Reviewer #1 (Public Review):

Romagosa, Nieukirk et al. present an interesting approach and interpretation to what is assumed to be a learned animal behavior. In this case, the observed behavior is fin whale (Balaenoptera physalus) singing and the analyses provide results indicating spatio-temporal variation in three fin whale song features at distinct locations within the Central and Northeast North Atlantic Ocean (ONA) region within a two-decade time period. The data set is a non-standardized collection of acoustic recordings obtained from multiple research scientists. Most of the acoustic recording samples are very sparse, with the majority of data coming from an area around the Azores and collected by Okeanos scientists. The senior author undertook the enormously demanding task of analyzing the acoustic data using non-automatic, standardized techniques and protocols. Songs from individual periods of singing on any given day were selected for analysis based on song quality. Song measurements included interval of time between successive 20-Hz song notes (INI), peak frequencies of those 20-Hz notes and peak frequencies of higher frequency notes (HF note). The resultant units of analysis are daily measures of INI (average and s.d.), 20-Hz note peak frequencies (average and s.d.), and HF note peak frequencies (average and s.d.). Several of the figures are confused by not representing the time axis in a typical, uniformly linear way (Fig. 2A and Fig. 3). This form of dynamic time warping smooths and distorts the time-varying features of the results and obscures the inherent sparseness of and high variability in the durations and locations of recordings in available data set. This fundamental characteristic of the available data (see Fig. S1), represents a form of sample aliasing, is not adequately addressed in the paper in terms of how it influences or restricts interpretation of the results. Another possible over-interpretation of results involves misrepresentation of the actual areas sampled. For example, data were collected on Dec 2007-Feb 2008 and Oct 2015 March from a recorder location off the southwest of the Iberian Peninsula. The acoustic sampling detection space is restricted to the ocean within some tens of kilometers of a single sensor, a very small dot on the maps in the manuscript, yet the data from this recorder are assigned to the relatively very large region referred to as the "Bay of Biscay & Iberian Coast". Within the two-decade period of the study (ca. 120 months), recordings were collected at this site (E in Figure 1) for 9 months (7.5%), and the two sampling periods occurred within the December 2007 through March 2018 time span (see Fig S1). It is scientifically inappropriate to translate this as data representing the Bay of Biscay & Iberian Coast as this kind of misrepresentation can lead to misinterpretation of the results.

Despite these spatial and temporal sampling issues, the analyses reveal several important features (Fig. 2 and Fig. 3) about fin whale song in the ONA. The import of the analytical results is that the time span and spatial scale over which recordings were collected provide a unique opportunity to observe whether or not there were variations in fin whale song features within a large ocean region, across a span of two decades. One can consider these spatial and temporal scales appropriately matched to the known scales of fin whale natural history and ecology. Thus, the study results, although confronted by some sampling issues, are not biased by inappropriately sized spatial and temporal scales.

This MS joins a small but growing list of papers documenting variability in baleen whale acoustic behaviors over ecologically appropriate spatial and temporal scales. These papers are primarily focused on singing, an acoustically obvious male reproductive display. As with several recent papers, the author takes advantage of a growing body of data collected during previous studies. The actual measurements utilized several established acoustic analysis software tools. The interpretation of the results focuses on evidence of vocal learning in fin whale singers (i.e. males performing reproductive displays) and wisely remains tangential to interpreting fin whale song through a cultural lens.

Reviewer #2 (Public Review):

This research brings togethor an impressively long timescale dataset of fin whale song vocalisations in the North Atlantic, measuring the note frequency content and inter-note intervals and thereby tracking shifts in both over time. Different time periods are covered in different regions of the north Atlantic during the course of the study. There are two principal results - the study documents a shift in the inter-note interval (INI) in an ICES eco-region termed 'Oceanic Northeast Atlantic' (although the relevance of this to fin whale populations is unclear) occuring relatively rapidly in the years 2000-2001. This shift is discontuous and appears to show an abrupt change in note intervals in most (though not all) of the songs recorded. The second key result is that this INI measure and also the peak frequency of song element termed the 'HF note' both show consistent directional change over timescales of 12 years. The INI measure begins to change back toward the value it held prior to the 2000/2001 shift, suggestive of a cyclical process of change coupled with resets. The average HF note peak frequency descended by about 5Hz during the study period but there was no evidence of abrupt shifts.

The research significance is largely in the description of these processes in a new area, similar changes in rorqual song have been examined in the Southern Ocean and Mediterranean, and the argued interpretation of these changes as evidence for cultural learning processes in song change - the debate over whether these changes have environmental causation or are due to learning processes similar to song change in humpbacks is ongoing and this study therefore contributes interesting evidence from a newly covered population.

I think the methods and analyses broadly support the claims but also that there are weaknesses in interpretation and presentation that should be addressed. I think perhaps the degree to which this is evidence of vocal learning may be a bit overplayed. Definitely there is change, but it is tricky to compare this to e.g. experimental demonstrations. For example, age-related changes in a changing post-whaling demographic scenario should at least be considered? Is there also any possibility for large-scale oceanographic variations to be included in some way - temperature shifts, for example? This could help understand the different roles of environment and learning in these processes. I think it is also important that these results be placed in a more detailed context of current knowledge of fin whale population structure in the north Atlantic - could population range shifts be a factor? The INI data show an interesting variation in the recordings from the Barents Sea and this could be discussed in the light of population structure knowledge also. It is unclear from the presentation whether the INI shift in 2000/2001 was coupled with any frequency shifts - if not, it suggests different trajectories and processes affecting these two aspects of the acoustic display.

I am not convinced the main story here is about conformity, and I think it would be a mistake to too easily reach for the humpback comparison but there are certainly questions to be asked about the 2000/2001 shift in terms of the processes that led to it.

Reviewer #3 (Public Review):

The authors used passive acoustic monitoring over a vast range of the North Atlantic to study the call rates of fin whales. They found a 'take over' of a new rythm (inter call intervals) during their study period. This was interpreted as a change in song production.

I am not completely convinced the authors are correct in describing this change in rate as a change in the song. Even though fin whale calls are evidently a male mating ground display, little is known about its function. Compared to humpback whales with their impressive repertoire of vocalizations, repeating themselves on the breeding grounds after some tens of minutes and therefore qualifying as a very slow 'song' similar to bird song, fin whale only emit a single type of call, which is remaining the same throughout the study period. It can be contested, I would assume, that a ,erely change the repetition rate of calls, even though seemingly done here in an 'overtake' fasion, can qualify as a change and learning of song,

Reviewer #1 (Public Review):

The hippocampus is a structure in the cerebral cortex known to be compartmentalised into regions with different functions. Dorsal hippocampus is involved in cognitive functions such as declarative memory and spatial navigation and interconnects chiefly with the neocortex. Ventral hippocampus interconnects with limbic structures such as amygdala and hypothalamus and is involved in affective states and anxiety. What specifies this functional regionalisation during development is not well understood. The present study focuses on the role of transcription factors COUPTFI and COUPTFII, confirming a previously observed dorsal to ventral gradient of expression of COUPTFI in both embryonic and adult mouse hippocampus, and reporting that expression of COUPTFII is strongest in ventral hippocampus. The aim of the authors was then to probe the role of these transcription factors with the use of conditional knockout of one or both factors using RxCre+ mice (sometimes Emx1Cre+ for comparison). As predicted, COUPTFI insufficiency resulted in failure of the CA1 subregion of the dorsal hippocampus to develop properly (with concomitant loss of performance in a spatial memory task) COUPTFII knockdown had even more marked effects upon the ventral hippocampus with ectopic CA1/CA3 domains forming, while a double knockout lead to a drastic reduction in size of the hippocampus with subsequent effects upon the appearance of hippocampal synaptic circuitry and the capacity for adult neurogenesis (a feature of rodent hippocampus). In order to help explain the role of COUPTFI/II a role in regulating expression of two transcription factors LHX2 and LHX5, known to be crucial to hippocampal development, was tested by examining gene and protein expression. Changes in LHX2 and LHX5 was observed and a role for COUPTFI/II in regulating expression of these genes was postulated.

I believe the authors have largely achieved their aims and the results mostly support the conclusions, but, as discussed further below, there are some weaknesses in the data and some areas that could be expanded upon and improved. The methods are mostly appropriate. The use of the transgenic mice and the application of histological methods, especially tyramide amplified immunohistochemistry, is exemplary. However, I'm not sure a wide enough range of tests to explore the phenotype of the transgenic mice was employed to back the conclusions drawn by the authors. The introduction and discussion are nicely written and explain the general concepts and conclusions well. The work makes an important contribution to our understanding of brain development in general and hippocampal development in particular.

Turning to more specific comments, I must first point out that specification of the ventral hippocampus by expression of COUPTFII is not an entirely original finding, as it was suggested for the developing human hippocampus following immunohistochemical experiments illustrating COUPTFII expression to be confined to the ventral hippocampal structures of the medial temporal cortex (doi: 10.1093/cercor/bhx185). Of course, this study, unlike the present study, was restricted to fetal cortex, not adult, and also reported expression of COUP-TFI throughout dorsal and ventral hippocampal structures but without observing any dorsal to ventral gradient, however I feel its contribution to the field has been overlooked by the present study, and should be incorporated into the introduction and/or discussion.

More information about Rx-cre mice would be informative and could help explain the different phenotype observed when EMX1-cre mice were used to conditionally knock down COUPTFI/II expression.

The demonstration of antagonistic gradients of COUP-TFI and -TFII across the hippocampus is more convincing in the immunohistochemical preparations than in the western blots. The qualitative data presented in Fig.1p does not convincingly represent the quantitative data presented in Fig.1q. There seem to be multiple bands for COUP-TFII and I wonder exactly how quantifying this was approached?

Behavioural testing is limited to one test of dorsal hippocampus function. other tests for non-spatial memory, e.g. novel object recognition, or ventral hippocampus function, e.g. step through passive avoidance, might have lead to some interesting discriminations between the various knock down animals (see doi: 10.3389/fnagi.2018.00091).

Abnormalities in the trisynaptic circuit. No studies of actual synapses, either physiological or morphological, were carried out. I wonder to what extent these immunohistochemical studies just further reflect the abnormalities in hippocampal morphology presented earlier in the manuscript without specifically telling us about synaptic circuits? Although the immunohistochemical preparations are beautiful, they are inadequate on their own in telling us much about what sort of synaptic circuitry exists in the transgenic animals.

LHX2/LHX5 interaction. The immunohistochemical study, which shows clear differences in LHX5 and LHX2 protein expression at E14.5 in double knockdown mice is more convincing than the qPCR study at E11.5, which show surprisingly small differences in mRNA expression. Could the authors expand upon whether this is due to stage of development, or differences between mRNA and protein expression? Why hasn't both mRNA and protein expression data at both time points been presented?

Response to the re-submission

I am happy that the western blot presentation has been improved, and my minor comments attended to. It is disappointing, although understandable given the timeframe, that the lack of qPCR data at 14.5 ED has not been rectified. The immunohistochemical data alone is qualitative and only indicative of LHX5 expression remaining depressed and LHX2 expression possibly increasing between E11.5 and E14.5. In the absence of qPCR data, a more quantitative immunohistochemical study, such as counting blind the number of LHX5+ Cajal-Retzius cells, or measuring optical density of LHX2 expression under rigorous experimental conditions regarding image collection and processing, would be required to support the hypothesis that COUPTFI/II expression modulates the LHX2/LHX5 axis.

Reviewer #2 (Public Review):

The Author's chose to limit their response to re-doing the Lhx5 immuno using the correct antibody which now displays the expected staining: Lhx5 expression is limited to the hem. They have not however presented a characterization of where the RxCre acts, although this was pointed out by other reviewers as well. It would have been useful to demonstrate the expression domain in particular with respect to the time of its initiation, to explain how it causes a phenotype close to that described for the Lhx5 knockout (Zhao et al., 1999). From the decrease of Lhx5 expression and the CR cells which arise from the hem, it appears that the RxCre does indeed act in the hem. However, the timing and spatial pattern is important to establish, as I had pointed out in my first review, "If [the expression of RxCre] it has a dorso-ventral bias in the early embryo, it could explain the regional difference in the COUPTF phenotypes."

The major interpretive criticisms I made have not been addressed even though these would have only required a re-writing and re-interpretation of the data. The revised manuscript continues to include major errors of interpretation such as the idea that Lhx2 and Lhx5 "inhibit each other", something that is unsupported since the expression domains of these two genes are mutually exclusive as is clear from the authors' own new data and the literature.Lines 355-360: "The expression of Lhx2 was comparable between the control and double-mutant mice at E11.5 (Figure 5Be-h, e'-h'). Interestingly, the expression of the Lhx2 protein was increased in the hippocampal primordium in the COUP-TF double-mutant mice at E13.5 and E14.5 (Figure 5Bm-p, m'-p', u-x, u'-x'). The upregulation of Lhx2 expression is most likely associated with the reduced expression of the Lhx5 gene"There's clearly no Lhx5 in the hippocampal primordium so how is this possible?

The authors have missed the insights from key papers that they cite, e.g. (lines 352-354) " The expression of Lhx2 was expanded ventrally into the choroid plexus in the Lhx5 null mutant mice (Zhao et al., 1999)" - this paper in fact shows there is no choroid plexus. Lhx2 appears to extend to the midline likely because the hem isn't specified. The authors would benefit from reading https://doi.org/10.1101/2022.10.25.513532 in which Lmx1a is shown to be the master regulator of the hem.<br /> A sentence like (lines 77-81) further blurs the literature: "Intriguingly, deficiency of either Lhx5 or Lhx2 results in agenesis of the hippocampus, and more particularly, these genes inhibit each other (Hébert & Fishell, 2008; Mangale et al., 2008; Roy, Gonzalez-Gomez, Pierani, Meyer, & Tole, 2014; Zhao et al., 1999), indicating that the Lhx5 and Lhx2 genes may generate an essential regulatory axis to ensure the appropriate hippocampal development"<br /> First, none of the papers they cite shows that these two factors inhibit each other. Second, the "agenesis of the hippocampus" in the Lhx2 knockout mentioned in Porter et al. (1997) was later shown to be due to a transformation of the hippocampal primordium into an EXPANDED hem (Mangale et al.) In contrast, the "agenesis of the hippocampus" in the Lhx5 mutant appears to be due to the near-complete LOSS of the hem and evidenced by the loss of its derivatives, the choroid plexus and the CR cells (Zhao et al., 1999). The fact that loss of these two factors have opposite effects on the hem (each resulting in loss of the hippocampus, one due to transformation of the hippocampal primordium into hem and the other because of a lack of hipopcampal induction) does not mean that there is an Lhx5-Lhx2 "axis" regulating hippocampal development.

I won't repeat my other comments here, but the majority of them were not addressed in any way.

In conclusion, I find it unfortunate that the authors have chosen not to use the detailed input provided by the reviewers which would have greatly improved their manuscript.

Reviewer #3 (Public Review):

The authors have made significant improvements in addressing my major concerns raised during the previous review. However, I still have some lingering concerns regarding the quantification and statistical analysis presented in the manuscript. Specifically, there is a lack of robust quantification and statistical analysis to support the conclusions drawn, particularly in relation to the numbers of DG, CA1, and CA3 neurons.

To strengthen the validity and reliability of the findings, I would strongly recommend the authors to incorporate a more rigorous quantitative approach in their research. This could involve implementing stereological methods or other appropriate techniques to accurately estimate the numbers of neurons in the DG, CA1, and CA3 regions. By doing so, the authors would enhance the credibility of their conclusions and provide more solid evidence for their claims.

Reviewer #1 (Public Review):

Barlow et al performed a viral insertion screen in larval zebrafish for sleep mutants. They identify a mutant named dreammist (dmist) that displayed defects in sleep, namely, decreased sleep both day and night, accompanied by increased activity. They find that dmist encodes a previously uncharacterized single-pass transmembrane protein that shows structural similarity to Fxyd1, a Na+K+-ATPase regulator. Disruption of fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit, decreased night-time sleep. By staining for sodium levels, the authors uncover a global increase of sodium in both dmist and atp1a3a mutants following PTZ treatment, consistent with defects in Na+K+-ATPase function. These genetic data from multiple mutant lines help establish the importance of sodium and/or potassium homeostasis in sleep regulation.

The conclusions of this paper are mostly well supported by data, with the following strengths and weaknesses as described below.

Strengths:<br /> Elegant use of CRISPR knockout methods to disrupt multiple genes that help establish the importance of regulating Na+K+-ATPase function in sleep.<br /> Data are mostly clearly presented.<br /> Double mutant analysis of dmist and atp1a3a help establish an epistatic relationship between these proteins.

Weaknesses:<br /> The authors emphasize the role of increased cellular sodium, but equally plausibly, the phenotypes could be due to decreased cellular potassium. The potassium channel shaker has been previously identified as a critical sleep regulator in Drosophila.<br /> Although the increased sleep rebound after PTZ treatment in the dmist mutant is interesting, I find it difficult to understand, especially in the context of the dmist mutant having decreased sleep.

The similar phenotype between dmist and Fxyd1 in sleep reduction yet very different expression patterns, with dmist being mostly neuronal while fxyd1 being mostly non-neuronal, raise many possible questions: 1) are the sleep phenotypes due to neuronal Na/K imbalance? Or 2) Are the sleep phenotypes due to extracellular Na/K imbalance? Or 3) both? Some feasible experiments may help achieve a better mechanistic understanding of the observed sleep defects.

Reviewer #2 (Public Review):

Barlow and colleagues describe a role for the Na+/K+ pump in sleep/wake regulation. They discovered this role starting with a forward genetic screen in which they tested a biased sample of virus insertion fish lines for sleep phenotypes. They found an insertion in a gene they named dreammist, which is homologous to the gene FXYD1 encoding single membrane-pass modifiers of Na/K pumps. They go on to show that genetic manipulations of either FXYD1 or the Na/K pump also reduce sleep. They use pharmacology and sleep deprivation experiments to provide further evidence that the NA/K pump regulates intracellular sodium and rebound sleep. This study provides additional evidence for the important role of membrane excitability in sleep regulation (prior studies have implicated K+ channel subunits as well as a sodium leak ion channel).

The study is well done and convincing with regard to its major conclusions. I had some minor comments/questions, which they properly addressed in their revision and rebuttal.

Reviewer #1 (Public Review):

In this manuscript, the authors present a valuable new method to represent animal behavior from video data using a variational autoencoder framework that disentangles individual-specific and background variance from variables that can be more reliably compared across individuals. They achieve this aim through the use of a novel Cauchy-Schwatz (C-S) regularization term in their loss function that leads to latents that model continuously varying features in the images. The authors present a variety of validations for the method, including testing across sessions and individuals for a head-fixed task. They also show how the methods could be used for behavioral decoding from neural data, quantifying social behavior in mice, demonstrating the applicability of the method outside of head-fixed environments and for different measurement modalities. While some areas of confusion and questions about the validation exist, this is an overall strong paper and an important contribution to this field.

Strengths:

- The use of the C-S regularizer is novel approach that has potential for wide use across experimental paradigms and model organisms<br /> - The extent of the validations performed was solid, although perhaps not as convincing in a couple of cases as might be ideal<br /> - The GitHub code demo worked well, and the code appears to be accessible and well-written

Weaknesses:

- Some of the validation figures were a bit unclear in their presentation, making it difficult to assess exactly what had been tested<br /> - It is possible that I missed this, but the authors didn't really provide a sense of how to pick a particular distribution to match using the CS term for a specific paradigm/modality and how the choice affects the results<br /> - While the authors' statements about individual training vs. transfer learning accuracy and efficiency in Figure 6 are technically true, the effect size is rather small ( a few percent at most in each case), thus I don't know how much of a big deal I would want to make out of these results<br /> - In general, I would have liked to have seen the Discussion section speak more to the choices and limitations inherent in applying the method. How does the choice of prior/metaparameters/architecture/etc affect the results? In what situations would this method to fail? What are the next advances that are necessary for the field to progress?

Reviewer #2 (Public Review):

This paper presents a valuable contribution to ongoing methods for understanding and modeling structure via latent variable models for neural and behavioral data. Building on the PS-VAE model of Whiteway et al. (2021), which posited a division of latent variables into unsupervised (i.e., useful for reconstruction) and supervised (useful for predicting selected labeled features) variables, the authors propose an additional set of "constrained subspace" latent variables that are regularized toward a prespecified prior via a Cauchy-Schwarz divergence previously proposed.

The authors contend that the added CS latents aid in capturing both patterns of covariance across the data and individual-specific features that are of particular benefit in multi-animal experiments, all without requiring additional labels. They substantiate these claims with a series of computational experiments demonstrating that their CS-VAE outperforms the PS-VAE in several tasks, particularly that of capturing differences between individuals, consistency in behavioral phenotyping, and predicting correlations with neural data.

Strengths of the present work include an extensive and rigorous set of validation experiments that will be of interest to those analyzing behavioral video. Weaknesses include a lack of discussion of key theoretical ideas motivating the design of the model, including the choice of a Cauchy-Schwarz divergence, the specific form of the prior, and arguments for sorts of information the CS latents might capture and why. In addition, the model makes use of a moderate number of key hyperparameters whose effect on training outcomes are not extensively analyzed. As a result, the model may be difficult for less experienced users to apply to their own data. Finally, as with many similar VAE approaches, the lack of a ground truth against which to validate means that much of evidence provided for the model is necessarily subjective, and its appeal lies in the degree to which the discovered latent spaces appear interpretable in particular applications.

In all, this work is a valuable contribution that is likely to have appeal to those interested in applying latent space methods, particularly to multi-animal video data.

Reviewer #3 (Public Review):

As naturalistic neuroscience becomes increasingly popular, the importance of new computational tools that facilitate the study of animals behaving in minimally constrained environments grows. Yi et al convincingly demonstrate the usefulness of their new method on data from neuroethological studies involving multiple animals, including those with social interactions. Briefly, their method improves upon prior semi-supervised machine learning methods in that extracted latent variables can be more cleanly separated into those representing the behavior of individual subjects and those representing social interactions between subjects. Such an improvement is broadly useful for downstream analysis tasks in multi-subject or social neuroethological studies.

Strengths:<br /> The authors tackle an important problem encountered in behavior analyses in an emerging subfield of neuroscience, naturalistic social neuroscience. They make a case for doing so using semi-unsupervised methods, a toolbox which balances competing scientific needs for building models using large neural-behavioral datasets and for model explainability. The paper is well written, with well-designed figures and relevant analyses that make for an enjoyable reading experience.

The authors provide a remarkable variety of examples that make a convincing case for the utility of their method when used by itself or in conjunction with other data analysis techniques commonly used in modern neuroscience (behavioral motif extraction, neural decoding, etc.). The examples show not just that the extracted latents are more disentangled, but also that the improvement in disentangling has positive effects in downstream analysis tasks.

Weaknesses:<br /> While the paper does a great job of applying the method to real world data, the components of the method itself are not as thoroughly investigated. For example, the contribution of the novel Cauchy-Schwarz regularization technique has not been systematically investigated. This could be done either by sharing additional data where hyperparameters control the contribution of the regularizer, or cite relevant papers where such an analysis have been carried out. It would also be valuable to understand what other regularization techniques might potentially have been applicable here.

The authors conclude from their empirical investigations that the specific prior distribution does not matter to the regularization process. This seems reasonable given that the neural network can learn a complex and arbitrary transformation of the data during training. It would be helpful if the authors could cite prior work where this type of prior distribution does matter and how their approach is different from such prior work. If there is a visualization/explainability related motivation for choosing one prior distribution over another, this could be clarified.

Reviewer #1 (Public Review):

In the manuscript by Urban et al., the authors attempt to further delineate the role which non-neuronal CNS cells play in the development of ALS. Toward this goal, the transmembrane signaling molecule ephrinB2 was studied. It was found that there is an increased expression of ephrinB2 in astrocytes within the cervical ventral horn of the spinal cord in a rodent model of ALS. Moreover, the reduction of ephrinB2 reduced motoneuron loss and prevented respiratory dysfunction at the NMJ. Further driving the importance of ephrinB2 is an increased expression in the spinal cords of human ALS individuals. Collectively, these findings present compelling evidence implicating ephrinB2 as a contributing factor towards the development of ALS.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediator of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied by the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function.

The major limitation of the manuscript as currently written is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many others that comprise the ventral gray matter, have not been investigated in this study. Thus, the primary conclusion that astrocytes drive ephrinB2-mediated pathogenesis in ALS mice is largely correlative. Further, it is interesting to note that no other functional outcomes were improved in this study. The C3-C5 region of the spinal cord consists of many motor pools that innervate forelimb muscles. CMAP recordings conducted at the diaphragm are a reflection of intact motor pools. This type of assessment of neuromuscular health is hard to re-capitulate in the kind of forelimb task that is being employed to test motor function (grip strength). Thus, it would be interesting to see if CMAP recordings of forelimb muscles would capture the kind of motor function preservation observed in the diaphragm muscle.

On a similar note, the functional impact of increased CMAP amplitude has not been presented. An increase in CMAP amplitude does not necessarily translate to improved breathing function or overall ventilation. Thus, the impact of this improvement in motor output should be clearly presented to the reader. Further, to the best of my knowledge, expression of Eph (or EphB) receptors has not been explicitly shown at the phrenic motor pool. It is thus speculative at best that the mechanism that the authors suggest in preserving diaphragm function is in fact mediated through Eph-EphrinB2 signaling at the phrenic motor pool. This aspect of the study would warrant a deeper discussion. Lastly, although authors include both male and female animals in this investigation, they do not have sufficient power to evaluate sex differences. Thus, this presents another exciting future of investigation, given that ALS has a slightly higher preponderance in males as compared to females.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Reviewer #1 (Public Review):

Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been previously published, if the authors can demonstrate that miR15/16 and/or Malat1 affect the clearance of Listeria or LCMV, this will significantly add to the current findings and provide physiological context to the study.

Reviewer #2 (Public Review):

This study connects prior findings on MicroRNA15/16 and Malat1 to demonstrate a functional interaction that is consequential for T cell activation and cell fate.

The study uses mice (Malat1scr/scr) with a precise genetic modification of Malat1 to specifically excise the sites of interaction with the microRNA, but sparing all other sequences, and mice with T-cell specific deletion of miR-15/16. The effects of genetic modification on in vivo T-cell responses are detected using specific mutations and shown to be T-cell intrinsic.

It is not known where in the cell the consequential interactions between MicroRNA15/16 and Malat1 take place. The authors depict in the graphical abstract Malat1 to be a nuclear lncRNA. Malat 1 is very abundant, but it is unclear if it can shuttle between the nucleus and cytoplasm. As the authors discuss future work defining where in the cell the relevant interactions take place will be important.

In addition to showing physiological phenotypic effects, the mouse models prove to be very helpful when the effects measured are small and sometimes hard to quantitate in the context of considerable variation between biological replicates (for example the results in Figure 4D).

The impact of the genetic modification on the CD28-IL2- Bcl2 axis is quantitatively small at the level of expression of individual proteins and there are likely to be additional components to this circuitry.

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the role of Elg1 in the regulation of telomere length. The main role of the Elg1/RLC complex is to unload the processivity factor PCNA, mainly after completion of synthesis of the Okazaki fragment in the lagging strand. They found that Elg1 physically interacts with the CST (Cdc13-Stn1-Ten1) and propose that Elg1 negatively regulates telomere length by mediating the interaction between Cdc13 and Stn1 in a pathway involving SUMOylation of both PCNA and Cdc13. Accumulation of SUMOylated PCNA upon deletion of ELG1 or overexpression of RAD30 leads to elongated telomeres. On the other hand, the interaction of Elg1 with Sten1 is SIM-dependent and occurs concurrently with telomere replication in late S phase. In contrast Elg1-Cdc13 interaction is mediated by PCNA-SUMO, is independent on the SIM of Elg1 but still dependent on Cdc13 SUMOylation. The authors present a model containing two main messages 1) PCNA-SUMO acts as a positive signal for telomerase activation 2) Elg1 promotes Cdc13/Stn1 interaction at the expense of Cdc13/Est1 interaction thus terminating telomerase action.

The manuscript contains a large amount of data that make a major inroads on a new type of link between telomere replication and regulation of the telomerase. Nevertheless, the detailed choreography of the events as well as the role of PCNA-SUMO remain elusive and the data do not fully explain the role of the Stn1/Elg1 interaction. The data presented do not convincingly support the claim that SUMO-PCNA is a positive signal for telomerase activation. This was partially addressed in the current version.

Reviewer #2 (Public Review):

This paper purports to unveil a mechanism controlling telomere length through SUMO modifications controlling interactions between PCNA unloader Elg1 and the CST complex that functions at telomeres. This is an extremely interesting mechanism to understand, and this paper indeed reveals some interesting genetic results, leading to a compelling model, with potential impact on the field. Overall, however, the data do not provide sufficient support for the claims. The model may be correct but it is not yet convincingly demonstrated.

The current version addressed some of the issues regarding language describing conclusions and more experimental detail has been provided. However, the authors have not provided new data supporting the model, so the overall evaluation is that the work remains inconclusive.

Reviewer #3 (Public Review):

This paper reveals interesting physical connections between Elg1 and CST proteins that suggest a model where Elg1-mediated PCNA unloading is linked to regulation of telomere length extension via Stn1, Cdc13, and presumably Ten1 proteins. Some of these interactions appear to be modulated by sumolyation and connected with Elg1's PCNA unloading activity. The strength of the paper is in the observations of new interactions between CST, Elg1, and PCNA. These interactions should be of interest to a broad audience interested in telomeres and DNA replication.

What is not well demonstrated from the paper is the functional significance of the interactions described. The model presented by the authors is one interpretation of the data shown, and proposes that the role of sumolyation is temporally regulate the Elg1, PCNA and CST interactions at telomeres. This model makes some assumptions that are not demonstrated by this work (such as Stn1 sumolyation, as noted) and are left for future testing. Alternative models that envision sumolyation as a key in promoting spatial localization could also be proposed based on the data here (as mentioned in the discussion), in addition to or instead of a role for sumolyation in enforcing a series of switches governing a tightly sequenced series of interactions and events at telomeres. Critically, the telomere length data from the paper indicates that the proposed model depicts interactions that are not necessary for telomerase activation or inhibition, as telomeres in pol30-RR strains are normal length and telomeres in elg1∆ strains are not nearly as elongated as in stn1 strains. One possibility mentioned in the paper is the PCNAS and Elg1 interactions are contributing to the negative regulation of telomerase under certain conditions that are not defined in this work. Could it also be possible that the role of these interactions is not primarily directed toward modulating telomerase activity? It will be of interest to learn more about how these interactions and regulation by Sumo function intersect with regulation of telomere extension.

Reviewer #1 (Public Review):

More than ten years ago, it was shown that activity in the primary visual cortex of mice substantially increases when mice are running compared to when they are sitting still. This finding 'revolutionised' our thinking about the visual cortex, turning away from it being a passive image processor and highlighting the influence of non-visual factors. The current study now for the first time repeats this experiment in a primate (the marmoset). The authors find that in contrast to mice, marmoset V1 activity is slightly suppressed during running, and they relate this to differences in gain modulations of V1 activity between the two species.

Strengths:

- Replication in primates of the original finding in mice partly took so long, because of the inherent difficulties with recording from the brain of a running primate. The treadmill for the marmosets in the current study is a very elegant solution to this problem. It allows for true replication of the 'running vs stationary' experiment and undoubtedly opens up many possibilities for other experiments recording from a head-fixed but active marmoset.<br /> - In addition to their own data on the marmoset, the authors run their analyses on a publicly available data set on the mouse. This allows them to directly compare mouse and marmoset findings, which significantly strengthens their conclusions.

Weaknesses:

- The main thing that is missing from the study is a good explanation as to why running has such a different effect on marmoset V1 compared to mouse V1. Differences in neuromodulatory inputs are cited in the discussion as a possible cause for the discrepancy, but an obvious influencing factor that the authors could investigate in their own data set is the retinal input. In Fig1b, the authors even show these data in the form of gaze and pupil size. In these example data, by eye, it looks like the pupil size is positively correlated with the run speed. This would of course have large consequences on the activity in V1, but the authors do not do anything with these data. The study would improve substantially if the authors would correlate their run speed traces with other factors that they have recorded too, such as pupil size and gaze.

- Fig2a shows the 'most correlated mouse session', i.e. the session where the relation between visual cortex activity and running speed was strongest. Looking at the raster plot, however, shows that this strong positive correlation must be due entirely to the lower half of the neurons significantly increasing their firing rate as the mouse starts to run; in fact, the upper 25% or so of the neurons show exactly the opposite (strong suppression of the neurons as the mouse starts running). It would be more balanced if this heterogeneity in the response is at least mentioned somewhere in the text.

Significance:

The paper provides interesting new evidence to the ongoing discussion about the influence of non-visual factors in general, and running in particular, on visual cortex activity. As such, it helps to pull this discussion out of the rodent field mainly and into the field of primate research. The elegant experimental set-up of the marmoset on a treadmill will certainly add new findings to this issue also in the years to come.

Reviewer #2 (Public Review):

This work aims at answering whether activity in the primate visual cortex is modulated by locomotion, as was reported for the mouse visual cortex. The finding that the activity in the mouse visual cortex is modulated by running has changed the concept of primary sensory cortical areas. However, it was an open question whether this modulation generalizes to primates.

