Reviewer #2 (Public review):

Summary:

This work by den Bakker and Kloosterman contributes to the vast body of research exploring the dynamics governing the communication between the hippocampus (HPC) and the medial prefrontal cortex (mPFC) during spatial learning and navigation. Previous research showed that population activity of mPFC neurons is replayed during HPC sharp-wave ripple events (SWRs), which may therefore correspond to privileged windows for the transfer of learned navigation information from the HPC, where initial learning occurs, to the mPFC, which is thought to store this information long term. Indeed, it was also previously shown that the activity of mPFC neurons contains task-related information that can inform about the location of an animal in a maze, which can predict the animals' navigational choices. Here, the authors aim to show that the mPFC neurons that are modulated by HPC activity (SWRs and theta rhythms) are distinct from those "encoding" spatial information. This result could suggest that the integration of spatial information originating from the HPC within the mPFC may require the cooperation of separate sets of neurons.

This observation may be useful to further extend our understanding of the dynamics regulating the exchange of information between the HPC and mPFC during learning. However, my understanding is that this finding is mainly based upon a negative result, which cannot be statistically proven by the failure to reject the null hypothesis. Moreover, in my reading, the rest of the paper mainly replicates phenomena that have already been described, with the original reports not correctly cited. My opinion is that the novel elements should be precisely identified and discussed, while the current phrasing in the manuscript, in most cases, leads readers to think that these results are new. Detailed comments are provided below.

Major concerns:

(1) The main claim of the manuscript is that the neurons involved in predicting upcoming choices are not the neurons modulated by the HPC. This is based upon the evidence provided in Figure 5, which is a negative result that the authors employ to claim that predictive non-local representations in the mPFC are not linked to hippocampal SWRs and theta phase. However, it is important to remember that in a statistical test, the failure to reject the null hypothesis does not prove that the null hypothesis is true. Since this claim is so central in this work, the authors should use appropriate statistics to demonstrate that the null hypothesis is true. This can be accomplished by showing that there is no effect above some size that is so small that it would make the effect meaningless (see https://doi.org/10.1177/070674370304801108).

(2) The main claim of the work is also based on Figure 3, where the authors show that SWRs-unmodulated mPFC neurons have higher spatial tuning, and higher directional selectivity scores, and a higher percentage of these neurons show theta skipping. This is used to support the claim that SWRs-unmodulated cells encode spatial information. However, it must be noted that in this kind of task, it is not possible to disentangle space and specific task variables involving separate cognitive processes from processing spatial information such as decision-making, attention, motor control, etc., which always happen at specific locations of the maze. Therefore, the results shown in Figure 3 may relate to other specific processes rather than encoding of space and it cannot be unequivocally claimed that mPFC neurons "encode spatial information". This limitation is presented by Mashoori et al (2018), an article that appears to be a major inspiration for this work. Can the authors provide a control analysis/experiment that supports their claim? Otherwise, this claim should be tempered. Also, the authors say that Jadhav et al. (2016) showed that mPFC neurons unmodulated by SWRs are less tuned to space. How do they reconcile it with their results?

(3) My reading is that the rest of the paper mainly consists of replications or incremental observations of already known phenomena with some not necessarily surprising new observations:<br /> a) Figure 2 shows that a subset of mPFC neurons is modulated by HPC SWRs and theta (already known), that vmPFC neurons are more strongly modulated by SWRs (not surprising given anatomy), and that theta phase preference is different between vmPFC and dmPFC (not surprising given the fact that theta is a travelling wave).<br /> b) Figure 4 shows that non-local representations in mPFC are predictive of the animal's choice. This is mostly an increment to the work of Mashoori et al (2018). My understanding is that in addition to what had already been shown by Mashoori et al here it is shown how the upcoming choice can be predicted. The author may want to emphasize this novel aspect.<br /> c) Figure 6 shows that prospective activity in the HPC is linked to SWRs and theta oscillations. This has been described in various forms since at least the works of Johnson and Redish in 2007, Pastalkova et al 2008, and Dragoi and Tonegawa (2011 and 2013), as well as in earlier literature on splitter cells. These foundational papers on this topic are not even cited in the current manuscript.<br /> Although some previous work is cited, the current narrative of the results section may lead the reader to think that these results are new, which I think is unfair. Previous evidence of the same phenomena should be cited all along the results and what is new and/or different from previous results should be clearly stated and discussed. Pure replications of previous works may actually just be supplementary figures. It is not fair that the titles of paragraphs and main figures correspond to notions that are well established in the literature (e.g., Figure 2, 2nd paragraph of results, etc.).<br /> d) My opinion is that, overall, the paper gives the impression of being somewhat rushed and lacking attention to detail. Many figure panels are difficult to understand due to incomplete legends and visualizations with tiny, indistinguishable details. Moreover, some previous works are not correctly cited. I tried to make a list of everything I spotted below.

Reviewer #2 (Public review):

Summary:

This study describes a novel method for mapping torsional stress in the genome of Saccharomyces cerevisiae using trimethylpsoralen (TMP). It introduces a procedure to establish a zero-torsion baseline while preserving the chromatin state by treating cells with formaldehyde before releasing torsion with restriction enzyme digestion.

This approach allows foer more accurate differentiation between torsional stress effects and accessibility effects in the psoralen signal. The results confirm that psoralen crosslinking is strongly affected by accessibility of the DNA and to a much more limited extent by the torsional stress of the DNA. Subtracting the baseline signal (no torsion) from the total signal allows detecting torsional stress, although TMP accessibility is still affecting the read out. The authors confirm the validity of the method by studying torsional stress in dependence of transcription levels, gene length and relative gene orientation. They propose that torsional stress may play a role in recruiting topoisomerases and regulating 3D genome architecture via cohesin. They also suggest that transcription factor binding might insulate negative supercoiling originated form transcription of neighboring divergent genes.

Strengths:

This paper offers a potentially interesting tool for future work.

Weaknesses:

The signal-to-background ratio, which represents the torsional fraction, appears to be quite limited relative to the overall signal (roughly 20x less, according to the scales in figs 2a and 2b, raising concerns about the robustness of the conclusions. It is clear from these figures, for instance, that a non-negligible fraction of the remaining signal is still dependent on DNA accessibility, revealing the nucleosomes footprints in spite of the fact that subtracting the zero-torsion signal should theoretically hinder the accessibility component. Because of this, some of the conclusions might be flawed, in that what is attributed to torsional stress might in reality be due, partially or fully, to accessibility issues.

Specific points:

Lines 226-227: "rotation may be more restricted with a lengthening in the RNA transcript, which is known to be associated with large machinery, such as spliceosomes". This argument is not appropriate to correlate torsional stress with gene length. Spliced genes are rare and generally short in yeast, generally in ribosomal proteins genes.

Lines 256-257 In discussing that torsional stress must hinder Pol II progression, the authors write: "Pol II has a minimal presence in the intergenic region between divergent genes and is enriched in the intergenic region between convergent genes, consistent with a previous finding that after termination, Pol II tends to remain on the DNA downstream of the terminator". The connection between Pol II distribution and torsional stress is unclear. Pol ii is depleted at promoters and is enriched at at 3'-end of convergent genes most likely because this ChIP signal is the sum of signals from the two convergent genes. The fact that positive torsional stress is observed in these region does not mean that polymerases accumulate because the torsional stress hinder Pol II progression. To claim elongation defects the authors should repeat the same analysis with stranded data (e.g. NET-seq or CRAC) and assess if polymerases transcribing these regions accumulate more when facing convergent genes compared to tandem genes. The claim that after termination the Pol II tends to remain on the DNA appears to be meaningless - the authors probably mean after RNA processing.

Lines 275-277: "These data provide evidence that the (+) supercoiling generated by transcription may facilitate genome folding in coordination with other participating proteins". This is an overstatement. It is known that cohesins accumulate between convergent genes. The fact that there is torsional stress in the same position does not imply that supercoiling participates in genome folding. These could be independent events, or even, supercoiling might depend on cohesins

Lines 289-290 "torsion generated from one gene can impact the expression of its neighboring gene, consistent with previous findings that the expression of these genes is coupled" the existence of negative torsional stress in a common intergenic region for two genes does not imply that torsion is causally associated to gene expression coupling

Lines 291-292: "Another large class of S. cerevisiae promoters (termed "TFO") are regulated by insulator ssTFs, such as Reb1 and Abf1, which decouple interactions between neighbouring genes" In these cases and others that depend on an activator binding the authors detect a region of accessibility interrupted by a valley, which they interpret as a topological insulator. However, the valley might be generated because of decreased TMP accessibility due to of TF binding.

Reviewer #2 (Public review):

Summary:

In this study, the authors build a statistical model that stochastically samples from a time-interval distribution of reorientation rates. The form of the distribution is extracted from a large array of behavioral data, and is then used to describe not only the dynamics of individual worms (including the inter-individual variability in behavior), but also the aggregate population behavior. The authors note that the model does not require assumptions about behavioral state transitions, or evidence accumulation, as has been done previously, but rather that the stochastic nature of behavior is "simply the product of stochastic sampling from an exponential function".

Strengths:

This model provides a strong juxtaposition to other foraging models in the worm. Rather than evoking a behavioral transition function (that might arise from a change in internal state or the activity of a cell type in the network), or evidence accumulation (which again maps onto a cell type, or the activity of a network) - this model explains behavior via the stochastic sampling of a function of an exponential decay. The underlying model and the dynamics being simulated, as well as the process of stochastic sampling, are well described and the model fits the exponential function (Equation 1) to data on a large array of worms exhibiting diverse behaviors (1600+ worms from Lopez-Cruz et al). The work of this study is able to explain or describe the inter-individual diversity of worm behavior across a large population. The model is also able to capture two aspects of the reorientations, including the dynamics (to switch or not to switch) and the kinetics (slow vs fast reorientations). The authors also work to compare their model to a few others including the Levy walk (whose construction arises from a Markov process) to a simple exponential distribution, all of which have been used to study foraging and search behaviors.

Weaknesses:

This manuscript has two weaknesses that dampen the enthusiasm for the results. First, in all of the examples the authors cite where a Gillespie algorithm is used to sample from a distribution, be it the kinetics associated with chemical dynamics, or a Lotka-Volterra Competition Model, there are underlying processes that govern the evolution of the dynamics, and thus the sampling from distributions. In one of their references, for instance, the stochasticity arises from the birth and death rates, thereby influencing the genetic drift in the model. In these examples, the process governing the dynamics (and thus generating the distributions from which one samples) is distinct from the behavior being studied. In this manuscript, the distribution being sampled is the exponential decay function of the reorientation rate (lines 100-102). This appears to be tautological - a decay function fitted to the reorientation data is then sampled to generate the distributions of the reorientation data. That the model performs well and matches the data is commendable, but it is unclear how that could not be the case if the underlying function generating the distribution was fit to the data.

The second weakness is somewhat related to the first, in that absent an underlying mechanism or framework, one is left wondering what insight the model provides. Stochastic sampling a function generated by fitting the data to produce stochastic behavior is where one ends up in this framework, and the authors indeed point this out: "simple stochastic models should be sufficient to explain observably stochastic behaviors." (Line 233-234). But if that is the case, what do we learn about how the foraging is happening? The authors suggest that the decay parameter M can be considered a memory timescale; which offers some suggestion, but then go on to say that the "physical basis of M can come from multiple sources". Here is where one is left for want: The mechanisms suggested, including loss of sensory stimuli, alternations in motor integration, ionotropic glutamate signaling, dopamine, and neuropeptides are all suggested: these are basically all of the possible biological sources that can govern behavior, and one is left not knowing what insight the model provides. The array of biological processes listed is so variable in dynamics and meaning, that their explanation of what governs M is at best unsatisfying. Molecular dynamics models that generate distributions can point to certain properties of the model, such as the binding kinetics (on and off rates, etc.) as explanations for the mechanisms generating the distributions, and therefore point to how a change in the biology affects the stochasticity of the process. It is unclear how this model provides such a connection, especially taken in aggregate with the previous weakness.

Providing a roadmap of how to think about the processes generating M, the meaning of those processes in search, and potential frameworks that are more constrained and with more precise biological underpinning (beyond the array of possibilities described) would go a long way to assuaging the weaknesses.

Reviewer #2 (Public review):

Summary:

This paper presents miniML as a supervised method for detection of spontaneous synaptic events. Recordings of such events are typically of low SNR, where state-of-the-art methods are prone to high false favourable rates. Unlike current methods, training miniML requires neither prior knowledge of the kinetics of events nor the tuning of parameters/thresholds.

The proposed method comprises four convolutional networks, followed by a bi-directional LSTM and a final fully connected layer, which outputs a decision event/no event per time window. A sliding window is used when applying miniML to a temporal signal, followed by an additional estimation of events' time stamps. miniML outperforms current methods for simulated events superimposed on real data (with no events) and presents compelling results for real data across experimental paradigms and species.

Strengths:

The authors present a pipeline for benchmarking based on simulated events superimposed on real data (with no events). Compared to five other state-of-the-art methods, miniML leads to the highest detection rates and is most robust to specific choices of threshold values for fast or slow kinetics. A major strength of miniML is the ability to use it for different datasets. For this purpose, the CNN part of the model is held fixed and the subsequent networks are trained to adapt to the new data. This Transfer Learning (TL) strategy reduces computation time significantly and more importantly, it allows for using a substantially smaller data set (compared to training a full model) which is crucial as training is supervised (i.e. uses labeled examples).

Weaknesses:<br /> The authors do not indicate how the specific configuration of miniML was set, i.e. number of CNNs, units, LSTM, etc. Please provide further information regarding these design choices, whether they were based on similar models or if chosen based on performance.

The data for the benchmark system was augmented with equal amounts of segments with/without events. Data augmentation was undoubtedly crucial for successful training.<br /> (1) Does a balanced dataset reflect the natural occurrence of events in real data? Could the authors provide more information regarding this matter?<br /> (2) Please provide a more detailed description of this process as it would serve users aiming to use this method for other sub-fields.

The benchmarking pipeline is indeed valuable and the results are compelling. However, the authors do not provide comparative results for miniML for real data (figures 4-8). TL does not apply to the other methods. In my opinion, presenting the performance of other methods, trained using the smaller dataset would be convincing of the modularity and applicability of the proposed approach.

Impact:

Accurate detection of synaptic events is crucial for the study of neural function. miniML has a great potential to become a valuable tool for this purpose as it yields highly accurate detection rates, it is robust, and is relatively easily adaptable to different experimental setups.

Comments on revisions:

The revised manuscript presents a compelling framework. The performance of mini ML is thouroughly explored and compared to several benchmarks. The training process along with other technical issues are now described in a satisfactory level of detail.<br /> I think the authors did a great job. They answered all claims and concerns raised by me and the other reviewers.

Reviewer #2 (Public review):

Summary:

The manuscript by Yu et al. describes a novel approach for collecting complex and different cognitive phenotypes in individually housed mice in their home cage. The authors report a simple yet elegant design that they developed for assessing a variety of complex and novel behavioral paradigms autonomously in mice.

Strengths:

The data are strong, the arguments are convincing, and I think the manuscript will be highly cited given the complexity of behavioral phenotypes one can collect using this relatively inexpensive ($100/box) and high throughput procedure (without the need for human interaction). Additionally, the authors include a machine learning algorithm to correct for erroneous strategies that mice develop which is incredibly elegant and important for this approach as mice will develop odd strategies when given complete freedom.

Weaknesses:

(1) A limitation of this approach is that it requires mice to be individually housed for days to months. This should be discussed in depth.

(2) A major issue with continuous self-paced tasks such as the autonomous d2AFC used by the authors is that the inter-trial intervals can vary significantly. Mice may do a few trials, lose interest, and disengage from the task for several hours. This is problematic for data analysis that relies on trial duration to be similar between trials (e.g., reinforcement learning algorithms). It would be useful to see the task engagement of the mice across a 24-hour cycle (e.g., trials started, trials finished across a 24-hour period) and approaches for overcoming this issue of varying inter-trial intervals.

(3) Movies - it would be beneficial for the authors to add commentary to the video (hit, miss trials). It was interesting watching the mice but not clear whether they were doing the task correctly or not.

(4) The strength of this paper (from my perspective) is the potential utility it has for other investigators trying to get mice to do behavioral tasks. However, not enough information was provided about the construction of the boxes, interface, and code for running the boxes. If the authors are not willing to provide this information through eLife, GitHub, or their own website then my evaluation of the impact and significance of this paper would go down significantly.

Minor concerns:

Learning rate is confusing for Figure 3 results as it actually refers to trials to reach the criterion, and not the actual rate of learning (e.g., slope).

Reviewer #2 (Public review):

Summary:

This study aimed to determine which spatial factors (conceived broadly as environmental, agronomic and socio-economic) explain greater avian influenza case numbers reported since 2020 (2020--2022) by comparing similar models built with data from the period 2015--2020. The authors have chosen an environmental niche modelling approach, where detected infections are modelled as a function of spatial covariates extracted at the location of each case. These covariates are available over the entire world so that the predictions can be projected back to space in the form of a continuous map.

Strengths:

The authors use boosted regression trees as the main analytical tool, which always feature among the best-performing models for environmental niche models (also known as habitat suitability models). They run replicate sets of the analysis for each of their model targets (wild/domestic x pathogen variant), which can help produce stable predictions. The authors take steps to ameliorate some forms of expected bias in the detection of cases, such as geographic variation in surveillance efforts, and in general more detections near areas of higher human population density.

Weaknesses:

The study is not altogether coherent with respect to time. Data sets for the response (N5H1 or N5Hx case data in domestic or wild birds ) are divided into two periods; 2015--2020, and 2020--2022. Each set is modelled using a common suite of covariates that are not time-varying. That suggests that causation is inferred by virtue of cases being in different geographic areas in those two time periods. Furthermore, important predictors such as chicken density appear to be informed (in the areas of high risk) from census data from before 2010. The possibility for increased surveillance effort *through time* is overlooked, as is the possibility that previously high-burden locations may implement practice changes to reduce vulnerability.

Reviewer #2 (Public review):

Summary:

The authors exposed naïve male flies to different groups of females, either mated or virgin. Male flies can successfully copulate with virgin females; however, they are rejected by mated females. This rejection reduces sugar preference and sensitivity in males. Investigating the underlying neural circuits, the authors show that dopamine signaling onto GR5a sensory neurons is required for reduced sugar preference. GR5a sensory neurons respond less to sugar exposure when they lack dopamine receptors.

Strengths:

The findings add another strong phenotype to the existing dataset about brain-wide neuromodulatory effects of mating. The authors use several state-of-the-art methods, such as activity-dependent GRASP to decipher the underlying neural circuitry. They further perform rigorous behavioral tests and provide convincing evidence for the local labellar circuit.

Weaknesses:

The authors focus on the circuit connection between dopamine and gustatory sensory neurons in the male SEZ. Therefore, it is still unknown how mating modulates dopamine signaling and what possible implications on other behaviors might result from a reduced sugar preference.

Reviewer #2 (Public review):

Summary:

In this manuscript, Krishnan et al devised three paradigms to perform contextual fear conditioning in head-fixed mice. Each of the paradigms relied on head-fixed mice running on a treadmill through virtual reality arenas. The authors tested the validity of three versions of the paradigms by using various parameters. As described below, I think there are several issues with the way the paradigms are designed and how the data are interpreted. Moreover, as Paradigm 3 was published previously in a study by the same group, it is unclear to me what this manuscript offers beyond the validations of parameters used for the previous publication. Below, I list my concerns point-by-point, which I believe need to be addressed to strengthen the manuscript.

Major comments

(1) In the analysis using the LME model (Tables 1 and 2), I am left wondering why the mice had increased freezing across recall days as well as increased generalization (increased freezing to the familiar context, where shock was never delivered). Would the authors expect freezing to decrease across recall days, since repeated exposure to the shock context should drive some extinction? This is complicated by the analysis showing that freeing was increased only on retrieval day 1 when analyzing data from the first lap only. Since reward (e.g., motivation to run) is removed during the conditioning and retrieval tests, I wonder if what the authors are observing is related to decreased motivation to perform the task (mice will just sit, immobile, not necessarily freezing per se). I think that these aspects need to be teased out.

(2) Related to point 1, the authors actually point out that these changes could be due to the loss of the water reward. So, in line 304, is it appropriate to call this freezing? I think it will be very important for the authors to exactly define and delineate what they consider as freezing in this task, versus mice just simply sitting around, immobile, and taking a break from performing the task when they realize there is no reward at the end.

(3) In the second paradigm, mice are exposed to both novel and (at the time before conditioning) neutral environments just before fear conditioning. There is a big chance that the mice are 'linking' the memories (Cai et al 2016) of the two contexts such that there is no difference in freezing in the shock context compared to the neutral context, which is what the authors observe (Lines 333-335). The experiment should be repeated such that exposure to the contexts does not occur on the conditioning day.

(4) On lines 360-361, the authors conclude that extinction happens rapidly, within the first lap of the VR trial. To my understanding, that would mean that extinction would happen within the first 5-10 seconds of the test (according to Figure S1E). That seems far too fast for extinction to occur, as this never occurs in freely behaving mice this quickly.

(5) Throughout the different paradigms, the authors are using different shock intensities. This can lead to differences in fear memory encoding as well as in levels of fear memory generalization. I don't think that comparisons can be made across the different paradigms as too many variables (including shock intensity - 0.5/0.6mA can be very different from 1.0 mA) are different. How can the authors pinpoint which works best? Indeed, they find Paradigm 3 'works' better than Paradigm 2 because mice discriminate better between the neutral and shock contexts. This can definitely be driven by decreased generalization from using a 0.6mA shock in Paradigm 3 compared to 1.0 mA shock in Paradigm 2.

(6) There are some differences in the calcium imaging dataset compared to other studies, and the authors should perform additional testing to determine why. This will be integral to validating their head-fixed paradigm(s) and showing they are useful for modeling circuit dynamics/behaviors observed in freely behaving mice. Moreover, the sample size (number of mice) seems low.

(7) It appears that the authors have already published a paper using Paradigm 3 (Ratigan et al 2023). If they already found a paradigm that is published and works, it is unclear to me what the current manuscript offers beyond that initial manuscript.

(8) As written, the manuscript is really difficult to follow with the averages and standard error reported throughout the text. This reporting in the text occurred heterogeneously throughout the text, as sometimes it was reported and other times it was not. Cleaning this reporting up throughout the paper would greatly improve the flow of the text and qualitative description of the results.

Reviewer #2 (Public review):

Summary:

This manuscript uses a recently published oxytocin receptor null prairie vole line to examine the effects of this mutation on pair bonding behavior and PVN gene expression. Results reveal that Oxtr sex specifically influences early courtship behavior and partner preference formation as well as suppressing promiscuity toward novel potential mates. PVN gene expression varies between Oxtr null and WT prairie voles.

Strengths:

Behavioral analyses extend beyond the typical reporting of frequency and duration. The gene expression models and analyses are well-done and convincing. The experimental designs and approaches are strong.

Weaknesses:

More details and background literature explaining the role of the Oxt system in pair bonding behaviors is necessary, particularly for the Introduction. The authors overstate several times that Oxtr expression is not necessary for partner preference formation, based on their previous findings. However, it does appear, particularly, in the short cohabitation that it is necessary. Thus, the nuanced answer may be that Oxt may accelerate partner preference formation. Improving the presentation of the statistics and figures will make the manuscript more reader-friendly.

Reviewer #2 (Public review):

Summary:

The authors developed a deep learning model based on a DenseNet CNN architecture to predict two cognitive functions: working memory and episodic memory, from functional connectivity matrices. These matrices were recorded under three conditions: during rest, a working memory task, and a movie, and were treated as images for the CNN algorithm. They tested their model's performance across different conditions and a separate dataset with a different age distribution (using the same MRI scanner, scanning configurations, and cognitive tests). They also calculated the "brain cognition gap" based on the model trained on resting functional connectivity to predict working memory. Extending from the commonly used index "brain age," the brain cognition gap was defined as the difference between the working memory score predicted by their model (predicted working memory) and the working memory score based on the working memory test itself (observed working memory). This brain cognition gap was found to be associated with physical activity, education, and cardiovascular risk. The authors also conducted additional mediation tests to examine whether regional functional variability mediated the relationship between PET-derived measures of dopamine and the brain cognition gap.

Strengths:

The major strength of this manuscript is the extensive effort the authors have put into creating a new 'biomarker' that links deep learning with fMRI, PET, physical activity, education, and cardiovascular risk across two studies. This effort is impressive.

Weaknesses:

There are several weaknesses in the current methods and results, making many of the claims unconvincing. These weaknesses include:

(1) The lack of baseline models to benchmark the predictive performance of their DenseNet models.

(2) The inappropriate calculation of the brain cognition gap due to the lack of control for regression-toward-the-mean and the influence of the working memory itself (a common practice in brain age studies).

(3) The lack of benchmarking of the brain cognition gap against the 'corrected' brain age gap and the direct prediction of physical activity, education, and cardiovascular risk.

(4) Minimal justification for their PET mediation analysis.

Regarding the impact of the work on the field and the utility of the methods and data to the community, I see its potential. However, addressing all the weaknesses listed above is crucial and likely to change the conclusions of the results.

It is important to note that many statements in the manuscript are overstated, making the contribution of the manuscript seem exaggerated.

For instance, the abstract claims "there is a lack of objective biomarkers to accurately predict cognitive function," and the discussion states, "across various studies, the correlation between predicted and actual fluid intelligence typically hovers around 0.25 (98-100)." However, a meta-analysis by Vieira and colleagues (2022 https://doi.org/10.1016/j.intell.2022.101654) found over 37 studies up to 2020 predicting cognitive abilities from fMRI with machine learning, with 24 studies published in 2019-20 alone. Since 2020, with the rise of machine learning and AI, even more studies have likely been published on this topic, all claiming to show objective biomarkers to accurately predict cognitive function. Vieira and colleagues also found an average performance of these objective biomarkers in predicting general cognition at r = .42, similar to what was found in this manuscript. Based on this alone, it is unclear how novel or superior their method is without a proper systematic benchmark.

Similarly, the authors claim superior performance of deep learning and mischaracterize machine learning algorithms: "In particular, deep neural networks (DNN) methods have been successfully applied to behavioral and disease prediction (24-26), and have been found to outperform other machine learning approaches (27-29)," and "Deep learning approaches overcome the limitation of predictive techniques that solely rely on linear associations between connectivity and behavioral phenotypes (17)." However, the superiority of deep learning is debatable. Studies show comparable performance between machine learning (such as kernel regression) and deep learning (such as fully-connected neural networks, BrainNetCNN, Graph CNN (GCNN), and temporal CNN), e.g., He and colleagues (2019) and Vieira and colleagues (2024) https://doi.org/10.1016/j.neuroimage.2019.116276 and Vieira and colleagues' https://doi.org/10.1101/2024.03.07.583858.

Moreover, many non-deep learning predictive techniques are non-linear, e.g., XGBoost, CatBoost, random forest, kernel ridge, and support vector regression with non-linear kernels (such as RBF and polynomial). Thus, stating that machine learning can only model linear relationships is incorrect. Moreover, for the small amount of data the authors had, some might argue that a linear algorithm might be more appropriate to balance the bias-variance trade-off in prediction. Again, without a proper systematic benchmark, it is unclear how well their DenseNet algorithm performs compared to other algorithms.

