. A method for systematically producing input with varying degrees of spatial tuning in both 1D and 2D environments has been previously used in (Fang et al 2023, eLife, see Figures 4 and 5), which could be readily adapted for the current study; and behaviorally plausible trajectories in 2D can be produced using the RatInABox package (George et al 2022, bioRxiv), which can also generate e.g. grid cell-like activity that could be used as physiologically plausible input to the network.

Finally, I was left wondering how the cells' spatial tuning relates to their cluster membership, and how the capacity of the network (number of different environments/locations that can be represented) relates to the number of clusters. It seems that if clusters of cells tend to code for nearby locations in the environment (as predicted by the results of Figure 5), then the number of encodable locations would be limited (by the number of clusters). Further, there should be a strong tendency for cells in the same cluster to encode overlapping locations in different environments, which is not seen in experimental data.

Reviewer #2 (Public review):

This work started with transcriptomic profiling of ductal cells to identify the upregulation of calcineurin in the zebrafish after beta-cell ablation. By suppressing calcineurin with its chemical inhibitor cyclosporin A and expressing a constitutively active form of calcineurin ubiquitously or specifically in ductal cells, the authors found that inhibited calcineurin activity promoted beta-cell regeneration transiently while ectopic calcineurin activity hindered beta-cell regeneration in the pancreatic tail. They also showed similar effects in the basal state but only when it was within a particular permissive window of Notch activity. To further investigate the roles of calcineurin in the ductal cells, the authors demonstrated that calcineurin inhibition additionally induced the proliferation of the ductal cells in the regenerative context or under a limited level of Notch activity. Interestingly, the enhanced proliferation was followed by a depletion of ductal cells, suggesting that calcineurin inhibition would exhaust the ductal cells. Based on the data, the authors proposed a very attractive and intriguing model of the role of calcineurin in maintaining the balance of the progenitor proliferation and the endocrine differentiation. However, the conclusions of this paper are only partially supported by the data as some evidence of the lineage between ductal cells and beta cells remains suggestive.

Reviewer #2 (Public Review):

In the manuscript Liu et al. observed that glucose and noncaloric monosaccharides can prompt an excessive formation of blood vessels, particularly intersegmental vessels (ISVs). They propose that these branched vessels arise from the ectopic activation of quiescent endothelial cells (ECs) into tip cells. Moreover, through single-cell transcriptome sequencing analysis of embryonic endothelial cells exposed to glucose, they noted an increased proportion of arterial and capillary endothelial cells, proliferative endothelial cells, along with a series upregulated genes in categories of blood vessel morphogenesis, development, and pro-angiogenesis. The authors provide evidence suggesting that caloric and noncaloric monosaccharides (NMS) induce excessive angiogenesis via the Foxo1a-Marcksl1a pathway.

Reviewer #2 (Public Review):

Summary:

In this important study, Baniulyte and Wade describe how the translation of an 8-codon uORF denoted toiL upstream of the topAI-yjhQP operon is responsive to different ribosome-targeting antibiotics, consequently controlling translation of the TopAI toxin as well as Rho-dependent termination with the gene.

Strengths:

I appreciate that the authors used multiple different approaches such as a genetic screen to identify factors such as 23S rRNA mutations that affect topA1 expression and ribosome profiling to examine the consequences of various antibiotics on toiL-mediated regulation. The results are convincing and clearly described.

Weaknesses:

I have relatively minor suggestions for improving the manuscript. These mainly relate to the figures.

Reviewer #2 (Public Review):

Summary:

Bonnifet et al. sought to characterize the expression pattern of L1 ORF1p expression across the entire mouse brain, in young and aged animals, and to corroborate their characterization with Western blotting for L1 ORF1p and L1 RNA expression data from human samples. They also queried L1 ORF1p interacting partners in the mouse brain by IP-MS.

Strengths:

A major strength of the study is the use of two approaches: a deep-learning detection method to distinguish neuronal vs. non-neuronal cells and ORF1p+ cells vs. ORF1p- cells across large-scale images encompassing multiple brain regions mapped by comparison to the Allen Brain Atlas, and confocal imaging to give higher resolution on specific brain regions. These results are also corroborated by Western blotting on six mouse brain regions. Extension of their analysis to post-mortem human samples, to the extent possible, is another strength of the paper. The identification of novel ORF1p interactors in the brain is also a strength in that it provides a novel dataset for future studies.

Weaknesses:

The main weakness of the study is that cell type specificity of ORF1p expression was not examined beyond neuron (NeuN+) vs non-neuron (NeuN-). Indeed, a recent study (Bodea et al. 2024, Nature Neuroscience) found that ORF1p expression is characteristic of parvalbumin-positive interneurons, and it would be very interesting to query whether other neuronal subtypes in different brain regions are distinguished by ORF1p expression. The data suggesting that ORF1p expression is increased in aged mouse brains is intriguing, although it seems to be based upon modestly (up to 27%, dependent on brain region) higher intensity of ORF1p staining rather than a higher proportion of ORF1+ neurons. Indeed, the proportion of NeuN+/Orf1p+ cells actually decreased in aged animals. It is difficult to interpret the significance and validity of the increase in intensity, as Hoechst staining of DNA, rather than immunostaining for a protein known to be stably expressed in young and aged neurons, was used as a control for staining intensity. The main weakness of the IP-MS portion of the study is that none of the interactors were individually validated or subjected to follow-up analyses. The list of interactors was compared to previously published datasets, but not to ORF1p interactors in any other mouse tissue.

The authors achieved the goals of broadly characterizing ORF1p expression across different regions of the mouse brain, and identifying putative ORF1p interactors in the mouse brain. However, findings from both parts of the study are somewhat superficial in depth.

This provides a useful dataset to the field, which likely will be used to justify and support numerous future studies into L1 activity in the aging mammalian brain and in neurodegenerative disease. Similarly, the list of ORF1p interacting proteins in the brain will likely be taken up and studied in greater depth.

Reviewer #2 (Public review):

Summary:

The manuscript reports the computational study of the dynamics of PROTAC-induced degradation complexes. The research investigates how different linkers within PROTACs affect the formation and stability of ternary complexes between the target protein BRD4BD1 and Cereblon E3 ligase, and the degradation machinery. Using computational modeling, docking, and molecular dynamics simulations, the study demonstrates that although all PROTACs form ternary complexes, the linkers significantly influence the dynamics and efficacy of protein degradation. The findings highlight that the flexibility and positioning of Lys residues are crucial for successful ubiquitination. The results also discussed the correlated motions between the PROTAC linker and the complex.

Strengths:

The field of PROTAC discovery and design, characterized by its limited research, distinguishes itself from traditional binary ligand-protein interactions by forming a ternary complex involving two proteins. The current understanding of how the structure of PROTAC influences its degradation efficacy remains insufficient. This study investigated the atomic-level dynamics of the degradation complex, offering potentially valuable insights for future research into PROTAC degradability.

Reviewer #2 (Public review):

Summary:

The inferior colliculus (IC) has been explored for its possible functions in behavioral tasks and has been suggested to play more important roles rather than simple sensory transmission. The authors revealed the climbing effect of neurons in IC during decision-making tasks, and tried to explore the reward effect in this condition.

Strengths:

Complex cognitive behaviors can be regarded as simple ideals of generating output based on information input, which depends on all kinds of input from sensory systems. The auditory system has hierarchic structures no less complex than those areas in charge of complex functions. Meanwhile, IC receives projections from higher areas, such as auditory cortex, which implies IC is involved in complex behaviors. Experiments in behavioral monkeys are always time-consuming works with hardship, and this will offer more approximate knowledge of how the human brain works.

Weaknesses:

These findings are more about correlation but not causality of IC function in behaviors. And I have a few major concerns.

Comparing neurons' spike activities in different tests, a 'climbing effect' was found in the oddball paradigm. The effect is clearly related to training and learning process, but it still requires more exploration to rule out a few explanations. First, repeated white noise bursts with fixed inter-stimulus-interval of 0.6 seconds was presented, so that monkeys might remember the sounds by rhymes, which is some sort of learned auditory response. It is interesting to know monkeys' responses and neurons' activities if the inter-stimuli-interval is variable. Second, the task only asked monkeys to press one button and the reward ratio (the ratio of correct response trials) was around 78% (based on the number from Line 302). so that, in the sessions with reward, monkeys had highly expected reward chances, does this expectation cause the climbing effect?

"Reward effect" on IC neurons' responses were showed in Fig. 4. Is this auditory response caused by physical reward action or not? In reward sessions, IC neurons have obvious response related to the onset of water reward. The electromagnetic valve is often used in water-rewarding system and will give out a loud click sound every time when the reward is triggered. IC neurons' responses may be simply caused by the click sound if the electromagnetic valve is used. It is important to find a way to rule out this simple possibility.

Reviewer #2 (Public review):

Summary:

RNAs can function across cell borders and animal generations as sources of epigenetic information for development and immunity. The specific mechanistic pathways how RNA travels between cells and progeny remains an open question. Here, Shugarts, et al. use molecular genetics, imaging, and genomics methods to dissect specific RNA transport and regulatory pathways in the C. elegans model system. Larvae ingesting double stranded RNA is noted to not cause continuous gene silencing throughout adulthood. Damage of neuronal cells expressing double stranded target RNA is observed to repress target gene expression in the germline. Exogenous supply of short or long double stranded RNA required different genes for entry into progeny. It was observed that the SID-1 double-stranded RNA transporter showed different expression over animal development. Removal of the sid-1 gene caused upregulation of two genes, the newly described sid-1-dependent gene sdg-1 and sdg-2. Both genes were observed to also be negatively regulated by other small RNA regulatory pathways. Strikingly, loss then gain of sid-1 through breeding still caused variability of sdg-1 expression for many, many generations. SDG-2 protein co-localizes with a Z-granule marker, an intracellular site for heritable RNA silencing machinery. Collectively, sdg-1 presents a model to study how extracellular RNAs can buffer gene expression in germ cells and other tissues.

Strengths:

(1) Very clever molecular genetic methods and genomic analyses, paired with thorough genetics, were employed to discover insights into RNA transport, sdg-1 and sdg-2 as sid-1-dependent genes, and sdg-1's molecular phenotype.

(2) The manuscript is well cited, and figures reasonably designed.

(3) The discovery of the sdg genes being responsive to the extracellular RNA cell import machinery provides a model to study how exogenous somatic RNA is used to regulate gene expression in progeny. The discovery of genes within retrotransposons stimulates tantalizing models how regulatory loops may actually permit the genetic survival of harmful elements.

Weaknesses:

(1) As presented, the manuscript is incredibly broad, making it challenging to read and consider the data presented. This concern is exemplified in the model figure, that requires two diagrams to summarize the claims made by the manuscript.

(2) The large scope of the manuscript denies space to further probe some of the ideas proposed. The first part of the manuscript, particularly Figures 1 and 2, presents data that can be caused by multiple mechanisms, some of which the authors describe in the results but do not test further. Thus, portions of the results text come across as claims that are not supported by the data presented.

(3) The manuscript focuses on the genetics of SDGs but not the proteins themselves. Few descriptions of the SDGs functions are provided nor is it clarified why only SDG-1 was pursued in imaging and genetic experiments. Additionally, the SDG-1 imaging experiments could use additional localization controls.

Reviewer #2 (Public review):

Summary:

Held et al. investigated the distinct activities of Insulin-Producing Cells (IPCs) by electrophysiological recordings and calcium imaging. In the brain of the fruit fly Drosophila melanogaster, there are approximately 14 IPCs that are analogous to mammalian pancreatic beta cells and provide a good model system for monitoring their activities in vivo. The authors performed single-nucleus RNA sequencing analysis to examine what types of neuromodulatory inputs are received by IPCs. A variety of neuromodulatory receptors are expressed heterogeneously in IPCs, which would explain the distinct activities of IPCs in response to the activations of neuromodulatory neurons. The authors also conducted the connectome analysis and G-protein prediction analysis to strengthen their hypothesis that the heterogeneity of IPCs may underlie the flexible insulin release in response to various environmental conditions.

Strengths:

The authors succeeded patch-clamp recordings and calcium imaging of individual IPCs in living animals at a single-cell resolution, which allows them to show the heterogeneity of IPCs precisely. They measured IPC activities in response to 9 types of neurons in patch-clamp recordings and 5 types of neurons in calcium imaging, comparing the similarities and differences in activities between two methods. These results support the idea that the neuromodulatory system affects individual IPC activities differently in a receptor-dependent manner.

Weaknesses:

One concern is how much extent the heterogeneity of IPC activities in a short time scale is relevant to the net output, a release of insulin-like peptides in response to metabolic demands in a relatively longer time scale. The authors can test their hypothesis by manipulating the heterogeneous expressions of receptor genes in IPCs and examining IPC activities on a longer time scale. Moreover, while the authors focus on IPC activities, they did not show the activation of the neuromodulatory inputs and the net output of insulin levels in the data. The readers might want to know which neurons are indeed activated to send signals to IPCs and how IPC activities result in the secretion of insulin peptides.

Reviewer #2 (Public Review):

As discussed in the original review, this manuscript is an important contribution to a mechanistic understanding of LRRK2 kinase. Kinetic parameters for the GTPase activity of the ROC domain have been determined in the absence/presence of kinase activity. A feedback mechanism from the kinase domain to GTP/GDP hydrolysis by the ROC domain is convincingly demonstrated through these kinetic analyses. However, a regulatory mechanism directly linking the T1343 phospho-site and a monomer/dimer equilibrium is not fully supported. The T1343A mutant has reduced catalytic activity and can form similar levels of dimer as WT. The revised manuscript does point out that other regulatory mechanisms can also play a role in kinase activity and GTP/GDP hydrolysis (Discussion section). The environmental context in cells cannot be captured from the kinetic assays performed in this manuscript, and the introduction contains some citations regarding these regulatory factors. This is not a criticism, the detailed kinetics here are rigorous, but it is simply a limitation of the approach.

Reviewer #2 (Public Review):

Summary:

Kreeger et.al provided mechanistic evidence for flexible coincidence detection of auditory nerve synaptic inputs by octopus cells in the mouse cochlear nucleus. The octopus cells are specialized neurons that can fire repetitively at very high rates (> 800 Hz in vivo), yield responses dominated by the onset of sound for simple stimuli, and integrate auditory nerve inputs over a wide frequency span. Previously, it was thought that octopus cells received little inhibitory input, and their integration of auditory input depended principally on temporally precise coincidence detection of excitatory auditory nerve inputs, coupled with a low input resistance established by high levels of expression of certain potassium channels and hyperpolarization-activated channels.

In this study, the authors used a combination of numerous genetic mouse models to characterize synaptic inputs and enable optogenetic stimulation of subsets of afferents, fluorescent microscopy, detailed reconstructions of the location of inhibitory synapses on the soma and dendrites of octopus cells, and computational modeling, to explore the importance of inhibitory inputs to the cells. They determined through assessment of excitatory and inhibitory synaptic densities that spiral ganglion neuron synapses are densest on the soma and proximal dendrite, while glycenergic inhibitory synaptic density is greater on the dendrites compared to the soma of octopus cells. Using different genetic lines, the authors further elucidated that the majority of excitatory synapses on the octopus cells are from type 1a spiral ganglion neurons, which have low response thresholds and high rates of spontaneous activity. In the second half of the paper, the authors employed electrophysiology to uncover the physiological response of octopus cells to excitatory and inhibitory inputs. Using a combination of pharmacological blockers in vitro cellular and computational modeling, the authors conclude that glycine in fact evokes IPSPs in octopus cells; these IPSPs are largely shunted by the high membrane conductance of the cells under normal conditions and thus were not clearly evident in prior studies. Pharmacological experiments point towards a specific glycine receptor subunit composition. Lastly, Kreeger et. al demonstrated with in vitro recordings and computational modeling that octopus cell inhibition modulates the amplitude and timing of dendritic spiral ganglion inputs to octopus cells, allowing for flexible coincidence detection.

Strengths:

The work combines a number of approaches and complementary observations to characterize the spatial patterns of excitatory and inhibitory synaptic input, and the type of auditory nerve input to the octopus cells. The combination of multiple mouse lines enables a better understanding of and helps to define, the pattern of synaptic convergence onto these cells. The electrophysiology provides excellent functional evidence for the presence of the inhibitory inputs, and the modeling helps to interpret the likely functional role of inhibition. The work is technically well done and adds an interesting dimension related to the processing of sound by these neurons. The paper is overall well written, the experimental tests are well-motivated and easy to follow. The discussion is reasonable and touches on both the potential implications of the work as well as some caveats.

Weaknesses:

While the conclusions presented by the authors are solid, a prominent question remains regarding the source of the glycinergic input onto octopus cells. In the discussion, the authors claim that there is no evidence for D-stellate, L-stellate, and tuberculoventral cell (all local inhibitory neurons of the ventral and dorsal cochlear nucleus) connections to octopus cells, and cite the relevant literature. An experimental approach will be necessary to properly rule out (or rule in) these cell types and others that may arise from other auditory brainstem nuclei. Understanding which cells provide the inhibitory input will be an essential step in clarifying its roles in the processing of sound by octopus cells.

The authors showed that type 1a SGNs are the most abundant inputs to octopus cells via microscopy. However, in Figure 3 they compare optical stimulation of all classes of ANFs, then compare this against stimulation of type 1b/c ANFs. While a difference in the paired-pulse ratio (and therefore, likely release probability) can be inferred by the difference between Foxg1-ChR2 and Ntng1-ChR2, it would have been preferable to have specific data with selective stimulation of type 1a neurons.

Reviewer #2 (Public Review):

Summary:

The authors have analyzed ethnogeographic differences in the comorbidity factors, such as a diabetes and heart disease, for the incidences of stroke and whether it leads to mortality.

Strengths:

The idea is interesting and data are compelling. The results are technically solid when presented, but in many cases statistical analyses are yet to be carried out to support statements of statistical significance.

The authors identify specific genetic loci that increase the risk of a stroke and how they differ by region.

Weaknesses:

The presentation is not focused. It is important to include p-values for all comparisons and focus the presentation on the main effects from the dataset analysis.

Reviewer #2 (Public Review):

This is a genome-wide association study of COVID-19 in individuals of admixed American ancestry (AMR) recruited from Brazil, Colombia, Ecuador, Mexico, Paraguay and Spain. After quality control and admixture analysis, a total of 3,512 individuals were interrogated for 10,671,028 genetic variants (genotyped + imputed). The genetic association results for these cohorts were meta-analyzed with the results from The Host Genetics Initiative (HGI), involving 3,077 cases and 66,686 controls. The authors found two novel genetic loci associated with COVID-19 at 2q24.2 (rs13003835) and 11q14.1 (rs77599934), and other two independent signals at 3p21.31 (rs35731912) and 6p21.1 (rs2477820) already reported as associated with COVID-19 in previous GWASs. Additional meta-analysis with other HGI studies also suggested risk variants near CREBBP, ZBTB7A and CASC20 genes.

Strengths:

These findings rely on state-of-the-art methods in the field of Statistical Genomics and help to address the issue of low number of GWASs in non-European populations, ultimately contributing to reduce health inequalities across the globe.

