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 #1 (Public review):

The work by Ginatt et al. uses genome-scale metabolic modeling to identify and characterize trophic interactions between rhizosphere-associated bacteria. Beyond identifying microbial species associated with specific host and soil traits (e.g., disease tolerance), a detailed understanding of the interactions underlying these associations is necessary for developing targeted microbiome-centered interventions for plant health. It has nonetheless remained challenging to define the roles of specific organisms and metabolic species in natural rhizobiomes. Here, the authors combine microbial compositional data obtained through metagenomic sequencing with a new collection of genome-scale models to predict interactions in the native rhizosphere communities of apple rootstocks. To do this, they have established processes to integrate these sources of data and model specific trophic exchanges, which they use to obtain testable hypotheses for targeted modulation of microbiota members in situ.

The authors carry out a careful model curation process based on metagenomic sequencing data and existing model generation tools, which, together with basing the in silico medium composition on known root exudates, strengthens their predictions of interaction network features. Moreover, its reliance on genome-scale models provides a broader basis for linking sequence-based information to predictions of function on a multispecies level beyond rhizosphere microbiomes.

Having generated a set of predicted trophic interactions, the authors carried out a detailed analysis linking features of these interactions to organism taxonomy and broader ecosystem properties. Intriguingly, the organisms predicted to grow in the first iteration of their framework (i.e., on only root exudates) broadly correspond to taxonomic groups experimentally shown to benefit from these compounds. Additionally, the simulations predicted some patterns of vitamin and amino acid secretion that are known to form the basis for interactions in the rhizosphere. Together, these outcomes underscore the applicability of this method to help disentangle trophic interaction networks in complex microbiomes.

The methodology described in this paper represents a useful and promising framework to better understand the complexity of microbial interaction networks in situ. In particular, the authors' simulation of trophic interactions based on cellulose degradation have generated predictions of interactions that can more readily be validated. While a more complete analysis of the method's sensitivity to environmental composition is still needed to fully interpret its conclusions - particularly those predicting the inability of many of the in silico organisms to produce biomass - it represents a valuable addition to the growing toolkit of computational and experimental methods for generating educated hypotheses on complex trophic networks.

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 #1 (Public review):

Summary:

The researchers demonstrated that when cytokine priming is combined with exposure to pathogens or pathogen-associated molecular patterns, human alveolar macrophages and monocyte-derived macrophages undergo metabolic adaptations, becoming more glycolytic while reducing oxidative phosphorylation. This metabolic plasticity is more in monocyte derived macrophages as compared to alveolar macrophages.

Strengths:

This study presents evidence of metabolic reprogramming in human macrophages, which significantly contributes to our existing understanding of this field primarily derived from murine models.

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 #3 (Public review):

Summary:

In this manuscript the authors explore the contribution of metabolism to the response of two subpopulations of macrophages to bacterial pathogens commonly encountered in the human lung, as well as the influence of priming signals typically produced at a site of inflammation. The two subpopulations are resident airway macrophages (AM) isolated via bronchoalveolar lavage and monocyte-derived macrophages (MDM) isolated from human blood and differentiated using human serum. The two cell types were primed using IFNγ and Il-4, which are produced at sites of inflammation as part of initiation and resolution of inflammation respectively, followed by stimulation with either heat-killed tuberculosis (Mtb) or LPS to simulate interaction with a bacterial pathogen that is either gram-negative in the case of Mtb or gram-positive in the case of LPS. The authors use human cells for this work, which makes use of widely reported and thoroughly described priming signals, as well as model antigens. This makes the observations on the functional response of these two subpopulations relevant to human health and disease to a greater extent that the mouse models typically used to interrogate theses interactions. To examine the relationship between metabolism and functional response, the authors measure rates of oxidative phosphorylation and glycolysis under baseline conditions, primed using IFNγ or IL-4, and primed and stimulated with Mtb or LPS.

The authors addressed most of the initial critiques. The dose of IFNγ used was justified, figure legends were harmonized, a contextual definition was provided for the term "functional plasticity," and the airway macrophage population was partially characterized by flow cytometry. However, some concerns remain relating to the clarity of methods and use of statistics. The authors have not adequately explained how % change was calculated in Figure 1, in either the figure legend or the methods section. Additionally, the use of multiple statistical analyses on the same data set in figures 4 and 5, with data exclusion resulting in lower p values, is not satisfactorily justified.

Strengths:

• The data indicate that both populations of macrophages increase metabolic rates when primed, but MDMs decrease their rates of oxidative phosphorylation after IL-4 priming and bacterial exposure while AMs do not.

• It is demonstrated that glycolysis rates are directly linked to the expression of surface molecules involved in T-cell stimulation and while secretion of TNFα in AM is dependent on glycolysis, in MDM this is not the case. IL-10 secretion does not appear regulated by glycolysis in either population. It is also demonstrated that Mtb and LPS stimulation produces responses that are not metabolically consistent across the two macrophage populations. The Mtb-induced response in MDMs differed from the LPS response, in that it relies on glycolysis, while this relationship is reversed in AMs. The difference in metabolic contributions to functional outcomes between these two macrophage populations is significant, despite acknowledgement of the reductive nature of the system by the authors.

• The observations that AM and MDM rely on glycolysis for production of cytokines during a response to bacterial pathogens in the lung, but that only AM shift to Warburg Metabolism following exposure to IL-4, are supported by the data and a significant contribution the study of the innate immune response.

Weaknesses:

Critiques:

• It is still difficult to interpret the metabolism data due to inconsistent normalization. It appears that in the case of rate measurement the data is normalized to unstimulated macrophages where values are set to one, but in the case of % change the values from unstimulated cells are not set to 100% and the methods say that values were calculated using primed controls, which is ambiguous. It is therefore unclear how exactly the % change values were determined. This makes it difficult to conclude whether the changes in glycolysis and oxidative phosphorylation in primed cells after stimulation are proportional to changes in unprimed cells. This would suggest that the majority of the observed effect on metabolism comes from priming itself and not from the subsequent stimulation as the authors claim.

• The use of repeated statistical analyses with different comparison groups in the same figure/data set (e.g., in Fig.4) is still not justified. The current approach, using two-way ANOVA, removing a third of the dataset, and then applying another two-way ANOVA, produces the desired p values, but is not appropriate.

Conclusion:

Overall, this study reveals how inflammatory and anti-inflammatory cytokine priming contributes to the metabolic reprogramming of AM and MDM populations. Their conclusions regarding the relationship between cytokine secretion and inflammatory molecule expression in response to bacterial stimuli are supported by the data. The involvement of metabolism in innate immune cell function is relevant when devising treatment strategies that target the innate immune response during infection. The data presented in this paper further our understanding of that relationship and advance the field of innate immune cell biology.

Reviewer #1 (Public review):

Summary:

It is suggested that for each limb, the RG (rhythm generator) can operate in three different regimes: a non-oscillating state-machine regime and a flexor driven and a classical half-center oscillatory regime. This means that the field can move away from the old concept that there is only room for the classic half-center organization

Strengths:

A major benefit of the present paper is that a bridge was made between various CPG concepts ( "a potential contradiction between the classical half-center and flexor-driven concepts of spinal RG operation"). Another important step forward is the proposal about the neural control of slow gait ("at slow speeds ({less than or equal to} 0.35 m/s), the spinal network operates in a state regime and requires external inputs for phase transitions, which can come from limb sensory feedback and/or volitional inputs (e.g. from the motor cortex").

Weaknesses:

Some references are missing

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 #3 (Public review):

General Comments

This work probes the control of walking in cats at different speeds and different states (split-belt and regular treadmill walking). Since the time of Sherrington there has been ongoing debate on this issue. The authors provide modeling data showing that they could reproduce data from cats walking on a specialized treadmill allowing for regular and split-belt walking. The data suggest that a non-oscillating state-machine regime best explains slow walking - where phase transitions are handled by external inputs into the spinal network. They then show at higher speeds a flexor-driven and then a classical half-center regime dominates. In spinal animals, it appears that a non-oscillating state-machine regime best explains the experimental data. The model is adapted from their previous work and raises interesting questions regarding the operation of spinal networks, that, at low speeds, challenge assumptions regarding central pattern generator function. This is an outstanding study which will be of general interest to the neuroscience community.

Strengths

The study has several strengths. Firstly the detailed model has been well established by the authors and provides details that relate to experimental data such as commissural interneurons (V0c and V0d), along with V3 and V2a interneuron data. Sensory input along with descending drive is also modelled and moreover the model reproduces many experimental data findings. Moreover, the idea that sensory feedback is more crucial at lower speeds, also is confirmed by presynaptic inhibition increasing with descending drive. The inclusion of experimental data from split-belt treadmills, and the ability of the model to reproduce findings here is a definite plus.

Weaknesses

Conceptually, this is a compelling study which provides interesting modeling data regarding the idea that the network can operate in different regimes, especially at lower speeds. The modelling data speaks for itself, but on the other hand, sensory feedback also provides generalized excitation of neurons which in turn project to the CPG. That is they are not considered part of the CPG proper. The authors have discussed this possibility in their revised paper.

Reviewer #1 (Public Review):

Summary:

In their paper, Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

This approach does fill an important gap in malaria epidemiology, namely estimating the force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

Major comments:

(1) Description and evaluation of FOI estimation procedure.

a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on lines 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At a minimum, all variables used in the equations should be clearly defined.

b. Additionally, the description in the main text of how the queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow.

c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2, and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether the authors are displaying the bootstrapped 95%CIs or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per-host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. Figure 10, Figure 1 under the mid-IRS panel). But it's not possible to conclude one way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates.

d. Furthermore the authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter-arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify the likelihood and include in an appendix an explanation of why their estimation procedure is in fact maximizing this likelihood, preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested is for their mean and variance since this could influence the overall quality of the estimation procedure.

(2) Limitation of FOI estimation procedure.

a. The authors discuss the importance of the duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5-year-olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4-year-old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population.

b. The evaluation of the capacity parameter c seems to be quite important and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if the carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increase will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation.

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 #3 (Public Review):

Summary:

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying it to simulated results from a previously published agent-based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

Strengths:

It would be great to be able to infer FOI from cross-sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross-sectional studies. They attempt to validate this process using a previously published agent-based model which helps us understand the complexity of parasite population genetics.

Weaknesses:

(1) I fear that the work could be easily over-interpreted as no true validation was done, as no field estimates of FOI (I think considered true validation) were measured. The authors have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

(2) Another aspect of the paper is adding greater realism to the previous agent-based model, by including assumptions on missing data and under-sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the latter before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

(5) The text is a little difficult to follow at times and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

Reviewer #3 (Public Review):

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

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

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

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

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

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

Reviewer #1 (Public Review):

Summary:

In this work, Ritchie and colleagues explore functional consequences of neuronal over-expression or deletion of the MAP3K DLK that their labs and others have strongly implicated in both axon degeneration, neuronal cell death, and axon regeneration. Their recent work in eLife (Li, 2021) showed that inducible over-expression of DLK (or the related LZK) induces neuronal death in the cerebellum. Here, they extend this work to show that inducible over-expression in Vglut1+ neurons also kills excitatory neurons in hippocampal CA1, but not CA3. They complement this very interesting finding with translatomics to quantify genes whose mRNAs are differentially translated in the context of DLK over-expression or knockout, the latter manipulation having little to no effect on the phenotypes measured. The authors note that several genes and pathways are differentially regulated according to whether DLK is over-expressed or knocked out. They note DLK-dependent changes in genes related to synaptic function and the cytoskeleton and ultimately relate this in cultured neurons to findings that DLK over-expression negatively impacts synapse number and changes microtubules and neurites, though with a less obvious correlation.

Strengths:

This work represents a conceptual advance in defining DLK-dependent changes in translation. Moreover, the finding that DLK may differentially impact neuronal death will become the basis for future studies exploring whether DLK contributes to differential neuronal susceptibility to death, which is a broadly important topic.

Weaknesses:

This seems like two works in parallel that the authors have not yet connected. First is that DLK affects the translation of an interesting set of genes, and second, that DLK(OE) kills some neurons, disrupts their synapses, and affects neurite growth in culture.

Specific questions:

(1) Is DLK effectively knocked out? The authors reference the floxxed allele in their 2016 work (PMID: 27511108), however, the methods of this paper say that the mouse will be characterized in a future publication. Has this ever been published? The major concern is that here the authors show that Cre-mediated deletion results in a smaller molecular weight protein and the maintenance of mRNA levels.

(2) Why does DLK(OE) not kill CA3 neurons? The phenomenon is clear but there is no link to gene expression changes. In fact, the highlighted transcript in this work, Stmn4, changes in a DLK-dependent manner in CA3.

(3) Why are whole hippocampi analyzed to IP ribosome-associated mRNAs? The authors nicely show a differential effect of DLK on CA1 vs CA3, but then - at least according to their methods ¬- lyse whole hippocampi to perform IP/sequencing. Their data are therefore a mix of cells where DLK does and does not change cell death. The key issue is whether DLK does/does not have an effect based on the expression changes it drives.

(4) Is the subtle decrease in synapse number (Basson/Homer co-loc.) in the DLK (OE) simply a function of neurons (and their synapses, presumably) having died? At the P15 time point that the authors choose because cell death is minimal, there is still a ~25% reduction in CA1 thickness (Figure 2B), which is larger than the ~15% change in synapses (Figure 5H) they describe.

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 #3 (Public Review):

Dr Jin and colleagues revisit DLK and its established multifactorial roles in neuronal development, axonal injury, and neurodegeneration. The ambitious aim here is to understand the DLK-dependent gene network in the brain and, to pursue this, they explore the role of DLK in hippocampal glutamatergic neurons using conditional knockout and induced overexpression mice. They produce evidence that dorsal CA1 and dentate gyrus neurons are vulnerable to elevated expression of DLK, while CA3 neurons appear unaffected. Then they identify the DLK-dependent translatome featured by conserved molecular signatures and cell-type specificity. Their evidence suggests that increased DLK signaling is associated with possible STMN4 disruptions to microtubules, among else. They also produce evidence on cultured hippocampal neurons showing that expression levels of DLK are associated with changes in neurite outgrowth, axon specification, and synapse formation. They posit that downstream translational events related to DLK signaling in hippocampal glutamatergic neurons are a generalizable paradigm for understanding neurodegenerative diseases.

Strengths

This is an interesting paper based on a lot of work and a high number of diverse experiments that point to the pervasive roles of DLK in the development of select glutamatergic hippocampal neurons. One should applaud the authors for their work in constructing sophisticated molecular cre-lox tools and their expert Ribotag analysis, as well as technical skill and scholarly treatment of the literature. I am somewhat more skeptical of interpretations and conclusions on spatial anatomical selectivity without stereological approaches and also going directly from (extremely complex) Ribotag profiling patterns to relevance based on immunohistochemistry and no additional interventions to manipulate (e.g. by knocking down or blocking) their top Ribotag profile hits. Also, it seems to this reviewer that major developmental claims in the paper are based on gene translational profiling dependent on DLK expression, not DLK activation, despite some evidence in the paper that there is a correlation between the two. Therefore, observed patterns and correlations may or may not be physiologically or pathologically relevant. Generalizability to neurodegenerative diseases is an overreach not justified by the scope, approach, and findings of the paper.

Weaknesses and Suggestions:

The authors state that the rationale for the translatomic studies is to "to gain molecular understanding of gene expression associated with DLK in glutamatergic neurons" and to characterize the "DLK-dependent molecular and cellular network", However, a problem with the experimental design is the selection of an anatomical region at a time point featured by active neurodegeneration. Therefore, it is not straightforward that the differentially expressed genes or pathways caused by DLK overexpression changes could be due to processes related to neurodegeneration. Indeed, the authors find enrichment of signals related to pathways involved in extracellular matrix organization, apoptosis, unfolded protein responses, the complement cascade, DNA damage responses, and depletion of signals related to mitochondrial electron transport, etc., all of which could be the consequence of neurodegeneration regardless of cause. A more appropriate design to discover DLK-dependent pathways might be to look at a region and/or a time point that is not confounded by neurodegeneration.

In a related vein, the authors ask "if the differentially expressed genes associated with DLK(iOE) might show correlation to neuronal vulnerability" and, to answer this question, they select the set of differentially expressed genes after DLK overexpression and assess their expression patterns in various regions under normal conditions. It looks to me that this selection is already confounded by neurodegeneration which could be the cause for their downregulation. Therefore, such gene profiles may not be directly linked to neuronal vulnerability. A similar issue also relates to the conclusion that "...the enrichment of DLK-dependent translation of genes in CA1 suggests that the decreased expression of these genes may contribute to CA1 neuron vulnerability to elevated DLK".

To understand the role and relevance of the DLK overexpression model, there should be a discussion of to what extent it corresponds to endogenous levels of DLK expression or DLK-MAPK pathway activation under baseline or pathological conditions.

The authors posit that "dorsal CA1 neurons are vulnerable to elevated DLK expression, while neurons in CA3 appear largely resistant to DLK overexpression". This statement assumes that DLK expression levels start at a similar baseline among regions. Do the authors have any such data? Ideally, they should show whether DLK expression and p-c-Jun (as a marker of downstream DLK signaling) are the same or different across regions in both WT and overexpression mice. For example, what are the DLK/p-c-Jun expression levels in regions other than CA1 in Supplementary Figures 2-3 and how do they compare with each other? Normalization to baseline for each region does not allow such a comparison. Also, in Supplementary Figure 6, analyses and comparisons between regions are done at a time point when degeneration has already started. Ideally, these should be done at P10.

Illustration of proposed selective changes in hippocampal sector volume needs to be very carefully prepared in view of the substantial claims on selective vulnerability. In 2A under P15 and especially P60, it is difficult to see the difference - this needs lower magnification and a lot of care that anteroposterior levels are identical because hippocampal sector anatomy and volumes of sectors vary from level to level. One wonders if the cortex shrinks, too. This is important.

One cannot be sure that there is selective death of hippocampal sectors with DLK overexpression versus, say, rearrangement of hippocampal architecture. One may need stereological analysis, otherwise this substantial claim appears overinterpreted.

Is the GFAP excess reflective of neuroinflammation? What do microglial markers show? The presence of neuroinflammation does not bode well with apoptosis. Speaking of which, TUNEL in one cell in Supplementary Figure 4E is not strong evidence of a more widespread apoptotic event in CA1.

In several places in the paper (as illustrated in Figure 4B, Supplementary Figure 2B, etc.): the unit of biological observation in animal models is typically not a cell, but an organism, in which averaged measures are generated. This is a significant methodological problem because it is not easy to sample neurons without involving stereological methods. With the approach taken here, there is a risk that significance may be overblown.

Other Comments and Questions:

Supplementary Figure 9: The authors state that data points are shown for individual ROIs - ideally, they should also show averages for biological replicates. Can the authors confirm that statistical analyses are based on biological replicates (mice) and not ROIs?

For in vitro experiments, what is the effect of DLK overexpression on neuronal viability and density? Could these variables confound effects on synaptogenesis/synapse maturation?

Correlations between c-jun expression and phosphorylation are extremely important and need to be carefully and convincingly documented. I am a bit concerned about Supplementary Figure 6 images, especially 6B-CA1 (no difference between control and KO, too small images) and 6D (no p-c-Jun expression at all anywhere in the hippocampus at P15?).

Reviewer #1 (Public Review):

This report contains two parts. In the first part, several experiments were carried out to show that CsoR binds to CheA, inhibits CheA phosphorylation, and impairs P. putida chemotaxis. The second part provides some evidence that CsoR is a copper-binding protein, binds to CheA in a copper-dependent manner, and regulates P. putida response to copper, a chemorepellent. Based on these results, a working model is proposed to describe how CsoR coordinates chemotaxis and resistance to copper in P. putida. While the second part of the study is relatively solid, there are some major concerns about the first part.

Critiques:

(1) The rigor from prior research is not clear. In addition to talking about other bacterial chemotaxis, the Introduction should briefly summarize previous work on P. putida chemotaxis and copper resistance.

(2) The rationale for identifying those CheA-binding proteins is vague. CheA has been extensively studied and its functional domains (P1 to P5) have been well characterized. Compared to its counterparts from other bacteria, does P. putida CheA contain a unique motif or domain? Does CsoR bind to other bacterial CheAs or only to P. putida CheA?