Regarding the Brain Age literature, the authors also misinterpreted recent findings: "However, a recent study suggests that brain age predictions contribute minimally compared to chronological age for explaining cognitive decline (65), implying that cognitive predictions are more reliable." In this study, Tetereva and colleagues (2024) (https://doi.org/10.7554/eLife.87297.4) showed that non-deep-learning machine learning can make good predictions from MRI on both chronological age (with r up to .88) and fluid cognition (with r up to .627). Using the combination of functional connectivity matrices across rest and tasks to predict fluid cognition, they found performance at r = .565, comparable to what was found in the current manuscript with deep learning. Nonetheless, while brain age predicted chronological age well (and brain cognition predicted fluid cognition well), it was problematic to predict fluid cognition from brain age. They showed that, because brain age, by design, shared so much common variance with chronological age, brain age and chronological age captured the same variance of fluid cognition. When chronological age was controlled for in the prediction of fluid cognition, brain age no longer had high predictive ability. In the case of the current manuscript, the brain cognition gap is not appropriately controlled for cognition (to be more precise, a working memory score). I expect the performance in predicting physical activity, education, and cardiovascular risk will drop dramatically once cognition is controlled for. There are at least two ways to control cognition according to Tetereva and colleagues' study (see more in the recommendations).

The authors mentioned, "The third aim of the current study is to uncover the contribution of dopamine (DA) integrity to brain-cognition gaps." However, I fail to see how mediation analysis would test this. The authors also mentioned, "Insufficient DA modulation can affect neurocognitive functions detrimentally (69, 74, 76-78)." They should test if DA levels are related to working memory scores in their study, and if so, whether the relationship is mediated by the "corrected" brain-cognition gaps. Note see more on the recommendation for the calculation of the "corrected" brain-cognition gaps.

Reviewer #2 (Public review):

Summary:

A rapid recovery of the ecosystems during the late Early Triassic, in the aftermath of the end-Permian mass extinction, is discussed based on different types of fossils.

Strengths:

The combined study of invertebrate trace fossils, tetrapod bones, and plant remains together with their stratigraphic distribution in different sections provides a convincing case to support a rapid recovery as the authors hypothesize.

Weaknesses:

The study is based on three regions with Triassic successions from the North China block. While a first-hand study of other localities of similar age would be ideal, this is of course a difficult task. Instead, the authors provide comparisons with other worldwide regions to build their case and support the initial hypothesis.

Reviewer #2 (Public review):

Summary:

Dr. Adam Kim and collaborators study the changes in chromatin structure in monocytes obtained from alcohol-associated hepatitis (AH) when compared to healthy controls (HC). Through the usage of high throughput chromatin conformation capture technology (Hi-C), they collected data on contact frequencies between both contiguous and distal DNA windows (100 kB each); mainly within the same chromosome. From the analyses of those data in the two cohorts under analysis, authors describe frequent pairs of regions subject to significant changes in contact frequency across cohorts. Their accumulation onto specific regions of the genome -referred to as hotspots- motivated authors to narrow down their analyses to these disease-associated regions, in many of which, authors claim, a number of key innate immune genes can be found. Ultimately, the authors try to draw a link between the changes observed in chromatin architecture in some of these hotspots and the differential co-expression of the genes lying within those regions, as ascertained in previous single-cell transcriptomic analyses.

Strengths:

The main strength of this paper lies in the generation of Hi-C data from patients, a valuable asset that, as the authors emphasize, offers critical insights into the role of chromatin architecture dysregulation in the pathogenesis of alcohol-associated hepatitis (AH). If confirmed, the reported findings have the potential to highlight an important, yet overlooked, aspect of cellular dysregulation-chromatin conformation changes - not only in AH but potentially in other immune-related conditions with a component of pathological inflammation.

Weaknesses:

In what I regard as the two most important weaknesses of the work, I feel that they are more methodological than conceptual. The first of these issues concerns the perhaps insufficient level of description provided on the definition of some key types of genomic regions, such as topologically associated domains, DNA hotspots, or even DNA loci showing significant changes in contact frequency between AH and HC. In spite of the importance of these concepts in the paper, no operational, explicit description of how are they defined, from a statistical point of view, is provided in the current version of the manuscript.

Without these definitions, some of the claims that authors make in their work become hard to sustain. Some examples are the claim that randomizing samples does not lead to significant differences between cohorts; the claim that most of the changes in contact frequency happen locally; or the claim that most changes do not alter the structure of TADs, but appear either within, or between TADs. In my viewpoint, specific descriptions and implementation of proper tests to check these hypotheses and back up the mentioned specific claims, along with the inclusion of explicit results on these matters, would contribute very significantly to strengthening the overall message of the paper.

The second notable weakness of the study pertains to the characterization of the changes observed around immune genes in relation to genome-wide expectations. Although the authors suggest that certain hotspots contain a high number of immune-related genes, no enrichment analysis is provided to verify whether these regions indeed harbor a higher concentration of such genes compared to other genomic areas. It would be important for readers to be promptly informed if no such enrichment is observed, for in that case, the presence of some immune genes within these hotspots would carry more limited implications.

Additionally, the criteria used to define a hotspot are not clearly outlined, making it difficult to assess whether the changes in contact frequencies around the immune genes highlighted in figures 5-8 are truly more pronounced than what would be expected genome-wide.

Reviewer #2 (Public review):

Summary:

This study examines the role of beta oscillations in motor control, particularly during rapid changes in movement direction among patients with Parkinson's disease. The researchers utilized magnetoencephalography (MEG) and local field potential (LFP) recordings from the subthalamic nucleus to investigate variations in beta band activity within the cortex and STN during the initiation, cessation, and reversal of movements, as well as the impact of external cue predictability on these dynamics. The primary finding indicates that beta oscillations more effectively signify the start and end of motor sequences than transitions within those sequences. The article is well-written, clear, and concise.

Strengths:

The use of a continuous motion paradigm with rapid reversals extends the understanding of beta oscillations in motor control beyond simple tasks. It offers a comprehensive perspective on subthalamo-cortical interactions by combining MEG and LFP.

Comments on revisions: I am satisfied with the revisions. I do not have further comments on the revised manuscript.

Reviewer #2 (Public review):

This manuscript builds on the authors' earlier work, most recently Wong et al. 2019, in which they showed the importance of the perirhinal cortex (PRh) during the first-order conditioning stage of sensory preconditioning. Sensory preconditioning requires learning between two neutral stimuli (S2-S1) and subsequent development of a conditioned response to one of the neutral stimuli after pairing of the other stimulus with a motivationally relevant unconditioned stimulus (S1-US). One highly debated question regarding the mechanisms of learning of sensory preconditioning has been whether conditioned responses evoked by the indirectly trained stimulus (S2) occur through a mediated representation at the time of the first-order US training, or whether the conditioned responses develop through a chained evoked representation (S2--> S1 --> US) at the time of test. The authors' prior findings provided strong evidence for PRh being involved in mediated learning during the first-order training. They showed that protein synthesis was required during the first-order S1-US learning to support the conditioned response to the indirectly trained stimulus (S2) at test.

One question remaining following the previous paper was whether certain conditions may promote a chaining mechanism over mediated learning, as there is some evidence for chained representations at the time of test. In this paper, the authors directly address this important question and find unambiguous results that the extent of training during the preconditioning stage impacts the involvement of PRh during the first-order conditioning or stage 2. They show that putative blockade of synaptic changes in PRh, using an NMDA antagonist, disrupts responding to the preconditioned cue at test during shorter duration preconditioning training (8 trials), but not during extended training (32 trials). They also show that this is the case for communication between the PRh and BLA during the same stage of training using a contralateral inactivation approach. This confirms their previous findings in 2019 of connectivity between these regions for the short duration training, while they observe here for the first time that this is not the case for extended training. Finally, they show that with extended training, communication between BLA and the PRh is required at the final test of the preconditioned stimulus, but not for the short duration training.

Strengths:

The results are clear and extremely consistent across experiments within this paper as well as with earlier work. The experiments here are thorough, well-conceived, and address an important and highly debated question in the field regarding the neural and psychological mechanisms underlying sensory preconditioning. This work is highly impactful for the field as the debate over mediated versus chaining mechanisms has been an important topic for more than 70 years.

Comments on revisions:

Thank you for addressing all of my concerns in considerable detail. I have no more suggestions for the authors. This is a fantastic paper both in the experimental design and the execution as well as in the high quality of writing.

Reviewer #2 (Public review):

Summary:

This well-written manuscript addresses an important but recalcitrant problem - molecular mechanism of protein misfolding in Ig light chain (LC) amyloidosis (AL), a major life-threatening form of systemic human amyloidosis. The authors use expertly recorded and analyzed small-angle X-ray scattering (SAXS) data as a restraint for molecular dynamics simulations (called M&M). Six patient-based LC proteins are explored, including four AL and two non-AL. The authors report a partially populated "H-state" determined computationally, wherein the two domains in an LC molecule acquire a straight rather than bent conformation, with an extended interdomain linker; this H-state distinguishes AL from non-AL LCs. H-D exchange mass spectrometry is used to support this conclusion. This is a novel and interesting finding with potentially important translational implications.

Strengths:

Expertly recorded and analyzed SAXS data combined with clever M&M simulations lead to a novel and interesting conclusion, which is supported by limited H-D exchange data.<br /> Stabilization of the CL-CL interface is a good idea that may help protect a subset of AL LCs from misfolding in amyloid.

Computational M&M evidence is convincing and is supported by SAXS data, which are used as restraints for simulations. Although Kratky plots reported in the main MS Fig. 1 show significant differences between the data and the structural model for only one AL protein, AL-55, H-state is also inferred for other AL proteins.

Apparent limitations:

HDX MS results show that residues 35-50 from VL-VL and VL-CL dimerization interface are less protected in AL vs. non-AL proteins, which is consistent with the H-state. However, the small number of proteins yielding useful HDX data (three AL and one non-AL) suggests that this conclusion should be treated with caution. It is unclear whether the conformational heterogeneity depicted in M&M simulations is consistent with HDX results, and whether prior HDX studies of AL and MM LCs are consistent with the conclusions that a particular domain-domain interface is weakened in AL vs. non-AL LCs. The butterfly plots in Fig. 5 could benefit from the X-axis labeling with the peptide fragments.

Reviewer #2 (Public review):

Summary:

In this set of studies, authors identify cFos activation in neurons in female mice that mated with males, and after experiencing male sexual behavior that is either restricted to appetitive behavior or including ejaculation. The medial preoptic nucleus was identified as an area with high cFos induction following ejaculation. Characterization of neurochemical phenotypes of cfos-expressing neurons showed a heterogenous distribution of activated neurons in the MPOA, including both inhibitory and excitatory cell types. Next, in vivo calcium imaging was used to show activation of Vgat and Vglut neurons in female mice MPOA after displaying sniffing of the male, experiencing male appetitive, or male consummatory sexual behavior, demonstrating significantly higher activation and of a greater subpopulation of Vgat neurons than Vglut neurons. Moreover, greatest activation of Vgat neurons was detected following experiencing ejaculation, and ejaculation activated different subpopulations of MPOA cells than consummatory or appetitive sexual behaviors experienced by the female. Finally, pharmaco-genetic activation of the subpopulation of MPOA neurons that were previously activated following ejaculation resulted in a significant reduction of approach behavior by the female mice towards the male, interpreted as suppression of female sexual motivation. In conclusion, a subpopulation of inhibitory cells in the MPOA is activated in female mice after experiencing ejaculation, in turn contributing to suppression of sexual approach behavior.

Strengths:

The current set of studies replicates previous findings that ejaculation causes longer latencies to initiate interactions with a male after receiving an ejaculation in a paced mating paradigm, which is widely validated and extensively used to investigate sexual behavior in female rodents. Studies also confirm that ejaculation increases cFos expression in the MPOA, while extending prior findings with a careful analysis of the neurochemical phenotype of activated neurons. A major strength of the studies is the use of cell-specific in vivo imaging and pharmaco-genetic activation to reveal a functional role of specific neuronal ensemble within the MPOA for post ejaculatory female sexual behavior.

Weaknesses:

The authors include an elegant manipulation of ejaculation-activated neurons in the MPOA using DREADD. However, this study was limited to show that activation of previously activated cells was sufficient to reduce approach behavior in a paced mating paradigm and receiving intromissions in a home cage mating paradigm. An inhibition approach using DREADD would have been a great complement to this study as it would have examined if activation of the cells was required. Moreover, additional tests for sexual motivation would have greatly strengthened the overall conclusions.

Reviewer #2 (Public review):

In this study, Wang et al., report the significance of XAP5L and XAP5 in spermatogenesis which are involved in transcriptional regulation of the ciliary gene in testes. In a previous study, the authors demonstrated that XAP5 is a transcription factor required for flagellar assembly in Chlamydomonas. Continuing from their previous study, the authors examined conserved role of the XAP5 and XAP5L, which are the orthologue pair in mammals.

XAP5 and XAP5L express ubiquitously and testis specifically, respectively, and their absence in testes causes male infertility with defective spermatogenesis. Interestingly, XAP5 deficiency arrest germ cell development at pachytene stage, whereas XAP5L absence causes impaired flagellar formation. RNA-seq analyses demonstrated that XAP5 deficiency suppresses ciliary gene expression including Foxj1 and Rfx family genes in early testis. By contrast, XAP5L deficiency abnormally remains Foxj1 and Rfx genes in mature sperm. From the results, the authors conclude that XAP5 and XAP5L are the antagonistic transcription factor to function at the upstream of Foxj1 and Rfx family genes.

The current version of the manuscript well represents this reviewer's initial concerns and supports author's claim. Key transcription factors for ciliogenesis, Foxj1 and Rfx2, are direct downstream targets for XAP5 and XAP5L and their common motifs well explain their antagonistic function in sperm flagellar development. All the results well demonstrate that ancient transcription factors, XAP5 and XAP5L, are upstream transcription factors to modulate flagellar development in male mammalian germ line.

Reviewer #2 (Public review):

Summary:

The Authors utilize biochemical approaches to determine and validate NRL protein-protein interactions to further understand the mechanisms by which the NRL transcription factor controls rod photoreceptor gene regulatory networks. Observations that NRL displays numerous protein-protein interactions with RNA-binding proteins, many of which are involved in R-loop biology, led the authors to investigate the role of RNA and R-loops in mediating protein-protein interactions and profile the co-localization of R-loops with NRL genomic occupancy.

Strengths:

Overall, the manuscript is well-written, providing succinct explanations of the observed results and potential implications. Additionally, the Authors use multiple orthogonal techniques and tissue samples to reproduce and validate that NRL interacts with DHX9 and DDX5. Experiments also utilize specific assays to understand the influence of RNA and R-loops on protein-protein interactions. The Authors also use state-of-the-art techniques to profile R-loop localization within the retina and integrate multiple previously established datasets to correlate R-loop presence with transcription factor binding and chromatin marks an attempt to understand the significance of R-loops in the retina.

Weaknesses:

In general, the Authors provide interpretations of the data that fit a narrative about NRL and the perceived significance of interactions with RNA binding proteins. Large-scale screens for NRL protein interactions were conducted but all of the data is not reported. For example, NRL IP-Mass Spec was performed, but the authors only provide interaction/detection data for identified interactions with known RNA binding proteins. We cannot assess the enrichment of interactions or specificity of interactions with RNA binding proteins based on the reported results. Additionally, the lack of experiments testing the functional significance of Nrl interactions with R-loops within the developing retina fails to provide novel biological insights into the regulation of gene regulatory networks. While this provides additional avenues for research in the future, it is unclear that NRL interaction with R-loops have physiological relevance for photoreceptor health or function.

Reviewer #2 (Public review):

Summary:

Generating biophysically detailed computational models that capture the characteristic physiological properties of biological neurons for diverse cell types is an important and difficult problem in computational neuroscience. One major challenge lies in determining the large number of parameters of such models, which are notoriously difficult to fit to experimental data. Thereby, the computational and energy costs can be significant. The study 'ElectroPhysiomeGAN: Generation of Biophysical Neuron Model Parameters from Recorded Electrophysiological Responses' by Kim et al. describes a computationally efficient approach for predicting model parameters of Hodgkin-Huxley neuron models using Generative Adversarial Networks (GANs) trained on simulation data. The method is applied to generate models for 9 non-spiking neurons in C. elegans based on electrophysiological recordings. While the generated models capture the responses of these neurons to some degree, they generally show significant deviations from the empirically observed responses in important features. Although EP-GAN shows clear benefits under limited compute, the results do not yet demonstrate the quality needed to match other state-of-the-art methods. Future work examining extended training, larger datasets, or hybrid approaches would help clarify whether EP-GAN can generate models of high quality. If so, this would indeed be a major step forward; if not, the computationally more expensive methods will remain essential.

Strengths:

The authors work on an important and difficult problem. A noteworthy strength of their approach is that once trained, the GANs can generate models from new empirical data with very little computational effort. The generated models reproduce the response to current injections reasonably well.

Weaknesses:

Major 1: Models do not faithfully capture empirical responses. While the models generated with EP-GAN reproduce the average voltage during current injections reasonably well, the dynamics of the response are generally not well captured. For example, for the neuron labeled RIM (Figure 2), the most depolarized voltage traces show an initial 'overshoot' of depolarization, i.e. they depolarize strongly within the first few hundred milliseconds but then fall back to a less depolarized membrane potential. In contrast, the empirical recording shows no such overshoot. Similarly, for the neuron labeled AFD, all empirically recorded traces slowly ramp up over time. In contrast, the simulated traces are mostly flat. Furthermore, all empirical traces return to the pre-stimulus membrane potential, but many of the simulated voltage traces remain significantly depolarized, far outside of the ranges of empirically observed membrane potentials. The authors trained an additional GAN (EP-GAN Extended) to improve the fit to the resting membrane potential. Interestingly, for one neuron (AWB), this improved the response during stimulation, which now reproduced the slowly raising membrane potentials observed empirically, however, the neuron still does not reliably return to its resting membrane potential. For the other two neurons, the authors report a decrease in accuracy in comparison to EP-GAN. While such deviations may appear small in the Root mean Square Error (RMSE), they likely indicate a large mismatch between the model and the electrophysiological properties of the biological neuron. The authors added a second metric during the revision - percentages of predicted membrane potential trajectories within empirical range. I appreciate this additional analysis. As the empirical ranges across neurons are far larger than the magnitude of dynamical properties of the response ('slow ramps', etc.), this metric doesn't seem to be well suited to quantify to which degree these dynamical properties are captured by the models.

Major 2: Comparison with other approaches is potentially misleading. Throughout the manuscript, the authors claim that their approach outperforms the other approaches tested. But compare the responses of the models in the present manuscript (neurons RIM, AFD, AIY) to the ones provided for the same neurons in Naudin et al. 2022 (https://doi.org/10.1371/journal. pone.0268380). Naudin et al. present models that seem to match empirical data far more accurately than any model presented in the current study. Naudin et al. achieved this using DEMO, an algorithm that in the present manuscript is consistently shown to be among the worst of all algorithms tested. I therefore strongly disagree with the authors claim that a "Comparison of EP-GAN with existing estimation methods shows EP-GAN advantage in the accuracy of estimated parameters". This may be true in the context of the benchmark performed in the study (i.e., a condition of very limited compute resources - 18 generations with a population size of 600, compare that to 2000 generations recommended in Naudin et al.), but while EP-GAN wins under these specific conditions (and yes, here the authors convincingly show that their EP-GAN produces by far the best results!), other approaches seem to win with respect to the quality of the models they can ultimately generate.

Major 3: As long as the quality of the models generated by the EP-GAN cannot be significantly improved, I am doubtful that it indeed can contribute to the 'ElectroPhysiome', as it seems likely that dynamics that are currently poorly captured, like slow ramps, or the ability of the neuron to return to its resting membrane potential, will critically affect network computations. If the authors want to motivate their study based on this very ambitious goal, they should illustrate that single neuron model generation with their approach is robust enough to warrant well-constrained network dynamics. Based on the currently presented results, I find the framing of the manuscript far too bold.

Major 4: The conclusion of the ablation study 'In addition the architecture of EP-GAN permits inference of parameters even when partial membrane potential and steady-state currents profile are given as inputs' does not seem to be justified given the voltage traces shown in Figure 3. For example, for RIM, the resting membrane potential stays around 0 mV, but all empirical traces are around -40mV. For AFD, all simulated traces have a negative slope during the depolarizing stimuli, but a positive slope in all empirically observed traces. For AIY, the shape of hyperpolarized traces is off. While it may be that by their metric neurons in the 25% category are classified as 'preserving baseline accuracy', this doesn't seem justified given the voltage traces presented in the manuscript. It appears the metric is not strict enough.

Reviewer #2 (Public review):

I thank the authors for their efforts in the revision. In general, I believe the main conclusion that Rab3A is required for TTX-induced homeostatic synaptic plasticity is well-supported by the data presented, and this is an important addition to the repertoire of molecular players involved in homeostatic compensations. I also acknowledge that the authors are more cautious in making conclusions based on the current evidence, and the structure and logic have been much improved.

The only major concern I have still falls on the interpretation of the mismatch between GluA2 cluster size and mEPSC amplitude. The authors argue that they are only trying to say that changes in the cluster size are more variable than those in the mEPSC amplitude, and they provide multiple explanations for this mismatch. It seems incongruous to state that the simplest explanation is a presynaptic factor when you have all these alternative factors that very likely have contributed to the results. Further, the authors speculate in the discussion that Rab3A does not regulate postsynaptic GluA2 but instead regulates a presynaptic contributor. Do the authors mean that, in their model, the mEPSC amplitude increases can be attributed to two factors- postsynaptic GluA2 regulation and a presynaptic contribution (which is regulated by Rab3A)? If so, and Rab3A does not affect GluA2 whatsoever, shouldn't we see GluA2 increase even in the absence of Rab3A? The data in Table 1 seems to indicate otherwise.

I also question the way the data are presented in Figure 5. The authors first compare 3 cultures and then 5 cultures altogether, if these experiments are all aimed to answer the same research question, then they should be pooled together. Interestingly, the additional two cultures both show increases in GluA2 clusters, which makes the decrease in culture #3 even more perplexing, for which the authors comment in line 261 that this is due to other factors. Shouldn't this be an indicator that something unusual has happened in this culture? Data in this figure is sufficient to support that GluA2 increases are variable across cultures, which hardly adds anything new to the paper or to the field. The authors further cite a study with comparable sample sizes, which shows a similar mismatch based on p values (Xu and Pozzo-Miller 2007), yet the effect sizes in this study actually match quite well (both ~160%). P values cannot be used to show whether two effects match, but effect sizes can. Therefore, the statement in lines 411-413 "... consistently leads to an increase in mEPSC amplitudes, and sometimes leads to an increase in synaptic GluA2 receptor cluster size" is not very convincing, and can hardly be used to support "the idea that there are additional sources contributing to the homeostatic increase in quantal size".

I would suggest simply showing mEPSC and immunostaining data from all cultures in this experiment as additional evidence for homeostatic synaptic plasticity in WT cultures, and leave out the argument for "mismatch". The presynaptic location of Rab3A is sufficient to speculate a presynaptic regulation of this form of homeostatic compensation.

Minor concerns:

(1) Line 214, I see the authors cite literature to argue that GluA2 can form homomers and can conduct currents. While GluA2 subunits edited at the Q/R site (they are in nature) can form homomers with very low efficiency in exogenous systems such as HEK293 cells (as done in the cited studies), it's unlikely for this to happen in neurons (they can hardly traffick to synapses if possible at all).

(2) Lines 221-222, the authors may have misinterpreted the results in Turrigiano 1998. This study does not show that the increase in receptors is most dramatic in the apical dendrite, in fact, this is the only region they have tested. The results in Figures 3b-c show that the effect size is independent of the distance from soma.

(3) Lines 309-310 (and other places mentioning TNFa), the addition of TNFa to this experiment seems out of place. The authors have not performed any experiment to validate the presence/absence of TNFa in their system (citing only 1 study from another lab is insufficient). Although it's convincing that glia Rab3A is not required for homeostatic plasticity here, the data does not suggest Rab3A's role (or the lack of) for TNFa in this process.

Reviewer #2 (Public review):

In their manuscript entitled "BEND2 is a crucial player in oogenesis and reproductive aging", the authors present their findings that full-length BEND2 is important for repair of meiotic double strand break repair in spermatocytes, regulation of LINE-1 elements in spermatocytes, and proper oocyte meiosis and folliculogenesis in females. The manuscript utilizes an elegant system to specifically ablate the full-length form of BEND2 which has been historically difficult to study due to its location on the X chromosome and male sterility of global knockout animals.

The authors have been extremely responsive to reviewer critiques and have presented strong data and appropriate conclusions, making it an excellent addition to the field.

Reviewer #2 (Public review):

The FGF receptor Heartless has previously been implicated in Drosophila peripheral glial growth and axonal wrapping. Here, the authors perform a large-scale screen of over 2600 RNAi lines to find factors that control the downstream signaling in this process. They identify a transmembrane protein Uninflatable to be necessary for the formation of plasma membrane domains. They further find that a Uif regulatory target, Notch, is necessary for glial wrapping. Interestingly, additional evidence suggests Notch itself regulates uif and htl, suggesting a feedback system. Together, they propose that Uif functions as a "switch" to regulate the balance between glial growl and wrapping of axons.

Little is known about how glial cell properties are coordinated with axons, and the identification of Uif is a promising link to shed light on this orchestration. The manuscript is well-written, and the experiments are generally well-controlled. The EM studies in particular are of outstanding quality and really help to mechanistically dissect the consequences of Uif and Notch signaling in the regulation of glial processes. Together, this valuable study provides convincing evidence of a new player coordinating the interactions controlling the glial wrapping of axons.

Reviewer #2 (Public review):

Summary:

Brooks et al. generate a compelling gene expression atlas of the early embryonic cranial neural plate. They generate single-cell transcriptome data from early cranial neural plate cells at 6 consecutive stages between E7.5 to E9. Utilizing computational analysis they infer temporal gene expression dynamics and spatial gene expression patterns along the anterior-posterior and mediolateral axis of the neural plate. Subsequent comparison with known gene expression patterns revealed a good agreement with their inferred patterns, thus validating their approach. They then focus on Sonic Hedgehog (Shh) signalling, a key morphogen signal, whose activities partition the neural plate into distinct gene expression domains along the mediolateral axis. Single-cell transcriptome analysis of embryos in which the Shh pathway was pharmacologically activated throughout the neural plate revealed characteristic changes in gene expression along the mediolateral axis and the induction of distinct Shh regulated gene expression programs in the developing fore-, mid- and hindbrain.

Strengths:

This manuscript provides a comprehensive transcriptomic characterisation of the developing cranial neural plate, a part of the embryo that to my knowledge has not been extensively analysed by single-cell transcriptomic approaches. The single-cell sequencing data appears to be of high quality and will be a great resource for the wider scientific community. Moreover, the computational analysis is well executed and the validation of the sequencing data using published gene expression patterns is convincing. In my opinion the authors completely achieved their aim of generating a reliable sequencing atlas of the early cranial neural plate. Conceptually, the findings that gene expression patterns differ along the rostrocaudal, mediolateral and temporal axes of the neural plate and that Shh signalling induces distinct target genes along the anterior-posterior axis of the nervous system are not completely unexpected. However, the comprehensive characterization of the spatiotemporal gene expression patterns and how they change upon ectopic activation of the Shh pathway will definitely contribute to a better understanding of neural plate patterning. Taken together, this is a well-executed study that describes a relevant scientific resource that will likely be of great use for the wider scientific community .

Weaknesses:

No weaknesses were identified.

Reviewer #3 (Public review):

Summary:

In this study, the authors analyzed the complex functional organization of the hippocampus using two separate adult lifespan datasets. They investigated how individual variations in the detailed connectivity patterns within the hippocampus relate to behavioral and molecular traits. The findings confirm three overlapping hippocampal gradients and reveal that each is linked to established functional patterns in the cortex, the arrangement of dopamine receptors within the hippocampus, and differences in memory abilities among individuals. By employing multivariate data analysis techniques, they identified older adults who display a hippocampal gradient pattern resembling that of younger individuals and exhibit better memory performance compared to their age-matched peers. This underscores the behavioral importance of maintaining a specific functional organization within the hippocampus as people age.