Weaknesses:

There is no replication cohort, as acknowledged by the authors (page 29, line 587) and no experimental validation to assess the biological effect of putative causal variants/genes. Thus, the study provides good evidence of association, rather than causation, between the genetic variants and COVID-19.

Comments on latest version:

The issues identified in the first round of review were well addressed by the authors in the revised version of the manuscript.

Reviewer #2 (Public review):

Summary:

The study by Rößling et al. addresses the link between the biochemical constitution of the cell wall, in particular the methylesterification state of pectin with signalling induced by the extracellular RALF peptide. The work suggests that only in the presence of demethylesterifies pectin, RALF is able to trigger activation of its receptor FERONIA (FER).<br /> Remarkably, the application of RALF peptides leads to rather dramatic FER-dependent changes in wall integrity and plasma membrane invaginations not observed before. Interestingly, RALF can be out-titrated from the wall by short pectin fragments. In addition, the study provides further evidence for multiple FER-dependent pathways by showing the presence of LRX proteins is not required for the pectin/RALF mediated signalling.

Strengths:

This work provides fundamental insight into a complex emerging pathway, or perhaps several pathways, linking pectin sensing, pectin structure and RALF/FER signalling. The study provides convincing evidence that pectin methylesterase activity is required for RALF sensing, indicating that the physical interaction of RALFs with the cell wall is important for signalling. Beyond that, the study documents very clearly how profoundly RALF signalling can affect cell wall integrity and membrane topology.

Weaknesses:

Not a weakness per se, as it cannot be avoided, but drawing conclusions from genetic material with altered pectin always suffers from the possibility of secondary effects as this cell wall component is under heavy surveillance and able to respond plastically to different cues. However, the authors take that into account and have performed adequate controls to minimize that possibility.

Reviewer #2 (Public review):

The reviewed manuscript "Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion" presents exciting results on the mechanisms governing the assembly and disassembly of the vimentin cytoskeleton. They show, using live-cell imaging, that changes in the intracellular ionic strength induce rapid and dramatic changes to the integrity of the vimentin cytoskeleton. Interestingly, mutants of vimentin with net positive or negative charges display notably different responses to hypotonic stress (and thus changes to the intracellular ionic strength). Even more interesting, the ionic strength-driven mechanism seems to generalize to the several other intermediate filaments explored here. These results are of high interest to the broader cytoskeleton field. A major caveat is that essentially every experiment in the paper is n=1, showing example images of a single cell. The experiments were not repeated, and the results were not quantified. Purported differences between experimental variables/conditions lack statistical significance. Generalization of the ionic strength-based mechanism is hindered by the fact that only one cell type was tested for each cytoskeletal protein. Another caveat is that the fluorescently tagged vimentin used thoroughly in this work is exogenous and overexpressed; it is unclear if the observed effects would also occur at endogenous concentrations of vimentin. As it is currently presented, it is my opinion that all four main figures in this work - although interesting and quite likely correct - should be interpreted as preliminary data by readers.

Reviewer #3 (Public review):

Summary:

The authors provide compelling evidence for the causal role of the subthalamic nucleus (STN) in perceptual decision-making. By recording from a large number of STN neurons and using microstimulation, they demonstrate the STN's involvement in setting decision bounds, scaling evidence accumulation, and modulating non-decision time.

Strengths:

The study tested three hypotheses about the STN's function and identified distinct STN subpopulations whose activity patterns support predictions from previous computational models. The experiments are well-designed, the analyses are rigorous, and the results significantly advance our understanding of the STN's multi-faceted role in decision formation.

Weaknesses:

While the study provides valuable insights into the STN's role in decision-making, there are a few areas that could be improved. First, the interpretation of the neural subpopulations' activity patterns in relation to the computational models should be clarified, as the observed patterns may not directly correspond to the specific signals predicted by the models. Second, a neural population model could be employed to better understand how the STN population jointly contributes to decision-making dynamics.

Reviewer #2 (Public Review):

Cryo_EM structures of the Kv1.2 channel in the open, inactivated, toxin complex and in Na+ are reported. The structures of the open and inactivated channels are merely confirmatory of previous reports. The structures of the dendrotoxin bound Kv1.2 and the channel in Na+ are new findings that will of interest to the general channel community.

Review of the resubmission:

I thank the authors for making the changes in their manuscript as suggested in the previous review. The changes in the figures and the additions to the text do improve the manuscript. The new findings from a further analysis of the toxin channel complex are welcome information on the mode of the binding of dendrotoxin.

Reviewing #2 (Public Review):

This paper reports the role of the Isoform II of RUNX2 in activating PRDX2 expression to suppress ferroptosis in oral squamous cell carcinoma (OSCC).<br /> The following major issues should be addressed.

A major postulate of this study is the specific role of RUNX2 isoform II compared to isoform I.

Figure 1F shows association between patient survival and Iso II expression, but nothing is shown for Iso I, this should be added, in addition the number of patients at risk in each category should be shown.<br /> The authors test Iso I and Iso II overexpression in CAL27 or SCC-9 model cell lines. In Fig. 2A in CAL27, the overexpression of Iso II is much stronger than Iso I so it seems premature to draw any conclusions. More importantly, however, no Iso I silencing is shown in either of the cell lines nor the xenografted tumours. This is absolutely essential for the authors hypothesis and should be tested using shRNA in cells and xenografted tumours.

A major conclusion of this study is that Iso II expression suppresses ferroptosis. To support this idea, the authors use the inhibitor Ferrostatin-1 (Fer-1). While Fer-1 typically does not lead to a 100% rescue, here the effect is only marginal and as shown in Figures 3F and G only marginally better than Z-VAD or Necrostatin 1. These data do not support the idea that the major cause of cell death is ferroptosis. Instead, Iso II silencing leads to cell death through different pathways. The authors should acknowledge this and rephrase the conclusion of the paper accordingly.<br /> Moreover, the authors consistently confound cell proliferation with cell death.

In Fig. 4A the authors investigate GPX1 expression, whereas GPX4 is often the key ferroprosis regulator, this has to be tested. This is important as the authors also test the effect of the GPX4 inhibitor RSL3, however, the authors do not determine IC50 values of the different cell lines with or without Iso II overexpression or silencing or compared to other RSL3 sensitive or resistant cells. Without this information, no conclusions can be drawn.

In summary, while the authors show that RUNX2 Iso II expression enhances cell survival, the idea that cell death is principally via ferroptosis is not fully established by the data. The authors should modify their conclusions accordingly.

Reviewer #2 (Public review):

Summary:

Mark and colleagues test the hypothesis that entorhinal cortical representations may contain abstract structural information that facilitates generalization across structurally similar contexts. To do so, they use a method called "subspace generalization" designed to measure abstraction of representations across different settings. The authors validate the method using hippocampal place cells and entorhinal grid cells recorded in a spatial task, then perform simulations that support that it might be useful in aggregated responses such as those measured with fMRI. Then the method is applied to fMRI data that required participants to learn relationships between images in one of two structural motifs (hexagonal grids versus community structure). They show that the BOLD signal within an entorhinal ROI shows increased measures of subspace generalization across different tasks with the same hexagonal structure (as compared to tasks with different structures) but that there was no evidence for the complementary result (ie. increased generalization across tasks that share community structure, as compared to those with different structures). Taken together, this manuscript describes and validates a method for identifying fMRI representations that generalize across conditions and applies it to reveal entorhinal representations that emerge across specific shared structural conditions.

Strengths:

I found this paper interesting both in terms of its methods and its motivating questions. The question asked is novel and the methods employed are new - and I believe this is the first time that they have been applied to fMRI data. I also found the iterative validation of the methodology to be interesting and important - showing persuasively that the method could detect a target representation - even in the face of a random combination of tuning and with the addition of noise, both being major hurdles to investigating representations using fMRI.

Weaknesses:

In part because of the thorough validation procedures, the paper came across to me as a bit of a hybrid between a methods paper and an empirical one. However, I have some concerns, both on the methods development/validation side, and on the empirical application side, which I believe limit what one can take away from the studies performed.

Regarding the methods side, while I can appreciate that the authors show how the subspace generalization method "could" identify representations of theoretical interest, I felt like there was a noticeable lack of characterization of the specificity of the method. Based on the main equation in the results section of the paper, it seems like the primary measure used here would be sensitive to overall firing rates/voxel activations, variance within specific neurons/voxels, and overall levels of correlation among neurons/voxels. While I believe that reasonable pre-processing strategies could deal with the first two potential issues, the third seems a bit more problematic - as obligate correlations among neurons/voxels surely exist in the brain and persist across context boundaries that are not achieving any sort of generalization (for example neurons that receive common input, or voxels that share spatial noise). The comparative approach (ie. computing difference in the measure across different comparison conditions) helps to mitigate this concern to some degree - but not completely - since if one of the conditions pushes activity into strongly spatially correlated dimensions, as would be expected if univariate activations were responsive to the conditions, then you'd expect generalization (driven by shared univariate activation of many voxels) to be specific to that set of conditions. A second issue in terms of the method is that there is no comparison to simpler available methods. For example, given the aims of the paper, and the introduction of the method, I would have expected the authors to take the Neuron-by-Neuron correlation matrices for two conditions of interest, and examine how similar they are to one another, for example by correlating their lower triangle elements. Presumably, this method would pick up on most of the same things - although it would notably avoid interpreting high overall correlations as "generalization" - and perhaps paint a clearer picture of exactly what aspects of correlation structure are shared. Would this method pick up on the same things shown here? Is there a reason to use one method over the other?

Regarding the fMRI empirical results, I have several concerns, some of which relate to concerns with the method itself described above. First, the spatial correlation patterns in fMRI data tend to be broad and will differ across conditions depending on variability in univariate responses (ie. if a condition contains some trials that evoke large univariate activations and others that evoke small univariate activations in the region). Are the eigenvectors that are shared across conditions capturing spatial patterns in voxel activations? Or, related to another concern with the method, are they capturing changing correlations across the entire set of voxels going into the analysis? As you might expect if the dynamic range of activations in the region is larger in one condition than the other? My second concern is, beyond the specificity of the results, they provide only modest evidence for the key claims in the paper. The authors show a statistically significant result in the Entorhinal Cortex in one out of two conditions that they hypothesized they would see it. However, the effect is not particularly large. There is currently no examination of what the actual eigenvectors that transfer are doing/look like/are representing, nor how the degree of subspace generalization in EC may relate to individual differences in behavior, making it hard to assess the functional role of the relationship. So, at the end of the day, while the methods developed are interesting and potentially useful, I found the contributions to our understanding of EC representations to be somewhat limited.

Reviewer #2 (Public review):

Summary:

Wolbachia are maternally transmitted bacteria that can manipulate host reproduction in various ways. Some Wolbachia induce male killing (MK), where the sons of infected mothers are killed during development. Several MK-associated genes have been identified in Homona magnanima, including Hm-oscar and wmk-1-4, but the mechanistic links between these Wolbachia genes and MK in the native host are still unclear.

In this manuscript, Arai et al. show that Hm-oscar is the gene responsible for Wolbachia-induced MK in Homona magnanima. They provide evidence that Hm-Oscar functions through interactions with the sex determination system. They also found that Hm-Oscar disrupts sex determination in male embryos by inducing female-type dsx splicing and impairing dosage compensation. Additionally, Hm-Oscar suppresses the function of Masc. The manuscript is well-written and presents intriguing findings. The results support their conclusions regarding the diversity and commonality of MK mechanisms, contributing to our understanding of the mechanisms and evolutionary aspects of Wolbachia-induced MK.

Strengths/weaknesses:

(1) The authors found that transient overexpression of Hm-oscar, but not wmk-1-4, in Wolbachia-free H. magnanima embryos induces female-biased sex ratios. These results are striking and mirror the phenotype of the wHm-t infected line (WT12). However, Table 1 lists the "male ratio," while the text presents the "female ratio" with standard deviation. For consistency, the calculation term should be uniform, and the "ratio" should be listed for each replicate.

(2) The error bars in Figure 3 are quite large, and the figure lacks statistical significance labels. The authors should perform statistical analysis to demonstrate that Hm-oscar-overexpressed male embryos have higher levels of Z-linked gene expression.

(3) The authors demonstrated that Hm-Oscar suppresses the masculinizing functions of lepidopteran Masc in BmN-4 cells derived from the female ovaries of Bombyx mori. They should clarify why this cell line was chosen and its biological relevance. Additionally, they should explain the rationale for evaluating the expression levels of the male-specific BmIMP variant and whether it is equivalent to dsx.

(4) Although the authors show that Hm-oscar is involved in Wolbachia-induced MK in Homona magnanima and interacts with the sex determination system in lepidopteran insects, the precise molecular mechanism of Hm-oscar-induced MK remains unclear. Further studies are needed to elucidate how Hm-oscar regulates Homona magnanima genes to induce MK, though this may be beyond the scope of the current manuscript.

Reviewer #2 (Public Review):

I have carefully reviewed the manuscript titled "Pronounced expression of extracellular matrix proteoglycans regulated by Ant pathway underlies the parallel evolution of lip hypertrophy in East African cichlids." I commend the authors for their work on elucidating the mechanism underlying lip thickening that has evolved in parallel across three lakes in Africa.

The use of histological comparison, proteomics, and transcriptomics methods to investigate this phenomenon is commendable and adds depth to the study. The findings indicate that the overexpression of proteoglycans is the cause of lip thickening and provides valuable insights into the evolutionary process.

I found the writing style to be clear and the explanations provided are easy to understand. Overall, I did not identify any significant issues with the manuscript.

Reviewer #2 (Public review):

Summary:<br /> The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

Strengths:<br /> The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. The use of state-of-the-art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

Weaknesses:<br /> Some of the data seems to contradict the conclusions. And it is unclear how direct the relationships are between Prmt1 and SFPQ.

Reviewer #3 (Public review):

Hall et al. benchmarked different variant calling methods on Nanopore reads of bacterial samples and compared the performance of Nanopore to short reads produced with Illumina sequencing. To establish a common ground for comparison, the authors first generated a variant truthset for each sample and then projected this set to the reference sequence of the sample to obtain a mutated reference. Subsequently, Hall et al. called SNPs and small indels using commonly used deep learning and conventional variant callers and compared the precision and accuracy from reads produced with simplex and duplex Nanopore sequencing to Illumina data. The authors did not investigate large structural variation, which is a major limitation of the current manuscript. It will be very interesting to see a follow-up study covering this much more challenging type of variation.

In their comprehensive comparison of SNPs and small indels, the authors observed superior performance of deep learning over conventional variant callers when Nanopore reads were basecalled with the most accurate (but also computationally very expensive) model, even exceeding Illumina in some cases. Not surprisingly, Nanopore underperformed compared to Illumina when basecalled with the fastest (but computationally much less demanding) method with the lowest accuracy. The authors then investigated the surprisingly higher performance of Nanopore data in some cases and identified lower recall with Illumina short read data, particularly from repetitive regions and regions with high variant density, as the driver. Combining the most accurate Nanopore basecalling method with a deep learning variant caller resulted in low error rates in homopolymer regions, similar to Illumina data. This is remarkable, as homopolymer regions are (or, were) traditionally challenging for Nanopore sequencing.

Lastly, Hall et al. provided useful information on the required Nanopore read depth, which is surprisingly low, and the computational resources for variant calling with deep learning callers. With that, the authors established a new state-of-the-art for Nanopore-only variant calling on bacterial sequencing data. Most likely these findings will be transferred to other organisms as well or at least provide a proof-of-concept that can be built upon.

As the authors mention multiple times throughout the manuscript, Nanopore can provide sequencing data in nearly real-time and in remote regions, therefore opening up a ton of new possibilities, for example for infectious disease surveillance. In these scenarios, computational resources can be very limited. The highest-performing variant calling method, as established in this study, requires the computationally very expensive sup and/or duplex nanopore basecalling, while the least computationally demanding basecalling method underperforms. To comprehensively guide users through the computational resources required for basecalling and variant calling, the authors provide runtime benchmarks assuming GPU access.

Reviewer #2 (Public Review):

Summary:

Fan et al. investigated the relationship between early acute myocardial infarction (eAMI) and disturbances in the gut microbiome using metabolomics and metagenomics analyses. They studied 30 eAMI patients and 26 healthy controls, finding elevated levels of long-chain fatty acids (LCFA) in the plasma of eAMI patients.

Strengths:

The research attributed a substantial portion of LCFA variance in eAMI to changes in the gut microbiome, as indicated by omics analyses. Computational profiling of gut bacteria suggested structural variations linked to LCFA variance. The authors also conducted molecular docking simulations and platelet assays, revealing that eAMI-associated LCFAs may enhance platelet aggregation.

Weaknesses:

The results should be validated using different assays, and animal models should be considered to explore the mechanisms of action.

Reviewer #2 (Public Review):

Summary:

The manuscript examines an important question about how an inaccessible, natural forgotten memory can be retrieved through engram ensemble reactivation. It uses a variety of strategies including optogenetics, behavioral and pharmacological interventions to modulate engram accessibility. The data characterize the time course of natural forgetting using an object recognition task, in which animals can retrieve 1 day and 1 week after learning, but not 2 weeks later. Forgetting is correlated with lower levels of cell reactivation (c-fos expression during learning compared to retrieval) and reduction in spine density and volume in the engram cells. Artificial activation of the original engram was sufficient to induce recall of the forgotten object memory while artificial inhibition of the engram cells precluded memory retrieval. Mice housed in an enriched environment had a slower rate of forgetting, and a brief reminder before the retrieval session promoted retrieval of a forgotten memory. Repeated reintroduction to the training context in the absence of objects accelerated forgetting. Additionally, activation of Rac1-mediated plasticity mechanisms enhanced forgetting, while its inhibition prolonged memory retrieval. Authors also reproduce the behavioral findings using a computational model inspired by Rescorla-Wagner model. In essence, the model proposes that forgetting is a form of adaptive learning that can be updated based on prediction error rules in which engram relevancy is altered in response to environmental feedback.

Strengths:

(1) The data presented in the current paper are consistent with the authors claim that seemingly forgotten engrams are, in fact, accessible. This suggests that retrieval deficits can lead to memory impairments rather than a loss of the original engram (at least in some cases).

(2) The experimental procedures and statistics are appropriate, and the behavioral effects appear to be very robust. Several key effects are replicated multiple times in the manuscript.

Comments on revised version:

The authors have adequately addressed my prior concerns.

Reviewer #2 (Public Review):

Summary:

This is an interesting and well-written manuscript that seeks to detail performance on two human psychophysical experiments designed to look at the relative contributions of transient and sustained components of a multisensory (i.e., audiovisual) stimulus to their integration. The work is framed within the context of a model previously developed by the authors and now somewhat revised to better incorporate the experimental findings. The major takeaway from the paper is that transient signals carry the vast majority of the information related to the integration of auditory and visual cues, and that the Multisensory Correlation Detector (MCD) model not only captures the results of the current study, but is also highly effective in capturing the results of prior studies focused on temporal and causal judgments.

Strengths:

Overall the experimental design is sound and the analyses well performed. The extension of the MCD model to better capture transients make a great deal of sense in the current context, and it is very nice to see the model applied to a variety of previous studies.