To answer this fundamental question the authors established a novel paradigm in which a head-fixed marmoset was able to run on a treadmill while watching a visual stimulus on a display. In addition, eye movements and running speed were monitored continuously and extracellular neuronal activity in the primary visual cortex was recorded using high-channel-count electrode arrays. This paradigm uniquely permitted investigation of whether locomotion modulates sensory-evoked activity in the visual cortex of a marmoset. Moreover, to directly compare the responses in the marmoset visual cortex to responses in the mouse visual cortex the authors made use of a publicly-available mouse dataset from the Allen Institute. In this dataset, the mouse was also running on a treadmill and observing a set of visual stimuli on a display. The authors took extra care to have the marmoset and mouse paradigms as comparable as possible.

To characterize the visually driven activity the authors present a series of moving gratings and estimate receptive fields with sparse noise. To estimate the gain modulation by running the authors split the dataset into epochs of running and non-running which allowed them to estimate the visually evoked firing rates in both behavioral states.

Strengths:<br /> The novel paradigm of head-fixed marmosets running on a treadmill while being presented with a visual stimulus is unique and ideally tailored to answering the question that the authors aimed to answer. Moreover, the authors took extra care to ensure that the paradigm in the marmoset matched as closely as possible to the conditions in the mouse experiments such that the results can be directly compared. To directly compare their data the authors re-analyzed publicly available data from the visual cortex of mice recorded at the Allen Institute. Such a direct comparison, and reuse of existing datasets, is another strong aspect of the work. Finally, the presented new marmoset dataset appears to be of high quality, the comparison between the mouse and marmoset visual cortex is well done and the results and interpretation are straightforward.

Weaknesses:<br /> While the presented results are clear and support the main conclusion of the authors, additional analysis and experimental details could have further strengthened and clarified some aspects of the results. For example, it is known that the locomotion gain modulation varies with layer in the mouse visual cortex, with neurons in the infragranular layers expressing a diversity of modulations (Erisken et al. 2014 Current Biology). However, for the marmoset dataset, it was not reported from which cortical layer the neurons are from, leaving this point unanswered.

Nonetheless, the aim of comparing the locomotion-induced modulation of activity in primate and mouse primary visual cortex was convincingly achieved by the authors. The results shown in the figures support the conclusion that locomotion modulates the activity in primate and mouse visual cortex differently. While mice show a profound gain increase, neurons in the primate visual cortex show little modulation or even a reduction in response strength.

This work will have a strong impact on the field of visual neuroscience but also on neuroscience in general. It revives the debate of whether results obtained in the mouse model system can be simply generalized to other mammalian model systems, such as non-human primates. Based on the presented results, the comparison between the mouse and primate visual cortex is not as straightforward as previously assumed. This will likely trigger more comparative studies between mice and primates in the future, which is important and absolutely needed to advance our understanding of the mammalian brain.

Moreover, the reported finding that neurons in the primary visual cortex of marmosets do not increase their activity during running is intriguing, as it makes you wonder why neurons in the mouse visual cortex do so. The authors discuss a few ideas in the paper which can be addressed in future experiments. In this regard, it is worth noting that the authors report an interesting difference between the foveal and peripheral parts of the visual cortex in marmoset. It will be interesting to investigate these differences in more detail in future studies. Likewise, while running might be an important behavioral state for mice, other behavioral states might be more relevant for marmosets and do modulate the activity of the primate visual cortex more profoundly. Future work could leverage the opportunities that the marmoset model system offers to reveal new insights about behavioral-related modulation in the primate brain.

Reviewer #3 (Public Review):

Prior studies have shown that locomotion (e.g., running) modulates mouse V1 activity to a similar extent as visual stimuli. However, it's unclear if these findings hold in species with more specialized and advanced visual systems such as nonhuman primates. In this work, Liska et al. leverage population and single neuron analyses to investigate potential differences and similarities in how running modulates V1 activity in marmosets and mice. Specifically, they discovered that although a shared gain model could describe well the trial-to-trial variations of population-level neural activity for both species, locomotion more strongly modulated V1 population activity in mice. Furthermore, they found that at the level of individual units, marmoset V1 neurons, unlike mice V1 neurons, experience suppression of their activity during running.

A major strength of this work is the introduction and completion of primate electrophysiology recordings during locomotion. Data of this kind was previously limited, and this work moves the field forward in terms of data collection in a domain previously inaccessible in primates. Another core strength of this work is that it adds to a limited collection of cross-species data collection and analysis of neural activity at the single-unit and population level, attempting to standardize analysis and data collection to be able to make inferences across species.

However, the authors did not take full advantage of the quantity and diversity of the marmoset visual cortex recordings in their analyses. They mention recording and analyzing the activity of peripheral V1 neurons but mainly present results involving foveal V1 neurons. Foveal neurons, with their small receptive fields strongly affected by precise eye position, would seem to be less likely to be comparable to rodent data. If the authors have a reason for not doing so, they should provide an explanation. Given that the marmosets are motivated to run with liquid rewards, the authors should provide more context as to how this may or may not affect marmoset V1 activity. Additionally, the lack of consideration of eye movements or position presents a major absence for the marmoset results, and fails to take advantage of one of the key differences between primate and rodent visual systems - the marmosets have a fovea, and make eye movements that fixate in various locations on the screen during the task. Finally, the model provides a strong basis for comparison at the level of neuronal populations, but some methodological choices are insufficiently described and may have an impact on interpreting the claims.

Overall, the methods and data are supportive of the main claims of the work. The use of single neuron and population level approaches demonstrate that the activity of V1 in mice and marmoset is categorically different. Since primate V1 is so diverse, this limits the interpretation of the cross-species comparison. Still, the work is a great step forward in the field, especially with the novel methodology of collecting neural activity from running primates.

Reviewer #2 (Public Review):

The pear psylla Cacopsylla chinensis has two morphologically different forms, winter- and summer-forms depending on the temperatures. The authors provided solid data showing that the cold sensor CcTRPM is responsible for switching summer- to winter forms, which is in turn regulated by the miRNA miR-252. This finding is interesting and novel.

Reviewer #1 (Public Review):

Here, the authors describe, in detail, the transition between the summer form and the winter form of the pear psyllid, Cacopsylla chinensis. While the authors explore many components of this transition, the central hypotheses they seek to test are (i) that a protein they deem CcTRPM is a cold-sensitive Transient Receptor Potential Melastatin (TRPM) channel, and (ii) that this channel is involved in the summer-to-winter transition, in response to cold.

The authors demonstrate that: both cold and menthol can initiate the summer-to-winter transition; that the protein of interest is required for the summer-to-winter transition (in vivo); that the protein of interest is involved in menthol-dependent Ca2+ transients (in vitro); that miR-252 expression is temperature-dependent, modulates the seasonal transition, and affects the expression of the transcript of interest; and finally, somewhat separately, that the chitin biosynthesis pathway is linked to the summer-to-winter transition.

Although I generally found the evidence to be convincing, I note a few critical weaknesses in the manuscript as it is currently presented. Firstly, there is insufficient methodological detail to understand how the genes/transcripts/proteins in this work were identified. Further, the structural and phylogenetic analyses are incompletely described and the results are inconsistent with our previous understanding of the structure and evolution of TRPMs. It is thus possible (although unlikely) that this protein has been misidentified. Alternatively, this could be a structurally aberrant TRPM from a lineage previously presumed to be lost in insects, but there is not sufficient evidence to conclude this. Perhaps more importantly, the authors conclude that the protein of interest is cold sensitive (i.e., a "temperature receptor") primarily based on menthol sensitivity. Although menthol and cold activate the same receptors in other species, there is no demonstrated reason to think that menthol sensitivity necessitates cold sensitivity, or vice-versa. Thus, the authors' conclusions are, in my opinion, incomplete and overstated. Below are specific comments giving further context to the criticisms summarized above:

1. The method used to identify the various genes/proteins described herein is not described. Relatedly, the alignment in Figure S1 lacks Trpms from non-hemipteran taxa, making it difficult to judge sequence similarity to other more well-characterized Trpms (e.g., from human, mouse, fly, nematode, etc.), and thus difficult to assess homology from the manuscript alone.

2. The authors suggest that the CcTrpm has ankyrin repeats. To my knowledge, this would be the first description of ankyrin repeats in TRPM. It's not stated how the authors identified these putative ankyrin repeats. There's also no description of the absence or presence of previously identified Melastatin Homology Regions (MHRs), a C-terminal coiled-coil that is typically present, other C-terminal domain motifs, or the TRP domain. In the absence of methodological detail, and given the proposed presence of ankyrin repeats, it seems possible that this may not be TRPM.

3. The authors suggest that, because mRNA abundance for CcTRPM is increased in response to cold, it is cold-sensitive. However, this says nothing as to whether cold actually activates the ion channel -- a critical distinction. The authors finally conclude that CcTRPM encodes a cold-sensitive ion channel because menthol elicits Ca2+ activity in vitro. However, this experiment only demonstrates that the protein is likely menthol sensitive. This experiment does not support the authors' conclusion that this is a cold-sensitive receptor (although their later knockdown experiments do, albeit indirectly).

4. The lack of taxonomic representation in the phylogenetic analysis makes it difficult to interpret, especially in the context of methodological detail concerning the initial identification of the gene/transcript/protein of interest. Further, it's not stated if the tree is rooted (if it is, the rooting methodology is not described), the branch lengths are not shown, and the branch support methodology is not described. Many previous phylogenetic analyses have concluded--implicitly or explicitly--that there are at least two ancestral animal TRPM paralogs. From the perspective of vertebrates, one ancestral copy went on to diversify into TRPMs 1, 3, 6, and 7, and the other ancestral copy went on to diversify into TRPMs 2, 8, 4, and 5. The insect Trpms are generally thought to be more closely related to vertebrate TRPMs 1,3,6, and 7. If this phylogeny is rooted, it implies that the hemipteran Trpms are more closely related to vertebrates 2, 8, 4, and 5 (or at least 8, since that is all that is present here), and quite distantly related to other insect Trpms (and presumably, to vertebrates 1,3,6, and 7, which are not present). To my knowledge, this would be the first description of this Trpm subfamily in insects, but there is insufficient evidence or phylogenetic rigor here to conclude that. The most likely explanation is that the tree is unrooted, incorrectly rooted, or that the protein of interest is not TRPM.

Reviewer #1 (Public Review):

The study titled "Distinct states of nucleolar stress induced by anti-cancer drugs" by Potapova and colleagues demonstrates that different chemotherapeutic agents can induce nucleolar stress, which manifests with varying cellular and molecular characteristics. The study also proposes a mechanism for how a novel type of nucleolar stress driven by CDK inhibitors may be regulated. As a reviewer, I appreciate the unbiased screening approach and I am enthusiastic about the novel insights into cell biology and the implications for cancer research and treatment. The study has several significant strengths: i) it highlights the understudied role of nucleolar stress in the on- and off-target effects of chemotherapy; ii) it defines novel molecular and cellular characteristics of the different types of nucleolar stress phenotypes; iii) it proposes novel modes of action for well-known drugs.

However, there are several important points that should be addressed:<br /> • The rationale behind choosing RPE cells for the screen is unclear. It might be more informative to use cancer cells to study the effects of chemotherapeutic agents. Alternatively, were RPE cells selected to evaluate the side effects of these agents on normal cells? Clarifying these points in the introduction and discussion would guide the reader.<br /> • Figure 2F indicates that DLD1 and HCT116 cells are less sensitive to nucleolar changes induced by several inhibitors, including CDK inhibitors. It would be crucial to correlate these differences with cell viability. Are these differences due to cell-type sensitivity or variations in intracellular drug levels? Assessing cell viability and intracellular drug concentration for the same drugs and cells would provide valuable insights.<br /> • Have the authors interpreted nucleolar stress as the primary cause of cell death induced by these drugs? When cells treated with CDK inhibitors exhibit the dissociated nucleoli phenotype, is this effect reversible? Is this phenotype indicative of cell death commitment? Conducting a washout experiment to measure the recovery of nucleolar function and cell viability would address these questions.<br /> • The correlation between the loss of Treacle phosphorylation and nucleolar stress upon CDK inhibition is intriguing. However, it remains unclear how these two events are related. Would Treacle knockdown yield the same nucleolar phenotype as CDK inhibition? Moreover, would point mutations that abolish Treacle phosphorylation prevent its interaction with Pol-I? Experiments addressing these questions would enhance our understanding of the correlation/causation between Treacle phosphorylation and the effects of CDK inhibition on nucleolar stress.

Overall, this study is significant and novel as it sheds light on the importance of nucleolar stress in defining the on-target and off-target effects of chemotherapy in normal and cancer cells.

Reviewer #2 (Public Review):

This is an interesting study with high-quality imaging and quantitative data. The authors devise a robust quantitative parameter that is easily applicable to any experimental system. The drug screen data can potentially be helpful to the wider community studying nucleolar architecture and the effects of chemotherapy drugs. Additionally, the authors find Treacle phosphorylation as a potential link between CDK9 inhibition, rDNA transcription, and nucleolar stress. Therefore I think this would be of broad interest to researchers studying transcription, CDKs, nucleolus, and chemotherapy drug mechanisms. However, the study has several weaknesses in its current form as outlined below.

1. Overall the study seems to suffer from a lack of focus. At first, it feels like a descriptive study aimed at characterizing the effect of chemotherapy drugs on the nucleolar state. But then the authors dive into the mechanism of CDK inhibition and then suddenly switch to studying biophysical properties of nucleolus using NPM1. Figure 6 does not enhance the story in any way; on the contrary, the findings from Fig. 6 are inconclusive and therefore could lead to some confusion.

2. The justification for pursuing CDK inhibitors is not clear. Some of the top hits in the screen were mTOR, PI3K, HSP90, Topoisomerases, but the authors fail to properly justify why they chose CDKi over other inhibitors.

3. In addition to poor justification, it seems like a very superficial attempt at deciphering the mechanism of CDK9i-mediated nucleolar stress. I think the most interesting part of the study is the link between CDK9, Pol I transcription, and nucleolar stress. But the data presented is not entirely convincing. There are several important controls missing as detailed below.

4. The authors did not test if inhibition of CDK7 and/or CDK12 also induces nucleolar stress. CDK7 and CDK12 are also major kinases of RNAPII CTD, just like CDK9. Importantly, there are well-established inhibitors against both these kinases. It is not clear from the text whether these inhibitors were included in the screen library.

5. In Figure 4E, the authors show that Pol I is reduced in nucleolus/on rDNA. The authors should include an orthogonal method like chromatin fractionation and/or ChIP

6. In Fig. 5D, in vitro kinase lacks important controls. The authors should include S to A mutants of Treacle S1299A/S1301A to demonstrate that CDK9 phosphorylates these two residues specifically.

7. To support their model, the authors should test if overexpression of Treacle mutants S1299A/S1301A can partially phenocopy the nucleolar stress seen upon CDK9 inhibition. This would considerably strengthen the author's claim that reduced Treacle phosphorylation leads to Pol I disassociation from rDNA and consequently leads to nucleolar stress.

8. Additionally, it would be interesting if S1299D/S1301D mutants could partially rescue CDK9 inhibition.

Reviewer #1 (Public Review):

In this paper by Lui and colleagues, the authors examine the role of locus coeruleus (LC)-noradrenaline (NA) neurons in the extinction of appetitive instrumental conditioning. They report that optogenetic activation of global LC-NA neurons during the conditioned stimulus (CS) period of extinction enhances long-term extinction memory without affecting within-session extinction. In contrast, LC-NA activation during the intertrial interval doesn't affect extinction and long-term memory. They then show that optogenetic activation of LC-NA neurons doesn't induce conditioned place preference/avoidance. Finally, they assess the necessity of LC-NA neurons in appetitive extinction and find that optogenetic inactivation of LC-NA neurons during the CS period results in the enhancement of within-session extinction. The experiments are well-designed, including offset control in the optogenetic activation study. I think this study adds new insight into the LC-NA system in the context of appetitive extinction.

Strengths:<br /> ・These studies identify that the artificial activation of LC-NA neurons enhances long-term memory of appetitive extinction, while this activation can't induce long-term conditioned place aversion. Thus, optogenetic activation of LC-NA neurons can inhibit spontaneous recovery of appetitive extinction without causing long-term aversive memory.<br /> ・Optoinhibition study demonstrates the reduction of a conditioned response of within-session extinction. Therefore, LC-NA neuronal activity at the CS period of extinction could act as anti-extinction or be important for the expression of the conditioned response.

Weaknesses:<br /> ・It is unclear how LC-NA neurons behave during the CS period of appetitive extinction from this study. This weakens the importance of the optogenetic inactivation result.<br /> ・While authors manipulate global LC-NA neurons, many people find functionally heterogeneous populations in the LC. It remains unsolved if there is a specific LC-NA subpopulation responsible for appetitive extinction.

Reviewer #2 (Public Review):

This study examines the role of the Locus Coeruleus (LC)/noradrenergic (NA) system in extinction in male and female rats. The behavioural task involves three phases i) training on a discriminative procedure in which operant responding was rewarded only during the presentation of a stimulus ii) extinction iii) testing for the expression of extinction at both short (1 day) or long (7 days) delays. Targeting LC/NA cells with optogenetic in TH::Cre rats, the authors found that photoexcitation during extinction led to an increase in the expression of extinguished responding at both short and long delays. By contrast, photo inhibition was found to be without an effect.

1. In such discrimination training, Pavlovian (CS-Food) and instrumental (LeverPress-Food) contingencies are intermixed. It would therefore be very interesting if the authors provided evidence of other behavioural responses (e.g. magazine visits) during extinction training and tests.<br /> 2. In Figure 1, the authors show the behavioural data of the different groups of control animals which were later collapsed in a single control group. It would be very nice if the authors could provide the data for each step of the discrimination training.<br /> 3. Inspection of Figures 2C & 2D shows that responding in control animals is about the same at test 2 as at the end of extinction training. Therefore, could the authors provide evidence for spontaneous recovery in control animals? This is of importance given that the main conclusion of the authors is that LC stimulation during extinction training led to an increased expression of extinction memory as expressed by reduced spontaneous recovery.<br /> 4. Current evidence suggests that there are differences in LC/NA system functioning between males and females. Could the authors provide details about the allocation of male and female animals in each group?<br /> 5. The histology section in both experiments looks a bit unsatisfying. Could the authors provide more details about the number of counted cells and also their distribution along the antero-posterior extent of the LC. Could the authors also take into account the sex in such an analysis?

Reviewer #3 (Public Review):

The introduction/background is excellent. It reviews evidence showing that the extinction of conditioned responding is regulated by noradrenaline and suggests that the locus coeruleus (LC) may be a critical locus of this regulation. This naturally leads to the aim of the study: to determine whether the locus coeruleus is involved in the extinction of an appetitive conditioned response. Overall, the study is well-designed, nicely conducted and the results advance our understanding of the role of the LC in the extinction of conditioned behaviour. As such, I believe that these results will be of interest to readers. I do, however, feel that the paper would benefit from the inclusion of additional data to clarify the impact of the LC manipulations (stimulation and inhibition) on performance in the task; and some comment regarding the likely source of differences between the groups at test.

Reviewer #1 (Public Review):

This is my first review of the article entitled "The canonical stopping network: Revisiting the role of the subcortex in response inhibition" by Isherwood and colleagues. This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.

In the current study, the authors compiled five datasets that aimed to investigate neural activity associated with stopping an already initiated action, as operationalized in the classic stop-signal paradigm. Three of these datasets are taken from their own 7T investigations, and two are datasets from the Poldrack group, which used 3T fMRI.

The authors make six chief points:<br /> 1. There does not seem to be a measurable BOLD response in the purportedly critical subcortical areas in contrasts of successful stopping (SS) vs. going (GO), neither across datasets nor within each individual dataset. This includes the STN but also any other areas of the indirect and hyperdirect pathways.<br /> 2. The failed-stop (FS) vs. GO contrast is the only contrast showing substantial differences in those nodes.<br /> 3. The positive findings of STN (and other subcortical) activation during the SS vs. GO contrast could be due to the usage of inappropriate smoothing kernels.<br /> 4. The study demonstrates the utility of aggregating publicly available fMRI data from similar cognitive tasks.<br /> 5. From the abstract: "The findings challenge previous functional magnetic resonance (fMRI) of the stop-signal task"<br /> 6. and further: "suggest the need to ascribe a separate function to these networks."

I strongly and emphatically agree with points 1-5. However, I vehemently disagree with point 6, which appears to be the main thrust of the current paper, based on the discussion, abstract, and - not least - the title.

To me, this paper essentially shows that fMRI is ill-suited to study the subcortex in the specific context of the stop-signal task. That is not just because of the issues of subcortical small-volume SNR (the main topic of this and related works by this outstanding group), but also because of its limited temporal resolution (which is unacknowledged, but especially impactful in the context of the stop-signal task). I'll expand on what I mean in the following.

First, the authors are underrepresenting the non-fMRI evidence in favor of the involvement of the subthalamic nucleus (STN) and the basal ganglia more generally in stopping actions.<br /> - There are many more intracranial local field potential recording studies that show increased STN LFP (or even single-unit) activity in the SS vs. FS and SS vs. GO contrast than listed, which come from at least seven different labs. Here's a (likely non-exhaustive) list of studies that come to mind:<br /> o Ray et al., NeuroImage 2012<br /> o Alegre et al., Experimental Brain Research 2013<br /> o Benis et al., NeuroImage 2014<br /> o Wessel et al., Movement Disorders 2016<br /> o Benis et al., Cortex 2016<br /> o Fischer et al., eLife 2017<br /> o Ghahremani et al., Brain and Language 2018<br /> o Chen et al., Neuron 2020<br /> o Mosher et al., Neuron 2021<br /> o Diesburg et al., eLife 2021<br /> - Similarly, there is much more evidence than cited that causally influencing STN via deep-brain stimulation also influences action-stopping. Again, the following list is probably incomplete:<br /> o Van den Wildenberg et al., JoCN 2006<br /> o Ray et al., Neuropsychologia 2009<br /> o Hershey et al., Brain 2010<br /> o Swann et al., JNeuro 2011<br /> o Mirabella et al., Cerebral Cortex 2012<br /> o Obeso et al., Exp. Brain Res. 2013<br /> o Georgiev et al., Exp Br Res 2016<br /> o Lofredi et al., Brain 2021<br /> o van den Wildenberg et al, Behav Brain Res 2021<br /> o Wessel et al., Current Biology 2022<br /> - Moreover, evidence from non-human animals similarly suggests critical STN involvement in action stopping, e.g.:<br /> o Eagle et al., Cerebral Cortex 2008<br /> o Schmidt et al., Nature Neuroscience 2013<br /> o Fife et al., eLife 2017<br /> o Anderson et al., Brain Res 2020

Together, studies like these provide either causal evidence for STN involvement via direct electrical stimulation of the nucleus or provide direct recordings of its local field potential activity during stopping. This is not to mention the extensive evidence for the involvement of the STN - and the indirect and hyperdirect pathways in general - in motor inhibition more broadly, perhaps best illustrated by their damage leading to (hemi)ballism.

Hence, I cannot agree with the idea that the current set of findings "suggest the need to ascribe a separate function to these networks", as suggested in the abstract and further explicated in the discussion of the current paper. For this to be the case, we would need to disregard more than a decade's worth of direct recording studies of the STN in favor of a remote measurement of the BOLD response using (provably) sub ideal imaging parameters. There are myriads of explanations of why fMRI may not be able to reveal a potential ground-truth difference in STN activity between the SS and FS/GO conditions, beginning with the simple proposition that it may not afford sufficient SNR, or that perhaps subcortical BOLD is not tightly related to the type of neurophysiological activity that distinguishes these conditions (in the purported case of the stop-signal task, specifically the beta band). But essentially, this paper shows that a specific lens into subcortical activity is likely broken, but then also suggests dismissing existing evidence from superior lenses in favor of the findings from the 'broken' lens. That doesn't make much sense to me.

Second, there is actually another substantial reason why fMRI may indeed be unsuitable to study STN activity, specifically in the stop-signal paradigm: its limited time resolution. The sequence of subcortical processes on each specific trial type in the stop-signal task is purportedly as follows: at baseline, the basal ganglia exert inhibition on the motor system. During motor initiation, this inhibition is lifted via direct pathway innervation. This is when the three trial types start diverging. When actions then have to be rapidly cancelled (SS and FS), cortical regions signal to STN via the hyperdirect pathway that inhibition has to be rapidly reinstated (see Chen, Starr et al., Neuron 2020 for direct evidence for such a monosynaptic hyperdirect pathway, the speed of which directly predicts SSRT). Hence, inhibition is reinstated (too late in the case of FS trials, but early enough in SS trials, see recordings from the BG in Schmidt, Berke et al., Nature Neuroscience 2013; and Diesburg, Wessel et al., eLife 2021).<br /> Hence, according to this prevailing model, all three trial types involve a sequence of STN activation (initial inhibition), STN deactivation (disinhibition during GO), and STN reactivation (reinstantiation of inhibition during the response via the hyperdirect pathway on SS/FS trials, reinstantiation of inhibition via the indirect pathway after the response on GO trials). What distinguishes the trial types during this period is chiefly the relative timing of the inhibitory process (earliest on SS trials, slightly later on FS trials, latest on GO trials). However, these temporal differences play out on a level of hundreds of milliseconds, and in all three cases, processing concludes well under a second overall. To fMRI, given its limited time resolution, these activations are bound to look quite similar.

Lastly, further building on this logic, it's not surprising that FS trials yield increased activity compared to SS and GO trials. That's because FS trials are errors, which are known to activate the STN (Cavanagh et al., JoCN 2014; Siegert et al. Cortex 2014) and afford additional inhibition of the motor system after their occurrence (Guan et al., JNeuro 2022). Again, fMRI will likely conflate this activity with the abovementioned sequence, resulting in a summation of activity and the highest level of BOLD for FS trials.

In sum, I believe this study has a lot of merit in demonstrating that fMRI is ill-suited to study the subcortex during the SST, but I cannot agree that it warrants any reappreciation of the subcortex's role in stopping, which are not chiefly based on fMRI evidence.

A few other points:<br /> - As I said before, this team's previous work has done a lot to convince me that 3T fMRI is unsuitable to study the STN. As such, it would have been nice to see a combination of the subsamples of the study that DID use imaging protocols and field strengths suitable to actually study this node. This is especially true since the second 3T sample (and arguably, the Isherwood_7T sample) does not afford a lot of trials per subject, to begin with.<br /> - What was the GLM analysis time-locked to on SS and FS trials? The stop-signal or the GO-signal?<br /> - Why was SSRT calculated using the outdated mean method?<br /> - The authors chose 3.1 as a z-score to "ensure conservatism", but since they are essentially trying to prove the null hypothesis that there is no increased STN activity on SS trials, I would suggest erring on the side of a more lenient threshold to avoid type-2 error.<br /> - The authors state that "The results presented here add to a growing literature exposing inconsistencies in our understanding of the networks underlying successful response inhibition". It would be helpful if the authors cited these studies and what those inconsistencies are.

Reviewer #2 (Public Review):

This work aggregates data across 5 openly available stopping studies (3 at 7 tesla and 2 at 3 tesla) to evaluate activity patterns across the common contrasts of Failed Stop (FS) > Go, FS > stop success (SS), and SS > Go. Previous work has implicated a set of regions that tend to be positively active in one or more of these contrasts, including the bilateral inferior frontal gyrus, preSMA, and multiple basal ganglia structures. However, the authors argue that upon closer examination, many previous papers have not found subcortical structures to be more active on SS than FS trials, bringing into question whether they play an essential role in (successful) inhibition. In order to evaluate this with more data and power, the authors aggregate across five datasets and find many areas that are *more* active for FS than SS, specifically bilateral preSMA, caudate, GPE, thalamus, and VTA, and unilateral M1, GPi, putamen, SN, and STN. They argue that this brings into question the role of these areas in inhibition, based upon the assumption that areas involved in inhibition should be more active on successful stop than failed stop trials, not the opposite as they observed.

As an empirical result, I believe that the results are robust, but this work does not attempt a new theoretical synthesis of the neuro-cognitive mechanisms of stopping. Specifically, if these many areas are more active on failed stop than successful stop trials, and (at least some of) these areas are situated in pathways that are traditionally assumed to instantiate response inhibition like the hyperdirect pathway, then what function are these areas/pathways involved in? I believe that this work would make a larger impact if the author endeavored to synthesize these results into some kind of theoretical framework for how stopping is instantiated in the brain, even if that framework may be preliminary.

I also have one main concern about the analysis. The authors use the mean method for computing SSRT, but this has been shown to be more susceptible to distortion from RT slowing (Verbruggen, Chambers & Logan, 2013 Psych Sci), and goes against the consensus recommendation of using the integration with replacement method (Verbruggen et al., 2019). Therefore, I would strongly recommend replacing all mean SSRT estimates with estimates using the integration with replacement method.

Reviewer #1 (Public Review):

In this manuscript, the authors describe an improved miniscope they name "E-scope", combining in vivo calcium imaging with electrophysiological recording. They use it to examine neural correlates of social interactions with respect to cerebellar and cortical circuits. Through correlations between electrophysiological single units of Purkinje cells and dentate nucleus neurons as well as with calcium signals imaging of neurons from the anterior cingulate cortex, the authors provide correlative data supporting the view that intracerebellar circuits and cerebello-cortical communications take part in the modulation of social behavior. In particular, the electrophysiological dataset reflects the PC-DN connection and strongly suggests its involvement in social interactions. Cross-correlations analyses between PC / DN single units and ACC calcium signals suggest that the recorded cerebellar and cortical structures both take part in the brain networks at play in social behavior.

Strengths:<br /> - This is a timely and important study with solid evidence for correlative conclusions that are not overstated in the manuscript, which is commendable.<br /> - Despite the technical challenge, the experiments presented in this study seem well performed and the quality of the dataset is appropriate.

Weaknesses:<br /> - While the novelty of the device is strongly emphasized, I find that its value is somewhat diminished by the wire-free device developed by the same group as it should thus be possible to perform calcium imaging wire-free and electrophysiological recording via a single conventional cable (or also via wireless headstages).<br /> - The role of the identified network activations in social interactions is not touched upon.

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however, the biological insight gained is rather limited.

Reviewer #3 (Public Review):

Complex behavior requires complex neural control involving multiple brain regions. The currently available tools to measure neural activity in multiple brain regions in small animals are limited and often involve obligatory head-fixation. The latter, obviously, impacts the behaviors under study. Hur and colleagues present a novel recording device, the E-Scope, that combines optical imaging of fluorescent calcium imaging in one brain region with high-density electrodes in another. Importantly, the E-Scope can be implanted and is, therefore, compatible with usage in freely moving mice. The authors used their new E-Scope to study neural activity during social interactions in mice. They demonstrate the presence of neural correlates of social interaction that happen simultaneously in the cerebellum and the anterior cingulate cortex.

The major accomplishment of this study is the development and introduction of the E-Scope. The evaluation of this part can be short: it works, so the authors succeeded.

The authors managed to reduce the weight of the implant to 4.5 g, which is - given all functionality - quite an accomplishment in my view. However, a mouse weighs between 20 and 40 g, so that an implant of 4.5 g is still quite considerable. It can be expected that this has an impact on the behavior and, possibly, the well-being of the animals. Whether this is the case or not, is not really addressed in this study. The authors suffice with the statement that "Recorded animals made more contact with the other mouse than with the object (Figure 2A), suggesting a normal preference for social contact with the E-Scope attached."

Overall, the description of animal behavior is rather sparse. The methods state only that stranger age-matched mice were used, but do not state their gender. The nature of the social interactions was not described? Was their aggressive behavior, sexual approach and/or intercourse? Did the stranger mice attack/damage the E-Scope? Were the interactions comparable (using which parameters?) with and without E-Scope attached? It is not even described what the authors define as an "interaction bout" (Figure 2A). The number of interaction bouts is counted per 7 minutes, I presume? This is not specified explicitly.

In Figure 1 D-G, the authors present raw data from the neurophysiological recordings. In panel D, we see events with vastly different amplitudes. It would be very insightful if the authors would describe which events they considered to be action potentials, and which not. Similarly, the raw traces of Figure 1E are declared to be single-unit recordings of Purkinje cells. Partially due to the small size of the traces (invisible in print and pixelated in the digital version), I have a hard time recognizing complex spikes and simple spikes in these traces. This is a bit worrisome, as the authors declare the typical duration of the pause in simple spike firing after a complex spike to be 20-100 ms. In my experience, such long pauses are rare in this region, and definitely not typical. In the right panel of Figure 1A, an example of a complex spike-induced pause is shown. This pause is around 15 ms, so not typical according to the text, and starts only around 4 ms after the complex spike, which should not be the case and suggests either a misalignment of the figure or the detection of complex spike spikelets as simple spikes, while the abnormally long pause suggests that the authors fail to detect a lot of simple spikes. The authors could provide more confidence in their data by including more raw data, making explicit how they analyzed the signals, and by reporting basic statistics of firing properties (like rate, cv or cv2, pause duration). In this respect, Figure 2 - figure supplement 3 shows quite a large percentage of cells to have either a very low or a very high firing rate.

The number of Purkinje cells recorded during social interactions is quite low: only 11 cells showed a modulation in their spiking activity (unclear whether in complex spikes, simple spikes or both. During object interaction, only 4 cells showed a significant modulation. Unclear is whether the latter 4 are a subset of the former 11, or whether "social cells" and "object cells" are different categories. Having so few cells, and with these having different types of modulation, the group of cells for each type of modulation is really small, going down to 2 cells/group. It is doubtful whether meaningful interpretation is possible here.