Strengths:

The evidence supporting the conclusions is compelling, based on a unique dataset, a rich set of carefully unpacked results, and a rigorous data analysis that is clearly explained and motivated. Possible confounds are carefully considered and ruled out.

Assessment after revision:

The authors improved the transparency of the statistical analyses by stating explicitly what tests and corrections were performed and clearly justifying the elected statistical approaches. They now also acknowledge and discuss the potential limitations of the presented PET analyses. Overall this is a rigorous and important contribution to the literature that will likely be of broad interest to basic and clinical neuroscience.

Reviewer #2 (Public review):

Summary:

Yue et al. set out to determine if the low but measurable level of DNMT3B expression that is observed prior to the major wave of de novo DNA methylation has a function (ie before the epiblast stage) . Re-analyzing existing DNA methylation data from Smith et al. (2012) they find a very modest DNA methylation gain over a subset of promoters, on the order of 1%, occurring between the 8-cell and blastocyst stages, and refer to this as "minor de novo DNA methylation". They attempt to assess the relevance/functionality of this minor DNA methylation gain, and intriguingly report reduced H3K27me3 in Dnmt3b knockdown (KD) trophoblast cells that normally undergo imprinted X-chromosome inactivation (iXCI) before the blastocyst stage. In addition, they assess proliferation, differentiation, metabolic function, implantation rate and live birth rate of Dnmt3b KD blastocysts, and assign specific phenotypes to the loss of DNA methylation at this early stage..

Strengths:

Working with early embryos is technically demanding and as such the relevance of disrupting epigenetic factors specifically at this stage in development is less well studied. The detailed analyses of published data as well as DNMT3B depletion experiments presented in this manuscript provides food for thought for the epigenetics community.

Weaknesses:

- Throughout the manuscript, please represent DNA methylation changes as delta DNA methylation instead of fold change. In many figures, it is not clear what the unit of DNA methylation presented actually is. Readers should be made aware that the changes in DNA methylation observed are very modest and the threshold applied to the delta in DNA methylation is just 1% "( Δ DNA methylation > 0.01)").<br /> - The minimum coverage threshold and threshold applied For DNA methylation should be presented in each relevant figure. Currently for example, the latter is only mentioned not in the methods section but rather once in "Figure 2, figure supplement 1"<br /> - Indirect effects of disrupting DNMT3B at the earlier stages in development, when de novo DNAme levels are very low in the promoter regions of interest, should be considered. For example, de novo DNA methylation in repetitive regions/pericentric heterochromatin at this stage (not studied here) could be much higher than 1%. Disruption of such methylation, could result in a "sink effect", with loss of H3K27me3 at promoter regions (including on the inactive X-chromosome), due to aberrant repositioning of Polycomb complexes/PRC2 to such ectopic sites from which they are normally excluded, rather than a direct positive effect of the very low DNA methylation gain observed on Polycomb recruitment.<br /> The impact of depletion of DNMT3B on the major wave of de novo DNA methylation that takes place at the peri-implantion stage of embryonic development may also play a role in some of the later phenotypes observed. In other words when the failure of de novo methylation is more profound as levels of DNA methylation are much higher at these later stages as a consequence of DNMT3B activity.

Reviewer #2 (Public review):

Summary:

The authors first conducted whole exome sequencing for infertile male patients and families where they co-segregated the biallelic mutations in the Dynein Axonemal Heavy Chain 12 (DNAH12) gene. Sperm from patients with biallelic DNAH12 mutations exhibited a wide range of morphological abnormalities in both tails and heads, reminiscing a prevalent cause of male infertility, asthenoteratozoospermia. To deepen the mechanistic understanding of DNAH12 in axonemal assembly, the authors generated two distinct DNAH12 knockout mouse lines via CRISPR/Cas9, both of which showed more severe phenotypes than observed in patients. Ultrastructural observations and biochemical studies revealed the requirement of DNAH12 in recruiting other axonemal proteins and that the lack of DNAH12 leads to the aberrant stretching in the manchette structure as early as stage XI-XII. At last, the authors proposed intracytoplasmic sperm injection as a potential measure to rescue patients with DNAH12 mutations, where the knockout sperm culminated in the blastocyst formation with a comparable ratio to that in WT.

Strengths:

The authors convincingly showed the importance of DNAH12 in assembling cilia and flagella in both human and mouse sperm. This study is not a mere enumeration of the phenotypes, but a strong substantiation of DNAH12's essentiality in spermiogenesis, especially in axonemal assembly.

The analyses conducted include basic sperm characterizations (concentration, motility), detailed morphological observations in both testes and sperm (electron microscopy, immunostaining, histology), and biochemical studies (co-immunoprecipitation, mass-spec, computational prediction). Molecular characterizations employing knockout animals and recombinant proteins beautifully proved the interactions with other axonemal proteins.

Many proteins participate in properly organizing flagella, but the exact understanding of the coordination is still far from conclusive. The present study gives the starting point to untangle the direct relationships and order of manifestation of those players underpinning spermatogenesis. Furthermore, comparing flagella and trachea provides a unique perspective that attracts evolutional perspectives.

Weaknesses:

Seemingly minor, but the discrepancies found in patients and genetically modified animals were not fully explained. For example, both knockout mice vastly reduced the count of sperm in the epididymis and the motility, while phenotypes in patients were rather milder. Addressing the differences in the roles that the orthologs play in spermatogenesis would deepen the comprehensive understanding of axonemal assembly.

Comments on revisions:

The reviewer is satisfied with the authors' response.

Reviewer #2 (Public review):

The manuscript investigates oviductal responses to the presence of gametes and embryos using a multi-omics and machine learning-based approach. By applying RNA sequencing (RNA-seq), single-cell RNA sequencing (sc-RNA-seq), and proteomics, the authors identified distinct molecular signatures in different regions of the oviduct, proximal versus distal. The study revealed that sperm presence triggers an inflammatory response in the proximal oviduct, while embryo presence activates metabolic genes that provide nutrients to the developing embryos. Overall, this study offers valuable insights and will likely be of great interest to reproductive biologists and researchers in oviduct biology.

Reviewer #2 (Public review):

This manuscript presents an interesting study of enalapril for its potential impact on senescence through the activation of Smad1/5/9 signaling with a focus on antioxidative gene expression. Repurposing enalapril in this context provides a fresh perspective on its effects beyond blood pressure regulation. The authors make a strong case for the importance of Smad1/5/9 in this process, and the inclusion of both in vitro and in vivo models adds value to the findings. Below, I have a few comments and suggestions which may help improve the manuscript.

A major finding in the study is that phosphorylated Smad1/5/9 mediates the effects of enalapril. However, the manuscript focused on the Smad pathway relatively abruptly, and the rationale behind targeting this specific pathway is not fully explained. What makes Smad1/5/9 particularly relevant to the context of this study?

Furthermore, their finding that activation of Smad1/5/9 leads to a reduction of senescence appears somewhat contradictory to the established literature on Smad1/5/9 in senescence. For instance, studies have shown that BMP4-induced senescence involves the activation of Smad1/5/8 (Smad1/5/9), leading to the upregulation of senescence markers like p16 and p21 (JBC, 2009, 284, 12153). Similarly, phosphorylated Smad1/5/8 has been shown to promote and maintain senescence in Ras-activated cells (PLOS Genetics, 2011, 7, e1002359). Could the authors provide more detailed mechanistic insights into why enalapril seems to reverse the typical pro-senescent role of Smad1/5/9 in their study?

While the authors showed that enalapril increases pSmad1/5/9 phosphorylation, what are the expression levels of other key and related factors like Smad4, pSmad2, pSmad3, BMP2, and BMP4 in both senescent and non-senescent cells? These data will help clarify the broader signaling effects.

They used BMP receptor inhibitor LDN193189 to pharmacologically inhibit BMP signaling, but it would be more convincing to also include genetic validation (e.g., knockdown or knockout of BMP2 or BMP4). This will help confirm that the observed effects are truly due to BMP-Smad signaling and not off-target effects of the pharmacological inhibitor LDN.

I don't see the results on the changes in senescence markers p16 and p21 in the mouse models treated with enalapril. Similarly, the effects of enalapril treatment on some key SASP factors, such as TNF-α, MCP-1, IL-1β, and IL-1α, are missing, particularly in serum and tissues. These are important data to evaluate the effect of enalapril on senescence.

Given that enalapril is primarily known as an antihypertensive, it would be helpful to include data on how it affects blood pressure in the aged mouse models, such as systolic and diastolic blood pressure. This will clarify whether the observed effects are independent of or influenced by changes in blood pressure.

Reviewer #2 (Public review):

Summary:

In this manuscript, authors use a combination of transgenic animals, intersectional viruses, retrograde tracing, and ex-vivo slice electrophysiology to show that VTA projections neurons synapse locally. First, the authors injected a cre-dependent channelrhodopsin into the VTA of PV, SST, MOR, and NTS-Cre mice. Importantly, PV, SST, MOR, and NTS are molecular markers previously used to describe VTA interneurons. Imaging of known VTA target regions identified that these neurons are not localized to the VTA and instead project to the PFC, NAc, VP, and LHb. Next, the authors used an intersectional viral strategy to label projections neurons with both GFP (membrane localized) and Syn:Ruby (release sites). These experiments identified that VTA projection neurons also make intra-VTA synapses. Finally, the authors use a combination of optogenetics and ex-vivo slice electrophysiology to show that neurons projecting from the VTA to the NAc/VP/PFC also synapse locally. Overall, the conclusions are well supported by the data.

Strengths:

Previous literature has described Pvalb, Sst, Oprm1, and Nts as selective markers of VTA interneurons. Here, the authors make use of cre driver lines to show that neurons defined by these genes are not classically-defined interneurons and project to known VTA target regions. Additionally, the authors convincingly use intersectional viral approaches and slice electrophysiology to show that projection neurons synapse onto neighboring cells within the VTA

Reviewer #2 (Public review):

The manuscript describes an interesting approach towards designing genetic circuits to sense different RAS mutants in the context of cancer therapeutics. The authors created sensors for mutant RAS and incorporated feed-forward control that leverages endogenous RAS/MAPK signaling pathways in order to dramatically increase the circuits' dynamic range. The modularity of the system is explored through the individual screening of several RAS binding domains, transmembrane domains, and MAPK response elements, and the author further extensively screened different combinations of circuit components. This is an impressive synthetic biology demonstration that took it all the way to cancer cell lines. However, given the sole demonstrated output in the form of fluorescent proteins, the authors' claims related to therapeutic implications require additional empirical evidence or, otherwise, expository revision.

Major comments:

"These therapies are limited to cancers with KRASG12C mutations" is technically accurate. However, in this fast-moving field, there are examples such as MRTX1133 which holds the promise to target the very G12D mutation that is the focus of this paper. There are broader efforts too. It would help the readers better appreciate the background if the authors could update the intro to reflect the most recent landscape of RAS-targeting drugs.

Only KRASG12D was used as a model in the design and optimization work of the genetic circuits. Other mutations should be quite experimentally feasible and comparisons of the circuits' performances across different KRAS mutations would allow for stronger claims on the circuits' generalizability. Particularly, the cancer cell line used for circuit validation harbored a KRASG13D mutation. While the data presented do indeed support the circuit's "generalizability," the model systems would not have been consistent in the current set of data presented.

In Figure 2a, the text claims that "inactivation of endogenous RAS with NF1 resulted in a lower YFP/RBDCRD-NarX expression," but Figure 2a does not show a statistically significant reduction in expression of SYFP (measured by "membrane-to-total signal ratio [RU]).

The therapeutic index of the authors' systems would be better characterized by a functional payload, other than florescent proteins, that for example induce cell death, immune responses, etc.

Regarding data presented in "Mechanism of action" (Figure 2), the observations are interesting and consistent across different fluorescent reporters. However, with regard to interpretations of the underlying molecular mechanisms, it is not clear whether the different output levels in 2b, 2c, and 2d are due to the pathway as described by the authors or simply from varied expression levels of RBDCRD-NarX itself (2a) that is nonlinearly amplified by the rest of the circuit. From a practical standpoint, this caveat is not critical with respect to the signal-to-noise ratios in later parts of the paper. From a mechanistic interpretation standpoint, claims made forth in this section are not clearly substantiated. Some additional controls would be nice. For example, if the authors express NarXs that constitutively dimerize on the membrane, what would the RasG12D-responsiveness look like? Does RasG12D alter the input-output curve of NarL-RE? How would Figure 4f compare to a NaxR constitutively dimerized control that only relies on transcriptional amplification of the Ras-dependent promoters? It's also possible that these Ras could affect protein production at the post-transcriptional or even post-translational levels, which were not adequately considered.

The text claims that "in contrast to what we saw in HEK293 overexpressing RAS (Figure 5d), the "AND-gate" RAS-targeting circuits do not generate higher output than the EF1a-driven, binding-triggered RAS sensor in HCT-116. Instead, the improved dynamic range results from decreased leakiness in HCT- 116k.o." Comparing the experiment from Figure 5d, which looks at activation in KRASG12D and KRASWT, to the experiments in Figure 6b-d, which looks at activation in HCT-116WT and HCT-116KO is misleading. In Fig 5d., cells are transfected with KRASG12D and KRASWT to emulate high levels of mutant RAS and high levels of wild-type RAS. In Figures 6b-d, HCT-116WT has endogenous levels of mutant RAS, while the KCT-116KO is a knock-out cell line, and does not have mutant or WT RAS. Therefore, the improved dynamic range or "decreased leakiness in HCT-116KO" in comparison to Figure 5d. is more comparable to the NF1 condition from Figure 2, which deactivates endogenous RAS. While this may not be feasible, the most accurate comparison would have been an HCT-116KO line with KRASWT stably integrated.

We couldn't locate the citation or discussion of Figure 4d in the text. Conversely, based on the text description, Figure 6g would contain exciting results. But we couldn't find Figure 6g anywhere ... unless it was a typo and the authors meant Figure 6f, in which case the cool results in Figure S8 could use more elaboration in the main text.

Reviewer #2 (Public review):

In this work the authors develop a mathematical model that incorporates three contributions to cellular force generation in 3D matrices: (1) actively generated contractile forces via myosin motors and consumption of ATP; (2) the energy stored in the extracellular matrix as it is deformed by the contractile cell; and (3), the energy associated with the interactions at the interface between the matrix and the cell, e.g. at focal adhesions. The authors make predictions about the dependence of cell shape on these three contributions.

The authors succeed in making a number of predictions of how cell shapes will depend on these contributions to force generation. However, these predictions seem to be largely buried in the supplemental material and come in a form that will be accessible to a certain type of physicist and modeler but will likely not be accessible to many experimentalists who may want to test the predictions of the model. The authors show a comparison between their expected cell shape distributions and those predicted by the model, under multiple regimes: cells in two different concentrations of collagen (Figure 4c), cells with inhibited myosin and therefore reduced contractility (Figure 4d), cells with impaired interactions with the ECM (Figure 4e), and for cells with both contractility and ECM interactions impaired. They find a strong agreement between the experiments and their predictions. However, it should be noted that there are multiple "tuning parameters" in their model, so the ability to match experiment and theory may not be ultimately so surprising.

While the authors do achieve their aim of building this modeling and testing it in comparison to experimental data, the text is frequently unclear and doesn't seem to have the right information at the right place and time to allow the reader to most clearly understand the motivation, the approach, or the results. A number of elements of this manuscript were confusing to this reviewer, and I discuss these below in the hopes that raising these points here can bring more clarity in future revisions, and/or that readers will be able to provide additional insight or attention to these questions.

There are certain elements of the writing that obscure, rather than clarify, the model and the results. For example, the authors frequently refer to "matrix stiffening" and "strain stiffening", which are typically used in the literature to describe the phenomenon whereby an applied force changes the mechanical properties of the substrate; here, for example in regard to the discussion of Figure 4C, these terms instead seem to be simply referring to the experimental intervention of exposing different cells to different concentrations of the collagen matrix. While there may be some element of classically understood strain stiffening, incorporated into the model as the function f(λ_i), this doesn't seem to match the experimental validation - which, as described above, is not about strain stiffening but instead simply uses softer vs. stiffer gels. Therefore, it is unclear what exactly is meant throughout the manuscript by strain stiffening - does it mean "difference in stiffness between two conditions" or does it mean "change in substrate stiffness upon application of force"?

Furthermore, while the introductory text emphasizes collective migration, the model itself focuses on the interactions between single cells and their environments. The emphasis on collective migration and cell shape in the introduction invokes previous literature focusing on collective phase transitions, but that is misleading. This paper is all about individual cell mechanics, not about collective migration or unjamming.

The experimental validation seems to have a significant flaw. The mechanics and interactions of the cellular extensions seem to be completely ignored. We see, in Figure 4, that cell bodies are outlined to determine cell shape, but that the extremely long extensions are simply ignored. We know from previous studies that these extensions are generating quite a bit of traction and are contractile, and yet they've been excluded from the analysis. This doesn't make physical sense or fit with previous literature, and would seem to indicate that the regimes predicted by the model are missing an essential component of force generation and cell-matrix interaction.

Reviewer #2 (Public review):

Summary:

This study by Kremer et al. investigates the impact of modulation of expression of TFAM, a key protein involved in mitochondrial DNA (mtDNA) packaging and expression, in mtDNA mutator mice, which carry random mtDNA mutations. While previous research suggested that increasing TFAM could counteract the pathological effects of mtDNA mutations, this study reveals that the effects of TFAM modulation are tissue-specific. These findings highlight the complexity of mtDNA copy number regulation and gene expression, emphasizing that TFAM alone is not the sole determinant of mtDNA levels in contexts where oxidative phosphorylation is impaired. Other factors likely play a significant role, underscoring the need for nuanced approaches when targeting TFAM for therapeutic interventions.

Strengths:

The data presented in the manuscript is of high quality and supports major conclusions.

Weaknesses:

The statistical methods used are not clearly described, and some marked non-significant results appear visually significant, which raises concerns about data analysis.

Data presentation requires improvement.

Reviewer #2 (Public review):

Summary:

Qi et al. determined the X-ray crystallographic structure of the methyltransferase core of the obligate heterodimeric complex METTL3-METTL14 in complex with methyladenosine monophosphate (m6A), a product mimic for the methylation of adenosine, to a resolution of 2.5 Å. Their structure appears to reveal a cryptic binding pocket for m6A that had not previously been identified. Using full-length protein produced in insect cells, Qi et al. determined the methyltransferase activity of wildtype METTL3-METTL14 and compared it to that of mutant forms of the protein that have been implicated in cancer. In addition to methyltransferase activity, the authors used both fluorescence polarization assays and surface plasmon resonance to investigate the affinities and kinetics of RNA binding to wildtype and mutant forms of the full-length complex. The results indicate that mutations in the methyltransferase core of two separate arginine residues alter the dynamics of RNA binding and enzyme specificity of METTL3-METTL14. The authors go on to use a combination of supervised molecular dynamics simulations and comparisons to recently published structures to propose a "swivelling" mechanism for the transfer of the methylated substrate from the catalytic site of the complex to the novel cryptic pocket.

Strengths:

I appreciated the inclusion of supplementary data showing the purity and monodispersity of the protein used for crystallization as well as the omit map and other electron density maps to support the placement of the product mimic in the cryptic site. The authors use a combination of complementary biophysical techniques to test the effects of mutations that were identified in the literature as being clinically important and to develop a hypothesis for the large-scale translocation required for the enzymatic product to move from the catalytic site to the cryptic pocket. The use of molecular dynamics simulations to attempt to indirectly visualize how this translocation might occur in vivo was well done.

Weaknesses:

Even taking into account the 2.5 Å resolution of the structure, the model is not refined to the point that it could be. Some waters seem to be built into blobs of density that aren't particularly convincing, and other seemingly obvious waters aren't built at all. The structure validation report supports this and shows that overall, and in the context of 2.5 Å resolution, this is not a great model. A good many parts of the structural analysis don't seem consistent with what I see when I look at the model and density in terms of proposed interactions in the cryptic pocket. Much of the language used in the manuscript is too strong when the model is quite speculative.

Reviewer #2 (Public review):

Summary:

In this manuscript, Li et al investigate the combined role of diacylglycerol (DAG) kinases (DGK) a and z in Foxp3+ Treg cells function that prevent autoimmunity. The authors generated DGK a and z Treg-specific double knockout mice (DKO) by crossing Dgkalpha-/- mice to DgKzf and Foxp3YFPCre/+ mice. The resulting "DKO" mice thus lack DGK a in all cells and DGK z in Foxp3+Treg cells. The authors show that the DKO mice spontaneously develop autoimmunity, characterized by multiorgan inflammatory infiltration and elevated anti-double-strand DNA (dsDNA), -single-strand DNA (ssDNA), and -nuclear autoantibodies. The authors attribute the DKO mice phenotype to Foxp3+Treg dysfunction, including accelerated conversion into "exTreg" cells with pathogenic activity. Interestingly, the combined deficiency of DGK a and z seems to release Treg cell dependence on CD28-mediated costimulatory signals, which the authors show by crossing their DKO mice to CD28-/- mice (TKO mice), which also develop autoimmunity.

Strengths:

The phenotypes of the mutant mice described in the manuscript are striking, and the authors provide a comprehensive analysis of the functional processes altered by the lack of DGKs.

Weaknesses:

One aspect that could be better explored is the direct role of "ex-Tregs" in causing pathogenesis in the models utilized.

However, overall, this is an important report that makes a significant addition to the understanding of DAG kinases in Treg cell biology.

Reviewer #2 (Public review):

Summary:

In this study, the authors have devised a novel assay to measure relative social rank in mice that is aimed at incorporating multiple aspects of social competition while minimizing direct contact between animals. Forming a hierarchy often involves complex social dynamics related to competitive drives for different fundamental resources including access to food, water, territory, and sexual mates. This makes the study of social dominance and its neural underpinnings hard, warranting the development of new tools and methods that can help understand both social functions as well as dysfunction.

Strengths:

This study showcases an assay called the Food Pellet Competition Test where cagemate mice compete for food, without direct contact, by pushing a block in a tube from opposite directions. The authors have attempted to quantify motivation to obtain the food independent of other factors such as age, weight, sex, etc. by running the assay under two conditions: one where the food is accessible and one where it isn't. This assay results in an impressive outcome consistency across days for females and males paired housed and for male groups of three. Further, the determined social ranks correlate strongly with two common assays: the tube test and the warm spot test.

Weaknesses:

This new assay has limited ethological validity since mice do not compete for food without touching each other with a block in the middle. In addition, the assay may only be valid for a single trial per day making its utility for recording neural recordings and manipulations limited to a single sample per mouse. Although the authors attempt to measure motivation as a factor driving who wins the social competition, the data is limited. This novel assay requires training across days with some mice reaching criteria before others. From the data reported, it is unclear what effects training can have on the outcome of social competition. Beyond the data shown, the language used throughout the manuscript and the rationale for the design of this novel assay is difficult to understand.

Reviewer #2 (Public review):

Summary:

Kargulyan et al. investigate the function of the transsynaptic adhesion molecule RTN4RL2 in the formation and function of ribbon synapses between type I spiral ganglion neurons (SGNs) and inner hair cells. For this purpose, they study constitutive RTN4RL2 knock-out mice. Using immunohistochemistry, they reveal defects in the recruitment of protein to ribbon synapses in the knockouts. Serial block phase EM reveals defects in SGN projections in mutants. Electrophysiological recordings suggest a small but statistically significant depolarized shift in the activation of Cav1.3 Ca2+ channels. Auditory thresholds are also elevated in the mutant mice. The authors conclude that RTN4RL2 contributes to the formation and function of auditory afferent synapses to regulate auditory function.

Strengths:

The authors have excellent tools to analyze ribbon synapses.

Weaknesses:

However, there are several concerns that substantially reduce my enthusiasm for the study.

(1) The analysis of the expression pattern of RTN4RL2 in Figure 1 is incomplete. The authors should show a developmental time course of expression up into maturity to correlate gene expression with major developmental milestones such as axon outgrowth, innervation, and refinement. This would allow the development of models supporting roles in axon outgrowth versus innervation or both.

(2) It would be important to improve the RNAscope data. Controls should be provided for Figure 1B to show that no signal is observed in hair cells from knockouts. The authors apparently already have the sections because they analyzed gene expression in SGNs of the knock-outs (Figure 1C).

(3) It is unclear from the immunolocalization data in Figure 1D if all type I SGNs express RTN4RL2. Quantification would be important to properly document the presence of RTN4RL2 in all or a subset of type I SGNs. If only a subset of SGNs express RTN4RL2, it could significantly affect the interpretation of the data. For example, SGNs selectively projecting to the pillar or modiolar side of hair cells could be affected. These synapses significantly differ in their properties.

(4) It is important to show proper controls for the RTN4RL2 immunolocalization data to show that no staining is observed in knockouts.

(5) The authors state in the discussion that no staining for RTN4RL2 was observed at synaptic sites. This is surprising. Did the authors stain multiple ages? Was there perhaps transient expression during development? Or in axons indicative of a role in outgrowth, not synapse formation?

(6) In Figure 2 it seems that images in mutants are brighter compared to wildtypes. Are exposure times equivalent? Is this a consistent result?

(7) The number of synaptic ribbons for wildtype in Figure 2 is at 10/IHCs, and in Figure 2 Supplementary Figure 2 at 20/IHCs (20 is more like what is normally reported in the literature). The value for mutant similarly drastically varies between the two figures. This is a significant concern, especially because most differences that are reported in synaptic parameters between wild-type and mutants are far below a 2-fold difference.

(8) The authors report differences in ribbon volume between wild-type and mutant. Was there a difference between the modiolar/pillar region of hair cells? It is known that synaptic size varies across the modiolar-pillar axis. Maybe smaller synapses are preferentially lost?

(9) The authors show in Figure 2 - Supplement 3 that GluA2/3 staining is absent in the mutants. Are GluA4 receptors upregulated? Otherwise, synaptic transmission should be abolished, which would be a dramatic phenotype. Antibodies are available to analyze GluA4 expression, the experiment is thus feasible. Did the authors carry out recordings from SGNs?

(10) The authors use SBEM to analyze SGN projections and synapses. The data suggest that a significant number of SGNs are not connected to IHCs. A reconstruction in Figure 3 shows hair cells and axons. It is not clear how the outline of hair cells was derived, but this should be indicated. Also, is this a defect in the formation of synapses and subsequent retraction of SGN projections? Or could RTN4RL2 mutants have a defect in axonal outgrowth and guidance that secondarily affects synapses? To address this question, it would be useful to sparsely label SGNs in mutants, for example with AAV vectors expression GFP, and to trace the axons during development. This would allow us to distinguish between models of RTN4RL2 function. As it stands, it is not clear that RTN4RL2 acts directly at synapses.

(11) The authors observe a tiny shift in the operation range of Ca2+ channels that has no effect on synaptic vesicle exocytosis. It seems very unlikely that this difference can explain the auditory phenotype of the mutant mice.

(12) ABR recordings were conducted in whole-body knockouts. Effects on auditory thresholds could be a secondary consequence of perturbation along the auditory pathway. Conditional knockouts or precisely designed rescue experiments would go a long way to support the authors' hypothesis. I realize that this is a big ask and floxed mice might not be available to conduct the study.