Comments on the revised version:

In the revised manuscript, the authors have done an excellent job of responding to the prior critiques. I have no additional concerns or comments.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

The researchers did not include a visual print task in the EEG task, which limits analysis of reading-specific regions such as the visual word form area, which is a commonly hypoactivated region in dyslexia. This region is a common one of interest in dyslexia, yet the researchers measured the I/E balance in only one region of interest, specific to the language network. Further, this work does not consider prior studies reporting neural inconsistency; a potential consequence of increased neural noise, which has been reported in several studies and linked with candidate-dyslexia gene variants (e.g., Centanni et al., 2018, 2022; Hornickel & Kraus, 2013; Neef et al., 2017). While E/I imbalance may not be a cause of increased neural noise, other potential mechanisms remain and should be discussed.

Reviewer #2 (Public Review):

Summary:

The results presented demonstrate that AAV2-CFI gene therapy delivers long-term and marginally higher FI protein in vitreous humor that results in a concomitant reduction in the FB activation product Ba. However, the lack of clinical efficacy in the phase I/II study, possibly due to lower in vitro potency when compared to currently approved pegcetacoplan, raises important considerations for the utility of this therapeutic approach. Despite the early termination of the PPY988 clinical development program, the study achieved significant milestones, including the implementation of subretinal gene therapy delivery in older adults, complement biomarker comparison between serial vitreous humor and aqueous humor samples and vitreous humor proteomic assessment via Olink.

Strengths:

Long-term augmentation of FI protein in vitreous humor over 96 weeks and reduction of FB breakdown product Ba in vitreous humor suggests modulation of the complement system. Developed a novel in vitro assay suggesting FI's ability to reduce C3 convertase activity is weaker than pegcetacoplan and FH and may suggest a higher dose of FI will be required for clinical efficacy. Warn of the poor correlation between vitreous humor and aqueous humor biomarkers and suggest aqueous humor may not be a reliable proxy for vitreous humor with regard to complement activation/inhibition studies.

Weaknesses:

The vitrectomy required for the subretinal route of administration causes a long-term loss of total protein and may influence the interpretation of complement biomarker results even with normalization. The modified in vitro assay of complement activation suggests a several hundred-fold increase in FI protein is required to significantly affect C3a levels. Interestingly, the in vitro assay demonstrates 100% inhibition of C3a with pegcetacoplan and FH therapeutics, but only a 50% reduction with FI even at the highest concentrations tested. This observation suggests FI may not be rate-limiting for negative complement regulation under the in vitro conditions tested and potentially in the eye. It is unclear if pharmacokinetic and pharmacodynamic properties in aqueous humor and vitreous humor compartments are reliable predictors of FI level/activity after subretinal delivery AAV2-CFI gene therapy.

Reviewer #2 (Public Review):

Summary:

Englert et al. use a novel modelling approach called functional connectome-based Hopfield Neural Networks (fcHNN) to describe spontaneous and task-evoked brain activity and the alterations in brain disorders. Given its novelty, the authors first validate the model parameters (the temperature and noise) with empirical resting-state function data and against null models. Through the optimisation of the temperature parameter, they first show that the optimal number of attractor states is four before fixing the optimal noise that best reflects the empirical data, through stochastic relaxation. Then, they demonstrate how these fcHNN-generated dynamics predict task-based functional activity relating to pain and self-regulation. To do so, they characterise the different brain states (here as different conditions of the experimental pain paradigm) in terms of the distribution of the data on the fcHNN projections and flow analysis. Lastly, a similar analysis was performed on a population with autism condition. Through Hopfield modeling, this work proposes a comprehensive framework that links various types of functional activity under a unified interpretation with high predictive validity.

Strengths:

The phenomenological nature of the Hopfield model and its validation across multiple datasets presents a comprehensive and intuitive framework for the analysis of functional activity. The results presented in this work further motivate the study of phenomenological models as an adequate mechanistic characterisation of large-scale brain activity.

Following up on Cole et al. 2016, the authors put forward a hypothesis that many of the changes to the brain activity, here, in terms of task-evoked and clinical data, can be inferred from the resting-state brain data alone. This brings together neatly the idea of different facets of brain activity emerging from a common space of functional (ghost) attractors.

The use of the null models motivates the benefit of non-linear dynamics in the context of phenomenological models when assessing the similarity to the real empirical data.

Weaknesses:

While the use of the Hopfield model is neat and very well presented, it still begs the question of why to use the functional connectome (as derived by activity flow analysis from Cole et al. 2016). Deriving the functional connectome on the resting-state data that are then used for the analysis reads as circular. If the fcHNN derives the basins of four attractors that reflect the first two principal components of functional connectivity, it perhaps suffices to use the empirically derived components alone and project the task and clinical data on it without the need for the fcHNN framework.

As presented here, the Hopfield model is excellent in its simplicity and power, and it seems suited to tackle the structure-function relationship with the power of going further to explain task-evoked and clinical data. The work could be strengthened if that was taken into consideration. As such the model would not suffer from circularity problems and it would be possible to claim its mechanistic properties. Furthermore, as mentioned above, in the current setup, the connectivity matrix is based on statistical properties of functional activity amongst regions, and as such it is difficult to talk about a certain mechanism. This contention has for example been addressed in the Cole et al. 2016 paper with the use of a biophysical model linking structure and function, thus strengthening the mechanistic claim of the work.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors report GCaMP fiber-photometry recordings from the GnRH neuron distal projections in the ventral arcuate nucleus. The recordings are taken from intact, male and female, freely behaving mice. The report three patterns of neuronal activity:

(1) Abrupt increases in the Ca2+ signals that are perfectly correlated with LH pulses.

(2) A gradual, yet fluctuating (with a slow ultradian frequency), increase in activity, which is associated with the onset of the LH surge in female animals.

(3) Clustered (high frequency) baseline activity in both female and male animals.

Strengths:

The GCaMP fiber-photometry recordings reported here are the first direct recordings from GnRH neurones in vivo. These recordings have uncovered a rich repertoire of activity suggesting the integration of distinct "surge" and "pulse" generation signals, and an ultradian rhythm during the onset of the surge.

Weaknesses:

The data analysis method used for the characterisation of the ultradian rhythm observed during the onset of the surge is not detailed enough. Hence, I'm left wondering whether this rhythm is in any way correlated with the clusters of activity observed during the rest of the cycle and which have similar duration.

Reviewer #2 (Public Review):

Summary:

This paper aimed to determine the role EP sst+ neurons play in a probabilistic switching task.

Strengths:

The in vivo recording of the EP sst+ neuron activity in the task is one of the strongest parts of this paper. Previous work had recorded from the EP-LHb population in rodents and primates in head-fixed configurations, the recordings of this population in a freely moving context is a valuable addition to these studies and has highlighted more clearly that these neurons respond both at the time of choice and outcome.

The use of a refined intersectional technique to record specifically the EP sst+ neurons is also an important strength of the paper. This is because previous work has shown that there are two genetically different types of glutamatergic EP neurons that project to the LHb. Previous work had not distinguished between these types in their recordings so the current results showing that the bidirectional value signaling is present in the EP sst+ population is valuable.

Weaknesses:

(1) One of the main weaknesses of the paper is to do with how the effect of the EP sst+ neurons on the behavior was assessed.

(a) All the manipulations (blocking synaptic release and blocking glutamatergic transmission) are chronic and more importantly the mice are given weeks of training after the manipulation before the behavioral effect is assessed. This means that as the authors point out in their discussion the mice will have time to adjust to the behavioral manipulation and compensate for the manipulations. The results do show that mice can adapt to these chronic manipulations and that the EP sst+ are not required to perform the task. What is unclear is whether the mice have compensated for the loss of EP sst+ neurons and whether they play a role in the task under normal conditions. Acute manipulations or chronic manipulations without additional training would be needed to assess this.

(b) Another weakness is that the effect of the manipulations was assessed in the 90/10 contingency version of the task. Under these contingencies, mice integrate past outcomes over fewer trials to determine their choice and animals act closer to a simple win-stay-lose switch strategy. Due to this, it is unclear if the EP sst+ neurons would play a role in the task when they must integrate over a larger number of conditions in the less deterministic 70/30 version of the task.

The authors show an intriguing result that the EP sst+ neurons are excited when mice make an ipsilateral movement in the task either toward or away from the center port. This is referred to as a choice response, but it could be a movement response or related to the predicted value of a specific action. Recordings while mice perform movement outside the task or well-controlled value manipulations within the session would be needed to really refine what these responses are related to.

(2) The authors conclude that they do not see any evidence for bidirectional prediction errors. It is not possible to conclude this. First, they see a large response in the EP sst+ neurons to the omission of an expected reward. This is what would be expected of a negative reward prediction error. There are much more specific well-controlled tests for this that are commonplace in head-fixed and freely moving paradigms that could be tested to probe this. The authors do look at the effect of previous trials on the response and do not see strong consistent results, but this is not a strong formal test of what would be expected of a prediction error, either a positive or negative. The other way they assess this is by looking at the size of the responses in different recording sessions with different reward contingencies. They claim that the size of the reward expectation and prediction error should scale with the different reward probabilities. If all the reward probabilities were present in the same session this should be true as lots of others have shown for RPE. Because however this data was taken from different sessions it is not expected that the responses should scale, this is because reward prediction errors have been shown to adaptively scale to cover the range of values on offer (Tobler et al., Science 2005). A better test of positive prediction error would be to give a larger-than-expected reward on a subset of trials. Either way, there is already evidence that responses reflect a negative prediction error in their data and more specific tests would be needed to formally rule in or out prediction error coding especially as previous recordings have shown it is present in previous primate and rodent recordings.

(3) There are a lot of variables in the GLM that occur extremely close in time such as the entry and exit of a port. If two variables occur closely in time and are always correlated it will be difficult if not impossible for a regression model to assign weights accurately to each event. This is not a large issue, but it is misleading to have regression kernels for port entry and exits unless the authors can show these are separable due to behavioral jitter or a lack of correlation under specific conditions, which does not seem to be the case.

Reviewer #2 (Public review):

Deciphering the metabolic alterations characterizing the prediabetes-diabetes spectrum could provide early time windows for targeted preventive measures to extend precision medicine while avoiding disproportionate healthcare costs. The authors identified a panel of 9 circulating metabolites combined with basic clinical variables that significantly improved the prediction from prediabetes to diabetes. These findings provided insights into the integration of these metabolites into clinical and public health practice.

Comments on the revised version:

Congratulations to the authors. I have no more comments.

Reviewer #3 (Public review):

Summary:

This study aims to understand the role of GABA-ergic inhibition in the human MT+ region in predicting visuo-spatial intelligence through a combination of behavioral measures, fMRI (for functional connectivity measurement), and MRS (for GABA/glutamate concentration measurement). It provides useful evidence that GABA levels in the sensory cortex, such as in the human MT+, are associated with visuo-spatial ability, thus highlighting the importance of GABA-ergic inhibition in complex cognition.

Strengths:

(1) Comprehensive Approach: The study adopts a multi-level approach, i.e., neurochemical analysis of GABA levels, functional connectivity, and behavioral measures to provide a holistic understanding of the relationship between GABA-ergic inhibition and visuo-spatial intelligence.<br /> (2) Sophisticated Techniques: The use of ultra-high field magnetic resonance spectroscopy (MRS) technology for measuring GABA and glutamate concentrations in the MT+ region is a recent development.

Weaknesses:

The authors have carefully addressed the major weaknesses previously mentioned.

Reviewer #2 (Public Review):

Summary:

Kisspeptin neurons of the arcuate nucleus (ARC) are thought to be responsible for the pulsatile GnRH secretory pattern and to mediate feedback regulation of GnRH secretion by estradiol (E2). Evidence in the literature, including the work of the authors, indicates that ARC kisspeptin coordinate their activity through reciprocal synaptic interactions and the release of glutamate and of neuropeptide neurokinin B (NKB), which they co-express. The authors show here that E2 regulates the expression of genes encoding different voltage-dependent calcium channels, calcium-dependent potassium channels and canonical transient receptor potential (TRPC5) channels and of the corresponding ionic currents in ARC kisspeptin neurons. Using computer simulations of the electrical activity of ARC kisspeptin neurons, the authors also provide evidence of what these changes translate into in terms of these cells' firing patterns. The experiments reveal that E2 upregulates various voltage-gated calcium currents as well as 2 subtypes of calcium-dependent potassium currents while decreasing TRPC5 expression (an ion channel downstream of NKB receptor activation), the slow excitatory synaptic potentials (slow EPSP) elicited in ARC kisspeptin neurons by NKB release and expression of the G protein-associated inward-rectifying potassium channel (GIRK). Based on these results, and on those of computer simulations, the authors propose that E2 promotes a functional transition of ARC kisspeptin neurons from neuropeptide-mediated sustained firing that supports coordinated activity for pulsatile GnRH secretion to a less intense burst-like firing pattern that could favor glutamate release from ARC kisspeptin. The authors suggest that the latter might be important for the generation of the preovulatory surge in females.

Strengths:

The authors combined multiple approaches in vitro and in silico to gain insights into the impact of E2 on the electrical activity of ARC kisspeptin neurons. These include patch-clamp electrophysiology combined with selective optogenetic stimulation of ARC kisspeptin neurons, reverse transcriptase quantitative PCR, pharmacology and CRISPR-Cas9-mediated knockdown of the Trpc5 gene. The addition of computer simulations for understanding the impact of E2 on the electrical activity of ARC kisspeptin cells is also a strength.<br /> The authors add interesting information on the complement of ionic currents in ARC kisspeptin neurons and on their regulation by E2 to what was already known in the literature. Pharmacological and electrophysiological experiments appear of the highest standards and robust statistical analyses are provided throughout. The impact of E2 replacement on calcium and potassium currents is compelling. Likewise, the results of Trpc5 gene knockdown do provide good evidence that the TRPC5 channel plays a key role in mediating the NKB-mediated slow EPSP. Surprisingly, this also revealed an unsuspected role for this channel in regulating the membrane potential and excitability of ARC kisspeptin neurons.

Weaknesses:

The manuscript also has weaknesses that obscure some of the conclusions drawn by the authors.

One is that the authors compare here two conditions, OVX versus OVX replaced with high E2, that may not reflect the physiological conditions under which the proposed transition between neuropeptide-dependent sustained firing and less intense burst firing might take place (i.e. the diestrous [low E2] and proestrous [high E2] stages of the estrous cycle). This is an important caveat to keep in mind when interpreting the authors' findings. Indeed, that E2 alters certain ionic currents when added back to OVX females, does not mean that the magnitude of all of these ionic currents will vary during the estrous cycle.

In addition, although the computational modeling indicates a role of the various E2-modulated conductances in causing a transition in ARC kisspeptin neuron firing pattern, their role is not directly tested in physiological recordings, weakening the link between these changes and the shift in firing patterns.

Overall, the manuscript provides interesting information about the effects of E2 on specific ionic currents in ARC kisspeptin neurons and some insights into the functional impact of these changes. However, some of the conclusions of the work, with regard, in particular, to the role of these changes in ion channels and their implications for the LH surge, are not fully supported by the findings.

Reviewer #2 (Public Review):

Summary:

This MR study by Zhao et al. provides a comprehensive hypothesis-free approach to identifying risk and protective factors causal to Alzheimer's Disease (AD).

Strengths:

The study employs a comprehensive, hypothesis-free approach, which is novel over traditional hypothesis-driven studies. Also, causal associations between risk/protective factors and AD were addressed using genetic instruments and analysis.

Reviewer #2 (Public Review):

The authors investigated the role of inflammatory molecules in diastolic dysfunction and screened antiviral and cardioprotective pharmacological agents for their potential to reverse inflammation-mediated diastolic dysfunction. This study focuses on heart failure with preserved ejection fraction (HFpEF) in people living with HIV (PLWH), a condition often challenging to study due to the lack of suitable animal models. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), researchers simulated HFpEF in vitro. They observed that inflammatory cytokines impaired cardiomyocyte relaxation, mimicking HFpEF, while SGLT2 inhibitors and mitochondrial antioxidants reversed this effect. Exposure to serum from HIV patients did not induce dysfunction in hiPSC-CMs. These findings suggest hiPSC-CMs as a promising model for understanding HFpEF mechanisms and testing potential treatments.

Comments on revised version:

The revised manuscript has been improved satisfactorily. The authors also have addressed all of my concerns.

Reviewer #2 (Public Review):

Summary:

This article explores the regenerative effects of recombinant PTH analogues on osteogenesis.

Strengths:

Although PTH has known to induce the activity of osteoclasts, accelerating bone resorption, paradoxically its intermittent use has become a common treat for osteoporosis. Previous studies successfully demonstrated this phenomenon in vivo, but most of them used rodent animal models, inevitably having a limitation. In this article, the authors tried to address this, using a beagle model, and assessed the osseointegrative effect of recombinant PTH analogues. As a result, the authors clearly observed the regenerative effects of PTH analogues, and compared the efficacy, using histologic, biochemical, and radiologic measurement for surgical-endocrinal combined large animal models. The data seem to be solid, and has potential clinical implications.

Weaknesses:

All the issues that I raised have been resolved in the revision process.

Overall, this paper is well-written and has clarity and consistency for a broader readership.

Reviewer #2 (Public Review):

The focus of this manuscript was to investigate whether Kv1.8 channels, which have previously been suggested to be expressed in type I hair cells of the mammalian vestibular system, are responsible for the potassium conductance gK,L. This is an important study because gK,L is known to be crucial for the function of type I hair cells, but the channel identity has been a matter of debate for the past 20 years. The authors have addressed this research topic by primarily investigating the electrophysiological properties of the vestibular hair cells from Kv1.8 knockout mice. Interestingly, gK,L was completely abolished in Kv1.8-deficient mice, in agreement with the hypothesis put forward by the authors based on the literature. The surprising observation was that in the absence of Kv1.8 potassium channels, the outward potassium current in type II hair cells was also largely reduced. Type II hair cells express the largely inactivating potassium conductance g,K,A, but not gK,L. The authors concluded that heteromultimerization of non-inactivating Kv1.8 and the inactivating Kv1.4 subunits could be responsible for the inactivating gK,A. Overall, the manuscript is very well written and most of the conclusions are supported by the experimental work. The figures are well described, and the statistical analysis is robust.

Reviewer #2 (Public Review):

Summary:

The authors provide evidence that helps resolve long-standing questions about the differential involvement of the frontal and posterior cortex in working memory. They show that whereas the early visual cortex shows stronger decoding of memory content in a memorization task vs a more complex categorization task, the frontal cortex shows stronger decoding during categorization tasks than memorization tasks. They find that task-optimized RNNs trained to reproduce the memorized orientations show some similarities in neural decoding to people. Together, this paper presents interesting evidence for differential responsibilities of brain areas in working memory.

Strengths:

This paper was strong overall. It had a well-designed task, best-practice decoding methods, and careful control analyses. The neural network modelling adds additional insight into the potential computational roles of different regions.

Weaknesses:

While the RNN model matches some of the properties of the task and decoding, its ability to reproduce the detailed findings of the paper was limited. Overall, the RRN model was not as well-motivated as the fMRI analyses.