This brings us to the next point: neural correlates of social interaction are notoriously difficult to interpret. Social behavior is complex, and involves the processing of sensory cues (olfaction, touch (whiskers), visual and auditory), the production of ultrasonic vocalizations (in specific contexts), movements, and emotional behavior (fear, pleasure, sexual interest). In other words, neural activity patterns observed during social interaction do not necessarily relate specifically to social interaction, but can also occur in a non-social context. The authors control this by comparing social interactions with object interactions, but I miss a direct comparison between the two conditions, both in terms of behavior (now only the number of interactions is counted, not their duration or intensity), and in terms of neural activity. There is some analysis done on the interaction between movement and cerebellar activity (Figure 2 - figure supplement 4), but it is unclear to what extent social interactions and movements are separated here. It would already help to indicate in the plots with trajectories (e.g., Fig. 2H) indicate the social interactions (e.g., social interaction-related movements in red, the rest of the trajectories in black).

The neuron count in the anterior cingulate cortex is much higher than for the cerebellum, but also here it is not so clear what is "social" and what is "non-social". In Figure 3G-H, the authors indicate a near-perfect separation between cells active during social encounters and those active during object encounters. This could indicate that there is here indeed a social aspect, but as we do not know to what extent the sensory and motor aspects differ between social and non-social interactions, this is still hard to interpret.

Finally, the authors show that there are correlations between the modulation in neurons of the anterior cingulate cortex and cerebellar neurons related to bouts of social activity. Here, it could be interesting to see whether there are differences in latency between the two brain areas.

In conclusion, the authors present a novel method to record neural activity with single cell-resolution in two brain regions in freely moving mice. Given the challenges associated with understanding of complex behaviors, this approach can be useful for many neuroscientists. The authors demonstrate the potential of their approach by studying social interactions in mice. Clearly, there are correlations in the activity of neurons in the anterior cingulate cortex and the cerebellum related to social interactions. To bring our understanding of these patterns to a higher level, more detailed analyses (and probably also larger group sizes of cerebellar neurons) are required, though.

Reviewer #1 (Public Review):

This study conducted a series of experiments to comprehensively support the allocentric rather than egocentric visual spatial reference updating for the path-integration mechanism in the control of target-oriented locomotion. Authors firstly manipulated the waiting time before walking to tease apart the influence from spatial working memory in guiding locomotion. They demonstrated that the intrinsic bias in perceiving distance remained constant during walking and that the establishment of a new spatial layout in the brain took a relatively longer time beyond the visual-spatial working memory. In the following experiments, the authors then uncovered that the strength of the intrinsic bias in distance perception along the horizontal direction is reduced when participants' attention is distracted, implying that world-centered path integration requires attentional effort. This study also revealed horizontal-vertical asymmetry in a spatial coding scheme that bears a resemblance to the locomotion control in other animal species such as desert ants.

The overall design of the behavioral experiments is elegant and statistics are well performed to support the authors' viewpoint in the allocentric rather than egocentric visual spatial coding scheme for distance perception along the horizontal line.

It is however worth noting the statement from Gibson in 1979 that for egocentric distances, tangible information arises from the effort required to walk a distance, thus, effort becomes associated through experience with visual distance cues. Accordingly, visual information alone is insufficient to support the awareness of distance. Perceived distance is rather specified by an invariant relationship between distal extent and a persons' potential to perform gross motion actions such as walking. This view is supported later by Proffitt et al. (2003) in which participants wore backpacks and their perceived distance increased compared with the baseline condition. Authors need to acknowledge the physical effort in addition to visual information for the spatial coding and may consider the manipulation of physical efforts in the future to support the robustness of constant intrinsic bias in ground-based spatial coding during walking.

Furthermore, it would be more comprehensive and fit into the Neuroscience Section if the authors can add in current understandings of the spatial reference frames in neuroscience in the introduction and discussion, and provide explanations on how the findings of this study supplement the physiological evidence that supports our spatial perception as well. For instance, world-centered representations of the environment, or cognitive maps, are associated with hippocampal formation while self-centered spatial relationships, or image spaces, are associated with the parietal cortex (see Bottini, R., & Doeller, C. F. (2020). Knowledge Across Reference Frames: Cognitive Maps and Image Spaces. Trends in Cognitive Sciences, 24(8), 606-619. https://doi.org/10.1016/j.tics.2020.05.008 for details)

Reviewer #2 (Public Review):

The study provides a valuable contribution by demonstrating the use of an allocentric spatial reference frame in the perception of the location of a dimly lit target in the dark. While the evidence presented in support of the authors' claims is solid and convincing, it would be beneficial for the study to address potential limitations, such as its ecological validity.

Strengths:<br /> Unlike previous research where observers were stationary during a visual-spatial perception task, this recent study expanded upon prior findings by incorporating bodily movements for the observers. This study is a valuable addition to the literature as it not only discovered that the intrinsic bias is grounded on the home base, but also identified several key characteristics through a series of follow-up experiments. The findings suggest that this "allocentric" spatial coding decays over time, requires attentional resources, can be based solely on vestibular signals, and is most effective in the horizontal direction. In general, this study is interesting, clearly presented, well-thought-out and executed. The results confirmed the conclusions and the study's comprehensive approach offers valuable insights into the nature of intrinsic bias in spatial perception.

The counter-intuitive results presented in the manuscript are intriguing and add to the study's overall appeal. Moreover, the manuscript draws an interesting parallel between human spatial navigation and that of desert ants. This comparison helps to underscore the importance of understanding spatial coding mechanisms across different species and highlights potential avenues for future research.

One aspect I particularly valued about this study was the authors' thorough description of the experimental methods. This level of detail not only highlights the rigor of the research but also enhances the reproducibility of the study, making it more accessible for future researchers.

Weaknesses:<br /> While the current study provides valuable insights into the nature of intrinsic bias in spatial perception, there is a concern regarding its ecological validity. The experimental design involved stringent precautions, such as a very dark room and a small target, to minimize the presence of depth cues. This is in contrast to the real world, where depth information is readily available from the ground and surrounding objects, aiding in our perception of space and depth. As a result, it is unclear to what extent this "allocentric" intrinsic bias is involved in our everyday spatial perception. To provide more context for the general audience, it would be beneficial for the authors to address this issue in their discussion.

The current findings on the "allocentric" coding scheme raise some intriguing questions as to why such a mechanism would be developed and how it could be beneficial. The finding that the "allocentric" coding scheme results in less accurate object localization and requires attentional resources seems counterintuitive and raises questions about its usefulness. However, this observation presents an opportunity for the manuscript to discuss the potential evolutionary advantages or trade-offs associated with this coding mechanism.

The manuscript lacks a thorough description of the data analysis process, particularly regarding the fitting of the intrinsic bias curve (e.g., the blue and gray dashed curve in Figure 3c) and the calculation of the horizontal separation between the curves. It would be beneficial for the authors to provide more detailed information on the specific function and parameters used in the fitting process and the formula used for the separation calculation to ensure the transparency and reproducibility of the study's results.

Reviewer #3 (Public Review):

This study investigated what kind of reference (allocentric or egocentric) frame we used for perception in darkness. This question is essential and was not addressed much before. The authors compared the perception in the walking condition with that in the stationary condition, which successfully separated the contribution of self-movement to the spatial representation. In addition, the authors also carefully manipulated the contribution of the waiting period, attentional load, vestibular input, testing task, and walking direction (forward or backward) to examine the nature of the reference frame in darkness systematically.

I am a bit confused by Figure 2b. Allocentric coordinate refers to the representation of the distance and direction of an object relative to other objects but not relative to the observer. In Figure 2, however, the authors assumed that the perceived target was located on the interception between the intrinsic bias curve and the viewing line from the NEW eye position to the target. This suggests that the perceived object depends on the observer's new location, which seems odd with the allocentric coordinate hypothesis.

According to Fig 2b, the perceived size should be left-shifted and lifted up in the walking condition compared to that in the stationary condition. However, in Figure 3C and Fig 4, the perceived size was the same height as that in the baseline condition.

Is the left-shifted perceived distance possibly reflecting a kind of compensation mechanism? Participants could not see the target's location but knew they had moved forward. Therefore, their brain automatically compensates for this self-movement when judging the location of a target. This would perfectly predict the left-shifted but not upward-shifted data in Fig 3C. A similar compensation mechanism exists for size constancy in which we tend to compensate for distance in computing object size.

According to Fig 2a, the target, perceived target, and eye should be aligned in one straight line. This means that connecting the physical targets and the corresponding perceived target results in straight lines that converge at the eye position. This seems, however, unlikely in Figure 3c.

Reviewer #1 (Public Review):

The manuscript by Lin et al describes a wide biophysical survey of the molecular mechanisms underlying full-length BTK regulation. This is a continuation of this lab's excellent work on deciphering the myriad levels of regulation of BTKs downstream of their activation by plasma membrane localised receptors.

The manuscript uses a synergy of cryo EM, HDX-MS and mutational analysis to delve into the role of how the accessory domains modify the activity of the kinase domain. The manuscript essentially has three main novel insights into BTK regulation.

1. Cryo EM and SAXS show that the PHTH region is dynamic compared to the conserved Src module.<br /> 2. A 2nd generation tethered PH-kinase construct crystal of BTK reveals a unique orientation of the PH domain relative to the kinase domain, that is different from previous structures.<br /> 3. A new structure of the kinase domain dimer shows how trans-phosphorylation can be achieved.

Excitingly these structural works allow for the generation of a model of how BTK can act as a strict coincidence sensor for both activated BCR complex as well as PIP3 before it obtains full activity. To my eye the most exciting result of this work is describing how the PH domain can inhibit activity once the SH3/SH2 domain is disengaged, allowing for an additional level of regulatory control.

I have very few experimental concerns as the methods and figures are well-described and clear. As the authors are potentially saying that the previously solved PH domain-kinase interface is artefactual, additional evidence strengthening their model would be helpful to resolve any possible controversies.

Reviewer #2 (Public Review):

In this study, multiple biophysical techniques were employed to investigate the activation mechanism of BTK, a multi-domain non-receptor protein kinase. Previous studies have elucidated the inhibitory effects of the SH3 and SH2 domains on the kinase and the potential activation mechanism involving the membrane-bound PIP3 inducing transient dimerization of the PH-TH domain, which binds to lipids.

The primary focus of the present study was on three new constructs: a full-length BTK construct, a construct where the PH-TH domain is connected to the kinase domain, and a construct featuring a kinase domain with a phosphomimetic at the autophosphorylation site Y551. The authors aimed to provide new insights into the autoinhibition and allosteric control of BTK.

The study reports that SAXS analysis of the full-length BTK protein construct, along with cryoEM visualization of the PH-TH domain, supports a model in which the N-terminal PH-TH domain exists in a conformational ensemble surrounding a compact/autoinhibited SH3-SH2-kinase core. This finding is interesting because it contradicts previous models proposing that each globular domain is tightly packed within the core.

Furthermore, the authors present a model for an inhibitory interaction between the N-lobe of the kinase and the PH-TH domain. This model is based on a study using a tethered complex with a longer tether than a previously reported construct where the PH-TH domain was tightly attached to the kinase domain (ref 5). The authors argue that the new structure is relevant. However, this assertion requires further explanation and discussion, particularly considering that the functional assays used to assess the impact of mutating residues within the PH-TH/kinase domain contradict the results of the previous study (ref 5).

Additionally, the study presents the structure of the kinase domain with swapped activation loops in a dimeric form, representing a previously unseen structure along the trans-phosphorylation pathway. This structure holds potential relevance. To better understand its significance, employing a structure/function approach like the one described for the PH-TH/kinase domain interface would be beneficial.

Overall, this study contributes to our understanding of the activation mechanism of BTK and sheds light on the autoinhibition and allosteric control of this protein kinase. It presents new structural insights and proposes novel models that challenge previous understandings. However, further investigation and discussion would significantly strengthen the study.

Reviewer #3 (Public Review):

Yin-wei Lin et al set out to visualize the inactive conformation of full-length Bruton's Tyrosine Kinase (BTK), a molecule that has evaded high-resolution structural studies in its full-length form to this date. An open question in the field is how the Pleckstrin Homology-Tec Homology (PHTH) domain inhibits BTK activity, with multiple competing models in the field. The authors used a complimentary set of biophysical techniques combined with well-thought-out stabilizing mutations to obtain structural insights into BTK regulation in its full-length form. They were able to crystallize the full-length construct of BTK but unfortunately, the PHTH was not resolved yielding a structure similar to that previously obtained in the field. The investigation of the same construct by SAXS yielded an elongated structural model, consistent with previous SAXS studies. Using cryo-EM the authors obtained a low-resolution model for the FL BTK with a loosely connected density assigned to the dynamic PHTH around the compact SH2-SH3-Kinase Domain (KD) core. To gain further molecular insights into PHTH-KD interactions the authors followed a previously reported strategy and generated a fusion of PHTH-KD with a longer linker, yielding a crystal structure with a novel PHTH-KD interface which they tested in biochemical assays. Lastly, Yin-wei Lin et al crystallized the BTK KD in a novel partially active state in a "face-to-face" dimer with kinases exchanging the activation loops, although partially disordered, being theoretically perfectly positioned for transphosphorylation. Overall this presents a valiant effort to gain molecular insights into what clearly is a dynamic regulatory motif on BTK and is a valuable addition to the field.

However, this work can be improved by considering these points:

1) The cryo-EM reconstructions are potentially over-interpreted. The reported resolution for all of the analyzed reconstructions is better than 8Å, at which point helices should be recognized as well-resolved structural elements. In the current view/depiction of the cryo-EM maps/models it is hard to see such structural features and it would be great if the authors could include a panel showing maps at higher thresholds to show correspondence between the helices in the kinase C lobe and the cryo-EM maps. Otherwise, the overall positioning of the models within the cryo-EM maps is hard to evaluate and may very well be wrong. (Fig 4, S2).

2) With the above in mind, if the maps are not at the point where helices are well resolved, it may be beneficial to low-pass filter the maps to a more conservative resolution for fitting, analysis, and representation. (Fig 4, S2).

3) It would be valuable to get a quantitative metric on the model/map fitting for the cryo-EM work. One good package for this is Situs which provides cross-correlation values for the top orthogonal fits, without user input for initial fitting. This would again increase the confidence in the correctness of model positioning on the map. (Fig 4, S2).

4) It would be great to see 2D class averages from the particles contributing to each of the 3D classes. Theoretically, a clear bright "blob" (hypothesized to be the PHTH domain) should be observable in the 2D class averages. In the current 2D class averages that region is unconvincingly weak. (Fig 4, S2).

5) It seems like there was quite a large circular mask applied during 2D classification. Are authors confident that the weak density attributed to the PHTH domain is not neighboring particles making their way into the extraction box? It would be great if the authors would trim their particle stack with a very stringent inter-particle distance cutoff (or report the cutoff in the manuscript if already done so) to minimize this possibility.

6) The cryo-EM processing may benefit from more stringent particle picking. The authors picked over 2M particles from 750 micrographs which likely represents very heavy overpicking. I would encourage the authors to re-pick the micrographs with 2D class averages and use more stringent metrics to reduce the overpicking. This may result in higher-resolution reconstructions. (Fig 4, S2).

7) The Dmax from SAXS for the Full Length BTK is at 190Å. It would be great if the authors could make a cartoon of what domain arrangement may satisfy this distance, as it is quite extended for such a small particle. Can the authors rule out dimerization at SAXS concentrations? (Fig 1).

8) In Figure S1 (C) it seems that the curves are just scattering curves with Guinier plots in the inserts, but are labeled as Guinier plots in the legend. The Guinier plots for some samples (FL 4P1F) show signs of aggregation, which may complicate the analysis, it could be beneficial to redo.

9) Have the authors verified that the activation loop mutations that they introduce do not disrupt the PHTH binding as they previously reported an activation loop on BTK to interact with PHTH, an interaction they do not see here? If so, a citation would be helpful in the text. If not, testing this would strengthen the paper.

10) Can the authors comment on the surfaces which are accessible and inaccessible to the PHTH in the crystal (Fig 3E)? The fact that PHTH doesn't adopt a stable conformation in the solvent channel to some degree indicates that the accessible interaction surfaces are not suitable for PHTH interactions, as the "effective concentration" of the PHTH would be quite high. Are these surfaces consistent with the cryo-EM analysis?

11) For the novel active state dimer of the Kinase Domain it would be great to see some functional validation of the dimerization interface. It is structurally certainly quite suggestive, but without such experiments the functional significance is unclear. If appropriate mutations have been published previously a citation would be helpful.

Reviewer #1 (Public Review):

First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.

The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.

The paper was well prepared.

Reviewer #2 (Public Review):

The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study. However, the present manuscript insists as a whole negatively that the experimental errors may hamper to provide the actual structural changes relevant to the electron transfer events. My concerns are the following two points:<br /> Is the premise of the authors for the electron transfer energetics obviously valid?<br /> Could the authors find any positive aspect(s) in the XFEL study?

The authors' argument is certainly due to their premise "Em(HL) is expected to be exclusively higher in the 5-ps and 20-ps structures than in the other XFEL structures due to the stabilization of the [PLPM]•+HL•- state by protein reorganization" as noted in the Results and Discussion (p. 12, lines 180-182); however, it is unknown whether this premise can be applied to the ps-timescale electron transfer events. The above premise is surely based on the Marcus theory, as the authors also noted in the Introduction "The anionic state formation induces not only reorganization of the protein environment (ref. 5: Marcus and Sutin, 1985) but also out-of-plane distortion of the chlorin ring (ref. 6: two of the authors, Saito and Ishikita, co-authored, 2012)"; however, it is unknown whether protein reorganization can follow the ps-timescale electron transfer events. Indeed, Dods et al. mentioned in the Nature paper (2021) "The primary electron-transfer step from SP (special pair PLPM) to BPhL (HL) occurs in 2.8 {plus minus} 0.2 ps across a distance of 10 Å by means of a two-step hopping mechanism via the monomeric BChL molecule and is more rapid than conventional Marcus theory". It was also mentioned, "By contrast, the 9 Å electron-transfer step from BPhL to QA has a single exponential decay time of 230 {plus minus} 30 ps, which is consistent with conventional Marcus theory". As for the primary electron-transfer step from PLPM to HL, Wang et al. (2007, Science 316, 747; cited as ref. 8 in the Nature paper 2021) reported, by monitoring tryptophan absorbance changes in various reaction centers in which the driving forces (namely, the Em gaps between PLPM and HL) are different, that the protein relaxation kinetics is independent of the charge separation kinetics on the picosecond timescale. On the other hand, in the EPR study cited by the authors as ref. 7 (Muh et al. (1998) Biochemistry 37, 13066), although the authors described "two distinct conformations of HL- were reported in spectroscopic studies" (p. 3, lines 44-45), it should be noted that conformation of HL- was formed by 1 or 45 s illumination prior to freezing, and hence the second-order reorganized conformations may differ from picosecond-order conformations observed by the XFEL study (Nature, 2021) and/or the transient absorption spectroscopy (Science, 2007).

Therefore, I consider there is a possibility that the authors' findings may reflect not experimental errors but the actual ps-timescale phenomena presented by the first-time XFEL study on the timescale of the primary charge-separation reactions of photosynthesis. Thus I would like to suggest that the authors reconsider the premise for the electron transfer energetics on the picosecond timescale.

In any case, to discuss the experimental errors in the XFEL study, it is better to calculate the Em(QA) changes in the 300-ps and 8-us XFEL structures, which showed distinctive structural changes even at the 2.8 Å resolution as discussed by Dods et al. Then, if the Em(QA) values are changed as expected from theoretical calculations, such calculated results may suggest a useful theoretical interpretation of the XFEL study as a positive aspect. If the Em(QA) values are not higher in the 300-ps and 8-us structures than in the other structures, it may be argued that the experimental errors would be so large that the XFEL structures are irrelevant to the electron transfer events expected from theoretical calculations.

Reviewer #1 (Public Review):

The authors aimed to establish a cell culture system to investigate muscle tissue development and homeostasis. They successfully developed a complex 3D cell model and conducted a comprehensive molecular and functional characterization. This approach represents a critical initial step towards using human cells, rather than animals, to study muscular disorders in vitro. Although the current protocol is time-consuming and the fetal cell model may not be mature enough to study adult-onset diseases, it nonetheless provides a valuable foundation for future disease modelling studies using isogenic iPSC lines or patient-derived cells with specific mutations. The manuscript does not explore whether or how this stem cell model can advance our understanding of muscular diseases, which would be an exciting avenue for future research. Overall, the detailed protocol presented in this paper will be useful for informing future studies and provides an important resource to the stem cells community. The inclusion of data on disease modelling using isogenic iPSC lines or patient-derived cells would further enhance the manuscript's impact.

Reviewer #2 (Public Review):

This paper illustrates that PSCs can model myogenesis in vitro by mimicking the in vivo development of the somite and dermomyotome. The advantages of this 3D system include (1) better structural distinctions, (2) the persistence of progenitors, and (3) the spatial distribution (e.g. migration, confinement) of progenitors. The finding is important with the implication in disease modeling. Indeed the authors tried DMD model although it suffered the lack of deeper characterization.

The differentiation protocol is based on a current understanding of myogenesis and compelling. They characterized the organoids in depth (e.g. many time points and immunofluorescence). The evidence is solid, and can be improved more by rigorous analyses and descriptions as described below.

Major comments:

1. Consistency between different cell lines.<br /> I see the authors used a few different PSC lines. Since organoid efficiency differ between lines, it is important to note the consistency between lines.

2. Heterogeneity among each organoid<br /> Let's say authors get 10 organoids in one well. Are they similar to each other? Does each organoid possess similar composition of cells? To determine the heterogeneity, the authors could try either FACS or multiple sectioning of each organoid.

3. Consistency of Ach current between organoids.<br /> Related to comment 2, are the currents consistent between each organoid? How many organoids were recorded in the figures? Also, please comment if the current differ between young and aged organoids.

4. Communication between neural cells and muscle?<br /> The authors did scRNAseq, but have not gone deep analysis. I would recommend doing Receptor-ligand mapping and address if neural cells and muscle are interacting.

5. More characterization of DMD organoids.<br /> One of the key applications of muscle organoids is disease model. They have generated DMD muscle organoids, but rarely characterized except for currents. I recommend conducting immunofluorescence of DMA organoids to confirm structure change. Very intriguing to see scRNAseq of DMD organoids and align with disease etiology.

6. More characterization of engraft.<br /> Authors could measure the size of myotube between mice and human. Does PAX7+ Sattelite cell exist in engraft? To exclude cell fusion events make up the observation, I recommend to engraft in GFP+ immunodeficient mice. Could the authors comment how long engraft survive.

Joint Public Review:

Throughout the study, there is insufficient information about how experiments were performed and how often (imaging, pull-downs etc), how data was acquired, modified and analysed (especially imaging data, see below), how statistical analyses were done and what is presented in the figures (single planes or maximum intensity projections etc). This makes it difficult to evaluate the data and results.

There is insufficient information about tools and reporters used. This is misleading and impacts the conclusions that can be made from the results presented. To give an example, in Figure 1D-F, the authors present data that HDA-1::GFP and LIN-53::mNeonGreen (both components of the nucleosome remodeling and deacetylation complex) but not the histone acetyltransferase MYS-1::GFP are 'asymmetrically segregated' during QR.a division. However, the authors do not mention that HDA-1::GFP and LIN-53::mNeonGreen are expressed at endogenous levels (they are CRISPR alleles) whereas MYS-1::GFP is overexpressed (integration of a multi-copy extrachromosomal array). The difference in 'segregation' could therefore be a consequence of different levels of expression rather than different modes of segregation ('asymmetric' versus 'symmetric').

There is insufficient information about the phenotypes of the animals used (RNAi knock-downs of hda-1, lin-53 RNAi, pig-1 etc). Again this is misleading and impacts the conclusions that can be made. To give some examples, (1) in Figure 3A-G, control RNAi embryos are compared to hda-1 RNAi and lin-53 RNAi embryos. What the authors do not mention is that hda-1 RNAi and lin-53 RNAi embryos have severe developmental defects and essentially cannot be compared to control RNAi embryos. The differences between the embryos can be seen in Figure S7B where bright-field images of control RNAi, hda-1 RNAi and lin-53 RNAi embryos are shown. At the 350 min time point, a normal embryo is visible for the control, a 'ball of cells' embryo for hda-1 RNAi and an embryo that seems to have arrested at an earlier developmental stage (and therefore have much larger cells) for lin-53 RNAi. Because of these pleiotropic phenotypes, it is unclear whether differences seen for example in sAnxV::GFP positive cells (Figure 3A) are the result of a direct effect of hda-1(RNAi) on cell death or whether they are the result of global changes in development and cell fate induced by hda-1(RNAi). hda-1(RNAi) and lin-53(RNAi) embryos are also used for the data shown in Figures S6 and S7, raising the same concerns; (2) the authors do not mention what the impact of Baf A1 treatment is on animals; however, the images provided in Figure 5E indicate that Baf A1 treatment causes pleiotropic effects in L1 larvae.

There is a lack of adequate controls. Because of this, some of the data presented must be considered as preliminary. To give some examples: (1) controls are lacking for the data shown in Figure 3D-G (i.e. genes other than egl-1). Since hda-1 RNAi has a pleiotropic effect and most likely affects H3K27 acetylation genome-wide, this is critical. Based on what is shown, it is unclear whether the results presented are specific to egl-1 or not; (2) the co-IP and mass spec data shown in Figure 4A, C and Figure S8 also lack a critical control, which is GFP only. Because of this, it is unclear whether subunits of the V-ATPase bind to HDA-1 or GFP. The co-IP and mass spec data forms the basis of Figures 5 and 6 as well as Figure S9. Data presented in these figures therefore has to be considered preliminary as well.

Inappropriate methods are used. For this reason, some of the data again must be considered preliminary. To give some examples: (1) in Figure 5A, B, the authors used super-ecliptic pHluorin to look at changes in pH in the daughter cells. However, the authors used quenching of super-ecliptic pHluorin fluorescence rather than a ratio-metric method to 'measure' changes in pH. Because of this, it is unclear whether the changes in fluorescence observed are due to changes in pH or changes in the amount of pHluorin protein. Figure 5A, B forms the basis for the experiments presented in the remaining parts of Figure 5 as well as in Figure 6 and Figure S9; (2) the authors' description of how some images were modified before quantitative analysis raises concerns. The figures of concern are particularly Figure 1 and Figure S4, where background subtraction with denoising and deconvolution was used. Background subtraction, with denoising and deconvolution is an image manipulation that enhances the contrast between background and what looks like foreground. Therefore, background subtraction should be applied primarily in experiments involving image segmentation not fluorescence intensity measurement. Not being provided any information by the authors about the kind of subtraction that was made, this processing could lead to an uneven subtraction across the image, which can easily lead to artefacts. Since the fluorescence intensity in the smaller daughter cell is lower, and thus closer to background, the algorithm the authors used may have misinterpreted the grey value information in the smaller daughter cell pixels. This could have led to an asymmetric subtraction of background in the two daughter cells, leading to a stronger subtraction in the smaller daughter cell. Ultimately, their processing could have artificially increased the intensity asymmetry between the two daughter cells in all their results.

The imaging data is of low quality (for example Figures 1, 2, 5, 6; Figures S2, S3, S5, S6, S9). Since much of the study and the findings are based on imaging, this is a major concern. Critical parameters are not mentioned (number of sections in z-stack, size of the field-of-view, laser power used etc), which makes it difficult to understand what was done and what one is looking at. To give some specific examples, (1) the images shown in Figure 2B are of very low quality with severe background from neighbouring cells. In addition, the outline of the cells (plasma membrane) or the nuclei of the daughter cells is unknown. Based on this it is not clear how the authors could have measured 'Fluorescence intensity ratio between sister nuclei' in an accurate and unbiased way (what is clear from these images is that there is an increase in HDA-1::GFP signal in ALL surviving daughters (asymmetric and symmetric divisions) post cytokinesis but not in the daughter cell that is about to die (asymmetric and unequal division); (2) the images in Figure 6A and Figure S9A on VHA-17 segregation and its colocalization to ER and lysosome segregation during QR.a division are of very low quality and it is unclear to the reviewer how such images were used to obtain the quantitative data shown.

In some cases, there is a discrepancy between what is shown in figures and what the authors state in the text. To give some examples: (1) on page 7, the authors state "..., we found that nuclear HDA-1 or LIN-53 asymmetry gradually increased from 1.1-fold at the onset of anaphase to 1.5 or 1.8-fold at cytokinesis, respectively (Figure 1D-E)." Looking at the images for HDA-1 and LIN-53 in Figure 1D, the increase in the ratio mainly occurs between 4 min and 6 min, which is post cytokinesis and NOT prior to cytokinesis; (2) these images (Figure 1D) also show that there is an increase in the HDA-1 and LIN-53 signals in the larger daughter cells (QR.ap), which suggests that the increase in ratios (Figure 1E) is the result of increased HDA-1 and LIN-53 synthesis post cytokinesis. However, on top of page 8, the authors state "The total fluorescence of HDA-1, LIN-53 and MYS-1 remained constant during ACDs, suggesting that protein redistribution may establish NuRD asymmetry (Figure S4C)." In Figure S4C, the authors present straight lines for 'relative total fluorescence' for imaging (probably z-stacks) that was done every min over the course of 7 min. If there was no increase in material as the authors claim, they should have seen significant photobleaching over the course of the 7 min and therefore reduced level of 'relative total fluorescence' over time. How the data presented in Figure S4C was generated is therefore unclear. (Despite the fact that the authors claim that the asymmetry seen is not due to new synthesis in the larger daughter cell post cytokinesis, it would be more consistent with the first experiment presented in this study (Figure S1) that shows that there is more hda-1 mRNA in egl-1(-) cells compared to egl-1(+) cells); (3) On page 12, the authors state "..., in Baf A1-treated animals, QRaa inherited similar levels of HDA-1::GFP as its sister cell,...". However, looking at the image provided in Figure 5E (0 min), there seems to be a similar ratio of HDA-1::GFP between the daughter cells in DMSO and Baf A1-treated animals.

Reviewer #1 (Public Review):

The study as a concept is well designed, although there are two issues I see in the methodology (these may be just needing further explanation or if I am correct in my interpretation of what was done, may need reanalysis to take into account). Both issues relate to the data that was extracted from the published literature on zoonotic malaria prevalence in the study area.

1. No limit was set on the temporal range<br /> With no temporal limit on the range of studies, the landscape in many cases will have changes between the study being conducted and the spatial data. This will be particularly marked in areas where there has been clearing since the zoonotic malaria prevalence study. Also, population changes (either through population growth, decline or movement) will have occurred. All research is limited in what it can do with the available data, so I realise that there may not be much the authors can do to correct this. One possible solution would be to look at the land use change at each site between the prevalence study and the remote sensing data. I'm not sure if this is feasible, but if it is I would recommend the authors attempt this as it will make their results stronger.

2. Most studies only gave a geographic area or descriptive location.<br /> The spatial analysis was based on a 5km and 20km radius of the 'study site' location, but for many of the studies the exact site is not known. Therefore the 'study site' was artificially generated using a polygon centroid. Considering that the polygon could be an administrative boundary (ie district/state/country), this is an extremely large area for which a 5km radius circle in the middle of the polygon is being taken as representative of the 'study site'. This doesn't make sense as it assumes that the landscape is uniform across the district, which in most cases it will not be (in rural areas it is going to be a mixture of villages, forest, plantation, crops etc which will vary across the landscape). This might just be a case of misunderstanding what was done (in which case the text needs rewording to make it clearer) or if I have interpreted it correctly the selection of the centroid to represent the study area does not make sense. I am not sure how to overcome this as it probably not possible to get exact locations for the study sites. One possibility could be to make the remote sensing data the same scale as the prevalence data ie if the study site is only identifiable at the polygon level, then the remote sensing data (fragmentation, cover and population) is used at the polygon level.

Both these issues could have an impact on the study's findings. I would think that in both cases it might make the relationship between the environmental variables and prevalence even clearer.

Reviewer #2 (Public Review):

This is the first comprehensive study aimed at assessing the impact of landscape modification on the prevalence of P. knowlesi malaria in non-human primates in Southeast Asia. This is a very important and timely topic both in terms of developing a better understanding of zoonotic disease spillover and the impact of human modification of landscape on disease prevalence.

This study uses the meta-analysis approach to incorporate the existing data sources into a new and completely independent study that answers novel research questions linked to geospatial data analysis. The challenge, however, is that neither the sampling design of previous studies nor their geospatial accuracy are intended for spatially-explicit assessments of landscape impact. On the one hand, the data collection scheme in existing studies was intentionally opportunistic and does not represent a full range of landscape conditions that would allow for inferring the linkages between landscape parameters and P. knowlesi prevalence in NHP across the region as a whole. On the other hand, the absolute majority of existing studies did not have locational precision in reporting results and thus sweeping assumptions about the landscape representation had to be made for the modeling experiment. Finally, the landscape characterization was oversimplified in this study, making it difficult to extract meaningful relationships between the NHP/human intersection on the landscape and the consequences for P. knowlesi malaria transmission and prevalence.