(3) Line 133-136, "Collectively, using pull-down, BTH, and BiFC assays, we identified 16 new CheA-interacting proteins in P. putida." It is surprising that so many proteins were identified but none of them were chemotaxis proteins, in particular those known to interact with CheA, such as CheW, CheY and CheZ, which raises a concern about the specificity of these methods. BTH and BiFC often give false-positive results and thus should be substantiated by other approaches such as co-IP, surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) along with mutagenesis studies.

(4) Line 147-149, "Fig. 2a, five strains (WT+pcsoR, WT+pispG, WT+pnfuA, WT+pphaD, and WT+pPP_1644) displayed smaller colony than the control strain (WT+pVec), indicating a weaker chemotaxis ability in these five strains." If copper is a chemorepellent, these strains should swim away from high concentrations of copper; thus, the sizes of colonies couldn't be used to measure this response. In the cited reference (reference 29), bacterial response to phenol was measured using a response index (RI).

(5) Figures 2 and 3 show both CsoR and PhaD bind to CheA and inhibit CheA autophosphorylation. Do these two proteins share any sequence or structural similarity? Does PhaD also bind to copper? Otherwise, it is difficult to understand these results.

(6) Line 195-196, "CsoR/PhaD had no apparent influence on the phosphate transfer between CheA and CheY". CheA controls bacterial chemotaxis through CheY phosphorylation. If this is true, how do CsoR and PhaD affect chemotaxis?

(7) Figure 3 shows that CsoR/PhaD bind to CheA through P1, P3, and P4. This result is intriguing. All CheA proteins contain these three domains. If this is true, CsoR/PhaD should bind to other bacterial CheAs too. That said, this experiment is premature and needs to be confirmed by other approaches.

(8) Figure 5, does PhaD contain these three residues (C40, H65, and C69)? If not, how does PhaD inhibit CheA autophosphorylation and chemotactic response to copper?

(9) Does deletion of cosR or cheA have any impact on P. putida resistance to high concentrations of copper?

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 #1 (Public Review):

Summary:

This work uses transgenic reporter lines to isolate entpd5a+ cells representing classical osteoblasts in the head and non-classical (osterix-) notochordal sheath cells. The authors also include entpd5a- cells, col2a1a+ cells to represent the closely associated cartilage cells. In a combination of ATAC and RNA-Seq analysis, the genome-wide transcriptomic and chromatin status of each cell population is characterized, validating their methodology and providing fundamental insights into the nature of each cell type, especially the less well-studied notochordal sheath cells. Using these data, the authors then turn to a thorough and convincing analysis of the regulatory regions that control the expression of the entpd5a gene in each cell population. Determination of transcriptional activities in developing zebrafish, again combined with ATAC data and expression data of putative regulators, results in a compelling and detailed picture of the regulatory mechanisms governing the expression of this crucial gene.

Strengths:

The major strength of this paper is the clever combination of RNA-Seq and ATAC analysis, further combined with functional transcriptional analysis of the regulatory elements of one crucial gene. This results in a very compelling story.

Weaknesses:

No major weaknesses were identified, except for all the follow-up experiments that one can think of, but that would be outside of the scope of this paper.

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #3 (Public Review):

Summary:

This study characterizes classical and nonclassical osteoblasts as both types were analyzed independently (integrated ATAC-seq and RNAseq). It was found that gene expression in classical and nonclassical osteoblasts is not regulated in the same way. In classical osteoblasts, Dlx family factors seem to play an important role, while Hox family factors are involved in the regulation of spinal ossification by nonclassical osteoblasts. In the second part of the study, the authors focus on the promoter structure of entpd5a. Through the identification of enhancers, they reveal complex modes of regulation of the gene. The authors suggest candidate transcription factors that likely act on the identified enhancer elements. All the results taken together provide comprehensive new insights into the process of bone development, and point to spatio-temporally regulated promoter/enhancer interactions taking place at the entpd5a locus.

Strengths:

The authors have succeeded in justifying a sound and consistent buildup of their experiments, and meaningfully integrating the results into the design of each of their follow-up experiments. The data are solid, insightfully presented, and the conclusion valid. This makes this manuscript of great value and interest to those studying (fundamental) skeletal biology.

Weaknesses:

The study is solidly constructed, the manuscript is clearly written and the discussion is meaningful - I see no real weaknesses.

Reviewer #1 (Public review):

Summary:

In this manuscript, Chen et al. used cryo-ET and in vitro reconstituted system to demonstrate that the autoinhibited form of LRRK2 can also assemble into filaments that wrap around the microtubule, although the filaments are typically shorter and less regular compared to the previously reported active-LRRK2 filaments. The structure revealed a new interface involving the N-terminal repeats that were disordered in the previous active-LRRK2 filament structure. The autoinhibited-LRRK2 filament also has different helical parameters compared to the active form.

Strengths:

The structure obtained in this study is the highest resolution of LRRK2 filaments done by subtomogram averaging, representing a major technical advance compared to the previous Cell paper from the same group. Overall, I think the data are well presented with beautiful graphic rendering, and valuable insights can be gained from this structural study.

Weaknesses:

(1) There are only three main figures, together with 9 supplemental figures. The authors may consider breaking the currently overwhelming Figures 1 and 3 into smaller figures and moving some of the supplemental figures to the main figure, e.g., Figure S7.

(2) The key analysis of this manuscript is to compare the current structure with the previous active-LRRK2 filament structure. Currently, such a comparison is buried in Figure 3H. It should be part of Figure 1.

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 #3 (Public review):

Summary:

The manuscript by Chen et al examines the structure of the inactive LRRK2 bound to microtubules using cryo-EM tomography. Mutations in this protein have been shown to be linked to Parkinson's Disease. It is already shown that the active-like conformation of LRRK2 binds to the MT lattice, but this investigation shows that full-length LRRk2 can oligomerize on MTs in its autoinhibited state with different helical parameters than were observed with the active-like state. The structural studies suggest that the autoinhibited state is less stable on MTs.

Strengths:

The protein of interest is very important biomedically and a novel conformational binding to microtubules in the proposed.

Weaknesses:

(1) The structures are all low resolution.

(2) There are no measurements of the affinity of the various LRRK2 molecules (with and without inhibitors) to microtubules. This should be addressed through biochemical sedimentation assay.

Reviewer #1 (Public Review):

Summary:

This research sheds light on the nuanced role of ABHD6 in the regulation of AMPARs, highlighting its interaction with TARP γ-2 as a critical factor in modulating receptor-gating kinetics. It is crucial to understand that while ABHD6 alone does not alter AMPAR kinetics, its presence alongside TARP γ-2 leads to accelerated deactivation and desensitization of AMPARs, impacting synaptic transmission dynamics.

Strengths:

Important findings in the research include:<br /> - ABHD6 does not affect the gating kinetics of GluA1 and GluA2(Q) homomeric receptors independently.<br /> - In the presence of TARP γ-2, ABHD6 accelerates deactivation and desensitization of these receptors, regardless of their splicing or editing isoforms.<br /> - The effect is consistent for both homomeric GluA1 and GluA2(Q) receptors and heteromeric GluA1i/GluA2(R)i-G receptors.<br /> - The recovery from desensitization of GluA1 with the flip splicing isoform is slowed by ABHD6 in the presence of TARP γ-2.

Weaknesses:

However, the study focuses on specific receptor subunits and isoforms, which may not fully represent the diversity of AMPAR compositions found in vivo (e.g. though the authors have claimed that TARP γ-2 failed to increase GluA3-induced currents significantly, the effect on GluA4 or the explanation was missing). Further research is needed to explore the implications of these findings in more complex neuronal environments.

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 #1 (Public review):

Summary:

Opioids and related drugs are powerful analgesics that reduce suffering from pain. Unfortunately, their use often leads to addiction and there is an opioid-abuse epidemic that affects people worldwide. This study represents an ongoing effort to develop non-opioid analgesics for pain management. The findings point to an alternative approach to control post-surgical pain in lieu of opioid medications.

Strengths:

(1) The study responds to the urgent need for the development of non-opioid analgesics.

(2) The study demonstrates the efficacy of Clarix Flo (FLO) and HC-HA/PTX3 from the human amniotic membrane (AM) in reducing pain in a mouse model without the adverse effects of opioids.

(3) The study further explored the underlying mechanisms of how HC-HA/PTX3 produces its effects on neurons, suggesting the molecules/pathways involved in pain relief.

(4) The potential use of naturally derived biologics from human birth tissues (AM) is safe and sustainable, compared to synthetic pharmaceuticals.

(5) The study was conducted with scientific rigor, involving purification of active components, comparative analysis with multiple controls, and mechanistic explorations.

Weaknesses:

(1) It should be cautioned that while the preclinical findings are promising, these results still need to be translated into clinical settings that are complex and often unpredictable.

(2) The study shows the efficacy of FLO and HC-HA/PTX3 in one preclinical model of post-surgical pain. The observed effect may be variable in other pain conditions.

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 #3 (Public review):

Summary:

Non-opioid analgesics derived from human amniotic membrane (AM) product represents a novel and unique approach to analgesia that may avoid the traditional harms associated with opioids. Here, the study investigators demonstrate that HC-HAPTX3 is the primary bioactive component of the AM product FLO responsible for anti-nociception in mouse-model and in-vitro dorsal root ganglion (DRG) cell culture experiments. The mechanism is demonstrated to be via CD44 with an acute cytoskeleton rearrangement that is induced that inhibits Na+ and Ca++ current through ion channels. Taken together, the studies reported in the manuscript provide supportive evidence clarifying the mechanisms and efficacy of HC-HAPTX3 antinociception and analgesia.

Strengths:

Extensive experiments including murine behavioral paw withdrawal latency and Catwalk test data demonstrating analgesic properties. The breadth and depth of experimental data are clearly supporting mechanisms and antinociceptive properties.

Weaknesses:

A few changes to the text of the manuscript would be recommended but no major weaknesses were identified.

Reviewer #1 (Public review):

This paper examines the role of MLCK (myosin light chain kinase) and MLCP (myosin light chain phosphatase) in axon regeneration. Using loss-of-function approaches based on small molecule inhibitors and siRNA knockdown, the authors explore axon regeneration in cell culture and in animal models. Their evidence shows that MLCK activity facilitates axon extension/regeneration, while MLCP prevents it.

Major concern:

A global inconsistency in the conclusions of the authors is evident when trying to understand the role of NMII in axon growth and to understand the present results in light of previous reports by the authors and many others on the role of NMII in axon extension. The discussion of the matter fails to acknowledge a vast literature on how NMII activity is regulated. The authors study enzymes responsible for the phosphorylation and dephosphorylation of NMII, referring to something that is strongly proven elsewhere, that phosphorylation activates NMII and dephosphorylation deactivates it. The authors mention their own previous evidence using inhibitors of NMII ATPase activity (blebbistatin, Bleb for short) and inhibitors of a kinase that phosphorylates NMII (ROCK), highlighting that Bleb increases axon growth. Since Bleb inhibits the ATPase activity of NMII, it follows that NMII is in itself an inhibitor of axon growth, and hence when NMII is inhibited, the inhibition on axon growth is relieved, and axonal growth takes place (REF1). It is known that NMII exists in an inactive folded state, and ser19 phosphorylation (by MLCK or ROCK) extends the protein, allowing NMII filament formation, ATPase activity, and force generation on actin filaments (REF2). From this, it is derived that if MLCK is inhibited, then there is no NMII phosphorylation, and hence no NMII activity, and, according to their previous work, this should promote axon growth. On the contrary, the authors show the opposite effect: in the lack of phospho-MLC, authors show axon growth inhibition.

Reporting evidence challenging previous conclusions is common business in scientific endeavors, but the problem with the current manuscript is that it fails to point to and appropriately discuss this contradiction. Instead, the authors refer to the fact that MLCK and Bleb inhibit NMII in different steps of the activation process. While this is true, this explanation does not solve the contradiction. There are many options to accommodate the information, but it is not the purpose of this revision to provide them. Since the manuscript is focused solely on phosphorylation states of MLC and axon extension, the claims are simply at odds with the current literature, and this important finding, if true, is not properly discussed.

What follows is a discussion of the merits and limitations of different claims of the manuscript in light of the evidence presented.

(1) Using western blot and immunohistochemical analyses, authors first show that MLCK expression is increased in DRG sensory neurons following peripheral axotomy, concomitant to an increase in MLC phosphorylation, suggesting a causal effect (Figure 1). The authors claim that it is common that axon growth-promoting genes are upregulated. It would have been interesting at this point to study in this scenario the regulation of MLCP, which is a main subject in this work, and expect its downregulation.

(2) Using DRG cultures and sciatic nerve crush in the context of MLCK inhibition and down-regulation, authors conclude that MLCK activity is required for mammalian peripheral axon regeneration both in vitro and in vivo (Figure 2).

The in vitro evidence is of standard methods and convincing. However, here, as well as in all other experiments using siRNAs, it is not clear what the control is about (the identity of the plasmids and sequences, if any).

Related to this, it is not helpful to show the same exact picture as a control example in Figures 2 and 3 (panels J and E, respectively). Either because they should not have received the same control treatment, or simply because it raises concern that there are no other control examples worth showing. In these images, it is not also clear where and how the crush site is determined in the GFP channel. This is of major importance since the axonal length is measured from the presumed crush site. Apart from providing further details in the text, the authors should include convincing images.

(3) The authors then examined the role of the phosphatase MLCP in axon growth during regeneration. The authors first use a known MLCP blocker, phorbol 12,13-dibutyrate (PDBu), to show that is able to increase the levels of p-MLC, with a concomitant increase in the extent of axon regrowth of DRG neurons, both in permissive as well as non-permissive. The authors repeat the experiments using the knockdown of MYPT1, a key component of the MLC-phosphatase, and again can observe a growth-promoting effect (Figure 3).

The authors further show evidence for the growth-enhancing effect in vivo, in nerve crush experiments. The evidence in vivo deserves more evidence and experimental details (see comment 2). Some key weaknesses of the data were mentioned previously (unclear RNAi controls and duplication of shown images), but in this case, it is also not clear if there is a change only in the extent of growth, or also in the number of axons that are able to regenerate.

(4) In the next set of experiments (presented in Figure 4) authors extend the previous observations in primary cultures from the CNS. For that, they use cortical and hippocampal cultures, and pharmacological and genetic loss-of-function using the above-mentioned strategies. The expected results were obtained in both CNS neurons: inhibition or knockdown of the kinase decreases axon growth, whereas inhibition or knockdown of the phosphatase increases growth. A main weakness in this set is that it is not indicated when (at what day in vitro, DIV) the treatments are performed. This is important to correctly interpret the results, since in the first days in vitro these neurons follow well-characterized stages of development, with characteristic cellular events with relevance to what is being evaluated. Importantly, this would be of value to understand whether the treatments affect axonal specification and/or axonal extension. Although these events are correlated, they imply a different set of molecular events.

The title of this section is misleading: line 241 "MLCK/MLCP activity regulated axon growth in the embryonic CNS"... the title (and the conclusion) implies that the experiments were performed in situ, looking at axons in the developing brain. The most accurate title and conclusion should mention that the evidence was collected in CNS primary cultures derived from embryos.

(5) Performing nerve crush injury in CNS nerves (optic nerve and spinal cord), and the local application of PBDu, the author shows contrasting results (Figure 5). In the ON nerve, they can see axons extending beyond the lesion site due to PBDu. On the contrary, the authors fail to observe so in the corticospinal tract present in the spinal cord. The authors fail to discuss this matter in detail. Also, they accommodate the interpretation of the evidence in light of a process known as axon retraction, and its prevention by MLCP inhibition. Since the whole paper is on axon extension, and it is known that mechanistically axon retraction is not merely the opposite of axon extension, the claim needs far more evidence.

In panel 5F and the supplementary data, the authors mention the occurrence of retraction bulbs, but the images are too small to support the claim, and it is not clear how these numbers were normalized to the number of axons labeled in each condition.

(6) The author combines MLCK and MLCP inhibitors with Bleb, trying to verify if both pairs of inhibitors act on the same target/pathway (Figure 6). The rationale is wrong for at least two reasons.<br /> a- Because both lines of evidence point to contrasting actions of NMII on axon growth, one approach could never "rescue" the other.<br /> b- Because the approaches target different steps on NMII activation, one could never "prevent" or rescue the other. For example, for Bleb to provide a phenotype, it should find any p-MLC, because it is only that form of MLC that is capable of inhibiting its ATPase site. In light of this, it is not surprising that Bleb is unable to exert any action in a situation where there is no p-MLC (ML-7, which by inhibiting the kinase drives the levels of p-MLC to zero, Figure 4A). Hence, the results are not possible to validate in the current general interpretation of the authors. (See 'major concern').

(7) In Figure 7, the authors argue that the scheme of replating and using ML7 before or after replating is evidence for a local cytoskeletal action of the drug. However, an alternative simpler explanation is that the drug acts acutely on its target, and that, as such, does not "survive" the replating procedure. Hence, the conclusion raised by the evidence shown is not supported.

(8) In Figure 8, the authors show that the inhibitory treatments on MLCK and MLCP (ML7 and PRBu) alter the morphology of growth cones. However, it is not clear how this is correlated with axon growth. The authors also mention in various parts of the text that a local change in the growth cone is evidence for a local action/activity of the drug or enzyme. However the local change<->local action is not a logical truth. It can well be that MLCK and MLCP activity trigger molecular events that ultimately have an effect elsewhere, and by looking at "elsewhere" one observes of course a local effect, but is not because the direct action of MLCK or MLCP are localized. To prove true localized effects there are numerous efforts that can be made, starting from live imaging, fluorescent sensors, and compartmentalized cultures, just to mention a few.

References:

(1) Eun-Mi Hur 1, In Hong Yang, Deok-Ho Kim, Justin Byun, Saijilafu, Wen-Lin Xu, Philip R Nicovich, Raymond Cheong, Andre Levchenko, Nitish Thakor, Feng-Quan Zhou. 2011. Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):5057-62. doi: 10.1073/pnas.1011258108.

(2) Garrido-Casado M, Asensio-Juárez G, Talayero VC, Vicente-Manzanares M. 2024. Engines of change: Nonmuscle myosin II in mechanobiology. Curr Opin Cell Biol. 2024 Apr;87:102344. doi: 10.1016/j.ceb.2024.102344.

(3) Karen A Newell-Litwa 1, Rick Horwitz 2, Marcelo L Lamers. 2015. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015 Dec;8(12):1495-515. doi: 10.1242/dmm.022103.

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 #1 (Public Review):

Summary of what the authors were trying to achieve:

In this manuscript, the authors investigated the role of β-CTF on synaptic function and memory. They report that β-CTF can trigger the loss of synapses in neurons that were transiently transfected in cultured hippocampal slices and that this synapse loss occurs independently of Aβ. They confirmed previous research (Kim et al, Molecular Psychiatry, 2016) that β-CTF-induced cellular toxicity occurs through a mechanism involving a hexapeptide domain (YENPTY) in β-CTF that induces endosomal dysfunction. Although the current study also explores the role of β-CTF in synaptic and memory function in the brain using mice chronically expressing β-CTF, the studies are inconclusive because potential effects of Aβ generated by γ-secretase cleavage of β-CTF were not considered. Based on their findings, the authors suggest developing therapies to treat Alzheimer's disease by targeting β-CTF, but did not address the lack of clinical improvement in trials of several different BACE1 inhibitors, which target β-CTF by preventing its formation.

Major strengths and weaknesses of the methods and results:

The conclusions of the in vitro experiments using cultured hippocampal slices were well supported by the data, but aspects of the in vivo experiments and proteomic studies need additional clarification.

(1) In contrast to the in vitro experiments in which a γ-secretase inhibitor was used to exclude possible effects of Aβ, this possibility was not examined in in-vivo experiments assessing synapse loss and function (Figure 3) and cognitive function (Figure 4). The absence of plaque formation (Figure 4B) is not sufficient to exclude the possibility that Aβ is involved. The potential involvement of Aβ is an important consideration given the 4-month duration of protein expression in the in vivo studies.

(2) The possibility that the results of the proteomic studies conducted in primary cultured hippocampal neurons depend in part on Aβ was also not taken into consideration.

Likely impact of the work on the field, and the utility of the methods and data to the community:

The authors' use of sparse expression to examine the role of β-CTF on spine loss could be a useful general tool for examining synapses in brain tissue.