Reviewer #2 (Public Review):

Summary:

The study proposes that many cancer driver mutations are not yet identified but could be identified if they harbor recurrent SNVs. The paper leverages the analysis from Paper #1 that used quantitative analysis to demonstrate that SNVs or CDNs seen 3 or more times are more likely to occur due to selection (ie a driver mutation) than they are to occur by chance or random mutation.

Strengths:

Empirically, mutation frequency is an excellent marker of a driver gene because canonical driver mutations typically have recurrent SNVs. Using the TCGA database, the paper illustrates that CDNs can identify canonical driver mutations (Figure 3) and that most CDNs are likely to disrupt protein function (Figure 2). In addition, CDNs can be shared between cancer types (Figure 4).

Weaknesses:

Driver alteration validation is difficult, with disagreements on what defines a driver mutation, and how many driver mutations are present in a cancer. The value proposed by the authors is that the identification of all driver genes can facilitate the design of patient-specific targeting therapies, but most targeted therapies are already directed towards known driver genes. There is an incomplete discussion of oncogenes (where activating mutations tend to target a single amino acid or repeat) and tumor suppressor genes (where inactivating mutations may be more spread across the gene). Other alterations (epigenetic, indels, translocations, CNVs) would be missed by this type of analysis.

The method could be more valuable when applied to the noncoding genome, where driver mutations in promoters or enhancers are relatively rare, or as yet to be discovered. Increasingly more cancers have had whole genome sequencing. Compared to WES, criteria for driver mutations in noncoding regions are less clear, and this method could potentially provide new noncoding driver CDNs. Observing the same mutation in more than one cancer specimen is empirically unusual, and the authors provide a solid quantitative analysis that indicates many recurrent mutations are likely to be cancer-driver mutations.

Reviewer #2 (Public Review):

The authors aim to investigate how voltage-gated calcium channel number, organization, and subunit composition lead to changes in synaptic activity at tonic and phasic motor neuron terminals, or type Is and Ib motor neurons in Drosophila. These neuron subtypes generate widely different physiological outputs, and many investigations have sought to understand the molecular underpinnings responsible for these differences. Additionally, these authors explore not only static differences that exist during the third-instar larval stage of development but also use a pharmacological approach to induce homeostatic plasticity to explore how these neuronal subtypes dynamically change the structural composition and organization of key synaptic proteins contributing to physiological plasticity. The Drosophila neuromuscular junction (NMJ) is glutamatergic, the main excitatory neurotransmitter in the human brain, so these findings not only expand our understanding of the molecular and physiological mechanisms responsible for differences in motor neuron subtype activity, but also contribute to our understanding of how the human brain and nervous system functions.

The authors employ state-of-the-art tools and techniques such as single-molecule localization microscopy 3D STORM and create several novel transgenic animals using CRISPR to expand the molecular tools available for exploration of synaptic biology that will be of wide interest to the field. Additionally, the authors use a robust set of experimental approaches from active zone level resolution functional imaging from live preparations to electrophysiology and immunohistochemical analyses to explore and test their hypotheses. All data appear to be robustly acquired and analyzed using appropriate methodology. The authors make important advancements to our understanding of how the different motor neuron subtypes, phasic and tonic-like, exhibit widely varying electrical output despite the neuromuscular junctions having similar ultrastructural composition in the proteins of interest, voltage gated calcium channel cacophony (cac) and the scaffold protein Bruchpilot (brp). The authors reveal the ratio of brp:cac appears to be a critical determinant of release probability (Pr), and in particular, the packing density of VGCCs and availability of brp. Importantly, the authors demonstrate a brp-dependent increase in VGCC density following acute philanthotoxin perfusion (glutamate receptor inhibitor). This VGCC increase appears to be largely responsible for the presynaptic homeostatic plasticity (PHP) observable at the Drosophila NMJ. Lastly, the authors created several novel CRISPR-tagged transgenic lines to visualize the spatial localization of VGCC subunits in Drosophila. Two of these lines, CaV5-C and stjV5-N, express in motor neurons and in the nervous system, localize at the NMJ, and most strikingly, strongly correlate with Pr at tonic and phasic-like terminals.

Reviewer #2 (Public Review):

Summary:

The authors aimed to investigate how microbial metabolites, such as hydrogen and short-chain fatty acids (SCFAs), influence feeding behavior and circadian gene expression in mice. Specifically, they sought to understand these effects in different microbial environments, including a reduced community model (EAM), germ-free mice, and SPF mice. The study was designed to explore the broader relationship between the gut microbiome and host circadian rhythms, an area that is not well understood. Through their experiments, the authors hoped to elucidate how microbial metabolism could impact circadian clock genes and feeding patterns, potentially revealing new mechanisms of gut microbiome-host interactions.

Strengths:

The manuscript presents a well-executed investigation into the complex relationship between microbial metabolites and circadian rhythms, with a particular focus on feeding behavior and gene expression in different mouse models. One of the major strengths of the work lies in its innovative use of a reduced community model (EAM) to isolate and examine the effects of specific microbial metabolites, which provides valuable insights into how these metabolites might influence host behavior and circadian regulation. The study also contributes to the broader understanding of the gut microbiome's role in circadian biology, an area that remains poorly understood. The experiments are thoughtfully designed, with a clear rationale that ties together the gut microbiome, metabolic products, and host physiological responses. The authors successfully highlight an intriguing paradox: the significant influence of microbial metabolites in the EAM model versus the lack of effect in germ-free and SPF mice, which adds depth to the ongoing exploration of microbial-host interactions. Despite some methodological concerns, the manuscript offers compelling data and opens up new avenues for research in the field of microbiome and circadian biology.

Weaknesses:

The manuscript, while providing valuable insights, has several methodological weaknesses that impact the overall strength of the findings. First, the process for stool collection lacks clarity, raising concerns about potential biases, such as the risk of coprophagia, which could affect the dry-to-wet weight ratio analysis and compromise the validity of these measurements. Additionally, the use of the term "circadian" in some contexts appears inaccurate, as "diurnal" might be more appropriate, especially given the uncertainty regarding whether the observed microbiome fluctuations are truly circadian. Another significant issue is the unexpected absence of an osmotic effect of lactulose in EAM mice, which contradicts the known properties of lactulose as an osmotic laxative. This finding requires further verification, including the use of a positive control, to ensure it is not artifactual. The presentation of qRT-PCR data as log2-fold changes, with a mean denominator, could introduce bias by artificially reducing variability, potentially leading to spurious findings or increased risk of Type I error. This approach may explain the unexpected activation of both the positive and negative limbs of the circadian clock. Moreover, the lack of detailed information on the primers and housekeeping genes used in the experiments is concerning, particularly given the importance of using non-circadian housekeeping genes for accurate normalization. The methods for measuring metabolic hormones, such as GLP-1 and GIP, are also not adequately described. If DPP-IV/protease inhibitor tubes were not used, the data could be unreliable due to the rapid degradation of these hormones by circulating proteases. Finally, the manuscript does not address the collection of hormone levels during both fasting and fed phases, a critical aspect for interpreting the metabolic impact of microbial metabolites. These methodological concerns collectively weaken the robustness of the study's results and warrant careful reconsideration and clarification by the authors.

Because of these weaknesses, the authors have partially achieved their aims by providing novel insights into the relationship between microbial metabolites and host circadian rhythms. The data do suggest that microbial metabolites can significantly influence feeding behavior and circadian gene expression in specific contexts. However, the unexpected absence of an osmotic effect of lactulose, the potential biases introduced by the log2-fold change normalization in qRT-PCR data, and the lack of clarity in critical methodological details weaken the overall conclusions. While the study provides valuable contributions to understanding the gut microbiome's role in circadian biology, the methodological weaknesses prevent a full endorsement of the authors' conclusions. Addressing these issues would be necessary to strengthen the support for their findings and fully achieve the study's aims.

Despite the methodological concerns raised, this work has the potential to make a significant impact on the field of circadian biology and microbiome research. The study's exploration of the interaction between microbial metabolites and host circadian rhythms in different microbial environments opens new avenues for understanding the complex interplay between the gut microbiome and host physiology. This research contributes to the growing body of evidence that microbial metabolites play a crucial role in regulating host behaviors and physiological processes, including feeding and circadian gene expression.

Reviewer #2 (Public Review):

The authors trained monkeys to discriminate peripheral visual cues and associate them with planning future saccades of an indicated direction. At the same time, the authors recorded single-unit neural activity in the cerebellar dentate nucleus. They demonstrated that substantial fractions of DN cells exhibited sustained modulation of spike rates spanning task epochs and carrying information about stimulus, response, and trial outcome. Finally, tracer injections demonstrated this region of the DN projects to a large number of targets including several known to interconnect the visual attention network. The data compellingly demonstrate the authors' central claims, and the analyses are well-suited to support the conclusions. Importantly, the study demonstrates that DN cells convey many motor and nonmotor variables related to task execution, event sequencing, visual attention, and arguably decision-making/working memory.

Reviewer #2 (Public Review):

Summary:<br /> This interesting study implicates the direct interaction between two multi-subunit complexes, known as the exocyst and septin complexes, in the function of both complexes during cytokinesis in fission yeast. While previous work from several labs had implicated roles for the exocyst and septin complexes in cytokinesis and cell separation, this study describes the importance of protein:protein interaction between these complexes in mediating the functions of these complexes in cytokinesis. Previous studies in neurons had suggested interactions between septins and exocyst complexes occur but the functional importance of such interactions was not known. Moreover, in baker's yeast where both of these complexes have been extensively studied - no evidence of such an interaction has been uncovered despite numerous studies which should have detected it. Therefore while exocyst:septin interactions appear to be conserved in several systems, it appears likely that budding yeast are the exception--having lost this conserved interaction.

Strengths:<br /> The strengths of this work include the rigorous analysis of the interaction using multiple methods including Co-IP of tagged but endogenously expressed proteins, 2 hybrid interaction, and Alphafold Multimer. Careful quantitative analysis of the effects of loss of function in each complex and the effects on localization and dynamics of each complex was also a strength. Taken together this work convincingly describes that these two complexes do interact and that this interaction plays an important role in post Golgi vesicle targeting during cytokinesis.

Weaknesses:<br /> The authors used Alphafold Multimer to predict (largely successfully) which subunits were most likely to be involved in direct interactions between the complexes. It would be very interesting to compare this to a parallel analysis on the budding yeast septin and exocyst complexes where it is quite clear that detectable interactions between the exocyst and septins (using the same methods) do not exist. Presumably the resulting pLDDT scores will be significantly lower. These are in silico experiments and should not be difficult to carry out.

Reviewer #2 (Public Review):

This interesting study focuses on the association between lifestyle factors and comprehensive and organ-specific biological aging in a multi-ethnic cohort from Southwest China. It stands out for its large sample size, longitudinal design, and robust statistical analysis.

Some issues deserve clarification to enhance this paper:

(1) How were the biochemical indicators for organ-specific biological ages chosen, and are these indicators appropriate? Additionally, a more detailed description of the multi-organ biological ages should be provided to help understand the distribution and characteristics of BAs.

(2) The authors categorized the HLI score into a dichotomous variable, which may cause a loss of information. How did the authors address this potential issue?

(3) Because lifestyle data are self-reported, they may suffer from recall bias. This issue needs to be addressed in the limitations section.

(4) It should be clarified whether the adjusted CA is the baseline value of CA. Additionally, why did the authors choose models with additional adjustments for time-invariant variables as their primary analysis? This approach does not align with standard FEM analysis (Lines 261-263).

(5) How is the relative contribution calculated in the QGC analysis? The relative contribution of some lifestyle factors is not shown in Figure 2 and the supplementary figures, such as Supplementary Figure 7. These omissions should be explained.

Reviewer #2 (Public Review):

Summary:

The manuscript by Ngo et al investigated how bacterial persisters form in early and late stationary phases and found that cAMP-Crp regulated metabolic reprogramming affects persister formation that occurs in the late but not early stationary phase. Further metabolomic, proteomic, and genomic screening studies point to TCA cycle, ATP synthesis, respiratory chains, and oxidative phosphorylation correlating with persister abundance. If these conclusions can be solidly drawn, the work would add some new understanding of the underexplored topic of how persisters form.

Strengths and weaknesses:

Although the topic of understanding how persisters form is interesting and thus can be counted as a strength of the paper, most of the conclusions drawn by the authors are, at best, on shaky ground due to the following weakness.

(1) The approaches used here are aimed at the major bacterial population, but yet the authors used the data reflecting the major population behavior to interpret the physiology of persister cells that comprise less than 1% of the major bacterial population. How they can pick up a needle from the hay without being fooled by the spill-over artifacts from the major population? Although it is probably very difficult to isolate and directly assay persister cells, firm conclusions for the type proposed by the authors cannot be firmly established without such assays. Perhaps introducing cyaA/crp mutation into the best example of persistence, the hipA-7 high persistence phenotype may clarify this issue to a certain extent.

(2) The authors overlooked/omitted a recently published work regarding cyaA and crp (PMID: 35648826). In that work, a deficiency in cyaA or crp confers tolerance to diverse types of lethal stressors, including all lethal antimicrobials tested. How a mutation conferring pan-tolerance to the major bacterial population would lead to a less protective effect with a minor subpopulation? The authors are kind of obligated to discuss such a paradox in the context of their work because that is the most relevant literature for the present work. It is also very interesting if the cyaA/crp deficiency really has an opposing effect on tolerance and persistence. As a note, most of the conclusions from the omics studies of the present work have been reached in that overlooked literature, which addresses mechanisms of tolerance, a major rather than a minor population behavior. That supports comment #1 above. The inability of the authors to observe tolerance phenotype with the cyaA or crp mutant possibly derived from extremely high antimicrobial concentrations used in the study prevents tolerance phenotype from being observed because tolerance is sensitive to antimicrobial concentration while persistence is not.

(3) The authors overly stressed the effect of cyaA/crp on persister formation but failed to test an alternative explanation of their effect on persister waking up after antimicrobial treatment. If the cyaA/crp-derived persisters are put into deeper sleep during antimicrobial treatment than wildtype-derived persisters, a 16-h recovery growth might have underestimated viable bacteria. This is often the case especially when extremely high concentrations of antimicrobials are used in performing persister assay. Thus, at least a longer incubation time (e.g. 48 and 72h) of agar plates for persister viable count needs to be performed to test such a scenario.

(4) The rationale for using extremely high drug concentrations to perform persister assay is unclear. There are 2 issues with using extremely high drug concentrations. First, when overly high concentrations are used, drug removal becomes difficult. For example, a two-time wash will not be able to bring drug concentration from > 100 x MIC to below MIC. This is especially problematic with aminoglycoside because drug removal by washing does not work well with this class of compound. Second, overly high concentrations of drug use may make killing so rapidly and severely that may mask the difference from being observed between mutants and the control wild-type strain. In such cases, you would need to kill over a wide range of drug concentrations to find the right window to show a difference. The gentamicin data in the present work is likely the case that needs to be carefully examined. The mutants and the wild-type strain have very different MICs for gentamicin, but a single absolute drug concentration rather than concentrations normalized to MIC was used. This is like to compare a 12-year-old with a 21-year-old to run a 100-meter dash, which is highly inappropriate.

Quotations and Literary Allusions spoken by Willy Wonka in the 1971 film, Willy Wonka and the Chocolate Factory<br /> by Thomas M. Brodhead<br /> https://bmt-systems.com/score/wonka.htm

Archived copy: https://web.archive.org/web/20200111135336/https://bmt-systems.com/score/wonka.htm

Reviewer #2 (Public Review):

Summary:<br /> Bryant et al. apply phenotypic profiling and saturating transposon mutagenesis to investigate the role of the non-essential lipoproteins BamB, BamC, and BamE, along with chaperones DegP, Skp, and SurA, in the biogenesis of the bacterial outer membrane. This generated a set of genetic interactions that revealed that changes in LPS and outer membrane fluidity impact Bam activity, and that the cyclic form of enterobacterial common antigen becomes essential in the absence of the chaperone surA. The study also uncovers that peptidoglycan crosslinking and DNA replication control are conditionally essential with the absence of certain Bam components, suggesting a coordination between outer membrane protein (OMP) biogenesis and other cellular processes such as lipid and peptidoglycan synthesis, as well as DNA replication.

Strengths:

(1) This is probably the first comprehensive analysis of genetic interactions involving Bam-associated proteins and should provide rich insight to refine the mechanistic understanding of this complex machine and the process of OM biogenesis.

(2) Good quality data and analysis. Well-presented manuscript.

Weaknesses:

(1) An important control in any genetic interaction study is to do complementation tests to demonstrate that the phenotype observed is indeed due to the missing gene under analysis. Although the Keio library was designed to avoid polar effects, it is impossible to predict other undesirable effects of the deletions (hitting of a non-annotated sRNA or RNA stability effects, for example). Thus, before one can safely conclude that a proposed genetic interaction is real, complementation tests should be carried out. This seems particularly important in the case of a new and surprising interaction, such as that between bamB and DNA replication and repair genes.

(2) Why not include the suppressor interactions in the work? There are probably plenty, and in principle, they should be as informative as the conditional essential (or synthetic lethal) ones. The only one highlighted in the paper is that between bamB and diaA, since it nicely fits with the synthetic lethal effects with initiation inhibitors seqA and hda. Even if the authors cannot make sense of the suppressor interactions, their inclusion in the paper should make the dataset richer and more valuable to the community.

(3) The enrichment analysis in Figure 2B deserves some clarification. What is the meaning of gene ratio? How can single genes of a pathway yield an enrichment signal? Why weren´t seqA and hda included in the DNA replication class in 2B?

(4) The writing puts too much emphasis on demonstrating that bam lipoproteins and chaperones are specialized instead of fully redundant. However, I have the impression this is a long-settled conclusion in the field, as the manuscript itself describes at several points when reviewing the literature.

Reviewer #2 (Public Review):

Summary:

Non-canonical Wnt signaling plays an important role in morphogenesis, but how different components of the pathway are required to regulate different developmental events remains an open question. This paper focuses on elucidating the overlapping and distinct functions of dact1 and dact2, two Dishevelled-binding scaffold proteins, during zebrafish axis elongation and craniofacial development. By combining genetic studies, detailed phenotypic analysis, lineage tracing, and single cell RNA-sequencing, the authors aimed to understand (1) the relative function of dact1/2 in promoting axis elongation, (2) their ability to modulate phenotypes caused by mutations in other non-canonical wnt components, and (3) pathways downstream of dact1/2.<br /> Corroborating previous findings, this paper showed that dact1/2 is required for convergent extension during gastrulation and body axis elongation. Strong qualitative evidence was also provided to support dact1/2's role in genetically modulating non-canonical wnt signaling to regulate body axis elongation and the morphology of the ethmoid plate (EP). However, the spatiotemporal function of dact1/2 remains unknown. The use of scRNA-seq identified novel pathways and targets downstream of dact1/2. Calpain 8 is one such example, and its overexpression in some of the dact1/2+/- embryos was able to phenocopy the dact1/2-/- mutant EP morphology, pointing to its sufficiency in driving the EP phenotype in a few embryos. However, the same effect was not observed in dact1-/-; dact2+/- embryos, leading to the question of how significant calpain 8 really is in this context. The requirement of calpain 8 in mediating the phenotype is unclear as well. This is the most novel aspect of the paper, but some weaknesses remain in convincingly demonstrating the importance of calpain 8.