Despite many study limitations, the authors point to the critical importance of understanding vector dynamics in fragmented forested landscapes as the likely primary driver in enhanced malaria transmission. This is an important conclusion particularly when taken together with the emerging evidence of substantially different mosquito biting behaviors than previously reported across various geographic regions.

Another important component of this study is its recognition and focus on the value of geospatial analysis and the availability of geospatial data for understanding complex human/environment interactions to enable monitoring and forecasting potential for zoonotic disease spillover into human populations. More multi-disciplinary focus on disease modeling is of crucial importance for current and future goals of eliminating existing and preventing novel disease outbreaks.

Reviewer #1 (Public Review):

The authors investigated the metabolic effects of ∆9-THC, the main psychoactive component of cannabis, on early mouse embryonic cell types. They found that ∆9-THC increases proliferation in female mouse embryonic stem cells (mESCs) and upregulates glycolysis. Additionally, primordial germ cell-like cells (PGCLCs) differentiated from ∆9-THC-exposed cells also show alterations to their metabolism. The study is valuable because it shows that physiologically relevant ∆9-THC concentrations have metabolic effects on cell types from the early embryo, which may cause developmental effects. However, the claim of "metabolic memory" is not justified by the current data, since the effects on PGCLCs could potentially be due to ∆9-THC persisting in the cultured cells over the course of the experiment, even after the growth medium without ∆9-THC was added.

The study shows that ∆9-THC increases the proliferation rate of mESCs but not mEpiLCs, without substantially affecting cell viability, except at the highest dose of 100 µM which shows toxicity (Figure 1). Treatment of mESCs with rimonabant (a CB1 receptor antagonist) blocks the effect of 100 nM ∆9-THC on cell proliferation, showing that the proliferative effect is mediated by CB1 receptor signaling. Similarly, treatment with 2-deoxyglucose, a glycolysis inhibitor, also blocks this proliferative effect (Figure 4G-H). Therefore, the effect of ∆9-THC depends on both CB1 signaling and glycolysis. This set of experiments strengthens the conclusions of the study by helping to elucidate the mechanism of the effects of ∆9-THC.

Although several experiments independently showed a metabolic effect of ∆9-THC treatment, this effect was not dose-dependent over the range of concentrations tested (10 nM and above). Given that metabolic effects were observed even at 10 nM ∆9-THC (see for example Figure 1C and 3B), the authors should test lower concentrations to determine the dose-dependence and EC50 of this effect. The authors should also compare their observed EC50 with the binding affinity of ∆9-THC to cellular receptors such as CB1, CB2, and GPR55 (reported by other studies).

The study also profiles the transcriptome and metabolome of cells exposed to 100 nM ∆9-THC. Although the transcriptomic changes are modest overall, there is upregulation of anabolic genes, consistent with the increased proliferation rate in mESCs. Metabolomic profiling revealed a broad upregulation of metabolites in mESCs treated with 100 nM ∆9-THC.

Additionally, the study shows that ∆9-THC can influence germ cell specification. mESCs were differentiated to mEpiLCs in the presence or absence of ∆9-THC, and the mEpiLCs were subsequently differentiated to mPGCLCs. mPGCLC induction efficiency was tracked using a BV:SC dual fluorescent reporter. ∆9-THC treated cells had a moderate increase in the double positive mPGCLC population and a decrease in the double negative population. A cell tracking dye showed that mPGCLCs differentiated from ∆9-THC treated cells had undergone more divisions on average. As with the mESCs, these mPGCLCs also had altered gene expression and metabolism, consistent with an increased proliferation rate.

My main criticism is that the current experimental setup does not distinguish between "metabolic memory" vs. carryover of THC (or its metabolites) causing metabolic effects. The authors assume that their PGCLC induction was performed "in the absence of continuous exposure" but this assumption may not be justified. ∆9-THC might persist in the cells since it is highly hydrophobic. In order to rule out the persistence of ∆9-THC as an explanation of the effects seen in PGCLCs, the authors should measure concentrations of ∆9-THC and THC metabolites over time during the course of their PGCLC induction experiment. This could be done by mass spectrometry. This is particularly important because 10 nM of ∆9-THC was shown to have metabolic effects (Figure 1C, 3B, etc.). Since the EpiLCs were treated with 100 nM, if even 10% of the ∆9-THC remained, this could account for the metabolic effects. If the authors want to prove "metabolic memory", they need to show that the concentration of ∆9-THC is below the minimum dose required for metabolic effects.

Overall, this study is promising but needs some additional work in order to justify its conclusions. The developmental effects of ∆9-THC exposure are important for society to understand, and the results of this study are significant for public health.

Reviewer #2 (Public Review):

In the study conducted by Verdikt et al, the authors employed mouse Embryonic Stem Cells (ESCs) and in vitro differentiation techniques to demonstrate that exposure to cannabis, specifically Δ9-tetrahydrocannabinol (Δ9-THC), could potentially influence early embryonic development. Δ9-THC was found to augment the proliferation of naïve mouse ESCs, but not formative Epiblast-like Cells (EpiLCs). This enhanced proliferation relies on binding to the CB1 receptor. Moreover, Δ9-THC exposure was noted to boost glycolytic rates and anabolic capabilities in mESCs. The metabolic adaptations brought on by Δ9-THC exposure persisted during differentiation into Primordial Germ Cell-Like Cells (PGCLCs), even when direct exposure ceased, and correlated with a shift in their transcriptional profile. This study provides the first comprehensive molecular assessment of the effects of Δ9-THC exposure on mouse ESCs and their early derivatives. The manuscript underscores the potential ramifications of cannabis exposure on early embryonic development and pluripotent stem cells. However, it is important to note the limitations of this study: firstly, all experiments were conducted in vitro, and secondly, the study lacks analogous experiments in human models.

Reviewer #3 (Public Review):

Verdikt et al. focused on the influence of Δ9-THC, the most abundant phytocannabinoid, on early embryonic processes. The authors chose an in vitro differentiation system as a model and compared the proliferation rate, metabolic status, and transcriptional level in ESCs, exposure to Δ9-THC. They also evaluated the change of metabolism and transcriptome in PGCLCs derived from Δ9-THC-exposed cells. All the methods in this paper do not involve the differentiation of ESCs to lineage-specific cells. So the results cannot demonstrate the impact of Δ9-THC on preimplantation developmental stages. In brief, the authors want to explore the impact of Δ9-THC on preimplantation developmental stages, but they only detected the change in ESCs and PGCLCs derived from ESCs, exposure to Δ9-THC, which showed the molecular characterization of the impact of Δ9-THC exposure on ESCs and PGCLCs.

Reviewer #1 (Public Review):

Trebino et al. investigated the BRAF activation process by analysing the interactions of BRAF N-terminal regulatory regions (CRD, RBD, and BSR) with the C-terminal kinase domain and with the upstream regulators HRAS and KRAS. To this end, they generated four constructs comprising different combinations of N-terminal domains of BRAF and analysed their interaction with HRAS as well as conformational changes that occur. By HDX-MS they confirmed that the RBD is indeed the main mediator of interaction with HRAS. Moreover, they observed that HRAS binding leads to conformational changes exposing the BSR to the environment. Next, the authors used OpenSPR to determine the binding affinities of HRAS to the different BRAF constructs. While BSR+RBD, RBD+CRD, and RBD bound HRAS with nanomolar affinity, no binding was observed with the construct comprising all three domains. Based on these experiments, the authors concluded that BSR and CRD negatively regulate binding to HRAS and hypothesised that BSR may confer some RAS isoform specificity. They corroborated this notion by showing that KRAS bound to BRAF-NT1 (BSR+RBD+CRD) while HRAS did not. Next, the authors analysed the autoinhibitory interaction occurring between the N-terminal regions and the kinase domain. Through pulldown and OpenSPR experiments, they confirm that it is mainly the CRD that makes the necessary contacts with the kinase domain. In addition, they show that the BSR stabilizes these interactions and that the addition of HRAS abolishes them. Finally, the D594G mutation within the KD of BRAF is shown to destabilise these autoinhibitory interactions, which could explain its oncogenic potential.

Overall, the in vitro study provides new insights into the regulation of BRAF and its interactions with HRAS and KRAS through a comprehensive in vitro analysis of the BRAF N-terminal region. Also, the authors report the first KD values for the N- and C-terminal interactions of BRAF and show that the BSR might provide isoform specificity towards KRAS. While these findings could be useful for the development of a new generation of inhibitors, the overall impact of the manuscript could probably be enhanced if the authors were to investigate in more detail how the BSR-mediated specificity of BRAF towards certain RAS isoforms is achieved. Moreover, though the very "clean" in vitro approach is appreciated, it also seems useful to examine whether the observed interactions and conformational changes occur in the full-length BRAF molecule and in more physiological contexts. Some of the results could be compared with studies including full-length constructs.

Reviewer #2 (Public Review):

In the manuscript entitled 'Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation', the authors conduct a series of in vitro experiments using N-terminal and C-terminal BRAF fragments (SPR, HDX-MS, pull-down assays) to interrogate BRAF domain-specific autoinhibitory interactions and engagement by H- and KRAS GTPases. Of the three RAF isoforms, BRAF contains an extended N-terminal domain that has yet to be detected in X-ray and cryoEM reconstructions but has been proposed to interact with the KRAS hypervariable region. The investigators probe binding interactions between 4 N-terminal (NT) BRAF fragments (containing one more NT domain (BRS, RBD, and CRD)), with full-length bacterial expressed HRAS, KRAS as well as two BRAF C-terminal kinase fragments to tease out the underlying contribution of domain-specific binding events. They find, consistent with previous studies, that the BRAF BSR domain may negatively regulate RAS binding and propose that the presence of the BSR domain in BRAF provides an additional layer of autoinhibitory constraints that mediate BRAF activity in a RAS-isoform-specific manner. One of the fragments studied contains an oncogenic mutation in the kinase domain (BRAF-KDD594G). The investigators find that this mutant shows reduced interactions with an N-terminal regulatory fragment and postulate that this oncogenic BRAF mutant may promote BRAF activation by weakening autoinhibitory interactions between the N- and C-terminus.

While this manuscript sheds light on B-RAF specific autoinhibitory interactions and the identification and partial characterization of an oncogenic kinase domain (KD) mutant, several concerns exist with the vitro binding studies as they are performed using tagged-isolated bacterial expressed fragments, 'dimerized' RAS constructs, lack of relevant citations, controls, comparisons and data/error analysis. Detailed concerns are listed below.

1. Bacterial-expressed truncated BRAF constructs are used to dissect the role of individual domains in BRAF autoinhibition. Concerns exist regarding the possibility that bacterial expression of isolated domains or regions of BRAF could miss important posttranslational modifications, intra-molecular interactions, or conformational changes that may occur in the context of the full-length protein in mammalian cells. This concern is not addressed in the manuscript.

2. The experiments employ BRAF NT constructs that retain an MBP tag and RAS proteins with a GST tag. Have the investigators conducted control experiments to verify that the tags do not induce or perturb native interactions?

3. The investigators state that the GST tag on the RAS constructs was used to promote RAS dimerization, as RAS dimerization is proposed to be key for RAF activation. However, recent findings argue against the role of RAS dimers in RAF dimerization and activation (Simanshu et al, Mol. Cell 2023). Moreover, while GST can dimerize, it is unclear whether this promotes RAS dimerization as suggested. In methods for the OpenSPR experiments probing NT BRAF:RAS interactions, it is stated that "monomeric KRAS was flowed...". This terminology is a bit confusing. How was the monomeric state of KRAS determined and what was the rationale behind the experiment? Is there a difference in binding interactions between "monomeric vs dimeric KRAS"?

4. The investigators determine binding affinities between GST-HRAS and NT BRAF domains (NT2 7.5 {plus minus} 3.5; NT3 22 {plus minus} 11 nM) by SPR, and propose that the BRS domain has an inhibitory role HRAS interactions with the RAF NT. However, it is unclear whether these differences are statistically meaningful given the error.

5. It is unclear why NT1 (BSR+RBD+CRD) was not included in the HDX experiments, which makes it challenging to directly compare and determine specific contributions of each domain in the presence of HRAS. Including NT1 in the experimental design could provide a more comprehensive understanding of the interplay between the domains and their respective roles in the HRAS-BRAF interaction. Further, excluding certain domains from the constructs, such as the BSR or CRD, may overlook potential domain-domain interactions and their influence on the conformational changes induced by HRAS binding.

6. The authors perform pulldown experiments with BRAF constructs (NT1: BSR+RBD+CRD, NT2: BSR+RBD, NT3: RBD+CRD, NT4: RBD alone), in which biotinylated BRAF-KD was captured on streptavidin beads and probed for bound His/MBP-tagged BRAF NTs. Western blot results suggest that only NT1 and NT3 bind to the KD (Figure 5). However, performing a pulldown experiment with an additional construct, CRD alone, it would help to determine whether the CRD alone is sufficient for the interaction or if the presence of the RBD is required for higher affinity binding. This additional experiment would strengthen the authors' arguments and provide further insights into the mechanism of BRAF autoinhibition.

7. While the investigators state that their findings indicate that H- and KRAS differentially interact with BRAF, most of the experiments are focused on HRAS, with only a subset on KRAS. As SPR & pull-down experiments are only conducted on NT1 and NT2, evidence for RAS isoform-specific interactions is weak. It is unclear why parallel experiments were not conducted with KRAS using BRAF NT3 & NT4 constructs.

8. The investigators do not cite the AlphaFold prediction of full-length BRAF (AF-P15056-F1) or the known X-ray structure of the BRAF BRS domain. Hence, it is unclear how Alpha-Fold is used to gain new structural information, and whether it was used to predict the structure of the N-terminal regulatory or the full-length protein.

9. In HDX-MS experiments, it is unclear how the authors determine whether small differences in deuterium uptake observed for some of the peptide fragments are statistically significant, and why for some of the labeling reaction times the investigators state " {plus minus} HRAS only" for only 3 time points?

10. The investigators find that KRAS binds NT1 in SPR experiments, whereas HRAS does not. However, the pull-down assays show NT1 binding to both KRAS and HRAS. SI Fig 5 attributes this to slow association, yet both SPR (on/off rates) and equilibrium binding measurements are conducted. This data should be able to 'tease' out differences in association.

11. The model in Figure 7B highlights BSR interactions with KRAS, however, BSR interactions with the KRAS HVR (proximal to the membrane) are not shown, as supported by Terrell et al. (2019).

12. The investigators state that 'These findings demonstrate that HRAS binding to BRAF directly relieves BRAF autoinhibition by disrupting the NT1-KD interaction, providing the first in vitro evidence of RAS-mediated relief of RAF autoinhibition, the central dogma of RAS-RAF regulation. However, in Tran et al (2005) JBC, they report pull-down experiments using N-and C-terminal fragments of BRAF and state that 'BRAF also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active HRAS'. This reference is not cited.

13. In Fig 2, panels A and C, it is unclear what the grey dotted line in is each plot.

14. In Fig 3, error analysis is not provided for panel E.

15. How was RAS GMPPNP loading verified?

Reviewer #1 (Public Review):

As this experience as a reviewer has been unusual, it may be helpful to outline some relevant parameters of the task at the outset. While I was invited to review the Fuentes et al. study only, two additional papers concerning the claimed engravings and burials associated with Homo naledi by Berger and colleagues were also provided as components of the reviewer package. The two manuscripts presenting the archaeological evidence are accessible as preprints in bioRxiv, by Lee Berger and colleagues ('2023a, 2023b').

Unfortunately, the arguments in the Fuentes et al manuscript hinge entirely on the strength of archaeological evidence for engravings and intentional burial by Homo naledi (presented in the abovementioned two preprints). All inferences regarding hominin behaviour and biology of Homo naledi, discussed by Fuentes and colleagues, are wholly dependent on the evidence presented in the archaeology preprints being true.

Yet both of the archaeological manuscripts are unfortunately weak. In short, the claims for engravings depend on the demonstration of several elements of association that are rather standard for linking material traces found in the archaeological record with particular hominin behaviours. For the particular arguments by Berger and colleagues to be demonstrated, the traces on the rock surface need to be linked causally with hominin agency, in other words, their anthropogenic nature need to be established. The author of the engravings needs to be demonstrated as a particular hominin species (Homo naledi in this case), and the activity of engraving needs to have taken place ~241-335 kya. After reading the manuscript on the engravings, however, what is clear is that the scratches could as easily have been made by a modern-day farmer 50 years ago, as Homo naledi ~335 kya. Berger and colleagues do not present any evidence to the contrary, they simply describe their narrative as the most parsimonious scenario. A particularly curious piece of information presented as evidence is a list of individuals known to have entered the Dinaledi system in recent times (and known not to have scratched the walls, one presumes, though this is not stated).

The question of intentional burial is more complex. What we know from other widely accepted early burials is that documenting the geoarchaeological context of the hominin remains is critical to assess the likelihood of an intentional burial - this needs to be established at the outset through high quality fieldwork. Yet even the boundaries of the excavation presented in the burial manuscript appear so angled or skew relative to one another (Fig. 2a) that the individual squares look to be aligned with different XY grids, which does not instill confidence in the quality of field documentation. One can make out very little from the sediment section images - which are key to identifying intrusive features associated with burials - and the multivariate geochemical analysis of sediments is unconvincing: a scatterplot (not a biplot) should have been provided showing the geochemistry of the burial sediment samples relative to the immediately surrounding sediment characteristics. While one remains excited about the potential for a spectacular archaeological discovery within the Dinaledi cave system, unfortunately, the three manuscripts provided do not present convincing evidence to that effect.

Reviewer #2 (Public Review):

Fuentes et al. provide a detailed and thoughtful commentary on the evolutionary and behavioral implications of complex behaviors associated with a small-brained hominin, Homo naledi. Within the Rising Star Cave of South Africa, Berger et al. 2023a,b proposed evidence that Homo naledi intentionally buried their dead through complex mortuary practices and engaged in symbolic expression by engraving the cave walls in cross-hatching motifs. Two burials were identified in the Rising Star cave subsystems: Feature 1 in the Dinaledi Chamber and a feature in the Hill Antechamber. The engravings are located in the Hill Antechamber near the passageway leading into the Dinaledi chamber. The authors aimed to provide evidence for burials by (1) testing sediment samples for mineral composition from within and outside the burial feature; (2) demonstrating an interruption in the stratigraphy indicative of a "bowl-shaped" feature; (3) evaluating the anatomical coherence of the skeletal remains; (4) demonstrate matrix-supported positioning of skeletal elements; and (5) determine the compatibility of non-articulated material with decomposition and subsequent collapse. Berger et al. 2023b evaluated the engravings through high resolution photography, cross-polarization, and 3D photogrammetry. Neither article involved radiometric dating of materials. While the review by Fuentes et al. highlights important assumptions about the relationship between hominin brain size, cognition, and complex behaviors, the evidence presented by Berger et al. 2023a,b does not support the claim that Homo naledi engaged in burial practices or symbolic expression through wall engravings.

The major weaknesses for Berger et al. 2023a are as follows:

1) The mineral composition from sediment sampled from within Dinaledi Feature 1 is not different compared to the surrounding sediment, which is one rationale proposed by the authors that would lead to the conclusion of a burial pit. An effort to replicate the multivariate statistical analysis using the data provided in SI Table 1 by this reviewer failed, and thus, the results are not replicable.

2) The authors failed to provide clear visualizations or analysis that showed an unambiguous interruption in the stratigraphy surrounding the Dinaledi Feature 1.

3) Attempts 1 and 2 were applied solely to Dinaledi Feature 1, not the Hill Antechamber Feature.

4) Skeletal cohesion does suggest that the bodies were likely covered or protected by external environment. However, given the geological context, there is minimal opportunity for scavengers or other agents to scatter the skeletal remains within such an isolated location. Thus, this alone cannot solely support intentional burials as this line of evidence is subject to equifinality.

5) Similar to the preceding statement, evidence for matrix-supported elements was inconclusive at best. There was no mention of sedimentary rate or expectations for how quickly sediments would naturally bury the remains of whole bodies in the chamber compared with the rate of decomposition of buried remains.

The major weaknesses for Berger et al. 2023b are as follows:

6) While this is incredibly difficult to accomplish, dating rock art or other cave wall engravings is the only method to ensure that the etchings were created during the time of Homo naledi. Unfortunately, this was not attempted. Instead, the authors state that "This description is intended to document the discovery and provide spatial and contextual information prior to any further analyses that may require invasive sampling." Yet, the authors assign a date to the engravings in the title of the paper. Here, the authors are generating interpretations before analyses are attempted.

7) The engravings are indeed very interesting and are likely anthropogenic in origin. However, the argument that these engravings were created by Homo naledi is based on the bold assumption that "No physical or cultural evidence of any other hominin population occurs within this part of the cave system, and there is no evidence that recent humans or earlier hominins ever entered any adjacent area of the cave until surveys by human cave explorers during the last 40 years." (page 6). To assume that no other individual entered the cave system from the time of Homo naledi until 40 years ago is an unrealistic and faulty assumption. This reviewer does not discount that the engravings could have been made by Homo naledi, but the evidence must be sufficient to support this statement or provide other alternatives as working hypotheses.

As a discipline, paleoanthropology aims to understand the evolutionary history of the hominin clade through fossil remains, material culture, and, most recently, ancient DNA. The methods and approaches that we as paleoanthropologists use to understand the past often bridge both the humanities and the hard sciences to create a unique understanding of our shared history. We are only limited by the conditions in which time and attrition has erased pieces of our collective story from the earth. Thus, it is our responsibility to ensure that our interpretations of the past are supported by measurable and testable means, to the best of our ability, and that hypotheses are not presented as conclusions.

Unfortunately, this is not the case for Berger et al. 2023a,b. The work presented by the authors is imprudent and incomplete and does not meet the requirements set forth by our discipline. While it is important that scholars publish their work in a dutiful timeline, it is arguably more critical for scholars to take the necessary time to ensure the integrity and resolution of the work. The consequences for rushing publications with such a significant unsubstantiated find will likely result in perilous ramifications, as it is more difficult to correct an idea than to introduce one.

Reviewer #3 (Public Review):

This paper presents the cognitive implications of claims made in two accompanying papers (Berger et al. 2023a, 2023b) about the creation of rock engravings, the intentional disposal of the dead, and fire use by Homo naledi. The importance of the paper, therefore, relies on the validity of the claims for the presence of socio-culturally complex and cognitively demanding behaviors that are presented in the associated papers. Given the archaeological, hominin, and taphonomic analyses in the associated papers are not adequate to enable the exceptional claims for naledi-associated complex behaviors, the inferences made in this paper are currently inadequate and incomplete.

The claimed behaviors are widely recognized as complex and even quintessential to Homo sapiens. The implications of their unequivocal association with such a small-brained Middle Pleistocene hominin are thus far reaching. Accordingly, the main thrust of the paper is to highlight that greater cognition and complex socio-cultural behaviors were not necessarily associated with a positively encephalized brain. This argument begs the obvious question of whether absolute brain size and/or encephalization quotient (i.e., the actual brain volume of a given species relative the expected brain size for a species of the same average body size) can measure cognitive capacity and the complexity of socio-cultural behaviors among late Middle Pleistocene hominins.

Claims for a positive correlation between absolute and/or relative brain size and cognitive ability are not common in discussions surrounding the evolution of Middle- and Late Pleistocene hominin behavior. Currently, the bulk of the evidence for early complex technological and social behaviors derives from multiple sites across South Africa and postdates the emergence of H. sapiens by more than 100,000 years. Such lag in the expression of complex technologies and behaviors within our species renders the brain size-implies-cognitive capacity argument moot. Instead, a rich body of research over the past several decades has focused on aspects related to socio-cultural, environmental, and even the wiring of the brain in order to understand factors underlying the expression of the capacity for greater behavioral variability. In this regard, even if the claimed evidence for complex behaviors among the small-brained naledi populations proves valid, the exploration of the specific/potential socio-cultural, neuro-structural, ecological and other factors will be more informative than the emphasis on absolute/relative brain size.

The paper presents as supporting evidence previous claims for the appearance of similar complex behaviors predating the emergence of our species, H. sapiens, although it does acknowledge their controversial nature. It then uses the current claims for the association of such behaviors with H. naledi as decisive. Given the inadequate analyses in the accompanying papers and the lack of evidence for stone tools in the naledi sites, the present claims for the expression of culturally and symbolically mediated behaviors by this small-brained hominin must be adequately established. The importance of the paper thus rests on the validity of the claimed evidence--including contextual aspects--for rock engraving, mortuary practices, and the use of fire presented in the associated two papers. The claims in both associated papers are inadequate, incomplete, and largely assumption- (rather than evidence) based. As responsible and ethical researchers, the team must return to the sites, conduct the required standard chronomoetric and taphonomic studies and weigh the strength of the evidence before proceeding with the current claims.

Reviewer #1 (Public Review):

I think it is important to note up front that I recognize that the goal of this paper was to announce the discovery of what appear to be intentionally-made marks in Rising Star cave in South Africa. This was not meant to be an in-depth analysis or a declaration of definitive results. With this in mind, I appreciate that the authors did not try to overstate this new discovery, but instead simply reported what had been observed, provided a little bit of background on the current state of the field in regards to the evolution of hominin visual mark-making, made a few tentative identifications, but then clearly acknowledged that a lot more documentation, sampling, and study would be needed before we could understand the full scope and potential importance of this find.

This is a big claim. If it proves to be true, it has the potential to be paradigm-shifting as the identification of intentional engraved marks, made by a small-brained distant human cousin 200,000+ years ago in South Africa, would completely change our understanding of where, when and who made the first graphic marks. Twenty years ago, this claim would probably have been dismissed out of hand as being too far-fetched to be taken seriously, but there have been some major shifts in the field in recent years, in regard to the age of the art and the identity of the artists, that means this is a claim that should be approached with a scientifically cautious, but open mind. There is now mounting evidence for Neanderthals, and potentially other closely related species as well, to have been engaging in similar art-making practices to our own Homo sapiens ancestors. What makes this particular claim so extraordinary is that these marks are some of the oldest in the world and that Homo naledi is a more distant relation with a smaller brain. This is also what makes the further study of this discovery such a fascinating exercise in scientific inquiry.

From a technical and methodological perspective, there is an excellent range of tools and technologies that can be used to study these engravings, so I have no doubt that further studies will help answer some of the "nuts and bolts" questions. Then there is also the opportunity created by this discovery to really open a broader dialogue in the field about who were the first artists and at what point does the hominin brain become "primed" for making visual marks. I look forward to all sorts of lively debates in the future and to seeing the results of further in-depth studies.

Reviewer #2 (Public Review):

Patterns scored into or painted on durable media have long been considered important markers of the cognitive capabilities of hominins. More specifically, the association of such markers with Homo sapiens has been used to argue that our evolutionary success was in part shaped by our unique ability to code, store and convey information through abstract conventions.

That singularity of association has been cast into doubt in the last decade with finds of designs apparently painted or carved by Neanderthals, and potentially by even earlier hominins. Even allowing for these developments, however, extending the capability to generate putatively abstract designs to a relatively small-brained hominin like Homo naledi is contentious. The evidential bar for such claims is necessarily high, and I don't believe that it has been cleared here.

The central issue is that the engravings themselves are not dated. As the authors themselves note, the minimum age constraint provided by U/Th on flowstone does not necessarily relate to the last occupation of the Dinaledi cave system, as the earlier ESR age on teeth does not necessarily document first use of the cave. The authors state that "At present we have no evidence limiting the time period across which H. naledi was active in the cave system". On those grounds though, assigning the age range of presently dated material within the cave system to the engravings - as the current title unambiguously does - is not justifiable.

Because we don't know when they were made, the association between the engravings and Homo naledi rests on the assertion that no humans entered and made alterations to the cave system between its last occupation by Homo naledi, and its recent scientific recording. This is argued on page 6 with the statement that "No physical or cultural evidence of any other hominin population occurs within this part of the cave system".

There is an important contrast between the quotes I have referred to in the last two paragraphs. In the earlier quote, the absence of evidence for Homo naledi in the cave system >335 ka and <241 ka is not considered evidence for their absence before or after these ages. Just because we have no evidence that Homo naledi was in the cave at 200 ka doesn't mean they weren't there, which is an argument I think most archaeologists would accept. When it comes to other kinds of humans, though - per the latter quote - the opposite approach is taken. Specifically, the present lack of physical evidence of more recent humans in the cave is considered evidence that no such humans visited the cave until its exploration by cavers 40 years ago. I don't think many archaeologists would consider that argument compelling. I can see why the authors would be drawn to make that assertion, but an absence of evidence cannot be used to argue in one way for use of the cave by Homo naledi and in another way for use of the cave by all other humans.

A second problem is with what Homo naledi might have made engravings. The authors state that "The lines appear to have been made by repeatedly and carefully passing a pointed or sharp lithic fragment or tool into the grooves". The authors then describe one rock with superficial similarities to a flake from the more recent site of Blombos to suggest that sharp-edge stones with which to make the engravings were available to Homo naledi. Blombos is considered relevant here presumably because it has evidence for Middle Stone Age engravings. The authors do not, however, demonstrate any usewear on that stone object such as might be expected if it was used to carve dolomite. Given that it is presented as the only such find in the cave system so far, this seems important.

My greater concern is that the authors did not compare the profile morphology of the Dinaledi engravings with the extensive literature on the morphology of scored lines caused by sharp-edge stone implements (e.g., Braun et al. 2016, Pante et al. 2017). I appreciate that the research group is reticent to undertake any invasive work until necessary, but non-destructive techniques could have been used to produce profiles with which to test the proposition that the engravings were made with a sharp edge stone.

One thing I noticed in this respect is that the engravings seem very wide, both in absolute terms and relative to their depth. The data I collected from the Middle Stone Age engraved ochre from Klein Kliphuis suggested average line widths typically around 0.1-0.2 mm (Mackay and Welz 2008). The engraved lines at Dinaledi appear to be much wider, perhaps 2-5 mm. This doesn't discount the possibility that the engravings in the Dinaledi system were carved with a sharp edge stone - the range of outcomes for such engravings in soft rock can be quite variable (Hodgskiss 2010) - only that detailed analysis should precede rather than follow any assertion about their mode of formation.

None of this is to say that the arguments mounted here are wrong. It should be considered possible that Homo naledi made the engravings in the Dinaledi cave system. The problem is that other explanations are not precluded.

As an example, the western end of the Dinaledi subsystem has a particular geometry to the intersection of its passages, with three dominant orientations, one vertical (which is to say, north-south), and two diagonal (Figure 1). The major lines on Panel A have one repeated vertical orientation and two repeated diagonal orientations (Figure 16), particularly in the upper area not impacted by stromatolites. The lines in both the cave system and engravings in Panel A appear to intersect at similar angles. Several of the cave features appear, superficially at least, to be replicated. In fact, scaled, rotated, and super-imposed, Figure 16 is a plausible 'mud map' of the western end of the Dinaledi system carved incrementally by people exploring the caves. A figure showing this is included here:

Of course, there are problems with this suggestion. The choice of the upper part of Panel A is selective, the similarity is superficial, and the scales are not necessarily comparable. (Note, btw, that all of those caveats hold equally well for the comparison the authors make between the unmodified rock from Dinaledi and the flake from Blombos in Figure 19). However, the point is that such a 'mud map hypothesis' is, as with the arguments mounted in this paper, both plausible and hard to prove.

Having read this paper a few times, I am intrigued by the engravings in the Dinaledi system and look forward to learning more about them as this research unfolds. Based on the evidence presently available, however, I feel that we have no robust grounds for asserting when these engravings were made, by whom they were made, or for what reason they were made.

References:

• Braun, D. R., et al. (2016). "Cut marks on bone surfaces: influences on variation in the form of traces of ancient behaviour." Interface Focus 6: 20160006.

• Hodgskiss, T. (2010). "Identifying grinding, scoring and rubbing use-wear on experimental ochre pieces." Journal of Archaeological Science 37: 3344-3358.

• Mackay, A. & A. Welz (2008). "Engraved ochre from a Middle Stone Age context at Klein Kliphuis in the Western Cape of South Africa." Journal of Archaeological Science 35: 1521-1532.

• Pante, M. C., et al. (2017). "A new high-resolution 3-D quantitative method for identifying bone surface modifications with implications for the Early Stone Age archaeological record." J Hum Evol 102: 1-11.

Reviewer #3 (Public Review):

Lee Berger and colleagues argue here that markings they have found in a dark isolated space in the Rising Star Cave system are likely over a quarter of a million years old and were made intentionally by Homo naledi, whose remains nearby they have previously reported. As in a European and much later case they reference ('Neanderthal engraved 'art' from the Pyrenees'), the entangled issues of demonstrable intentionality, persuasive age and likely authorship will generate much debate among the academic community of rock art specialists. The title of the paper and the reference to 'intentional designs', however, leave no room for doubt as to where the authors stand, despite avoidance of the word art, entering a very disputed terrain. Iain Davidson's (2020) 'Marks, pictures and art: their contributions to revolutions in communication', also referenced here, forms a useful and clearly articulated evolutionary framework for this debate. The key questions are: 'are the markings artefactual or natural?', 'how old are they?' and 'who made them?, questions often intertwined and here, as in the Pyrenees, completely inseparable. I do not think that these questions are definitively answered in this paper and I guess from the language used by the authors (may, might, seem etc) that they do not think so either.