Additional context that might help readers interpret or understand the significance of the work:

The discovery of BACE1 stimulated an international effort to develop BACE1 inhibitors to treat Alzheimer's disease. BACE1 inhibitors block the formation of β-CTF which, in turn, prevents the formation of Aβ and other fragments. Unfortunately, BACE1 inhibitors not only did not improve cognition in patients with Alzheimer's disease, they appeared to worsen it, suggesting that producing β-CTF actually facilitates learning and memory. Therefore, it seems unlikely that the disruptive effects of β-CTF on endosomes plays a significant role in human disease. Insights from the authors that shed further light on this issue would be welcome.

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 #3 (Public Review):

Summary:

Most previous studies have focused on the contributions of Abeta and amyloid plaques in the neuronal degeneration associated with Alzheimer's disease, especially in the context of impaired synaptic transmission and plasticity which underlies the impaired cognitive functions, a hallmark in AD. But processes independent of Abeta and plaques are much less explored, and to some extent, the contributions of these processes are less well understood. Luo et all addressed this important question with an array of approaches, and their findings generally support the contribution of beta-CTF-dependent but non-Abeta-dependent process to the impaired synaptic properties in the neurons. Interestingly, the above process appears to operate in a cell-autonomous manner. This cell-autonomous effect of beta-CTF as reported here may facilitate our understanding of some potentially important cellular processes related to neurodegeneration. Although these findings are valuable, it is key to understand the probability of this process occurring in a more natural condition, such as when this process occurs in many neurons at the same time. This will put the authors' findings into a context for a better understanding of their contribution to either physiological or pathological processes, such as Alzheimer's. The experiments and results using the cell system are quite solid, but the in vivo results are incomplete and hence less convincing (see below). The mechanistic analysis is interesting but primitive and does not add much more weight to the significance. Hence, further efforts from the authors are required to clarify and solidify their results, in order to provide a complete picture and support for the authors' conclusions.

Strengths:

(1) The authors have addressed an interesting and potentially important question

(2) The analysis using the cell system is solid and provides strong support for the authors' major conclusions. This analysis has used various technical approaches to support the authors' conclusions from different aspects and most of these results are consistent with each other.

Weaknesses:

(1) The relevance of the authors' major findings to the pathology, especially the Abeta-dependent processes is less clear, and hence the importance of these findings may be limited.

(2) In vivo analysis is incomplete, with certain caveats in the experimental procedures and some of the results need to be further explored to confirm the findings.

(3) The mechanistic analysis is rather primitive and does not add further significance.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

While the aim of the study is well-motivated and analyses rigorously conducted, the overall structure of the manuscript, as it stands now, is partially misleading. Some of the described results are not well-embedded and lack discussion.

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 #3 (Public review):

Summary:

Schmidt et al., aimed to provide an extremely comprehensive demonstration of the influence cardiac electromagnetic fields have on the relationship between age and the aperiodic slope measured from electroencephalographic (EEG) and magnetoencephalographic (MEG) data.

Strengths:

Schmidt et al., used a multiverse approach to show that the cardiac influence on this relationship is considerable, by testing a wide range of different analysis parameters (including extensive testing of different frequency ranges assessed to determine the aperiodic fit), algorithms (including different artifact reduction approaches and different aperiodic fitting algorithms), and multiple large datasets to provide conclusions that are robust to the vast majority of potential experimental variations.

The study showed that across these different analytical variations, the cardiac contribution to aperiodic activity measured using EEG and MEG is considerable, and likely influences the relationship between aperiodic activity and age to a greater extent than the influence of neural activity.

Their findings have significant implications for all future research that aims to assess aperiodic neural activity, suggesting control for the influence of cardiac fields is essential.

Weaknesses:

Figure 4I: The regressions explained here seem to contain a very large number of potential predictors. Based on the way it is currently written, I'm assuming it includes all sensors for both the ECG component and ECG rejected conditions?

I'm not sure about the logic of taking a complete signal, decomposing it with ICA to separate out the ECG and non-ECG signals, then including these latent contributions to the full signal back into the same regression model. It seems that there could be some circularity or redundancy in doing so. Can the authors provide a justification for why this is a valid approach?

I'm not sure whether there is good evidence or rationale to support the statement in the discussion that the presence of the ECG signal in reference electrodes makes it more difficult to isolate independent ECG components. The ICA algorithm will still function to detect common voltage shifts from the ECG as statistically independent from other voltage shifts, even if they're spread across all electrodes due to the referencing montage. I would suggest there are other reasons why the ICA might lead to imperfect separation of the ECG component (assumption of the same number of source components as sensors, non-Gaussian assumption, assumption of independence of source activities).

The inclusion of only 32 channels in the EEG data might also have reduced the performance of ICA, increasing the chances of imperfect component separation and the mixing of cardiac artifacts into the neural components, whereas the higher number of sensors in the MEG data would enable better component separation. This could explain the difference between EEG and MEG in the ability to clean the ECG artifact (and perhaps higher-density EEG recordings would not show the same issue).

In addition to the inability to effectively clean the ECG artifact from EEG data, ICA and other component subtraction methods have also all been shown to distort neural activity in periods that aren't affected by the artifact due to the ubiquitous issue of imperfect component separation (https://doi.org/10.1101/2024.06.06.597688). As such, component subtraction-based (as well as regression-based) removal of the cardiac artifact might also distort the neural contributions to the aperiodic signal, so even methods to adequately address the cardiac artifact might not solve the problem explained in the study. This poses an additional potential confound to the "M/EEG without ECG" conditions.

Literature Analysis, Page 23: was there a method applied to address studies that report reducing artifacts in general, but are not specific to a single type of artifact? For example, there are automated methods for cleaning EEG data that use ICLabel (a machine learning algorithm) to delete "artifact" components. Within these studies, the cardiac artifact will not be mentioned specifically, but is included under "artifacts".

Statistical inferences, page 23: as far as I can tell, no methods to control for multiple comparisons were implemented. Many of the statistical comparisons were not independent (or even overlapped with similar analyses in the full analysis space to a large extent), so I wouldn't expect strong multiple comparison controls. But addressing this point to some extent would be useful (or clarifying how it has already been addressed if I've missed something).

Methods:

Applying ICA components from 1Hz high pass filtered data back to the 0.1Hz filtered data leads to worse artifact cleaning performance, as the contribution of the artifact in the 0.1Hz to 1Hz frequency band is not addressed (see Bailey, N. W., Hill, A. T., Biabani, M., Murphy, O. W., Rogasch, N. C., McQueen, B., ... & Fitzgerald, P. B. (2023). RELAX part 2: A fully automated EEG data cleaning algorithm that is applicable to Event-Related-Potentials. Clinical Neurophysiology, result reported in the supplementary materials). This might explain some of the lower frequency slope results (which include a lower frequency limit <1Hz) in the EEG data - the EEG cleaning method is just not addressing the cardiac artifact in that frequency range (although it certainly wouldn't explain all of the results).

It looks like no methods were implemented to address muscle artifacts. These can affect the slope of EEG activity at higher frequencies. Perhaps the Riemannian Potato addressed these artifacts, but I suspect it wouldn't eliminate all muscle activity. As such, I would be concerned that remaining muscle artifacts affected some of the results, particularly those that included high frequency ranges in the aperiodic estimate. Perhaps if muscle activity were left in the EEG data, it could have disrupted the ability to detect a relationship between age and 1/f slope in a way that didn't disrupt the same relationship in the cardiac data (although I suspect it wouldn't reverse the overall conclusions given the number of converging results including in lower frequency bands). Is there a quick validity analysis the authors can implement to confirm muscle artifacts haven't negatively affected their results? I note that an analysis of head movement in the MEG is provided on page 32, but it would be more robust to show that removing ICA components reflecting muscle doesn't change the results. The results/conclusions of the following study might be useful for objectively detecting probable muscle artifact components: Fitzgibbon, S. P., DeLosAngeles, D., Lewis, T. W., Powers, D. M. W., Grummett, T. S., Whitham, E. M., ... & Pope, K. J. (2016). Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis. Clinical neurophysiology, 127(3), 1781-1793.

Reviewer #1 (Public Review):

Summary:

Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

Strengths:

Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

Weaknesses:

However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

(1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

(2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.

(3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

(4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.

(5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

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.

for - paper review - Hard Problem and Free Will: An Information-Theoretical Approach - Giacomo Mauro D’Ariano & Federico Faggin

annotation by time

5:54 - what is awareness (or consciousness)? - it is the "feeling of the information processing

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 #3 (Public Review):

Summary:

This work offers a novel perspective on the question of how hippocampal networks can adaptively generate different spatial maps and replays/preplays of the corresponding place cells, without any such maps pre-existing in the network architecture or its inputs. Unlike previous modeling attempts, the authors do not pre-tune their model neurons to any particular place fields. Instead, they build a random, moderately-clustered network of excitatory (and some inhibitory) cells, similar to CA3 architecture. By simulating spatial exploration through border-cell-like synaptic inputs, the model generates place cells for different "environments" without the need to reconfigure its synaptic connectivity or introduce plasticity. By simulating sleep-like random synaptic inputs, the model generates sequential activations of cells, mimicking preplays. These "preplays" require small-world connectivity, so that weakly connected cell clusters are activated in sequence. Using a set of electrophysiological recordings from CA1, the authors confirm that the modeled place cells and replays share many features with real ones. In summary, the model demonstrates that spontaneous activity within a small-world structured network can generate place cells and replays without the need for pre-configured maps.

Strengths:

This work addresses an important question in hippocampal dynamics. Namely, how can hippocampal networks quickly generate new place cells when a novel environment is introduced? And how can these place cells preplay their sequences even before the environment is experienced? Previous models required pre-existing spatial representations to be artificially introduced, limiting their adaptability to new environments. Other models depended on synaptic plasticity rules which made remapping slower than what is seen in recordings. This modeling work proposes that quickly-adaptive intrinsic spiking sequences (preplays) and spatially tuned spiking (place cells) can be generated in a network through randomly clustered recurrent connectivity and border-cell inputs, avoiding the need for pre-set spatial maps or plasticity rules. The proposal that small-world architecture is key for place cells and preplays to adapt to new spatial environments is novel and of potential interest to the computational and experimental community.

The authors do a good job of thoroughly examining some of the features of their model, with a strong focus on excitatory cell connectivity. Perhaps the most valuable conclusion is that replays require the successive activation of different cell clusters. Small-world architecture is the optimal regime for such a controlled succession of activated clusters.

The use of pre-existing electrophysiological data adds particular value to the model. The authors convincingly show that the simulated place cells and preplay events share many important features with those recorded in CA1 (though CA3 ones are similar).

Weaknesses:

To generate place cell-like activity during a simulated traversal of a linear environment, the authors drive the network with a combination of linearly increasing/decreasing synaptic inputs, mimicking border cell-like inputs. These inputs presumably stem from the entorhinal cortex (though this is not discussed). The authors do not explore how the model would behave when these inputs are replaced by or combined with grid cell inputs which would be more physiologically realistic.

Even though the authors claim that no spatially-tuned information is needed for the model to generate place cells, there is a small location-cue bias added to the cells, depending on the cluster(s) they belong to. Even though this input is relatively weak, it could potentially be driving the sequential activation of clusters and therefore the preplays and place cells. In that case, the claim for non-spatially tuned inputs seems weak. This detail is hidden in the Methods section and not discussed further. How does the model behave without this added bias input?

Unlike excitation, inhibition is modeled in a very uniform way (uniform connection probability with all E cells, no I-I connections, no border-cell inputs). This goes against a long literature on the precise coordination of multiple inhibitory subnetworks, with different interneuron subtypes playing different roles (e.g. output-suppressing perisomatic inhibition vs input-gating dendritic inhibition). Even though no model is meant to capture every detail of a real neuronal circuit, expanding on the role of inhibition in this clustered architecture would greatly strengthen this work.

For the modeling insights to be physiologically plausible, it is important to show that CA3 connectivity (which the model mimics) shares the proposed small-world architecture. The authors discuss the existence of this architecture in various brain regions but not in CA3, which is traditionally thought of and modeled as a random or fully connected recurrent excitatory network. A thorough discussion of CA3 connectivity would strengthen this work.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Shao et al. investigate the contribution of different cortical areas to working memory maintenance and control processes, an important topic involving different ideas about how the human brain represents and uses information when it is no longer available to sensory systems. In two fMRI experiments, they demonstrate that the human frontal cortex (area sPCS) represents stimulus (orientation) information both during typical maintenance, but even more so when a categorical response demand is present. That is, when participants have to apply an added level of decision control to the WM stimulus, sPCS areas encode stimulus information more than conditions without this added demand. These effects are then expanded upon using multi-area neural network models, recapitulating the empirical gradient of memory vs control effects from visual to parietal and frontal cortices. In general, the experiments and analyses provide solid support for the authors' conclusions, and control experiments and analyses are provided to help interpret and isolate the frontal cortex effect of interest. However, I suggest some alternative explanations and important additional analyses that would help ensure an even stronger level of support for these results and interpretations.

Strengths:

- The authors use an interesting and clever task design across two fMRI experiments that is able to parse out contributions of WM maintenance alone along with categorical, rule-based decisions. Importantly, the second experiment only uses one fixed rule, providing both an internal replication of Experiment 1's effects and extending them to a different situation when rule-switching effects are not involved across mini-blocks.

- The reported analyses using both inverted encoding models (IEM) and decoders (SVM) demonstrate the stimulus reconstruction effects across different methods, which may be sensitive to different aspects of the relationship between patterns of brain activity and the experimental stimuli.

- Linking the multivariate activity patterns to memory behavior is critical in thinking about the potential differential roles of cortical areas in sub-serving successful working memory. Figure 3 nicely shows a similar interaction to that of Figure 2 in the role of sPCS in the categorization vs. maintenance tasks.

- The cross-decoding analysis in Figure 4 is a clever and interesting way to parse out how stimulus and rule/category information may be intertwined, which would have been one of the foremost potential questions or analyses requested by careful readers. However, I think more additional text in the Methods and Results to lay out the exact logic of this abstract category metric will help readers better interpret the potential importance of this analysis and result.

Weaknesses:

- Selection and presentation of regions of interest: I appreciate the authors' care in separating the sPCS region as "frontal cortex", which is not necessarily part of the prefrontal cortex, on which many ideas of working memory maintenance activity are based. However, to help myself and readers interpret these findings, at a minimum the boundaries of each ROI should be provided as part of the main text or extended data figures. Relatedly, the authors use a probabilistic visual atlas to define ROIs in the visual, parietal, and frontal cortices. But other regions of both lateral frontal and parietal cortices show retinotopic responses (Mackey and Curtis, eLife, 2017: https://elifesciences.org/articles/22974) and are perhaps worth considering. Do the inferior PCS regions or inferior frontal sulcus show a similar pattern of effects across tasks? And what about the middle frontal gyrus areas of the prefrontal cortex, which are most analogous to the findings in NHP studies that the authors mention in their discussion, but do not show retinotopic responses? Reporting the effects (or lack thereof) in other areas of the frontal cortex will be critical for readers to interpret the role of the frontal cortex in guiding WM behavior and supporting the strongly worded conclusions of broad frontal cortex functioning in the paper. For example, to what extent can sPCS results be explained by visual retinotopic responses? (Mackey and Curtis, eLife, 2017: https://elifesciences.org/articles/22974).

- When looking at the time course of effects in Figure 2, for example, the sPCS maintenance vs categorization effects occur very late into the WM delay period. More information is needed to help separate this potential effect from that of the response period and potential premotor/motor-related influences. For example, are the timecourses shifted to account for hemodynamic lag, and if so, by how much? Do the sPCS effects blend into the response period? This is critical, too, for a task that does not use a jittered delay period, and potential response timing and planning can be conducted by participants near the end of the WM delay. Regardless, parsing out the timing and relationship to response planning is important, and an ROI for M1 or premotor cortex could also help as a control comparison point, as in reference (24).

- Interpreting effect sizes of IEM and decoding analysis in different ROIs. Here, the authors are interested in the interaction effects across maintenance and categorization tasks (bar plots in Figure 2), but the effect sizes in even the categorization task (y-axes) are always larger in EVC and IPS than in the sPCS region... To what extent do the authors think this representational fidelity result can or cannot be compared across regions? For example, a reader may wonder how much the sPCS representation matters for the task, perhaps, if memory access is always there in EVC and IPS? Or perhaps late sPCS representations are borrowing/accessing these earlier representations? Giving the reader some more intuition for the effect sizes of representational fidelity will be important. Even in Figure 3 for the behavior, all effects are also seen in IPS as well. More detail or context at minimum is needed about the representational fidelity metric, which is cited in ref (35) but not given in detail. These considerations are important given the claims of the frontal cortex serving such an important for flexible control, here.

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 #1 (Public review):

Induction of beta cell regeneration is a promising approach for the treatment of diabetes. In this study, Massoz et.al., identified calcineurin (CaN) as a new potential modulator of beta cell regeneration by using zebrafish as model. They also showed that calcineurin (CaN) works together with Notch signaling calcineurin (CaN) to promote the beta cell regeneration. Overall, the paper is well organized, and technically sound. However, some evidences seem weak to get the conclusion.

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 #1 (Public Review):

Dong Liu et al. successfully established a short-term zebrafish model by treating the embryos with high concentrations of monosaccharides, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy. The authors found that excessive angiogenesis induced by glucose and noncaloric monosaccharides can be achieved by activating the quiescent endothelial cells into proliferating tip cells. Importantly, the authors further confirmed the effects of monosaccharides on inducing excessive angiogenesis were mediated by the foxo1a-marcksl1a pathway. These results demonstrate the potentially detrimental effects of the noncaloric monosaccharides on blood vessel function and provided novel insights into the underlying mechanisms.

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 #3 (Public Review):

The authors have investigated the effect of noncaloric monosaccharides on angiogenesis in the zebra fish embryo. These compounds are used as substitutes of sugars to sweeten beverages and they are commonly used by diabetic patients. The authors show that noncaloric monosaccharides and glucose similarly induce excessive blood vessels formation due to increased formation of tip cells by endothelial cells. The authors show that this excessive angiogenesis involved the foxo1a-marcksl1a pathway.

A limitation of the study is that the mechanism of angiogenesis in the retinal circulation and in peripheral vasculature is certainly different.

This result suggests that these noncaloric monosaccharides share common side effects with glucose. Consequently, more caution should be taken as regard to the use of these artificial sweeteners. This work is of interest for a better management of diabetes.

Reviewer #1 (Public Review):

Summary:

The manuscript reports that expression of the E. coli operon topAI/yjhQ/yjhP is controlled by the translation status of a small open reading frame, that authors have discovered and named toiL, located in the leader region of the operon. The authors propose the following model for topAI activation: Under normal conditions, toiL is translated but topAI is not expressed because of Rho-dependent transcription termination within the topAI ORF and because its ribosome binding site and start codon are trapped in an mRNA hairpin. Ribosome stalling at various codons of the toiL ORF, caused by the presence of some ribosome-targeting antibiotics, triggers an mRNA conformational switch which allows translation of topAI and, in addition, activation of the operon's transcription because the presence of translating ribosomes at the topAI ORF blocks Rho from terminating transcription. Even though the model is appealing and several of the experimental data support some aspects of it, several inconsistencies remain to be solved. In addition, even though TopAI was shown to be an inhibitor of topoisomerase I (Yamaguchi & Inouye, 2015, NAR 43:10387), the authors suggest, without offering any experimental support, that, because ribosome-targeting antibiotics act as inducers, expression of the topAI/yjhQ/yjhP operon may confer resistance to these drugs.

Strengths:

- There is good experimental support of the transcriptional repression/activation switch aspect of the model, derived from well-designed transcriptional reporters and ChIP-qPCR approaches.

- There is a clever use of the topAI-lacZ reporter to find the 23S rRNA mutants where expression topAI was upregulated. This eventually led the authors to identify that translation events occurring at toiL are important to regulate the topAI/yjhQ/yjhP operon. This section can be strengthened if the authors suggest an explanation for how mutant ribosomes translating toiL increased topAI expression. Is there any published evidence that ribosomes with the identified mutations translate slowly (decreased fidelity does not necessarily mean slow translation, does it?)?

- Authors incorporate relevant links to the antibiotic-mediated expression regulation of bacterial resistance genes. Authors can also mention the tryptophan-mediated ribosome stalling at the tnaC leader ORF that activates the expression of tryptophan metabolism genes through blockage of Rho-mediated transcriptional attenuation.

Weaknesses:

The main weaknesses of the work are related to several experimental results that are not consistent with the model, or related to a lack of data that needs to be included to support the model.