Reviewer #2 (Public review):

Summary:

Sphingosine-1-phosphate (S1P) metabolic and signaling genes are expressed highly in retinal Müller glia (MG) cells. This study tested how S1P signaling regulates glial phenotype, dedifferentiation of, reprogramming into proliferating MG-derived progenitor cells (MGPCs), and neuronal differentiation of the progeny of MGPCs using in vivo chick retina. Major techniques used are Sc-RNASeq and immunohistochemistry to determine the gene expression and proliferation of MG cells that co-label with signaling antibodies or mRNA FISH following treating the in vivo eyes with various S1P signaling antagonists, agonists, and signal modulators. The major conclusions drawn are supported by the results presented. However, the methodology they have used to modulate the S1P pathway using various chemical drugs raises questions about the outcomes and whether those are the real effects of S1P receptor modulation or S1P synthesis inhibition.

Strengths:

- Use of elaborated single-cell RNAseq expression data.<br /> - Use of FISH for S1P receptors and kinase as a good quality antibody is not available.<br /> - Use of EdU assay in combination with IHC<br /> - Comparison with human and Zebrafish Sc-RNA data

Reviewer #2 (Public review):

A large number of ovarian experiments have been conducted - especially in morphological and molecular biology studies - specifically removing the ovarian membrane. This experiment is a good supplement to existing knowledge and plays an important role in early ovarian development and the regulation of ovarian homeostasis during the estrous cycle. There are also innovations in research ideas and methods, which will meet the requirements of experimental design and provide inspiration for other researchers.

Comments on revisions: I don't have any further opinions and suggest to accept.

Reviewer #2 (Public review):

Summary:

This is a compelling study that systematically characterized and identified clonal MSC populations derived from normal and osteoarthritis human synovium. There is immense growth in the focus on synovial-derived progenitors in the context of both disease mechanisms and potential treatment approaches, and the authors sought to understand the regenerative potential of synovial-derived MSCs.

Strengths:

This study has multiple strengths. MSC cultures were established from an impressive number of human subjects, and rigorous cell surface protein analyses were conducted, at both pre-culture and post-culture timepoints. In vivo experiments using a rat DMM model showed beneficial therapeutic effects of MSCs vs non-MSCs, with compelling data demonstrating that only "real" MSC clones incorporate into cartilage repair tissue and express Prg4. Proteomics analysis was performed to characterize non-MSC vs MSC cultures, and high CD47 expression was identified as a marker for MSC. Injection of CD47-Hi vs CD47-Low cells in the same rat DMM model also demonstrated beneficial effects, albeit only based on histology. A major strength of these studies is the direct translational opportunity for novel MSC-based therapeutic interventions, with high potential for a "personalized medicine" approach.

Weaknesses:

Weaknesses of this study include the rather cursory assessment of the OA phenotype in the rat model, confined entirely to histology (i.e. no microCT, no pain/behavioral assessments, no molecular readouts). It is somewhat unclear how the authors converged on CD47 vs the other factors identified in the proteomics screen, and additional information is needed to understand whether true MSCs only engraft in articular cartilage or also in ectopic cartilage (in the context of osteophyte/chondrophyte formation). Some additional discussion and potential follow-up analyses focused on other cell surface markers recently described to identify synovial progenitors is also warranted. A conceptual weakness is the lack of discussion or consideration of the multiple recent studies demonstrating that DPP4+ PI16+ CD34+ stromal cells (i.e. the "universal fibroblasts") act as progenitors in all mesenchymal tissues, and their involvement in the joint is actively being investigated. Thus, it seems important to understand how the MSCs of the present study are related to these DPP4+ progenitors. Despite these areas for improvement, this is a strong paper with a high degree of rigor, and the results are compelling, timely, and important.

Overall, the authors achieved their aims, and the results support not just the therapeutic value of clonally-isolated synovial MSCs but also the immense heterogeneity in stromal cell populations (containing true MSCs and non-MSCs) that must be investigated further. Of note, the authors employed the ISCT criteria to characterize MSCs, with mixed results in pre-culture and post-culture assessments. This work is likely to have a long-term impact on methodologies used to culture and study MSCs, in addition to advancing the field's knowledge about how synovial-derived progenitors contribute to cartilage repair in vivo.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors develop an exciting new hemogenic gastruloid (hGX) system, which they claim reproduces the sequential generation of various blood cell types. The key advantage of this cellular system would be its potential to more accurately recapitulate the spatiotemporal emergence of hematopoietic progenitors within their physiological niche compared to other available in vitro systems. The authors present a large set of data and also validate their new system in the context of investigating infant leukemia.

Strengths:

The development of this new in vitro system for generating hematopoietic cells is innovative and addresses a significant drawback of current in vitro models. The authors present a substantial dataset to characterize this system, and they also validate its application in the context of investigating infant leukemia.

Weaknesses:

The thorough characterization and full demonstration that the cells produced truly represent distinct waves of hematopoietic progenitors are incomplete. The data presented to support the generation of late yolk sac (YS) progenitors, such as lymphoid cells, and aortic-gonad-mesonephros (AGM)-like progenitors, including pre-hematopoietic stem cells (pre-HSCs), by this system are not entirely convincing. Given that this is likely the manuscript's most crucial claim, it warrants further scrutiny and direct experimental validation. Ideally, the identity of these progenitors should be further demonstrated by directly assessing their ability to differentiate into lymphoid cells or fully functional HSCs. Instead, the authors primarily rely on scRNA-seq data and a very limited set of markers (e.g., Ikzf1 and Mllt3) to infer the identity and functionality of these cells. Many of these markers are shared among various types of blood progenitors, and only a well-defined combination of markers could offer some assurance of the lymphoid and pre-HSC nature of these cells, although this would still be limited in the absence of functional assays.

The identification of a pre-HSC-like CD45⁺CD41⁻/lo c-Kit⁺VE-Cadherin⁺ cell population is presented as evidence supporting the generation of pre-HSCs by this system, but this claim is questionable. This FACS profile may also be present in progenitors generated in the yolk sac such as early erythro-myeloid progenitors (EMPs). It is only within the AGM context, and in conjunction with further functional assays demonstrating the ability of these cells to differentiate into HSCs and contribute to long-term repopulation, that this profile could be strongly associated with pre-HSCs. In the absence of such data, the cells exhibiting this profile in the current system cannot be conclusively identified as true pre-HSCs.

The engraftment data presented are also not fully convincing, as the observed repopulation is very limited and evaluated only at 4 weeks post-transplantation. The cells detected after 4 weeks could represent the progeny of EMPs that have been shown to provide transient repopulation rather than true HSCs.

Reviewer #2 (Public review):

Previously, the authors published a Leishmania cytosine base editor (CBE) genetic tool that enables the generation of functionally null mutants. This works by utilising a CAS9-cytidine deaminase variant that is targeted to a genetic locus by a small guide RNA (sgRNA) and causes a cytosine to thymine conversion. This has the potential to generate a premature stop codon and therefore a loss of function mutant.

CBE has advantages over existing CAS-based knockout tools because it allows the targeting of multicopy gene families and, potentially, the easier generation of pooled loss of function mutants in complex population experiments. Although successful, the first generation of this genetic tool had several limitations that may have prevented its wider adoption, especially in complex genome-wide screens. These include nonspecific toxicity of the sgRNAs, low transfection efficiencies, low editing efficiencies, a proportion of transfectants that express multiple different sgRNAs, and insufficient effectivity in some Leishmania species.

Here, the authors set out to systematically solve each of these limitations. By trialling different transfection conditions and different CAS12a cut sites to promote sgRNA expression cassette integration, they increase the transfection efficiency 400-fold and ensure that only a single sgRNA expression cassette integrates that edits with high efficiencies. By trialling different T7 promoters, they significantly reduce the non-specific toxicity of sgRNA expression whilst retaining high editing efficiencies in several Leishmania species (Leishmania major, L. mexicana and L. donovani). By improving the sgRNA design, the authors predict that null mutants will be more efficiently produced after editing. They validate this tool in a small-scale loss of function screen incorporating essential and non-essential genes, identifying the expected growth phenotypes.

This tool will find adoption for producing null mutants of single-copy genes, multicopy gene families, and genome-wide mutational analyses.

This is an impressive and thorough study that significantly improves the previous iteration of the CBE. The approach is careful and systematic and reflects the authors excellent experience developing CRISPR tools. The quality of data and analysis is high and data are clearly presented.

Reviewer #2 (Public review):

In this article, the authors examined the organization of misplaced retinal inputs in the visual thalamus of albino mice at electron-microscopic (EM) resolution to determine whether these synaptic inputs are segregated from the rest of the retinogeniculate circuitry.

The study's major strengths include its high resolution, achieved through serial EM and confocal microscopy, which enabled the identification of all synaptic inputs onto neurons in the dorsolateral geniculate nucleus (dLGN).<br /> The experiments are very precise and demanding thus, only the synaptic inputs of a few neurons were fully reconstructed in one animal.

Despite this, the authors clearly demonstrate the synaptic segregation of misrouted retinal axons onto dLGN neurons, separate from the rest of the retinogeniculate circuitry.

This finding is impactful because retinal inputs typically do not segregate within the mouse dLGN, and it was previously thought that this was due to the nucleus's small size, which might prevent proper segregation. The study shows that in cases where axons are misrouted and exhibit a different activity pattern than surrounding retinal inputs, segregation of inputs can indeed occur. This suggests that the normal system has the capacity to segregate inputs, despite the limited volume of the mouse dLGN.

Reviewer #2 (Public review):

Summary:

While it is often assumed that the cerebellar cortex connects, via its sole output neuron, Purkinje cell, exclusively to the cerebellar nuclei, axonal projections of the Purkinje cells to dorsal brainstem regions have been well documented. This paper provides comprehensive mapping and quantification of such extracerebellar projections of the Purkinje cells, most of which are confirmed with electrophysiology in slice preparation. A notable methodological strength of this work is the use of highly Purkinje cell-specific transgenic strategies, enabling selective and unbiased visualization of Purkinje terminals in the brainstem. By utilizing these selective mouse lines, the study offers compelling evidence challenging the general assumption that Purkinje cell targets are limited to the cerebellar nuclei. While the individual connections presented are not entirely novel, this paper provides a thorough and unambiguous demonstration of their collective significance. Regarding another major claim of this paper, "characterization of direct Purkinje cell outputs (Title)", however, the depth of electrophysiological analysis is limited to presence/absence of physiological Purkinje input to postsynaptic brainstem neurons whose known cell types are mostly blinded. Overall, conceptual advance is largely limited to confirmatory or incremental, although it would be useful for the field to have the comprehensive landscape presented.

Strengths:

Unsupervised comprehensive mapping and quantification of the Purkinje terminals in the dorsal brainstem are enabled, for the first time, by using the current state-of-the-art mouse lines, BAC-Pcp2-Cre and synaptophysin-tdTomato reporter (Ai34).

Combinatorial quantification with vGAT puncta and synaptophysin-tdTomato labeled Purkinje terminals clarifies the anatomical significance of the Purkinje terminals as an inhibitory source in each dorsal brainstem region.

Electrophysiological confirmation of the presence of physiological Purkinje synaptic input to 7 out of 9 dorsal brainstem regions identified.

Pan-Purkinje ChR2 reporter provides solid electrophysiological evidence to help understand the possible influence of the Purkinje cells onto LC.

Weaknesses:

The present paper is largely confirmatory to what is presented in a previous paper published by the author's group (Chen et al., 2023, Nat Neurosci). In this preceding paper, the author's group used AAV1-mediated anterograde transsynaptic strategy to identify postsynaptic neurons of the Purkinje cells. The experiments performed in the present paper is, by nature, complementary to the AAV1 tracing which can also infect retrogradely and thus is not able to demonstrate the direction of synaptic connections between reciprocally connected regions. Anatomical findings are all consistent with the preceding paper.

While the authors appear to assume uniform cell type and postsynaptic response in each of the dorsal brainstem nuclei (as noted in the Discussion, "PCs likely function similarly to their inputs to the cerebellar nuclei, where a very brief pause in firing can lead to large and rapid elevations in target cell firing"), we know that the responses to the Purkinje cell input are cell type dependent, which vary in neurotransmitter, output targets, somata size, and distribution, in the cerebellar and vestibular nuclei (Shin et al., 2011, J Neurosci; Najac and Raman, 2015, J Neurosci; Özcan et al., 2020, J Neurosci). Also, whether 23 % (for PCG), for example, is "a small fraction" would be subjective: it might represent a numerically small but functionally important cell type population. From a functional perspective, the cell type-blind physiological characterization in this manuscript remains superficial compared to existing cell type-specific analyses, although the authors commented on these issues in the manuscript.

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 16 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. This work explores the fundamental properties of IPCs that may contribute to the neuroendocrine regulation of insulin-like peptides in maintaining metabolic homeostasis.

Weaknesses:

It remains unknown 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 heterogenous expressions of receptor genes in IPCs and examine IPC activities in the future. Moreover, while the authors focus on IPC activities, they did not show the activation of the neuromodulatory inputs and the net output of insulin levels in the data. The readers might want to know which neurons are indeed activated to send signals to IPCs and how IPC activities result in the secretion of insulin peptides.

Reviewer #2 (Public review):

Summary:

The study by Fisher et al investigates a therapeutic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy of promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well-designed study with thorough investigation using multiple disease models.

Strengths:

(1) Well-written manuscript with clear design, robust methods, and discussion.

(2) Using multiple models strengthens the findings and expands beyond ALD.

(3) Identification of SZN-043 as a novel potent drug for liver regeneration.

Weaknesses:

(1) The introduction needs to be re-structured with an emphasis on liver regeneration. It seems that the entire manuscript is focused on liver regeneration, however, only the last two sentences or so describe liver regeneration. The frequency of liver transplants owing to a reduced ability for liver regeneration in AH patients needs to be highlighted.

(2) In Figure 4, it appears that the humanized mice liver was injected with the SZN-043. Is it possible that using a partial hepatectomy model will be beneficial for assessing the effects of SZN-043 rather than using them in mice without any hepatocyte damage?

(3) Figure 4B. Panel 3 has 10mpk merged inside the figure. Please correct this.

(4) Figure 4B. DAPI staining will be vital to show the Ki67 staining specific to hepatocytes (at least visually we can do co-localization with a double nucleus in each cell). The current image shows some cells show Ki67 staining which shows some cells which are not binuclear.

(5) The alcohol feeding was performed for 8 weeks and is described as NIAAA model in the methods section. NIAAA model is 11 days of alcohol+ one binge. Please correct this or clarify it in the methods section, as this is not reflected. ASGR1 may be also expressed by macrophages so it's important to show the specificity.

(6) Is it possible that the SZN-043 also has effect on macrophages promoting an anti-inflammatory state? This should be discussed.

(7) Potential off-target effects of SZN-043, particularly in stellate cell activation in the context of fibrosis should be discussed.

(8) Discuss the limitations of current models and how they might influence the interpretation of the results.

(9) Clearly explain how SZN-043 overcomes limitations of prior RSPO-based therapies.

Reviewer #2 (Public review):

Summary:

This interesting study compared two different intensities of aerobic exercise (low-intensity, high-intensity) and their efficacy in inducing a hypoalgesic reaction (i.e. exercise-induced hypoalgesia; EIH). fMRI was used to identify signal changes in the brain, with the infusion of naloxone used to identify hypoalgesia mechanisms. No differences were found in post-exercise pain perception between the high-intensity and low-intensity conditions, with naloxone infusion causing increased pain perception across both conditions which was mirrored by activation in the medial frontal cortex (identified by fMRI). However, the primary conclusion made in this manuscript (i.e. that aerobic exercise has no overall effect on pain in a mixed population sample) cannot be supported by this study design, because the methodology did not include a baseline (i.e. pain perception following no exercise) to compare high/low-intensity exercise against. Therefore, some of the statements/implications of the findings made in this manuscript need to be very carefully assessed.

Strengths:

(1) The use of fMRI and naloxone provides a strong approach by which to identify possible mechanisms of EIH.

(2) The infusion of naloxone to maintain a stable concentration helps to ensure a consistent effect and that the time course of the protocol won't affect the consistency of changes in pain perception.

(3) The manipulation checks (differences in intensity of exercise, appropriate pain induction) are approached in a systematic way.

(4) Whilst the exploratory analyses relating to the interactions for fitness level and sex were not reported in the study pre-registation, they do provide some interesting findings which should be explored further.

Weaknesses:

(1) Given that there is no baseline/control condition, it cannot be concluded that aerobic exercise has no effect on pain modulation because that comparison has not been made (i.e. pain perception at 'baseline' has not been compared with pain perception after high/low-intensity exercise). Some of the primary findings/conclusions throughout the manuscript state that there is 'No overall effect of aerobic exercise on pain modulation', but this cannot be concluded.

(2) Across the manuscript, a number of terms are used interchangeably (and applied, it seems, incorrectly) which makes the interpretation of the manuscript difficult (e.g. how the author's use the term 'exercise-induced pain').

(3) There is a lack of clarity on the interventions used in the methods, for example, it is not exactly clear the time and order in which the exercise tasks were implemented.

(4) The exercise test (functional threshold power) used to set the intensity of the low/high exercise bouts is not an accurate means of demarcating steady state and non-steady state exercise. As a result, at the intensity selected for the high-intensity exercise in this study, it is likely that the challenge presented for the high-intensity exercise would have been very different between participants (e.g. some would have been in the 'heavy' domain, whereas others would be in the 'severe' domain).

(5) It is likely that participants did not properly understand how to use the 6-20 Borg scale to rate their perceived effort, and so caution must be taken in how this RPE data is used/interpreted.

(6) Although interesting, the secondary analyses (relating to the interaction effects of fitness level and sex) were not included in the study pre-registration, and so the study was not designed to undertake this analysis. These findings should be taken with caution.

Reviewer #2 (Public review):

The manuscript investigates the relationship between sleep, DNA damage, and aging in the Mexican cavefish (Astyanax mexicanus), a species that exhibits significant differences in sleep patterns between surface-dwelling and cave-dwelling populations. The authors aim to understand whether these evolved sleep differences influence the DNA damage response (DDR) and oxidative stress levels in the brain and gut of the fish.

Summary of the Study:

The primary objective of the study is to determine if the reduced sleep observed in cave-dwelling populations is associated with increased DNA damage and altered DDR. The authors compared levels of DNA damage markers and oxidative stress in the brains and guts of surface and cavefish. They also analyzed the transcriptional response to UV-induced DNA damage and evaluated the DDR in embryonic fibroblast cell lines derived from both populations.

Strengths of the Study:

Comparative Approach: The study leverages the unique evolutionary divergence between surface and cave populations of A. mexicanus to explore fundamental biological questions about sleep and DNA repair.

Multifaceted Methodology: The authors employ a variety of methods, including immunohistochemistry, RNA sequencing, and in vitro cell line experiments, providing a comprehensive examination of DDR and oxidative stress.<br /> Interesting Findings: The study presents intriguing results showing elevated DNA damage markers in cavefish brains and increased oxidative stress in cavefish guts, alongside a reduced transcriptional response to UV-induced DNA damage.

Weaknesses of the Study:

Link to Sleep Physiology: The evidence connecting the observed differences in DNA damage and DDR directly to sleep physiology is not convincingly established. While the study shows distinct DDR patterns, it does not robustly demonstrate that these are a direct result of sleep differences.

Causal Directionality: The study fails to establish a clear causal relationship between sleep and DNA damage. It is possible that both sleep patterns and DDR responses are downstream effects of a common cause or independent adaptations to the cave environment.

Environmental Considerations: The lab conditions may not fully replicate the natural environments of the cavefish, potentially influencing the results. The impact of these conditions on the study's findings needs further consideration.

Photoreactivity in Albino Fish: The use of UV-induced DNA damage as a primary stressor may not be entirely appropriate for albino, blind cavefish. Alternative sources of genotoxic stress should be explored to validate the findings.

Assessment of the Study's Achievements:

The authors partially achieve their aims by demonstrating differences in DNA damage and DDR between surface and cavefish. However, the results do not conclusively support the claim that these differences are driven by or directly related to the evolved sleep patterns in cavefish. The study's primary claims are only partially supported by the data.

Impact and Utility:

The findings contribute valuable insights into the relationship between sleep and DNA repair mechanisms, highlighting potential areas of resilience to DNA damage in cavefish. While the direct link to sleep physiology remains unsubstantiated, the study's data and methods will be useful to researchers investigating evolutionary biology, stress resilience, and the molecular basis of sleep.

Comments on revisions:

The manuscript should tone down claims of a direct causal relationship between sleep differences and DDR outcomes, acknowledging the possibility that both are independent or downstream adaptations to the cave environment. To strengthen the study, the authors should adopt additional genotoxic stressors, such as chemical agents (e.g., cisplatin or hydrogen peroxide) or physical stress (e.g., ionizing radiation), to validate findings beyond UV-induced DNA damage, which may not be ideal for albino cavefish. Explicitly discussing the influence of laboratory conditions, such as water quality, lighting, and diet, on oxidative stress and DDR phenotypes, and comparing lab-reared and wild-caught fish if feasible, would bolster ecological relevance. The study should clarify that the current data do not establish a causal link between sleep and DNA damage, instead proposing this as a hypothesis for future research. Expanding the evolutionary context by linking DDR differences to other cavefish traits, such as metabolic efficiency or hypoxia tolerance, could provide a more integrative perspective. Additionally, proposing future experiments involving pharmacological or behavioral manipulation of sleep, as well as incorporating comparative genomics or transcriptomics to identify DDR-related genetic adaptations, would enhance the study's depth.

Reviewer #2 (Public review):

Summary:

This study employed voltage imaging in the CA1 region of the mouse hippocampus during the exploration of a novel environment. The authors report synchronous activity, involving almost half of the imaged neurons, occurred during periods of immobility. These events did not correlate with SWRs, but instead, occurred during theta oscillations and were phased locked to the trough of theta. Moreover, pairs of neurons with high synchronization tended to display non-overlapping place fields, leading the authors to suggest these events may play a role in binding a distributed representation of the context.

Strengths:

Technically this is an impressive study, using an emerging approach that allow single-cell resolution voltage imaging in animals, that while head-fixed, can move through a real environment. The paper is written clearly and suggests novel observations about population-level activity in CA1.

Weaknesses:

The evidence provided is weak, with the authors making surprising population-level claims based on a very sparse data set (5 data sets, each with less than 20 neurons simultaneously recorded) acquired with exciting, but less tested technology. Further, while the authors link these observations to the novelty of the context, both in the title and text, they do not include data from subsequent visits to support this. Detailed comments are below:

(1) My first question for the authors, which is not addressed in the discussion, is why these events have not been observed in the countless extracellular recording experiments conducted in rodent CA1 during exploration of novel environments. Those data sets often have 10x the neurons simultaneously recording compared to these present data, thus the highly synchronous firing should be very hard to miss. Ideally, the authors could confirm their claims via the analysis of publicly available electrophysiology data sets. Further, the claim of high extra-SWR synchrony is complicated by the observation that their recorded neurons fail to spike during the limited number of SWRs recorded during behavior- again, not agreeing with much of the previous electrophysiological recordings.<br /> (2) The authors posit that these events are linked to the novelty of the context, both in the text, as well as in the title and abstract. However they do not include any imaging data from subsequent days to demonstrate the failure to see this synchrony in a familiar environment. If these data are available it would strengthen the proposed link to novelty is they were included.<br /> (3) In the discussion the authors begin by speculating the theta present during these synchronous events may be slower type II or attentional theta. This can be supported by demonstrating a frequency shift in the theta recording during these events/immobility versus the theta recording during movement.<br /> (4) The authors mention in the discussion that they image deep layer PCs in CA1, however this is not mentioned in the text or methods. They should include data, such as imaging of a slice of a brain post-recording with immunohistochemistry for a layer specific gene to support this.

Comments on revisions:

I have no further major requests and thank the authors for the additional data and analyses.

Reviewer #3 (Public review):

Summary:

The mechanisms by which short-term isolation influences the brain to promote social behavior remain poorly understood. The authors observed that acute isolation enhanced social behaviors, including increased investigation, mounting, and ultrasonic vocalizations (USVs). These effects were evident in same-sex interactions among females and in male-female interactions. Concurrently, cFos expression in the preoptic area (POA) of the hypothalamus was selectively elevated in single-housed females. To further investigate, the authors used an innovative tagging strategy (TRAP2) to manipulate these neurons. Overall, the study identifies a population of hypothalamic neurons that promote various aspects of social behavior after short-term isolation, with effects that are sex- and context-dependent.

Strengths:

Understanding the neural circuit mechanisms underlying acute social isolation is an important and timely topic. By employing state-of-the-art techniques to tag neurons active during specific behavioral epochs, the authors identified the preoptic area (POA) as a key locus mediating the effects of social isolation. The experimental design is sound, and the data are of high quality. Notably, the control experiments, which show that chemogenetic inactivation of other hypothalamic regions (AH and VMH) does not affect social behavior, strongly support the specificity of the POA's role within the hypothalamus. Through a combination of behavioral assays, activity-dependent neural tagging, and circuit manipulation techniques, the authors provide compelling evidence for the POA's involvement in behaviors following social isolation. These findings represent a valuable contribution to understanding how hypothalamic circuits adapt to the challenges of social isolation.

Weaknesses:

The authors conducted several circuit perturbation experiments, including chemogenetics, ablation, and optogenetics, to investigate the effects of POA-social neurons. They observed that the outcomes of these manipulations varied depending on whether the intervention was chronic (e.g., ablations) or acute (e.g., DREADDs), potentially due to compensatory mechanisms in other brain regions. Furthermore, their additional experiments revealed that the robustness of the manipulations was influenced by the heterozygosity or homozygosity of TRAP2 animals. While these findings suggest that POA neurons contribute to multiple behavioral responses to social isolation, further experiments are needed to clarify their precise roles.

Reviewer #2 (Public review):

Summary:

This study makes a significant contribution to understanding the microenvironment of megakaryocytes (MKs) in the bone marrow, identifying an extracellular matrix (ECM) cage structure that influences MK localization and maturation. The authors provide compelling evidence for the presence of this ECM cage and its role in MK homeostasis, employing an array of sophisticated imaging techniques and molecular analyses. While the work is innovative and impactful, there are several points that require clarification or further data to fully support the conclusions.

Major Strengths:

Novelty: The identification of an ECM cage as a regulator of MK localization and maturation in the bone marrow is a novel and exciting finding.

Imaging Techniques: The use of advanced microscopy to visualize the 3D structure of the ECM cage and its role in MK homeostasis provides a strong visual foundation for the study's claims.

Comprehensive Analysis: The integration of in vivo and ex vivo approaches enhances the significance of the findings, offering valuable insights into the molecular mechanisms involved in ECM cage formation.

Areas for Improvement and Clarifications:

(1) ECM cage imaging:<br /> a) The value or additional information provided by the staining on nano-sections (A) is not clear, especially considering that the thick vibratome sections already display the entirety of the laminin γ1 cage structure effectively. Further clarification on the unique insights gained from each approach would help justify its inclusion.<br /> b) The sMK shown in Supplementary Figure 1C appears to be linked to two sinusoids, releasing proplatelets to the more distant vessels. Is this observation representative, and if so, can further discussion be provided?<br /> c) Freshly isolated BM-derived MKs are reported to maintain their laminin γ1 cage. Are the proportions of MKs with/without cages consistent with those observed in microscopy?

(2) ECM cage formation:<br /> a) The statement "the full assembly of the 3D ECM cage required megakaryocyte interaction with the sinusoidal basement membrane" on page 7 is too strong given the data presented at this stage of the study. Supplemental Figure 1C shows that approximately 10% of pMKs form cages without direct vessel contact, indicating that other factors may also play a role in cage formation.<br /> b) The data supporting the statement that "pMK represent a small fraction of the total MK population" (cell number or density) could be shown to help contextualize the 10% of them with a cage.<br /> c) How "the full assembly of the 3D ECM cage" is defined at this stage of the study should be clarified, specifically regarding the ECM components and structural features that characterize its completion.