First, a few referencing issues: the key reference quoted for distinguishing natural from artefactual markings (Fernandez-Jalvo et al. 2014), whilst mentioned in the text, is not included in the references. In the acknowledgements, the claim that "permits to conduct research in the Rising Star Cave system are provided by the South African National Research Foundation" should perhaps refer rather to SAHRA? In the primary description of their own markings from Rising Star and their presumed significance, there are, oddly, several unacknowledged quotes from the abstract of one of the most significant European references (Rodriguez-Vidal et al. 2014). These need attention.

Before considering the specific arguments of the authors to justify the claims of the title, we should recognise the shift in the academic climate of those concerned with 'ancient markings' that has taken place over the past two or three decades. Before those changes, most specialists would probably have expected all early intentional markings to have been made by Homo sapiens after the African diaspora as part of the explosion of innovative behaviours thought to characterise the 'origins of modern humans'. Now, claims for earlier manifestations of such innovations from a wider geographic range are more favourably received, albeit often fiercely challenged as the case for Pyrenean Neanderthal 'art' shows (White et al. 2020). This change in intellectual thinking does not, however, alter the strict requirements for a successful assertion of earlier intentionality by non-sapiens species. We should also note that stone, despite its ubiquity in early human evolutionary contexts, is a recalcitrant material not easily directly dated whether in the form of walling, artefact manufacture or potentially meaningful markings. The stakes are high but the demands are no less so.

Why are the markings not natural? Berger and co-authors seem to find support for the artefactual nature of the markings in their location along a passage connecting chambers in the underground Rising Star Cave system. The presumption is that the hominins passed by the marked panel frequently. I recognise the thinking but the argument is weak. More confidently they note that "In previous work researchers have noted the limited depth of artificial lines, their manufacture from multiple parallel striations, and their association into clear arrangement or pattern as evidence of hominin manufacture (Fernandez-Jalvo et al. 2014)". The markings in the Rising Star Cave are said to be shallow, made by repeated grooving with a pointed stone tool that has left striations within the grooves and to form designs that are "geometric expressions" including crosshatching and cruciform shapes. "Composition and ordering" are said to be detectable in the set of grooved markings. Readers of this and their texts will no doubt have various opinions about these matters, mostly related to rather poorly defined or quantified terminology. I reserve judgement, but would draw little comfort from the similarities among equally unconvincing examples of early, especially very early, 'designs'. Two or even three half-convincing arguments do not add up to one convincing one.

The authors draw our attention to one very interesting issue: given the extensive grooving into the dolomite bedrock by sharp stone objects, where are these objects? Only one potential 'lithic artefact' is reported, a "tool-shaped rock [that] does resemble tools from other contexts of more recent age in southern Africa, such as a silcrete tool with abstract ochre designs on it that was recovered from Blombos Cave (Henshilwood et al. 2018)", also figured by Berger and colleagues. A number of problems derive from this comparison. First, 'tool-shaped rock' is surely a meaningless term: in a modern toolshed 'tool-shaped' would surely need to be refined into 'saw-shaped', 'hammer-shaped' or 'chisel-shaped' to convey meaning? The authors here seem to mean that the Rising Star Cave object is shaped like the Blombos painted stone fragment. But the latter is a painted fragment, not a tool and so any formal similarity is surely superficial and offers no support to the 'tool-ness' of the Rising Star Cave object. Does this mean that Homo naledi took (several?) pointed stone tools down the dark passageways, used them extensively and, whether worn out or still usable, took them all out again when they left? Not impossible, of course. And the lighting?

The authors rightly note that the circumstance of the markings "makes it challenging to assess whether the engravings are contemporary with the Homo naledi burial evidence from only a few metres away" and more pertinently, whether the hominins did the markings. Despite this honest admission, they are prepared to hypothesise that the hominin marked, without, it seems, any convincing evidence. If archaeologists took juxtaposition to demonstrate authorship, there would be any number of unlikely claims for the authorship of rock paintings or even stone tools. The idea that there were no entries into this Cave system between the Homo naledi individuals and the last two decades is an assertion, not an observation, and the relationship between hominins and designs no less so. In fact, the only 'evidence' for the age of the markings is given by the age of the Homo naledi remains, as no attempt at the, admittedly very difficult, perhaps impossible, task of geochronological assessment, has been made.

The claims relating to artificiality, age and authorship made here seem entangled, premature and speculative. Whilst there is no evidence to refute them, there isn't convincing evidence to confirm them.

References:

• Davidson, I. 2020. Marks, pictures and art: their contribution to revolutions in communication. Journal of Archaeological Method and Theory 27: 3 745-770.

• Henshilwood, C.S. et al. 2018. An abstract drawing from the 73,000-year-old levels at Blombos Cave, South Africa. Nature 562: 115-118.

• Rodriguez-Vidal, J. et al. 2014. A rock engraving made by Neanderthals in Gibralter. Proceedings of the National Academy of Sciences.

• White, Randall et al. 2020. Still no archaeological evidence that Neanderthals created Iberian cave art.

Reviewer #4 (Public Review):

This is potentially a landmark study with far-reaching consequences for archaeology, palaeoanthropology, and more widely. The antiquity of intentional human mark marking is a hot topic but this study – understood as initial – has as yet incomplete sources of evidence and methods; and it will be interesting to follow how the study develops in subsequent studies.

Strengths and points to build on:

* Heuristic potential: As knowledge advances it poses a risk to accepted knowledge – and we should accept that one such risk is moving on from long-held disciplinary tenets. In this case, there has been a growing quantum of evidence – all hotly debated – for the deep antiquity of mark-making and even symbolism by species other than ourselves. Most researchers now accept Neanderthal symbolic capacity actualised in burials, intentional mark-making and the like. The evidence here presented is not unequivocal but is very suggestive and an ideal test case for applying multi-disciplinary techniques of analysis and interpretation beyond the expertise of the listed authors *see comments in 'weaknesses'). This work by itself may be equivocal but when taken together with other such work, points to a 'human' sensu lato past that is as complex as it is long. This work then helps all researchers to at least be alive to the possibility of things like anthropic marks and residues in a context not normally thought to have it.

* Decentering speciesism: As per the above comment, I appreciate empirical studies that erode speciesism – in particular studies that open up our minds to the possibility that multiple members of the Genus Homo were capable of intentional mark-making and even 'symbolic' behaviour, though this latter term is not well understood or uniformly used. This is probably because of continuous unconscious bias on our part as currently the only exemplar of our genus living - in contrast to most of the past in which different species and genera co-existed - if not on the same landscape and/or at exactly the same time, then with enough overlap that people would have realised 'others' were about either by sight and/or by encountering their physical remains and artefacts.

* Problematising 'firsts' and deep time: A strength – but which needs to be developed in this manuscript – is our understanding of time and change. We have a plethora of dating techniques but relatively few substantive monographs, articles, and think tanks on time – and especially on how change comes about and what causes it. This leads us to privilege 'firsts' and the 'oldest' finds in 'deep' time above those that are more recent and in 'shallow' time. I would suggest in addition to the claims for the oldest of the reported marks, the authors develop nascent remarks on the possibility the suite of marks may have been made over time. This will help counter criticism that these marks – if established to be anthropic – were not just a singularity, but part of patterned behaviour, which would move it towards the realm of 'symbolic' cognitive behaviour. And indeed, it would be good to hear more about why in this place, these marks were made to establish a replicable model for identifying early anthropic marks.

Ultimately, this manuscript presents evidence that those who are pro the deep antiquity of intentional mark-making by Homo (and possibly even other genera) will find enough evidence to support; while those sceptical of such claims will find enough methodological flaws and evidential limits to refute those claims. The next decade of work will likely be definitive and this article makes a key contribution to the debate.

Weaknesses and points to attend to:

* Definitions: The term 'rock engraving' is used rather uncritically and also the term 'etching' – and it would be useful to have a short definition of how the authors understand the term. Rock art scholars regularly debate these terms and whether they are or are not 'rock art' with its overwhelmingly visual bias; which this discovery may usefully help overthrow and advance.

* Dating: There is no evidence provided for dating the marks found in the cave system. They could, for example, have been made more recently than the dates claimed – and by another species (if we accept their anthropogenic authorship). This is a perennial problem of much rock art research – especially when it comes to understanding the wider archaeological/palaeoanthropological context. More crucially, accurate dating allows a more reliable understanding of authorship and who/what was responsible for a particular artefact or feature. This has not been demonstrated in this case, though we do have fossil evidence of Homo naledi in the cave system. The article title is this incorrect / and unsupported claim as the marks, if they are anthropic, have not been dated and are of unknown age. The authors allow that there may have been multiple episodes, but not that the marks can belong to a time other than they posit – either earlier, later, or distributed over a long period as the authors allow for in their concluding remarks.

* Authorship: The study does not utilise either a geoscientist as one of the authorial team, or a rock art specialist. These are key oversights as the former would help better contextualise the dating of the marks reported on, as well as explore alternative non-anthropogenic agents that may have created the marks reported on. For example, the marks and 'pitting' etc may be the result of water bringing abrasive agents during times of flooding, hitting prominent rock features in the cave system. Some explanation is given from lines 114-124, but are uncited. The overlying 'sediment' may be similar to the mondmilch found in cave systems and which is of natural origin. It may be that these non-anthropogenic causes are easy to discount; but the arguments do need to be made. Or, that the polishing was made by Homo naledi brushing against the surfaces as they moved in the cave system, independent of any mark-making. A Table showing the pros and cons of intentional anthropic versus natural authorship would be very effective - as well as showing some of the natural linear marks in the cave system to avoid any confirmation or similar bias. FTIR analysis of the panel A-C would be more than useful to determine whether an additional layer of material has been added. This is mentioned for future work, but this seems a rather post-hoc research programme.

* Use-wear analysis: If the marks are anthropic in origin; they are likely to have been made by a stone tool, which would leave characteristic marks, directionality and sequencing, distinct from natural causes. It is vital this work – such as was done on the Blombos engraved ochre – is done here – for example, linking to the chert and other tools described on lines 152-158. Note Figure 19, of such a tool, is very hard to make out. The Blombos – and Klasies River Mouth engraved ochres (curiously not referenced) – have very similar geometric markings and there is a real opportunity to compare these in securely dated contexts of 70-120 kya –which could support the argument made here for Homo naledi's cognitive capacity. On figure 16 it would be good to know on what basis some marks were selected as anthropic – and why others were not; this would help demonstrate the methodology and ability to distinguish between the two kinds of marks.

* Viewshed: The rock art specialist would have added essential expertise on how to study anthropic marks. For example, the images of the marks shown are all of individual or small collections of motifs rather than showing each panel as well as all panels together, to help understand the iconographic context as an ensemble – a 'feature' rather than isolated 'artefacts' or 'motifs'. Line 60 mentions being able to see these as a 'triptych' but the reader is not able to have this view in this manuscript. From the cave map, it is not clear whether all three 'panels' (an unfortunate art historical term that suggests a framed entity - better to use a term like 'cluster') can be viewed simultaneously or in sequence. The view shed in relation to the area where the bodies were recovered is vaguely stated as 'only a few metres away' and is worth developing. I understand 3D scans have been made so it would be useful to have a version showing the marks in relation to where the bodies were recovered and as a 3-cluster ensemble.

* Image enhancements: Also, in addition to polarised images, have colour enhancement tools like DStretch been tried to see if, for example, attempts at colouring with different coloured sands were made? Similarly, a 3D scan of the motif and panel – (Metashape is mentioned but not shown) – might assist in understanding how the marks and the rock they are on might relate to each other- as research in European upper Palaeolithic contexts has shown. Here, experimenting with different kinds of lighting - or in the absence of lighting, of tactility and how these marks and their rock support may have been experienced by those who may have made and interacted with them? As a note, it would be useful to have a scale in each image of the 'engravings' and it is a pity the one in situ photograph with the scale is not a standard rock art colour-corrected scale as is commonly used in rock art research.

Reviewer #1 (Public Review):

The discovery of Homo naledi fossils and the rising star cave system is unquestionably important for paleoanthropology. The fossils themselves hold a wealth of information about the diversity and complexity of morphological and evolutionary change on the hominin family tree. It is a truly amazing find and important site and it is important that information about this site continues to be produced so that more can be known. It is equally important that the papers produced from the site be fully reviewed for scientific rigor. I hope to assist with this to the best of my ability.

In its current form the paper, "Evidence for the deliberate burial of the dead by Homo naledi," does not meet the standards of our field. The paper is hard to follow. It lacks key citations, contextual background information to inform the reader about the geological and depositional structure of the caves, and concise understandable descriptions of the methods and the significance of the results.

The main point of the paper is to describe three possible burial features. The working hypothesis is that the features are intentional burials, and the authors seek to support this hypothesis throughout rather than test it. The authors do this by noting mineralogical differences in sediment and possible bowl-shaped sedimentological distinctions where fossil bones occur. As stated above, this evidence needs to be elaborated on the in text, contextualized, and edited for clarity. In addition, throughout the paper, the authors only consider two depositional scenarios for burial and body decomposition: 1) a body was intentionally buried in a pit that was dug into the cave sediments, and then buried in sediment (without detailing in the main text what sediment was used to backfill the pit); and 2) the body was left in a natural pit and decayed in the open. A major problem with only considering these two scenarios for body decomposition is that previous reports about cave geology and sedimentology show that it is a dynamic system involving erosion, sediment slumping and drainage, and contraction of clay, which is a major component of the sediment, etc. The authors are very clear that flooding is not a viable option for the movement of skeletal elements in the cave. However, they do not mention other processes such as erosion or sediment slumping, that are known to occur and could be responsible for moving sediment and fossils in each chamber of the cave. They also do not consider carnivore involvement which has been suggested by Val (2016) and Egeland et al. (2018). Such processes could naturally transport bodies, shift them around, and sediment erosion could bury them. The articulation of some skeletal elements is a major argument for intentional burial, yet within the cave substructure, articulated bones are often commingled with disarticulated elements from the same or different individuals. This same situation exists in the features included in this paper. It does appear that some skeletal material was covered in sediment before decomposition and remains in articulation, but bodies decompose at different rates, and can decompose slowly, especially in environments that lack insects (see Simmons et al. 2010 Journal of Forensic Sciences https://doi-org.aurarialibrary.idm.oclc.org/10.1111/j.1556-4029.2009.01206.x). Wiersma et al., 2019 describe the cave system as very humid, but dry due to little standing water, mildly acidic, with an average temperature today of 18{degree sign}C and a minimum of 12{degree sign}C over the last million years. The starting null hypothesis should be that the bodies were naturally covered in sediment. Intentional burial requires extraordinary circumstances and requires multiple lines of solid evidence to support the hypothesis. In testing for natural burial processes, the rate of body decomposition should be reconstructed given the environmental parameters of the cave.

In keeping with supporting their starting hypothesis that Homo naledi intentionally buried individuals in the cave, the authors conclude that "A parsimonious explanation for this configuration of skeletal remains is that these remains may be a palimpsest of burials that have sequentially disrupted each other. In this hypothesis, early burials were disturbed when pits were dug for subsequent burials. Other occurrences of remains outside of the Dinaledi Chamber and Hill Antechamber (Hawks et al., 2017; Brophy et al., 2021) are discussed as possible evidence of mortuary practices in SI 4.2. Instances where parts of individuals occur in remote narrow passages cannot be explained as a result of carnivore or water transport (Elliott et al., 2021; Brophy et al., 2021), making it necessary to consider that H. naledi may have placed these partial remains in these locations, possibly representing a form of funerary caching." After reviewing the evidence presented in the current manuscript, it is not clear why this is a parsimonious explanation. The authors have repeatedly described how incredibly challenging it is to get into and out of this cave system and all of its chambers. How could any species, even small bodied species, drag/pull/shove dead bodies through small crevasses, shove or drop them down a narrow shoot, continue to move through the hill antechamber to the Dineledi chamber and bury bodies? It is not impossible but given the previously published descriptions of the dynamic process of sedimentation movement in the cave it is certainly not a parsimonious explanation. To support this will take many more lines of evidence than presented here such as micromorphological analysis of the overall cave system and each feature (discussed in the supplementary information but briefly), full detailed reconstruction of sediment, water, fossil, and debris movement throughout the cave system coupled with reconstructions of body decomposition rates. Scientifically precise computer-generated reconstructions of all of this are possible working with specialists affiliated with National Geographic. An analysis also needs to start by testing a null hypothesis, not deciding on the conclusion and setting out to "prove" it.

Reviewer #2 (Public Review):

In this study (Berger et al.), geological and fossil data from the Rising Star Cave System in South Africa are presented to provide evidence for intentional burials of Homo naledi individuals. The authors focus on describing and interpreting what they refer to as "delimited burial features." These features include two located on the floor of the Dinaledi Chamber (referred to as 'Dinaledi Features' 1 and 2) and one from the floor of the Hill Antechamber.

'Dinaledi Feature 1' consists of a collection of 108 skeletal elements recovered from sub-unit 3b deposits. These remains are believed to primarily represent the remains of a single adult individual, along with at least one additional juvenile individual. Although additional anatomical elements associated with 'Dinaledi Feature 1' are mentioned, they are not described as they remain unexcavated. The study states that the spatial arrangement of the skeletal remains is indicative of the primary burial of a fleshed body. On the other hand, 'Dinaledi Feature 2' is not extensively discussed, and its complete extent was not thoroughly investigated.

Regarding the Hill Antechamber feature, it was divided into three separate plaster jackets for removal from the excavation. Through micro-CT and medical CT scans of these plaster jackets, a total of 90 skeletal elements and 51 dental elements were identified. From these data, three individuals were identified, along with a fourth individual described as significantly younger. Individuals 1 and 2 are classified as juveniles.

I feel that there is a significant amount of missing information in the study presented here, which fails to convince me that the human remains described represent primary burials, i.e. singular events where the bodies are placed in their final resting places. Insufficient evidence is provided to differentiate between natural processes and intentional funerary practices. In my opinion, the study should include a section that distinguishes between taphonomic changes and deliberate human modifications of the remains and their context, as well as reconstruct the sequence and timeline of events surrounding death and deposition. A deliberate burial involves a complex series of changes, including decomposition of soft tissues, disruption of articulations between bones, and the sequence of skeletonization. While the geological information is detailed, the archaeothanatological reasoning (see below) is largely absent and, when presented, it lacks clarity and unambiguousness.

My main concern is that the study does not apply or cite the basic principles of archaeothanatology, which combines taphonomy, anatomy, and knowledge of human decomposition to interpret the arrangement of human bones within the Dinaledi Chamber and the Hill Antechamber. Archaeothanatology has been developed since the 1970s (see Duday et al., 1990; Boulestin and Duday, 2005; Duday and Guillon, 2006) and has been widely used by archaeologists and osteologists to reconstruct various aspects such as the original treatment of the body, associated mortuary practices, the sequence of body decomposition, and the factors influencing changes in the skeleton within the burial.

Specifically, the study lacks a description of the relative sequence of joint disarticulation during decomposition and the spatial displacement of bones. A detailed assessment of the anatomical relationships of bones, both articulated and disarticulated, as well as the direction and extent of bone displacement, is missing. For instance, while it is mentioned that "many elements are in articulation or sequential anatomical position," a comprehensive list of these articulated elements and their classification (as labile or not) is not provided.

Furthermore, the patterns described are not illustrated in sufficient detail. If Homo naledi was deliberately buried, it would be crucial to present illustrations depicting the individuals in their burial positions, as well as the representation and proportions of the larger and smaller anatomical elements for each individual. While Figure 2B provides an overall view of 'Dinaledi Feature 1,' it is challenging to determine the relationships of bones, whether articulated or disarticulated, in Figures 2C or 2D. Such information is essential to determine whether the bones are in a primary or secondary position, differentiate between collective and multiple burials, ascertain the body's stage of decomposition at the time of burial, identify postmortem and post-depositional manipulation of the body and grave (e.g., intentional removal of bodies/body parts), and establish whether burial occurred immediately after death or was delayed.

Moreover, the study does not address bone displacements within secondary voids created after the decomposition of soft tissues, nor does it provide assessments of the position of bones within or outside of the original body volume. Factors such as variations in soft tissue volume between individuals of different sizes/corpulence, and the progressive filling (i.e., sediment continually fills newly formed voids) or delayed filling (causing the 'flattening' of the ribcage and 'hyper-flexed' burials, for instance) of secondary open spaces with sediment over time should also be discussed.

In conclusion, while I acknowledge the importance of investigating potential deliberate burials in Homo naledi, I do not think that in its present form, the evidence presented in this study is as robust as it should be.

Reviewer #3 (Public Review):

This paper provides new information on the Dinaledi Chamber at the Rising Star Cave System. In short, a previously excavated area was expanded and resulted in the discovery of a cluster of bones appearing to be of one individual, a second similar cluster, and a third cluster with articulated elements (though with several individuals). Two of these clusters are argued to be intentionally buried individuals (the third one has not been investigated) and thus Homo naledi not only placed conspecifics in deep and hard to reach parts of caves but also buried them (apparently in shallow graves). This would be the oldest evidence of intentional burial. The main issue with the paper is that the purported burials were not fully excavated. Two are still in the ground, and one was removed in blocks but left unexcavated. As burials are mostly about sediments, it means the authors are lacking important lines of evidence. Instead, they bring other lines of argument as outlined below. While their preferred scenario is possible, there are important issues with the evidence as presented and they are severely hampered by the lack of detailed archaeological and geoarchaeological information both from the specific skeletal contexts and more generally from the chamber (because in fact the amount of excavation conducted here is still quite limited in scope). I also found that while the presentations of the various specialists in the team was quite good, the integration of these contributions into the main text was not. In particular, the geology of the cave system and the chamber need (especially what is known of the depositional and post-depositional processes) need to be better integrated into the presentation of the archaeology and the interpretation of the finds.

Often times the presence of articulated or mostly articulated skeletons is used to argue for intentional burial. This argument, however, is based on the premise that if not buried, these skeletons would have otherwise become disarticulated. Normally disarticulation would happen as a result of subsequent use of the site by hominins (e.g. purported burials in Neandertal cave sites) or by carnivores scavenging the body. Indeed this latter point is why bodies are buried so deeply in many Western societies (i.e. beyond the reach and smell of carnivores). Bodies can also be disarticulated by natural processes of deposit and erosion.

However, here in the case of the Dinaledi Chamber, we apparently don't have any of these other processes. The chamber was not used by carnivores and it was not a living area where H. naledi would have frequently returned and cleared out the space. As for depositional processes, it is more complex, but it is clear from Wiersma et al. that there is a steady, constant movement of these sediments towards drains. They also think that this process can account for the mix of articulated and non-articulated elements in the cave. Importantly, that same paper makes the argument that the formation of these sediments is not the result of water movement and that the cave has been dry since the formation of this deposit. So bodies lying on the surface and slowly covered by the formation of the deposit and slowly moving towards the drains could perhaps account for the pattern observed, meaning burial is not needed to account for articulations (note that more information on fabrics would be good in this context - orientation analysis of surface finds or of excavated finds is either completely lacking or minimal - see figure 13b and c report orientations on 79 bones of unknown context that appear to show perhaps elevated plunge angles and some slightly patterning in bearing but there is no associated statistics or text explaining the significance).

So, unless the team can provide some process that would have otherwise disarticulated these skeletons after the bodies arrived here and decomposed, their articulated state is not evidence of burial (no more than finding an articulated or mostly articulated bear skeleton deep in a European cave would suggest that it was buried).

As for the elemental analysis, what I understood from the paper is that the sediment associated with bones is different from the sediment not associated with bones. It is therefore unsurprising that the sediment associated with the reported skeletons clusters with sediments with bones. The linking argument for why this makes this sediment pit fill is unclear to me. Perhaps it is there, but as written I didn't follow it.

What the elemental analysis could suggest, I think, is that there has not been substantial reworking of the sediments (as opposed to the creep suggested by Wiersma et al.) since the bones leached these minerals into the sediment. What I don't know, and what is not reported, is how long after deposition we can expect the soil chemistry to change. If this elemental analysis were extended in a systematic way across the chamber (both vertically and horizontally) after more extensive excavations, I could see it perhaps being useful for better understanding the site formation processes and depositional context. As it is now, I did not see the argument in support of a burial pit.

The other line of evidence here is that some bones are sediment supported. The argument here is that when a body decomposes, bones that were previously held in place by soft tissues will be free to move and will shift their position. How the bones shift will differ depending on whether the body is surrounded by matrix (as they argue here in an excavated burial pit) or whether it is in the open (say, for instance, in a coffin) (and there are other possibilities as well - for instance wrapped in a shroud). Experiments have also shown the order in which the tendons, for instance, decompose and therefore which bones are likely to be free to move first or last.

I will note that this literature is poorly cited. I think the only two papers cited for how bodies decompose are Roksandic 2002 and Mickleburgh and Wescott 2019. The former is a review paper that summarizes a great many contexts that are clearly not appropriate here, and it generally makes the point that it is difficult to sort out, and it notes that progressively filled is an additional alternative to not buried/buried. The other looks at experimental data of bodies decomposing without being buried. In the paper here, this citation is used to argue that the body must have been buried. I don't see the linking argument at all. And the cited paper is mostly about how complicated it is to figure this all out and how many variables are still unaccounted for (including the initial positioning of the body and the consumption of the body by insects - something that is attested to at Naledi - plus snails - see not just Val but also Wiersma et al. and I think the initial Dirk et al. paper).

So the team here instead simply speaks of how the body decomposes in burials as if it is known. For the Feature 1 skeleton, the authors note that the ribs are "apparently" sediment supported and that a portion of the partial cranium is vertical or subvertical and sediment supported. For both of these, the figures show it very poorly. We really have to take their word for it. Second, I would have liked to have seen some reference and comparison to the literature for how the ribs should be in sediment burial cases. For the cranium, seems like a broken cranium resting on a surface will have vertical aspects regardless of sediment support. To the contrary, the orientation of the cranium will change depending on whether there is sediment holding it in place or not. But that argument is not made here. It is very hard from the figures to have a detailed idea of how these skeletons are oriented in the sediments, to know which elements are in articulation, which are missing, etc.

In the case of the Hill Antechamber Feature, an additional argument is made about the orientation of the finds in relation to the natural stratigraphy in this location. The team argues that the skeleton is lying more horizontally than the sediments and that in fact the foot is lying against the slope. First, there is no documentation of the slope of the layers here (e.g. a stratigraphic profile with the layers marked or a fabric analysis). There is a photo in the SI that says it shows sloping, but it needs some work. Second, this skeleton was removed in three blocks and then scanned. So the position of the skeleton is being worked out separate from its context. This is doable, but I would have liked to have seen some mention of how the blocks were georeferenced in the field and then subsequently in the lab and of how the items inside the block (i.e. the data coming from the CT scanner) were then georeferenced. I can think of ways I would try to do this, but without some discussion of this critical issue, the argument presented in Figure 10c is difficult to evaluate. Further, even if we accept this work, it is hard for me to see how the alignment of the foot is 15 degrees opposite the slope (the figure in the SI is better). It is also hard to understand the argument that the sediment separating the lower limb from the torso means burial. The team gives the explanation that if the body was in an open pit it would have been flat with no separation. Maybe. I mean I guess if the pit was flat. But there is no evidence here of a pit (at all). And what if the body was stuffed down the chute and was resting on a slope and covered with additional sediments from the chute (or additional bodies) as it decomposed? It seems that this should be the starting point here rather than imagining a pit.

One of the key pieces of evidence for demonstrating deliberate burial is the recognition of a pit. Pits can be identified because of the rupture they create in the stratigraphy when older sediments are brought to the surface, mixed, and then refilled into the pit with a different color, texture, compaction, etc. In some homogenous sediments a pit can be hard to detect and in some instances post-depositional processes (e.g. burrowing) can blur the distinction between the pit and the surrounding sediments. But the starting point of any discussion of deliberate burial has to be the demonstration of a pit. And I don't see it here. It might just be that the figures need to be improved. But I am skeptical because the team has taken the view that these finds can't be excavated. While I appreciate the scanning work done on the Antechamber find, it is not the same as excavating. Same comment for Features 1 and 2.

In short, my view is that they have an extremely interesting dataset. That H. naledi buried their dead here can't be excluded based on the data, but neither is it supported here. My view is that this paper is premature and that more excavation and the use of geoarchaeological techniques (especially micromorphology) are required to sort this out (or go a long way towards sorting it out).

Reviewer #4 (Public Review):

Berger et al. 2023a argues that Homo naledi intentionally buried their dead within the Rising Star cave system by digging pits and covering the bodies with infilled sediment. The authors identified two burials: Dinaledi Feature 1 from the Dinaledi Chamber, and the Hill Antechamber Feature from the Hill Antechamber. The evolutionary and behavioral implications for such behavior are highly significant and would be the first instance of a relatively small-brained hominin engaging is complex behavior that is often found in association with Homo sapiens and Homo neanderthalensis. Thus, the scientific rigor to validate these findings should be of the highest quality, and thus, provide clear documentation of intentional burial. In an attempt to meet these standards, the authors stated a series of tests that would support their hypothesis of intentional burials in the Rising Star Cave system:

"The key observations are (1) the difference in sediment composition within the feature compared to surrounding sediment; (2) the disruption of stratigraphy; (3) the anatomical coherence of the skeletal remains; (4) the matrix-supported position of some skeletal elements; and (5) the compatibility of non-articulated material with decomposition and subsequent collapse." (page 5)

To find support for the first (1) test, the authors collected sediment samples from various locations within the Rising Star Cave system, including sediment from within and outside Dinaledi Feature 1. However:

• The authors did not select sediment samples from within the Hill Antechamber Feature, so this test was only used to assess Dinaledi Feature 1.

• The sediment samples were analyzed using x-ray diffraction (XRD) and x-ray fluorescence (XRF) to test the mineralogy and chemistry of the samples from within and outside the feature. The XRF results were presented as weighted percentages (not intensities) with no control source reported. The weighted percentages were analyzed using a principal components analysis (PCA) while the particle-size distribution was analyzed using GRADISTAT statistics package and the Folk and Ward Method to summarize "mean grain size, sorting, skewness and kurtosis in addition to the percentages of clay, silt and sand in each sample." (page 28).

• The PCA results were reported solely as a biplot without showing the PC scores projected into the loading space, which is unusual and does not present the data accurately. Instead, the authors present the scores of a single component (PC2, figure 3) because the authors interpreted this component as "distinctly delineates fossil-bearing sediments from sterile sediments based on the positive loadings of P and S" (Page 6). However, the supplementary table that reports XRF bulk chemistry results as a weighted percentage of minerals within each sample (SI Table 1) shows mostly an absence of data for both Na and S. Since Na is at the lower end of detection limits for the method, and S seems to just be absent from the list, the intentions of the authors for showing the inclusion of these elements in their PCA results is unclear. Given that this is the author's primary method for demonstrating a burial, this issue is particularly concerning and requires additional attention.

• Regardless of the missing data, this reviewer attempted to replicate the XRF PCA results using the data provided in SI Table 1 and was unsuccessful. The samples that were collected from within the feature (SB) cluster with samples collected from sterile sediments and other locations around the cave system. Thus, these results are not replicable as currently reported.

• Visual comparisons of sediment grain size, shape, and composition were qualitatively summarized. Grain size was plotted as a line graph and is buried as supplemental Figure S13 showing sample by color and area, but these results do not distinguish samples from WITHIN the burial compared to OUTSIDE the burial as the authors state in the methods as a primary goal.

To test the second (2) aim, the "stratigraphy" was primarily described in text.

• For Dinaledi Feature 1, the authors state that the layer around Feature 1 "is continuous in the profile immediately to the east of the feature; it is disrupted in the sediment profile at the southern extent of the feature (fig. 3b)." Upon examination of figure 3b, the image shows an incredibly small depiction of the south (?) profile view with an extremely large black box overlaying a large portion of the photograph containing a small 5 cm scale. Visually, there is no difference in the profile that would suggest a disruption in the form of a pit. The LORM (orange-red mud layer) does seem to become fragmentary, but no micromorphological analysis was conducted on this section to provide an evaluation of stratigraphic composition. Also, by only excavating a portion of the feature, the authors were unable to adequately demonstrate the full extent of this feature.

• The authors attempt to describe "a bowl-shaped concave layer of clasts and sediment-free voids make up the bottom of the feature" (page 13) and refer to figures and supplementary information that do not depict any stratigraphic profile. Moreover, the authors state that "the leg, foot, and adjacent [skeletal?] material cut across stratigraphy" indicating that the skeleton is orientated on a flat plane against the surrounding stratigraphy that is "30{degree sign} slope of floor and underlying strata" (page 51, fig. 10c captions). There is no mention of infilled sediment from a pit and how this relates to the skeleton or the slope of the floor. It is therefore extremely unclear what the authors are meaning to describe without any visual or micromorphological supplementation to demonstrate a "bowl-shaped concave layer".

The third (3) test was to evaluate the anatomical coherence of the skeletal remains using macro- and micro-CT (computed tomography) of the Hill Antechamber Feature that was removed during excavation. To visually assess the anatomy of the Dinaledi Feature 1 burial, the authors describe the spatial relationship of skeletal elements as they were being excavated but halted partway through the excavation.

• The authors do not provide any documentation (piece-plotting, 3D rendering of stages of excavation, etc.) of the elements that were removed from the Dinaledi Feature. Figure 4 and SI Fig. S22 show the spatial relationship between identifiable skeletal elements that remain in the Feature. However, in Fig. 4, it is unclear why the authors chose to plot 2023-2014 excavated material along with material reported here, and it's even more difficult to understand the anatomical positioning of the elements given their color and point size choices. Although, the authors do provide a 3D rendering of the unexcavated remains showing some skeletal cohesion, apart from the mandible and teeth being re-located near the pelvis (Fig. 9). That said, it is very difficult to visually confirm the elements from this model or understand the original placement of the skeleton.