The following are a few examples:

- It is surprising that authors do not mention that several published Ribo-seq data from E. coli cells show active translation of toiL (for example Li et al., 2014, Cell 157: 624). Therefore, it is hard to reconcile with the model that starts codon/Shine-Dalgarno mutations in the toiL-lux reporter have no effect on luciferase expression (Figure 2C, bar graphs of the no antibiotic control samples).

- The SHAPE reactivity data shown in Figure 5A are not consistent with the toiL ORF being translated. In addition, it is difficult to visualize the effect of tetracycline on mRNA conformation with the representation used in Figure 5B. It would be better to show SHAPE reactivity without/with Tet (as shown in panel A of the figure).

- The "increased coverage" of topAI/yjhP/yjhQ in the presence of tetracycline from the Ribo-seq data shown in Figure 6A can be due to activation of translation, transcription, or both. For readers to know which of these possibilities apply, authors need to provide RNA-seq data and show the profiles of the topAI/yjhQ/yjhP genes in control/Tet-treated cells.

- Similarly, to support the data of increased ribosomal footprints at the toiL start codon in the presence of Tet (Figure 6B), authors should show the profile of the toiL gene from control and Tet-treated cells.

- Representation of the mRNA structures in the model shown in Figure 5, does not help with visualizing 1) how ribosomes translate toiL since the ORF is trapped in double-stranded mRNA, and 2) how ribosome stalling on toiL would lead to the release of the initiation region of topAI to achieve expression activation.

- The authors speculate that, because ribosome-targeting antibiotics act as expression inducers [by the way, authors should mention and comment that, more than a decade ago, it had been reported that kanamycin (PMID: 12736533) and gentamycin (PMID: 19013277) are inducers of topAI and yjhQ], the genes of the topAI/yjhQ/yjhP operon may confer resistance to these antibiotics. Such a suggestion can be experimentally checked by simply testing whether strains lacking these genes have increased sensitivity to the antibiotic inducers.

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 #3 (Public Review):

Summary:

The authors nicely show that the translation and ribosome stalling within the ToiL uORF upstream of the co-transcribed topAI-yjhQ toxin-antitoxin genes unmask the topAI translational initiation site, thereby allowing ribosome loading and preventing premature Rho-dependent transcription termination in the topAI region. Although similar translational/transcriptional attenuation has been reported in other systems, the base pairing between the leader sequence and the repressed region by the long RNA looping is somehow unique in toiL-topAI-yjhQP. The experiments are solidly executed, and the manuscript is clear in most parts with areas that could be improved or better explained. The real impact of such a study is not easy to appreciate due to a lack of investigation on the physiological consequences of topAI-yjhQP activation upon antibiotic exposure (see details below).

Strengths:

>Conclusion/model is supported by the integrated approaches consisting of genetics, in vivo SHAPE-seq and Ribo-Seq.

>Provide an elegant example of cis-acting regulatory peptides to a growing list of functional small proteins in bacterial proteomes.

Reviewer #1 (Public Review):

The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

Reviewer #1 (Public Review):

Summary:

In this study, Bonnifet et al. profile the presence of L1 ORF1p in the mouse and human brain. They claim that ORF1p is expressed in the human and mouse brain at a steady state and that there is an age-dependent increase in expression. This is a timely report as two recent papers have extensively documented the presence of full-length L1 transcripts in the mouse and human brain (PMID: 38773348 & PMID: 37910626). Thus, the finding that L1 ORF1p is consistently expressed in the brain is not surprising, but important to document.

Strengths:

Several parts of this manuscript appear to be well done and include the necessary controls. In particular, the evidence for steady-state expression of ORF1p in the mouse brain appears robust.

Weaknesses:

Several parts of the manuscript appear to be more preliminary and need further experiments to validate their claims. In particular, the data suggesting expression of L1 ORF1p in the human brain and the data suggesting increased expression in the aged brain need further validation. Detailed comments:

(1) The expression of ORF1p in the human brain shown in Figure 1j is not convincing. Why are there two strong bands in the WB? How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? Additional validations and controls are needed to verify the specificity of this signal.

(2) The data shown in Figure 2g are not convincing. How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? Extensive additional validations and controls are needed to verify the specificity of this signal.

(3) The data showing a reduction in ORF1p expression in the aged mouse brain is confusing and maybe even misleading. Although there is an increase in the intensity of the ORF1p signal in ORF1p+ cells, the data clearly shows that fewer cells express ORF1p in the aged brain. If these changes indicate an overall loss or gain of ORF1p, expression in the aged brain is not resolved. Thus, conclusions should be more carefully phrased in this section. It is important to show the quantification of NeuN+ and NeuN- cells in young vs aged (not only the proportions as shown in Figure 3b) to determine if the difference in the number of ORF1p+ cells is due to loss of neurons or perhaps a sampling issue. More so, it would be essential to perform WB and/or proteomics experiments to complement the IHC data for the aged mouse samples.

(4) The transcriptomic data presented in Figure 4 and Figure 5 are not convincing. Quantification of transposon expression on short read sequencing has important limitations. Longer reads and complementary approaches are needed to study the expression of evolutionarily young L1s (see PMID: 38773348 & PMID: 37910626 for examples of the current state of the art). Given the read length and the unstranded sequencing approach, I would at least ask the authors to add genome browser tracks of the upregulated loci so that we can properly assess the clarity of the results. I would also suggest adding the mappability profile of the elements in question. In addition, since this manuscript focuses on ORF1p, it would be essential to document changes in protein levels (and not just transcripts) in the ageing human brain.

(5) More information is needed on RNAseq of microdissections of dopaminergic neurons from 'healthy' post-mortem samples of different ages. No further information on these samples is provided. I would suggest adding a table with the clinical information of these samples (especially age, sex, and cause of death). The authors should also discuss whether this experiment has sufficient power. The human ageing cohort seems very small to me.

(6) The findings in this manuscript apply to both human and mouse brains. However, the landscape of the evolutionarily young L1 subfamilies between these two species is very different and should be part of the discussion. For example, the regulatory sequences that drive L1 expression are quite different in human and mouse L1s. This should be discussed.

(7) On page 3 the authors write: "generally accepted that TE activation can be both, a cause and consequence of aging". This statement does not reflect the current state of the field. On the contrary, this is still an area of extensive investigation and many of the findings supporting this hypothesis need to be confirmed in independent studies. This statement should be revised to reflect this reality.

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 #3 (Public Review):

The question about whether L1 exhibits normal/homeostatic expression in the brain (and in general) is interesting and important. L1 is thought to be repressed in most somatic cells (with the exception of some stem/progenitor compartments). However, to our knowledge, this has not been authoritatively / systematically examined and the literature is still developing with respect to this topic. The full gamut of biological and pathobiological roles of L1 remains to be shown and elucidated and this area has garnered rapidly increasing interest, year-by-year. With respect to the brain, L1 (and repeat sequences in general) have been linked with neurodegeneration, and this is thought to be an aging-related consequence or contributor (or both) of inflammation. This study provides an impressive and apparently comprehensive imaging analysis of differential L1 ORF1p expression in mouse brain (with some supporting analysis of the human brain), compatible with a narrative of non-pathological expression of retrotransposition-competent L1 sequences. We believe this will encourage and support further research into the functional roles of L1 in normal brain function and how this may give way to pathological consequences in concert with aging. However, we have concerns with conclusions drawn, in some cases regardless of the lack of statistical support from the data. We note a lack of clarity about how the 3rd party pre-trained machine learning models perform on the authors' imaging data (validation/monitoring tests are not reported), as well as issues (among others) with the particular implementation of co-immunoprecipitation (ORF1p is not among the highly enriched proteins and apparently does not reach statistical significance for the comparison) - neither of which may be sufficiently rigorous.

Reviewer #1 (Public review):

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

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

Strengths:

(1) Comprehensive computational analysis of PROTAC-related protein complexes.

(2) Focus on critical aspects: linker role, protein-protein interaction stability, and lysine accessibility.

Weaknesses:

(1) Limited examination of lysine accessibility despite its stated importance.

(2) Use of RMSD as the primary metric for conformational assessment, which may overlook important local structural changes.

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 #3 (Public review):

The authors offer an interesting computational study on the dynamics of PROTAC-driven protein degradation. They employed a combination of protein-protein docking, structural alignment, atomistic MD simulations, and post-analysis to model a series of CRBN-dBET-BRD4 ternary complexes, as well as the entire degradation machinery complex. These degraders, with different linker properties, were all capable of forming stable ternary complexes but had been shown experimentally to exhibit different degradation capabilities. While in the initial models of the degradation machinery complex, no surface Lys residue(s) of BRD4 were exposed sufficiently for the crucial ubiquitination step, MD simulations illustrated protein functional dynamics of the entire complex and local side-chain arrangements to bring Lys residue(s) to the catalytic pocket of E2/Ub for reactions. Using these simulations, the authors were able to present a hypothesis as to how linker property affects degradation potency. They were able to roughly correlate the distance of Lys residues to the catalytic pocket of E2/Ub with observed DC50/5h values. This is an interesting and timely study that presents interesting tools that could be used to guide future PROTAC design or optimization.

Reviewer #1 (Public review):

Summary:

This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activitity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1）IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, which are not traditionally associated with this structure.

Strengths:

The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

Weaknesses:

(1) The authors cited several papers focusing on dopaminergic inputs in the IC to suggest the involvement of this brain region in cognitive functions. However, all those cited work were done in rodents. Whether monkey's IC shares similar inputs is not clear.<br /> (2) The authors confused the two terms, novelty and deviation. According to their behavioral paradigm, deviation rather than novelty should be used in the paper because all the stimuli have been presented to the monkeys during training. Therefore, there is actually no novel stimuli but only deviant stimuli. This reflects that the author has misunderstood the basic concept.<br /> (3) Most of the conclusions were made based on correlational analysis or speculation without providing causal evidences.<br /> (4) Results are presented in a very "straightforward" manner with too many detailed descriptions of phenomena but lack of summary and information synthesis. For example, the first section of Results is very long but did not convey clear information.<br /> (5) The logic between different sections of Results is not clear.<br /> (6) In the Discussion, there is excessive repetition of results, and further comparison with and discussion of potentially related work are very insufficient. For example, Metzger, R.R., et al. (J Neurosc, 2006) have shown similar firing patterns of IC neurons and correlated their findings with reward.

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 #3 (Public review):

Summary:

The authors aimed to investigate the multifaceted roles of the Inferior Colliculus (IC) in auditory and cognitive processes in monkeys. Through extracellular recordings during a sound duration-based novelty detection task, the authors observed a "climbing effect" in neuronal firing rates, suggesting an enhanced response during sensory prediction. Observations of reward prediction errors within the IC further highlight its complex integration in both auditory and reward processing. Additionally, the study indicated IC neuronal activities could be involved in decision-making processes.

Strengths:

This study has the potential to significantly impact the field by challenging the traditional view of the IC as merely an auditory relay station and proposing a more integrative role in cognitive processing. The results provide valuable insights into the complex roles of the IC, particularly in sensory and cognitive integration, and could inspire further research into the cognitive functions of the IC.

Weaknesses:

Major Comments:

(1) Structural Clarity and Logic Flow:<br /> The manuscript investigates three intriguing functions of IC neurons: sensory prediction, reward prediction, and cognitive decision-making, each of which is a compelling topic. However, the logical flow of the manuscript is not clearly presented and needs to be well recognized. For instance, Figure 3 should be merged into Figure 2 to present population responses to the order of sounds, thereby focusing on sensory prediction. Given the current arrangement of results and figures, the title could be more aptly phrased as "Beyond Auditory Relay: Dissecting the Inferior Colliculus's Role in Sensory Prediction, Reward Prediction, and Cognitive Decision-Making."

(2) Clarification of Data Analysis:<br /> Key information regarding data analysis is dispersed throughout the results section, which can lead to confusion. Providing a more detailed and cohesive explanation of the experimental design would significantly enhance the interpretation of the findings. For instance, including a detailed timeline and reward information for the behavioral paradigms shown in Figures 1C and D would offer crucial context for the study. More importantly, clearly presenting the analysis temporal windows and providing comprehensive statistical analysis details would greatly improve reader comprehension.

(3) Reward Prediction Analysis:<br /> The conclusion regarding the IC's role in reward prediction is underdeveloped. While the manuscript presents evidence that IC neurons can encode reward prediction, this is only demonstrated with two example neurons in Figure 6. A more comprehensive analysis of the relationship between IC neuronal activity and reward prediction is necessary. Providing population-level data would significantly strengthen the findings concerning the IC's complex functionalities. Additionally, the discussion of reward prediction in lines 437-445, which describes IC neuron responses in control experiments, does not sufficiently demonstrate that IC neurons can encode reward expectations. It would be valuable to include the responses of IC neurons during trials with incorrect key presses or no key presses to better illustrate this point.

Reviewer #1 (Public review):

Summary:

In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes" Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. They induce oxidative damage in worms, blocking dsRNA import into cells (and potentially affecting the worms in other ways). Oxidative stress inhibits dsRNA import and the associated heritable regulation of gene expression in the adult germline (Fig. 2). The authors identify a novel gene, sid-1-dependent gene-1 (sdg-1), which is induced upon inhibition of SID-1 (Fig. 3). Both transient inhibition and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). The expression of SDG-1 is variable, potentially indicating buffering regulation. While the expression of Sdg-1 could be consistent with a role in intergenerational transport of dsRNA, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing (Fig. 7) in the germline. It would be interesting to test if sdg-2 functions redundantly.

In summary, the authors have identified a novel worm-specific protein (sdg-1) that is induced upon loss of dsRNA import via SID-1, but is not required to mediate SID-1 RNA regulatory effects.

Remaining Questions:

• The authors use an experimental system that induces oxidative damage specifically in neurons to release dsRNAs into the circulation. Would the same effect be observed if oxidative damage were induced in other cell types?

• Besides dsRNA, which other RNAs and cellular products (macromolecules and small signalling molecules) are released into the circulation that could affect the observed changes in germ cells?

• SID-1 modifies RNA regulation within the germline (Fig. 7) and upregulates sdg-1 and sdg-2 (Fig. 5). However, SID-1's effects do not appear to be mediated via sdg-1. Testing the role of sdg-2 would be intriguing.

• Are sdg-1 or sdg-2 conserved in other nematodes or potentially in other species? Sdg-1 appears to be encoded or captured by a retro-element in the C. elegans genome and exhibits stochastic expression in different isolates. Is this a recent adaptation in the C. elegans genome, or is it present in other nematodes? Does loss-of-function of sdg-1 or sdg-2 have any observable effect?

Clarification for Readability:

To enhance readability and avoid misunderstandings, it is crucial to specify the model organism and its specific dsRNA pathways that are not conserved in vertebrates:

• In the first sentence of the paragraph "Here, we dissect the intergenerational transport of extracellular dsRNA ...", the authors should specify "in the nematode C. elegans". Unlike vertebrates, which recognise dsRNA as a foreign threat, worms and other invertebrates pervasively use dsRNA for signalling. Additionally, worms, unlike vertebrates and insects, encode RNA-dependent RNA polymerases that generate dsRNA from ssRNA substrates, enabling amplification of small RNA production. Especially in dsRNA biology, specifying the model organism is essential to avoid confusion about potential effects in humans.

• Similarly, the authors should specify "in C. elegans" in the sentence "Therefore, we propose that the import of extracellular dsRNA into the germline tunes intracellular pathways that cause heritable RNA silencing." This is important because C. elegans small RNA pathways differ significantly from those in other organisms, particularly in the PIWI-interacting RNA (piRNA) pathways, which depend on dsRNA in C. elegans but uses ssRNA in vertebrates. Specification is crucial to prevent misinterpretation by the reader. It is well understood that mechanisms of transgenerational inheritance that operate in nematodes or plants are not conserved in mammals.

• The first sentence of the discussion, "Our analyses suggest a model for ...", would also benefit from specifying "in C. elegans". The same applies to the figure captions. Clarification of the model organism should be added to the first sentence, especially in Figure 1.

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 #1 (Public review):

Summary:

Insulin is crucial for maintaining metabolic homeostasis, and its release is regulated by various pathways, including blood glucose levels and neuromodulatory systems. The authors investigated the role of neuromodulators in regulating the dynamics of the adult Drosophila IPC population. They showed that IPCs express various receptors for monoaminergic and peptidergic neuromodulators, as well as synaptic neurotransmitters with highly heterogeneous profiles across the IPC population. Activating specific modulatory inputs, e.g. dopaminergic, octopaminergic or peptidergic (Leucokinin) using an optogenetic approach coupled with in vivo electrophysiology unveiled heterogeneous responses of individual IPCs resulting in excitatory, inhibitory or no responses. Interestingly, calcium imaging of the entire IPC population with or without simultaneous electrophysiological recording of individual cells showed highly specific and stable responses of individual IPCs suggesting their intrinsic properties are determined by the expressed receptor repertoire. Using the adult fly connectome they further corroborate the synaptic input of excitatory and inhibitory neuronal subsets of IPCs. The authors conclude that the heterogeneous modulation of individual IPC activity is more likely to allow for flexible control of insulin release to adapt to changes in metabolic demand and environmental cues.

Strengths:

This study provides a comprehensive, multi-level analysis of IPC properties utilizing single-nucleus RNA sequencing, anatomical receptor expression mapping, connectomics, electrophysiological recordings, calcium-imaging and an optogenetics-based 'intrinsic pharmacology' approach. It highlights the heterogeneous receptor profiles of IPCs, demonstrating complex and differential modulation within the IPC population. The authors convincingly showed that different neuromodulatory inputs exhibit varied effects on IPC activity and simultaneous occurrence of heterogeneous responses in IPCs with some populations exciting a subset of IPCs while inhibiting others, showcasing the intricate nature of IPC modulation and diverse roles of IPC subgroups. The temporal dynamic of IPC modulation showed that polysynaptic and neuromodulatory connections play a major role in IPC response. The authors demonstrated that certain neuromodulatory inputs, e.g. dopamine, can shift the overall IPC population activity towards either an excited or inhibited state. The study thus provides a fundamental entry point to understanding the complex influence of neuromodulatory inputs on the insulinergic system of Drosophila.

Weakness:

GPCRs are typically expressed at low levels and while the transcriptomic and reporter expression analysis was comprehensive, both approaches have the caveat that they do not allow validating protein level expression. Thus, some receptors might have been missed while others might be false positives. The authors acknowledged the challenges in accurately accessing receptor expression in complex modulatory systems indicating there are limitations in full understanding of the receptor profiles of IPCs.

While this study provides valuable insights into the heterogeneity of IPC responses and receptor expression, it will require future studies to elucidate how these modulatory inputs affect insulin release and transcriptional long-term changes.<br /> The authors further analyzed male and female snRNAseq data and claimed that the differences in receptor expression were minimal. The experimental analyses used mated females only and while the study is very complete in this respect, it would have been extremely interesting to compare male flies in terms of their response profiles.<br /> Lastly as also pointed out by the authors, their approach of using optogenetically driven excitation of modulatory neuronal subsets limits the interpretation of the results due to the possibly confounding direct or indirect effect of fast synaptic transmission on IPC excitation/inhibition, and the broad expression of some neuromodulatory lines used in this analysis.

Overall, however, the conclusions of this study are well supported by the data provided by the authors. Moreover, their detailed and thorough analysis of IPC modulation will have a significant impact on the field of metabolic regulation to understand the complex regulatory mechanism of insulin release, which can now be studied further to provide insight about metabolic homeostasis and neural control of metabolic processes.

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.

Die Fossilindustrie finanziert seit Jahrzehten Universitäten und fördert damit Publikationen in ihrem Interesse, z.B. zu false solutions wie #CCS. Hintergrundbericht anlässlich einer neuen Studie: https://www.theguardian.com/business/article/2024/sep/05/universities-fossil-fuel-funding-green-energy

Studie: https://doi.org/10.1002/wcc.904

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 #1 (Public Review):

Kreeger and colleagues have explored the balance of excitation and inhibition in the cochlear nucleus octopus cells of mice using morphological, electrophysiological, and computational methods. On the surface, the conclusion, that synaptic inhibition is present, does not seem like a leap. However, the octopus cells have been in the past portrayed as devoid of inhibition. This view was supported by the seeming lack of glycinergic fibers in the octopus cell area and the lack of apparent IPSPs. Here, Kreeger et al. used beautiful immunohistochemical and mouse genetic methods to quantify the inhibitory and excitatory boutons over the complete surface of individual octopus cells and further analysed the proportions of the different subtypes of spiral ganglion cell inputs. I think the analysis stands as one of the most complete descriptions of any neuron, leaving little doubt about the presence of glycinergic boutons.