Strengths:

(1) The generation of dact1/2 germline mutants and the use of genetic approaches to dissect their genetic interactions with wnt11f2 and gpc4 provide unambiguous and consistent results that inform the relative functions of dact1 and dact2, as well as their combined effects.<br /> (2) Because the ethmoid plate exhibits a spectrum of phenotypes in different wnt genetic mutants, it is a useful system for studying how tissue morphology can be modulated by different components of the wnt pathway, as demonstrated in this study.<br /> (3) The authors leveraged lineage tracing by photoconversion to dissect how dact1/2 differentially impacts the ability of different cranial neural crest populations to contribute to the anterior neurocranium. This revealed that distinct mechanisms via dact1/2 and shh can lead to similar phenotypes.<br /> (4) The use of scRNA-seq was a powerful approach and identified potential novel pathways and targets downstream of dact1/2.

Weaknesses:

(1) Expression of dact1/2 and wnt11f2: Certain claims regarding the expression similarity between dact2 and wnt11f2 is not clearly demonstrated in figures and the text description of dact1/2 and wnt11f2 expression for the Daniocell scRNA-seq tool is also somewhat confusing. As the paper makes claim that dact1/2 may function in the same pathway as wnt11f2, their expression should be accurately described and used to draw conclusion on what tissue types such a signaling may take place.<br /> (2) Spatiotemporal function of dact1/2: Germline mutations limit the authors' ability to study a gene's spatiotemporal functional requirement. They, therefore, cannot concretely attribute nor separate early-stage phenotypes (during gastrulation) to/from late stage phenotypes (EP morphological changes), which the authors postulated to result from secondary defects in floor plate and eye field morphometry.<br /> (3) The functional significance of calpain 8: The authors showed that calpain 8 was upregulated in the mutant and subsequently tested its function by overexpressing dact1/2 mRNA in embryos. While only 1 out of 142 calpain-overexpressing wild type animals phenocopied dact1/2 mutants, 7.5% of dact1/2+/- embryos did exhibit the phenotype. However, the same effect was not observed in dact1-/-; dact2+/- embryos and the requirement of calpain 8 in driving the phenotype remains unclear.

Reviewer #2 (Public Review):

Raymond Laboy et.al explored how transcriptional Mondo/Max-like complex (MML-1/MXL-2) is regulated by glucose metabolic signals using germ-line removal longevity model. They believed that MML-1/MXL-2 integrated multiple longevity pathways through nutrient sensing and therefore screened the glucose metabolic enzymes that regulated MML-1 nuclear localization. Hexokinase 1 and 2 were identified as the most vigorous regulators, which function through mitochondrial beta-oxidation and the pentose phosphate pathway (PPP), respectively. MML-1 localized to mitochondria associated with lipid droplets (LD), and MML-1 nuclear localization was correlated with LD size and metabolism. Their findings are interesting and may help us to further explore the mechanisms in multiple longevity models. The data support their proposed working model. Nonetheless, the roles of hxk-1 and lipid oxidation in regulating LD, as proposed in the working model, are not clear.

Reviewer #2 (Public Review):

Summary:

This is a tour de force study that aims to understand the genetic basis of male germ cell development across three animal species (human, mouse and flies) by performing a genetic program conservation analysis (using phylostratigraphy and network science) with a special emphasis on genes that peak or decline during mitosis-to-meiosis. This analysis, in agreement with previous findings, reveals that several genes active during and before meiosis are deeply conserved across species, suggesting ancient regulatory mechanisms. To identify critical genes in germ cell development, the investigators integrated clinical genetics data, performing gene knockdown and knockout experiments in both mice and flies. Specifically, over 900 conserved genes were investigated in flies, with three of these genes further studied in mice. Of the 900 genes in flies, ~250 RNAi knockdowns had fertility phenotypes. The fertility phenotypes for the fly data can be viewed using the following browser link: https://pages.igc.pt/meionav. The scope of target gene validation is impressive. Below are a few minor comments.

(1) In Supplemental Figure 2, it is notable that enterocyte transcriptomes are predominantly composed of younger genes, contrasting with the genetic age profile observed in brain and muscle cells. This difference is an intriguing observation and it would be curious to hear author comments.

(2) Regarding the document, the figures provided only include supplemental data; none of the main text figures are in the full PDF.

(3) Lastly, it would be great to section and stain mouse testis to classify the different stages of arrest during meiosis for each of the mouse mutants in order to compare more precisely to flies.

This paper serves as a vital resource, emphasizing that only through the analysis of hundreds of genes can we prioritize essential genes for germ cell development. its remarkable that about 60% of conserved genes have no apparent phenotype during germ cell development.

Strengths:

High-throughput screening was conducted on a conserved network of 920 genes expressed during the mitosis-to-meiosis transition. Approximately 250 of these genes were associated with fertility phenotypes. Notably, mutations in 5 of the 250 genes have been identified in human male infertility patients. Furthermore, 3 of these genes were modeled in mice, where they were also linked to infertility. This study establishes a crucial groundwork for future investigations into germ cell development genes, aiming to delineate their essential roles and functions.

Weaknesses:

The fertility phenotyping in this study is limited, yet dissecting the mechanistic roles of these proteins falls beyond its scope. Nevertheless, this work serves as an invaluable resource for further exploration of specific genes of interest.

Reviewer #3 (Public Review):

While progressive and also hyperactivated motility are required for sperm to reach the site of fertilization and to penetrate oocyte's outer vestments, during fusion with the oocyte's plasma membrane it has been observed that sperm motility ceases. Identifying the underlying molecular mechanisms would provide novel insights into a crucial but mostly overlooked physiological change during the sperm's life cycle. In this publication the authors aim to provide evidence that the helical actin structure surrounding the sperm mitochondria in the midpiece plays a role in regulating sperm motility, specifically the motility arrest during sperm fusion but also during earlier cessation of motility in a subpopulation of sperm post acrosomal exocytosis.

The main observation the authors make is that in a subpopulation of sperm undergoing acrosomal exocytosis and sperm that fuse with the plasma membrane of the oocyte display a decrease in midpiece parameter of 30 nm. The authors propose the decrease in midpiece diameter via various microscopy techniques based on membrane dyes and bright-field images. In the revised version of the manuscript, a change in midpiece diameter is now confirmed via electron microscopy, even though the difference is not significant. The authors also propose that the midpiece diameter decrease is driven by changes in sperm intracellular Ca2+ and structural changes of the actin helix network. Future studies are still needed to confirm the casualty of these events and explore the discrepancy between fluorescence microscopy results and SEM. Overall, the authors should further tone down their conclusions.

Reviewer #2 (Public Review):

This revised manuscript attempts to explore the underlying chromatin accessibility landscape of spermatogonia from the developing and adult mouse testis. The key criticism of the first version of this manuscript was that bulk preparations of mixed populations of spermatogonia were used to generate the data that form the basis of the entire manuscript. To address this concern, the authors applied a deconvolution strategy (CIBERSORTx (Newman et al., 2019)) in an attempt to demonstrate that their multi-parameter FACS isolation (from Kubota 2004) of spermatogonia enriched for PLZF+ cells recovered spermatogonial stem cells (SSCs). PLZF (ZBTB16) protein is a transcription factor known to mark all or nearly all undifferentiated spermatogonia and some differentiating spermatogonia (KIT+ at the protein level) - see Niedenberger et al., 2015 (PMID: 25737569). The authors' deconvolution using single-cell transcriptomes produced at postnatal day 6 (P6) argue that 99% of the PLZF+ spermatogonia at P8 are SSCs, 85% at P15 and 93% in adults. Quite frankly given the established overlap between PLZF and KIT and known identity of spermatogonia at these developmental stages, this is impossible. Indeed - the authors' own analysis of the reference dataset demonstrates abundant PLZF mRNA in P6 progenitor spermatogonia - what is the authors' explanation for this observation? The same is essentially true in the use of adult references for celltype assignment. The authors found 63-82% of SSCs using this different definition of types (from a different dataset), begging the question of which of these results is true.

In their rebuttal, the authors also raise a fair point about the precision of differential gene expression among spermatogonial subsets. At the mRNA level, Kit is definitely detectable in undifferentiated spermatogonia, but it is never observed at the protein level until progenitors respond to retinoic acid (see Hermann et al., 2015). I agree with the authors that the mRNAs for "cell type markers" are rarely differentially abundant at absolute levels (0 or 1), but instead, there are a multitude of shades of grey in mRNA abundance that "separate" cell types, particularly in the male germline and among the highly related spermatogonial subtypes of interest (SSCs, progenitor spermatogonia and differentiating spermatogonia). That is, spermatogonial biology should be considered as a continuous variable (not categorical), so examining specific cell populations with defined phenotypes (markers, function) likely oversimplifies the underlying heterogeneity in the male germ lineage. But, here, the authors have ignored this heterogeneity entirely by selecting complex populations and examining them in aggregate. We already know that PLZF protein marks a wide range of spermatogonia, complicating the interpretation of aggregate results emerging from such samples. In their rebuttal, the authors nicely demonstrate the existence of these mixtures using deconvolution estimation. What remains a mystery is why the authors did not choose to perform single-cell multiome (RNA-seq + ATAC-seq) to validate their results and provide high-confidence outcomes. This is an accessible technique and was requested after the initial version, but essentially ignored by the authors.

A separate question is whether these data are novel. A prior publication by the Griswold lab (Schleif et al., 2023; PMID: 36983846) already performed ATAC-seq (and prior data exist for RNA-seq) from germ cells isolated from synchronized testes. These existing data are higher resolution than those provided in the current manuscript because they examine germ cells before and after RA-induced differentiation, which the authors do not base on their selection methods. Another prior publication from the Namekawa lab extensively examined the transcriptome and epigenome in adult testes (Maezawa et al., 2000; PMID: 32895557; and several prior papers). The authors should explain how their results extend our knowledge of spermatogonial biology in light of the preceding reports.

The authors are also encouraged to improve their use of terminology to describe the samples of interest. The mitotic male germ cells in the testis are called spermatogonia (not spermatogonial cells, because spermatogonia are cells). Spermatogonia arise from Prospermatogonia. Spermatogonia are divisible into two broad groups: undifferentiated spermatogonia (comprised of few spermatogonial stem cells or SSCs and many more progenitor spermatogonia - at roughly 1:10 ratio) and differentiating spermatogonia that have responded to RA. The authors also improperly indicate that SSCs directly produce differentiating spermatogonia - indeed, SSCs produce transit-amplifying progenitor spermatogonia, which subsequently differentiate in response to retinoic acid stimulation. Further, the use of Spermatogonial cells (and SPGs) is imprecise because these terms do not indicate which spermatogonia are in question. Moreover, there have been studies in the literature which have used similar terms inappropriately to refer to SSCs, including in culture. A correct description of the lineage and disambiguation by careful definition and rigorous cell type identification would benefit the reader.

Overall, my concern from the initial version of this manuscript stands - critical methodological flaws prevent interpretation of the results and the data are not novel. Readers should take note that results in essentially all Figures do not reflect the biology of any one type of spermatogonium.

Reviewer #2 (Public Review):

Summary:<br /> The authors built a tool to extract the timing and location of mouse urine and fecal deposits in their laboratory set up. They indicate that they are happy with the results they achieved in this effort.

The authors note urine is thought to be an important piece of an animal's behavioral repertoire and communication toolkit so methods that make studying these dynamics easier would be impactful.

Strengths:<br /> With the proposed method, the authors are able to detect 79% of the urine that is present and 84% of the feces that is present in a mostly automated way.

Weaknesses:<br /> The method proposed has a large number of design choices across two detection steps that aren't investigated. I.e. do other design choices make the performance better, worse, or the same? Are these choices robust across a range of laboratory environments? How much better are the demonstrated results compared to a simple object detection pipeline (i.e. FasterRCNN or YOLO on the raw heat images)?

The method is implemented with a mix of MATLAB and Python.

One proposed reason why this method is better than a human annotator is that it "is not biased." While they may mean it isn't influenced by what the researcher wants to see, the model they present is still statistically biased since each object class has a different recall score. This wasn't investigated. In general there was little discussion of the quality of the model. Precision scores were not reported. Is a recall value of 78.6% good for the types of studies they and others want to carry out? What are the implications of using the resulting data in a study? How do these results compare to the data that would be generated by a "biased human?"

5 out of the 6 figures in the paper relate not to the method but to results from a study whose data was generated from the method. This makes a paper, which, based on the title, is about the method, much longer and more complicated than if it focused on the method. Also, even in the context of the experiments, there is no discussion of the implications of analyzing data that was generated from a method with precision and recall values of only 70-80%. Surely this noise has an effect on how to correctly calculate p-values etc. Instead, the authors seem to proceed like the generated data is simply correct.

Reviewer #2 (Public Review):

The authors frame the MS-spectrum-based prediction of antimicrobial resistance prediction as a drug recommendation task. Weis et al. introduced the dataset this model is tested on and benchmark models which take as input a single species and are trained to predict resistance to a single drug. Instead here, a pair of drugs and spectrum are fed to 2 neural network models to predict a resistance probability. In this manner, knowledge from different drugs and species can be shared through the model parameters. Questions asked: 1. what is the best way to encode the drugs? 2. does the dual NN outperform the single spectrum-drug?

Overall the paper is well-written and structured. It presents a novel framework for a relevant problem.

Reviewer #2 (Public review):

Wnt signaling is the name given to a cell-communication mechanism that cells employ to inform on each other's position and identity during development. In cells that receive the Wnt signal from the extracellular environment, intracellular changes are triggered that cause the stabilization and nuclear translocation of β-catenin, a protein that can turn on groups of genes referred to as Wnt targets. Typically these are genes involved in cell proliferation. Genetic mutations that affect Wnt signaling components can therefore affect tissue expansion. Loss of function of APC is a drastic example: APC is part of the β-catenin destruction complex, and in its absence, β-catenin protein is not degraded and constitutively turns on proliferation genes, causing cancers in the colon and rectum. And here lies the importance of the finding: β-catenin has for long been considered to be regulated almost exclusively by tuning its protein turnover. In this article, a new aspect is revealed: Ctnnb1, the gene encoding for β-catenin, possesses tissue-specific regulation with transcriptional enhancers in its vicinity that drive its upregulation in intestinal stem cells. The observation that there is more active β-catenin in colorectal tumors not only because the broken APC cannot degrade it, but also because transcription of the Ctnnb1 gene occurs at higher rates, is novel and potentially game-changing. As genomic regulatory regions can be targeted, one could now envision that mutational approaches aimed at dampening Ctnnb1 transcription could be a viable additional strategy to treat Wnt-driven tumors.

Reviewer #2 (Public Review):

Summary:

The authors propose that cancer-driver mutations can be identified by Cancer Driving Nucleotides (CDNs). CDNs are defined as SNVs that occur frequently in genes. There are many ways to define cancer driver mutations, and the strengths and weaknesses are the reliance on statistics to define them.

Strengths:

There are many well-known approaches and studies that have already identified many canonical driver mutations. A potential strength is that mutation frequencies may be able to identify as yet unrecognized driver mutations. They use a previously developed method to estimate mutation hotspots across the genome (Dig, Sherman et al 2022). This publication has already used cancer sequence data to infer driver mutations based on higher-than-expected mutation frequencies. The advance here is to further illustrate that recurrent mutations (estimated at 3 or more mutations (CDNs) at the same base) are more likely to be the result of selection for a driver mutation (Figure 3). Further analysis indicates that mutation sequence context (Figure 4) or mutation mechanisms (Figure 5) are unlikely to be major causes for recurrent point mutations. Finally, they calculate (Figure 6) that most driver mutations identifiable by the CDN approach could be identified with about 100,000 to one million tumor coding genomes.

Weaknesses:

The manuscript does provide specific examples where recurrent mutations identify known driver mutations but do not identify "new" candidate driver mutations. Driver mutation validation is difficult and at least clinically, frequency (ie observed in multiple other cancer samples) is indeed commonly used to judge if an SNV has driver potential. The method would miss alternative ways to trigger driver alterations (translocations, indels, epigenetic, CNVs). Nevertheless, the value of the manuscript is its quantitative analysis of why mutation frequencies can identify cancer driver mutations.

Reviewer #2 (Public Review):

This work explores the connection between glioblastoma, mito-RQC, and msiCAT-tailing. They build upon previous work concluding that ATP5alpha is CAT-tailed and explore how CAT-tailing may affect cell physiology and sensitivity to chemotherapy. The authors conclude that when ATP5alpha is CAT-tailed, it either incorporates into the proton pump or aggregates and that these events dysregulate MPTP opening and mitochondrial membrane potential and that this regulates drug sensitivity. This work includes several intriguing and novel observations connecting cell physiology, RQC, and drug sensitivity. This is also the first time this reviewer has seen an investigation of how a CAT tail may specifically affect the function of a protein. However, some of the conclusions in this work are not well supported. This significantly weakens the work but can be addressed through further experiments or by weakening the text.

Reviewer #2 (Public Review):

Summary:

This study reports on the existence of subpopulations of isogenic E. coli and P. aeruginosa cells that are tolerant to the antimicrobial peptide tachyplesin and are characterized by the accumulation of low levels of a fluorescent tachyplesin-NBD conjugate. The authors then set out to address the molecular mechanisms, providing interesting insights even though the mechanism remains incompletely defined: The work suggests that amongst others changes in membrane lipid composition and increased drug efflux may cause this phenotype and it demonstrates that pharmacological manipulation can prevent generation of tolerance. The authors are cautious in their interpretation and the claims made are largely justified by the data.

Strengths:

Going beyond the commonly used bulk techniques for studying susceptibility to AMPs , Lee et al. used fluorescent antibiotic conjugates in combination with flow cytometry analysis to study variability in drug accumulation at the single-cell level. This powerful approach enabled the authors to expose bimodal drug accumulation patterns that were condition-dependent, but conserved across a variety of E. coli clinical isolates. Using cell sorting in combination with colony-forming unit assays as well as quantitative fluorescence microscopic analysis in a microfluidics setup the authors compellingly demonstrate that low accumulators (where the fluorescence signal is mostly restricted to the membrane), can survive antibiotic treatment, whereas high accumulators (with high intracellular fluorescence) were killed. Comparative transcriptomics analysis of sorted ´low accumulator´ and ´high accumulator´ subpopulations suggest that changes in the lipid composition, increased efflux, and other mechanisms may contribute to tachyplesin-tolerance in this subpopulation. Lipidomics analysis of bulk untreated vs. tachyplesin-NBD treated cells confirmed changes in the lipid composition in accordance with the transcriptomics data. Intriguingly, a time-course experiment on tachyplesin-NBD accumulation revealed that all cells initially were high accumulators, before a subpopulation of cells subsequently managed to reduce the signal intensity (most likely through efflux), demonstrating that the ´low accumulator´ phenotype is an induced response and not a pre-existing property.

Finally, the demonstration that treatment with efflux pump inhibitors (although some caution needs to be taken regarding the selectivity of these inhibitors, see comment on weaknesses below) prevents the generation of low accumulators and enhances tachyplesin-based killing is an important basis for developing combination therapies.