• 3D renderings of the Hill Antechamber feature skeletal material is clearly shown in SI Fig. S26. Contrary to what the authors state in text, there is a rather wide dispersal and rearrangement of elements for a "burial" that is theoretically protected from scavengers and other agents that would aid in dispersing bone from the surface. The authors do not offer any alternatives to explain disturbance, such as human activity, which clearly took place.

• Moreover, there does not appear to be any intentional arrangement of limbs that may suggest symbolic orientation of the dead (another line of evidence often used to support intentional burial but omitted by the authors). Thus, skeletal cohesion is not enough evidence to support the hypothesis of an intentional burial.

The fourth (4) test was attempted by evaluating whether some elements were vertically aligned from 3D reconstructed models of Hill Antechamber Feature and a photogrammetric model of the Dinaledi Feature 1. The authors state that "the spatial arrangement of the skeletal remains is consistent with primary burial of the fleshed body" (page 8 in reference to Dinaledi Feature 1) without providing any evidence, qualitative or quantitative, that this is the case for either burial.

Since this reviewer was unable to understand the fifth (5) test as it was written by the authors, I am unable to comment on the evidence to support this test and will default to the other reviewers for evaluation of this claim.

In addition to a lack of evidence to support the claims of intentional burial, this paper was also written extremely poorly. For example, the authors often overused 'persuasive communication devices' (see eLife article, https://elifesciences.org/articles/88654) to mislead readers:

"During this excavation, we recognized that the developing evidence was suggestive of a burial, due to the spatial configuration of the feature and the evidence that the excavated material seemed to come from a single body." (page 5)

As an opening statement to introduce Dinaledi Feature 1, the authors state the interpretation and working hypothesis as fact before the authors present any evidence. This is known as "HARKing" and "gives the impression that a hypothesis was formulated before data were collected" (Corneille et al. 2023). This type of writing is pervasive throughout the manuscript and requires extensive editing. I recommend that the authors review the article provided by eLife (https://elifesciences.org/articles/88654) and carefully review the manuscript. Moreover, as this text demonstrates, the authors’ word choice is indicative of storytelling for a popular news article instead of a scientific paper. I highly suggest that the authors review the manuscript carefully and present the data prior to giving conclusions in a clear and concise manner.

Moreover, the writing structure is inconsistent. Information that should be included in results is included in the methods, text in the results should be in discussions, and so forth. This inconsistency is pervasive throughout the entire manuscript, making it incredibly difficult to adequately understand what the authors had done and how the results were interpreted.

Finally, the "artifact" that was described and visualized using CT models is just that - a digitally colored model. The object in question has not been analyzed. Until this object is removed from the dirt and physically analyzed, this information needs to be removed from the manuscript as there is nothing to report before the object is physically examined.

Overall, there is not enough evidence to support the claim that Homo naledi intentionally buried their dead inside the Rising Star Cave system. Unfortunately, the manuscript in its current condition is deemed incomplete and inadequate, and should not be viewed as finalized scholarship.

Public Review:

In this article, a novel technique allowing the linking of viral transcription levels and progeny virion production is presented. Barcoded libraries of an H1N1 influenza virus (two genes were barcoded near the 3'end) were used to infect cells using an experimental approach ensuring that, in the low multiplicity of infection condition, each cell is infected by one virion and that nearly every virion has a unique barcode. This allows then, upon single-cell RNA sequencing and sequencing of the supernatants, to infer back the cells that were producing certain barcoded viruses. Assessing detection frequencies of barcodes in the single-cell sequencing and in the sequencing of the supernatants allows us to compare the levels of viral transcription and progeny virion production.

Observations that viral transcription levels are very heterogenous at the single-cell level are not novel, but reinforce those from previous studies. The major findings of this study are (i) progeny virion production is also very heterogenous, i.e., a few cells produce most of the progeny virions and (ii) there is a poor correlation between viral transcription levels and progeny virion production at the single-cell level.

Strengths:<br /> The article is very well written, the experimental choices are very well justified and the methods are very detailed, allowing the possibility of reproducing the work performed in this study. The conclusions are very well supported by the data and the limitations of the study and how those might influence the conclusions are also clearly explained. In addition, several experimental caveats, such as PCR cross-overs in next-generation sequencing and cell multiplets in single-cell sequencing, were well accounted for, which is not always the case in studies using these techniques.

Weaknesses:<br /> It seems that the results presented here are from one single experiment. How reproducible are the results?

As explained in the article, it is important that nearly every virion has a unique barcode. This was assessed by sequencing the barcodes in the virus libraries. Between 92% to 96% of the barcodes were unique. With this information, it should be possible to assess whether non-unique barcodes were detected in infected cells, and if yes, remove these from the downstream analysis.

It seems like all the information available in this very rich dataset was not fully exploited. For instance, Figure 5C suggests that cells missing the expression of one viral gene might still be able to produce progeny viruses. It would be interesting to have the information regarding which gene was not expressed in these cells.

The introduction and discussion are rather short and the article could benefit from expanding them. Additional speculations about viral or cellular factors (e.g. differences in innate immune responses, differences in cell division status) that might explain the observed heterogeneity, both at the viral transcription and viral progeny virus production levels, would be interesting.

Reviewer #3 (Public Review):

This study explores how condensin and telomere proteins cooperate to facilitate sister chromatid disjunction at chromosome ends during anaphase. Building upon previous results published by the same group (Reyes et al. 2015, Berthezene et al. 2020), the authors demonstrate that condensin is essential for sister telomere disjunction in anaphase in fission yeast. The primary role of condensin appears to be counteracting cohesin, which holds sister telomeres together. Furthermore, condensin is found to be enriched at telomeres, and this enrichment partially relies on Taz1, the principal telomere factor in S. pombe. The loss of Taz1 does not cause an obvious defect in sister telomere disjunction, which prevents drawing strong conclusions about its role in this process.

Reviewer #1 (Public Review):

Colin et al demonstrated that condensin is a key factor for the disjunction of sister-telomeres during mitosis and proposed that it is due to that condensin restrains the telomere association of cohesin. The authors first showed that condensin binds telomeres in mitosis evidenced by ChIP-qPCR and calibrated ChIP-seq. They further demonstrated that compromising condensin's activity leads to a failure in the disjunction of telomeres, with convincing cytological and HI-seq evidence. Two telomeric proteins Taz1 and Mit1 were identified to specifically regulate the telomere association of condensin. Deletion of these genes decreased/increased condensin's telomere association and exacerbated/remedied the defected telomere disjunction in a condensin mutant, echoing the role of condensin in telomere disjunction. They proposed that the underlying mechanism is that condensin inhibits cohesin's accumulation at telomeres. However, the evidence for this claim might need to be further strengthened. Nevertheless, this study uncovered a novel role of condensin in the separation of telomeres of sister chromosomes and open a question of how condensin regulates the structure of chromosomal ends.

Reviewer #2 (Public Review):

This manuscript presents a comprehensive investigation into the role of condensin complexes in telomere segregation in fission yeast. The authors employ chromatin immunoprecipitation analysis to demonstrate the enrichment of condensin at telomeres during anaphase. They then use condensin conditional mutants to confirm that this complex plays a crucial role in sister telomere disjunction as well as the unclustering of telomeric regions from the preceding Rabl configuration. Interestingly, they show that condensin's role in telomere disjunction is unlikely related to catenation removal but rather related to the organization of telomeres in cis and/or the elimination of structural constraints or proteins that hinder separation.

The authors also investigate the regulation of condensin localization to telomeres and reveal the involvement of the shelterin subunit Taz1 in promoting condensin's association with telomeres while demonstrating that the chromatin remodeler Mit1 prevents excessive loading of condensin onto telomeres. Finally, they show that cohesin acts as a negative regulator of telomere separation, counteracting the positive effects of condensin.

Overall, the manuscript is well-executed, and the authors provide sufficient supporting evidence for their claims. There are a couple of aspects that arise from this study that when fully elucidated will lead to a mechanistic understanding of important biological processes. For instance, the exact mechanism by which Taz1 affects condensin loading or the mechanistic link between cohesin and condensin, especially in the context of their opposing roles, are exciting prospects for the future and it is possible that future work within the context of telomeres might provide valuable insights into this question.

Another crucial point emphasized by the manuscript is that the role of condensin in telomere segregation extends beyond facilitating catenation removal.

Reviewer #2 (Public Review):

The authors have addressed most of the concerns. Yet, I still think the authors should at least mention in the article the residues involved in the intra-pore lipid binding pockets for further experimental validation (not only for those residues involve in disease). This is important because the lipid-like density information usually does not come integrated into the PDB structures, so it is not easily accessible for non-structural biologists. The structural data seems solid, and the MD data supports the notion that the GJC is in a putative close state.

Reviewer #1 (Public Review):

Gap junctions, formed from connexins, are important in cell communication, allowing ions and small molecules to move directly between cells. While structures of connexins have previously been reported, the structure of Connexin 43, which is the most widely expressed connexin and is important in many physiological processes was not known. Qi et al used cryo-EM to solve the structure of Connexin 43. They then compared this structure to structures of other connexins. Connexin gap junctions are built from two "hemichannels" consisting of hexamers of connexins. Hemichannels from two opposing cells dock together to form a complete channel that allows the movement of molecules between cells. N-terminal helices from each of the 6 subunits of each hemichannel allow control of whether the channels are open or closed. Previously solved structures of Cx26 and Cx46/50 have the N-termini pointing down into the pore of the protein leaving a central pore and so these channels have been considered to be open. The structure that Qi et al observed has the N-termini in a more raised position with a narrower pore through the centre. This led them to speculate whether this was the "closed" form of the protein. They also noted that, if only the protein was considered, there were gaps between the N-terminal helices, but these gaps were filled with lipid-like molecules. They, therefore, speculated that lipids were important in the closure mechanism. To address whether their structure was open or closed with respect to ions they carried out molecular dynamics studies, and demonstrated that under the conditions of the molecular dynamics ions did not traverse the channel when the lipids were present.

Strengths:

The high resolution cryo-EM density maps clearly show the structure of the protein with the N-termini in a lateral position and lipid density blocking the gaps between the neighbouring helices. The conformation that they observe when they have solved the structure from protein in detergent is also seen when they reconstitute the protein into nanodiscs, which is ostensibly a more membrane-like environment. They, therefore, would appear to have trapped the protein in a stable conformational state.<br /> The molecular dynamics simulations are consistent with the channel being closed when the lipid is present and raises the possibility of lipids being involved in regulation.<br /> A comparison of this structure with other structures of connexin channels and hemichannels gives another representation of how the N-terminal helix of connexins can variously be involved in the regulation of channel opening.

Weaknesses:

While the authors have trapped a relatively stable state of the protein and shown that, under the conditions of their molecular dynamics simulations, ions do not pass through, it is harder to understand whether this is physiologically relevant. Determining this would be beyond the scope of the article. To my knowledge there is no direct evidence that lipids are involved in regulation of connexins in this way, but this is also an interesting area for future exploration. It is also possible that lipids were trapped in the pore during the solubilisation process making it non-physiological. The authors acknowledge this and they describe the structure as a "putative" closed state.

The positions of the mutations in disease shown in Figure 4 is interesting. However, the authors don't discuss/speculate how any of these mutations could affect the binding of the lipids or the conformational state of the protein.

It should also be noted that a structure of the same protein has recently been published. This shows a very similar conformation of the N-termini with lipids bound in the same way, despite solubilising in a different detergent.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #2 (Public Review):

The Xerces Blue is an iconic species, now extinct, that is a symbol for invertebrate conservation. Using genomic sequencing of century-old specimens of the Xerces Blue and its closest living relatives, the authors hypothesize about possible genetic indicators of the species' demise. Although the limited range and habitat destruction are the most likely culprits, it is possible that some natural reasons have been brewing to bring this species closer to extinction.

The importance of this study is in its generality and applicability to any other invertebrate species. The authors find that low effective population size, high inbreeding (for tens of thousands of years), and higher fraction of deleterious alleles characterize the Xerces colonies prior to extinction. These signatures can be captured from comparative genomic analysis of any target species to evaluate its population health.

It should be noted that it remains unclear if these genomic signatures are indeed predictive of extinction, or populations can bounce back given certain conditions and increase their genetic diversity somehow.

Methods are detailed and explained well, and the study could be replicated. I think this is a solid piece of work. Interested researchers can apply these methods to their chosen species and eventually, we will assemble datasets to study extinction process in many species to learn some general rules.

Several small questions/suggestions:

1) The authors reference a study concluding that Shijimiaeoides is Glaucopsyche. Their tree shows the same, confirming previous publications. And yet they still use Shijimiaeoides, which is confusing. Why not use Glaucopsyche for all these blues?

2) Plebejus argus is a species much more distant from P. melissa than Plebejus anna (anna and melissa are really very close to each other), and yet their tree shows the opposite. What is the problem? Misidentification? Errors in phylogenetic analyses?

3) Wouldn't it be nicer to show the underside of butterfly pictures that reveals the differences between xerces and others? Now, they all look blue and like one species, no real difference.

4) The authors stated that one of five xerces specimens failed to sequence, and yet they show 5 specimens in the tree. Was the extra specimen taken from GenBank?

Reviewer #1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Reviewer #2 (Public Review):

Accumulating data suggests that the presence of immune cell infiltrates in the meninges of the multiple sclerosis brain contributes to the tissue damage in the underlying cortical grey matter by the release of inflammatory and cytotoxic factors that diffuse into the brain parenchyma. However, little is known about the identity and direct and indirect effects of these mediators at a molecular level. This study addresses the vital link between an adaptive immune response in the CSF space and the molecular mechanisms of tissue damage that drive clinical progression. In this short report the authors use a spatial transcriptomics approach using Visium Gene Expression technology from 10x Genomics, to identify gene expression signatures in the meninges and the underlying brain parenchyma, and their interrelationship, in the PLP-induced EAE model of MS in the SJL mouse. MRI imaging using a high field strength (11.7T) scanner was used to identify areas of meningeal infiltration for further study. They report, as might be expected, the upregulation of genes associated with the complement cascade, immune cell infiltration, antigen presentation, and astrocyte activation. Pathway analysis revealed the presence of TNF, JAK-STAT and NFkB signaling, amongst others, close to sites of meningeal inflammation in the EAE animals, although the spatial resolution is insufficient to indicate whether this is in the meninges, grey matter, or both.

UMAP clustering illuminated a major distinct cluster of upregulated genes in the meninges and smaller clusters associated with the grey matter parenchyma underlying the infiltrates. The meningeal cluster contained genes associated with immune cell functions and interactions, cytokine production, and action. The parenchymal clusters included genes and pathways related to glial activation, but also adaptive/B-cell mediated immunity and antigen presentation. This again suggests a technical inability to resolve fully between the compartments as immune cells do not penetrate the pial surface in this model or in MS. Finally, a trajectory analysis based on distance from the meningeal gene cluster successfully demonstrated descending and ascending gradients of gene expression, in particular a decline in pathway enrichment for immune processes with distance from the meninges.

Although these results confirm what we already know about processes involved in the meninges in MS and its models and gradients of pathology in sub-pial regions, this is the first to use spatial transcriptomics to demonstrate such gradients at a molecular level in an animal model that demonstrates lymphoid like tissue development in the meninges and associated grey matter pathology. The mouse EAE model being used here does reproduce many, although not all, of the pathological features of MS and the ability to look at longer time points has been exploited well. However, this particular spatial transcriptomics technique cannot resolve at a cellular level and therefore there is a lot of overlap between gene expression signatures in the meninges and the underlying grey matter parenchyma.

The short nature of this report means that the results are presented and discussed in a vague way, without enough molecular detail to reveal much information about molecular pathogenetic mechanisms.

The trajectory analysis is a good way to explore gradients within the tissues and the authors are to be applauded for using this approach. However, the trajectory analysis does not tell us much if you only choose 2 genes that you think might be involved in the pathogenetic processes going on in the grey matter. It might be more useful to choose some genes involved in pathogenetic processes that we already know are involved in the tissue damage in the underlying grey matter in MS, for which there is already a lot of literature, or genes that respond to molecules we know are increased in MS CSF, although the animal models may be very different. Why were C3 and B2m chosen here?

Strengths:<br /> - The mouse model does exhibit many of the features of the compartmentalized immune response seen in MS, including the presence of meningeal immune cell infiltrates in the central sulcus and over the surface of the cortex, with the presence of FDC's HEVs PNAd+ vessels and CXCL13 expression, indicating the formation of lymphoid like cell aggregates. In addition, disruption of the glia limitans is seen, as in MS. Increased microglial reactivity is also present at the pial surface.<br /> - Spatial transcriptomics is the best approach to studying gradients in gene expression in both white matter and grey matter and their relationship between compartments.<br /> - It would be useful to have more discussion of how the upregulated pathways in the two compartments fit with what we know about the cellular changes occurring in both, for which presumably there is prior information from the group's previous publications.

Limitations:<br /> - EAE in the mouse is not MS and may be far removed when one considers molecular mechanisms, especially as MS is not a simple anti-myelin protein autoimmune condition. Therefore, this study could be following gene trajectories that do not exist in MS. This needs a significant amount of discussion in the manuscript if the authors suggest that it is mimicking MS.<br /> - The model does not have the cortical subpial demyelination typical of MS and it is unknown whether neuronal loss occurs in this model, which is the main feature of cytokine-mediated neurodegeneration in MS. If it does not then a whole set of genes will be missing that are involved in the neuronal response to inflammatory stimuli that may be cytotoxic.<br /> - Visium technology does not get down to single cell level and does not appear to allow resolution of the border between the meninges and the underlying grey matter.<br /> - Neuronal loss in the MS cortex is independent of demyelination and therefore not related to remyelination failure. There does not appear to be any cortical grey matter demyelination in these animals, so it is difficult to relate any of the gene changes seen here to demyelination.<br /> - No mention of how the ascending and descending patterns of gene expression may be due to the gradient of microglial activation that underlies meningeal inflammation, which is a big omission.

Reviewer #3 (Public Review):

In this study, Gadani et al. induced EAE in SJL/J mice and performed a comprehensive spatial transcriptomic analysis in areas of meningeal inflammation during the relapse phase of the disease. The authors found specific enrichment in spatial gene signatures (cluster 11) in the regions of increased contrast-enhancement by MRI (where meningeal extravasation of activated immune cells is observed) that overlap with signatures in the adjacent brain parenchyma, namely the thalamus. Several pathways were similarly upregulated in the meningeal-associated cluster 11 and adjacent parenchymal clusters (like adaptive mediated immunity, and antigen processing and presentation), suggestive of a "leakage" of inflammatory mediators from the meninges into the brain during the re-activation of disease. The tested hypothesis, as well as the data presented in this study, is quite interesting and novel.

Reviewer #1 (Public Review):

In this paper, the authors developed an image analysis pipeline to automatically identify individual ‎neurons within a population of fluorescently tagged neurons. This application is optimized to deal with ‎multi-cell analysis and builds on a previous software version, developed by the same team, to resolve ‎individual neurons from whole-brain imaging stacks. Using advanced statistical approaches and ‎several heuristics tailored for C. elegans anatomy, the method successfully identifies individual ‎neurons with a fairly high accuracy. Thus, while specific to C. elegans, this method can become ‎instrumental for a variety of research directions such as in-vivo single-cell gene expression analysis ‎and calcium-based neural activity studies.‎

The analysis procedure depends on the availability of an accurate atlas that serves as a reference map ‎for neural positions. Thus, when imaging a new reporter line without fair prior knowledge of the ‎tagged cells, such an atlas may be very difficult to construct. Moreover, usage of available reference ‎atlases, constructed based on other databases, is not very helpful (as shown by the authors in Fig 3), ‎so for each new reporter line a de-novo atlas needs to be constructed.‎

I have a few comments that may help to better understand the potential of the tool to become handy:

‎1) I wonder the degree by which strain mosaicism affects the analysis (Figs 1-4) as it was performed on ‎a non-integrated reporter strain. As stated, for constructing the reference atlas, the authors used ‎worms in which they could identify the complete set of tagged neurons. But how sensitive is the ‎analysis when assaying worms with different levels of mosaicism? Are the results shown in the paper ‎stem from animals with a full neural set expression? Could the authors add results for which the ‎assayed worms show partial expression where only 80%, 70%, 50% of the cells population are ‎observed, and how this will affect identification accuracy? This may be important as many non-‎integrated reporter lines show high mosaic patterns and may therefore not be suitable for using this ‎analytic method. In that sense, could the authors describe the mosaic degree of their line used for ‎validating the method.‎<br /> ‎<br /> 2) For the gene expression analysis (Fig 5), where was the intensity of the GFP extracted from? As it has ‎no nuclear tag, the protein should be cytoplasmic (as seen in Fig 5a), but in Fig 5c it is shown as if the ‎region of interest to extract fluorescence was nuclear. If fluorescence was indeed extracted from the ‎cytoplasm, then it will be helpful to include in the software and in the results description how this was ‎done, as a huge hurdle in dissecting such multi-cell images is avoiding crossreads between ‎adjacent/intersecting neurons.‎<br /> ‎<br /> 3) In the same matter: In the methods, it is specified that the strain expressing GCAMP was also used ‎in the gene expression analysis shown in Figure 5. But the calcium indicator may show transient ‎intensities depending on spontaneous neural activity during the imaging. This will introduce a ‎significant variability that may affect the expression correlation analysis as depicted in Figure 5.‎

Reviewer #2 (Public Review):

The authors succeed in generalizing the pre-alignment procedure for their cell identification method to allow it to work effectively on data with only small subsets of cells labeled. They convincingly show that their extension accurately identifies head angle, based on finding auto fluorescent tissue and looking for a symmetric l/r axis. They demonstrate that the method works to identify known subsets of neurons with varying accuracy depending on the nature of underlying atlas data. Their approach should be a useful one for researchers wishing to identify subsets of head neurons in C. elegans, for example in whole brain recording, and the ideas might be useful elsewhere.

The authors also strive to give some general insights on what makes a good atlas. It is interesting and valuable to see (at least for this specific set of neurons) that 5-10 ideal examples are sufficient. However, some critical details would help in understanding how far their insights generalize. I believe the set of neurons in each atlas version are matched to the known set of cells in the sparse neuronal marker, however this critical detail isn't explicitly stated anywhere I can see. In addition, it is stated that some neuron positions are missing in the neuropal data and replaced with the (single) position available from the open worm atlas. It should be stated how many neurons are missing and replaced in this way (providing weaker information). It also is not explicitly stated that the putative identities for the uncertain cells (designated with Greek letters) are used to sample the neuropal data. Large numbers of openworm single positions or if uncertain cells are misidentified forcing alignment against the positions of nearby but different cells would both handicap the neuropal atlas relative to the matched florescence atlas. This is an important question since sufficient performance from an ideal neuropal atlas (subsampled) would avoid the need for building custom atlases per strain.

Reviewer #1 (Public Review):

Summary of the major findings -

1. The authors used saturation mutagenesis and directed evolution to mutate the highly conserved fusion loop (98 DRGWGNGCGLFGK 110) of the Envelope (E) glycoprotein of Dengue virus (DENV). They created 2 libraries with parallel mutations at amino acids 101, 103, 105-107, and 101-105 respectively. The in vitro transcribed RNA from the two plasmid libraries was electroporated separately into Vero and C6/36 cells and passaged thrice in each of these cells. They successfully recovered a variant N103S/G106L from Library 1 in C6/36 cells, which represented 95% of the sequence population and contained another mutation in E outside the fusion loop (T171A). Library 2 was unsuccessful in either cell type.

2. The fusion loop mutant virus called D2-FL (N103S/G106L) was created through reverse genetics. Another variant called D2-FLM was also created, which in addition to the fusion loop mutations, also contains a previously published, evolved, and optimized prM-furin cleavage sequence that results in a mature version of the virus (with lower prM content). Both D2-FL and D2-FLM viruses grew comparably to wild type virus in mosquito (C6/36) cells but their infectious titers were 2-2.5 log lower than wild<br /> type virus when grown in mammalian (Vero) cells. These viruses were not compromised in thermostability, and the mechanism for attenuation in Vero cells remains unknown.

4. Next, the authors probed the neutralization of these viruses using a panel of monoclonal antibodies (mAbs) against fusion loop and domain I, II and III of E protein, and against prM protein. As intended, neutralization by fusion loop mAbs was reduced or impaired for both D2-FL and D2-FLM, compared to wild type DENV2. D2-FLM virus was equivalent to wild type with respect to neutralization by domain I, II, and III antibodies tested (except domain II-C10 mAb) suggesting an intact global antigenic landscape of the mutant virion. As expected, D2-FLM was also resistant to neutralization by prM mAbs (D2-FL was not tested in this batch of experiments).

5. Finally, the authors evaluated neutralization in the context of polyclonal serum from convalescent humans (n=6) and experimentally infected non-human primates (n=9) at different time points (27 total samples). Homotypic sera (DENV2) neutralized D2-FL, D2-FLM, and wild type DENV similarly, suggesting that the contribution of fusion loop and prM epitopes is insignificant in a serotype-specific neutralization response. However, heterotypic sera (DENV4) neutralized D2-FL and D2-FLM less potently than wild type DENV2, especially at later time points, demonstrating the contribution of fusion loop- and prM-specific antibodies to heterotypic neutralization.

Impact of the study-

1. The engineered D2-FL and D2-FLM viruses are valuable reagents to probe antibodies targeting the fusion loop and prM in the overall polyclonal response to DENV.

2. Though more work is needed, these viruses can facilitate the design of a new generation of DENV vaccine that does not elicit fusion loop- and prM-specific antibodies, which are often poorly neutralizing and lead to antibody-dependent enhancement effect (ADE).

3. This work can be extended to other members of the flavivirus family.

4. A broader impact of their work is a reminder that conserved amino acids may not always be critical for function and therefore should not be immediately dismissed in substitution/mutagenesis/protein design efforts.

Evaluating this study in the context of prior literature -

The authors write "Although the extreme conservation and critical role in entry have led to it being traditionally considered impossible to change the fusion loop, we successfully tested the hypothesis that massively parallel directed evolution could produce viable DENV fusion-loop mutants that were still capable of fusion and entry, while altering the antigenic footprint."<br /> ".....Previously, a single study on WNV successfully generated a viable virus with a single mutation at the fusion loop, although it severely attenuated neurovirulence. Otherwise, it has not been generated in DENV or other mosquito-borne flaviviruses"

The above claims are a bit overstated. In the context of other flaviviruses:

- A previous study applied a similar saturation mutagenesis approach to the *full length* E protein of Zika virus and found that while the conserved fusion loop was mutationally constrained, some mutations, including at amino acid residue 106 were tolerated (PMID 31511387).<br /> - The Japanese encephalitis virus (JEV) SA14-14-2 live vaccine strain contains a L107F mutation in the fusion loop (in addition to other changes elsewhere in the genome) relative to the parental JEV SA14 strain (PMID: 25855730).<br /> - For tickborne encephalitis virus (TBEV-DENV4 chimera), H104G/L107F double mutant has been described (PMID: 8331735)

There have also been previous examples of functionally tolerated mutations within the DENV fusion loop:

- Goncalvez et al., isolated an escape variant of DENV 2 using chimpanzee Fab 1A5, with a mutation in the fusion loop G106V (PMID: 15542644). G106 is also mutated in D2-FL clone (N103S/G106L) described in the current study.<br /> - In the context of single-round infectious DENV, mutation at site 102 within the fusion loop has been shown to retain infectivity (PMID 31820734).

Appraisal of the results -

The data largely support the conclusions, but some improvements and extensions can benefit the work.

1. Line 92-93: "This major variant comprised ~95% of the population, while the next most populous variant comprised only 0.25% (Figure 1C)".<br /> What is the sequence of the next most abundant variant?

2. Lines 94-95: "Residues W101, C105, and L107 were preserved in our final sequence, supporting the structural importance of these residues."<br /> L107F is viable in other flaviviruses.

3. Figure 2c: The FLM sample in the western blot shows hardly any E protein, making E/prM quantitation unreliable.

4. Lines 149 -151: "Importantly, D2-FL and D2-FLM were resistant to antibodies targeting the fusion loop. While neutralization by 1M7 is reduced by ~2-logs, no neutralization was observed for 1N5, 1L6, and 4G2 for either variant (Figure 3 A)".

a) Partial neutralization was observed for 1N5, for D2-FL.<br /> b) Do these mAbs cover the full spectrum of fusion loop antibodies identified thus far in the field?<br /> c) Are the epitopes known for these mAbs? It would be useful to discuss how the epitope of 1M7 differs from the other mAbs? What are the critical residues?<br /> d) Maybe the D2-FL mutant can be further evolved with selection pressure with fusion loop mAbs 1M7 +/-1N5 and/or other fusion loop mAbs.

5. It would have been useful to include D2-M for comparison (with evolved furin cleavage sequence but no fusion loop mutations).

6. Data for polyclonal serum can be better discussed. Table 1 is not discussed much in the text. For the R1160-90dpi-DENV4 sample, D2-FL and D2-FLM are neutralized better than wild type DENV2? The authors' interpretation in lines 181-182 is inconsistent with the data presented in Figure 3C, which suggests that over time, there is INCREASED (not waning) dependence on FL- and prM-specific antibodies for heterotypic neutralization.

Suggestions for further experiments-

1. It would be interesting to see the phenotype of single mutants N103S and G106L, relative to double mutant N103S/G106L (D2-FL).<br /> 2. The fusion capability of these viruses can be gauged using liposome fusion assay under different pH conditions and different lipids.<br /> 3. Correlative antibody binding vs neutralization data would be useful.

Reviewer #2 (Public Review):

Antibody-dependent enhancement (ADE) of Dengue is largely driven by cross-reactive antibodies that target the DENV fusion loop or pre-membrane protein. Screening polyclonal sera for antibodies that bind to these cross-reactive epitopes could increase the successful implementation of a safe DENV vaccine that does not lead to ADE. However, there are few reliable tools to rapidly assess the polyclonal sera for epitope targets and ADE potential. Here the authors develop a live viral tool to rapidly screen polyclonal sera for binding to fusion loop and pre-membrane epitopes. The authors performed a deep mutational scan for viable viruses with mutations in the fusion loop (FL). The authors identified two mutations functionally tolerable in insect C6/36 cells, but lead to defective replication in mammalian Vero cells. These mutant viruses, D2-FL and D2-FLM, were tested for epitope presentation with a panel of monoclonal antibodies and polyclonal sera. The D2-FL and D2-FLM viruses were not neutralized by FL-specific monoclonal antibodies demonstrating that the FL epitope has been ablated. However, neutralization data with polyclonal sera is contradictory to the claim that cross-reactive antibody responses targeting the pre-membrane and the FL epitopes wane over time.

Overall the central conclusion that the engineered viruses can predict epitopes targeted by antibodies is supported by the data and the D2-FL and D2-FLM viruses represent a valuable tool to the DENV research community.

Reviewer #1 (Public Review):

Masson et al. leveraged the natural genetic diversity presented in a large cohort of the Diversity Outbred in Australia (DOz) mice (n=215) to determine skeletal muscle proteins that were associated with insulin sensitivity. The hits were further filtered by pQTL analysis to construct a proteome fingerprint for insulin resistance. These proteins were then searched against Connectivity Map (CMAP) to identify compounds that could modulate insulin sensitivity. In parallel, many of these compounds were screened experimentally alongside other compounds in the Prestwick library to independently validate some of the compound hits. These two analyses were combined to score for compounds that would potentially reverse insulin resistance. Thiostrepton was identified as the top candidate, and its ability to reverse insulin resistance was validated using assays in L6 myotubes.

Below are several comments made on the original version of this study, addressed by the authors in the current version:

(1) Please describe the rationale of trypsinizing the tissue prior to mitochondrial isolation.

(2) The authors mentioned that the proteomics data were Log2 transformed and median-normalized. Please provide a bit more details on this, including whether the subjects were randomized.

(3) In Figure 1D, please give the numbers of mice the authors used for the CV comparisons in each group, whether they were of similar age and sex, and whether the differences in CV values were statistically significant

(4) The authors stated in lines 155-157 that proteins negatively associated with Matsuda index were further filtered by presence of their cis-pQTLs. Please provide more explanations to justify this filtering criterion.

(5) Please explain why the first half of the paper focused extensively on the authors' discoveries in the mitochondrial proteome, and how proteins involved in mitochondrial processes (such as complex I) were associated with Matsuda Index, but the final fingerprint list of insulin resistance, which contained 76 proteins, only had 7 mitochondrial proteins.

(6) The authors found that thiostrepton-induced insulin resistance reversal effects were not through insulin signalling. Please list the proteins in the fingerprint list that led to identification of thiostrepton on CMAP, and discuss whether you think that thiostrepton directly or indirectly acts on these protein targets.