Kreeger et al then examined inhibition physiologically, but here I felt that the study was incomplete. Specifically, no attempt was made to assess the actual, biological values of synaptic conductance for AMPAR and GlyR. Thus, we don't really know how potent the GlyR could be in mediating inhibition. Here are some numbered comments:

(1) "EPSPs" were evoked either optogenetically or with electrical stimulation. The resulting depolarizations are interpreted to be EPSPs. However previous studies from Oertel show that octopus cells have tiny spikes, and distinguishing them from EPSPs is tricky. No mention is made here about how or whether that was done. Thus, the analysis of EPSP amplitude is ambiguous.

(2) For this and later analysis, a voltage clamp of synaptic inputs would have been a simple alternative to avoid contaminating spikes or shunts by background or voltage-gated conductances. Yet only the current clamp was employed. I can understand that the authors might feel that the voltage clamp is 'flawed' because of the failure to clamp dendrites. But that may have been a good price to pay in this case. The authors should have at least justified their choice of method and detailed its caveats.

(3) The modeling raised several concerns. First, there is little presentation of assumptions, and of course, a model is entirely about its assumptions. For example, what excitatory conductance amplitudes were used? The same for inhibitory conductance? How were these values arrived at? The authors note that EPSGs and IPSGs had peaks at 0.3 and 3 ms. On what basis were these numbers obtained? The model's conclusions entirely depend on these values, and no measurements were made here that could have provided them. Parenthetical reference is made to Figure S5 where a range of values are tested, but with little explanation or justification.

(4) In experiments that combined E and I stimulation, what exactly were time timecourses of the conductance changes, and how 'synchronous' were they, given the different methods to evoke them? (had the authors done voltage clamp they would know the answers).

(5) Figure 4G is confusing to me. Its point, according to the text, is to show that changes in membrane properties induced by a block of Kv and HCN channels would not be expected to alter the amplitudes of EPSCs and IPSCs across the dendritic expanse. Now we are talking about currents (not shunting effects), and the presumption is that the blockers would alter the resting potential and thus the driving force for the currents. But what was the measured membrane potential change in the blockers? Surely that was documented. To me, the bigger concern (stated in the text) is whether the blockers altered exocytosis, and thus the increase in IPSP amplitude in blockers is due BOTH to loss of shunting and increase in presynaptic spike width. Added to this is that 4AP will reduce the spike threshold, thus allowing more ChR2-expressing axons to reach the threshold. Figure 4G does not address this point.

(6) Figure 5F is striking as the key piece of biological data that shows that inhibition does reduce the amplitude of "EPSPs" in octopus cells. Given the other uncertainties mentioned, I wondered if it makes sense as an example of shunting inhibition. Specifically, what are the relative synaptic conductances, and would you predict a 25% reduction given the actual (not modeled) values?

(7) Some of the supplemental figures, like 4 and 5, are hardly mentioned. Few will glean anything from them unless the authors direct attention to them and explain them better. In general, the readers would benefit from more complete explanations of what was done.

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 #1 (Public Review):

Summary:

This paper conducted a GWAS meta-analysis for COVID-19 hospitalization among admixed American populations. The authors identified four genome-wide significant associations, including two novel loci (BAZ2B and DDIAS), and an additional risk locus near CREBBP using cross-ancestry meta-analysis. They utilized multiple strategies to prioritize risk variants and target genes. Finally, they constructed and assessed a polygenic risk score model with 49 variants associated with critical COVID-19 conditions.

Strengths:

Given that most of the previous studies were done in European ancestries, this study provides unique findings about the genetics of COVID-19 in admixed American populations. The GWAS data would be a valuable resource for the community. The authors conducted comprehensive analyses using multiple different strategies, including Bayesian fine mapping, colocalization, TWAS, etc., to prioritize risk variants and target genes. The polygenic risk score (PGS) result demonstrated the ability of cross-population PGS model for COVID-19 risk stratification.

Weaknesses:

(1) One of the major limitations of this study is that the GWAS sample size is relatively small, which limits its power.<br /> (2) Lack of replication cohort.<br /> (3) Colocalization and TWAS used eQTL data from GTEx data, which are mainly from European ancestries.

Comments on latest version:

The authors addressed most of my concerns.

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 #3 (Public Review):

Summary:

In the context of the SCOURGE consortium's research, the authors conduct a GWAS meta-analysis on 4,702 hospitalized individuals of admixed American descent suffering from COVID-19. This study identified four significant genetic associations, including two loci initially discovered in Latin American cohorts. Furthermore, a trans-ethnic meta-analysis highlighted an additional novel risk locus in the CREBBP gene, underscoring the critical role of genetic diversity in understanding the pathogenesis of COVID-19.

Strengths:

(1) The study identified two novel severe COVID-19 loci (BAZ2B and DDIAS) by the largest GWAS meta-analysis for COVID-19 hospitalization in admixed Americans.<br /> (2) With a trans-ethnic meta-analysis, an additional risk locus near CREBBP was identified.

Weaknesses:

(1) The GWAS power is limited due to the relatively small number of cases.

(2) There is no replication study for the novel severe COVID-19 loci, which may lead to false positive findings.

(3) The variants selected for the PGS appear arbitrary and may not leverage the GWAS findings.

(4) The TWAS models were predominantly trained on European samples, and there is no replication study for the findings as well.

Reviewer #1 (Public review):

Summary:

Rößling et al., report in this study that the perception of RALF1 by the FER receptor is mediated by the association of RALF1 with deesterified pectin, contributing to the regulation of the cell wall matrix and plasma membrane dynamics. In addition, they report that this mode of action is independent from the previously reported cell wall sensing mechanism mediated by the FER-LRX complex.

This manuscript reproduces and aligns with the results from a recently published study (Liu et al., Cell) where they also report that RALF1 can interact with deesterified pectin, forming coacervates and promoting the recruitment of LLG-FER at the membrane.

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 #3 (Public review):

In this important work, the authors show compelling evidence that the Rapid Alkalinisation Factor1 (RALF1) peptide acts as an interlink between pectin methyl esterification status and FERONIA receptor-like kinase in mediating extracellular sensing. Moreover, the RALF1-mediated pectin perception is surprisingly independent of LRX-mediated extracellular sensing in roots. The authors also show that the peptide directly binds demethylated pectin and the positively charged amino acids are required for pectin binding as well as for its physiological activity.

Some present findings are surprising; previously, the FERONIA extracellular domain was shown to bind pectin directly, and the mode of operation in the pollen tube involves the LRX8-RALF4 complex, which seems not the case for RALF1 in the present study. Although some aspects remain controversial, this work is a very valuable addition to the ongoing debate about this elusive complex regulation and signaling.

The authors drafted the manuscript well, so I do not have a lot of criticism or suggestions. The experiments are well-designed, executed, and presented, and they solidly support the authors' claims.

Reviewer #1 (Public review):

Summary:

The authors investigate the mechanism behind the widely observed but poorly understood phenomenon of reversible vimentin disassembly upon hypotonic challenge. Using permeabilized COS-7 cells expressing vimentin-mEos3.2, the authors demonstrate that vimentin disassembly is not due to lower osmotic pressure but rather due to decreased intracellular ionic strength. They propose a model in which vimentin filament stability is predicted by the protein's net charge and support this idea through approaches that involve (i) manipulating buffer ionic strength, (ii) manipulating buffer pH, or (iii) introducing charged amino acids into the linker of the exogenously expressed vimentin-mEos3.2.

Strengths & Weaknesses:

While the discovery is intriguing and presents an interesting concept, significant shortcomings in experimental design and numerous inconsistencies prevent it from reaching the high standards expected. The lack of reproducibility, inadequate controls, and insufficient quantification make the findings feel very preliminary. Additionally, the authors need to address the apparent discrepancies between their current results and their previous work implicating calpains and altered calcium levels in vimentin disassembly upon hypotonic challenge (which has led to much confusion in the field). This discrepancy should be thoroughly addressed in the discussion with the authors citing their prior work and explaining why it was incorrect.

An additional concern is the relevance of the findings to vimentin biology inside cells. The most important insight in this work is the observation that an isotonic buffer balanced with non-electrolytes (glucose or sorbitol) is sufficient to drive vimentin disassembly. The authors show that vimentin disassembly is not due to changes in osmotic pressure but rather due to a change in the concentration of critical dissolved ions, specifically the number of charged states on vimentin. What is missing is when and how this is controlled within cells under physiological conditions - not just when cells are permeabilized with detergents (conditions that cells rarely survive). Without this deeper dive into vimentin states within cells and how it is controlled, the paper seems very narrow in its focus.

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:

This report analyzes the structure of vimentin, GFAP, and keratin intermediate filament networks in cells that have been subjected to hypotonic stress and other treatments that either alter the ionic strength of the cytoplasm or change the charge density of the intermediate filament.

Strengths:

These experiments expand on the work of references 8 and 9, which showed that the vimentin network rapidly dissociates after hypotonic shock. The cellular imaging uses sophisticated super-resolution techniques and produces some striking images.

Weaknesses:

A fundamental weakness of this study lies in the interpretation, and lack of biochemical evidence for the provocative hypothesis raised that the assembly state of intermediate filaments in a cell is governed by coulomb interactions between charged filaments. Several essential experiments need to be done before this striking hypothesis can be plausibly supported.

(1) First the assembly and disassembly of vimentin filaments needs to be done in vitro systems. If the hypothesis is correct then these same effects will happen with purified vimentin intermediate filaments. These proteins are readily purified from bacterial expression systems and there's a wealth of biophysical data on them, so verifying the predictions of this cell-based model can be realistically tested with purified proteins.

(2) Interpretation of these results explicitly avoids a role for post-translational modifications of vimentin, which are well described and related to filament assembly state. For example, reference 9, which is one of the first discoveries that the vimentin network dissociates under osmotic stress explicitly shows that the vimentin network remains intact if either calcium influx or calpain proteolytic activity is prevented. Hypoosmotic stress will still lead to the same dilution, but the filaments remain intact, apparently contradicting the major interpretation of this paper.

(3) The third issue is that the role of polyelectrolyte effects and especially the importance of divalent cations is scarcely mentioned in the interpretation. There are many studies of how strongly vimentin intermediate filaments interact with divalent cations, in a manner that is relatively insensitive to ionic strength, and these effects need to be taken into account to interpret the cellular data or the hypothesis that coulomb repulsion is the major driver for vimentin disassembly.

Reviewer #2 (Public review):

This study uses single-unit recordings in the monkey STN to examine the evidence for three theoretical models that propose distinct roles for the STN in perceptual decision-making. Importantly, the proposed functional roles are predictive of unique patterns of neural activity. Using k-means clustering with seeds informed by each model's predictions, the current study identified three neural clusters with activity dynamics that resembled those predicted by the described theoretical models. The authors are thorough and transparent in reporting the analyses used to validate the clustering procedure and the stability of the clustering results. To further establish a causal role for the STN in decision-making, the researchers applied microsimulation to the STN and found effects on response times, choice preferences, and latent decision parameters estimated with a drift-diffusion model. Overall, the study provides strong evidence for a functionally diverse population of STN neurons that could indeed support multiple roles involved in perceptual decision-making. The manuscript would benefit from stronger evidence linking each neural cluster to specific decision roles in order to strengthen the overall conclusions.

The interpretation of the results, and specifically, the degree to which the identified clusters support each model, is largely dependent on whether the artificial vectors used as model-based clustering seeds adequately capture the expected behavior under each theoretical model. The manuscript would benefit from providing further justification for the specific model predictions summarized in Figure 1B. Further, although each cluster's activity can be described in the context of the discussed models, these same neural dynamics could also reflect other processes not specific to the models. That is, while a model attributing the STN's role to assessing evidence accumulation may predict a ramping up of neural activity, activity ramping is not a selective correlate of evidence accumulation and could be indicative of a number of processes, e.g., uncertainty, the passage of time, etc.. This lack of specificity makes it challenging to infer the functional relevance of cluster activity and should be acknowledged in the discussion.

Additionally, although the effects of STN microstimulation on behavior provide important causal evidence linking the STN to decision processes, the stimulation results are highly variable and difficult to interpret. The authors provide a reasonable explanation for the variability, showing that neurons from unique clusters are anatomically intermingled such that stimulation likely affects neurons across several clusters. It is worth noting, however, that a substantial body of literature suggests that neural populations in the STN are topographically organized in a manner that is crucial for its role in action selection, providing "channels" that guide action execution. The authors should comment on how the current results, indicative of little anatomical clustering amongst the functional clusters, relates to other reports showing topographical organization.

Overall, the association between the identified clusters and the function ascribed to the STN by each of the models is largely descriptive and should be interpreted accordingly. For example, Figure 3 is referenced when describing which cluster activity is choice/coherence dependent, yet it is unclear what specific criteria and measures are being used to determine whether activity is choice/coherence "dependent." Visually, coherence activity seems to largely overlap in panel B (top row). Is there a statistically significant distinction between low and high coherence in this plot? The interpretation of these plots and the methods used to determine choice/coherence "dependence" needs further explanation.

In general, the association between cluster activity and each model could be more directly tested. At least two of the models assume coordination with other brain regions. Does the current dataset include recordings from any of these regions (e.g., mPFC or GPe) that could be used to bolster claims about the functional relevance of specific subpopulations? For example, one would expect coordinated activity between neural activity in mPFC and Cluster 2 according to the Ratcliff and Frank model. Additionally, the reported drift-diffusion model (DDM) results are difficult to interpret as microsimulation appears to have broad and varied effects across almost all the DDM model parameters. The DDM framework could, however, be used to more specifically test the relationships between each neural cluster and specific decision functions described in each model. Several studies have successfully shown that neural activity tracks specific latent decision parameters estimated by the DDM by including neural activity as a predictor in the model. Using this approach, the current study could examine whether each cluster's activity is predictive of specific decision parameters (e.g., evidence accumulation, decision thresholds, etc.). For example, according to the Ratcliff and Frank model, activity in cluster 2 might track decisions thresholds.

Review of revision

The authors have sufficiently addressed the concerns raised in the initial reviews and have revised their manuscript accordingly. We commend the authors for these efforts and feel that the revisions have strengthened the major claims of the manuscript.

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 #3 (Public Review):

Summary:

The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of time points, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delivered FI was much less efficacious than Pegceptacoplan (FDA-approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.

Strengths:

The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.

Weaknesses:

Perhaps the conclusions drawn regarding the lack of observed efficacy are not fully justified. The authors focus on the hypothesis that not enough FI was synthesised in these patients receiving the PPY988 gene therapy, suggesting a delivery/transduction/expression issue. But beyond rare CFI genetic variants, most genetic associations with AMD imply that it is a FI-cofactor disease. A hypothesis supported by the authors' own experiments when they supplement their artificial VH matrix with FH and achieve a significantly greater breakdown of C3b than achieved with PPY988 treatment alone. Justification around doubling FI levels driving complement turnover refers to studies conducted in blood, which has an entirely different complement protein profile than VH. In Supplemental Table 5 we see there is approx. 10-fold more FH than FI (533ug/ml vs 50ug/ml respectively) so increasing FI levels will have a direct effect. Yet in Supplemental Table 3 we see there is more FI than FH in VH (608ng/ml vs 466ng/ml respectively). Therefore, adding more FI without more co-factors would have a very limited effect. Surely this demonstrates that the study was delivering the wrong payload, i.e. FI, which hit a natural ceiling of endogenous co-factors within the eye?

Reviewer #1 (Public Review):

Summary:

This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in the vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for a thorough biomarker review to determine optimal dosing in future studies.

Strengths:

This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making sharing this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.

Weaknesses:

No particular weaknesses. The study was carefully performed and limitations are discussed.

I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?

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 #1 (Public Review):

In this manuscript by Wu et al., the authors present the high resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure, the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

Another interesting structure is the complex of Kv1.2 with the pore blocking toxin Dendrotoxin 1. The results shown in the revised version indicate that the mechanism of block is similar to that of related blocking-toxins, in which a lysine residue penetrates in the pore. Surprisingly, in these new structures, the bound toxin results in a pore with empty external potassium binding sites.

The quality of the structural data presented in this revised manuscript is very high and allows for unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. In the revised version, the authors have addressed my previous specific comments.

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.

Reviewer #3 (Public Review):

Wu et al. present cryo-EM structures of the potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and presumably sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9 Å. The work builds on a large body of structural work on Kv1.2 and related voltage-gated potassium channels. The manuscript presents a plethora of structural work, and the authors are commended on the breadth of the studies. The structural studies are well-executed. Although the findings are mostly confirmatory, they do add to the body of work on this and related channels. Notably, the authors present structures of DTx-bound Kv1.2 and of Kv1.2 in a low concentration of potassium (which may contain sodium ions bound within the selectivity filter). These two structures add considerable new information. The DTx structure has been markedly improved in the revised version and the authors arrive at well-founded conclusions regarding its mechanism of block. Overall, the manuscript is well-written, a nice addition to the field, and a crowning achievement for the Sigworth lab.

Reviewer #1 (Public Review):

Summary:<br /> In this paper, authors investigated the role of RUNT-related transcription factor 2 (RUNX2) in oral squamous carcinoma (OSCC) growth and resistance to ferroptosis. They found that RUNX2 suppresses ferroptosis through transcriptional regulation of peroxiredoxin-2. They further explored the upstream positive regulator of RUNX2, HOXA10 and found that HOXA10/RUNX2/PRDX2 axis protects OSCC from ferroptosis.

Strengths:<br /> The study is well designed and provides a novel mechanism of HOXA10/RUNX2/PRDX2 control of ferroptosis in OSCC.

Weaknesses:

According to the data presented in (Figure 2F, Figure 3Fand G, Figure 5D and Figure 6E and F), apoptosis seems to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis, which raises questions on the authors' specific focus on ferroptosis in this study. Reasonably, authors should adapt the title and the abstract in a way that recapitulates the whole data, which is HOXA10/RUNX2/PRDX2 axis control of cell death, including ferroptosis and apoptosis in OSCC.

Comments:

- In the description of the result section related to Figure 3E, the author wrote "In addition, we found that isoform II-knockdown induced shrunken mitochondria with vanished cristae with transmission electron microscopy (Figure 3E). These results suggest that RUNX2 isoform II may suppress ferroptosis." The interpretation provided here is not clear to the reviewer. How shrunken mitochondria and vanished cristae can be linked to ferroptosis?<br /> - The electron microscopy images show more elongated mitochondria in the RUNX2 isoform II-KO cells than in RUNX2 isoform II positive cells, which might result from the fusion of mitochondria. These images should completed with a fluorescent mitochondria staining of these cells.<br /> - What is the oxygen consumption rate in RUNX2 KO cells?<br /> - The increase in cell proliferation after RUNX2 overexpression in Figure 2A is not convincing, is there any differences in their migration or invasion capacity?<br /> - The in vivo study shows 50% reduction in primary tumor growth after RUNX2 inhibition by shRNA in CAL 27 xenografts, but only one shRNA is shown. Is this one shRNA clone? At least 2 shRNA clones should be used.<br /> - Apoptosis and necroptosis seem to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis. This is evident from experiments in Figure 3E, F and from Figure 6E, F and Figure 3G. Either Fer-1, Z-VAD,or Nec-1 used alone, were not able to fully restore cell proliferation to control cell level, which implies an additive effect of ferroptosis, apoptosis and necrosis. The author should verify potential additive or synergistic effect of the combination of Fer-1 and Z-VAD in these assays after si-RUNX2 in Figure 3 F and G and after si-HOX assays.<br /> - What is the effect of PRDX2 or HOXA10 depletion on tumor growth?<br /> - What is the clinical relevance of HOXA10 in OSCC patients?

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 #1 (Public review):

Summary:

This study develops and validates a neural subspace similarity analysis for testing whether neural representations of graph structures generalize across graph size and stimulus sets. The authors show the method works in rat grid and place cell data, finding that grid but not place cells generalize across different environments, as expected. The authors then perform additional analyses and simulations to show that this method should also work on fMRI data. Finally, the authors test their method on fMRI responses from the entorhinal cortex (EC) in a task that involves graphs that vary in size (and stimulus set) and statistical structure (hexagonal and community). They find neural representations of stimulus sets in lateral occipital complex (LOC) generalize across statistical structure and that EC activity generalizes across stimulus sets/graph size, but only for the hexagonal structures.