The study convincingly illustrates how susceptibility to tachoplesin adaptively changes in a heterogeneous way dependent on the growth phases/ environments and availability of nutrients. This is highly relevant also beyond the presented example of tachyplesin and similar subpopulation-based adaptive changes to the susceptibility towards antimicrobial peptides or other drugs that may occur during infections in vivo and they would likely be missed out by standardized in vitro susceptibility testing.

Weaknesses:

Some questions regarding the mechanism remain. One shortcoming of the setup of the transcriptomics experiment is that the tachyplesin-NBD probe itself has antibiotic efficacy and induces phenotypes (and eventually cell death) in the ´high accumulator´cells. This makes it challenging to interpret whether any differences seen between the two groups are causative for the observed accumulation pattern or if they are a consequence of differential accumulation and downstream phenotypic effects. The role of efflux systems is further supported by the finding that efflux pump inhibitors sensitize E. coli to tachyplesin and prevent the occurrence of the tolerant ´low accumulator´ subpopulations. In principle, this is a great way of validating the role of efflux pumps, but the limited selectivity of these inhibitors (CCCP is an uncoupling agent, and for sertraline direct antimicrobial effects on E. coli have been reported by Bohnert et al.) leaves some ambiguity as to whether the synergistic effect is truly mediated via efflux pump inhibition. It would be relevant to test and report the MIC of sertraline for the strain tested, particularly since in Figure 4G an initial reduction in CFUs is observed for sertraline treatment, which suggests the existence of biological effects in addition to efflux inhibition.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

(1) The selection of strains is not very clear. Nothing is known about the sequence types of the strains or how representative they are for strains circulating in general. Thus, it is difficult to generalize from this limited number of isolates, although the studies done in these isolates are comprehensive.

(2) Nothing is known about the susceptibility of the strains to other novel antimicrobial agents. Colistin has a limited role in the treatment of gram-negative infections, and although it can be used sometimes in combination, it is not clear why it would be combined with two other nephrotoxic agents and how this could have relevance in a clinical setting.

(3) It is not clear whether their transcriptomics analysis should at least be carried out in duplicate for reasons of being able to assess reproducibility. It is also not clear why the samples were incubated for 6 hours - no discussion is presented on the selection of a time point for this.

(4) Discussion is lacking on the reproducibility and selection of details for the methodology.

Reviewer #2 (Public Review):

Summary:

The authors address an important question in cardiovascular science that is very topical. The use of exogenous mitochondrial transplantation is assessed after cardiac arrest to determine if these exogenous mitochondria can enhance cardiac function. Given the role of mitochondria in the energy expenditure of the heart, this is an important question to study.

Strengths:

The strength lies mainly in the hypothesis being addressed as it is highly relevant in the quest for more strategies to enhance cardiac function.

Weaknesses:

There is further refinement needed in experimental details and transparency. Also, additional experiments need to be performed such as the seahorse experiment for oxygen consumption. Improvements in the text and in figures are needed and these comments are directed to the authors in our recommendations to the authors.

Reviewer #2 (Public Review):

In the present study, authors report the role of virus-induced apoptosis in positively regulating the innate immune response. Upon infection, host cell apoptosis is triggered as a defence mechanism against virus replication. Culmination of infected-cell death impairs replicative potential for viruses, hence attenuating virus propagation. Reports exist denoting the inhibitory effect of apoptosis upon innate immune signalling. Contrary to that, the findings of this manuscript underscore the possible role of apoptosis in enhancing innate immune signalling and effector response. Infection-induced activation of caspases (3, 6, 7, and 8) has been demonstrated to cleave DRP1 protein. DRP1, a positive regulator for mitochondrial fission, degradation leads to altered mitochondrial morphology (elongation).

Mitochondria, being a hub for innate immune signalling (via operation of RLR-MAVS-downstream effector molecule-axis), upon elongation as a result of DRP1 depletion, results in greater innate immune signal flux and interferon induction. Increased interferon induction thus acts to inhibit virus propagation, as demonstrated by the authors using cell-culture models.

Strengths:

(1) The findings presented by the authors have been validated by employing elaborate biochemical experimental approaches. The study entails extensive biochemical characterization of DRP1 residues targeted by activated caspases, in vitro assays validating caspase-mediated DRP1 cleavage & caspase-DRP1 interaction.

(2) This study possesses broad implications since the authors demonstrate the role of caspase-mediated DRP1 cleavage in promoting innate immunity in the context of infection by diverse viruses (both RNA and DNA viruses).

Weaknesses:

Although the authors undertook a thorough experimental approach attempting to validate their findings, all the experiments were performed using either cell-culture models for infection or in vitro biochemical assays (cleavage and protein-protein interaction). Additional experimentation using animal models (in vivo) will further help strengthen the biological significance of their findings under more physiological settings.

Reviewer #2 (Public Review):

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria and plays a critical role in bacterial virulence. The LPS export mechanism is a potential target for new antibiotics. Inhibiting this process can render bacteria more susceptible to the host immune system or other antibacterial agents. Given the rise of antibiotic-resistant bacteria, novel targets are urgently needed. The seven LPS transport (Lpt) proteins, A-G, move LPS from the inner to the outer membrane. This study investigated the conformational changes in the LptB2FG-LptC complex using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, revealing how ATP binding and hydrolysis affect the LptF β-jellyroll domain and lateral gates. The findings highlight the role of LptC in regulating LPS entry, ensuring efficient and unidirectional transport across the periplasm.

The β-jellyrolls are not fully resolved in the vanadate-trapped structure of LptB2FG and LptB2FGC. Therefore, the current study provides valuable information on the functional dynamics of these periplasmic domains, their interactions, and their roles in the unidirectional transport of LPS. Additionally, the dynamic perspective of the lateral gates in LptFG in the presence and absence of LptC is another strength of this study. Moreover, at least in detergent samples, more comprehensive intermediates of the ATP turnover cycle are studied than in the available structures, providing crucial missing mechanistic details.

Other major strengths of the study include high-quality DEER/PELDOR distance measurements in both detergent and proteoliposomes, the latter providing valuable dynamics information in the lipid environment. The proteoliposome study is crucial since the previous structural study (Li, Orlando & Liao 2019) was done in rather small-diameter nanodiscs, which might affect the overall dynamics of the complex. It would have been beneficial if the investigators had reconstituted the complex in lipid nanodiscs with the same composition as proteoliposomes. The mixed lipid/detergent micelles provide an alternative. It seems the ATPase activity of the protein complex is much lower in detergent compared with lipid nanodiscs (Li, Orlando & Liao 2019). It is unclear how ATPase activity in proteoliposomes compares to that in detergent micelles.

Additionally, from previous structural studies and the mass spectrometry data presented here, LPS co-purifies and is already bound to the complex, thus the Apo state may represent the LPS-bound state without nucleotides.

Reviewer #2 (Public Review):

Summary:

The current work by Banwait et al. reports a fluorescence-based single turnover method based on protein-induced fluorescence enhancement (PIFE) to show that ClpB is a processive motor. The paper is a crucial finding as there has been ambiguity on whether ClpB is a processive or non-processive motor. Optical tweezers-based single-molecule studies have shown that ClpB is a processive motor, whereas previous studies from the same group hypothesized it to be a non-processive motor. As co-chaperones are needed for the motor activity of the ClpB, to isolate the activity of ClpB, they have used a 1:1 ratio ATP and ATPgS, where the enzyme is active even in the absence of its co-chaperones, as previously observed. A sequential mixing stop-flow protocol was developed, and the unfolding and translocation of RepA-TitinX, X = 1,2,3 repeats was monitored by measuring the fluorescence intensity with time of Alexa F555 that was labelled at the C-terminal Cysteine. The observations were a lag time, followed by a gradual increase in fluorescence due to PIFE, and then a decrease in fluorescence plausibly due to the dissociation from the substrate allowing it to refold. The authors observed that the peak time depends on the substrate length, indicating the processive nature of ClpB. In addition, the lag and peak times depend on the pre-incubation time with ATPgS, indicating that the enzyme translocates on the substrates even with just ATPgS without the addition of ATP, which is plausible due to the slow hydrolysis of ATPgS. From the plot of substrate length vs peak time, the authors calculated the rate of unfolding and translocation to be ~0.1 aas-1 in the presence of ~1 mM ATPgS and increases to 1 aas-1 in the presence of 1:1 ATP and ATPgS. The authors have further performed experiments at 3:1 ATP and ATPgS concentrations and observed ~5 times increase in the translocation rates as expected due to faster hydrolysis of ATP by ClpB and reconfirming that processivity is majorly ATP driven. Further, the authors model their results to multiple sequential unfolding steps, determining the rate of unfolding and the number of amino acids unfolded during each step. Overall, the study uses a novel method to reconfirm the processive nature of ClpB.

Strengths:

(1) Previous studies on understanding the processivity of ClpB have primarily focused on unfolded or disordered proteins; this study paves new insights into our understanding of the processing of folded proteins by ClpB. They have cleverly used RepA as a recognition sequence to understand the unfolding of titin-I27 folded domains.<br /> (2) The method developed can be applied to many disaggregating enzymes and has broader significance.<br /> (3) The data from various experiments are consistent with each other, indicating the reproducibility of the data. For example, the rate of translocation in presence of ATPgS, ~0.1 aas-1 from the single mixing experiment and double mixing experiment are very similar.<br /> (4) The study convincingly shows that ClpB is a processive motor, which has long been debated, describing its activity in the presence of only ATPgS and a mixture of ATP and ATPgS.<br /> (5) The discussion part has been written in a way that describes many previous experiments from various groups supporting the processive nature of the enzyme and supports their current study.

Weaknesses:

(1) The authors model that the enzyme unfolds the protein sequentially around 60 aa each time through multiple steps and translocates rapidly. This contradicts our knowledge of protein unfolding, which is generally cooperative, particularly for titinI27, which is reported to unfold cooperatively or utmost through one intermediate during enzymatic unfolding by ClpX and ClpA.<br /> (2) It is also important to note that the unfolding of titinI27 from the N-terminus (as done in this study) has been reported to be very fast and cannot be the rate-limiting step as reported earlier(Olivares et al, PNAS, 2017). This contradicts the current model where unfolding is the rate-limiting step, and the translocation is assumed to be many orders faster than unfolding.<br /> (3) The model assumes the same time constant for all the unfolding steps irrespective of the secondary structural interactions.<br /> (4) Unlike other single-molecule optical tweezer-based assays, the study cannot distinguish the unfolding and translocation events and assumes that unfolding is the rate-limiting step.

Reviewer #2 (Public Review):

Summary:

Karim et al investigated the regulation of ACSS2 by SIRT2. The authors identified a previously undescribed acetylation that they then show is important for the regulation and stability of ACSS2 in cells. The authors show that ACSS2 ubiquitination and degradation by the proteasome is regulated by SIRT2-mediated deacetylation of ACSS2 and that stabilizing ACSS2 by blocking SIRT2 can alter lipid accumulation in adipocytes.

Strengths:

Identification of a novel acetylation site on ACSS2 that regulates its protein stability and that has consequences on its activity in adipocytes. Multiple standard approaches were used to manipulate the expression and function of SIRT2 and ACSS2 (i.e., overexpression, knockdown, inhibitors).

Weaknesses:

Throughout the manuscript, normalizing the data to 1 and then comparing the fold-change using a t-test is not the best statistical approach in that situation since every normalized value for control is 1 with zero standard deviation. The authors should consider an alternative statistical approach.

Though not necessary, using 13C-acetate or D3-acetate tracing would be better for understanding the impact of acetylation on the activity of ACSS2 and its impact on lipogenesis.

Reviewer #2 (Public Review):

Clément Mazeaud et al. identified the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a proviral cellular protein that regulates Zika virus (ZIKV) RNA replication by modulating the biogenesis of virus-induced replication organelles. Based on their findings and previously published data, the authors propose a model outlining the role of IGF2BP2 in the ZIKV infectious cycle. This model details the changes in IGF2BP2 interactions with both cellular and viral proteins and RNAs during viral infection.

Strengths:

This revised manuscript presents an interesting and convincing mechanism by which a cellular RNA-binding protein alters its protein and RNA interactome during viral infection. Using various molecular biology methods, proteomic analysis and a newly described replication-independent vesicle packets induction system, the authors describe the relevance of IGF2BP2 protein during Zika virus infection.

Weaknesses:

In the proposed model, the IGF2BP2 protein specifically binds to the 3' nontranslated region (NTR) of the ZIKV genome, while excluding binding to the 5' NTR. However, the authors cannot rule out the possibility that this host protein associates with other regions of the viral genome, a topic which is discussed in the manuscript.

In this study, the physiological cellular consequences of altering the interaction of IGF2BP2 with its endogenous mRNA ligands due to ZIKV infection remain unexplored. This aspect would be of interest for future studies.

Reviewer #2 (Public Review):

The central theme of the manuscript is to report on the structure of SBPase - an enzyme central to the photosynthetic Calvin-Benson-Bassham cycle. The authors claim that the structure is the first of its kind from a chlorophyte Chlamydomonas reinhardtii, a model unicellular green microalga. The authors use a number of methods like protein expression, purification, enzymatic assays, SAXS, molecular dynamics simulations and x-ray crystallography to resolve a 3.09 A crystal structure of the oxidized and partially reduced state. The results are supported by the claims made in the manuscript. While the structure is the first from a chlorophyte, it is not unique. Several structures of SBPase are available and a comparison has been made between the structure reported here and others that have been previously published.

Reviewer #2 (Public Review):

Summary:

This manuscript investigates the role of the abundant NK cells that are observed in colon cancer liver metastasis using sequencing and spatial approaches in an effort to clarify the pro and anti-tumourogenic properties of NK cells. This descriptive study characterizes different categories of NK cells in tumor and tumor adjacent tissues and some correlations. An attempt has been made using pseudotime trajectory analysis but no models around how these NK cells might be regulated is provided.

Strengths:

This study integrates multiomics data to attempt to resolve correlates of protection that might be useful in understanding NK cell diversity and activation. The authors have strengthened the study in revision by demonstrating the very strong correlation between Granzyme+ NK cells and the poor prognosis, but the main claims are only partially supported.

Weaknesses:

While this work is interesting, the power of such studies are in taking the discovered information and applying this to other cohorts to determine the strength and predictive power of the genes identified. It is also clear that these 'snapshots' analysed poorly take account of the dynamic temporal changes that occur within a tumour. It would have been good to see a proposed model of NK cell regulation as it might occur in the tumour (accounting for turnover and recruitment) beyond the static data. Further evidence linking mechanistic causality to prognostic outcome would provide significant data for approaches forward.

Reviewer #3 (Public Review):

Summary and Strength:

The manuscript by Amir et al. describes that Sertoli-specific inactivation of the mTORC1 and mTORC2 complex by KO of either Raptor or Rictor, respectively, resulted in progressive changes in blood-testis-barrier (BTB) function, testis weight, and sperm parameters, including counts, morphology, mtDNA content and sperm DNA methylation.

The described studies are based on the hypothesis that a decline of BTB function with increasing chronological age of a male contributes to the DNA methylation changes that are known to occur in sperm DNA of old males when compared to sperm DNA from isogenic young males. In order to demonstrate the relevance of a functioning BTB for the maintenance of sperm methylation patterns, the authors generated mice with genetically disrupted mTORC2 complex or mTORC1 complex in Sertoli cells and determined sperm methylation patterns in comparison to isogenic wild-type males. In line with previously published scientific literature (e.g. Mok et al., 2013; Dong et al, 2015; and others), the manuscript corroborates that a Sertoli-cell specific deletion of mTORC2 caused a loss of BTB function and a progressive spermatogenic defect. The authors further show that sperm DNA is differentially methylated (DMRs) as a consequence of either a mTORC2 disruption (associated with a loss of BTB function) or following a mTORC1 disruption (BTB function either increased or not leaky) when compared to their isogenic age-matched wt controls. Those DMRs overlap partially with changes in sperm DNA methylation that were found when comparing sperm from 8-week males with sperm isolated from 22-week-old male mice.

The authors interpret the observed changes as representative of the sperm DNA methylation changes that occur during normal chronological aging of the male. For an aged control group, the authors use sperm DNA of 22-week-old wild-type mates from the mTORC2 and mTORC2 KO breeding and compare the sperm methylation patterns found in sperm from those 22-week males to 8-week young males, that are intended to represent an old and a young cohort, respectively. DNA methylation analysis indicates that a disruption of mTORC2 (& decrease of BTB function) results in increased DNA methylation of sperm DNA, while a disruption of mTORC1 (and proposed increase of BTB tightness, not shown in the manuscript, though) resulted in increased hypomethylation.

Weaknesses:

While the hypothesis and experimental system are interesting and the data demonstrating the relevance of the mTORC2 complex for BTB function is convincing, several open questions limit the evidence that supports the hypothesis that the sperm DNA methylation changes seen in old males are caused by BTB failure following an imbalance of mTOR signaling complexes. The major critique points are the lack of a chronologically old group and the choice of 8 weeks & 22 weeks age of age:

- Data illustrating the degree of BTB decline and sperm DNA methylation changes from chronologically "old" male mice is missing. 22-week-old mice are not considered old but are of good and mature breeding age, equivalent to humans in their mid-late twenties. (In the manuscript, the 22-week-old wildtype mice show no evidence of BTB breakdown (Figure 3), so why are their sperm used to represent "aged" sperm?

- Adding a group of "old" wild-type mice of 12-14 months of age, which is closer to the end of effective reproduction in mice, more equivalent to 45-59 year-old humans) could be used to illustrate that (a) aging causes a marked decrease in BTB function at this time in mouse life, and that this BTB breakdown chronologically aligns with the age-associated DNA hypermethylation seen in old sperm. Age-matched "old" mTORC1 KO, with a (supposedly) tighter BTB barrier, could then be expected to have a sperm DMA methylation profile closer to that of younger wild-type animals. Such data are currently missing. While the progressive testicular decline observed in the mTORC1 KO (Fig.5) could make it difficult to obtain the appropriately aged mTORC1 KO tissues, it is completely feasible to obtain data from chronologically old wild-type males. (The progressive testicular decline further raises the question of what additional defects the KO causes, and how such additional defects would influence the sperm DNA methylation profile.) The addition of data from an old group to the currently included groups could strengthen the interpretation that the observations in the BTB-defective mTORC2 KO mice are modelling an age-related testicular decline, provided that the DMRs seen in the chronologically old group significantly overlap with the BTB-defective changes.

- In the current form, the described differences in sperm DNA methylation are based on comparisons between pubertal mice (8 weeks) and mature but not old adult males (22 weeks), while a chronologically "old" group is missing from the data sets and comparisons. Thus, it appears that the described sperm methylation changes reflect developmental changes associated with normal maturation and not necessarily declining sperm quality due to aging. (Sperm obtained from 8-week-old mice likely were generated, at least in part, during the 1st wave of spermatogenesis, which is known to differ from the continuously proceeding spermatogenesis during the remained of the mature life. During the 1st wave of spermatogenesis, Sertoli cells are known to undergo gene expression changes which could contribute to varying degrees of BTB function, and thus have effects on the sperm DNA methylation profiles of such 1st wave sperm.)