Reviewer #2 (Public Review):

In the present study, Masson et al. provide an elegant and profound demonstration of utilization of systems genetics data to fuel discovery of actionable therapeutics. The strengths of the study are many: generation of a novel skeletal muscle genetics proteomic dataset which is paired with measures of glucose metabolism in mice, systematic utilization of these data to yield potential therapeutic molecules which target insulin resistance, cross-referencing library screens from connectivity map with an independent validation platform for muscle glucose uptake and preclinical data supporting a new mechanism for thiostrepton in alleviating muscle insulin resistance. Future studies evaluating similar integrations of omics data from genetic diversity with compound screens, as well as detailed characterization of mechanisms such as thiostrepton on muscle fibers will further inform some remaining questions. In general, the thorough nature of this study not only provides strong support for the conclusions made but additionally offers a new framework for analysis of systems-based data. I had made several comments on the prior submission, all of which have been fully addressed and incorporated.

Reviewer #1 (Public Review):

The authors investigated the molecular correlates in potential neural centers in the Japanese quail brain associated with photoperiod-induced life-history states. The authors simulated photoperiod to attain winter and summer-like physiology and samples of neural tissues at spring, and autumn life-history states, daily rhythms in transcripts in solstices and equinox, and lastly studies FSHb transcripts in the pituitary. The experiments are based on a series of changes in photoperiod and gave some interesting results. The experiment did not have a control for no change in photoperiod so it seems possible that endogenous rhythms could be another aspect of seasonal rhythms that lack in this study. The short-day group does not explain the endogenous seasonal response.

The manuscript would benefit from further clarity in synthesizing different sections. Additionally, there are some instances of unclear language and numerous typos throughout the manuscript. A thorough revision is recommended, including addressing sentence structure for improved clarity, reframing sentences where necessary, correcting typos, conducting a grammar check, and enhancing overall writing clarity.

Data analysis needs more clarity particularly how transcriptome data explains different physiological measures across seasonal life-history states. It seems the discussion is built around a few genes that have been studied in other published literature on quail seasonal response. Extending results on the promotor of DEGs and building discussion is an extrapolating discussion on limited evidence and seems redundant.

Last, I wondered if it would be possible to add an ecological context for the frequent change in the photoperiod schedule and not take account of the endogenous annual response. Adding discussion on ecological relevance would make more sense.

Reviewer #2 (Public Review):

This study is carefully designed and well executed, including a comprehensive suite of endpoint measures and large sample sizes that give confidence in the results. I have a few general comments and suggestions that the authors might find helpful.

1) I found it difficult to fully grasp the experimental design, including the length of light treatment in the three different experiments (which appears to extend from 2 weeks up to 8 weeks). A graphical description of the experimental design along a timeline would be very helpful to the reader. I suggest adding the respective sample sizes to such a graphic, because this information is currently also difficult to keep track of.

2) The authors use a lot of terminology that is second nature to a chronobiologist but may be difficult for the general reader to keep track of. For example, what is the difference between "photoinducibility" and "photosensitivity"? Similarly, "vernal" and "autumnal" should be briefly explained at the outset, or maybe simply say "spring equinox" and "fall equinox."

3) What was the rationale for using only male birds in this study? The authors may want to include a brief discussion on whether the expected results for females might be similar to or different from what they found in males, and why.

4) The authors used the Bonferroni correction method to account for multiple hypothesis testing of measures of testes mass, body mass, fat score, vimentin immunoreactivity and qPCR analyses in Study 1. I don't think Bonferroni is ever appropriate for biological data: these methods assume that all variables are independent of each other, an assumption that is almost never warranted in biology. In fact, the data show clear relationships between these endpoint measures. Alternatively, one might use Benjamini-Hochberg's FDR correction or various methods for calculating the corrected alpha level.

5) The graphical interpretations of the results shown in Figure 1n and Figure 3e, along with the hypothesized working model shown in Figure S5, might best be combined into a single figure that becomes part of the Discussion. As is, I do not think these interpretative graphics (which are well done and super helpful!) are appropriate for the Results section.

Reviewer #3 (Public Review):

It is well known that as seasonal day length increases, molecular cascades in the brain are triggered to ready an individual for reproduction. Some of these changes, however, can begin to occur before the day length threshold is reached, suggesting that short days similarly have the capacity to alter aspects of phenotype. This study seeks to understand the mechanisms by which short days can accomplish this task, which is an interesting and important question in the field of organismal biology and endocrinology.

The set of studies that this manuscript presents is comprehensive and well-controlled. Many of the effects are also strong and thus offer tantalizing hints about the endo-molecular basis by which short days might stimulate major changes in body condition. Another strength is that the authors put together a compelling model for how different facets of an animal's reproductive state come "on line" as day length increases and spring approaches. In this way, I think the authors broadly fulfill their aims.

I do, however, also think that there are a few weaknesses that the authors should consider, or that readers should consider when evaluating this manuscript. First, some of the molecular genetic analyses should be interpreted with greater caution. By bioinformatically showing that certain DNA motifs exist within a gene promoter (e.g., FSHbeta), one is not generating robust evidence that corresponding transcription factors actually regulate the expression of the gene in question. In fact, some may argue that this line of evidence only offers weak support for such a conclusion. I appreciate that actually running the laboratory experiments necessary to generate strong support for these types of conclusions is not trivial, and doing so may even be impossible. I would therefore suggest a clear admission of these limitations in the paper.

Second, I have another issue with the interpretation of data presented in Figure 3. The data show that FSHbeta increases in expression in the 8Lext group, suggesting that endogenous drivers likely act to increase the expression of this gene despite no change in day length. However, more robust effects are reported for FSHbeta expression in the 10v and 12v groups, even compared to the 8Lext group. Doesn't this suggest that both endogenous mechanisms and changes in day length work together to ramp up FSHbeta? The rest of the paper seemed to emphasize endogenous mechanisms and gloss over the fact that such mechanisms likely work additively with other factors. I felt like there was more nuance to these findings than the authors were getting into.

Third, studies 1 - 3 are well controlled; however, I'm left wondering how much of an effect the transitions in day length might have on the underlying molecular processes that mediate changes in body condition. While the changes in day length are themselves ecologically relevant, the transitions between day length states are not. How do we know, for example, that more gradual changes in day length that occur over long timespans do not produce different effects at the levels of the brain and body? This seemed especially relevant for study 3, where animals experience a rather sudden change in day length. I recognize that these experimental methods are well described in the literature, and they have been used by endocrinologists for a long time; nonetheless, I think questions remain.

Reviewer #1 (Public Review):

In this study, the authors set out to investigate spatial RNA processing events, specifically alternative splicing and 3' UTR usage, in mouse brain and kidney tissues using ReadZS and SpliZ methodologies on spatial transcriptomics data. The research contributes to understanding tissue-specific gene expression regulation from a spatial perspective. The study introduces a novel approach for analyzing spatial transcriptomics data, allowing for the identification of RNA processing and regulation patterns directly from 10X Visium data. The authors present convincing evidence supporting the identification of novel RNA processing patterns using their methodology, which holds significant implications for researchers in the field of spatial transcriptomics and the study of alternative splicing and 3' UTR usage

The conclusions of the study are mostly well-supported by the data; however, certain aspects could be improved to strengthen the findings.<br /> 1) The conclusions of this study would be strengthened by conducting a more extensive tissue sample analysis and including biological replicates. Additionally, appropriate batch effect corrections should be applied when dealing with biological replicates.<br /> 2) The 3' UTR usage and alternative splicing should be compared among clearly labeled clusters for a more comprehensive analysis.<br /> 3) The authors should clarify their rationale for choosing ReadZS and SpliZ approaches and provide comparisons with other methods to demonstrate the advantages and potential limitations of their chosen methodologies.

Reviewer #2 (Public Review):

The authors applied existing ReadZS and the SpliZ methods, previously developed to analyze RNA process in scRNA-seq data, to Visium data to study spatial splicing and RNA processing events in tissues by Moran's I. The authors showed several example genes in mouse brain and kidney, whose processing are spatially regulated, such as Rps24, Myl6, Gng13.

The paper touches on an important question in RNA biology about how RNA processing is regulated spatially. Both experimental and computational challenges remain to address it. Despite some potentially interesting findings, most claims remain to be validated by orthogonal methods such as RNA FISH and simulations. In addition, the percentage of spatial processing events (splicing in 0.8-2.2% of detected genes, i.e. 8-17 genes and RNA processing in 1.1-5.5% of detected genomic windows, i.e. 57-161 windows) discovered is low. Does it suggest that most of RNA processing events were not spatially regulated across the tissue? Or does it question the assumption of treating spatial transcriptomics data similar to scRNA-seq data? The unique features for ST data, such as mixture of neighboring cells, different capture biases and much smaller number of spots (pseudo cells here), may have significant effects on the power of scRNA-seq based methods, but it is not discussed in the manuscript. The lack of careful evaluation and low discovery rates could limit application of the approach to other tissues and subcellular data.

Reviewer #1 (Public Review):

Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) have shown that PIP5Pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5Pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5Pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5Pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5Pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch". Therefore, the title should be revised.

There are serious concerns about many conclusions made by Touray et al., according to their experimental approaches:<br /> 1. The authors have been studying RAP1's chromatin association pattern by ChIPseq in cells expressing a C-terminal HA tagged RAP1. According to data from tryptag.org, RAP1 with an N-terminal or a C-terminal tag does not seem to have identical subcellular localization patterns, suggesting that adding tags at different positions of RAP1 may affect its function. It is therefore essential to validate that the C-terminally HA-tagged RAP1 still has its essential functions. However, this data is not available in the current study. RAP1 is essential. If RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. In addition, these cells should have the WT VSG expression pattern, and RAP1-HA should still interact with TRF. Without these validations, it is impossible to judge whether the ChIPseq data obtained on RAP1-HA reflect the true chromatin association profile of RAP1.

2. Touray et al. expressed and purified His6-tagged recombinant RAP1 fragments from E. coli and used these recombinant proteins for EMSA analysis: The His6 tag has been used for purifying various recombinant proteins. It is most likely that the His6 tag itself does not convey any DNA binding activities. However, using His6-tagged RAP1 fragments for EMSA analysis has a serious concern. It has been shown that His6-tagged human RAP1 protein can bind dsDNA, but hRAP1 without the His6 tag does not. It is possible that RAP1 proteins in combination with the His6 tag can exhibit certain unnatural DNA binding activities. To be rigorous, the authors need to remove the His6 tag from their recombinant proteins before the in vitro DNA binding analyses are performed. This is a standard procedure for many in vitro assays using recombinant proteins.

3. It is unclear why Nanopore sequencing was used for RNAseq and ChIPseq experiments. The greatest benefit of Nanopore sequencing is that it can sequence long reads, which usually helps with mapping, particularly at genome loci with repetitive sequences. This seems beneficial for RAP1 ChIPseq analysis as RAP1 is expected to bind telomere repeats. However, for ChIPseq, the chromatin needs to be fragmented. Larger DNA fragments from ChIPseq experiments will decrease the accuracy of the final calculated binding sites. Therefore, ChIPseq experiments are not supposed to have long reads to start with, so Nanopore sequencing does not seem to bring any advantage. In addition, compared to Illumina sequencing, Nanopore sequencing usually yields smaller numbers of reads, and the sequencing accuracy rate is lower. The Nanopore sequencing accuracy may be a serious concern in the current study. All telomeres have the perfect TTAGGG repeats, all VSG genes have a very similar 3' UTR, and all 70 bp repeats have very similar sequences. In fact, the active and silent ESs have 90% sequence identity. Are sequence reads accurately mapped to different ESs? How is the sequencing and mapping quality controlled? Furthermore, it is unclear whether the read depth for RNAseq is deep enough.

4. Many statements in the discussion section are speculations without any solid evidence. For example, lines 218 - 219 "likely due to RAP1 conformational changes", no data have been shown to support this at all. In lines 224-226, the authors acknowledged that more experiments are necessary to validate their observations, so it is important for the authors to first validate their findings before they draw any solid conclusions. Importantly, RAP1 has been shown to help compact telomeric and subtelomeric chromatin a long time ago by Pandya et al. (2013. NAR 41:7673), who actually examined the chromatin structure by MNase digestion and FAIRE. The authors should acknowledge previous findings. In addition, the authors need to revise the discussion to clearly indicate what they "speculate" rather than make statements as if it is a solid conclusion.

There are also minor concerns:

1. In the PIP5Pase conditional knockout system, the WT or mutant PIP5Pase with a V5 tag is constitutively expressed from the tubulin array. What's the relative expression level of this allele and the endogenous PIP5Pase? Without a clear knowledge of the mutant expression level, it is hard to conclude whether the mutant has any dominant negative effects or whether the mutant phenotype is simply due to a lower than WT PIP5pase expression level.

2. In EMSA analysis, what are the concentrations of the protein and the probe used in each reaction? The amount of protein used in the binding assay appears to be very high, and this can contribute to the observation that many complexes are stuck in the well. Better quality EMSA data need to be shown to support the authors' claims.

Reviewer #2 (Public Review):

This manuscript by Touray, et al. provides a significant new twist to our understanding of how antigenic variation may be regulated in T. brucei. Key aspects of antigenic variation are the mutually exclusive expression of a single antigen per cell and the periodic switching from expression of one antigen isoform to another. In this manuscript, the authors show, as they have previously shown, that depletion of the nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) results in a loss of mutually exclusive VSG expression. Furthermore, using ChIP-seq, the authors show that the repressor/activator protein 1 (RAP1) binds to regions upstream and downstream of VSG genes located in transcriptionally repressed expression sites and that this binding is lost in the absence of a functional PIP5Pase. Importantly, the authors decided to further investigate this link between PIP5Pase and RAP1, a protein that has previously been implicated in antigenic variation in T. brucei, and found that inactivation of PIP5Pase results in the accumulation of PI(3,4,5)P3 bound to the RAP1 N-terminus and that this binding impairs the ability of RAP1 to bind DNA. Based on these observations, the authors suggest that the levels of PI(3,4,5)P3 may determine the cellular function of RAP1, either by binding upstream of VSG genes and repressing their function, or by not binding DNA and allowing the simultaneous expression of multiple VSG genes in a single parasite.

While I find most of the data presented in this manuscript compelling, there are aspects of Figure 1 that are not clear to me. Based on Figure 1F, the authors claim that transient inactivation of PIP5Pase results in a switch from the expression of one VSG isoform to another. However, I am not exactly sure what the authors are showing in this panel, nor do the data in Figure 1F seem to be consistent with those shown in Figure 1C. Based on Figure 1F, a transient inactivation of PIP5Pase appears to result in an almost exclusive switch to a VSG located in BES12. However, based on Figure 1E, the VSG transcripts most commonly found after a transient inactivation of PIP5Pase are those from the previously active VSG (BES1) and VSGs located on chr 1 and 6 (I believe). The small font and the low resolution make it impossible to infer the location of the expressed VSG genes, nor to confirm that ALL VSG genes located in expression sites are activated, as the authors claim. Also, I was not able to access the raw ChIP-seq and RNA-seq reads. Thus, could not evaluate the quality of the sequencing data.

Reviewer #3 (Public Review):

In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei.

The methods used in the study are rigorous and well-controlled. The authors convincingly demonstrate that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript.

There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome that does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be misassigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

The authors state that "our data imply that antigenic variation is not exclusively stochastic." I am not sure this is true. While I also favor the idea that switching is not exclusively stochastic, evidence for a signaling pathway does not necessarily imply that antigenic variation is not stochastic. This pathway could be important solely for lifecycle-related control of VSG expression, rather than antigenic variation during infection. Nevertheless, these data are critical for establishing a potential pathway that could control antigenic variation and thus represent a fundamental discovery.

Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

Overall, this is an excellent study that represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

Reviewer #1 (Public Review):

Bierman et al. have developed a set of metrics for measuring the spatial patterning of mRNAs in high-throughput fluorescence in situ hybridisation experiments and applied these to identify a subset of mRNAs whose spatial patterning correlates with 3'UTR length. A strength of the study is the clarity and honesty with which the authors have outlined the strengths and weaknesses of their own approach and reported negative results. A key benefit of the tool is that the methodological choices allow wide applicability to existing datasets. However, these choices also feed into a limitation of the method, which is the difficulty in interpreting the biology underpinning the metrics - raising the question of how users will understand the output of the tool.

Reviewer #2 (Public Review):

The authors develop SPRAWL (Subcellular Patterning Ranked Analysis With Labels), a statistical framework to identify cell-type specific subcellular RNA localization from multiplexed imaging datasets. The tool is able to assign to each gene and in each annotated cell type, a score (with a p-value) that measures:<br /> - Peripheral/central localization of RNAs within the cell, based on a previous segmentation step defining cell boundaries and the centroid coordinate.<br /> - Radial/punctuate localization of RNAs within the cell

The method is applied to three multiplexed imaging datasets, identifying defined and cell-type specific patterns for several transcripts.

In the second part of the manuscript, the authors couple SPRAWL with ReadZS, a computational tool developed by the same group and recently published (Meyer et al, 2022). Starting from single-cell datasets, ReadZS is able to quantify 3'UTR length in each cell type. The authors find a subset of genes showing a positive, or negative correlation between the predicted localization and the predicted 3'UTR length across cell types.

Strengths:<br /> As the authors state in the introduction, the study of subcellular RNA localization, with the characterization of organizational principles and of molecular regulation mechanisms, is extremely relevant. The authors develop a strategy to detect statistically significant and non-random patterns of RNA sub-cellular localization in MERFISH and SeqFISH+ datasets, i.e. emerging platforms producing spatially resolved maps of hundreds of transcripts with cellular resolution.

Weaknesses:<br /> Although the method and the presented results have strengths in principle, the main weakness of the paper is that these strengths are not directly demonstrated. That is, insufficient validations are performed to show the biological significance of the results and to fully support the key claims in the manuscript by the data presented.

In particular, the authors imply that their tool is unique and not comparable to any other method. Therefore there is no comparison of SPRAWL with any other method. For example, a comparison could be made with Baysor (Petukhov, V et al. Nat Biotechnol. https://doi.org/10.1038/s41587-021-01044-w). According to the authors, this method is able to identify "small molecular neighbourhoods with stereotypical transcriptional composition" and provides a "General approach for statistical labeling of spatial data".

The authors claim that SPRAWL is able to identify spatial patterns of localization and generated relevant hypotheses to be tested, yet the manuscript contains little proof that the results have biological significance (for example association of RNAs with specific subcellular compartments) and there is no experimental validation for the results obtained applying this method.

The correlation between localization scores and 3'UTR length across cell types for certain genes is also not experimentally validated: results are based on inference from single-cell or imaging data, with no complementary experimental validation.

It is therefore very difficult to assess the biological relevance of the results produced by SPRAWL.

Reviewer #3 (Public Review):

Bierman et al. present a novel statistical framework for examining the subcellular localisation of RNA molecules. Subcellular Patterning Ranked Analysis With Labels, SPRAWL, uses the data available in multiplexed single-cell imaging datasets to assign four metrics of localisation patterns to RNA at a gene per cell level. These easy-to-understand scores, ranging from -1 to 1, can be averaged to detect cell-type specific spatial patterns or used in tandem with tools for RNA 3' UTR length or splicing state to determine the correlation between subcellular localisation and RNA isoforms. Such quantitive association between RNA isoforms and localisation provides a useful tool to determine candidate genes for future studies.

The peripheral and central scores indicate the proximity of RNA molecules to the cell boundary and centre of the cell respectively in relation to other RNA present in the cell. Whilst understanding whether a gene tends to be localised to the cellular membrane is important, it is unclear what biological benefits the central metric gives compared to high "anti-peripheral" scores considering that no single organelle (eg. the nucleus) is located specifically at the centre of the cell in all cell-types.

The punctate and radial patterning scores provide information on the spatial aggregation of RNA molecules of a given gene within a cell. Whilst the punctate score is easy to understand as simply the distance between RNA, the radial score, the angle between RNA, is harder to understand from the main text and would benefit from a schematic showing how this is in respect to the cell-boundary centroid.

Despite endeavouring to create a robust statistical measure of RNA subcellular localisation, this paper is full of inconsistencies. Values (eg. Pearson correlation coefficient values, number of significant genes, number of total genes) and names (eg. cell types, gene names) stated throughout the main text and figures/table do not match repeatedly and without fixing these disparities, the conclusions from this paper are hard to believe.

Reviewer #1 (Public Review):

This study focuses on molecular and cellular mechanisms underlying the sorting of miRNAs into exosomes originating from multivesicular bodies (MVBs). Following up on their previous work, the authors analysed the biochemical basis of miRNA selection by the RNA-binding protein YBX1 which is known to participate in this sorting. Using electrophoretic mobility shift assays (EMSA) involving a series of YBX1 constructs, they pinpointed the key role of the cold shock domain of YBX1 (supported by the C-terminal domain) in miRNA binding. By comparing a secreted model miRNA (miR223), a control cytoplasmic miRNA that is not enriched in exosomes (miR190), and a series of their swap mutants, the authors identified what could be a sequence motif enabling YBX1 to discriminate - through direct binding - between miRNAs to be secreted or to be retained.

The authors then wondered from which subcellular pool miR223 could be mobilised for secretion. They turned their attention to the mitochondria and found evidence of miR223 association with these organelles. Interestingly, when mitochondria were depleted by Parkin overexpression and CCCP treatment, the cellular level of miR223, but not of miR190, increased, whereas its enrichment in extracellular vesicles dropped. This observation permitted to forward a hypothesis whereby mitochondria could be involved in miR223 mobilisation into exosomes. This process would be mediated by YBX1 which shuttles between mitochondria and endosomes, as was elegantly shown in live imaging experiments.

Finally, the authors provide initial data implicating in this process the mitochondrial matrix protein YBAP1, broadly known as C1QBP, or p32. YBAP1 was found to interact with YBX1 and miR223 in pull-down assays. Moreover, direct and moderately strong miR223 binding by YBAP1 was confirmed by EMSA. Interestingly, just like YBX1, YBAP1 seems to prefer this substrate over miR190, indicating certain binding specificity. The observation that YBAP1 knockout resulted in the decreased association of miR223 with mitochondria, paralleled by its correspondingly better mobilisation into exosomes, enabled the authors to propose that YBAP1 could negatively control miR223 secretion at the level of mitochondria.

Strengths

This is a very interesting study proposing an elegant hypothesis and featuring a creative panel of methods, many of which will certainly be of interest to biochemists and cell biologists working with extracellular RNA and mitochondria (e.g. the Parkin/CCCP-mediated mitochondria depletion and the time-lapse imaging of RNA-binding proteins against cellular organelles).

The authors did a good job of dissecting the YBX1 interaction with miR223 versus miR190. These experiments are performed at a high technical level, and their interpretation is straightforward and convincing. The nearly two orders of magnitude difference in affinity provides a plausible means by which YBX1 could recognise and funnel one, but not the other, miRNA into the secretion pathway.<br /> Another valuable piece of data is related to YBAP1. This important, deeply conserved protein, strongly implicated in severe mitochondrial diseases and cancer, remains poorly understood at the level of basic molecular mechanisms, and even its subcellular localisation is debated. The data presented by the authors reinforce the idea of its primarily mitochondrial localisation, in agreement with earlier studies. They also provided new information about the RNA-binding activity of YBAP1. First proposed to interact with RNA by Yagi et al., Nucleic Acids Res 2012 (doi:10.1093/nar/gks774), YBAP1 is confirmed in the present study as a reasonably affine RNA-binding protein, based on direct EMSA experiments involving a highly purified protein and natural RNAs. These data should encourage the community to explore the full RNA-binding potential of YBAP1/C1QBP/p32 in a wider variety of models, especially in the context of mitochondrial gene expression.

Weaknesses

While the authors might be right about the existence of a sequence motif that specifies miRNAs for exosome sorting by YBX1, it is at present difficult to disentangle the sequence and structure contributions to YBX1 binding within the variants described in the paper. RNA structure predictions, however imperfect, suggest that miR223-3p is a fully single-stranded transcript (ensemble ΔG = -0.33 kcal/mol, RNAfold), while miR190-5p is a tightly base-paired one (ensemble ΔG = -2.85 kcal/mol). This likely explains the differential affinity to YBX1, known to strongly prefer single-stranded RNAs. When mutating the putative sorting motif in miR223 (UCAGU>AGACA), the authors introduced some amount of secondary structure (ΔG = -1.04 kcal/mol), which could have impeded YBX1 binding. By contrast, the mutation of miR190 (AUAUG>UCAGU) significantly weakened the structure (ensemble ΔG = -2.21 kcal/mol), which might explain the improvement in YBX1 interaction.

Mitochondria appear to be a plausible location for mobilisable RNAs, given their multiple associations with ribosomes, RNA-containing condensates, and other organelles. However, the presented evidence of the mitochondrial localisation of miR223 is limited. The colocalisation pattern of the ATTO 647-labelled miR223 with the well-behaved mitochondrial marker Tom22 is remarkable; such a neat overlap has so far only been observed for some abundant mtDNA-encoded transcripts, but not for an extraneous transcript. The interpretation of this result will depend a great deal on experimental details which, unfortunately, are missing for this section. ATTO 647N is known to be quantitatively recruited to mitochondria, producing just the same kind of complete colocalisation, making it a perfect tool to visualise mitochondria in the cell (Han et al., Nat Commun 2017, doi:10.1038/s41467-017-01503-6). There is a worry that the colocalisation observed here might have been driven by the dye alone.

Furthermore, the definition of the topology of RNA localisation with respect to the mitochondrial membranes remains challenging, and a number of more robust methods have been recently proposed to address this contentious issue. At minima, one would expect that the authors would use RNase treatment, with or without Triton X-100 (like they did in the in vitro packaging assay), to see whether miR223 is indeed protected by the mitochondrial membranes and, therefore, resides in the interior of the organelles. As for now, based on the presented data, one can safely conclude that miR223 is associated with the mitochondria, without claiming that it is necessary inside them.

The Parkin/CCCP method is very powerful, which is its strength and weakness at the same time. miR223 secretion does decrease when the mitochondria are depleted. However, it is unclear how direct and specific this effect is. The destruction of mitochondria likely crashed the cellular ATP levels, which could have generally affected vesicular transport, not only miR223 sorting. A more detailed analysis of the overall abundance of extracellular vesicles and their cargo under these conditions could reveal the true scope of the mitochondrial contribution to RNA secretion.

YBAP1 is a difficult, indeed "treacherous", protein to work with. Its strong negative charge (pI = 4) makes it easily stick to positively charged proteins, such as YBX1 (pI = 9.9). Such interactions are routinely observed in pulldown assays from cell lysates, where all components are intermixed (but often cannot be corroborated by in situ or in vivo approaches). The authors carefully showed that YBX1 and YBAP1 do not significantly colocalise in the cell, which makes the interplay between the two proteins in miR223 sorting difficult to stage. They also studied the miR223 distribution between mitochondria and extracellular vesicles using YBAP1 knockout cells. However, such cells are known to be very sick and have an extremely pleiotropic mitochondrial and metabolic phenotype. Therefore, the apparent implication of YBAP1 in miR223 sorting might be less direct than currently envisaged.

Reviewer #2 (Public Review):

The manuscript by Ma et al, "Two RNA-binding proteins mediate the sorting of miR223 from mitochondria into exosomes" examines the contribution of two RNA-binding proteins on the exosomal loading of miR223. The authors conclude that YBX1 and YBAP1 work in tandem to traffic and load miR223 into the exosome. The manuscript is interesting and potentially impactful. It proposes the following scenario regarding the exosomal loading of miR223: (1) YBAP1 sequesters miR223 in the mitochondria, (2) YBAP1 then transfers miR223 to YBX1, and (3) YBX1 then delivers miR223 into the early endosome for eventual secretion within an exosome. While the authors propose plausible explanations for this phenomenon, they do not specifically test them and no mechanism by which miR223 is shuttled between YBAP1 and YBX1, and the exosome is shown. Thus, the paper is missing critical mechanistic experiments that could have readily tested the speculative conclusions that it makes.

Comments:<br /> 1. The major limitation of this paper is that it fails to explore the mechanism of any of the major changes it describes. For example, the authors propose that miR223 shuttles from mitochondrially localized YBAP1 to P-body-associated YBX1 to the exosome. This needs to be tested directly and could be easily addressed by showing a transfer of miR223 from YBAP1 to YBX1 to the exosome.<br /> 2. If YBAP1 retains miR223 in mitochondria, what is the trigger for YBAP1 to release it and pass it off to YBX1? The authors speculate in their discussion that sequestration of mito-miR223 plays a "role in some structural or regulatory process, perhaps essential for mitochondrial homeostasis, controlled by the selective extraction of unwanted miRNA into RNA granules and further by secretion in exosomes...". This is readily testable by altering mitochondria dynamics and/or integrity.<br /> 3. Much of the miRNA RT-PCR analysis is presented as a ratio of exosomal/cellular. This particular analysis assumes that cellular miRNA is unaffected by treatments. For example, Figure 1a shows that the presence of exosomal miR223 is significantly reduced when YBX1 is knocked out. This analysis does not consider the possibility that YBX1-KO alters (up or down-regulates) intracellular miR223 levels. Should that be the case, the ratiometric analysis is greatly skewed by intracellular miRNA changes. It would be better to not only show the intracellular levels of the miRs but also normalize the miRNA levels to the total amount of RNA isolated or an irrelevant/unchanged miRNA.<br /> 4. In figure 1, the authors show that in YBX1-KO cells, miR223 levels are decreased in the exosome. They further suggest this is because YBX1 binds with high affinity to miR223. This binding is compared to miR190 which the authors state is not enriched in the exosome. However, no data showing that miR190 is not present in the exosome is shown. A figure showing the amount of cellular and exosomal miR223 and 190 should be shown together on the same graph.<br /> 5. Figure 2 Supplement 1 - As to determine the nucleotides responsible for interacting with YBX1, the authors made several mutations within the miR223 sequence. However, no explanation is given regarding the mutant sequences used or what the ratios mean. Mutant sequences need to be included. How do the authors conclude that UCAGU is important when the locations of the mutations are unclear? Also, the interpretation of this data would benefit from a binding affinity curve as shown in Fig 2C.<br /> 6. While the binding of miR223mut to YBX1 is reduced, there is still significant binding. Does this mean that the 5nt binding motif is not exact? Do the authors know if there are multiple nucleotide possibilities at these positions that could facilitate binding? Perhaps confirming binding "in vivo" via RIP assay would further solidify the UCAGU motif as critical for binding to YBX1.<br /> 7. Figures 2g, h - It would be nice to show that miR190mut also packages in the cell-free system. This would confirm that the sequence is responsible. Also, to confirm that the sorting of miR223 is YBX1-dependent, a cell-free reaction using cytosol and membranes from YBX1 KO cells is needed.<br /> 8. In Figure 3a, the authors show that miR223 is mitochondrially localized. Does the sequence of miR223 (WT or Mut) matter for localization? Does it matter for shuttling between YBAP1 and YBX1?<br /> 9. Supplement 3c - Is it strange that miR190 is not localized to any particular compartment? Is miR190 present ubiquitously and equally among all intracellular compartments?<br /> 10. Figure 3h - Why would the miR223 levels increase if you remove mitochondria? Does CCCP also cause miR223 upregulation? I would have thought miR223 would just be mis-localized to the cytosol.<br /> 11. Figure 3i - What is the meaning of "Urd" in the figure label? This isn't mentioned anywhere.<br /> 12. Figure 3j - The data is presented as a ratio of EV/cell. Again, this inaccurately represents the amount of miR223 in the EV. This issue is apparent when looking at Figures 3h and 3j. In 3h, CCCP causes an upregulation of intracellular miR223. As such, the presumed decrease in EV miR233 after CCCP (3j) could be an artifact due to increased levels of intracellular miR223. Both intracellular and EV levels of miRs need to be shown.<br /> 13. In Figure 4, the authors show that when overexpressed, YBX1 will pulldown YBAP1. Can the authors comment as to why none of the earlier purifications show this finding (Figure 1 for example)? Even more curious is that when YBAP1 is purified, YBX1 does not co-purify (Figure 4 supplement 1a, b).<br /> 14. Figure 4f, g - The text associated with these figures is very confusing, as is the labeling for the input. Also, what is "miR223 Fold change" in this regard? Seeing as your IgG should not have IP'd anything, normalizing to IgG can amplify noise. As such, RIP assays are typically presented as % input or fold enrichment.<br /> 15. Figure 4h - The authors show binding between miR223 and YBAP1 however it is not clear how significant this binding is. There is more than a 30-fold difference in binding affinity between miR223 and YBX1 than between miR223 and YBAP1. Even more, when comparing the EMSAs and fraction bound from figures 1 and 2 to those of Figure 4h, the binding between miR223 and YBAP1 more closely resembles that of miR190 and YBX1, which the authors state is a non-binder of YBX1. The authors will need to reconcile these discrepancies.<br /> 16. Can the authors present the Kd values for EMSA data?<br /> 17. Figure 5 - Does YBAP1-KO affect mitochondrial protein integrity or numbers?<br /> 18. Figure 6a - Are the authors using YBAP1 as their mitochondrial marker? Please include TOM20 and/or 22.<br /> 19. Figure 6b - Rab5 is an early endosome marker and may not fully represent the organelles that become MVBs. Co-localization at this point does not suggest that associating proteins will be present in the exosome, and it is possible that the authors are looking at the precursor of a recycling endosome. Even more, exosome loading does not occur at the early endosome, but instead at the MVB. Perhaps looking at markers of the late endosome such as Rab7 or ideally markers of the MVB such as M6P or CD63 would help draw an association between YBX1, YBAP1, and the exosome. Also, If the authors want to make the claim that interactions at the early endosome leads to secretion as an exosome, the authors should show that isolated EVs from Rab5Q79L-expressing cells contain miR223.<br /> 20. The mentioning of P-bodies is interesting but at no time is an association addressed. This is therefore an overly speculative conclusion. Either show an association or leave this out of the manuscript.<br /> 21. In lines 55-58, the authors make the comment "However, many of these studies used sedimentation at ~100,000 g to collect EVs, which may also collect RNP particles not enclosed within membranes which complicates the interpretation of these data." Do RNPs not dissolve when secreted? Can the authors give a reference for this statement?