(3) Data on MK Circulation and Cage Integrity: Does the cage require full component integrity to prevent MK release in circulation? Are circulating MKs found in Lama4-/- mice? Is the intravasation affected in these mice? Are the ~50% sinusoid associated MK functional?

(4) Methodology:<br /> a) Details on fixation time are not provided, which is critical as it can impact antibody binding and staining. Including this information would improve reproducibility and feasibility for other researchers.<br /> b) The description of 'random length measuring' is unclear, and the rationale behind choosing random quantification should be explained. Additionally, in the shown image, it appears that only the branching ends were measured, which makes it difficult to discern the randomness in the measurements.

(5) Figures:<br /> a) Overall, the figures and their corresponding legends would benefit from greater clarity if some panels were split, such as separating images from graph quantifications.

Reviewer #2 (Public review):

Summary:

The authors used rats to determine the receptor for a food-related perception (kokumi) that has been characterized in humans. They employ a combination of behavioral, electrophysiological, and immunohistochemical results to support their conclusion that ornithine-mediated kokumi effects are mediated by the GPRC6A receptor. They complemented the rat data with some human psychophysical data. I find the results intriguing, but believe that the authors overinterpret their data.

Strengths:

The authors provide compelling evidence that ornithine enhances the palatability of several chemical stimuli (i.e., IMP, MSG, MPG, Intralipos, sucrose, NaCl, quinine). Ornithine also increases CT nerve responses to MSG. Additionally, the authors provide evidence that the effects of ornithine are mediated by GPRC6A, a G-protein-coupled receptor family C group 6 subtype A, and that this receptor is expressed primarily in fungiform taste buds. Taken together, these results indicate that ornithine enhances the palatability of multiple taste stimuli in rats and that the enhancement is mediated, at least in part, within fungiform taste buds. This is an important finding that could stand on its own. The question of whether ornithine produces these effects by eliciting kokumi-like perceptions (see below) should be presented as speculation in the Discussion section.

Weaknesses:

I am still unconvinced that the measurements in rats reflect the "kokumi" taste percept described in humans. The authors conducted long-term preference tests, 10-min avidity tests and whole chorda tympani (CT) nerve recordings. None of these procedures specifically model features of "kokumi" perception in humans, which (according to the authors) include increasing "intensity of whole complex tastes (rich flavor with complex tastes), mouthfulness (spread of taste and flavor throughout the oral cavity), and persistence of taste (lingering flavor)." While it may be possible to develop behavioral assays in rats (or mice) that effectively model kokumi taste perception in humans, the authors have not made any effort to do so. As a result, I do not think that the rat data provide support for the main conclusion of the study--that "ornithine is a kokumi substance and GPRC6A is a novel kokumi receptor."

Why are the authors hypothesizing that the primary impacts of ornithine are on the peripheral taste system? While the CT recordings provide support for peripheral taste enhancement, they do not rule out the possibility of additional central enhancement. Indeed, based on the definition of human kokumi described above, it is likely that the effects of kokumi stimuli in humans are mediated at least in part by the central flavor system.

The authors include (in the supplemental data section) a pilot study that examined the impact of ornithine on variety of subjective measures of flavor perception in humans. The presence of this pilot study within the larger rat study does not really mice sense. While I agree with the authors that there is value in conducting parallel tests in both humans and rodents, I think that this can only be done effectively when the measurements in both species are the same. For this reason, I recommend that the human data be published in a separate article.

The authors indicated on several occasions (e.g., see Abstract) that ornithine produced "synergistic" effects on the CT nerve response to chemical stimuli. "Synergy" is used to describe a situation where two stimuli produce an effect that is greater than the sum of the response to each stimulus alone (i.e., 2 + 2 = 5). As far as I can tell, the CT recordings in Fig. 3 do not reflect a synergism.

Reviewer #2 (Public review):

Summary:

The authors set out to determine how the microbiome and host genotype impact host protein-based nutrition.

Strengths:

The quantification of protein uptake dynamics is a major strength of this work and the sensitivity of this assay shows that the microbiome and even mono-associated bacterial strains dampen protein uptake in the host by causing down-regulation of genes involved in this process rather than a change in cell type.

The use of fluorescent proteins in combination with transcript clustering in the single cell seq analysis deepens our understanding of the cells that participate in protein uptake along the intestine. In addition to the lysozome-rich enterocytes (LRE), subsets of enteroendocrine cells, acinar, and goblet cells also take up protein. Intriguingly, these non-LRE cells did not show lysosomal-based protein degradation; but importantly analysis of the transcripts upregulated in these cells include dab2 and cubn, genes shown previously as being essential to protein uptake.

The derivation of zebrafish mono-associated with single strains of microbes paired with HCR to localize and quantify the expression of host protein absorption genes shows that different bacterial strains suppress these genes to variable extents.

The analysis of microbiome composition, when host protein absorption is compromised in cubn-/- larvae or by reducing protein in the food, demonstrates that changes to host uptake can alter the abundance of specific microbial taxa like Aeramonas.

Weaknesses:

The finding that neurons are positive for protein uptake in the single-cell data set is not adequately discussed. It is curious because the cldn:GFP line used for sorting does not mark neurons and if the neurons are taking up mCherry via trans-synaptic uptake from EECs, those neurons should be mCherry+/GFP-; yet methods indicate GFP+ and GFP+/mCherry+ cells were the ones collected and analyzed.

for - longterminism - 2 fundamental technologies - rockets and AI

Reviewer #2 (Public review):

The authors investigate the phosphotransfer capacity of Ser/Thr kinase IκB kinase (IKK), a mediator of cellular inflammation signaling. Canonically, IKK activity is promoted by activation loop phosphorylation at Ser177/Ser181. Active IKK can then unleash NF-κB signaling by phosphorylating repressor IκBα at residues Ser32/Ser26. Noting the reports of other IKK phosphorylation sites, the authors explore the extent of autophosphorylation.

Semi-phosphorylated IKK purified from Sf9 cells, exhibits the capacity for further autophosphorylation. Anti-phosphotyrosine immunoblotting indicated unexpected tyrosine phosphorylation. Contaminating kinase activity was tested by generating a kinase-dead K44M variant, supporting the notion that the unexpected phosphorylation was IKK-dependent. In addition, the observed phosphotyrosine signal required phosphorylated IKK activation loop serines.

Two candidate IKK tyrosines were examined as the source of the phosphotyrosine immunoblotting signal. Activation loop residues Tyr169 and Tyr188 were each rendered non-phosphorylatable by mutation to Phe. The Tyr variants decreased both autophosphorylation and phosphotransfer to IκBα. Likewise, Y169F and Y188F IKK2 variants immunoprecipitated from TNFa-stimulated cells also exhibited reduced activity in vitro.

The authors further focus on Tyr169 phosphorylation, proposing a role as a phospho-sink capable of phosphotransfer to IκBα substrate. This model is reminiscent of the bacterial two-component signaling phosphotransfer from phosphohistidine to aspartate. Efforts are made to phosphorylate IKK2 and remove ATP to assess the capacity for phosphotransfer. Phosphorylation of IκBα is observed after ATP removal, although there are ambiguous requirements for ADP.

Strengths:

Ultimately, the authors draw together the lines of evidence for IKK2 phosphotyrosine and ATP-independent phosphotransfer to develop a novel model for IKK2-mediated phosphorylation of IκBα. The model suggests that IKK activation loop Ser phosphorylation primes the kinase for tyrosine autophosphorylation. With the assumption that IKK retains the bound ADP, the phosphotyrosine is conformationally available to relay the phosphate to IκBα substrate. The authors are clearly aware of the high burden of evidence required for this unusual proposed mechanism. Indeed, many possible artifacts (e.g., contaminating kinases or ATP) are anticipated and control experiments are included to address many of these concerns. The analysis hinges on the fidelity of pan-specific phosphotyrosine antibodies, and the authors have probed with two different anti-phosphotyrosine antibody clones. Taken together, the observations are thought-provoking, and I look forward to seeing this model tested in a cellular system.

Weaknesses:

Multiple phosphorylated tyrosines in IKK2 were apparently identified by mass spectrometric analyses. LC-MS/MS spectra are presented, but fragments supporting phospho-Y188 and Y325 are difficult to distinguish from noise. It is common to find non-physiological post-translational modifications in over-expressed proteins from recombinant sources. Are these IKK2 phosphotyrosines evident by MS in IKK2 immunoprecipitated from TNFa-stimulated cells? Identifying IKK2 phosphotyrosine sites from cells would be especially helpful in supporting the proposed model.

Reviewer #2 (Public review):

Summary:

Chlamydial cell division is a peculiar event, whose mechanism was mysterious for many years. C. trachomatis division was shown to be polar and involve a minimal divisome machinery composed of both homologues of divisome and elongasome components, in absence of an homologue of the classical division organizer FtsZ. In this paper, Harpring et al., show that FtsK is required at an early stage of the chlamydial divisome formation.

Strengths:

The manuscript is well-written and the results are convincing. Quantification of divisome component localization is well performed, number of replicas and number of cells assessed are sufficient to get convincing data. The use of a CRISPRi approach to knock down some divisome components is an asset and allows a mechanistic understanding of the hierarchy of divisome components

Weaknesses

Despite advances in the understanding of the importance of FtsK for chlamydial division, this manuscript does not show by which mechanism FtsK specifically localizes at the division site and how it mediates recruitment of other divisome members. Moreover, the potential link with DNA partitioning is not addressed.

Reviewer #2 (Public review):

Summary:

The authors set out to determine how the microbiome and host genotype impact host protein-based nutrition.

Strengths:

The quantification of protein uptake dynamics is a major strength of this work and the sensitivity of this assay shows that the microbiome and even mono-associated bacterial strains dampen protein uptake in the host by causing down-regulation of genes involved in this process rather than a change in cell type.

The use of fluorescent proteins in combination with transcript clustering in the single cell seq analysis deepens our understanding of the cells that participate in protein uptake along the intestine. In addition to the lysozome-rich enterocytes (LRE), subsets of enteroendocrine cells, acinar, and goblet cells also take up protein. Intriguingly, these non-LRE cells did not show lysosomal-based protein degradation; but importantly analysis of the transcripts upregulated in these cells include dab2 and cubn, genes shown previously as being essential to protein uptake.

The derivation of zebrafish mono-associated with single strains of microbes paired with HCR to localize and quantify the expression of host protein absorption genes shows that different bacterial strains suppress these genes to variable extents.

The analysis of microbiome composition, when host protein absorption is compromised in cubn-/- larvae or by reducing protein in the food, demonstrates that changes to host uptake can alter the abundance of specific microbial taxa like Aeramonas.

Reviewer #2 (Public review):

Summary:

The interplay between the medial prefrontal cortex and ventral hippocampal system is critical for many cognitive processes, including memory and its consolidation over time. A prominent idea in recent research is that this relationship is mediated at least in part by the midline nucleus reuniens with respect to consolidation in particular. Whereas the bulk of evidence has focused on neuroanatomy and the effects of temproary or permanent lesions of the nucleus reuniens, the current work examined the electrophysiology of these three structures and how they inter-relate, especially during sleep, which is anticipated to be critical for consolidation. They provide evidence from intercellualr recordings of the bi-directional functional connectivity among these structures. There is an emphasis on the interactions between these regions during sleep, especially slow wave sleep. They provide evidence, in cats, that cortical slow waves precede reuniens slow waves and hippocampal sharp-wave ripples, which may reflect prefrontal control of the timing of thalamic and hippocampal events, They also find evidence that hippocampal sharp wave ripples trigger thalamic firing and precede the onset of reuniens and medial prefrontal cortex spindles. The authors suggest that the effectiveness of bidirectional connections between the reuniens and the (ventral) CA1 is particularly strong during non-rapid eye movement sleep in the cat. This is a very interesting, complex study on a highly topical subject.

Strengths:

An excellent array of different electrophysiological techniques and analyses are conducted. The temporal relationships described are novel findings that suggest mechanisms behind the interactions between the key regions of interest. These may be of value for future experimental studies to test more directly their association with memory consolidation.

Weaknesses:

The number of findings provided is complex and some readers may struggle to follow all the details. The fact that bidirectional connections exist in the model system is not new per se. How and why the specific findings add to existing literature could still be presented with a little more impact. However, I am not sure this can be done more easily than is currently presented. I leave that to the authors to consider.

Reviewer #2 (Public review):

Summary:

Marylin Alves de Almeida et al. developed a novel mouse cross via conditionally depleting functional SMN protein in the liver (AlbCre/+;Smn2B/F7). This mouse model retains a proportion of SMN in the liver, which better recapitulates SMN deficiency observed in SMA patients and allows further investigation into liver-specific SMN deficiency and its systemic impact. They show that AlbCre/+;Smn2B/F7 mice do not develop an apparent SMA phenotype as mice did not develop motor neuron death, neuromuscular pathology or muscle atrophy, which is observed in the Smn2B/- controls. Nonetheless, at P19 and P60, these mice develop mild liver steatosis, and interestingly, this conditional depletion of SMN in the liver impacts cells in the pancreas.

Strengths:

The current model has clearly delineated the apparent metabolic perturbations which involve a significantly increased lipid accumulation in the liver and pancreatic cell defects in AlbCre/+;Smn2B/F7 mice at P19 and P60. Standard methods like H&E and Oil Red-O staining show that in AlbCre/+;Smn2B/F7 mice, their livers closely mimic the livers of Smn2B/- mice, which have the full body knockout of SMN protein. Unlike previous work, this liver-specific conditional depletion of SMN is superior in that it is not lethal to the mouse, which allows an opportunity to investigate the long-term effects of liver-specific SMN on the pathology of SMA.

Weaknesses:

Given that SMA often involves fatty liver, dyslipidemia and insulin resistance, using the current mouse model, the authors could have explored the long-term effects of liver-specific depletion of SMN on metabolic phenotypes beyond P19, as well as systemic effects like glucose homeostasis. Given that the authors also report pancreatic cell defects, the long-term effect on insulin secretion and resistance could be further explored. This has been addressed in the revised manuscript. The mechanistic link between a liver-specific SMN depletion and apparent pancreatic cell defects has been made clearer.

Discussion:

This current work explores a novel mouse cross in order to specifically deplete liver SMN using an Albumin-Cre driver line. This provides insight into the contribution of liver-specific SMN protein to the pathology of SMA, which is relevant for understanding metabolic perturbations in SMA patients. Nonetheless, given that SMA in patients involve a systemic deletion or mutation of the SMN gene, the authors could emphasize the utility of this liver-specific mouse model, as opposed to using in vitro models, which have been recently reported (Leow et al, 2024, JCI).

Comments on current version:

No further suggestions. Previous recommendations have been addressed by the authors.

Reviewer #2 (Public review):

Summary:

This study provides novel insights into the neurotransmitter release mechanisms employed by two distinct subclasses of dopaminergic neurons in the olfactory bulb (OB). The findings suggest that anaxonic neurons primarily release neurotransmitters through their dendrites, whereas axon-bearing neurons predominantly release neurotransmitters via their axons. Furthermore, the study reveals that anaxonic neurons exhibit self-inhibitory behavior, indicating that closely related neuronal subclasses may possess specialized roles in sensory processing.

Strengths:

This study introduces a novel and significant concept, demonstrating that two closely related neuron subclasses can exhibit distinct patterns of neurotransmitter release. Therefore, this finding establishes a valuable framework for future investigations into the functional diversity of neuronal subclasses and their contributions to sensory processing. Furthermore, these findings offer fundamental insights into the neural circuitry of the olfactory bulb, enhancing our understanding of sensory information processing within this critical brain region.

Weaknesses:

While this study offers novel insights, it is hindered by several limitations. The experimental approaches sometimes lack comprehensive justification and often rely on citations without providing adequate explanatory context. The small sample sizes (n values) compromise the statistical reliability and generalizability of the findings. Furthermore, the reliance on synaptophysin-based presynaptic structures raises concerns regarding whether these structures represent functional synapses. These shortcomings highlight the need for further refinement and additional data to substantiate the study's central conclusions. Addressing these concerns would improve the rigor and impact of the study's findings while ensuring the validity of its conclusions.

Reviewer #2 (Public review):

Summary:

The authors aimed to improve the detection of enkephalins, opioid peptides involved in pain modulation, reward, and stress. They used optogenetics, microdialysis and mass spectrometry to measure enkephalin release during acute stress in freely moving rodents. Their study provided detection and quantitation of enkephalins due to implementation of previously reported derivatization reaction combined with improved sample collection and offered insights into the dynamics and relationship between Met- and Leu-Enkephalin in the Nucleus Accumbens shell during stress.

Strengths:

A strength of this work is the quantitative Enk measurements resulted from an optimized microdialysis technique coupled with established derivatization approach and sensitive and quantitative nLC-MS measurements. This approach allowed basal and stimulated peptide release with higher temporal resolution, lower detection thresholds, and native-state endogenous peptide measurement.

Weaknesses:

The optimization of the previously published customizable microdialysis probe and the Met-Enk oxidation derivatization are included in the results, but these adjustments seem more like refinements or practical adaptations rather than significant innovations.

Another consideration is the use of log transformation for quantitation of peptides. Transforming data to achieve normality for parametric tests does not guarantee that all assumptions of normality are met, especially in small and variable datasets reported here. Visual checks like QQ-plots can help verify the appropriateness of transformations. In biological contexts, log transformation can obscure the relationship between measured values and underlying processes.

Reviewer #2 (Public review):

Summary:

In this paper, Oláh and colleagues introduce new research data on the cellular and biophysical elements involved in transmission within the pyramidal circuits of the human neocortex. They gathered a comprehensive set of patch-clamp recordings from human and rat pyramidal neurons to compare how the temporal aspect of neuronal processing is maintained in the larger human neocortex. A range of experimental techniques have been used, including two-photon guided dual whole-cell recordings, electron microscopy, complemented by theoretical and computational methods.

The authors find that synaptically connected pyramidal neurons within the human neocortex have longer intercellular path lengths. They go on to show that the short soma to soma latencies is not due to propagation velocity along the axon but instead reflects a higher propagation speed of synaptic potentials from dendrite to soma. Next, in a series of extensive computational modeling studies focusing on the synaptic potentials, the authors show that the shorter latency may be explained by larger diameters, affecting the cable properties and resulting is relatively faster propagation of EPSPs in the human neuron. The manuscript is well-written, and the physiological experiments and in-depth theoretical steps for the simulations are clear. Whether passive cable properties of the dendrites alone are responsible for higher velocities remains to be further investigated. Based on the present data the contribution of active membrane properties cannot be excluded.

Strengths:

The authors used complex 2P-guided dual whole-cell recordings in human neurons. In combination with detailed reconstructions, these approaches represent the next steps in unravelling the information processing in human circuits.

The computational modelling and cable theory application to the experimentally constrained simulations provide an integrated view of the passive membrane properties of human neurons.

Weaknesses:

There are concerns with the statistical analyses of the experimental data. The two-way analyses are not supporting that the backpropagation speed in human neurons is more affected by TTX-induced or after ZD remains higher. Significance of interaction is required, and the authors make errors in the interpretation and application of separate additional t-tests. Whether the cable properties alone are the main explanation for speeding the electrical signaling in human pyramidal neurons deserves further studies.

Comments on the latest version:

In my previous review I suggested the author read upon the need to perform two-way ANOVA for their experiments. Although I am glad this has now been done, I'm surprised to read the interpretation remains flawed and we are not provided with all the analyses. We need to know all the covariates and results of the post-hoc comparisons. What is written in the results is incomplete.

One cannot perform two-way ANOVA and subsequently performing t-tests on computed differences. Figures 3C and F are irrelevant. Instead, we need to know the Bonferroni post-hoc results for all comparisons.

If there is an interaction significance then the authors will have to conclude the Na+ channels have a larger contribution to the backpropagation.

Line187 "It therefore be argued that HCN channels may contribute to the higher conduction velocities in human dendrites, but do not by themselves explain the differences between the two species."

One wonders whether supplementary figures are required.

Reviewer #2 (Public review):

In this study, Nartker et al. examine how much observers are conscious of using variations of classic inattentional blindness studies. The key idea is that rather than simply ask observers if they noticed a critical object with one yes/no question, the authors also ask follow-up questions to determine if observers are aware of more than the yes/no questions suggest. Specifically, by having observers make forced choice guesses about the critical object, the authors find that many observers who initially said "no" they did not see the object can still "guess" above chance about the critical object's location, color, etc. Thus, the authors claim, that prior claims of inattentional blindness are mistaken and that using such simple methods has led numerous researchers to overestimate how little observers see in the world. To quote the authors themselves, these results imply that "inattentionally blind subjects consciously perceive these stimuli after all... they show sensitivity to IB stimuli because they can see them."

Before getting to a few issues I have with the paper, I do want to make sure to explicitly compliment the researchers for many aspects of their work. Getting massive amounts of data, using signal detection measures, and the novel use of a "super subject" are all important contributions to the literature that I hope are employed more in the future.

Main point 1: My primary issue with this work is that I believe the authors are misrepresenting the way people often perform inattentional blindness studies. In effect, the authors are saying, "People do the studies 'incorrectly' and report that people see very little. We perform the studies 'correctly' and report that people see much more than previously thought." But the way previous studies are conducted is not accurately described in this paper. The authors describe previous studies as follows on page 3:

"Crucially, however, this interpretation of IB and the many implications that follow from it rest on a measure that psychophysics has long recognized to be problematic: simply asking participants whether they noticed anything unusual. In IB studies, awareness of the unexpected stimulus (the novel shape, the parading gorilla, etc.) is retroactively probed with a yes/no question, standardly, "Did you notice anything unusual on the last trial which wasn't there on previous trials?". Any subject who answers "no" is assumed not to have any awareness of the unexpected stimulus.

If this quote were true, the authors would have a point. Unfortunately, I do not believe it is true. This is simply not how many inattentional blindness studies are run. Some of the most famous studies in the inattentional blindness literature do not simply as observes a yes/no question (e.g., the invisible gorilla (Simons et al. 1999), the classic door study where the person changes (Simons and Levin, 1998), the study where observers do not notice a fight happening a few feet from them (Chabris et al., 2011). Instead, these papers consistently ask a series of follow-up questions and even tell the observers what just occurred to confirm that observers did not notice that critical event (e.g., "If I were to tell you we just did XYZ, did you notice that?"). In fact, after a brief search on Google Scholar, I was able to relatively quickly find over a dozen papers that do not just use a yes/no procedure, and instead as a series of multiple questions to determine if someone is inattentionally blind. In no particular order some papers:

(1) Most et al. (2005) Psych Review<br /> (2) Drew et al. (2013) Psych Science<br /> (3) Drew et al. (2016) Journal of Vision<br /> (4) Simons et al. (1999) Perception<br /> (5) Simons and Levin (1998) Perception<br /> (6) Chabris et al. (2011) iPerception<br /> (7) Ward & Scholl (2015) Psych Bulletin and Review<br /> (8) Most et al. (2001) Psych Science<br /> (9) Todd & Marois (2005) Psych Science<br /> (10) Fougnie & Marois (2007) Psych Bulletin and Review<br /> (11) New and German (2015) Evolution and Human Behaviour<br /> (12) Jackson-Nielsen (2017) Consciousness and cognition<br /> (13) Mack et al. (2016) Consciousness and cognition<br /> (14) Devue et al. (2009) Perception<br /> (15) Memmert (2014) Cognitive Development<br /> (16) Moore & Egeth (1997) JEP:HPP<br /> (17) Cohen et al. (2020) Proc Natl Acad Sci<br /> (18) Cohen et al. (2011) Psych Science

This is a critical point. The authors' key idea is that when you ask more than just a simple yes/no question, you find that other studies have overestimated the effects of inattentional blindness. But none of the studies listed above only asked simple yes/no questions. Thus, I believe the authors are mis-representing the field. Moreover, many of the studies that do much more than ask a simple yes/no question are cited by the authors themselves! Furthermore, as far as I can tell, the authors believe that if researchers do these extra steps and ask more follow-ups, then the results are valid. But since so many of these prior studies do those extra steps, I am not exactly sure what is being criticized.

To make sure this point is clear, I'd like to use a paper of mine as an example. In this study (Cohen et al., 2020, Proc Natl Acad Sci USA) we used gaze-contingent virtual reality to examine how much color people see in the world. On the critical trial, the part of the scene they fixated on was in color, but the periphery was entirely in black and white. As soon as the trial ended, we asked participants a series of questions to determine what they noticed. The list of questions included:

(1) "Did you notice anything strange or different about that last trial?"<br /> (2) "If I were to tell you that we did something odd on the last trial, would you have a guess as to what we did?"<br /> (3) "If I were to tell you we did something different in the second half of the last trial, would you have a guess as to what we did?"<br /> (4) "Did you notice anything different about the colors in the last scene?"<br /> (5) We then showed observers the previous trial again and drew their attention to the effect and confirmed that they did not notice that previously.<br /> In a situation like this, when the observers are asked so many questions, do the authors believe that "the inattentionally blind can see after all?" I believe they would not say that and the reason they would not say that is because of the follow-up questions after the initial yes/no question. But since so many previous studies use similar follow-up questions, I do not think you can state that the field is broadly overestimating inattentional blindness. This is why it seems to me to be a bit of a straw-man: most people do not just use the yes/no method.

Main point 2: Let's imagine for a second that every study did just ask a yes/no question and then would stop. So, the criticism the authors are bringing up is valid (even though I believe it is not). I am not entirely sure that above chance performance on a forced choice task proves that the inattentionally blind can see after all. Could it just be a form of subliminal priming? Could there be a significant number of participants who basically would say something like, "No I did not see anything, and I feel like I am just guessing, but if you want me to say whether the thing was to the left or right, I will just 100% guess"? I know the literature on priming from things like change and inattentional blindness is a bit unclear, but this seems like maybe what is going on. In fact, maybe the authors are getting some of the best priming from inattentional blindness because of their large sample size, which previous studies do not use.<br /> I'm curious how the authors would relate their studies to masked priming. In masked priming studies, observers say the did not see the target (like in this study) but still are above chance when forced to guess (like in this study). Do the researchers here think that that is evidence of "masked stimuli are truly seen" even if a participant openly says they are guessing?

Main point 3: My last question is about how the authors interpret a variety of inattentional blindness findings. Previous work has found that observers fail to notice a gorilla in a CT scan (Drew et al., 2013), a fight occurring right in front of them (Chabris et al., 2011), a plane on a runway that pilots crash into (Haines, 1991), and so forth. In a situation like this, do the authors believe that many participants are truly aware of these items but simply failed to answer a yes/no question correctly? For example, imagine the researchers made participants choose if the gorilla was in the left or right lung and some participants who initially said they did not notice the gorilla were still able to correctly say if it was in the left or right lung. Would the authors claim "that participant actually did see the gorilla in the lung"? I ask because it is difficult to understand what it means to be aware of something as salient as a gorilla in a CT scan, but say "no" you didn't notice it when asked a yes/no question. What does it mean to be aware of such important, ecologically relevant stimuli, but not act in response to them and openly say "no" you did not notice them?

Overall: I believe there are many aspects of this set of studies that are innovative and I hope the methods will be used more broadly in the literature. However, I believe the authors misrepresent the field and overstate what can be interpreted from their results. While I am sure there are cases where more nuanced questions might reveal inattentional blindness is somewhat overestimated, claims like "the inattentionally blind can see after all" or "Inattentionally blind subjects consciously perceive thest stimuli after all" seem to be incorrect (or at least not at all proven by this data).