- It is unclear why the aging-related DMRs between the 8 and 22-week-old wild-type mice vary so dramatically between the two wild-type groups derived from the mTORC1 and the mTORC2 breeding (Fig. S4). If the main difference was due to mTORC1 or mTORC2 activity, both wildtype groups should behave very similarly. Changes seen in a truly "old" mouse (e.g. 20 weeks to 56 weeks), changes in "young mTORC1" and in "old mTORC2" are missing. How do those numbers and profiles compare to the shown samples?

Comments on latest version:

The rebuttal letter and public response indicate the authors' reluctance to consider the limitations of their study, i.e. having chosen chronologically young animals to demonstrate a sperm aging effect and indicate that they are not willing to include adequate controls.

Since there is no evidence that mice at this young age have a deteriorating blood-testis-barrier (indeed, normal intact BTB is clearly visible in the figures included in this study from animals of the relevant age group), the whole central hypothesis that the study is built upon (i.e. that increasing age causes deteriorating BTB integrity which in turn causes age-related changes in sperm DNA methylation), appears irrelevant or invalid.

The authors' claim that age-related DNA methylation changes in sperm occur in linear fashion and that the changes are somewhat proportional with chronological age is in stark contrast of the claim that a decline of the BTB in old animals is causative for age-related sperm epigenetic changes, putting the relevance of the whole study in question.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

We have only few comments to improve an already strong manuscript.

Comments:

(1) Can the authors comment on how they justify K+ ion positions in their models (e.g. the K+ ion bridging G-1 and G+1 nucleotides)? How do they discriminate e.g. in the 'G-1 and G+1' case K+ from water?<br /> (2) The authors comment on Yju2 and Fyv6 assignments in all yeast structures except for the ILS. Can the authors comment on if they have also looked into the assignment of Yju2 in the yeast ILS structure in the same manner? While it is possible that Fyv6 could dissociate and Yju2 reassociate at the P to ILS transition, this would merit a closer look given that in the yeast P complex Yju2 had been misassigned previously.<br /> (3) For accessibility to a general reader, figures 1c, d, e, 2a, b, would benefit from additional headings or labels, to immediately convey what is being displayed. It is also not clear to us if Fig 1e might fit better in the supplement and be instead replaced by Supplementary Figure 1a (wt) , b (delta upf1), and a new c (delta fyv6) and new d (delta upf1, delta fyv6). This may allow the reader to better follow the rationale of the authors' use of the Fyv6/Upf1 double deletion.<br /> (4) The authors carefully interpret the various suppressor mutants, yet to a general reader the authors may wish to focus this section on only the most critical mutants for a better flow of the text.

Reviewer #2 (Public Review):

Summary:

The authors have tested the effects of partial- or whole-chromosome aneuploidy on the m6A RNA modification in Drosophila. The data reveal that overall m6A levels trend up but that the number of sites found by meRIP-seq trend down, which seems to suggest that aneuploidy causes a subset of sites to become hyper-methylated. Subsequent bioinformatic analysis of other published datasets establish correlations between the activity of the H4K16 acetyltransferase dosage compensation complex (DCC) and the expression of m6A components and m6A abundance, suggesting that DCC and m6A can act in a feedback loop on each other. Overall, this paper uses bioinformatic trends to generate a candidate model of feedback between DCC and m6A. It would be improved by functional studies that validate the effect in vivo.

Strengths:

• Thorough bioinformatic analysis of their data.

• Incorporation of other published datasets that enhance scope and rigor.

• Finds trends that suggest that a chromosome counting mechanism can control m6A, as fits with pub data that the Sxl mRNA is m6A modified in XX females and not XY males.

• Suggests this counting mechanism may be due to the effect of chromatin-dependent effects on the expression of m6A components.

Weaknesses:

• The linkage between H4K16 machinery and m6A is indirect and based on bioinformatic trends with little follow-up to test the mechanistic bases of these trends.

• The paper lacks sufficient in vivo validation of the effects of DCC alleles on m6A and vice versa. For example, Is the Ythdc1 genomic locus a direct target of the DCC component Msl-2 ? (see Figure 7).

• Quite a bit of technical detail is omitted from the main text, making it difficult for the reader to interpret outcomes.

(1) Please add the tissues to the labels in Figure 1D.

(2) In the main text, please provide detail on the source tissues used for meRIP; was it whole larvae? adult heads? Most published datasets are from S2 cells or adult heads and comparing m6A across tissues and developmental stages could introduce quite a bit of variability, even in wt samples. This issue seems to be what the authors discuss in lines 197-199.

(3) In the main text, please identify the technique used to measure "total m6A/A" in Fig 2A. I assume it is mass spec.

(4) Line 190-191: the text describes annotating m6A sites by "nearest gene" which is confusing. The sites are mapped in RNAs, so the authors must unambiguously know the identity of the gene/transcript, right?

Reviewer #2 (Public Review):

Summary:<br /> This work aims to characterize the neural signaling cascade underlying the initiation of metamorphosis in Ciona larvae. Combining gene-specific functional analyses, pharmacological experiments, and live imaging approaches, the authors identify the molecular players downstream of GABA to initiate Ciona metamorphosis. The results of this study may serve as a useful framework for future research on animal metamorphosis.

Strengths:<br /> The authors did a great job in connecting their experiments with previous findings on Ciona metamorphosis. Taking advantage of the Ciona model system, they meticulously conducted genetic manipulation and pharmacological experiments to test the epistatic relationships among the signaling players controlling the initiation of Ciona metamorphosis.

Weaknesses:<br /> The causal relationship between cAMP accumulation and the initiation of metamorphosis was not clearly demonstrated by the life-imaging observation with the fluorescent cAMP indicator (Pink Flamindo). It is a pity that this experiment was only conducted using normal larvae to compare those who underwent metamorphosis versus those who failed to initiate metamorphosis. This approach should be applied to some of the genetic manipulation and pharmacological experiments, to strengthen their main thesis on the "cAMP timer" mechanism.<br /> On several occasions, the interpretation of the results seems to be imprecise and may lead to misunderstanding. This should be improved by rewriting the descriptions of those results and carefully comparing the differences in results from various treatments and experiments.

Reviewer #2 (Public Review):

Summary:

The authors provide the first (to my knowledge) detailed characterization of cell wall b-1,6 glucan in the pathogen Candida albicans. The approaches range from biochemistry to genetics to immunology. The study provides fundamental information and will be a resource of exceptional value to the field going forward. Highlights include the construction of a mutant that lacks all b-1,6 glucan and the characterization of its cell wall composition and structure. Figure 5a is a feast for the eyes, showing that b-1,6 glucan is vital for the outer fibrillar layer of the cell wall. Also much appreciated was the summary figure, Figure 7, which presents the main findings in digestible form.

Strengths:

The work is highly significant for the fungal pathogen field especially, and more broadly for anyone studying fungi, antifungal drugs, or antifungal immune responses.

The manuscript is very readable, which is important because most readers will be cell wall nonspecialists.

The authors construct a key quadruple mutant, which is not trivial even with CRISPR methods, and validate it with a complemented strain. This aspect of the study sets the bar high.

The authors develop new and transferable methods for b-1,6 glucan analysis.

Weaknesses:

The one "famous" cell type that would have been interesting to include is the opaque cell. This could be included in a future paper.

Reviewer #2 (Public Review):

Summary: Padder et al. demonstrate that ATG5 mediates lysosomal repair via the recruitment of the retromer components during LLOMe-induced lysosomal damage and that mAtg8-ylation contributes to retromer-dependent cargo sorting of GLUT1. Although previous studies have suggested that during glucose withdrawal, classical autophagy contributes to retromer-dependent GLUT1 surface trafficking via interactions between LC3A and TBC1D5, the experiments here demonstrate that during basal conditions or lysosomal damage, ATGs that are not involved in mATG8ylation, such as FIP200, are not functionally required for retromer-dependent sorting of GLUT1. Overall, these studies suggest a unique role for ATG5 in the control of retromer function, and that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes.

Strengths:

(1) Overall, these studies suggest a unique non-autophagic role for ATG5 in the control of retromer function. They also demonstrate that conjugation of ATG8 to single membranes (CASM) is a partial contributor to these phenotypes. Overall, these data point to a new role for ATG5 and CASM-dependent mATG8ylation in lysosomal membrane repair and trafficking.

(2) Although the studies are overall supportive of the proposed model that the retromer is controlled by CASM-dependent mATG8-ylaytion, it is noteworthy that previous studies of GLUT1 trafficking during glucose withdrawal (Roy et al. Mol Cell, PMID: 28602638) were predominantly conducted in cells lacking ATG5 or ATG7, which would not be able to discriminate between a CASM-dependent vs. canonical autophagy-dependent pathway in the control of GLUT1 sorting. Is the lack of GLUT1 mis-sorting to lysosomes observed in FIP200 and ATG13KO cells also observed during glucose withdrawal? Notably, deficiencies in glycolysis and glucose-dependent growth have been reported in FIP200 deficient fibroblasts (Wei et al. G&D, PMID: 21764854) so there may be differences in regulation dependent on the stress imposed on a cell.

Weaknesses:

(1) Additional controls are needed to clarify the role of CASM in the control of retromer function. Because the manuscript proposes both CASM-dependent and independent pathways in the ATG5 mediated regulation of the retromer, it is important to provide robust evidence that CASM is required for retromer-dependent GLUT1 sorting to the plasma membrane vs. lysosome. The experiments with monsensin in Fig. 7C-E are consistent with but not unequivocally corroborative of a role for CASM. Based on the results shown with ATG16KO in Fig 4A-D, rescue experiments of these 16KO cells with WT vs. C-terminal WD40 mutant versions of ATG16 will specifically assess the requirement for CASM and potentially provide more rigorous support for the conclusions drawn.

(2) Also, the role of TBC1D5 should be further clarified. In Fig S7, are there any changes in the interactions between TBC1D5 and VPS35 in response to LLOMe or other agents utilized to induce CASM? Does TBC1D5 loss-of-function modulate the numbers of GLUT1 and Gal3 puncta observed in ATG5 deficient cells in response to LLOMe?

(3) Finally, the studies here are motivated by experiments in Fig. S1 (as well as other studies from the Deretic and Stallings labs) suggesting unique autophagy-independent functions for ATG5 in myeloid cells and neutrophils in susceptibility to Mycobacterium tuberculosis infection. However, it is curious that no attempt is made to relate the mechanistic data regarding the retromer or GLUT1 receptor mis-sorting back to the infectious models. Do myeloid cells or neutrophils lacking ATG5 have deficiencies in glucose uptake or GLUT1 cell surface levels?

Reviewer #2 (Public Review):

Chong Wang et al. investigated the role of H3K4me2 during the reprogramming processes in mouse preimplantation embryos. The authors show that H3K4me2 is erased from GV to MII oocytes and re-established in the late 2-cell stage by performing Cut & Run H3K4me2 and immunofluorescence staining. Erasure and re-establishment of H3K4me2 have not been studied well, and profiling of H3K4me2 in germ cells and preimplantation embryos is valuable to understanding the reprogramming process and epigenetic inheritance.

(1) The authors claim that the Cut & Run worked for MII oocytes, zygotes, and the 2-cell embryos. However, it is unclear if H3K4me2 is erased during the stage or if the Cut & Run did not work for these samples. To support the hypothesis of the erasure of H3K4me2, the authors conducted immunofluorescence staining, and H3k4me2 was undetected in the MII oocyte, PN5, and 2-cell stage. However, the published papers showed strong staining of H3K4me2 at the zygote stage and 2-cell stage ((Ancelin et al., 2016; Shao et al., 2014)). The authors need to cite these papers and discuss the contradictory findings.

The authors used 165 MII oocytes and 190 GV oocytes for the Cut & Run. The amount of DNA in MII oocytes is halved because of the emission of the first polar body. Would it be a reason that H3K4me2 has fewer H3K4me2 peaks in MII oocytes than GV oocytes?

In Figure 3C, 98% (13,183/13,428) of H3K4me2 marked genes in GV oocytes overlap with those in the 4-cell stage. Furthermore, 92% (14,049/15,112) of H3K4me2 marked genes in sperm overlap with those in the 4-cell stage. Therefore, most regions maintain germ line-derived H3K4me2 in the 4-cell stage. The authors need to clarify which regions of germ line-derived H3K4me2 are maintained or erased in preimplantation embryos. Additionally, it would be interesting to investigate which regions show the parental allele-specific H3K4me2 in preimplantation embryos since the authors used hybrid preimplantation embryos (B6 x DBA).

(2) The authors claim that Kdm1a is rarely expressed during mouse embryonic development (Figure 4A). However, the published paper showed that KDM1a is present in the zygote and 2-cell stage using immunostaining and western blotting ((Ancelin et al., 2016)). Additionally, this paper showed that depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage, and therefore, KDM1a is functionally important in early development. The authors should have cited the paper and described the role of KDM1a in early embryos.

(3) The authors used the published RNA data set and interpreted that KDM1B (LSD2) was highly expressed at the MII stage (Figure S3A). However, the heat map shows that KDM1B expression is high in growing oocytes but not at 8w_oocytes and MII oocytes. The authors need to interpret the data accurately.

(4) All embryos in the TCP group were arrested at the four-cell stage. Embryos generated from KDM1b KO females can survive until E10.5 (Ciccone et al., 2009); therefore, TCP-treated embryos show a more severe phenotype than oocyte-derived KDM1b deleted embryos. Depletion of maternal KDM1A protein results in developmental arrest at the two-cell stage ((Ancelin et al., 2016)). The authors need to examine whether TCP treatment affects KDM1a expression. Western blotting would be recommended to quantify the expression of KDM1A and KDM1B in the TCP-treated embryos.

(5) H3K4me2 is increased dramatically in the TCP-treated embryos in Figure 4 (the intensity is 1,000 times more than the control). However, the Cut & Run H3K4me2 shows that the H3K4me2 signal is increased in 251 genes and decreased in 194 genes in the TCP-treated embryos (Fold changes > 2, P < 0.01). The authors need to explain why the gain of H3K4me2 is less evident in the Cut & Run data set than in the immunofluorescence result.

References

Ancelin, K., ne Syx, L., Borensztein, M., mie Ranisavljevic, N., Vassilev, I., Briseñ o-Roa, L., Liu, T., Metzger, E., Servant, N., Barillot, E., Chen, C.-J., Schü le, R., & Heard, E. (2016). Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation. https://doi.org/10.7554/eLife.08851.001

Ciccone, D. N., Su, H., Hevi, S., Gay, F., Lei, H., Bajko, J., Xu, G., Li, E., & Chen, T. (2009). KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature, 461(7262), 415-418. https://doi.org/10.1038/nature08315

Shao, G. B., Chen, J. C., Zhang, L. P., Huang, P., Lu, H. Y., Jin, J., Gong, A. H., & Sang, J. R. (2014). Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development. In Vitro Cellular and Developmental Biology - Animal, 50(7), 603-613. https://doi.org/10.1007/s11626-014-9741-6

Reviewer #2 (Public Review):

Summary:<br /> This article, titled "A multi-gene predictive model for the radiation sensitivity of nasopharyngeal carcinoma based on machine learning," utilizes machine learning methods and transcriptomic data from nasopharyngeal carcinoma (NPC) patients to construct a biomarker called NPC-RSS that can predict the radiosensitivity of NPC patients. The authors further explore the biological mechanisms underlying the relationship between NPC-RSS and radiotherapy response in NPC patients. The main objective of this study is to guide the selection of radiotherapy strategies for NPC patients, thereby improving their clinical outcomes and prognosis.

Strengths:<br /> (1) The combination of multiple machine learning algorithms and cross-validation was used to select the best predictive model for radiotherapy sensitivity from 71 differentially expressed genes, enhancing the robustness and reliability of the predictions.<br /> (2) Functional enrichment analysis revealed close associations between NPC-RSS key genes and immune characteristics, expression of radiotherapy sensitivity-related genes, and signaling pathways related to disease progression, providing a biological basis for NPC-RSS in predicting radiotherapy sensitivity.<br /> (3) Grouping NPC samples according to NPC-RSS showed that the radiotherapy-sensitive group exhibited a more enriched and activated state of immune infiltration compared to the radioresistant group. In single-cell samples, NPC-RSS was higher in the radiotherapy-sensitive group, with immune cells playing a dominant role. These results clarify the mechanism of NPC-RSS in predicting radiotherapy sensitivity from an immunological perspective.<br /> (4) The study used public datasets and in-house cohort data for validation, confirming the good predictive performance of NPC-RSS and increasing the credibility of the results.

Limitation:<br /> (1) The study focuses on a specific type of nasopharyngeal carcinoma (NPC) and may not be generalizable to other subtypes or related head and neck cancers. The applicability of NPC-RSS to a broader range of patients and tumor types remains to be determined.<br /> (2) The study does not account for potential differences in radiotherapy protocols, doses, and techniques between the training and validation cohorts, which could influence the performance of the predictive model. Standardization of treatment parameters would be important for future validation studies.<br /> (3) The binary classification of patients into radiotherapy-sensitive and resistant groups may oversimplify the complex spectrum of treatment responses. A more granular stratification system that captures intermediate responses could provide more nuanced predictions and better guide personalized treatment decisions.<br /> (4) The study does not address the potential impact of other relevant factors, such as tumor stage, histological subtype, and concurrent chemotherapy, on the predictive performance of NPC-RSS. Incorporating these clinical variables into the model could enhance its accuracy and clinical utility.

Reviewer #2 (Public Review):

Summary:

This study aims to describe the single-cell transcriptomes of H pylori-associated (Hp) gastric cancers and tumour microenvironment (TME), as a starting point to understand TME diversity stratified by Hp status.

RNAseq was performed for gastric cancers with current Hp+ (from N=9 people), ex-Hp+ (N=6), non-Hp (N=6), and healthy gastric tissue (N=6).

The study expands on previous single-cell transcriptomic studies of gastric cancers and was motivated by previous observations about the effect of H pylori status on therapeutic outcomes. The study includes a brief review of previous work and provides valuable context for this study.

Strengths:

The observations are supported by solid RNAseq study design and analysis. The authors describe correlations between Hp status and inferred molecular characteristics including cell lineages, enrichment for cell subclusters identified as tumour-infiltrating lyphocyte cell types, tumour-infiltrating myeloid cells, and cancer-associated fibroblasts.

The observed correlations between Hp status and enrichment of cell subclusters were broadly corroborated using comparisons to deconvolved bulk RNAseq from publicly available gastric cancer data, providing a convincing starting point for understanding the diversity of tumour microenvironment by Hp-status.

Weaknesses:

The authors acknowledge several limitations of this study.

The correlations with HP-status are based on a small number of participants per Hp category (N=9 with current Hp+; N=6 for ex-HP+ and non-HP), and would benefit from further validation to establish reproducibility in other cohorts.

The ligand-receptor cross-talk analysis and the suggestion that suppressive T cells could interact with the malignant epithelium through TIGIT-NECTIN2/PVR pairs, are preliminary findings based on transcriptomic analysis and immunostaining and will require further validation.

Reviewer #2 (Public Review):

Summary:

This study proposes that the microRNA cluster miR-277/34 controls the generation of sexual dimorphism in Drosophila melanogaster during metamorphosis by acting on specific hormonal and developmental gene pathways.