Strengths:

(1) The overall topic is very interesting and timely and the manuscript is well-written.

(2) The method is clever and powerful. It could be important for future research testing whether neural representations are aligned across problems with different state manifestations.

(3) The findings provide new insights into generalizable neural representations of abstract task states in the entorhinal cortex.

Weaknesses:

(1) The manuscript would benefit from improving the figures. Moreover, the clarity could be strengthened by including conceptual/schematic figures illustrating the logic and steps of the method early in the paper. This could be combined with an illustration of the remapping properties of grid and place cells and how the method captures these properties.

(2) Hexagonal and community structures appear to be confounded by training order. All subjects learned the hexagonal graph always before the community graph. As such, any differences between the two graphs could thus be explained (in theory) by order effects (although this is practically unlikely). However, given community and hexagonal structures shared the same stimuli, it is possible that subjects had to find ways to represent the community structures separately from the hexagonal structures. This could potentially explain why the authors did not find generalizations across graph sizes for community structures.

(3) The authors include the results from a searchlight analysis to show the specificity of the effects of EC. A better way to show specificity would be to test for a double dissociation between the visual and structural contrast in two independently defined regions (e.g., anatomical ROIs of LOC and EC).

(4) Subjects had more experience with the hexagonal and community structures before and during fMRI scanning. This is another confound, and possible reason why there was no generalization across stimulus sets for the community structure.

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 #3 (Public review):

Summary:

The article explores the brain's ability to generalize information, with a specific focus on the entorhinal cortex (EC) and its role in learning and representing structural regularities that define relationships between entities in networks. The research provides empirical support for the longstanding theoretical and computational neuroscience hypothesis that the EC is crucial for structure generalization. It demonstrates that EC codes can generalize across non-spatial tasks that share common structural regularities, regardless of the similarity of sensory stimuli and network size.

Strengths:

(1) Empirical Support: The study provides strong empirical evidence for the theoretical and computational neuroscience argument about the EC's role in structure generalization.

(2) Novel Approach: The research uses an innovative methodology and applies the same methods to three independent data sets, enhancing the robustness and reliability of the findings.

(3) Controlled Analysis: The results are robust against well-controlled data and/or permutations.

(4) Generalizability: By integrating data from different sources, the study offers a comprehensive understanding of the EC's role, strengthening the overall evidence supporting structural generalization across different task environments.

Weaknesses:

A potential criticism might arise from the fact that the authors applied innovative methods originally used in animal electrophysiology data (Samborska et al., 2022) to noisy fMRI signals. While this is a valid point, it is noteworthy that the authors provide robust simulations suggesting that the generalization properties in EC representations can be detected even in low-resolution, noisy data under biologically plausible assumptions. I believe this is actually an advantage of the study, as it demonstrates the extent to which we can explore how the brain generalizes structural knowledge across different task environments in humans using fMRI. This is crucial for addressing the brain's ability in non-spatial abstract tasks, which are difficult to test in animal models.

While focusing on the role of the EC, this study does not extensively address whether other brain areas known to contain grid cells, such as the mPFC and PCC, also exhibit generalizable properties. Additionally, it remains unclear whether the EC encodes unique properties that differ from those of other systems. As the authors noted in the discussion, I believe this is an important question for future research.

Reviewer #1 (Public review):

Summary:

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

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

Strengths:

The authors use a broad number of approaches to implicate oscar, and to dissect its mechanism of male lethality. These approaches include:<br /> (1) Overexpressing oscar (and wmk) by injecting RNA into moth eggs.<br /> (2) Determining the sex of embryos by staining female sex chromosomes.<br /> (3) Determining the consequences of oscar expression by assaying sex-specific splice variants of doublesex, a key sex determination gene, and by quantifying gene expression and dosage of sex chromosomes, using RNASeq.<br /> (4) Expressing oscar along with masculinizer from various moth and butterfly species, in a silkmoth cell line.

This extends recently published studies implicating oscar in male-killing by Wolbachia in Ostrinia corn borer moths, although the Homona and Ostrinia oscar proteins are quite divergent. Combined with other studies, there is now broad support for oscar as the male-killing gene in moths and butterflies (i.e. order Lepidoptera). So an outstanding question is to understand the role of wmk. Is it the master male-killing gene in insects other than Lepidoptera and if so, how does it operate?

Weaknesses:

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

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 #3 (Public review):

Summary:

Overall, this is a clearly written manuscript with nice hypothesis testing in a non-model organism that addresses the mechanism of Wolbachia-mediated male killing. The authors aim to determine how five previously identified male-killing genes (encoded in the prophage region of the wHm Wolbachia strain) impact the native host, Homona magnanima moths. This work builds on the authors' previous studies in which:<br /> (1) They tested the impact of these same wHm genes via heterologous expression in Drosophila melanogaster.<br /> (2) They examined the activity of other male-killing genes (e.g., from the wFur Wolbachia strain in its native host: Ostrinia furnacalis moths).

Advances here include identifying which wHm gene most strongly recapitulates the male-killing phenotype in the native host (rather than in Drosophila), and the finding that the Hm-Oscar protein has the potential for male-killing in a diverse set of lepidopterans, as inferred by the cell-culture assays.

Strengths:

Strengths of the manuscript include the reverse genetics approaches to dissect the impact of specific male-killing loci, and the use of a "masculinization" assay in Lepidopteran cell lines to determine the impact of interactions between specific masc and oscar homologs.

Weaknesses:

My major comments are related to the lack of statistics for several experiments (and the data normalization process), and opportunities to make the manuscript more broadly accessible.

The manuscript I think would be much improved by providing more details regarding some of the genes and cross-lineage comparisons. I know some of this is reported in previous publications, but some summary and/or additional analysis would make this current manuscript much more approachable for a broader audience, and help guide readers to specific important findings. For example, a graphic and/or more detail on how the wmk/oscar homologs (within and across Wolbachia strains) differ (e.g., domains, percent divergence, etc) would be helpful for contextualizing some of the results. I recognize the authors discuss this in parts (e.g., lines 223-227), but it does require some bouncing between sections to follow. Similarly, the experiments presented in Figure 4 indicate that Hm-oscar has broad spectrum activity: how similar are the masc proteins from these various lepidopterans? Are they highly conserved? Rapidly evolving? Do the patterns of masc protein evolution provide any hints at how Oscar might be interacting with masc?

It is clear from Figure 1 that the combinations of wmk homologs do not cause male killing on their own. Did the authors test if any of the wmk homologs impact the MK phenotype of oscar? It looks like a previous study tested this in wFur (noted in lines 250-252), but given that the authors also highlight the differences between the wFur-oscar and Hm-oscar proteins, this may be worth testing in this system. Related to this, what is the explanation for why there would be 4 copies of wmk in Hm?

Why are some of the broods male-biased (2/3) rather than ~50:50? (Lines 170-175, Figure 2a). For example, there is a strong male bias in un-hatched oscar-injected and naturally infected embryos, whereas the control uninfected embryos have normal 50:50 sex ratios. It is difficult to interpret the rate of male-killing given that the sex ratios of different sets of zygotes are quite variable.

Figure 2b - it appears there are both male and female bands in the HmOsc male lane. I think this makes sense (likely a partial phenotype due to the nature of the overexpression approach), but this is worth highlighting, especially in the context of trying to understand how much of the MK phenotype might be recapitulated through these methods. Related, there is no negative control for this PCR.

It appears the RNA-seq analysis (Figure 3) is based on a single biological replicate for each condition. And, there are no statistical comparisons that support the conclusions of a shift in dosage compensation. Finally, it is unclear what exactly is new data here: the authors note "The expression data of the wHm-t-infected and non-infected groups were also calculated based on the transcriptome data included in Arai et al. (2023a)" - So, are the data in Figure 3c and 3d a re-print of previous data? The level of dosage compensation inferred by visually comparing the control conditions in 3b and 3d does not appear consistent. With only one biological replicate library per condition, what looks like a re-print of previous data, and no statistical comparisons, this is a weakly supported conclusion.

In Figure 4: There are no statistics to support the conclusions presented here. Additionally, the data have gone through a normalization process, but it is difficult to follow exactly how this was done. The control conditions appear to always be normalized to 100 ("The expression levels of BmImpM in the Masc and Hm-Oscar/Oscar co-transfected cells were normalized by setting each Masc-transfected cell as 100"). I see two problems with this approach:<br /> (1) This has eliminated all of the natural variation in BmImpM expression, which is likely not always identical across cells/replicates.<br /> (2) How then was the percentage of BmImpM calculated for each of the experimental conditions? Was each replicate sample arbitrarily paired with a control sample? This can lead to very different outcomes depending on which samples are paired with each other. The most appropriate way to calculate the change between experimental and control would be to take the difference between every single sample (6 total, 3 control, 3 experimental) and the mean of the control group. The mean of the control can then be set at 100 as the authors like, but this also maintains the variability in the dataset and then eliminates the issue of arbitrary pairings. This approach would also then facilitate statistical comparisons which is currently missing.

Reviewer #1 (Public Review):

Summary:

Machii et al. reported a possible molecular mechanism underlying the parallel evolution of lip hypertrophy in African cichlids. The multifaceted approach taken in this manuscript is highly valued, as it uses histology, proteomics, and transcriptomics to reveal how phylogenetically distinct thick-lips have evolved in parallel. Findings from histology and proteomics connected to wnt signaling through the transcriptome are very exciting.

Strengths:

There is consistency between the results and it is possible to make a strong argument from the results.

Weaknesses:

The authors do not discuss based on genomic information; the genomes of the cichlids from the three lakes have been decoded and are therefore available. However, indeed, the species in Lake Tanganyika and Lake Malawi/Victoria are genetically distant from each other, so a comparative genome analysis would not have yielded the results presented here. I recommend adding such a discussion to the Discussion.

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 #1 (Public review):

The central finding of the current manuscript is that embryonic ablation of PRMT1 results in a craniofacial phenotype that is primarily linked to downstream intron splicing defects. This manuscript is one of several to underscore the relative importance of intron splicing to gene expression regulation during development, and moreover, to recapitulate splicing-related craniofacial defects. Specifically, authors introduce a regulatory axis consisting of PRMT1-SFPQ that directs mechanisms of long intron retention. This finding represents a significant contribution to our understanding of splicing regulation, in the sense that it highlights the regulatory impact that post-translational modification of splicing-related proteins can have on intron processing. Further, it emphasizes the importance of extending the study of splicing regulation beyond core components of the spliceosome, to include their upstream regulators as well.

The significance of neural crest cells in the development of craniofacial structures has long been considered a major contributor to developmental phenotypes. This specific symptomology is heavily associated with spliceosomopathies, wherein disruption of spliceosome components is the primary mechanism of disease pathogenesis. Thus, the PRMT1 associated phenotype is noteworthy. The role of PRMT1 in methylating downstream splicing factors introduces a new avenue of research focused on the mechanisms of spliceosome component activation and their effects on splicing. The strength of the current study lies in their establishing the molecular mechanism through which PRMT1 could alter craniofacial development through regulation of the transcriptome, but the data presented to support the claim that a PRMT1-SFPQ axis directly regulates intron retention of the relevant gene networks should be robust and with multiple forms of clear validation. For example, elevated intron retention findings are based on the intron retention index, and according to the manuscript, are assessed considering the relative expression of exons and introns from a given transcript. However, delineating between intron retention and other forms of alternative splicing (i.e., cryptic splice site recognition) requires a more comprehensive consideration of the intron splicing defects that could be represented in data. A certain threshold of intron read coverage (i.e., the percent of an intron that is covered by mapped reads) is needed to ascertain if those that are proximal to exons could represent alternative introns ends rather than full intron retention events. In other words, intron retention is a type of alternative splicing that can be difficult to analyze in isolation given the confounding influence of cryptic splicing and cryptic exon inclusion. If other forms of alternative splicing were assessed and not detected, more confident retention calls can be made.

While data presented to support the PRMT1-SFPQ activation axis is quite compelling, that this is directly responsible for the elevated intron retention remains enigmatic. First, in characterizing their PRMT1 knockout model, it is unclear whether the elevated intron retention events directly correspond to downregulated genes. Moreover, intron splicing is a well-documented node for gene regulation during embryogenesis and in other proliferation models, and craniofacial defects are known to be associated with 'spliceosomopathies'. However, reproduction of this phenotype does not suggest that the targets of interest are inherently splicing factors, and a more robust assessment is needed to determine the exact nature of alternative splicing in this system. Because there are several known splicing factors downstream of PRMT1 and presented in the supplemental data, the specific attribution of retention to SFPQ would be additionally served by separating its splicing footprint from that of other factors that are primed to cause alternative splicing.

Clarifying the relationship between SFPQ and splicing regulation is important given that the observed splicing defects are incongruous with published data presented by Takeuchi et al., (2018) regarding SFPQ control of neuronal apoptosis in mice. In this system, SFPQ was more specifically attributed to the regulation of transcription elongation over long introns and its knockout did not result in significant splicing changes. Thus, to establish the specificity for the SFPQ in regulating these retention events, authors would need to show that the same phenotype is not achieved by mis-regulation of other splicing factors. That the authors chose SFPQ based on its binding profile is understandable but potentially confounding given its mechanism of action in transcription of long introns (Takeuchi 2018). Because mechanisms and rates of transcription can influence splicing and exon definition interactions, the role of SFPQ as a transcription elongation factor versus a splicing factor is inadequately disentangled by authors.

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 #2 (Public review):

Summary:

Hall et al describe the superiority of ONT sequencing and deep learning-based variant callers to deliver higher SNP and Indel accuracy compared to previous gold-standard Illumina short-read sequencing. Furthermore, they provide recommendations for read sequencing depth and computational requirements when performing variant calling.

Strengths:

The study describes compelling data showing ONT superiority when using deep learning-based variant callers, such as Clair3, compared to Illumina sequencing. This challenges the paradigm that Illumina sequencing is the gold standard for variant calling in bacterial genomes. The authors provide evidence that homopolymeric regions, a systematic and problematic issue with ONT data, are no longer a concern in ONT sequencing.

Weaknesses:

The study is limited in the number of samples included, even though it covers different species with divergent genome sequences, likely covering major evolutionary changes. The methods section could be more detailed. A structural variation analysis would be an interesting next step.

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 #1 (Public Review):

Summary:

In this manuscript, Rohde et al. discuss how single cells isolated from the presomitic mesoderm of the zebrafish embryo follow a cell-autonomous differentiation "programme", which is dependent on the initial anteroposterior position in the embryo.

Strengths:

This work and, in particular, the comparison to cellular behaviour in vivo presents a detailed description of the oscillatory system that brings the developmental biology forward in their understanding of somitogenesis.<br /> The main novelty lies in the direct comparison of these isolated single cells to single cells tracked within the developing embryo. This allows them to show that isolated cells follow a similar path of differentiation without direct contact to neighbours or the presence of external morphogen gradients. Based on this, the authors propose an internal timer that starts ticking as cells traverse the presomitic mesoderm, while external signals modify this behaviour.

There are a few direct questions that follow up from this study, for instance, intercellular synchronization influences the variability of the timer. However, I agree with the authors that such experiments are out of the scope of this study.

Reviewer #1 (Public Review):

Summary:

The authors aimed to identify potential biomarkers for acute myocardial infarction (AMI) through blood metabolomics and fecal microbiome analysis. They found that long chain fatty acids (LCFAs) could serve as biomarkers for AMI and demonstrated a correlation between LCFAs and the gut microbiome. Additionally, in silico molecular docking and in vitro thrombogenic assays showed that these LCFAs can induce platelet aggregation.

Strengths:

The study utilized a comprehensive approach combining blood metabolomics and fecal microbiome analysis.

The findings suggest a novel use of LCFAs as biomarkers for AMI.

The correlation between LCFAs and the gut microbiome is a significant contribution to understanding the interplay between gut health and heart disease.

The use of in silico and in vitro assays provides mechanistic insights into how LCFAs may influence platelet aggregation.

Weaknesses:

The evidence is incomplete as it does not definitively prove that gut dysbiosis contributes to fatty acid dysmetabolism.

The study primarily shows an association between the gut microbiome and fatty acid metabolism without establishing causation.

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.

Joint Public Review

The molecular mechanisms that mediate the regulated exocytosis of neuropeptides and neurotrophins from neurons via large dense-core vesicles (LDCVs) are still incompletely understood. Motivated by their earlier discovery that the Rab3-RIM1 pathway is essential for neuronal LDCV exocytosis, the authors now examined the role of the Rab3 effector Rabphilin-3A in neuronal LDCV secretion. Based on live, confocal, and super-resolution imaging approaches, the authors provide evidence for a synaptic enrichment of Rabphilin-3A and for independent trafficking of Rabphilin-3A and LDCVs. Using an elegant NPY-pHluorin imaging approach, they show that genetic deletion of Rabphilin-3A causes an increase in electrically triggered LDCV fusion events and increased neurite length. Finally, knock-out-replacement studies, involving Rabphilin-3A mutants deficient in either Rab3- or SNAP25-binding, indicate that the synaptic enrichment of Rabphilin-3A depends on its Rab3 binding ability, while its ability to bind to SNAP25 is required for its effects on LDCV secretion and neurite development. The authors conclude that Rabphilin-3A negatively regulates LDCV exocytosis and propose that this mechanism also affects neurite growth, e.g. by limiting neurotrophin secretion. These are important findings that advance our mechanistic understanding of neuronal large dense-core vesicle (LDCV) secretion.

The major strengths of the present paper:

(i) The use of a powerful Rabphilin-3A KO mouse model.<br /> (ii) Stringent lentiviral expression and rescue approaches as a strong genetic foundation of the study.<br /> (iii) An elegant FRAP imaging approach.<br /> (iv) A cutting-edge NPY-pHluorin-based imaging approach to detect LDCV fusion events.

Weaknesses of the present paper:

(i) It remains unclear why a process that affects a general synaptic SNARE fusion protein - SNAP25 - would specifically affect LDCV but not synaptic vesicle fusion.<br /> (iii) The mechanistic links between Rabphilin-3A function, LDCV density in neurites, neurite outgrowth, and the proposed underlying mechanisms involving trophic factor release remain unresolved.

Reviewer #1 (Public Review):

O'Leary and colleagues sought to understand the factors that underlie memory processes, including formation, retrieval, and forgetting. The present data identify time, environmental enrichment, Rac-1, context reexposure, and brief reminders of the familiar object as factors that alter discrimination between novel and familiar objects. This is complimented with an engram approach to quantify cells that are active during learning to examine how their activation is impacted with each of the above factors at test. There are many strengths in the manuscript, including systematic testing of several factors that contribute to poor discrimination between novel and familiar objects. These results are interesting and outline essential boundaries of incidental, nonaversive memory. With this behavioral data, authors apply a modeling approach to understand the factors that contribute to good and poor object memory recall.

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 #3 (Public Review):

Summary:

The manuscript by Ryan and colleagues uses a well-established object recognition task to examine memory retrieval and forgetting. They show that memory retrieval requires activation of the acquisition engram in the dentate gyrus and failure to do so leads to forgetting. Using a variety of clever behavioural methods, the authors show that memories can be maintained and retrieval slowed when animals are reared in environmental enrichment and that normally retrieved memories can be forgotten if exposed to the environment in which the expected objects are no longer presented. Using a series of neural methods, the authors also show that activation or inhibition of the acquisition engram is key to memory expression and that forgetting is due to Rac1.

Strengths:

This is an exemplary examination of different conditions that affect successful retrieval vs forgetting of object memory. Furthermore, the computational modelling that captures in a formal way how certain parameters may influence memory provides an important and testable approach to understanding forgetting.<br /> The use of the Rescorla-Wanger model in the context of object recognition and the idea of relevance being captured in negative prediction error are novel (but see below).<br /> The use of gain and loss of function approaches are a considerable strength and the dissociable effects on behaviour eliminate the possibility of extraneous variables such as light artifacts as potential explanations for the effects.

Weaknesses:

A closer examination of the process that governs the behavioural effect in the present investigation would have been of even greater significance. The authors acknowledge the distinction between object familiarity vs object recognition, but a direct assessment would benefit the field's understanding of the current role of engrams on behaviour.