Reviewer #2 (Public review):

Summary:

This paper presents a very interesting use of a causal graph framework to identify the "root genes" of a disease phenotype. Root genes are the genes that cause a cascade of events that ultimately leads to the disease phenotype, assuming the disease progression is linear.

Strengths:

- The methodology has a solid theoretical background.<br /> - This is a novel use of the causal graph framework to infer root causes in a graph

Comments on revisions:

The authors addressed all of my comments.

Reviewer #2 (Public review):

Summary: This study proposes a novel role for ensheathing glia (EG) in a Pink1-model of Parkinson's disease and shows that this cell population exibits the highest number of DEG in a pre-symptomatic stage. In the olfactory system, there seems to be morphological changes in this cell-type that resembles an 'activated' state and the authors further show that the neuronal loss of Pink1 is responsible for this defect. The authors go on to show that manipulation of Pink1 in EG also leads to some defects in the visual system and in the dopaminergic neurons (DAN) that innervate the mushroom body (MB), and performed a screen based on the 'on-transient' defect of the ERG to identify potential genes that may modulate the function of EG in synaptic regulation. They focus on several genes related to Rab7/Vps13, and performed some additional experiments in the visual system and MB to propose the role of vesicle/lipid trafficking in EG as a important factor for PD pathogenesis.

Strengths: The study proposes functional and mechanistic connections between several genes that have been linked to PD (PINK1, VPS13A/C). I feel that the data presented in Figure 1 and Fig3A-C are performed with rigor and are convincing/novel. The selection of Drosophila to study the questions is also a strength and the lab has extensive experiences in this field and model organism.

Weaknesses: There is one fundamental concern I have with the genetic experiments performed in this paper (especially in Fig 3D and Fig4, see major issue #1), and I feel that there is a bit of a disconnect between the EG 'activation' phenotype the author show in the olfactory system and the other two neuronal systems (visual system, MB DAN) that the authors investigate see major issue #2). Also, there are quite a bit of information that is not provided in the manuscript (see major issues #3 and #4), which makes me difficult to judge the rigor and interpretation of several experiments.

Major Concern #1: A number of lines used in this study are referred to as "RNAi" lines but when I look at the actual genotypes of reagents listed in the table in the METHODS section, many are actually NOT RNAi lines. Quite a few lines, including lines that the authors use as RNAi against Ccz1, Rab7 and Mon1, are gRNA lines for the TKO (TRiP-CRISPR knockout) system. While these reagents can theoretically knock-out these genes in somatic cells if used in combination with UAS-Cas9, there is no mention that UAS-Cas9 was used in this work throughout the manuscript. Hence, when these lines are just crossed to GAL4 with or without the Pink1 mutant, they shouldn't be having any effects. Similarly, the strongest hit from their screen was a TOE (TRiP-CRISPR Over Expression) gRNA against PIG-A, which could allow overexpression of PIG-A if there is a UAS-dCas9::VP64. However, I also do not see any mention that such activator was introduced into the crossing scheme. Considering that 3 of the 4 'hits' from their screen are not RNAi lines, I am quite skeptical of the study. Similarly, except for Vps13, all reagents used in Fig4 are TKO gRNA lines. Therefore, if this experiment was conducted without an UAS-Cas9, most of the data shown here are problematic. Also, note that several of the 'RNAi' lines listed in the Table in the METHODS section are actually MiMIC alleles. While some MiMIC lines could function as strong LOF alleles (if they are inserted in the exon or in an intron of the gene in the same orientation as the gene), some of the lines are not expected to affect gene function (e.g. FASN2 and CG17712, MiMICs are in introns and face the opposite orientation). Hence, the rationale of including these reagents in the screen doesn't make much sense. The description of the modifier screen should be much more detailed in the RESULTS and METHODS section and if the UAS-Cas9/dCas9::VP64 transgenes were not introduced when the TKO/TOE reagents were utilized, what can be concluded?

In addition, for the 4 genes that the authors further study in Fig4, there are many other reagents that the authors can use, including mutant alleles, previously characterized RNAi lines (e.g. Vps13) and dominant negative/constitute active lines (e.g. especially for Rab7). The authors should validate their results with independent reagents to really convincingly show that the same conclusions can be drawn for the Vps13/Rab7 related genes since this is the key takeaway message of this paper.

Also, they do not show whether the manipulation of these genes in a wild-type background (they only show what happens in Pink1 mutants) affect ERG and MB DAN synapse morphology. If these manipulations alone dramatically affect these phenotypes, it would be very difficult to interpret their data.

Major Concern #2: In Figure 1, the authors show some morphological evidence that EG are 'activated' in Pink1 mutants, but whether the same phenomenon occurs in the visual system and in the MB is not shown. Since all of the studies in Fig3D and Fig4 are done in the visual system and MB, it is not clear whether the visual system and MB phenotypes are related to 'activation' of EG.

Also, in the RNA-seq data in Fig1A and Fig3C, is there any molecular evidence that EG are indeed 'activated'? The only evidence that the authors show to state that EG are 'activated' in young Pink1 null animals is based on increased CD8::GFP staining in the olfactory system.

The authors cannot draw a strong conclusion that indeed EG are 'activated' based on these data (e.g. perhaps the expression level of CD8::GFP is just increased). Additional evidence that the EG are 'activated' could be provided by looking at the increase in Draper intensity (as reported by Doherty et al. and MacDonald et al. that the authors cite), not only in the olfactory system, but also in the visual system and in the MB. It would also be informative if the authors can look at morphology of the EG in the visual system and MB to convincingly that the data shown in Fig4 is relevant to EG 'activation'.

Major Concern #3: In Fig3, there is no clear explanation why they focus on the ON transients and ignore the OFF transients, and also why the difference in the depolarization is not quantified in Fig4.

Major Concern #4: While the authors claim that mz709-GAL4 is a EG specific driver, do the authors know that this is indeed true in the tissues and stages that are studied here? The Ito et al,. paper that is cited in the METHOD section has only looked at the expression of this reporter in embryonic and larval stages. The authors need to that the authors should validate their findings with an additional EG specific driver and/or provide additional data that mz709-GAL4 is indeed specific to EG in the adult fly brain and eye. If mz709-GAL4 is expressed in other cell-types, the interpretation of many of the data in this paper becomes quite questionable. I believe the data in Fig3B is suggesting that mz709-GAL4 is indeed specific to glia cells and not expressed in neurons, but whether this driver is truly specific to EG (and not in other glial types), especially in the visual system (including the lamina as well as in the eye), is not obvious.

Reviewer #2 (Public review):

The authors inject, into the rete testes, mRNA and plasmids encoding mRNAs for GFP and then ARMC2 (into infertile Armc2 KO mice) in a gene therapy approach to express exogenous proteins in male germ cells. They do show GFP epifluorescence and ARMC2 protein in KO tissues, although the evidence presented is weak. Overall, the data do not necessarily make sense given the biology of spermatogenesis and more rigorous testing of this model is required to fully support the conclusions, that gene therapy can be used to rescue male infertility.

In this revision, the authors attempt to respond to the critiques from the first round of reviews. While they did address many of the minor concerns, there are still a number to be addressed. With that said, the data still do not support the conclusions of the manuscript.

(1) The authors have not satisfactorily provided an explanation for how a naked mRNA can persist and direct expression of GFP or luciferase for ~3 weeks. The most stable mRNAs in mammalian cells have half-lives of ~24-60 hours. The stability of the injected mRNAs should be evaluated and reported using cell lines. GFP protein's half-life is ~26 hours, and luciferase protein's half-life is ~2 hours.

(2) There is no convincing data shown in Figs. 1-8 that the GFP is even expressed in germ cells, which is obviously a prerequisite for the Armc2 KO rescue experiment shown in the later figures! In fact, to this reviewer the GFP appears to be in Sertoli cell cytoplasm, which spans the epithelium and surrounds germ cells - thus, it can be oft-confused with germ cells. In addition, if it is in germ cells, then the authors should be able to show, on subsequent days, that it is present in clones of germ cells that are maturing. Due to intracellular bridges, a molecule like GFP has been shown to diffuse readily and rapidly (in a matter of minutes) between adjacent germ cells. To clarify, the authors must generate single cell suspensions and immunostain for GFP using any of a number of excellent commercially-available antibodies to verify it is present in germ cells. It should also be present in sperm, if it is indeed in the germline.

Other comments:

70-1 This is an incorrect interpretation of the findings from Ref 5 - that review stated there were ~2,000 testis-enriched genes, but that does not mean "the whole process involves around two thousand of genes"

74 would specify 'male'

79-84 Are the concerns with ICSI due to the procedure itself, or the fact that it's often used when there is likely to be a genetic issue with the male whose sperm was used? This should be clarified if possible using references from the literature, as this reviewer imagines this could be a rather contentious issue with clinicians who routinely use this procedure, even in cases where IVF would very likely have worked

199 Codon optimization improvement of mRNA stability needs a reference; in one study using yeast transcripts, optimization improved RNA stability on the order of minutes (e.g., from ~5 minutes to ~17 minutes); is there some evidence that it could be increased dramatically to days or weeks?

472-3 The reported half-life of EGFP is ~36 hours - so, if the mRNA is unstable (and not measured, but certainly could be estimated by qRT-PCR detection of the transcript on subsequent days after injection) and EGFP is comparatively more stable (but still hours), how does EGFP persist for 21 days after injection of naked mRNA??

Curious why the authors were unable to get anti-GFP to work in immunostaining?

In Fig. 3-4, the GFP signals are unremarkable, in that they cannot be fairly attributed to any structure or cell type - they just look like blobs; and why, in Fig. 4D-E, why does the GFP signal appear stronger at 21 days than 15 days? And why is it completely gone by 28 days? This data is unconvincing. If the authors did a single cell suspension, what types or percentage of cells would be GFP+? Since germ cells are not adherent in culture, a simple experiment could be done whereby a single cell suspension could be made, cultured for 4-6 hours, and non-adherent cells "shaken off" and imaged vs adherent cells. Cells could also be fixed and immunostained for GFP, which has worked in many other labs using anti-GFP.

In Fig. 5, what is the half-life of luciferase? From this reviewer's search of the literature, it appears to be ~2-3 h in mammalian cells. With this said, how do the authors envision detectable protein for up to 20 days from a naked mRNA? The stability of the injected mRNAs should be shown in a mammalian cell line - perhaps this mRNA has an incredibly long half-life, which might help explain these results. However, even the most stable endogenous mRNAs (e.g., globin) are ~24-60 hrs.

527-8 The Sertoli cell cytoplasm is not just present along the basement membrane as stated, but also projects all the way to the lumina

529-30 This is incorrect, as round spermatids are never "localized between the spermatocytes and elongated spermatids" - if elongated spermatids are present, rounds are not - they are never coincident in the same testis section

Fig. 7 To this reviewer, all of the GFP appears to be in Sertoli cell cytoplasm

In Figs 1-8 there is no convincing evidence presented that GFP is expressed in germ cells! In fact, it appears to be in Sertoli cells

Fig. 9 - alpha-tubuline?

Fig. 11 - how was sperm morphology/motility not rescued on "days 3, 6, 10, 15, or 28 after surgery", but it was in some at 21 and 35? How does this make sense, given the known kinetics of male germ cell development?? And at least one of the sperm in the KO in Fig. B5 looks relatively normal, and the flagellum may be out-of-focus in the image? With only a few sperm for reviewers to see, how can we know these represent the population?

Reviewer #2 (Public review):

Summary:

Zhang et al. present a methodology to model protein-DNA interactions via learning an optimizable energy model, taking into account a representative bound structure for the system and binding data. The methodology is sound and interesting. They apply this model for predicting binding affinity data and binding sites in vivo. However, the manuscript lacks discussion of/comparison with state-of-the-art and evidence of broad applicability. The interpretability aspect is weak, yet over-emphasized.

Strengths:

The manuscript is well organized with good visualizations and is easy to follow. The methodology is discussed in detail. The IDEA energy model seems like an interesting way to study a protein-DNA system in the context of a given structure and binding data. The authors show that an IDEA model trained on one system can be transferred to other structurally similar systems. The authors show good performance in discriminating between binding-vs-decoy sequences for various systems, and binding affinity prediction. The authors also show evidence of the ability to predict genome-wide binding sites.

Weaknesses:

An energy-based model that needs to be optimized for specific systems is inherently an uncomfortable idea. Is this kind of energy model superior to something like Rosetta-based energy models, which are generally applicable? Or is it superior to family-specific knowledge-based models? It is not clear.

Prediction of binding affinity is a well-studied domain and many competitors exist, some of which are well-used. However, no quantitative comparison to such methods is presented. To understand the scope of the presented method, IDEA, the authors should discuss/compare with such methods (e.g. PMID 35606422).

The term "interpretable" has been used lavishly in the manuscript while providing little evidence on the matter. The only evidence shown is the family-specific residue-nucleotide interaction/energy matrix and speculations on how these values are biologically sensible. Recent works already present more biophysical, fine-grained, and sometimes family-independent interpretability (e.g. PMID 39103447, 36656856, 38352411, etc.). The authors should put into context the scope of the interpretability of IDEA among such works.

The manuscript disregards subtle yet important differences in commonly used terminology in the field. For example, the authors use the term "specificity" and "affinity" almost interchangeably (for example, the caption for Figure 3A uses "specificity" although the Methods text describes the prediction as about "affinity"). If the authors are looking to predict specificity, IDEA needs to be put in the context of the corresponding state-of-the-art (PMID 36123148, 39103447, 38867914, 36124796, etc).

It is not clear how much the learned energy model is dependent on the structural model used for a specific system/family. It would be interesting to see the differences in learned model based on different representative PDB structures used. Similarly, the supplementary figures show a lack of discriminative power for proteins like PDX1 (homeodomain family), POU, etc. Can the authors shed some light on why such different performances?

It is also not clear if IDEA's prediction for reverse complement sequences is the same for a given sequence. If so, how is this property being modelled? Either this description is lacking or I missed it.

Reviewer #2 (Public review):

Summary:

The posterior parietal cortex (PPC) has been identified as an integrator of multiple sensory streams and guides decision-making. Hira et al observe that dissection of the functional specialization of PPC subregions requires simultaneous measurement of neuronal activity throughout these areas. To this end, they use wide-field calcium imaging to capture the activity of thousands of neurons across the PPC and surrounding areas. They begin by delineating the boundaries between the primary sensory and higher visual areas using intrinsic imaging and validate their mapping using calcium imaging. They then conduct imaging during a visually guided task to identify neurons that respond selectively to visual stimuli or choices. They find that vision and choice neurons intermingle primarily in the anterior medial (AM) area, and that AM uniquely encodes information regarding both the visual stimulus and the previous choice, positioning AM as the main site of integration of behavioral and visual information for this task.

Strengths:

There is an enormous amount of data and results reveal very interesting relationships between stimulus and choice coding across areas and how network dynamics relate to task coding.

Weaknesses:

The enormity of the data and the complexity of the analysis make the manuscript hard to follow. Sometimes it reads like a laundry list of results as opposed to a cohesive story.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate how IL-4 modulates the reactive state of microglia in the context of neuropathic pain. Specifically, they sought to determine whether IL-4 drives an increase in CD11c+ microglial cells, a population associated with anti-inflammatory responses and whether this change is linked to the suppression of neuropathic pain. The study employs a combination of behavioral assays, pharmacogenetic manipulation of microglial populations, and characterization of microglial markers to address these questions.

Strengths:

The methodological approach in this study is robust, providing convincing evidence for the proposed mechanism of IL-4-mediated microglial regulation in neuropathic pain. The experimental design is well thought out, utilizing two distinct neuropathic pain models (SpNT and SNI), each yielding different outcomes. The SpNT model demonstrates spontaneous pain remission and an increase in the CD11c+ microglial population, which correlates with pain suppression. In contrast, the SNI model, which does not show spontaneous pain remission, lacks a significant increase in CD11c+ microglia, underscoring the specificity of the observed phenomenon. This design effectively highlights the role of the CD11c+ microglial population in pain modulation. The use of behavioral tests provides a clear functional assessment of IL-4 manipulation, and pharmacogenetic tools allow for precise control of microglial populations, minimizing off-target effects. Notably, the manipulation targets the CD11c promoter, which presumably reduces the risk of non-specific ablation of other microglial populations, strengthening the experimental precision. Moreover, the thorough characterization of microglial markers adds depth to the analysis, ensuring that the changes in microglial populations are accurately linked to the behavioral outcomes.

Weaknesses:

One potential limitation of the study is that the mechanistic details of how IL-4 induces the observed shift in microglial populations are not fully explored. While the study demonstrates a correlation between IL-4 and CD11c+ microglial cells, a deeper investigation into the specific signaling pathways and molecular processes driving this population shift would greatly strengthen the conclusions. Additionally, the paper does not clearly integrate the findings into the broader context of microglial reactive state regulation in neuropathic pain.

Reviewer #2 (Public review):

Summary:

Despite several methodological strengths, the major and highly significant drawback is the confound of arousal with movement. This confound is not resolved, so the results could be explained by previously established relationships between orexin and arousal/wakefulness.

Strengths:

The authors show that orexin neuron activity is associated with body movement and that this information is conveyed irrespective of the fasted state. They also report differences in different orexin target brain regions for orexin release during movement.

This paper contains an impressive array of cutting-edge techniques to examine a very important brain system, the orexin-hypocretin system. The authors offer an original perspective on the function of this system. The authors showed that orexin neuron activity scales to some degree with the magnitude of body movement change; this is unaffected by a fasted state and seems to be somewhat unique to orexin neurons.

The investigation of other genetically-defined subcortical neuron populations to determine the specificity of findings is also a strength, as is the ability to quantify movement and use deep learning to classify specific behaviors adds sophistication to analysis. The authors also show heterogeneity in orexin projections to specific target nuclei, which is interesting.

The authors "speculate that narcolepsy-cataplexy, caused by HON loss-of-function, is perhaps explained by oscillations into unwanted sleep-states and motor programs due to impaired control loops for wakefulness and movement". This is quite an interesting aspect of their work, and deserving of further study.

Weaknesses:

Despite the strengths, there are several major and minor weaknesses that detract significantly from the study.

Weaknesses - Major

My main concern with this work is the confound of arousal with movement so that correlations with one might reflect a relationship instead with the other. The orexin system is well known to play an important role in arousal, with elevated activity of orexin neurons reported for waking and high arousal. Orexin signaling has also been strongly associated with motivation, which also is associated with arousal and movement. The authors offer no compelling evidence that the relationships they describe between different movements and orexin signaling do not simply reflect the known relationship between arousal and motivation.

The authors could address this concern by including classical arousal measurements, eg, cortical EEG recorded simultaneously with movements. Often, EEG arousal occurs independently of movement, so this could provide one approach to disentangling this confound. The idea that orexin signaling plays a role in arousal rather than movement is supported by their finding that orexin lesions using the orexin-DTR mouse model did not impact movements. In contrast, prior lesion and pharmacologic studies have found that decreased orexin signaling significantly decreases arousal and waking.

Another way they could test their idea would be to paralyze and respirate animals so that orexin activity could be recorded without movement. Alternatively, animals could be trained to remain motionless to receive a reward. Thus, there are several ways to test the overall hypothesis of this work that have not been examined here.

The authors propose that "a simple interpretation of their results is that, via HON movement tracking, the brain creates a "wake up" signal in proportion to movement". This seems to argue for the role of the orexin system in arousal and motivation rather than in movement per se.

There are several studies that have examined the effect of orexin antagonist treatment in rodents on locomotor and other motor activities. These studies have largely found no consistent effect of antagonizing orexin signaling, especially at the OxR1 receptor, on simple motor activity. These studies are not referenced here but should be taken into account in the authors' conclusions.

Figure 3, panel F: I understand HON-DTR is a validated model but a picture of HONs ablation is necessary, including pictures of HONs outputs ablation within the SNc and LC.

The discussion lacks a more extensive paragraph on the distinct signal and role of Ox->SNc and Ox-LC projections.

Reviewer #2 (Public review):

The aim of the investigation was to find out more about the mechanism(s) by which the structural protein vimentin can facilitate the epithelial-mesenchymal transition in breast cancer cells.

The authors focussed on a key amino acid of vimentin, C238, its role in the interaction between vimentin and actin microfilaments, and the downstream molecular and cellular consequences. They model the binding between vimentin and actin in silico to demonstrate the potential involvement of C238, but the outcome is described vaguely. The phenotype of a non-metastatic breast cancer cell line MCF7, which doesn't express vimentin, could be changed to a metastatic phenotype when mutant C238S vimentin, but not wild-type vimentin, was expressed in the cells. Expression of vimentin was confirmed at the level of mRNA, protein, and microscopically. Patterns of expression of vimentin and actin reflected the distinct morphology of the two cell lines. Phenotypic changes were assessed through assay of cell adhesion, proliferation, migration, and morphology and were consistent with greater metastatic potential in the C238S MCF7 cells. Changes in the transcriptome of MCF7 cells expressing wild-type and C238S vimentins were compared and expression of Xist long ncRNA was found to be the transcript most markedly increased in the metastatic cells expressing C238S vimentin. Moreover changes in expression of many other genes in the C238S cells are consistent with an epithelial mesenchymal transition. Tumourigenic potential of MCF7 cells carrying C238S but not wild-type, vimentin was confirmed by inoculation of cells into nude mice. This assay is a measure of the stem-cell quality of the cells and not a measure of metastasis. It does demonstrate phenotypic changes that could be linked to metastasis.

shRNA was used to down-regulate vimentin or Xist in the MCF7 C238S cells. The description of the data is limited in parts and data sets require careful scrutiny to understand the full picture. Down-regulation of vimentin reversed the morphological changes to some degree, but down-regulation of Xist didn't. Conversely, down-regulation of Xist inhibited cell growth, a sign of reversing metastatic potential, but down-regulation of vimentin had no effect on growth. Down-regulation of either did inhibit cell migration, another sign of metastatic reversal. The interpretation of this type of experiment is handicapped when full reversal of expression is not achieved, as was the case in this study.

Overall the study describes an intriguing model of metastasis that is worthy of further investigation, especially at the molecular level to unravel the connection between vimentin and metastasis. The identification of a potential role for Xist in metastasis, beyond its normal role in female cells to inactivate one of the X chromosomes, corroborates the work of others demonstrating increased levels in a variety of tumours in women and even in some tumours in men. It would be of great interest to see where in metastatic cells Xist is expressed and what it binds to.

Reviewer #2 (Public review):

Summary:

This manuscript describes a comprehensive analysis of signalling downstream of the chemokine receptor CCR7. A comprehensive dataset supports the authors' hypothesis that G protein and beta arrestin signalling can occur simultaneously at CCR7 with implications for continued signalling following receptor endocytosis.

Strengths:

The experiments are well controlled and executed, employing a wide range of assay, using in the main, CCR7 transfectants. Data are well presented, with the authors claims supported by the data. The paper also has an excellent narrative which makes it relatively easy to follow. I think this would certainly be of interest to the readership of the journal.

Weaknesses:

The experiments are currently representative of signalling events in HEK293 transfectants and await verification in more relevant systems e.g. T-cells and dendritic cells.

Appraisal and Discussion

Overall, the authors appear to have achieved their experimental aims and provide substantial evidence that chemokine receptors can stimulate G proteins from within endosomes to regulate signalling pathways involved in cell migration. This builds upon earlier studies from the Legler group which showed that endocytosed CCR7 could activate Rac1 and influence lamellipodia formation. An unbiased mass spectrometry-based proteome profiling approach was used by the authors of this study to identify several candidate proteins which appear to play a role in receptor trafficking and signalling downstream of CCR7. These data may provide clues as to how other chemokine receptors are regulated post endocytosis in various leukocyte subsets.

Reviewer #3 (Public review):

In this work, the authors conducted a large-scale field trial of 130 indica accessions in normal vs. moderate salt stress conditions. The experiment consists of 3 replicates for each accession in each treatment, making it 780 plants in total. Leaf transcriptome, plant traits, and final yield were collected. Starting from a quantitative genetics framework, the authors first dissected the heritability and selection forces acting on gene expression. After summarizing the selection force acting on gene expression (or plant traits) in each environment, the authors described the difference in gene expression correlation between environments. The final part consists of eQTL investigation and categorizing cis- and trans-effects acting on gene expression.

Building on the group's previous study and using a similar methodology (Groen et al. 2020, 2021), the unique aspect of this study is in incorporating large-scale empirical field works and combining gene expression data with plant traits. Unlike many systems biology studies, this study strongly emphasizes the quantitative genetics perspective and investigates the empirical fitness effects of gene expression data. The large amounts of RNAseq data (one sample for each plant individual) also allow heritability calculation. This study also utilizes the population genetics perspective to test for traces of selection around eQTL. As there are too many genes to fit in multiple regression (for selection analysis) and to construct the G-matrix (for breeder's equation), grouping genes into PCs is a very good idea.

In the previous review, three major points were mentioned. The manuscript was modified, and here I briefly summarize them as a reference for future works:

(1) The separate sections (selection analysis, transcript correlation structure change, and eQTL) could use better integration.<br /> (2) It would be worth considering joint analyses integrating the two environments together.<br /> (3) Whether gene expression PCs or unique expression modules should be used in selection analyses.

Regarding whether to use PCs or WGCNA eigengenes to summarize gene expression for selection analyses, the authors reported that only a few WGCNA eigengenes were under selection, citing this observation as the rationale for choosing PC over eigengenes. However, as the relative false positive-negative rates of these choices likely require another dedicated study to explore, at this stage, it might be premature to state which method is better based on which gives more positive results. On one hand, one could easily imagine that plants screwed up by salinity have erratic genomewide expression and become extreme data points on the PCs, making the PCs a good proxy to correlate with fitness. On the other, it remains to be discussed whether this genomewide screwed-up-ness is what we want to measure in this study or whether we should focus on more dedicated gene modules instead. I suggest the authors acknowledge both possibilities. In this revision, I do not see relevant WGCNA results (as mentioned in the previous response letter) reported.

Figure 4: The observation that chlorophyll a content is under negative selection under BOTH conditions is a bit counterintuitive. The manuscript only mentioned "consistent with the general trend for reduced photosynthesis under salinity stress" (line 329) but did not mention why this increased fitness, even in normal conditions.

Reviewer #2 (Public review):

Summary:

This paper has some intriguing data regarding the different potential roles of Pch-2 in ensuring crossing over. In particular the alterations in crossover distribution and Msh-5 foci are compelling. My main issue is that some of the models are confusingly presented and would benefit from some reframing. The role of Pch-2 across organisms has been difficult to determine, the ability to separate pairing and synapsis roles in worms provides a great advantage for this paper.

Strengths:

Beautiful genetic data, clearly made figures. Great system for studying the role of Pch-2 in crossing over.

Comments on revisions: The authors have responded to all major and minor critiques.

Reviewer #2 (Public review):

Summary:

In the manuscript de la Fuente et al analyze the species specificity of sperm-egg recognition by looking at sperm binding and penetration of zonae pellucidae from different mammalian species and find a role for the oviductal protein OVGP1 in determining species specificity.

Strengths:

By combining sperm, oocytes, zona pellucida (ZP), and oviductal fluid from different mammalian species, they elucidate the essential role of OVGP1 in conferring species-specific fertilization.

Weaknesses:

Mice with OVGP1 deletion are viable and fertile. It would be quite interesting to investigate the species-specificity of sperm-ZP binding in this model. That would indicate whether OVGP1 is the only glycoprotein involved in determining species-specificity. Alternatively, the authors could immunodeplete OVGP1 from oviductal fluid and then ascertain whether this depleted fluid retains the ability to impede cross-species fertilization.