Strengths:

Using a combination of mRNA and small RNA sequencing together with genome-wide in silico and in vitro analyses the authors identified a microRNA cluster that may be involved in metamorphosis and the generation of sexual dimorphism in Drosophila melanogaster.

Weaknesses:

Biological validation of the identified sexually dimorphic genes and a detailed understanding of how the microRNA cluster miR-277/34 might be involved in the regulation of sesquiterpenoids are needed.

Major suggestions:

(1) If AstC-R1 and Kr-h1 are targets of the miR-277/34 cluster and cause their downregulation, it is not clear why there would also be a decrease in the levels of these genes in the miR-277/34 mutants. This would suggest that the mechanism is not straightforward and that further epistatic experiments should be carried out in order to clarify this issue.

(2) The changes in the expression levels of AstC-R1 in pupae of miR-277-KO and mir-34-KO flies must be accompanied by photos of the respective larvae and pupae, as well as an analysis of the larvae-pupa transition on the mutants by gender.

(3) Biological validation of the identified sexually dimorphic genes in vivo will be necessary for the support of this work.

Reviewer #2 (Public Review):

Summary:

This manuscript addresses the intriguing topic of the potential roles of germline-specific proteins in early development. While this issue is quite interesting and generally under-explored, the work falls short of making truly tangible inroads.

Strengths:

The strength of the study is in new proteomic datasets.

Weaknesses:

The manuscript makes some strong statements, beginning with the title "STAG3 (1) promotes exit from pluripotency (2) through post-transcriptional mRNA regulation in the cytoplasm".

Upon reviewing the data it appears that neither (1) or (2) here have strong foundations based on experiments presented. While intriguing, the experimental evidence is still rather inconclusive.

The potential involvement of STAG3 in PGC specification is the most intriguing aspect of this study. Unfortunately, it is not going far enough to derive a fully meaningful biological conclusion. In fact, DPPPA3-GFP, PRDM-GFP, and PGC marker expression results are contradictory and do not form a coherent picture of the biological effect of STAG3 depletion. No effect of the knock-down in PGC specification when PRDM is scored (line 167) is particularly worrisome. As for finding a cytoplasmic role of STAG3, the data also remain inconclusive.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, Lin and colleagues aim to understand the role of different salts on the phase behavior of a model protein of significant biological interest, Caprin1, and its phosphorylated variant, pY-Caprin1. To achieve this, the authors employed a variety of methods to complement experimental studies and obtain a molecular-level understanding of ion partitioning inside biomolecular condensates. A simple theory based on rG-RPA is shown to capture the different salt dependencies of Caprin1 and pY-Caprin1 phase separation, demonstrating excellent agreement with experimental results. The application of this theory to multivalent ions reveals many interesting features with the help of multicomponent phase diagrams. Additionally, the use of CG model-based MD simulations and FTS provides further clarity on how counterions can stabilize condensed phases.

Strengths:<br /> The greatest strength of this study lies in the integration of various methods to obtain complementary information on thermodynamic phase diagrams and the molecular details of the phase separation process. The authors have also extended their previously proposed theoretical approaches, which should be of significant interest to other researchers. Some of the findings reported in this paper, such as bridging interactions, are likely to inspire new studies using higher-resolution atomistic MD simulations.

Weaknesses:<br /> The paper does not have any major issues.

Reviewer #2 (Public Review):

Summary:

During vertebrate gastrulation, the mesoderm and endoderm arise from a common population of precursor cells and are specified by similar signaling events, raising questions as to how these two germ layers are distinguished. Here, Cheng and colleagues use zebrafish gastrulation as a model for mesoderm and endoderm segregation. By reanalyzing published single-cell sequencing data, they identify a common progenitor population for the anterior endoderm and the mesodermal prechordal plate (PP). They find that expression levels of PP genes Gsc and ripply are among the earliest differences between these populations and that their increased expression suppresses the expression of endoderm markers. Further analysis of chromatin accessibility and Ripply cut-and-tag is consistent with direct repression of endoderm by this PP marker. This study demonstrates the roles of Gsc and Ripply in suppressing anterior endoderm fate, but this role for Gsc was already known and the effect of Ripply is limited to a small population of anterior endoderm. The manuscript also focuses extensively on the function of Nodal in specifying and patterning the mesoderm and endoderm, a role that is already well known and to which the current analysis adds little new insight.

Strengths:

Integrated single-cell ATAC- and RNA-seq convincingly demonstrate changes in chromatin accessibility that may underlie the segregation of mesoderm and endoderm lineages, including Gsc and ripply. Identification of Ripply-occupied genomic regions augments this analysis. The genetic mutants for both genes provide strong evidence for their function in anterior mesendoderm development, although these phenotypes are subtle.

Weaknesses:

The use of zebrafish embryonic explants for cell fate trajectory analysis (rather than intact embryos) is not justified. In both transcriptomic comparisons between the two fate trajectories of interest and Ripply cut-and-tag analysis, the authors rely too heavily on gene ontology which adds little to our functional understanding. Much of the work is focused on the role of Nodal in the mesoderm/endoderm fate decision, but the results largely confirm previous studies and again provide few new insights. Some experiments were designed to test the relationship between the mesoderm and endoderm lineages and the role of epigenetic regulators therein, but these experiments were not properly controlled and therefore difficult to interpret.

Reviewer #2 (Public Review):

Summary, strengths, and weaknesses:

This article by Rice et al focuses on the study of limbal epithelial stem cells (LESCs). To obtain high resolution of stem/progenitor cell populations by single-cell RNA sequencing (scRNA-seq), the authors enriched the stem/progenitors by capturing GFP+ epithelial cells undergoing mitosis using the Cyclin-B-GFP transgene. The key novelty in the paper is that they identified Sox9 as a new LSC gene that is important for the health of the cornea. They show that Sox9 is expressed by LSCs at the mRNA and protein level, and that the Sox9-GFP transgene is useful to identify LSCs in live animals. They performed lineage tracing of Sox9-Cre mice that clearly demonstrated that Sox9+ cells are true LSCs. Further, Sox9-knockouts display severe opacification of the cornea, accompanied by the transformation of the central cornea into hyperplastic epidermis - a phenotype similar to previously described Notch1 conditional knockout (cKO). By studying the lifetimes of Notch dominant-negative transgenic individual basal corneal epithelial cells, they suggest that the thickening of the central zone in the Notch model is due to the loss of asymmetric division, and from that, they infer the phenotype of the Sox2-KO. In other words, it is suggested that the increased rate and type of division in the central Sox9-null cornea produce this dramatic epithelial thickening phenotype. This claim makes sense but suggests a role for the Notch, not Sox9, and this role is relevant for the central corneal epithelial cells, and not in LESCs. So I suggest considering revising the conclusions (title of the paper) on this aspect. This would not affect the impact of the paper.

In general, I believe that this is a very interesting and novel study that will be of interest to a broad readership. The methodology and study design are robust and elegant.

However, in some cases, typos, text modifications, additional controls, and new experiments are suggested.

Reviewer #2 (Public Review):

Summary:

The aim was to identify the mechanisms that underlie a form of long-term potentiation (LTP) that requires the activation of dopamine (DA).

Strengths:

The authors have provided multiple lines of evidence that support their conclusions; namely that this pathway involves the activation of a cAMP / PKA pathway that leads to the insertion of calcium-permeable AMPA receptors.

Weaknesses:

Some of the experiments could have been conducted in a more convincing manner.

Reviewer #2 (Public Review):

This study compares neural responses to speech in normal-hearing and hearing-impaired listeners, investigating how different levels of the linguistic hierarchy are impacted across the two cohorts, both in a single-talker and multi-talker listening scenario. It finds that, while normal-hearing listeners have a comparable cortical encoding of speech-in-quiet and attended speech from a multi-talker mixture, participants with hearing impairment instead show a reduced cortical encoding of speech when it is presented in a competing listening scenario. When looking across the different levels of the speech processing hierarchy in the multi-talker condition, normal-hearing participants show a greater cortical encoding of the attended compared to the unattended stream in all speech processing layers - from acoustics to sentence-level information. Hearing-impaired listeners, on the other hand, only have increased cortical responses to the attended stream for the word and phrase levels, while all other levels do not differ between attended and unattended streams.<br /> The methods for modelling the hierarchy of speech features (HM-LSTM) and the relationship between brain responses and specific speech features (ridge-regression) are appropriate for the research question, with some caveats on the experimental procedure. This work offers an interesting insight into the neural encoding of multi-talker speech in listeners with hearing impairment, and it represents a useful contribution towards understanding speech perception in cocktail-party scenarios across different hearing abilities. While the conclusions are overall supported by the data, there are limitations and certain aspects that require further clarification.<br /> (1) In the multi-talker section of the experiment, participants were instructed to selectively attend to the male or the female talker, and to rate the intelligibility, but they did not have to perform any behavioural task (e.g., comprehension questions, word detection or repetition), which could have demonstrated at least an attempt to comply with the task instructions. As such, it is difficult to determine whether the lack of increased cortical encoding of Attended vs. Unattended speech across many speech features in hearing-impaired listeners is due to a different attentional strategy, which might be more oriented at "getting the gist" of the story (as the increased tracking of only word and phrase levels might suggest), or instead it is due to hearing-impaired listeners completely disengaging from the task and tuning back in for selected key-words or word combinations. Especially the lack of Attended vs. Unattended cortical benefit at the level of acoustics is puzzling and might indicate difficulties in performing the task. I think this caveat is important and should be highlighted in the Discussion section.<br /> (2) In the EEG recording and preprocessing section, you state that the EEG was filtered between 0.1Hz and 45Hz. Why did you choose this very broadband frequency range? In the literature, speech responses are robustly identified between 0.5Hz/1Hz and 8Hz. Would these results emerge using a narrower and lower frequency band? Considering the goal of your study, it might also be interesting to run your analysis pipeline on conventional frequency bands, such as Delta and Theta, since you are looking into the processing of information at different temporal scales.<br /> (3) A paragraph with more information on the HM-LSTM would be useful to understand the model used without relying on the Chung et al. (2017) paper. In particular, I think the updating mechanism of the model should be clarified. It would also be interesting to modify the updating factor of the model, along the lines of Schmitt et al. (2021), to assess whether a HM-LSTM with faster or slower updates can better describe the neural activity of hearing-impaired listeners. That is, perhaps the difference between hearing-impaired and normal-hearing participants lies in the temporal dynamics, and not necessarily in a completely different attentional strategy (or disengagement from the stimuli, as I mentioned above).<br /> (4) When explaining how you extracted phoneme information, you mention that "the inputs to the model were the vector representations of the phonemes". It is not clear to me whether you extracted specific phonetic features (e.g., "p" sound vs. "b" sound), or simply the phoneme onsets. Could you clarify this point in the text, please?

Reviewer #3 (Public Review):

In this manuscript, Picard et al. propose a Facial Expression Pain Signature (FEPS) as a distinctive marker of pain processing in the brain. Specifically, they attempt to use functional magnetic resonance imaging (fMRI) data to predict facial expressions associated with painful heat stimulation.

The main strengths of the manuscript are that it is built on an extensive foundation of work from the research group, and that experience can be observed in the analysis of fMRI data and the development of the machine learning model. Additionally, it provides a comparative account of the similarities of the FEPS with other proposed pain signatures. The main weaknesses of the manuscript are the absence of a proper control condition to assess the specificity of the facial pain expressions, as well as several limitations in the experimental setup.

I believe that the authors partially succeed in their aims, as described in the introduction, which are to assess the association between pain facial expression and existing pain-relevant brain signatures, and to develop a predictive brain activation model of the facial responses to painful thermal stimulation. However, they list several limitations in the study that should be addressed in future research in order to establish whether FEPS truly conveys distinctive information about the brain response to nociceptive stimuli.

Reviewer #2 (Public Review):

Summary:

The authors set out to establish the role of the rice LEC1 homolog OsNF-YB7 in embryo development, especially as it pertains to the development of photosynthetic capacity, with chlorophyll production as a primary focus.

Strengths:

The results are well-supported and each approach used complements each other. There are no major questions left unanswered and the central hypothesis is addressed in every figure.

Weaknesses:

There are a handful of sections which could use clarifying for readers, but overall this is a solidly composed manuscript.

The authors clearly achieved their aims; the results compellingly establish a disparity between how this system operates in rice and Arabidopsis. Conclusions are thoroughly supported by the provided data and interpretations. This work will force a reconsideration of the value of Arabidopsis as a model organism for embryo chlorophyll biosynthesis and possibly photosynthesis during embryo maturation more broadly, as rice is a major crop organism and it very clearly does not follow the Arabidopsis model. It will thus be useful to carry out similar tests in other organisms rather than relying on Arabidopsis and attempt to more fully establish the regulatory mechanism in rice.

Reviewer #2 (Public Review):

Summary:

In this manuscript by Cannarsa et. al., the authors describe the engineering of a light-entrainable synthetic biological oscillator in bacteria. It is based on an upgraded version of one of the first synthetic circuits to be constructed, the repressilator. The authors sought to make this oscillator entrainable by an external forcing signal, analogous to the way natural biological oscillators (like the circadian clock) are synchronized. They reasoned that an optogenetic system would provide a convenient and flexible means of manipulation. To this end, the authors exploited the CcaS-CcaA light-switchable system, which allows activation and deactivation of transcription by green and red light, respectively. They used this system to make the expression of one of the repressilator's transcription factors (lacI) light-controlled, from a construct separated from the main repressilator plasmid. This way, under red light the oscillator runs freely, but exposure to green light causes overexpression of the lacI, pushing the system into a specific state. Consequently, returning to red light will restore the oscillations from the same phase in all cells, effectively synchronizing the cell population.

After demonstrating the functionality of the basic concept, the authors combined modeling and experiments to show how periodic exposure to green light enables efficient entrainment, and how the frequency of the forcing signal affects the oscillatory behavior (detuning).

This work provides an important demonstration of engineering tunability into a foundational genetic circuit, expands the synthetic biology toolbox, and provides a platform to address critical questions about synchronization in biological oscillators. Due to the flexibility of the experimental system, it is also expected to provide a fertile ground for future testing of theoretical predictions regarding non-linear oscillators.

Strengths:

* The study provides a simple and elegant mechanism for the entertainment of a synthetic oscillator. The design relies on optogenetic proteins, which enable efficient experimentation compared to alternative approaches (like using chemical inducers). This way, a static culture (without microfluidics or change of growth media) can be easily exposed to flexible temporal sequences of the zeitgeber, and continuously measured through time.

* The study makes use of both plate-reader-based population-level readout and mother-machine single-cell measurements. Synchronization through entrainment is a single cell level phenomenon, but with a clear population-level manifestation. Thus, this experimental approach combination provides a strong validation to their system. At the same time, differences between the readout from the two systems have emerged, and provided a further opportunity for model refinement and testing.

* The authors correctly identified the main optimization goal, namely the effective leakiness of their construct even under red light. Then, they successfully overcame this issue using synthetic biology approaches.

* The work is supported by a simplified model of the repressilator, which provides a convenient analytical and numerical means to draw testable predictions. The model predictions are well aligned with the experimental evidence.

Weaknesses:

* Even after optimizing the expression level of the light-sensitive gene, the system is very sensitive, i.e., a very short exposure is sufficient to elicit the strongest entertainment. This limited dynamic range might hamper some model testing and future usage.

* As a result of the previous point, the system is entrained by transiently "breaking" the oscillator: each pulse of green light represents a Hopf bifurcation into a single attractor. it means that the system cannot oscillate in constant green light. In comparison, this is generally not the case for natural zeitgebers like light and temperature for the circadian rhythms. Extreme values might prevent oscillations (not necessarily due to breaking the core oscillator), but usually, free running is possible in a wide range of constant conditions. In some cases, the free-running period length will vary as a function of the constant value.

While the approach presented in this manuscript is valid, a comprehensive analysis of more subtle modes of repressilator entrainment could also be of value.

* The entire work makes use of a single intensity and single duration of the green pulse to force entrainment. While the model has clear predictions for how different modalities should affect entrainment, more experiments are needed to validate those predictions.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Bradley and his colleagues represented the cryo-EM structure of the nuclear cap-binding complex (CBC) in complex with an mRNA export factor, ALYREF, providing a structural basis for understanding CBC regulating gene expression.

Strengths:

The authors successfully modeled the N-terminal region and the RRM domain of ALYREF (residues 1-183) within the CBC-ALYREF structure, which revealed that both the NCBP1 and NCBP2 subunits of the CBC interact with the RBM domain of ALYREF. Further mutagenesis and pull-down studies provided additional evidence to the observed CBC-ALYREF interface. Additionally, the authors engaged in a comprehensive discussion regarding other cellular complexes containing CBC and/or ALYREF components. They proposed potential models that elucidated coordinated events during mRNA maturation. This study provided structural evidence to show how CBC effectively recruits mRNA export factor machinery, enhancing our understanding of CBC regulating gene expression during mRNA transcription, splicing, and export.

Weaknesses:

Absence of functional data to support the proposed models in this study.

Reviewer #2 (Public Review):

Summary:

In this study, the author demonstrates that deficiency or pharmacological inhibition of O-glcNac transferase (OGT) enhances tumor immunity in colorectal cancer models. The authors propose that OGT deficiency triggers a DNA damage response, activating the cGAS-STING innate immunity pathway and promoting a Type I interferon response. They suggest that OGT-mediated processing of HSF1 is crucial in maintaining genomic integrity. This research is significant as it identifies OGT inhibition as a potential immunomodulatory target in cancer treatment.

Strengths:

The strength of the paper lies primarily in the in vivo data, demonstrating the impact of OGT deficiency or inhibition on modulating tumor growth and anti-tumor immunity. The experiments are well-controlled. However, there are several unresolved questions:

Weaknesses:

The mechanisms of how OGT deficiency can trigger DNA damage and the role of this response in promoting immunity are only partially addressed in the manuscript.

Reviewer #2 (Public Review):

Summary:

An analysis of images in the biology literature that are problematic for people with a color-vision deficiency (CVD) is presented, along with a machine learning-based model trained on an eLife dataset to identify such images and a web application that uses the model to flag problematic images. Their analysis reveals that about 13% of the images could be problematic for people with CVD and that the frequency of such images decreased over time. Their best model (convolutional neural network, CNN) yields 0.89 AUROC score and 0.77 AUPRC on held-out eLife articles, but lower scores (0.78 and 0.39, respectively). It is proposed that their approach could help making biology literature accessible to diverse audiences.

Strengths:

The manuscript focuses on an important yet mostly overlooked problem and makes contributions both in expanding our understanding of the extent of the problem and in developing solutions to mitigate the problem. The paper is generally well-written and clearly organized. Their CVD simulation combines five different metrics. The dataset has been assessed by two researchers and is likely to be of high-quality. Machine learning algorithm used (CNN) is an appropriate choice for the problem. The evaluation of various hyperparameters for the CNN model is extensive.

Weaknesses:

While the study has significant strengths, it also has some limitations. Specifically, the focus on one type of CVD (deuteranopia) and selecting images from a single journal (eLife) for training limit the generalizability of the models. This is, to some extent, shown by applying the model to PMC articles, which yields lower performance. "Probably problematic" and "probably okay" classes are excluded from the analysis.