Relatedly, while relevance is an interesting concept that has been operationalized in the paper, it is unclear how distinct it is from extinction. Specifically, in the case where the animals are exposed to the context in the absence of the object, the paper currently expresses this as a process of relevance - the objects are no longer relevant in that context. Another way to think about this is in terms of extinction - the association between the context and the objects is reduced resulting in a disrupted ability of the context to activate the object engram. The authors have noted the potential role of extinction in their studies.

The impact of the paper is somewhat limited by the use of only one sex. Do the authors expect an identical process to be engaged in females in the present set of studies?

Reviewer #1 (Public Review):

The authors present a model for multisensory correlation detection that is based on the neurobiologically plausible Hassenstein Reichardt detector (Parise & Ernst, 2016). They demonstrate that this model can account for human behaviour in synchrony or temporal order judgements and related temporal tasks in two new data sets (acquired in this study) and a range of previous data sets. While the current study is limited to the model assessment for relatively simple audiovisual signals, in future communications, the authors demonstrate that the model can also account for audiovisual integration of complex naturalistic signals such as speech and music.

The significance of this work lies in its ability to explain multisensory perception using fundamental neural mechanisms previously identified in insect motion processing.

Strengths:

(1) The model goes beyond descriptive models such as cumulative Gaussians for TOJ and differences in cumulative Gaussians for SJ tasks by providing a mechanism that builds on the neurobiologically plausible Hassenstein-Reichardt detector.<br /> (2) This model can account for results from two new experiments that focus on the detection of correlated transients and frequency doubling. The model also accounts for several behavioural results from experiments including stochastic sequences of A/V events and sine wave modulations (and naturalistic Av signals such as speech and music as shown in future communications).

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 #1 (Public Review):

Summary:

"Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

Strengths:

The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials according to the experts' consensus-recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on the rigor, the EEG methods is beyond my expertise as a reviewer.

Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.

The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.

The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

Weaknesses:

Though the methods employed in the paper are generally strong, there are certain aspects that are not clearly described in the Materials & Methods section, such as a description of the statistical analyses used for hypothesis testing.

With regard to metabolite quantification, it is unclear why the authors chose to analyze and report metabolite values in terms of creatine ratios rather than quantification based on a water reference given that the MRS acquisition appears to support using a water reference. GABA is typically quantified using J-editing sequences as lower field strengths (~3T), and there is some evidence that the GABA signal can be reliably measured at 7T without editing, however, the authors should discuss potential limitations, such as reliability of Glu and GABA measurements with short-TE semi-laser at 7T. In addition, MRS measurements of GABA are known to be influenced by macromolecules, and GABA is often denoted as GABA+ to indicate that other compounds contribute to the measured signal, especially at a short TE and in the absence of symmetric spectral editing. A general discussion of the strengths and limitations of unedited Glu and GABA quantification at 7T is warranted given the interest of this work to researchers who may not be experts in MRS.

Further, the single MRS voxel location is a limitation of the study as neurochemistry can vary regionally within individuals, and the putative excitatory/inhibitory imbalance in dyslexia may appear in regions outside the left temporal cortex (e.g., network-wide or in frontal regions involved in top-down executive processes). While the functional localization of the MRS voxel is a novelty and a potential advantage, it is unclear whether voxel placement based on left-lateralized reading-related neural activity may bias the experiment to be more sensitive to small, activity-related fluctuations in neurotransmitters in the CON group vs. the DYS group who may have developed an altered, compensatory reading strategy.

As the authors note in the discussion, sex could serve as a moderator of associations between neural noise and reading abilities and should be considered in future studies.

Appraisal:

The authors present a thorough evaluation of the neural noise hypothesis of developmental dyslexia in a sample of adolescents and young adults using multiple methods of measuring excitatory/inhibitory imbalances as an indicator of neural noise. The authors concluded that there was no support for the neural noise hypothesis of dyslexia in their study based on null significance and Bayes factors. This conclusion is justified, and further research is called for to more broadly evaluate the neural noise hypothesis in developmental dyslexia.

Impact:

This study provides an exemplary foundation for the evaluation of the neural noise hypothesis of dyslexia. Other researchers may adopt the model applied in this paper to examine neural noise in various populations with/without dyslexia, or across a continuum of reading abilities, to more thoroughly examine the evidence (or lack thereof) for this hypothesis. Notably, the lack of evidence here does not rule out the possibility of a role for neural noise in dyslexia, and the authors point out that presentation with co-occurring conditions, such as ADHD, may contribute to neural noise in dyslexia. Dyslexia remains a multi-faceted and heterogenous neurodevelopmental condition, and many genetic, neurobiological, and environmental factors play a role. This study demonstrates one step toward evaluating neurobiological mechanisms that may contribute to reading difficulties.

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 #3 (Public Review):

Summary:

This study by Glica and colleagues utilized EEG (i.e., Beta power, Gamma power, and aperiodic activity) and 7T MRS (i.e., MRS IE ratio, IE balance) to reevaluate the neural noise hypothesis in Dyslexia. Supported by Bayesian statistics, their results show solid 'no evidence' of EI balance differences between groups, challenging the neural noise hypothesis. The work will be of broad interest to neuroscientists, and educational and clinical psychologists.

Strengths:

Combining EEG and 7T MRS, this study utilized both the indirect (i.e., Beta power, Gamma power, and aperiodic activity) and direct (i.e., MRS IE ratio, IE balance) measures to reevaluate the neural noise hypothesis in Dyslexia.

Weaknesses:

The authors may need to provide more data to assess the quality of the MRS data.

Reviewer #1 (Public Review):

Summary:

Englert et al. proposed a functional connectome-based Hopfield artificial neural network (fcHNN) architecture to reveal attractor states and activity flows across various conditions, including resting state, task-evoked, and pathological conditions. The fcHNN can reconstruct characteristics of resting-state and task-evoked brain activities. Additionally, the fcHNN demonstrates differences in attractor states between individuals with autism and typically developing individuals.

Strengths:

(1) The study used seven datasets, which somewhat ensures robust replication and validation of generalization across various conditions.

(2) The proposed fcHNN improves upon existing activity flow models by mimicking artificial neural networks, thereby enhancing the representational ability of the model. This advancement enables the model to more accurately reconstruct the dynamic characteristics of brain activity.

(3) The fcHNN projection offers an interesting visualization, allowing researchers to observe attractor states and activity flow patterns directly.

Weaknesses:

(1) The fcHNN projection can offer low-dimensional dynamic visualizations, but its interpretability is limited, making it difficult to make strong claims based on these projections. The interpretability should be enhanced in the results and discussion.

(2) The presentation of results is not clear enough, including figures, wording, and statistical analysis, which contributes to the overall difficulty in understanding the manuscript. This lack of clarity in presenting key findings can obscure the insights that the study aims to convey, making it challenging for readers to fully grasp the implications and significance of the research.

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 #1 (Public Review):

Summary:

The authors aimed to investigate the oscillatory activity of GnRH neurones in freely behaving mice. By utilising GCaMP fiber photometry, they sought to record real-time neuronal activity to understand the patterns and dynamics of GnRH neuron firing and their implications for reproductive physiology.

Strengths:

(1) The use of GCaMP fiber photometry allows for high temporal resolution recordings of neuronal activity, providing real-time data on the dynamics of GnRH neurones.

(2) Recording in freely behaving animals ensures that the findings are physiologically relevant and not artifacts of a controlled laboratory environment.

(3) The authors used statistical methods to characterise the oscillatory patterns, ensuring the reliability of their findings.

Weaknesses:

(1) While the study identifies distinct oscillatory patterns in GnRH neurones' calcium dynamics, it falls short in exploring the functional implications of these patterns for GnRH pulsatility and overall reproductive physiology.

(2) The study lacks a broader discussion to include comparisons with existing studies on GnRH neurone activity and pulsatility and highlight how the findings of this study align with or differ from previous research and what novel contributions are made.

(3) The authors aimed to characterise the oscillatory activity of GnRH neurons and successfully identified distinct oscillatory patterns. The results support the conclusion that GnRH neurons exhibit complex oscillatory behaviours, which are critical for understanding their role in reproductive physiology. However, it has not been made clear what exactly the authors mean by "multi-dimensional oscillatory patterns" and how has this been shown.

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 #1 (Public Review):

Summary:

In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modeling to demonstrate that EPNSst+ neural activity represents movement, choice direction, and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

Strengths and Weaknesses:

Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modeling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Figures 1 F-J, Supplementary Figures 1 F, K, etc i.e via the inclusion of faint hash lines connecting individual data points across variables. Additionally, Figure 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, Figure 3C clearly shows that both SXcontra and SXipsi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high vs. low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results is striking. However, this relies on the co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters, etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well-learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitation is transparently discussed by the authors.

Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization, it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduce generalizability and to some extent the impact of their findings.

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 #3 (Public Review):

Summary:

The authors find that Sst-EPN neurons, which project to the lateral habenula, encode information about response directionality (left vs right) and outcome (rewarded vs unrewarded). Surprisingly, impairment of vesicular signaling in these neurons onto their LHb targets did not impair probabilistic choice behavior.

Strengths:

Strengths of the current work include extremely detailed and thorough analysis of data at all levels, not only of the physiological data but also an uncommonly thorough analysis of behavioral response patterns.

Weaknesses:

Overall, I saw very few weaknesses, with only two issues, both of which should be possible to address without new experiments:

(1) The authors note that the neural response difference between rewarded and unrewarded trials is not an RPE, as it is not affected by reward probability. However, the authors also show the neural difference is partly driven by the rapid motoric withdrawal from the port. Since there is also a response component that remains different apart from this motoric difference (Figure 2, Supplementary Figure 1E), it seems this is what needs to be analyzed with respect to reward probability, to truly determine whether there is no RPE component. Was this done?

(2) The current study reaches very different conclusions than a 2016 study by Stephenson-Jones and colleagues despite using a similar behavioral task to study the same Sst-EPN-LHb circuit. This is potentially very interesting, and the new findings likely shed important light on how this circuit really works. Hence, I would have liked to hear more of the authors' thoughts about possible explanations of the differences. I acknowledge that a full answer might not be possible, but in-depth elaboration would help the reader put the current findings in the context of the earlier work, and give a better sense of what work still needs to be done in the future to fully understand this circuit.

For example, the authors suggest that the Sst-EPN-LHb circuit might be involved in initial learning, but play less of a role in well-trained animals, thereby explaining the lack of observed behavioral effect. However, it is my understanding that the probabilistic switching task forces animals to continually update learned contingencies, rendering this explanation somewhat less persuasive, at least not without further elaboration (e.g. maybe the authors think it plays a role before the animals learn to switch?).

Also, as I understand it, the 2016 study used manipulations that likely impaired phasic activity patterns, e.g. precisely timed optogenetic activation/inhibition, and/or deletion of GABA/glutamate receptors. In contrast, the current study's manipulations - blockade of vesicle release using tetanus toxin or deletion of VGlut2, would likely have blocked both phasic and tonic activity patterns. Do the authors think this factor, or any others they are aware of, could be relevant?

Reviewer #1 (Public review):

Using the UK Biobank, this study assessed the value of nuclear magnetic resonance measured metabolites as predictors of progression to diabetes. The authors identified a panel of 9 circulating metabolites that improved the ability in risk prediction of progression from prediabetes to diabetes. In general, this is a well-performed study, and the findings may provide a new approach to identifying those at high risk of developing diabetes.

Comments on the revised version:

Thanks so much for carefully addressing my comments.

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 #1 (Public review):

Summary:

The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

Strengths:

(1) 7T high-resolution MRS is used<br /> (2) This study combines the behavioral tests, MRS, and fMRI.

Major<br /> I have no further comments. The approach and experiment are sound. The only overall drawback is the relatively low sample size.

Weaknesses:<br /> (1) Line 138, "This finding supports the hypothesis that motion perception is associated with neural activity in MT+ area". This sentence is strange because it is a well-established finding in numerous human fMRI papers. I think the authors should be more specific about what this finding implies.

Response: We thank reviewer for pointing this out. We have revised it to:" This finding is in line with prior results, which indicates that motion perception is associated with neural activity in hMT+ area, but not in EVC (primarily in V1)" (lines 156-158)

Reply: This argument should be refined. Numerous studies have shown the key role of V1 in motion perception. V1 contains a vast proportion of direction selective neurons. I am asking how your results here are related to existing literature. This argument is incorrect and too rough. Can you please revise this?

(9) Line 213, as far as I know, the study (Melnick et al., 2013) is a psychophysical study and did not provide evidence that the spatial suppression effect is associated with MT+.

Response: We thank reviewer for pointing this out. It was a mistake to use this reference, and we have revised it accordingly. (line 242)

Reply: Thanks. New citation is good. But that paper is a modeling study. The direct empirical evidence on humans should be as follow:

Tadin, D., Silvanto, J., Pascual-Leone, A. & Battelli, L. (2011) Improved motion perception and impaired spatial suppression following disruption of cortical area MT/V5. Journal of Neuroscience, 31, 1279-1283.

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 #1 (Public Review):

Summary:

In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment. The manuscript has been substantially improved from the initial version by the addition of new experiments and clarification of important figures. Importantly, the overlap of data with previous reports from the same group has been corrected.

Strengths:

(1) Examination of multiple types of calcium and potassium currents, both through electrophysiology and molecular biology.

(2) Focus on arcuate kisspeptin neurons during the surge is relatively conceptually novel as the anteroventral periventricular nucleus (AVPV) kisspeptin neurons have received much more attention as the "surge generator" population.

(3) The modeling studies allow for direct examination of manipulation of single and multiple conductances, whereas the electrophysiology studies necessarily require examination of each current in isolation. Construction of an arcuate kisspeptin neuron model promises to be of value to the reproductive neuroendocrinology field.

Weaknesses:

A remaining weakness in this revised version of the manuscript is that the relevance of the CRISPR experiments is still rather tenuous given that the goal is to understand what happens in the estrogen-treatment condition, and these experiments were performed only in OVX mice. Similar concerns reflect that the computational model examining the effect of E2 infers multiple conductances based on qPCR data and an assumption that the conductances are directionally proportional to the level of gene expression, and then tunes these to the current recordings obtained from OVX mice, without a direct confirmation in OVX+E2 conditions that the model parameters accurately reflect the properties of these currents in the presence of estrogen.

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 #1 (Public Review):

Summary:

An online database called MRAD has been developed to identify the risk or protective factors for AD.

Strengths:

This study is a very intriguing study of great clinical and scientific significance that provided a thorough and comprehensive evaluation with regard to risk or protective factors for AD. It also provided physicians and scientists with a very convenient, free as well as user-friendly tool for further scientific investigation.

Comments on revised version:

The authors have resolved all of my previous comments. It's a decent paper worth to be published in this field.

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 #1 (Public Review):

Summary:

This is a reviewed manuscript submission to better understand mechanisms for why HIV individuals have diastolic dysfunction. Due to a lack of robust animal models, the team developed iPS-CM models to study HFpEF. The revised manuscript has toned down claims regarding diastolic function given the lack of mechanical testing. The team has focused on the altered Ca2+ phenotype, which improves the precision of the claims of the team. There remain questions on the functional relevance of the altered calcium handling given the lack of physiological assays. There also remain some questions about whether SGLT2 protein is expressed in these models without testing it, and whether the effects of SGLT2i could be off-target.

Overall, the revised manuscript is improved. I have no major remaining concerns except that the lack of biomechanical assessments diminishes the significance of the study as altered calcium alone would not be considered sufficient evidence for diastolic dysfunction, which was major task set out to answer by the group.

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 #1 (Public Review):

Summary:

This study, titled "Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and Dimeric R25CPTH(1-34) in an Osteoporotic Beagle Model," provides valuable insights into the therapeutic effects of two parathyroid hormone (PTH) analogs on bone regeneration and osseointegration. The research is methodologically sound, employing a robust animal model and a comprehensive array of analytical techniques, including micro-CT, histological/histomorphometric analyses, and serum biochemical analysis.

Strengths:

The use of a large animal model, which closely mimics postmenopausal osteoporosis in humans, enhances the study's relevance to clinical applications. The study is well-structured, with clear objectives, detailed methods, and a logical flow from introduction to conclusion. The findings are significant, demonstrating the potential of rhPTH(1-34) and dimeric R25CPTH(1-34) in enhancing bone regeneration, particularly in the context of osteoporosis.

Weaknesses: There are no major weaknesses.

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 #3 (Public Review):

Summary:

The work submitted by Dr. Jeong-Oh Shin and co-workers aims to investigate the therapeutic efficacy of rhPTH(1-34) and R25CPTH(1-34) on bone regeneration and osseointegration of titanium implants using a postmenopausal osteoporosis animal model.

In my opinion the findings presented are not strongly supported by the provided data since the methods utilized do not allow to significantly support the primary claims.

Strengths:

Strengths include certain good technologies utilized to perform histological sections (i.e. the EXAKT system).

Reviewer #2 (Public Review):

Summary:

The authors performed a systematic review and meta-analysis to investigate whether the frequency of emergence of resistance is different if combination antibiotic therapy is used compared to fewer antibiotics. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed.

Strengths:

The strength of the manuscript is that the article addresses a relevant research question which is often debated. The article is well-written and the methodology used is valid. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed. I have several comments and suggestions for the manuscript.

Weaknesses:

Weaknesses of the manuscript are the large clinical and statistical heterogeneity and the lack of clear definitions of acquisition of resistance. Both these weaknesses complicate the interpretation of the study results.

Comments on latest version:

The authors adressed all the comments that were shared in the previous peer review. I still believe that both clinical and statistical heterogeneity remains a problem with the interpretation of the meta-analysis. However, as the authors state, this is in line with the original research question as formulated on Prospero.

Reviewer #1 (Public Review):

Summary:

In this paper the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

Strengths:

The experiments are comprehensive and clearly described, both in text and in the figures. Statistical analyses are provided throughout.

Weaknesses:

None.

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 #3 (Public Review):

Summary:

This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts and heterozygotes) are examined here and whole cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

(1) From previous work it is known that GK,L in type I hair cells has unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+ and modulated by cyclic GMP. The question arises-if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells. However, hair cell specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

(2) Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

(3) A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be really interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Reviewer #1 (Public Review):

The study investigates Cancer Driving Nucleotides (CDNs) using the TCGA database, finding that these recurring point mutations could greatly enhance our understanding of cancer genomics and improve personalized treatment strategies. Despite identifying 50-150 CDNs per cancer type, the research reveals that a significant number remain undiscovered, limiting current therapeutic applications, and underscoring the need for further larger-scale research.

Strengths:

The study provides a detailed examination of cancer-driving mutations at the nucleotide level, offering a more precise understanding than traditional gene-level analyses. The authors found a significant number of CDNs remain undiscovered, with only 0-2 identified per patient out of an expected 5-8, indicating that many important mutations are still missing. The study indicated that identifying more CDNs could potentially significantly impact the development of personalized cancer therapies, improving patient outcomes.

Weaknesses:

The study is constrained by relatively small sample sizes for each cancer type, which reduces the statistical power and robustness of the findings. ICGC and other large-scale WGS datasets are publicly available but were not included in this study.

To be able to identify rare driver mutations, more samples are needed to improve the statistical power, which is well-known in cancer research.

The challenges in direct functional testing of CDNs due to the complexity of tumor evolution and unknown mutation combinations limit the practical applicability of the findings.

The QC of the TCGA data was not very strict, i.e, "patients with more than 3000 coding region point mutations were filtered out as potential hypermutator phenotypes", it would be better to remove patients beyond +/- 3*S.D from the mean number of mutations for each cancer type. Given some point mutations with >3 hits in the TCGA dataset, they were just false positive mutation callings, particularly in the large repeat regions in the human genome.

The codes for the statistical calculation (i.e., calculation of Ai_e, et al) are not publicly available, which makes the findings hard to be replicated.

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 #1 (Public Review):

Calcium channels are key regulators of synaptic strength and plasticity, yet how these channels are differentially utilized to enable synaptic diversity is not clear. In this manuscript, the authors use new endogenous tagging of the Drosophila CaV2 channel Cac and three auxiliary subunits to investigate distinct calcium channel functions at two motor neuron subtypes at the fly NMJ, Is and Ib. Although it is clear from previous studies that Pr is higher at Is over Ib, it is not clear why. The authors confirm these differences using postsynaptic calcium imaging combined with post-hoc Cac-TdTomato imaging. Then, through a series of confocal and super resolution imaging studies, the authors describe differences in calcium channel and active zone structure between Is and Ib motor neuron terminals, and the role of Brp and homeostatic plasticity in regulating channel abundance. Finally, the authors show that while the CaBeta subunit is present at similar levels at Is and Ib active zones, there is an interesting reduction in Stj at Is active zones. The authors conclude that these differences in active zone structure and architecture contribute to the generation of the observed heterogeneity in synaptic strength.

Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. The key factors generating diversity in synaptic strength beyond simple Ca2+ influx are well articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

Comments on revision:

This is a much improved revised manuscript, where the authors have done an excellent job of responding to my initial concerns. In particular, the key factors generating diversity in synaptic strength beyond simple Ca2+ influx are better articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

Upon reflection, I think my initial review came across as a bit harsh, and I am happy to now update my original evaluation to better reflect the importance and impact of this very nice study. I commend the authors on an outstanding study.

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 #1 (Public Review):

Greter et al. provide an interesting and creative use of lactulose as a "microbial metabolism" inducer, combined with tracking of H2 and other fermentation end products. The topic is timely and will likely be of broad interest to researchers studying nutrition, circadian rhythm, and gut microbiota. However, a couple of moderate to major concerns were noted that may impact the interpretation of the current data:

(1) Much of the data relies on housing gnotobiotic mice in metabolic cages, but I couldn't find any details of methods to assess contamination during multiple days of housing outside of gnotobiotic isolators/cages. Given the complexity of the metabolic cage system used, sterility would likely be incredibly challenging to achieve. More details needed to be included about how potential contamination of the mice was assessed, ideally with 16S rRNA gene sequencing data of the endpoint samples and/or qPCR for total colonization levels relative to the more targeted data shown.

(2) The language could be softened to provide a more nuanced discussion of the results. While lactulose does seem to induce microbial metabolism it also could have direct effects on the host due to its osmotic activity or other off-target effects. Thus, it seems more precise to just refer to lactulose specifically in the figure titles and relevant text. Additionally, the degree to which lactulose "disrupts the diurnal rhythm" isn't clear from the data shown, especially given that the markers of circadian rhythm rapidly recover from the perturbation. It is probably more precise to instead state that lactulose transiently induces fermentation during the light phase or something to that effect. The discussion could also be expanded to address what methods are available or could be developed to build upon the concepts here; for example, the use of genetic inducers of metabolism which may avoid the more complex responses to lactulose.

Despite these concerns, this was still an intriguing and valuable addition to the growing literature on the interface of the microbiome and circadian fields.

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 #3 (Public Review):

Summary:

In the manuscript by Greter, et al., entitled "Acute targeted induction of gut-microbial metabolism affects host clock genes and nocturnal feeding" the authors are attempting to demonstrate that an acute exposure to a non-nutritive disaccharide (lactulose) promotes microbial metabolism that feeds back onto the host to impact circadian networks. The premise of the study is interesting and the authors have performed several thoughtful experiments to dissect these relationships, providing valuable insights for the field. However, the work presented does not necessarily support some of the conclusions that are drawn. For instance, lactulose is administered during the fasting period to mimic the impact of a feeding bout on the gut microbiota, but it would be important to perform this treatment during the fed state as well to show that the effects on food intake, etc. do not occur. To truly draw the conclusion that the current outcomes are directly connected to and mediated via an impact on the host circadian clock, it would be ideal to perform these studies in a circadian gene knock-out animal (i.e., Cry1 or Cry2 KO mice, or perhaps Bmal-VilCre tissue-specific KO mice). If the effects are lost in these animals, this would more concretely connect the current findings to the circadian clock gene network. Despite these reservations, the work is promising.

Strengths:

Attempting to disentangle nutrient acquisition from microbial fermentation and its impact on diurnal dynamics of gut microbes on host circadian rhythms is an important step for providing insights into these host-microbe interactions.

The authors utilize a novel approach in leveraging lactulose coupled with germ-free animals and metabolic cages fitted with detectors that can measure microbial byproducts of fermentation, particularly hydrogen, in real-time.

The authors consider several interesting aspects of lactulose delivery, including how it shifts osmotic balance as well as provides calculations that attempt to explain the caloric contribution of fermentation to the animal in the context of reduced food intake. This provides interesting fundamental insights into the role of microbial outputs on host metabolism.

Weaknesses:

While the authors have done a large amount of work to examine the osmotic vs. metabolic influence of lactulose delivery, the authors have not accounted for the enlarged cecum and increased cecal surface area in germ-free mice. The authors could consider an additional control of cecectomy in germ-free mice.

The authors have examined GI hormones as one possible mechanism for how food intake is altered by microbial fermentation of lactulose. However, the authors measure PYY and GLP-1 only at a single time point, stating that there are no differences between groups. Given the goal of the studies is to tie these findings back into circadian rhythms, it would be important to show if the diurnal patterns of these GI hormones are altered.

Considerations of other factors, such as conjugated vs. deconjugated bile acids, microbial bile salt hydrolase activity, and bile acid resorption, might be an important consideration for how lactulose elicits more influence on ileal circadian clock genes relative to cecum and colon.

Measurements of GI transit time (both whole gut and regional) would be an important for consideration for how lactulose might be impacting the ileum vs. cecum vs. colon.

Reviewer #1 (Public Review):

Summary:

Recordings were made from the dentate nucleus of two monkeys during a decision-making task. Correlates of stimulus position and stimulus information were found to varying degrees in the neuronal activities.

Strengths:

A difficult decision-making task was examined in two monkeys.

Weaknesses:

One of the monkeys did not fully learn the task. The manuscript lacked a coherent hypothesis to be tested, and no attempt was made to consider the possibility that this part of the brain may have little to do with the task that was being studied.

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 #1 (Public Review):

Summary:<br /> In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments.

Strengths:<br /> The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear.

Weaknesses:<br /> I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments. Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.

The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis.

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 #3 (Public Review):

Septins in several systems are thought to guide the location of exocytosis, and they have been found to interact with the exocyst vesicle-tethering complex in some cells. However, it is not known whether such interactions are direct or indirect. Moreover, septin-exocyst physical associations were not detected in several other systems, including yeasts, making it unclear whether such interactions reflect a conserved septin-exocytosis link or whether they may missed if they depend on septin polymerization or association into higher-order structures. Singh et. al., set out to define whether and how septins influence the exocyst during S. pombe cytokinesis. Based on three lines of evidence, the authors conclude that septins directly bind to exocyst subunits to regulate localization of the exocyst and vesicle secretion during cytokinesis.<br /> The conclusions are consistent with the data presented, but some interpretations need to be clarified and extended:

(1) The first line of evidence examines septin and exocyst localization during cytokinesis in wild-type and septin-mutant or exocyst-mutant yeast. Quantitative imaging convincingly shows that the detailed localization of the exocyst at the division site is perturbed in septin mutants, and that this is accompanied by modest accumulation of vesicles and vesicle cargos. Whether that is sufficient to explain the increased thickness of the division septum in septin mutants remains unclear.

(2) The second line of evidence involves a comprehensive Alphafold2 analysis of potential pair-wise interactions between septin and exocyst subunits. This identifies several putative interactions in silico, but it is unclear whether the identified interaction surfaces would be available in the full septin or exocyst complexes.

(3) The third line of evidence uses co-immunoprecipitation and yeast two hybrid assays to show that several physical interactions predicted by Alphafold2 can be detected, leading the authors to conclude that they have identified direct interactions. However, both methods leave open the possibility that the interactions are indirect and mediated by other proteins in the fission yeast extract (co-IP) or budding yeast cell (two-hybrid).

(4) Based on prior studies it would be expected that the large majority of both septins and exocyst subunits are present in cells and extracts as stoichiometric complexes. Thus, one would expect any septin-exocyst interaction to yield associations detectable with multiple subunits, yet co-IPs were not detectded in some combinations. It is therefore unclear whether the interactions reflect associations between fully-formed functional complexes or perhaps between transient folding intermediates.

Reviewer #1 (Public Review):

Summary:

This study was to examine the associations of a healthy lifestyle with comprehensive and organ-specific biological ages. It emphasized the importance of lifestyle factors in biological ages, which were defined using common blood biomarkers and body measures.

Strengths:

The data were from a large cohort study and defined comprehensive and six-specified biological ages.

Weaknesses:

(1) Since only 8.5% of participants from the CMEC (China Multi-Ethnic Cohort Study) were included in the study, has any section bias happened?

(2) The authors should specify the efficiency of FFQ. How can FFQ genuinely reflect the actual intake? Moreover, how was the aMED calculated?

(3) HLI (range) and HLI (category) should be clearly defined.

(4) The comprehensive rationale and each specific BA construction should be clearly defined and discussed. For example, can cardiopulmonary BA be reflected only by using cardiopulmonary status? I do not think so.

(5) The lifestyle index is defined based on an equal-weight approach, but this does not reflect reality and cannot fully answer the research questions it raises.

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 #1 (Public Review):

Summary:

The authors set out to understand the role played by a key global metabolic regulator called Crp/cAMP in the formation of persister Escherichia coli that survive antibiotic treatment without acquiring genetic mutations.

In order to achieve this aim, the authors employ an interdisciplinary approach exquisitely integrating standard microbiology assays with cutting-edge genomic, metabolomic, and proteomics screening.

The data presented by the authors convincingly demonstrate that the deletion of two key genes that are part of the Crp/cAMP complex (i.e. crp and cyaA) leads to a significant decrease in the number of persisters, thus pointing towards a key role played by the Crp/cAMP complex in the formation of persisters in E. coli.

The data presented also demonstrate that deletion of the crp gene leads to an overall decrease in energy metabolism and an overall increase in anabolic metabolism at the population level. It is not clear either what the contribution of the cyaA gene is in this respect, or why the deletion of cyaA has an opposite effect on cAMP concentration compared to crp deletion, although the authors present two reasonable untested hypotheses in the discussion. The authors might also want to explicitly acknowledge that these key data are obtained at the whole population level rather than at the level of the persister subpopulation.

Finally, the authors convincingly show that the persisters they investigated are non-growing and have a higher redox activity and that the deletion of key genes involved in energy metabolism leads to a decrease in the number of persisters.

These data will be key for future investigations on the biochemical mechanisms that allow bacteria to adapt to stressors such as nutrient depletion or exposure to antibiotics. As such this work will likely have an impact in a variety of fields such as bacterial biochemistry, antimicrobial resistance research, and environmental microbiology.

Strengths:

Interdisciplinary approach.<br /> Excellent use of replication and ensuring reproducibility.<br /> Excellent understanding and presentation of the biochemical mechanisms underpinning bacterial physiology via an integrated genomic, metabolomic, and proteomic screening.

Weaknesses:

Two genes from the Crp/cAMP complex (crp and cyaA) are hypothesised to be key for persistence but key metabolomics and proteomics data are obtained from only one deletion mutant in the crp gene.

The deletion of crp and cyaA have opposite effects on the concentration of cAMP, a comparison of metabolomics and proteomics data obtained using both mutants might aid in understanding this difference.

Metabolomics, proteomics, and metabolic activity data are obtained at the whole population level rather than at the level of the persister sub-population.

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.

Reviewer #3 (Public Review):

Summary:

The authors describe how E. coli in the late stationary phase have an active TCA cycle and respiration. Mutation of crp results in the down-regulation of TCA cycle genes and an upregulation of anabolic pathways and reduced persisters. Mutation of a variety of metabolic genes also resulted in fewer persisters in the late-stationary phase.

Strengths:

The work is vast, including metabolomic analysis and characterization of a large number of mutant strains. The identification of active respiration being required for persister cell survival in the late stationary phase is interesting. The induction of anabolic pathways resulting in the sensitization of bacteria to antibiotics is possibly the most interesting part of the paper.

Weaknesses:

The authors try to draw too many conclusions and it's difficult to identify what their actual findings are. For instance, they do not have any interesting findings with aminoglycosides but include the data and spend a lot of time discussing it, but it is really a distraction. The correlation between the induction of anabolic pathways in the crp mutant in the late stationary phase and the reduction in persisters is potentially very interesting but is buried in the paper with the vast quantities of data, and observations and conclusions that are often not well substantiated.

The discussion section is particularly difficult to read and I recommend a large overhaul to increase clarity. For instance, what are the authors trying to conclude in section (iii) of the discussion? That persisters in the stationary phase have higher energy than other cells? Is there data to support that? All sections are similarly lacking in clarity.

The large number of mutants characterized is a strength, but the quality of the data provided for those experiments is poor. Did some of these mutants lose fitness in the deep stationary phase in the absence of antibiotics? Did some reach a far lower cfu/ml in the stationary phase? These details are important and without them, it is difficult to interpret the data.

There is ample analysis of persister formation in mutants in the pts/CRP pathway that is not discussed (Zeng et al PNAS 2022, Parsons et al PNAS, 2024).

The authors do not discuss ROS production and antibiotic killing in these experiments. Presumably, the WT would have a greater propensity to produce ROS in response to antibiotics than the crp mutant, but it survives better. Is ROS not involved in antibiotic killing in these conditions?

Joint Public Review:

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

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

Reviewer #1 (Public Review):

Summary:

The overall goal of the manuscript is to delineate pathways that are conditionally essential with the Bam complex and associated chaperones. The Bam complex is made of several proteins, including BamA and BamD, which are essential. The protein complex works to insert proteins in the asymmetric outer membrane. Substrates are translated in the cytoplasm prior to transport across the cell envelope to the Bam complex. Transport includes non-essential periplasmic chaperones, SurA, Skp, and DegP. According to the authors, the pathways were assumed to be redundant. The Bam complex also includes non-essential components, BamBCE. These were thought to be accessory components that interact with BamA and BamD to coordinate optimal activity. While some roles have been assigned to BamE and BamB, a detailed understanding of the role of each accessory Bam protein is lacking. In this study, more specific roles for each non-essential Bam component are proposed.

Strengths:

The overall findings are intriguing and could advance our understanding as to how the Gram-negative cell envelope is assembled. These studies could provide new targets for antimicrobial treatment. In general, the manuscript was well-written.

Weaknesses:

While the overall findings are interesting, I had some concerns with the data analysis, presentation, and conclusions. Not all the conclusions are supported by data. The proposed revisions include experimental and editorial work. The manuscript is generally well-written and could provide impactful data to advance the field if the concerns are addressed.

Major concerns:

Overall Comments:

(1) The cutoffs the authors used to define "conditionally essential" mutants are not reported. The results also lack validation for lethality using a titratable system. It would be ideal to validate several genes in each dataset to determine cutoffs (i.e. 5-fold decrease in insertion mutants) for conditional lethality. It was not done (or described) here.

(2) Also, two mutations that both make the cells sick could provide an additive effect (i.e. dapF and BamB), which doesn't necessarily mean the pathways are linked. The authors should revise their wording. They have not shown genetic linkage in some cases.

(3) Mutations throughout the manuscript are not complemented. It would be ideal to add complementation data to show the gene-phenotype relationship is specific.

(4) Also, I would argue the term "conditionally essential genes" should be replaced with "synthetically lethal". Strains were compared in the same conditions but with different genetic backgrounds.

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 #3 (Public Review):

In this work, Bryant, et al. investigate genetic interactions between non-essential members of the outer membrane protein biogenesis pathway and other genes in the genome using a transposon-directed insertion sequencing (TraDIS) approach in E. coli K-12. The authors identify interactions with other components of the envelope including LPS, peptidoglycan, and enterobacterial common antigen biogenesis, and they tie these interactions to specific members of the outer membrane biogenesis pathway. Although many of these interactions are known and have been previously investigated in the field, the study provides several synthetic phenotypes that could be useful for further investigations.

The strengths of the paper include their unbiased, TraDIS approach, and follow up on the interactions they observe. The interactions with genes of unknown function also are of interest as they may suggest experiments to find the functions of these genes. The largest weakness of this paper is the use of a gene deletion allele for bamB that is known to be polar leading to decreased expression of an essential gene. This largely invalidates all results related to DNA replication. In addition, it is a weakness that the paper does not adequately address its place in the field through discussion of existing results on the interactions they investigate.

Reviewer #1 (Public Review):

Summary:

This study explores the roles of dact1 and dact2 in zebrafish embryonic axis formation and craniofacial morphogenesis. The researchers aim to uncover the mechanisms by which dact1/2 modulates Wnt signaling during embryonic development and patterning. They propose distinct spatiotemporal roles for Dact1 and Dact2 proteins in zebrafish embryonic development, particularly their involvement in modulating noncanonical Wnt signaling during convergent extension events. The findings demonstrate that dact1 and dact2 have unique spatiotemporal expression domains during development and that mutations in dact1/2 lead to convergent extension defects. Furthermore, the study attempts to link these defects to craniofacial abnormalities resulting from dact1/2 mutations. Compound mutants were used to investigate the connection between dact1 and dact2, as single mutants did not exhibit craniofacial phenotypes. The research also includes comprehensive transcriptomics and pathway analyses of differentially expressed genes in dact1/2 mutants, revealing the overexpression of calpain 8, a calcium-dependent cysteine protease. The study suggests that the upregulation of calpain 8 is linked to the observed craniofacial dysmorphology in dact1/2 mutants, implying a potential connection between calpain 8 expression and craniofacial abnormalities.

Strengths:

• The study effectively recapitulates previous findings on the role of dact1/2 in modulating convergent extension during zebrafish embryogenesis.<br /> • A combination of multiple approaches, including in vivo time-lapse imaging, is used to elucidate the etiology of the rod-like neurocranial phenotype in dact1/2 double mutants.<br /> • The study utilizes both traditional and newly created mutant lines, analyzing them through single-cell transcriptomics.

Weaknesses:

(1) The authors successfully addressed reviewers' suggestions with revised experiments and explanations. However, the overall narrative struggles to build a more coherent storyline.<br /> (2) The potential activity of truncated and upregulated dact mRNAs (Fig S2) and partially functional dact proteins needs further clarification.<br /> (3) Data-rich figures, specifically Figs 6, 7, and 8D, could be simplified for better clarity.

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 #3 (Public Review):

Summary:

In this manuscript the authors explore the roles of dact1 and dact2 during zebrafish gastrulation and craniofacial development. Previous studies used morpholino (MO) knockdowns to show that these scaffolding proteins, which interact with dishevelled (Dsh), are expressed during zebrafish gastrulation and suggested that dact1 promotes canonical Wnt/B-catenin signaling, while dact2 promotes non-canonical Wnt/PCP-dependent convergent-extension (Waxman et al 2004). This study goes beyond this work by creating loss-of-function mutant alleles for each gene and unlike the MO studies finds little (dact2) to no (dact1) phenotypic defects in the homozygous mutants. Interestingly, dact1/2 double mutants have a more severe phenotype, which resembles those reported with MOs as well as homozygous wnt11/silberblick (wnt11/slb) mutants that disrupt non-canonical Wnt signaling (Heisenberg et al., 1997; 2000). Further analyses in this paper try to connect gastrulation and craniofacial defects in dact1/2 mutants with wnt11/slb and other wnt-pathway mutants. scRNAseq conducted in mutants identifies calpain 8 as a potential new target of dact1/2 and Wnt signaling.

Previous comments:

Strengths:

When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:

However, the hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.

Comment on the revised version:

The authors addressed some of our comments, but not our main criticisms, which we reiterate here:

(1) The authors argue that morpholino studies are unreliable and here they made new mutants to solve this uncertainty for dap 1/2. However, creating stable mutant lines to largely confirm previous results obtained by using morpholino knock-down phenotypes does not justify publication in eLife.

(2) The authors argue that since it has not been shown conclusively that craniofacial defects in wnt11 and dap1/2 mutants are secondary to gastrulation defects there is no solid evidence preventing them from investigating these craniofacial defects. However, since it is extremely likely that the rod-like ethmoid plates of wnt11f2- and dact1/2 mutants focused on here are secondary to gastrulation defects previously described by others (Heisenberg and NussleinVolhard 1997; Waxman et al., 2004), the burden of proof is on the authors to provide much stronger evidence against this interpretation.

(3) The data for calpain overexpression remains too preliminary.

Reviewer #1 (Public Review):

In this manuscript, Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

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 #1 (Public Review):

Summary:

By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to the identification of a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

Strengths:

In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

Weaknesses:

The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as, for example, species-specific gamete recognition. Additionally, the use of a TPM >1 threshold for protein-coding transcripts - which, as the authors pointed out, was a necessary compromise due to the high transcriptional noise of the system under study - may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis. Moreover, as mentioned in the Discussion, future variations of the pipeline described in the manuscript may allow us to extend the reach of the present analysis.