Reviewer #2 (Public Review):

Summary:

Environmental influences on development are ubiquitous, affecting many phenotypes in organisms. However molecular genetic and cellular mechanisms transducing environmental signals are still only barely understood. This study examines part of one such intracellular mechanism in a polyphenic (or dimorphic) aphid.

Strengths:

While other published reports have linked phenotypic plasticity to RNA editing before, this study reports such an interaction in insects. The study uses a wide array of molecular tools to identify connections upstream and downstream of the RNA editing to elucidate the regulatory mechanism, which is illuminating.

Weaknesses:

While this system is intriguing, this report does not foster confidence in its conclusions. Many of the analyses seem based on very small sample sizes. It is itself problematic that sample sizes are not obvious in most figures, although based on Methods section covering RNAseq, they seem to be either 3, 6 or 9, depending on whether stages were pooled, but that point is not made clear. With such small sample sizes, statistical tests of any kind are unreliable. Besides the ambiguity on sample sizes, it's unclear what error bars or whiskers show in plots throughout this study. When sample sizes are small estimates of variance are not reliable. Student's t-test is not appropriate for comparisons with such small sample sizes. Presently, it is not possible to replicate the tests shown in Figures 3, 4 and 6. (Besides the HT-seq reads, other data should also be made publicly available, following the journal's recommendations.) Regardless, effect sizes in some comparisons (Fig 3J, 4A-C, 6E,H) are clearly not large, making confidence in conclusions low. The authors should be cautious about over-interpreting these data.

[Editors' note: The authors made a great effort to address the reviewers' concerns. The current manuscript is significantly improved with additional data and clarification.]

Reviewer #2 (Public review):

Summary:

In the manuscript "Mapping HIV-1 RNA Structure, Homodimers, Long-Range Interactions and 1 persistent domains by HiCapR" Zhang et al report results from an omics-type approach to mapping RNA crosslinks within the HIV RNA genome under different conditions i.e. in infected cells and in virions. Reportedly, they used a previously published method which, in the present case, was improved for application to RNAs of low abundance.

Their claims include the detection of numerous long-range interactions, some of which differ between cellular and virion RNA. Further claims concern the detection and analysis of homodimers.

Strengths:

(1) The method developed here works with extremely little viral RNA input and allows for the comparison of RNA from infected cells versus virions.

(2) The findings, if validated properly, are certainly interesting to the community.

Weaknesses:

(1) On the communication level, the present version of the manuscript suffers from a number of shortcomings. I may be insufficiently familiar with habits in this community, but for RNA afficionados just a little bit outside of the viral-RNA-X-link community, the original method (reference 22) and the presumed improvement here are far too little explained, namely in something like three lines (98-100). This is not at all conducive to further reading.

(2) Experimentally, the manuscript seems to be based on a single biological replicate, so there is strong concern about reproducibility.

(3) The authors perform an extensive computational analysis from a limited number of datasets, which are in thorough need of experimental validation

Comments on revisions:

The authors have made cosmetic changes with regards to the problems I raised. 1 - Reproducibilty: the rebuttal letter says there are now 3 replicates, but there is only data for 2 in the supplement. The generation of biological replicates needs to be precisely stated, e.g. taken on different days, from separate cultures, or from neighbouring dishes on the same day etc. I think, the manuscript would greatly benefit from the comparison of at least 3 replicates that were not generated on the same day. Given that the authors report a r2 of 0.99 between the sets they have, this seems quite plausible.

The validation of the dimerisation sites is marginally better, but the authors should read up on significant digits and how precise Kd values can be determined.

The authors state that they want to make several of the experimeriments that would address my issues in the future in the context of another study. I find that disappointing, and correspondingly the present datasets insufficient for further endorsement.

Reviewer #2 (Public Review):

Summary:

In this work, the authors show that the camelid single-chain antibody sdAb42 selectivity inhibits Trypanosome pyruvate kinase (PYK) but not human PYK. Through the determination of the crystal structure and biophysical experiments, the authors show that the nanobody binds to the inactive T-state of the enzyme, and in silico analysis shows that the binding site coincides with an allosteric hotspot, suggesting that nanobody binding may affect the enzyme active site. Binding to the T-state of the enzyme is further supported by non-linear inhibition kinetics. PYK is an important enzyme in the glycolytic pathway, and inhibition is likely to have an impact on organisms such a trypanosomes, that heavily rely on glycolysis for their energy production. The nanobody was generated against Trypanosoma congolense PYK, but for technical reasons the authors progressed to testing its impact on cell viability in Trypanosoma brucei brucei. First, they show that sdA42 is able to inhibit Tbb PYK, albeit with lower potency. Cell-based experiments next show that expression of sdA42 has a modest, and dose-dependent effect on the growth rate of Tbb. The authors conclude that their data indicates that targeting this allosteric site affects cell growth and is a valuable new option for the development of new chemotherapeutics for trypanosomatid diseases.

Strengths:

The work clearly shows that sdA42A inhibits Trypanosome and Leishmania PYK selectively, with no inhibition of the human orthologue. The crystal structure clearly identifies the binding site of the nanobody, and the accompanying analysis supports that the antibody acts as an allosteric inhibitor of PYK, by locking the enzyme in its apo state (T-state).

Weaknesses:

(1) The most impactful claim of this work is that sdAb42-mediated inhibition of PYK negatively affects parasite growth and that this presents an opportunity to develop novel chemotherapeutics for trypanosomatid diseases. For the following reasons I think this claim is not sufficiently supported:

- The authors do not provide evidence of target-engagement in cells, i.e. they do not show that sdA42A binds to, or inhibits, Tbb PYK in cells and/or do not provide a functional output consistent with PYK inhibition (e.g. effect on ATP production). Measuring the extent of target engagement and inhibition is important to draw conclusions from the modest effect on growth.

- The authors do not explore the selectivity of sdA42A in cells. Potentially sdA42A may cross-react with other proteins in cells, which would confound interpretation of the results.

- sdA42A only affects minor growth inhibition in Tbb. The growth defect is used as the main evidence to support targeting this site with chemotherapeutics, however based on the very modest effect on the parasites, one could reasonably claim that PYK is actually not a good drug target. The strongest effect on growth is seen for the high expressor clone in Figure 4a, however here the uninduced cells show an unusual profile, with a sudden increase in growth rate after 4 days, something that is not seen for any of the other control plots. This unexplained observation accentuates the growth difference between induced and uninduced, and the growth differences seen in all other experiments, including those with the highest expressors (clones 54 and 55) are much more modest. The loss of expression of sdA42A over time is presented as a reason for the limited effect, and used to further support the hypothesis that targeting the allosteric site is a suitable avenue for the development of new drugs. However, strong evidence for this is missing.

- For chemotherapeutic interventions to be possible, a ligandable site is required. There is no analysis provided of the antibody binding site to indicate that small molecule binding is indeed feasible.

(2) The authors comment on the modest growth inhibition, and refer to the need to achieve over 88% reduction in Vmax of PYK to see a strong effect, something that may or may not be achieved in the cell-based model (no target-engagement or functional readout provided). The slow binding model and switch of species are also raised as potential explanations. While these may be plausible explanations, they are not tested which leaves us with limited evidence to support targeting the allosteric site on PYK.

(3) The evidence to support an allosteric mechanism is derived from structural studies, including the in silico allosteric network predictions. Unfortunately, standard enzyme kinetics mode of inhibition studies are missing. Such studies could distinguish uncompetitive from non-competitive behaviour and strengthen the claim that sdAb42 locks the enzyme complex in the apo form.

(4) As general comment, the graphical representation of the data could be improved in line with recent recommendations: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128, https://elifesciences.org/inside-elife/5114d8e9/webinar-report-transforming-data-visualisation-to-improve-transparency-and-reproducibility.

- Bar-charts for potency are ideally presented as dot plots, showing the individual data points, or box plots with datapoints shown.

- Images in Figure 7 show significant heterogeneity of nanobody expression, but the extent of this can not be gleaned from Figure 7B. It would be much better to use box plots or violin plots for each cell line on this figure panel. The same applies to Figure 10.

Comments on revision:

The authors have reduced the emphasis on the potential drug discovery applications. They are now referring to opportunities using a so called "chemo-superior" approach. This is not a commonly used term, and the newly added text seems to indicate that "chemo-superiors" target sites exposed by antibody binding, whereas the paper that the authors refer to (Lawson, 2012), defines "chemo-superiors" as small-molecules that induce similar effects to antibodies. I suggest removing the term "chemo-superior" altogether, as it has not been used since being coined in 2012, and instead simply point out the examples where antibodies have successfully informed small molecule design.

Unfortunately, the authors were unable to carry out additional experiments. Any experimental data to support their hypotheses as to why the observed growth defect is only marginal, and how the effect on growth could be increased, would have been very useful. As such, the evidence to support embarking on a drug discovery campaign for this allosteric site remains very limited.

The authors do provide some evidence of a druggable allosteric pocket, that partially overlaps with the antibody binding site, which is useful. However, I also ran the APOP tool on TcoPYK and it reveals 217 potential allosteric pockets all over the protein. The authors should provide the rank and APOP confidence score for the pocket that they have selected, to show that this is a high confidence allosteric pocket.

Reviewer #2 (Public review):

Summary:

This study by Pradhan et al. offers critical insights into the mechanisms by which antimony-resistant Leishmania donovani (LD-R) parasites alter host cell lipid metabolism to facilitate their own growth and, in the process, acquire resistance to amphotericin B therapy. The authors illustrate that LD-R parasites enhance LDL uptake via fluid-phase endocytosis, resulting in the accumulation of neutral lipids in the form of lipid droplets that surround the intracellular amastigotes within the parasitophorous vacuoles (PV) that support their development and contribute to amphotericin B treatment resistance. The evidence provided by the authors supporting the main conclusions is compelling, presenting rigorous controls and multiple complementary approaches. The work represents an important advance in understanding how intracellular parasites can modify host metabolism to support their survival and escape drug treatment.

Strengths:

(1) The study utilizes clinical isolates of antimony-resistant L. donovani and provides interesting mechanistic information regarding the increased LD-R isolate virulence and emerging amphotericin B resistance.

(2) The authors have used a comprehensive experimental approach to provide a link between antimony-resistant isolates, lipid metabolism, parasite virulence, and amphotericin B resistance. They have combined the following approaches:<br /> (a) In vivo infection models involving BL/6 and Apoe-/- mice.<br /> (b) Ex-vivo infection models using primary Kupffer cells (KC) and peritoneal exudate macrophages (PEC) as physiologically relevant host cells.<br /> (c) Various complementary techniques to ascertain lipid metabolism including GC-MS, Raman spectroscopy, microscopy.<br /> (d) Applications of genetic and pharmacological tools to show the uptake and utilization of host lipids by the infected macrophage resident L. donovani amastigotes.

(3) The outcome of this study has clear clinical significance. Additionally, the authors have supported their work by including patient data showing a clear clinical significance and correlation between serum lipid profiles and treatment outcomes.

(4) The present study effectively connects the basic cellular biology of host-pathogen interactions with clinical observations of drug resistance.

(5) Major findings in the study are well-supported by the data:<br /> (a) Intracellular LD-R parasites induce fluid-phase endocytosis of LDL independent of LDL receptor (LDLr).<br /> (b) Enhanced fusion of LDL-containing vesicles with parasitophorous vacuoles (PV) containing LD-R parasites both within infected KCs and PECs cells.<br /> (c) Intracellular cholesterol transporter NPC1-mediated cholesterol efflux from parasitophorous vacuoles is suppressed by the LD-R parasites within infected cells.<br /> (d) Selective exclusion of inflammatory ox-LDL through MSR1 downregulation.<br /> (e) Accumulation of neutral lipid droplets contributing to amphotericin B resistance.

Weaknesses:

The weaknesses are minor:

(1) The authors do not show how they ascertain that they have a purified fraction of the PV post-density gradient centrifugation.

(2) The study could have benefited from a more detailed analysis of how lipid droplets physically interfere with amphotericin B access to parasites.

Impact and significance:

This work makes several fundamental advances:

(1) The authors were able to show the link between antimony resistance and enhanced parasite proliferation.

(2) They were also able to reveal how parasites can modify host cell metabolism to support their growth while avoiding inflammation.

(3) They were able to show a certain mechanistic basis for emerging amphotericin B resistance.

(4) They suggest therapeutic strategies combining lipid droplet inhibitors with current drugs.

Reviewer #2 (Public review):

Summary:

In this study, the authors use a mechanical model to investigate how the geometry and deformations of myosin II filaments influence their force generation. They introduce a force generation efficiency that is defined as the ratio of the total generated force and the maximal force that the motors can generate. By changing the architecture of the myosin II filaments, they study the force generation efficiency in different systems: two filaments, a disorganized bundle, and a 2D network. In the simple two-filament systems, they found that in the presence of actin cross-linking proteins motors cannot add up their force because of steric hindrances. In the disorganized bundle, the authors identified a critical overlap of motors for cooperative force generation. This overlap is also influenced by the arrangement of the motor on the filaments and influenced by the length of the bare zone between the motor heads.

Strengths:

The strength of the study is the identification of organizational principles in myosin II filaments that influence force generation. It provides a complementary mechanistic perspective on the operation of these motor filaments. The force generation efficiency and the cooperative overlap number are quantitative ways to characterize the force generation of molecular motors in clusters and between filaments. These quantities and their conceptual implications are most likely also applicable in other systems.

Weaknesses:

The detailed model that the authors present relies on over 20 numerical parameters that are listed in the supplement. Because of this vast amount of parameters, it is not clear how general the findings are. On the other hand, it was not obvious how specific the model is to myosin II, meaning how well it can describe experimental findings or make measurable predictions. The model seems to be quantitative, but the interpretation and connection to real experiments are rather qualitative in my point of view.

It was often difficult for me to follow what parameters were changed and what parameters were set to what numerical values when inspecting the curve shown in the figures. The manuscript could be more specific by explicitly giving numbers. For example, in the caption for Figure 6, instead of saying "is varied by changing the number of motor arms, the bare zone length, the spacing between motor arms", the authors could be more specific and give the ranges: ""is varied by changing the number of motor arms form ... to .., the bare zone length from .. to..., and the spacing between motor arms from .. to ..".

This unspecificity is also reflected in the text: "We ran simulations with a variation in either Lsp or Lbz" What is the range of this variation? "When LM was similar" similar to what? "despite different NM." What are the different values for NM? These are only a few examples that show that the text could be way more specific and quantitative instead of qualitative descriptions.

In the text, after equation (2) the authors discuss assumptions about the binding of the motor to the actin filament. I think these model-related assumptions and explanations should be discussed not in the results section but rather in the "model overview" section.

The lines with different colors in Figure 2A are not explained. What systems and parameters do they represent?

Reviewer #2 (Public review):

Frangos et al present a set of studies aiming to determine mechanisms underlying initiation and tumour progression. Overall, this work provides some useful insights into the involvement of mitochondrial dysfunction during the cellular transformation process. This body of work could be improved in several possible directions to establish more mechanistic connections.

(1) The interesting point of the paper: the contrast between suppressed ETC components and activated OXPHOS function is perplexing and should be resolved. It is still unclear if activated mitochondrial function triggers gene down-regulation vs compensatory functional changes (as the title suggests). Have the authors considered reversing the HER2-derived signals e.g. with PI3K-AKT-MTOR or ERK inhibitors to potentially separate the expression vs. functional phenotypes? The root of the OXPHOS component down-regulation should also be traced further, e.g. by probing into levels of core mitochondrial biogenesis factors. Are transcript levels of factors encoded by mtDNA also decreased?

(2) The second interesting aspect of this study is the implication of mitochondrial activation in tumours, despite the downregulation of expression signatures, suggestive of a positive role for mitochondria in this tumour model. To address if this is correlative or causal, have the authors considered testing an OXPHOS inhibitor for suppression of tumorigenesis?

(3) A number of issues concerning animal/ tumour variability and further pathway dissection could be explored with in vitro approaches. Have the authors considered deriving tumour-derived cell cultures, which could enable further confirmations, mechanistic drug studies and additional imaging approaches? Culture systems would allow alternative assessment of mitochondrial function such as Seahorse or flow cytometry (mitochondrial potential and ROS levels).

(4) The study could be greatly improved with further confirmatory studies, eg immunoblotting for mitochondrial components with parallel blots for phospho-signalling in the same samples. It would be interesting if trends could be maintained in tumour-derived cell cultures. It is notable that OXPHOS protein/transcript changes are more consistent (Figure 5, Supplementary Figure 4) than mitochondrial dynamics /mitophagy factors (Figure 8). Core regulatory factors in these pathways should be confirmed by conventional immunoblotting.

Reviewer #2 (Public review):

This manuscript builds from the interesting observation that local recruitment of the DHPH domain of the RhoGEF PRG can induce local retraction, protrusion, or neither. The authors convincingly show that these differential responses are tied to the level of expression of the PRG transgene. This response depends on the Rho-binding activity of the recruited PH domain and is associated with and requires (co?)-activation of Cdc42. This begs the question of why this switch in response occurs. They use a computational model to predict that the timing of protein recruitment can dictate the output of the response in cells expressing intermediate levels and found that, "While the majority of cells showed mixed phenotypes irrespectively of the activation pattern, in few cells (3 out of 90) we were able to alternate the phenotype between retraction and protrusion several times at different places of the cell by changing the frequency while keeping the same total integrated intensity (Figure 6F and Supp Movie)."

Reviewer #2 (Public review):

Summary

The authors characterize the cell-cycle arrest induced by HIV-1 Vif in infected cells. They show this arrest is not at G2/M as previously thought but during metaphase. They show that the metaphase plate forms normally but progression to anaphase is massively delayed, and chromosome segregation is dysregulated in a manner consistent with impaired assembly of microtubules at the kinetochore. This correlates with the lack of recruitment of B56-subunits of PP2 phosphatase which are known degradation targets of Vif, suggesting that this weakens and unbalances the microtubule-mediated forces on the separating chromosomes.

Strengths

The authors present a very well-performed set of quantitative live cell imaging experiments that convincingly show a difference between Vif and Vpr-mediated cell cycle arrests. Through an in-depth characterization of the Vif-mediated block in metaphase, they make a strong case for this phenotype being tied to the degradation of PP2-B56 by Vif. Furthermore, it is important that they have performed most of these experiments with virally infected cells, meaning that their observations are observable at relevant viral expression levels of Vif.

Comments on revisions:

The authors have addressed the concerns and have discussed them accordingly. I hope they pursue the in vivo relevance in their future work

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors ran a dual task. Subjects monitored a peripheral location for a target onset (to generate a saccade to), and they also monitored a foveal location for a foveal probe. The foveal probe could be congruent or incongruent with the orientation of the peripheral target. In this study, the authors manipulated the conspicuity of the peripheral target, and they saw changes in performance in the foveal task. However, the changes were somewhat counterintuitive.

Strengths:

The authors use solid analysis methods and careful experimental design.

Comments on revisions:

The authors have addressed my previous comments.

One minor thing is that I am confused by their assertion that there was no smoothing in the manuscript (other than the newly added time course analysis). Figure 3A and Figure 6 seem to have smoothing to me.

Another minor comment is related to the comment of Reviewer 1 about oscillations. Another possible reason for what looks like oscillations is saccadic inhibition. when the foveal probe appears, it can reset the saccade generation process. when aligned to saccade onset, this appears like a characteristic change in different parameters that is time-locked to saccade onset (about a 100 ms earlier). So, maybe the apparent oscillation is a manifestation of such resetting and it's not really an oscillation. so, I agree with Reviewer 1 about removing the oscillation sentence from the abstract.

Reviewer #2 (Public review):

Summary:

The manuscript by Nohara et al. presents a novel 3D assay that allows for stratification of epithelia, active EMT through small pores, and active MET. They show that 3um pores allow for keratinocytes to sample the pore through filopodia and up-regulate EMT genes to transverse the pores to the other side of the membrane where EMT genes are downregulated as the cells re-establish stratified epithelia. The TGFbeta pathway and actin polymerization promote the movement of cells into the pores and Piezo1 and KRT6 actively block this movement. This work provides a novel 3D assay that is likely to become a benchmark to analyze these processes using a more complex system than other current culture-specific EMT and MET assays.

Strengths:

The strengths of the manuscript include the foundational analysis of the pathways involved in establishing the tri-phasic epithelium. The authors have incorporated live imaging, drug studies, KO analysis, and RNA sequencing to show the relevant pathways involved.

Weaknesses:

While the authors provide strong evidence that the tri-phasic epithelium represents the EMT process, the MET process is largely relegated to the absence of EMT genes. It would be interesting to know how the stratified MET epithelia submerged in the media is similar or different from the stratified epithelia at the air-liquid interface.

Reviewer #2 (Public review):

Summary:

In this study, the authors suggest that the structure of Piezo2 in a tensionless simulation is flatter compared to the electron microscopy structure. This is an interesting observation and highlights the fact that the membrane environment is important for Piezo2 curvature. Additionally, the authors calculate the excess area of Piezo2 and Piezo1, suggesting that it is significantly smaller compared to the area calculated using the EM structure or simulations with restrained Piezo2. Finally, the authors propose an elastic model for Piezo proteins. Those are very important findings, which would be of interest to the mechanobiology field.

Whilst I like the suggestion that the membrane environment will change Piezo2 flatness, could this be happening because of the lower resolution of the MARTINI simulations? In other words, would it be possible that MARTINI is not able to model such curvature due to its lower resolution?

Related to my comment above, the authors say that they only restrained the secondary structure using an elastic network model. Whilst I understand why they did this, Piezo proteins are relatively large. How can the authors know that this type of elastic network model restrains, combined with the fact that MARTINI simulations are perhaps not very accurate in predicting protein conformations, can accurately represent the changes that happen within the Piezo channel during membrane tension?

Modelling or Piezo1, seems to be based on homology to Piezo2. However, the authors need to further evaluate their model, e.g. how it compares with an Alphafold model.

To calculate the tension-induced flattening of the Piezo channel, the authors "divide all simulation trajectories into 5 equal intervals and determine the nanodome shape in each interval by averaging over the conformations of all independent simulation runs in this interval.". However, probably the change in the flattening of Piezo channel happens very quickly during the simulations, possibly within the same interval. Is this the case? and if yes does this affect their calculations?

Finally, the authors use a specific lipid composition, which is asymmetric. Is it possible that the asymmetry of the membrane causes some of the changes in the curvature that they observe? Perhaps more controls, e.g. with a symmetric POPC bilayer are needed to identify whether membrane asymmetry plays a role in the membrane curvature they observe.

Reviewer #2 (Public review):

This study analyzes the interaction among the gut microbiota, lipid metabolism, and the host in type 2 diabetes (T2DM) using rhesus macaques. The authors first identified 8 macaques with T2DM from 1698 individuals. Then, they observed in T2DM macaques: dysbiosis by 16S rRNA gene amplicon analysis and shotgun sequencing, imbalanced tryptophan metabolism and fatty acid beta oxidization in the feces by metabolome analysis, increased plasma concentration of palmitic acid by MS analysis, and sn inflammatory gene signature of blood cells by transcriptomic analysis. Finally, they transplanted feces of T2DM macaques into mice and fed them with palmitic acid and showed that those mice became diabetic through increased absorption of palmitic acid in the ileum.

This study clearly shows the interaction among gut microbiota, lipid metabolism, and the host in T2DM. The experiments were well designed and performed, and the data are convincing. One point I would suggest is that in the experiments of mice with FMT, control mice should be those colonized with feces of healthy macaques, but not with no FMT.

Reviewer #3 (Public review):

Arbués and colleagues describe the impact of mycobacterial genetic diversity on host-infection phenotypes. The authors evaluate Mtb infection and contextualize host-responses, bacterial growth and metabolic transitioning in vitro using their previously established model of blood-derived, primary-human-cells cultured within a collagen/fibronectin matrix. They seek to demonstrate the effectiveness of the model in determining mycobacterial strain specific granuloma-dependent host-pathogen interactions.

Understanding the way mycobacterial genetic diversity impacts granuloma biology in tuberculosis is an important goal. One of this works strengths is the use of primary human cells and two constituents of pulmonary extracellular matrix to model Mtb infection. The authors and others have previously shown that Mtb infected PBMC aggregates share important characteristics with early pulmonary TB granulomas. Use of multiple genetically distinct strains of Mtb defines this work and further bolsters it potential impact. However, the study is not comprehensive as lineages 6 and 7 are not tested. Experiments are primarily descriptive, and the methodologies are conventional. Correlative relationships are the manuscripts focus and effect sizes are generally small.

The main aim of this work is to extend an in vitro granuloma model to the study of a large collection of well characterized, genetically diverse representatives of the mycobacterium tuberculosis complex (MTBC). I believe that they accomplish that aim. The work does investigate MTBC infection of aggregated PBMCs using three strains each of Mtb lineages 1-5 and H37Rv, which is not a trivial undertaking. The experimental aims are to show that MTBC genetic diversity impacts growth and dormancy of granuloma bound bacteria and, the host responses of granulomatous aggregation as well as macrophage apoptosis, lymphocyte activation and soluble mediator release within granulomas. The methodologies employed are sufficient to test most of these aims. The authors conclusions regarding their results are mostly supported by the data. The conclusion that lineage impacts growth within granulomas is likely true and the data as presented reflect such a relationship. Their conclusions regarding lineage's impact on dormancy are partially supported, as their findings demonstrate that assays for dormancy identify strain-specific metabolic changes in the bacteria consistent with a dormancy-like state but also identify replicating bacteria as being dormant. The data strongly supports the impact of mycobacterial genetic diversity on a spectrum of granulomatous responses in their model system. Those findings are a highlight of the publication. The data further supports the idea that strain diversity impacts macrophage apoptosis but a relationship of apoptosis to the granulomatous response is not effectively evaluated. The association of lymphocyte activation with reduced mycobacterial growth as an aspect of granulomas is well documented in the literature and a negative correlation between T cell activation and growth is supported by the authors results. Their data also support the conclusion that soluble mediator production by PBMCs is different based on the infecting strain of mycobacteria and that IL1b modulates aggregate phenotypes in their model.

The authors contribute some valuable insights, particularly in figure 3. Their model is higher echelon relative to others in the field, but I don't believe that it possesses all the components necessary to replicate formation of mycobacterial granulomas in vivo. That being said, their identification of donor-dependent aggregation phenotypes by mycobacterial strain has the potential to enable future investigations of human and mycobacterial genetic components that are involved in the formation of TB granulomas.

Disease: Von-willebrand Disorder Type 2B, Noncanonical

Patient: 55 YO, Italian male

Note: these heterozygous variants are in cis mutations

Variant1: VWF NM_000552.5 c.2771G>A p.(R924Q), in exon 21, D'D3 domain affected

Variant2: VWF NM_000552.5 c.6532G>T p.(A2178S), in exon 37, D4 domain affected

Phenotypes: Mucosal and cutaneous bleeding, low VWF antigent and VWF activity, mild thrombocytopenia, increased ristocetin-induced platelet aggregation, deficiency in high-molecular-weight multimers. Epistaxis, repeated GI bleeding, easy bruising, ISTH BAT score of 7.

Note: Proband initially diagnosed with VWD type 1 in 2010 but later re-diagnosed as VWD-Type 2.

Family: No reported family history of bleeding, parents unable to be included in family work-up as they were deceased by time of study. Paternity test was performed for family, parental link established. Patient's daughter does not present the same mutation noted in proband but has heterozygous polymorphic variant inherited from mother which is: VWF NM_000552.5 c.3379C>T p.(P1127S), in exon 25. Daughter did not show menorrhagia or hemorrhagic disorder except for large hematoma on the thigh after trauma of moderate intensity.

Molecular workup: Paternity test, Sanger-sequencing to validate genetic variants, multimer analysis with electrophoresis, platelet aggregation analysis with Ristocetin, electron micrographs to observe conformation differences.

Prediction workup: Structural analysis with I-TASSER modeling program to identify perturbed structure. A2178S is predicted as tolerated in all in-silico systems used.

Database Information: Effect of the R924Q mutation is noted in literature database but has contradicting reports of effects altering VWF levels. Some studies do report it as a polymorphic variant

Variant is present in dbSNP database (rs33978901). MAF in European population 0.01< MAF< 0.02

Effect of A2178S variant is present in dbSNP database (rs34230288), MAF = 0.02 in European population.

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.

Comments on the latest version:

The authors have sufficiently addressed all issues raised with the initial manuscript.

Reviewer #2 (Public review):

This is an excellent and timely study from the Rao lab investigating the interactions of enteric glia with the intestinal epithelium. Two early studies in the late 90's and early 2000's had previously suggested that enteric glia play a pivotal role in control of the intestinal epithelial barrier, as their ablation using mouse models resulted in severe and fatal intestinal inflammation. However, it was later identified that these inflammatory effects could have been an indirect product of the transgenic mouse models used, rather than due to the depletion of enteric glia. In previous studies from this lab, the authors had identified expression of PLP1 in enteric glia, and its use in CRE driver lines to label and ablate enteric glia.

In the current paper, the authors carefully examine the role of enteric glia by first identifying that PLP1-creERT2 is the most useful driver to direct enteric glial ablation, in terms of the quantity of glial cells targeted, their proximity to the intestinal epithelium, and the relevance for human studies (GFAP expression is rather limited in human samples in comparison). They examined gene expression changes in different regions of the intestine using bulk RNA-seq following ablation of enteric glia by driving expression of diptheria toxin A (PLP1-creERT2;Rosa26-DTA). Alterations in gene expression were observed in different regions of the gut, with specific effects in different regions. Interestingly, while there were gene expression changes in the epithelium, there were limited changes to the proportions of different epithelial cell types identified using immunohistochemistry in control vs glial-ablated mice. The authors then focused on investigation of Paneth cells in the ileum, identifying changes in the ultrastructural morphology and lysozyme activity. In addition, they identified alterations in gut microbiome diversity. As Paneth cells secrete antimicrobial peptides, the authors conclude that the changes in gut microbiome are due to enteric glia-mediated impacts on Paneth cell activity.

Overall, the study is excellent and delves into the different possible mechanisms of action, including investigation of changes in enteric cholinergic neurons innervating the intestinal crypts. The use of different CRE-drivers to target enteric glial cells has led to varying results in the past, and the authors should be commended on how they address this in the Discussion.

Comments on the latest version:

Thanks to the authors for addressing my concerns. The additional stratification of male vs female microbiome data was very helpful.

Reviewer #2 (Public review):

Piersma et al. continue to work on deciphering the role and function of Ly49 NK cell receptors. This manuscript shows that a single inhibitory Ly49 receptor is sufficient to license NK cells and eliminate MHC-I-deficient target cells in mice. In short, they refined the mouse model ∆Ly49-1 (Parikh et al., 2020) into the Ly49KO model in which all Ly49 genes are disrupted. Using this model, they confirmed that NK cells from Ly49KO mice cannot be licensed, produce lower levels of IFN-gamma, and cannot reject MHC-I-deficient cells. To study the effect of a single Ly49 receptor in the function of NK cells, the authors backcrossed Ly49KO mice to H-2Dd transgenic KODO (D8-KODO) Ly49A knock-in mice in which a single inhibitory Ly49A receptor that recognizes H-2Dd ligands is expressed. By doing so, they demonstrate that a single inhibitory Ly49 receptor expressed by all NK cells is sufficient for licensing and missing-self killing.

While the results of the study are largely consistent with the conclusions, it is important to address some discrepancies. For instance, in the title of Figure 1, the authors state that NK cells in Ly49KO mice compared to WT mice have a less mature phenotype , which is not consistent with the corresponding text in the Results section (lines 170-171) that states there is no difference in maturation. These differences are not evident in Figure 1, panel D. It is crucial to acknowledge these inconsistencies to ensure a comprehensive understanding of the research findings.

In the legend of Figure 2. the text related to panel C indicates the use of dyes to label the splenocytes, and CFSE, CTV, and CTFR were mentioned. However, only CTV and CTFR are shown on the plots and mentioned in the corresponding text in the Results section. Similarly, in the legend of Figure 4, which is related to panel C, the authors write that splenocytes were differentially labeled with CFSE and CTV as indicated; however, in Figure 4, C and the Results section text, there is no mention of CFSE.

The authors should clarify why they assume that KLRG1 expression is influenced by the expression of inhibitory Ly49 receptors and not by manipulations on chromosome 6, where the genes for both KLRG1 and Ly49 receptors are located. However, a better explanation for the possible influence of other inhibitory NK cell receptors still needs to be included. In the study by Zhang et al. (doi: 10.1038/s41467-019-13032-5 the authors showed the synergized regulation of NK cell education by the NKG2A receptor and the specific Ly49 family members. Although in this study, Piersma and colleagues show the control of MHC-I deficient cells by Ly49A+ NKG2A-NK cells in Figure 4., this receptor is not mentioned in the Results or in the Discussion section, so its role in this story needs to be clarified. Therefore, the reader would benefit from more information regarding NKG2A receptor and NKG2A+/- populations in their results.

Comments on revisions: The authors have successfully answered all my questions and edited the manuscript accordingly.

Reviewer #3 (Public review):

Summary:

infectious bursal disease virus (IBDV) is a birnavirus and an important avian pathogen. Interestingly, IBDV appears to be a unique dsRNA virus that uses early endosomes for RNA replication that is more common for +ssRNA viruses such as for example SARS-CoV-2.

This work builds on previous studies showing that IBDV VP3 interacts with PIP3 during virus replication. The authors provide further biophysical evidence for the interaction and map the interacting domain on VP3.

Strengths:

Detailed characterization of the interaction between VP3 and PIP3 identified R200D mutation as critical for the interaction. Cryo-EM data show that VP3 leads to membrane deformation.

Comments on revisions:

I have no further comments. The authors have addressed my questions and concerns. I congratulate the authors on their work!

Reviewer #2 (Public Review):

When people help others is an important psychological and neuroscientific question. It has received much attention from the psychological side, but comparatively less from neuroscience. The paper translates some ideas from a social Psychology domain to neuroscience using a neuroeconomically oriented computational approach. In particular, the paper is concerned with the idea that people help others based on perceptions of merit/deservingness, but also because they require/need help. To this end, the authors conduct two experiments with an overlapping participant pool:

(1) A social perception task in which people see images of people that have previously been rated on merit and need scales by other participants. In a blockwise fashion, people decide to whether the depicted person a) deserves help, b) needs help, and c) whether the person uses both hands (== control condition)

(2) In an altruism task, people make costly helping decisions by deciding between giving a certain amount of money to themselves or another person. It is manipulated how much the other person needs and deserves the money.

The authors use sound and robust computational modelling approach for both tasks using evidence accumulation models. They analyse behavioural data for both tasks, showing that the behaviour is indeed influenced, as expected, by the deservingness and the need of the shown people. Neurally, the authors use a block-wise analysis approach to find differences in activity levels across conditions of the social perception task. The authors do find large activation clusters in areas related to theory of mind. Interestingly, they also find that activity in TPJ that relates to the deservingness condition correlates with people's deservingness ratings while they do the task, but also with computational parameters related to helping others in the second task, the one that was conducted many months later. Also some behavioural parameters correlate across the two tasks, suggesting that how deserving of help others are perceived reflects a relatively stable feature that translates into concrete helping decisions later-on.

The conclusions of the paper are overall well supported by the data.

(1) I found that the modelling was done very thoroughly for both tasks. Overall, I had the impression that the methods are very solid with many supplementary analyses. The computational modelling is done very well.

(2) A slight caveat, however, regarding this aspect, is that, in my view, the tasks are relatively simplistic, so that even the complex computational models do not as much as they can in the case of more complex paradigms. For example, the bias term in the model seems to correspond to the mean response rate in a very direct way (please correct me if I am wrong).

(3) Related to the simple tasks: The fMRI data is analysed in a simple block-fashion. This is in my view not appropriate to discern the more subtle neural substrates of merit/need-based decision making or person perception. Correspondingly, the neural activation patterns (merit > control, need > control) are relatively broad and unspecific. They do not seem to differ in the classic theory of mind regions, that are the focus of the analyses.

(4) However, the relationship between neural signal and behavioural merit sensitivity in TPJ is noteworthy.

(5) The latter is even more the case, as the neural signal and aspects of the behaviour are correlated across subjects with the second task that is conducted much later. Such a correlation is very impressive and suggests that the tasks are sensitive for important individual differences in helping perception/behaviour.

(6) That being said, the number of participants in the latter analyses are at the lower end of the number of participants that are these days used for across-participant correlations.

Reviewer #2 (Public review):

In this manuscript, Hua et al. proposed SLC7A11, a protein facilitating cellular cystine uptake, as a potential target for the treatment of trastuzumab-resistant HER2-positive breast cancer. If this claim holds true, the finding would be of significance and might be translated to clinical practice. Nevertheless, this reviewer finds that the conclusion was poorly supported by the data.

Notably, most of the data (Figures 2-6) were based on two cell lines - JIMT1 as a representative of trastuzumab-resistant cell line, and SKBR3 as a representative of trastuzumab sensitive cell line. As such, these findings could be cell-line specific while irrelevant to trastuzumab sensitivity at all. Furthermore, the authors claimed ferroptosis simply based on lipid peroxidation (Figure 3). Cell viability was not determined, and the rescuing effects of ferroptosis inhibitors were missing. The xenograft experiments were also suspicious (Figure 4). The description of how cysteine starvation was performed on xenograft tumors was lacking, and the compound (i.e., erastin) used by the authors is not suitable for in vivo experiments due to low solubility and low metabolic stability. Finally, it is confusing why the authors focused on epigenetic regulations (Figures 5 & 6), without measuring major transcription factors (e.g., NRF2, ATF4) which are known to regulate SLC7A11.

To sum up, this reviewer finds that the most valuable data in this manuscript is perhaps Figure 1, which provides unbiased information concerning the metabolic patterns in trastuzumab-sensitive and primary resistant HER2-positive breast cancer patients.

Reviewer #2 (Public review):

Summary:

This paper investigates the mode of action of GPR55, a relatively understudied type of cannabinoid receptor, in presynaptic terminals of Purkinje cells. The authors use demanding techniques of patch clamp recording of the terminals, sometimes coupled with another recording of the postsynaptic cell. They find a lower release probability of synaptic vesicles after activation of GPR55 receptors, while presynaptic voltage-dependent calcium currents are unaffected. They propose that the size of a specific pool of synaptic vesicles supplying release sites is decreased upon activation of GPR55 receptors.

Strengths:

The paper uses cutting-edge techniques to shed light on a little-studied, potentially important type of cannabinoid receptor. The results are clearly presented, and the conclusions are for the most part sound.

Weaknesses:

The nature of the vesicular pool that is modified following activation of GPR55 is not definitively characterized.

Reviewer #2 (Public review):

Summary:

The authors describe the development and implementation of hamFISH, a sensitive multiplexed ISH method. They leverage a pre-existing scRNA-seq dataset for the MeA to design 32 probes that combinatorically represent MeA neuronal populations - ~80% of MeA neurons express three of these markers. Using these markers to assess the spatial organization of the MeA, the authors identify a novel population of Ndnf+ projection neurons and characterize their connectivity with anterograde and retrograde labeling. They additionally combine hamFISH with CTB labeling of three principal MeA projection sites to show that 75% of MeA neurons have only a single projection target. Finally, they engage adult male mice in encounters with other adult males (aggression), females (mating), and pups (infanticide), followed by hamFISH and c-fos labeling to relate cell identity to behavior. Their overall conclusion is that hamFISH-defined cell types are broadly active to multiple sensory stimuli. However, the data presented are not sufficient to conclude that no selectivity exists within the MeA. A weakness of the study is that the selected hamFISH genes contain only Lhx6 as a lineage-marking transcription factor. Instead, the authors predominately use neuropeptides as markers. Genes such as Tac1, Cartpt, Adcyap1, Calb1, and Gal are expressed throughout the MeA, and many other brain regions; they are not restricted to a single transcriptomic cell type and they do not denote any developmental origins. By design, the panel has low cell type specificity as all MeA neurons express at least three of the genes. Therefore, the authors' conclusions may not hold with a more stringent classification of cell type or cell identity.

Reviewer #2 (Public review):

Summary:

Using the well-studied oxalate-microbiome-host system, the authors propose a novel conceptual and experimental framework for developing targeted bacteriotherapies using a three-phase pre-clinical workflow. The third phase is based on a 'complex system theoretical approach' in which multi-omics technologies are combined in independent in vivo and in vitro models to successfully identify the most pertinent variables that influence specific phenotypes in diet-host-microbe systems. The innovation relies on the third phase since phase I and phase II are the dominant approaches everyone in the microbiome field uses.

Strengths:

The authors used a multidisciplinary approach which included:

(1) fecal transplant of two distinct microbial communities into Swiss-Webster mice (SWM) to characterize the host response (hepatic response-transcriptomics) and microbial activity (untargeted metabolomics of the stool samples) to different oxalate concentrations;

(2) longitudinal analysis of the N. albigulia gut microbiome composition in response to varying concentrations of oxalate by shotgun metagenomics, with deep bioinformatic analyses of the genomes assembled; and

(3) development of synthetic microbial communities around oxalate metabolisms and evaluation of these communities' activity in oxalate degradation in vivo.

Weaknesses:

However, I have concerns about the frame the authors tried to provide for a 'complex system theoretical approach' and how the data are interpreted within this frame. Several of the conclusions the authors provide do not seem to have sufficient data to support them.

Reviewer #2 (Public review):

Summary:

This manuscript by Bisson et al describes the role GATA6 to regulate cardiac progenitor cell (CPC) specification and cardiomyocyte (CM) generation using human embryonic stem cells (hESCs). The authors found that GATA6 loss-of-function hESC exhibit early defects in mesendoderm and lateral mesoderm patterning stages. Using RNA-seq and CUT&RUN assays the genes of the Wnt and BMP programs were found to be affected by the loss of GATA6 expression. Modulating Wnt and BMP during early cardiac differentiation can partially rescue CPC and CM defects in GATA6 hetero- and homozygous mutant hESCs.

Strengths:

The studies performed were rigorous and the rationale for the experimental designed were logical. The results obtained were clear and supports the conclusions that the authors made regarding the role of GATA6 on Wnt and BMP pathway gene expression.

Weaknesses:

Given the wealth of studies that have been performed in this research area previously, the amount of new information provided in this study is relatively modest. Nevertheless, the results and quite clear and should make a strong contribution to the field.

Comments on revised version:

The authors have addressed the prior request to assess genes expression representing each stage of development/differentiation from mesoderm to cardiac progenitor to cardiomyocytes and confirmed that the differentiation defect lies at the cardiac progenitor and cardiomyocyte stages and not in mesodermal differentiation. This work has significantly improved the robustness of the study.

Reviewer #2 (Public review):

Summary:

In this article, the authors study the function of TEDC1 and TEDC2, two proteins previously reported to interact with TUBD1 and TUBE1. Previous work by the same group had shown that TUBD1 and TUBE1 are required for centriole assembly and that human cells lacking these proteins form abnormal centrioles that only have singlet microtubules that disintegrate in mitosis. In this new work, the authors demonstrate that TEDC1 and TEDC2 depletion results in the same phenotype with abnormal centrioles that also disintegrate into mitosis. In addition, they were able to localize these proteins to the proximal end of the centriole, a result not previously achieved with TUBD1 and TUBE1, providing a better understanding of where and when the complex is involved in centriole growth.

Strengths:

The results are very convincing, particularly the phenotype, which is the same as previously observed for TUBD1 and TUBE1. The U-ExM localization is also convincing: despite a signal that's not very homogeneous, it's clear that the complex is in the proximal region of the centriole and procentriole. The phenotype observed in U-ExM on the elongation of the cartwheel is also spectacular and opens the question of the regulation of the size of this structure. The authors also report convincing results on direct interactions between TUBD1, TUBE1, TEDC1, and TEDC2, and an intriguing structural prediction suggesting that TEDC1 and TEDC2 form a heterodimer that interacts with the TUBD1- TUBE1 heterodimer.

Comments on revisions:

I would like to thank the authors for their work and for thoroughly addressing most of my questions. I extend my congratulations to the authors for this excellent and impactful article.

Reviewer #2 (Public review):

Summary:

In this study, the authors identified and characterized a regulatory mechanism based on transcriptional anti-termination that connects the two gene clusters, capsid and run-off replication (ROR) locus, of the bipartite Bartonella gene transfer agent (GTA). Among genes essential for GTA functionality identified in a previous transposon sequencing project, they found a potential antiterminatior of phage origin within the ROR locus. They employed fluorescence reporter and gene transfer assays of overexpression and knockout strains in combination with ChiPSeq and promoter-fusions to convincingly show that this protein indeed acts as an antiterminator counteracting attenuation of the capsid gene cluster expression.

Impact on the field:

The results provide valuable insights into the evolution of the chimeric BaGTA, a unique example of phage co-domestication by bacteria. A similar system found in the other broadly studied Rhodobacterales/Caulobacterales GTA family suggests that antitermination could be a general mechanism for GTA control.

Strengths:

Results of the selected and carefully designed experiments support the main conclusions.

Weaknesses:

It remains open why overexpression of the antiterminator does not increase the gene transfer frequency.

Reviewer #2 (Public review):

In the present manuscript, Huang et.al. revealed the significant roles of the DNA methylome in regulating virulence and metabolism within Pseudomonas syringae, with a particular focus on the HsdMSR system in this model strain. The authors used SMRT-seq to profile the DNA methylation patterns (6mA, 5mC, and 4mC) in three P. syringae strains (Psph, Pss, and Psa) and displayed the conservation among them. They further identified the type I restriction-modification system (HsdMSR) in P. syringae, including its specific motif sequence. The HsdMAR participated in the process of metabolism and virulence (T3SS & Biofilm formation), as demonstrated through RNA-seq analyses. Additionally, the authors revealed the mechanisms of the transcriptional regulation by 6mA. Strictly from the point of view of the interest of the question and the work carried out, this is a worthy and timely study that uses third-generation sequencing technology to characterize the DNA methylation in P. syringae. The experimental approaches were solid, and the results obtained were interesting and provided new information on how epigenetics influences the transcription in P. syringae. The conclusions of this paper are mostly well supported by data.

Comments on revisions:

The authors have successfully addressed all the comments from the reviewers in their revised manuscript.

Reviewer #3 (Public review):

Hong et al. used a model they previously developed to study the impact of plasmid transfer on microbial multispecies communities. They investigated the effect of plasmid transfer on the existence of alternative stable states in a community. The model most closely resembles plasmid conjugation, where the transferred genes confer independent growth-related fitness effects and different plasmids do not affect each other's transfer or growth effects. For this process, the authors find that increasing the rate of plasmid transfer leads to an increasing number of stable states, as long as the model includes a constant death/dilution term.

This is an interesting and important topic, and I welcome the authors' efforts to explore these topics with mathematical modeling. The addition of sensitivity analyses also strengthens the usefulness for quantitative microbial ecologists. However, the additional sections have made the main text harder to read. Between the effect of the dilution rate, the increase in subpopulations with HGT, and the modulation of interspecies competition, the reviewers have suggested a number of factors that may explain the way plasmid transfer modulates multistability. I think it would be helpful if the authors could summarize some of these effects/interactions between different parameters in their model more. I personally continue to find the model very unintuitive, especially in the way it averages over subpopulations carrying more than one foreign plasmid. Additional sentences that give the reader intuition for the sensitivity analyses and how these interplay with the results would be good.

Specific points

(1) The model makes strong assumptions about the biology of HGT, that could be spelled out even more. Since the model is primarily applicable to HGT driven by the exchange of plasmids, I believe the abstract (and perhaps even the title of the paper) should be updated to reflect that.

(2) I am not surprised that a mechanism that creates diversity will lead to more alternative stable states. Specifically, the null model for the absence of HGT is to set gamma to zero, resulting in pij=0 for all subpopulations (line 454). This means that a model with N^2 classes is effectively reduced to N classes. It seems intuitive that an LV-model with many more species would also allow for more alternative stable states. For a fair comparison one would really want to initialize these subpopulations in the model (with the same growth rates - e.g. mu1(1+lambda2)) but without gene mobility.<br /> [Update:] It is good that it seems that initializing pij with non-zero abundance did not seem to affect the conclusion that higher amounts of HGT increases multi stability. However, rather than listing it as one control for a specific condition, I would argue that this is the appropriate null model across the board (where HGT rate is varied from 0 to a high value), including figures S9 and S10.

(3) The possibility that the same cell may be counted in different pij runs counter to all intuition that researchers coming from a background of compartmental /epidemiological modeling may have. The associated assumption that plasmids do not affect each other's dynamics or (growth/interaction) effects at all is also a very strong assumption. This should be signaled much earlier in the manuscript, possibly already in line 106 when the model is introduced.

Reviewer #2 (Public review):

In this study the authors developed a framework to investigate the export rates of Influenza viral RNAs translocating from the nucleus to the cytoplasm. This model suggests that the influenza virus may control gene expression at the RNA export level, namely, the retention of certain transcripts in the nucleus for longer times, allows the generation of other viral encoded proteins that are exported regularly, and only later on do certain mRNAs get exported. These encode proteins that alert the cell to the presence of viral molecules, hence keeping their emergence to very end, might help the virus to avoid detection as late as possible in the infection cycle.

The study is of limited scope. The notion that some mRNAs are retained in the nucleus after transcription is concluded early on from the FISH data. The model does not contribute much to the understanding and is mostly confirming the FISH data. The export rate is an ambiguous number and this part is not elaborated upon. One is left with more questions since no mechanistic knowledge emerges, and no additional experimentation is attempted to try drive to a deeper understanding.

Comments on revisions:

The authors have implemented the comments that required textual rewriting, which does make the paper clearer. On the experimental side, very little was done. It is fine to answer that the suggested experiments are not relevant or feasible for one reason or another, but one would expect to see some effort in providing other experimental sets to address key comments, and not only to modify a sentence in the text. So in my mind this round of revision feels more like some kind of intellectual discussion, which is fine, but I would have expected more, particularly after so much time has passed. I am still not satisfied with the way the analysis is presented in Fig. 2B, and writing a line about what is not analyzed in the legend, does not seem clear enough.

Reviewer #2 (Public review):

Summary:

Giménez-Orenga carried out this study to assess whether human endogenous retroviruses (HERVs) could be used to improve the diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Fibromyalgia (FM). To this end, they used the HERV-V3 array developed previously, to characterize the genome-wide changes in the expression of HERVs in patients suffering from ME/CFS, FM, or both, compared to controls. In turn, they present a useful repertoire of HERVs that might characterize ME/CFS and FM. For the most part, the paper is written in a manner that allows a natural understanding of the workflow and analyses carried out, making it compelling. The figures and additional tables present solid support for the findings. However, some statements made by the authors seem incomplete and would benefit from a more thorough literature review. Overall, this work will be of interest to the medical community seeking in better understanding of the co-occurrence of these pathologies, hinting at a novel angle by integrating HERVs, which are often overlooked, into their assessment.

Strengths:

(1) The work is well-presented, allowing the reader to understand the overall workflow and how the specific aims contribute to filling the knowledge gap in the field.

(2) The analyses carried out to understand the potential impact on gene expression mediated by HERVs are in line with previous works, making it solid and robust in the context of this study.

Weaknesses:

(1) The authors claim to obtain genome-wide HERV expression profiles. However, the array used was developed using hg19, while the genomic analysis of this work are carried out using a liftover to hg38. It would improve the statement and findings to include a comparison of the differences in HERVs available in hg38, and how this could impact the "genome-wide" findings.

(2) The authors in some points are not thorough with the cited literature. Two examples are:<br /> a) Lines 396-397 the authors say "the MLT1, usually found enriched near DE genes (Bogdan et al., 2020)". I checked the work by Bogdan, and they studied bacterial infection. A single work in a specific topic is not sufficient to support the statement that MLT1 is "usually" in close vicinity to differentially expressed genes. More works are needed to support this.<br /> b) After the previous statement, the authors go on to mention "contributing to the coding of conserved lncRNAs (Ramsay et al., 2017)". First, lnc = long non-coding, so this doesn't make sense. Second, in the work by Ramsay they mention "that contributed a significant amount of sequence to primate lncRNAs whose expression was conserved", which is different from what the authors in this study are trying to convey. Again, additional work and a rephrasing might help to support this idea.

(3) When presenting the clusters, the authors overlook the fact that cluster 4 is clearly control-specific, and fail to discuss what this means. Could this subset of HERV be used as bona fide markers of healthy individuals in the context of these diseases? Are they associated with DE genes? What could be the impact of such associations?

Appraisals on aims:

The authors set specific questions and presented the results to successfully answer them. The evidence is solid, with some weaknesses discussed above that will methodologically strengthen the work.

Likely impact of work on the field:

This work will be of interest to the medical community looking for novel ways to improve clinical diagnosis. Although future works with a greater population size, and more robust techniques such as RNA-Seq, are needed, this is the first step in presenting a novel way to distinguish these pathologies.

It would be of great benefit to the community to provide a table/spreadsheet indicating the specific genomic locations of the HERVs specific to each condition. This will allow proper provenance for future researchers interested in expanding on this knowledge, as these genomic coordinates will be independent of the technique used (as was the array used here).

Reviewer #2 (Public review):

Summary:

The authors have examined the effects of two parameters that could improve their clade forecasting predictions for A(H3N2) seasonal influenza viruses based solely on analysis of haemagglutinin gene sequences deposited on the GISAID Epiflu database. Sequences were analysed from viruses collected between April 1, 2005 and October 1, 2019. The parameters they investigated were various lag periods (0, 1, 3 months) for sequences to be deposited in GISAID from the time the viruses were sequenced. The second parameter was the time the forecast was accurate over projecting forward (for 3,6,9,12 months). Their conclusion (not surprisingly) was that "the single most valuable intervention we could make to improve forecast accuracy would be to reduce the forecast horizon to 6 months or less through more rapid vaccine development". This is not practical using conventional influenza vaccine production and regulatory procedures. Nevertheless, this study does identify some practical steps that could improve the accuracy and utility of forecasting such as a few suggested modifications by the authors such as "..... changing the start and end times of our long-term forecasts. We could change our forecasting target from the middle of the next season to the beginning of the season, reducing the forecast horizon from 12 to 9 months.'

Strengths:

The authors are very familiar with the type of forecasting tools used in this analysis (LBI and mutational load models) and the processes used currently for influenza vaccine virus selection by the WHO committees having participated in a number of WHO Influenza Vaccine Consultation meetings for both the Southern and Northern Hemispheres.

Weaknesses:

The conclusion of limiting the forecasting to 6 months would only be achievable from the current influenza vaccine production platforms with mRNA. However, there are no currently approved mRNA influenza vaccines, and mRNA influenza vaccines have also yet to demonstrate their real-world efficacy, longevity, and cost-effectiveness and therefore are only a potential platform for a future influenza vaccine. Hence other avenues to improve the forecasting should be investigated.

While it is inevitable that more influenza HA sequences will become available over time a better understanding of where new influenza variants emerge would enable a higher weighting to be used for those countries rather than giving an equal weighting to all HA sequences.

Also, other groups are considering neuraminidase sequences and how these contribute to the emergence of new or potentially predominant clades.