Reviewer #3 (Public Review):

The article by Ma et al pursues the previous work of the Schekman group, exploring the mechanisms of targeting of miRNAs into extracellular vesicles (EVs), or possibly exosomes, in HEK293 and U2OS cells. The authors had identified YBX1 as an RNA-binding protein required for the sorting of miR223 into CD63-expressing small EVs, probably mainly exosomes. Here they further observed that YBX1 directly binds miR223, which also binds to another protein, YBAP1, localized in mitochondria, where it sequesters miR223, thus preventing its targeting to MVBs' intraluminal vesicles. They observe the association of YBX1-containing P-bodies in the cytoplasm with mitochondria and with enlarged Rab5-endosomes and propose that this step is required for the exchange of miR223 for its loading into MVBs intraluminal vesicles and future exosomes.

The biochemical parts of the article, with quantitative experiments to decipher the molecular interactions of YBX1 and YBAP1 with miR223, are nicely performed and convincing. By contrast, the parts on the involvement of YBX1 and of YBAP1 in the release of miR223 in EVs or exosomes are more correlative than demonstrative and lack some controls. In particular, it is far-fetched to conclude from the observed movement (which may be serendipitous) of 2 P-bodies between mitochondria and enlarged endosomes (without any visualization of the miR) that this movement may be instrumental in the transfer of miR223 between mitochondria and putative exosomes (figures 6 and model in figure 7).

The experiments designed to evidence the mechanisms of miR223 release in EVs are also not sufficiently controlled and analysed to really support the interpretations. And the EV isolation steps are not performed in a way that supports the actual exosomal nature (i.e. exclusive origin from multivesicular endosome) of the EV analysed.

Another experimental weakness is that the authors make strong conclusions on MVBs and exosomes when they only analyse artificially-enlarged endosomes induced by overexpression of mutant Rab5. Although this approach has been used previously and shown CD63 in these induced enlarged compartments, it is an artificial blocking of normal endosomal trafficking, and may not reflect the situation of intracellular trafficking of miR223 in normal cells.

Reviewer #1 (Public Review):

This study is based on the hypothesis that tumor treating fields, a form of cancer therapy that exposes tumors to alternating electrical fields, has an effect on tunneling microtubes, fine actin-rich protrusions that connect cancer cells and allow intercellular communication, contributing to the tumor microenvironment and therapeutic resistance. This is an interesting hypothesis and may be of importance. To prove their hypothesis better data presentation and mechanistic studies are needed, as it is not clear based on this study how the proposed effect is working.

Reviewer #2 (Public Review):

The authors tested TTFields' effect on TNT formation in two mesothelioma cell lines, MSTO-211H and VMAT. The MSTO-211H is a biphasic cell line with epithelioid and sarcomatoid features while VMAT only has sarcomatoid morphology. They treated their cell lines at 150 or 200 kHz either unidirectionally or bidirectionally. The experiments took place within 72 hours of plating, after which the cells will become confluent on coverslips and their TNT formation drops.

Under these experimental conditions, they found: (i) Unidirectional is more effective than bidirectional TTFields in reducing TNT formation, (ii) TNT formation was markedly reduced after 48 hours of treatment in MSTO-211H but not VMAT cells, (iii) no difference in actin polymerization or actin filament bundling after one hour of TTFields treatment, (iv) reduced TNT formation when TTFields were combined with cisplatin but not with both cisplatin and pemetrexed, (v) analysis TNT cargo transport using markers of gondolas and mitochondria did not show changes in transport velocity, and (vi) in vivo spatial transcriptomic analysis revealed EMT markers and immunogenic markers.

Reviewer #3 (Public Review):

Sarkari et al. describe the effects of TTFields on inter-cellular communication structures called tunneling nanotubes in malignant pleural mesothelioma cells. Recent studies have implicated these F-actin-based nanotubes in promoting malignant transformation and biology by allowing long-range communications between malignant cells. The authors suggest that TTFields disrupt these structures by impacting the expression of genes involved in nanotube formation and cell proliferation. Although TTFields are thought to affect tubulin-based structures, recent studies suggest that TTFields also impact actin-based structures. Therefore, the authors' findings are in keeping with this new understanding. They also found that TTFields upregulated marker genes in immunity. This is one of the first studies that implicate TTFields in these tunneling nanotube structures. Overall, the study adds to our understanding of TTFields on various cellular structures. However, conclusions are only partially supported by the data presented. The study is largely descriptive and there are many areas that need to be addressed to substantively improve the premise and rigors and strengthen the conclusions.

Reviewer #1 (Public Review):

In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

Reviewer #2 (Public Review):

I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition.<br /> The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes. Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza). Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)? Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.

Reviewer #3 (Public Review):

The authors investigate the potential effect of OGlcNacylation on the activity of the DNA methyltransferase DNMT1.

Some results that are convincingly obtained include:<br /> - There is more overall OGlcNacylation when Glucose concentration in the culture medium or the feed is high;<br /> - DNMT1 is OGlcNacylated, and more so in high glucose or on rich chow;<br /> - The position S878 can be OGlcNacylated;<br /> - The activity of transfected DNMT1 is decreased in high glucose conditions. This effect is lessened when S878 is mutated to A or D.

Some results that are suggested but not fully backed by experimental data include:<br /> - This process happens to the endogenous protein under physiologically relevant conditions;<br /> - This process is responsible for changes in DNA methylation, leading to changes in gene expression, leading to increased ROS and increased apoptosis.

Studying the connection between cellular metabolism and epigenetic phenomena is interesting. However, I feel that the article falls short of its aims because of the limits of the experimental system, some missing controls, and some data overinterpretation.

Reviewer #1 (Public Review):

Overall, this manuscript exposes key gaps in patient care resulting from the pandemic, as well as the challenges and unmet needs felt by healthcare workers in cervical cancer screening. The authors' findings on the struggles while regaining screening volume across the nation in a sustainable way, demonstrate that pre-existing weaknesses in the cancer control system were exacerbated by the pandemic and are integral to amend. The authors were able to identify these gaps in care and work environments through their synthesis of qualitative interviews. I applaud the use of such mixed methods, which emphasizes the complementary need for both quantitative and qualitative data. What could be better strengthened in the manuscript is the authors' justification for statistical analyses within the context of the research question, and reporting of survey administration and management.

Reviewer #2 (Public Review):

Fuzzell et al. conducted a mixed-method study looking into the possible impact of COVID-19 on clinician perceptions of cervical cancer screening. The authors examined how the pandemic-related staffing changes might have affected the screening and abnormal results follow-up during the period October 2021 through July 2022.

They found that 80% of the clinicians experienced decreased screening during the start of the pandemic and that ≈67% reported a return to pre-pandemic levels. The general barriers for not returning to pre-pandemic levels were staffing shortages and problems with structural systems for tracking overdue patients and those in need of follow-up after abnormal screening tests.

Strengths:

There is a high focus on the consequences and the need for action to prevent the ongoing impact of COVID-19 on cervical cancer screening. Some of the actions mentioned by the authors could be the use of HPV self-sampling kits, and it is interesting to be provided knowledge on the clinicians' views on HPV self-sampling. Both are of high interest to the general population in the US. Throughout the discussion, the authors and their claims are supported by other studies.

Weaknesses:

The lack of a National representative sample, where 63% of the responding clinicians were practicing in the Northeast, affects the possibility of generalization of the results found in the study. The overrepresentation of white females is not addressed in the discussion. This composition could have affected the results, especially when the authors report a need to look at higher salaries and better childcare to maintain adequate staffing.

The conclusions are mostly supported by the data, however, some aspects of the data analysis need to be clarified.

Reviewer #3 (Public Review):

This US study presents findings from an online survey and in-person interviews of healthcare providers regarding themes associated with cervical screening in federally qualified health centres (FQHCs). The study provides insights during the post-acute phase of the pandemic into a range of areas, including perceived changes in the provision of cervical cancer screening services and the impact of the pandemic, staffing and systems barriers to cervical cancer screening, strategies for tracking missed screens and catch-ups, follow-up of abnormal screening results, as well as attitudes towards HPV self-sampling. Results indicate persisting pandemic-related impacts on patient engagement and staffing, as well as system barriers to effective screening, catch-up of missed screens and follow-ups. Taken together, these issues may lead to increases in cervical cancer in the long-term in populations serviced by these centres, if measures are not taken to adequately support them. Participants were recruited from various regions in the US, however, the study was not conducted using a nationally-representative sample. Although highlighted issues are informative, findings cannot be generalised and larger studies are warranted in the future to monitor cervical screening provision and outcomes in FQHCs.

Reviewer #1 (Public Review):

Flowers et al. studied requirements for the persistence and clearance of mutant mtDNA in C. elegans using the uaDf5 deletion in mtDNA. This mutant mtDNA persists at relatively constant levels, despite clearly having detrimental effects. Surprisingly, no mutations were found in the existing wt copies, which would have otherwise explained the persistence of mutant DNA by complementation. The authors then investigated the potential role of programmed cell death in the removal of mutant mtDNA from the germline using crosses with existing cell death mutants. They observed increased amounts of uaDf5 DNA in 1 day old progeny in strains with mutations in the caspases ced-3 and csp-1 and in several other cell death genes, showing that a significant amount of uaDF5 is removed by PCD in the germline. The authors also observed increased uaDf5 over time in the germline, and effects lifespan mutations on the mount of uaDf5. This was true both for the insulin signaling pathway and the clk-1 pathway, suggesting that both pathways regulate uaDf5 levels consistent with the connection between longevity and mitochondrial homeostasis. Finally, the authors discuss results showing that PCD mutants with high amounts of uaDf5 in the germline, have surprisingly low amounts of uaDf5 in their progeny, which would suggest that PCD can be replaced by another clearance method.

This manuscript is of general interest because it demonstrates the importance of PCD for clearance of mutant mtDNA. The evidence for this mechanism of removal is strong. The effects of the aging mutants are more difficult to understand and the discussion of these effects is therefore somewhat speculative.

Reviewer #2 (Public Review):

In this study, the authors sought to elucidate regulators of mitochondrial DNA (mtDNA) quality control in the germline. To this end, the authors used Caenorhabditis elegans as a model organism and 3.1kb mtDNA deletion mutation uaDf5 that is stably transmitted across generations. The key data presented were the heteroplasmy level of mtDNA, specifically the molar ratio of mutant vs. wildtype (WT) mtDNA molecules, at different ages. The authors specifically focused on the role of programmed cell death (PCD) signaling and a few well-known aging pathways in C. elegans. The data showed that attenuation of PCD has the general effect of increasing the steady-state mutant-to-WT ratio, while increasing PCD does not reduce this ratio. The data also showed that this mutant-to-WT ratio increases with age, an effect that is transmitted to progenies, and that perturbations to well-known insulin signaling and CLK-1 aging pathways affect the rate of this increase, where a longer lifespan is correlated with a slower increase. Finally, the data demonstrated an intergenerational reduction in mutant-to-WT ratio and that the degree of this reduction has a nonlinear ultrasensitive-like dependence on the ratio.

A strength of the study is the comprehensive exploration of the role of key molecules of the PCD machinery in mtDNA quality control in the germline. Also, the data on the effects of age and aging pathways on the maintenance of mtDNA quality in the germline, as well as on intergenerational mtDNA quality control, are extremely interesting and have the potential to trigger transformative studies that connect mtDNA purifying selection and aging.

A major weakness of the study is that the key findings are predominantly based on data of the mutant-to-WT mtDNA ratio. But, a higher mutant-to-WT ratio does not necessarily equate to an increase/accumulation of mutant mtDNA in the cell population, since the same increase can also be caused by a decrease in WT mtDNA. No data for copy numbers of WT and mutant mtDNA or their proxies were analyzed. As a consequence, some of the major findings, such as the non-canonical/non-apoptotic role of PCD machinery in mediating mitochondrial purifying selection and the accumulation of mutant mtDNA with age, cannot be uniquely concluded from the data. Alternative explanations could be given to explain the observed trends of mutant-to-WT ratios.

Another weakness is that the connection between the two pathways in this study: PCD and aging, in regulating mtDNA quality control was not more deeply explored. The study did not delve into how the interplay of aging and PCD if any, affects mtDNA quality control in the germline.

Finally, as the authors noted, the important role of stochasticity in purifying selection against pathogenic mtDNA is established. Yet, this aspect of purifying selection is not explored in this study (e.g., how such stochasticity is working with PCD in mtDNA quality control in the germline), nor it is accounted for in the analysis of the data and the discussion of the observation.

Reviewer #1 (Public Review):

The work presented here uses a large collection of PET data to discover the principle axes of neurotransmitter receptor/transporter molecule (NTRM) variation in the human cortex and subcortex. These spatial axes are then systematically annotated for their alignment with diverse other measures of brain organization. The work is valuable for providing a systematic description and annotation of a new "layer" of brain organization that has been relatively poorly integrated with the wider neuroimaging literature to date. The methods used are state-of-the-art and the findings generated by these methods are sound. The discovered NTRM gradients will allow others in the field to more easily incorporate information of neurotransmitter maps in their analyses - helping to advance integration between different views of the human brain. A fundamental challenge to this goal of cross-modal integration, however - which doesn't just impact this work, but the field more broadly - is that we are often left to work with spatial correlations between modalities in humans. The lack of access to experimental methods means that the biological basis for observed spatial correlations between different brain features in humans is typically poorly understood. It is therefore hard to know what newly-reported spatial correlations are telling us about brain organization that was not already captured in prior work. Nevertheless, the new resources and results presented here are important because they can guide the future work needed to unpick the biology behind spatially correlated features of the human brain

Reviewer #2 (Public Review):

In this work, Hänisch and colleagues investigate the relationship between neurotransmitter transporter and receptor's spatial heterogeneity and well-studied functional and structural brain gradients in the human brain. They calculate the spatial similarity between the distribution of the neurotransmitter transporters and receptors for each parcel, thus obtaining a new brain distribution comprising a similarity index of all neurotransmitters mapped to each brain area. They employ a nonlinear dimensionality reduction on this neurotransmitter similarity map to reveal three spatial gradients for cortical and subcortical levels, respectively. Based on this, they characterize their significance by comparing them with functional fMRI meta-analytic activations, MRI microstructure, architectural contextualization, MRI-based structural and functional connectivity, and gray matter atrophy-derived disease maps.

The claim of the work is broad, and the motivation is general, but the data presented is specific and biologically diverse. The neurotransmitter system operates at different pre- and post-synaptic synaptic levels, and the general assumption that transporters are equivalent to receptors lacks appropriate discussion for supporting this claim. The motivations of the work are very broad, and the analysis used is sufficient for the general claims, but the data presented is specific and biologically diverse.

Besides these conceptual issues, I find this work interesting as it jointly characterizes the cortical and subcortical PET neurotransmitter's distribution maps and their structural and functional meaning for the first time. In essence, the study presents several arguments to consider the organization of the characterized maps as an additional layer of brain organization. The results are convincing and clearly presented. Although this is a correlative study using unconnected datasets, I appreciate the use of multiple brain maps. I also appreciate that the authors made the data and code available for reproducibility. The data and analysis used in the current draft enable a powerful set of tools for hypothesis testing in the human brain's natural distribution of neurotransmitters beyond the usual pharmacological intervention strategy traditionally used in neurotransmitters' brain mapping area.

Reviewer #1 (Public Review):

The aim of this study was to evaluate the increased prevalence of mental health (MH) disorders such as depression, anxiety, eating disorders, and postpartum depression in patients with polycystic ovary syndrome (PCOS) the most common reproductive disorder affecting about one in seven reproductive-aged women worldwide. The consequences of excess economic burden were estimated.

Meta-analyses were performed using the Der Simonian-Laird random-effects model to compute pooled estimates of prevalence ratios for the associations between PCOS and these MH disorders, and then the excess direct costs in U.S. dollars (USD) for women suffering from PCOS were estimated.

After screening the articles by title/abstract, 25 articles were selected for their quality according to the Newcastle-Ottawa scale. These studies included a control group. The data showed an increase in the prevalence ratios for each of the selected mental health disorder items: anxiety 1.42, depression 1.65, and eating disorders 1.48. The additional direct health care costs associated with these disorders were estimated to be $4.261 billion per year in 2021 USD.

The authors extended their previous report that the total cost of evaluating and providing care to reproductive-aged PCOS women in the United States was $4.36 billion. Interestingly, the cost for diagnostic evaluation including laboratory accounted for a relatively minor part of the total costs (approximately 2%). In the present study, mental health disorders were clearly identified as a part of the excess economic burden. Their cost is estimated at $4.261 billion/year. These results were not anticipated intuitively and are of value for prioritization of the disorder as a public health priority.

Provided that the study is validated for extraction of a meta-analysis, the data are of great interest not only for economic issues but also for early consideration of the mental distress of PCOS patients that has long been underestimated. Several studies have expressed patient resentment of delayed diagnosis and imperfect management, including the physical damage of hyperandrogenism and the associated metabolic syndrome. This medico-economic approach to chronic diseases with a strong impact on quality of life contributes to the global management of PCOS, which is a primary demand of patients.

Reviewer #2 (Public Review):

Yadav et al have performed a careful systematic review and meta-analysis of mental health disorder prevalence ratios in PCOS to estimate the mental health-related excess economic burden associated with this common endocrine disorder. Using random effect modelling of prevalence ratios from quality-assessed, peer-reviewed publications, they determine the excess PCOS-related prevalence and healthcare costs associated with anxiety, depression, and eating disorders to be greater than $4 billion USD per year. In conjunction with previously reported direct economic burden estimates for PCOS, they determine that PCOS healthcare costs exceed $15 billion USD per year (in the US alone) and that mental health disorder-related costs account for nearly one-third of these costs. The findings of this paper will be impactful for a broad field of clinical and bench scientists investigating PCOS, endocrinologists, general practitioners, health economists, and policymakers. The findings of this paper demonstrate the significant contribution that mental health-related pathology makes to the total economic burden associated with PCOS and present a strong case for additional research and policy investment into this underfunded area.

The important findings and claims presented in this paper are mostly clearly presented and well supported by strong evidence and careful analysis. However, some additional clarity and rationalisation of referenced healthcare cost input to the model would strengthen the conclusions.

Strengths:<br /> This paper clearly describes the inclusion criteria and characteristics of the included studies. The papers included were quality assessed using a well-regarded assessment tool and only those with high-quality information were included in subsequent meta-analyses. Publication bias was assessed by multiple methods and data were interpreted accordingly.

The authors combine their mental health-related findings with previously reported economic burden estimates for specific PCOS-related care and treatment to provide a comprehensive estimation of PCOS-related healthcare costs in the US. They discuss these findings in relation to healthcare-related costs reported for other prevalent disorders and make a compelling case for prioritising research and investment into PCOS.

An important observation made by the authors is the relatively small contribution to PCOS economic burden made by diagnostic evaluation, supporting quality diagnosis and evaluation as a cost-effective measure to improve PCOS patient treatment.

Weaknesses:<br /> The systematic review includes data from some studies where PCOS is self-reported. While self-reported PCOS information has been found to be largely sensitive and specific, it would be of interest to know if prevalence ratios of mental health-related were impacted by self-reporting. Likewise, the screening vs self-reported nature of the mental health disorders is not clear from the information included in the characteristics table.

Calculated prevalence ratios were compared with prevalence values for the general population to determine the excess prevalence. However, the source of these general population statistics (i.e., whether these figures come from the control data in the included studies or other sources) is not clear. The estimated costs for anxiety-, depression- and eating disorder-related care are accessed in published papers and used to calculate the excess costs. Conclusions would be strengthened by a defence of these figures, particularly for anxiety where the source paper is from 1999. An inflation tool is used to adjust the figure, but this does not take into account changes in treatment or practice since this estimate was made. The accuracy of these estimated figures is central to the final conclusions.

Reviewer #1 (Public Review):

GSK3 is a multi-tasking kinase that recognises primed (i.e. phosphorylated) substrates. One of the mechanisms by which the activity of GSK3 can be regulated is through N-terminal (pSer9) phosphorylation. In this case, the phosphorylated N-terminus turns into a pseudo-substrate that occupies the substrate binding pocket and thus inhibits the activity of GSK3 towards its real substrates.

One outstanding question is how this autoinhibitory mechanism can affect some, but not all signaling pathways that GSK3 is involved in. One example is WNT/CTNNB1 signaling. Here, GSK3 plays a central role in the turnover of CTNNB1 in the absence of WNT, but this pool of GSK3 is not affected by pSer9 phosphorylation.

Gavagan et al. address this question using an in vitro approach with purified proteins. They identify a role for AXIN1 in protecting the "WNT signaling pool" of GSK3 from the auto-inhibition that occurs upon pSer9 phosphorylation.<br /> Specifically, they show that i) GSK3-pSer9 is less capable of binding and phosphorylating primed CTNNB1 - thus suggesting that GSK3-pSer9 does not contribute to WNT signaling, ii) in the presence of AXIN1, GSK3-pSer9 becomes more capable of binding and phosphorylating CTNNB1 - suggesting that Axin can promote binding of GSK3 and CTNNB1 even when the primed binding pocket on GSK3 is blocked initially, iii) AXIN1 specifically prevents the PKA mediated phosphorylation of GSK3B on pSer9 - while leaving the phosphorylation of other PKA substrates unaffected.

Strengths:<br /> - The authors use an in vitro system in which they can reconstitute different interactions and reactions using purified proteins, thus allowing them to zoom in on specific biochemical events in isolation.<br /> - The authors measure the phosphorylation of primed substrates (pSer45-CTNNB1 or WNT-independent substrates) and quantify specific kinetic parameters (kcat, KM, and kcat/KM) - of wildtype non-phosphorylated GSK3B, pSer9GSK3B, or the non-phosphorylatable S9A-GSK3B, either in the presence or absence of AXIN1 (or an AXIN1 fragment).<br /> - The experiments appear to be well-controlled and the results appear to be interpreted correctly.

Weaknesses:<br /> - Key experiments (e.g. Figures 2 and 3) are described as being performed as n=3 technical replicates rather than independent/biological replicates.<br /> - The validation in a biologically relevant setting (i.e. a cellular context) is limited to Figure 4C, which shows that over-expression of AXIN1 reduces the total levels of pSer9-GSK3.

The authors convincingly show that AXIN1 can play a role in shielding GSK3 from auto-inhibition. As it stands, the impact of this work on the field of WNT/CTNNB1 signaling is likely to remain limited. This is mainly due to the reason that the mechanism by which AXIN1 shields the WNT/CTNNB1 signaling pool of GSK3 from pSer9 inhibition remains unresolved. Based on the fact that a mini AXIN1 (i.e. an AXIN1 fragment) behaves the same as WT AXIN1, the authors conclude that AXIN1 likely causes allosteric changes on GSK3 but is less likely to block PKA from binding. They cannot conclusively show this, however, as they do not have evidence in favour of one or the other explanation.

However, this study does offer more insight into the compartmentalisation of GSK3 and the quantitative parameters may be used in computational models describing the different cellular activities of GSK3.

This work also has conceptual significance: Scaffold proteins are known to promote signal transduction by bringing proteins together (often: kinases and substrates). Here, Gavagan et al. show that AXIN1 also plays a second role, namely in protecting one of its binding kinases (GSK3) from inhibitory signals. This could potentially hold for other scaffolding proteins as well.

Reviewer #2 (Public Review):

Gavagan et al. investigated the role of the scaffolding protein, Axin, in the cross-pathway inhibition of GSK3b. The authors utilize reconstituted Axin, b-catenin, GSK3b, and protein kinase A to test 2 models. In the first model, the formation of the complex consisting of Axin, b-catenin, and GSK3b overcomes inhibitory phosphorylation of serine 9 of GSK3b. In the second model, the binding of Axin to GSK3b inhibits serine 9 phosphorylation through allosteric effects.

Previous literature has established that the phosphorylation of serine 9 of GSK3b inhibits its kinase activity. To provide a quantitative measure of inhibition, the authors determine the binding affinity and catalytic efficiency of GSK3b in comparison to GSK3b phosphoS9 towards b-catenin. Interestingly, the data demonstrate a 200-fold decrease in Kcat/Km and 7 fold increase in Km. It is unclear why serine 9 mutation to alanine increases the rate of B-catenin phosphorylation more than the GSK unphosphorylated protein in figure S10. Next, the authors tested if the addition of Axin could overcome this inhibition. Although the addition of Axin decreases the Km, thereby producing a 20-fold increase in catalytic efficiency, the addition of Axin does not rescue the catalytic turnover of the phosphorylated GSK3b. Hence, the authors propose that Axin does not rescue the kinase activity of GSK3b from the inhibitory effects of serine 9 phosphorylation.

Next, the authors test if Axin protects GSK3b from phosphorylation by the upstream kinase PKA. Excitingly, the data show a decrease in binding affinity and catalytic efficiency of PKA with GSK3b phosphoS9 in comparison to GSK3b. The binding of Axin inhibits GSK3b serine 9 phosphorylation by PKA but does not inhibit the phosphorylation of other PKA substrates such as Creb. The authors demonstrate that a fragment of Axin, residues 384-518, behaves similarly to the full-length Axin to shield GSK3b from phosphorylation. However, it is unclear how this fragment may bind in the destruction complex and if Axin has allosteric effects on GSK3b.

Reviewer #1 (Public Review):

In this study, Wang et al performed structural peripheral and central imaging of the auditory pathway using high-resolution MR. For the first time, they evaluated children with congenital severe to profound sensorineural hearing loss with and without cochlear nerve deficiency and cochlear malformations. The authors evaluated 13 children with severe to profound congenital hearing loss (6 with cochlear nerve deficiency) and 10 typically-hearing controls. They found significant differences in the central auditory pathway that were influenced by the status of the peripheral auditory pathway. Determination of outcomes after cochlear implantation or auditory brainstem implantation is critical and we currently have no good methods for this, so this study is very promising in that regard.

The authors have achieved their aim of evaluating these children with high-resolution imaging and identifying differences in auditory pathways. My primary issues are that some of their claims for clinical potential are not justified as of yet and the authors did not determine a diagnosis for the patients' hearing loss.

Reviewer #2 (Public Review):

The aim of this work is to introduce a new pipeline for mapping the human auditory pathway using structural and diffusional MRI, and to examine the brain structural development of children with profound congenital sensorineural hearing loss (SNHL) at both the acoustic processing level and the speech perception level. The authors use this pipeline to investigate the structural development of the auditory-language network for profound SNHL children with normal peripheral structure and those with inner ear malformations and/or cochlear nerve deficiency (IEM&CND). The authors successfully developed a new pipeline for reconstructing the human auditory pathway and used it to investigate the structural development of the auditory-language network in children with profound SNHL. They segmented the subcortical auditory nuclei using super-resolution track density imaging (TDI) maps and T1-weighted images and tracked the auditory and language pathways using probabilistic tractography. The authors found that the language pathway was more sensitive to peripheral auditory condition than the central auditory pathway, highlighting the importance of early intervention for profound SNHL children to provide timely speech inputs. The authors also proposed a comprehensive pre-surgical evaluation extending from the cochlea to the auditory-language network, which has promising clinical potential.

The major strengths of this work are the use of a new pipeline for mapping the human auditory pathway, the inclusion of children with profound SNHL with and without IEM&CND, and the finding that the language pathway is more sensitive to peripheral auditory condition than the central auditory pathway. However, a limitation of this study is the small sample size, which may limit the generalizability of the findings.

The results support the conclusions that the language pathway is more sensitive to peripheral auditory condition than the central auditory pathway, highlighting the importance of early intervention for profound SNHL children to provide timely speech inputs.

This work has the potential to have a significant impact on the field by providing new insights into the structural development of the auditory-language network in children with profound SNHL. The methods and data presented in this work may be useful to the community in developing comprehensive pre-surgical evaluation for children with profound SNHL extending from the cochlea to the auditory-language network.

Reviewer #3 (Public Review):

This study presents a new pipeline for mapping the auditory-language pathway in children with profound congenital sensorineural hearing loss (SNHL), focusing on those with inner ear malformations and/or cochlear nerve deficiency (IEM&CND). Using structural and diffusional MRI, the researchers investigated the structural fiber properties of the auditory-language networks in affected children under six years old. Findings suggest that the language pathway is more sensitive to peripheral auditory than the central auditory pathway, emphasizing the need for early intervention to provide speech inputs. The study also proposes a comprehensive pre-surgical evaluation from the cochlea to the auditory-language network.

Strengths:

1. Investigating fiber properties across various brain network levels (from peripheral structures to central auditory and higher-level language pathways) using high-resolution diffusion imaging and an innovative pipeline.

2. Evaluating presurgical fiber properties in two subgroups of SNHL children (cochlear implant and auditory brainstem implant candidates) to demonstrate the relationship between peripheral auditory structure damage and the development of auditory-language structural pathways.

Weaknesses:

1. Limited sample size: The study analyzed data from 13 SNHL children and 10 normal-hearing children, potentially restricting the validity and reproducibility of the findings, particularly in correlation results based on individual differences.

2. Lack of speech and language behavioral measures: Although the researchers collected behavioral data post-CI/ABI surgery for most participants, no such data was reported. Consequently, the association between presurgical fiber measures and postsurgical outcomes remains unclear.

3. Unclear practical implications: The relevance of the presurgical evaluation of the auditory-language network for surgical decision-making and prognosis estimation is not evident, as fiber measures may not correlate with behavioral outcomes.

Reviewer #1 (Public Review):

Various parts of the premotor cortex have been implicated in choices underlying decision-making tasks. Further, norepinephrine has been implicated in modulating behavior during various decision-making tasks. Less work has been done on how noradrenergic modulation would affect M2 activity to alter decision-making, nor is it clear whether noradrenergic modulation effects on activity would differ between the male and female sexes.

This manuscript addresses some of these questions.<br /> - In particular, clear sex differences in task engagement are seen.<br /> - May also show some interesting differences and distributions of β2 adrenergic receptors in M2 between males and females.

Less clear is the specificity of systemic antagonism of β adrenergic receptors on the changes in M2 activity reported. As propranolol was given systemically, changes in M2 firing rates could also be due to broader circuit (indirect) activity changes. As it was not given locally, nor were local receptor populations manipulated, one is unable to make the conclusion that changes in neural activity are due to the direct effects of adrenergic receptors within M2 populations.

Also not clear, is the contribution of M2 to this task, and whether the changes in M2 activity patterns observed are directly responsible for the behavioral disruptions measured.

Reviewer #2 (Public Review):

This paper by Rodbarg et al describes an interesting study on the role of beta noradrenergic receptors in action-related activity in the premotor cortex of behaving rats. This work is precious because even if the action of neuromodulatory systems in the cortex is thought to be critical for cognition, there is very little data to actually substantiate the theories. The study is well conducted and the paper is well written. I think, however, that the paper could benefit from several modifications since I can see 3 major issues:

Both from a theoretical and from a practical point of view, the emphasis on 'cue-related' activity and the potential influence of NA on sensory processing is problematic. First, recent studies in rodents and primates have clearly demonstrated that LC activation is more closely related to actions than to stimulus processing (see Poe et al, 2020 for review). Second, the analysis of neural activity around cue onset should be examined with spikes aligned on the action, since M2 is a motor region and raster plots suggest that activity is strongly related to action (I'll be more specific below).

The distinction between neural activity and behavior or cognition is not always clear. I understand that spike count can be related to motor preparation or decision, but it should not be taken for granted that neuronal activity is action planning. The analysis should be clarified and the relation between neural activity, behavior, and potential hidden cognitive operations should be explicated more clearly.<br /> The sex difference is interesting, but at the moment it seems anecdotal. From a theoretical point of view, is there any ecological/ biological reason for a sex dependency of noradrenergic modulation of the cortex? Is there any background literature on sex differences in motor functions in rats, or in terms of NA action? If not, why does it matter (how does it change the way we should interpret the data?) From a practical point of view, is there a functional sex difference in absence of treatment, or is it that the drug has a distinct effect on males vs females? This has very distinct consequences, I think.

These issues could be clarified both in the introduction and in the discussion, but the authors might have a different view on what is theoretically relevant here. In the result section, however, I think that both the lack of specificity in the description of behavior and cognitive operation and the confusion between 'sensory' and 'motor' functions make it very difficult to figure out what is going on in these experiments, both at a behavioral and at a neurophysiological level.

First, the description of the behavior in the task is clearly not sufficient, which makes the interpretation of the measures very difficult. One possible interpretation of the effects of the drug is a decrease in motivation, for instance, due to a decrease in reward sensitivity or an increase in sensitivity to effort. But there are others. More importantly, none of these measures can be used to tease apart action preparation from action execution, even though the study is supposed to be about the former.<br /> Also, but this is less critical: In Figures 2C and D, it looks like there is a bimodal distribution for the effect of propranolol in females. Is there something similar in the neuronal effects of the drug? And in the distribution of receptors? Can it be accounted for by hormonal cycles/ anything else?

The description of neural activity is also very superficial.<br /> In general, it is not clear how spike count measures have been extracted. For example, legend and figure C are not clear, is the (long) period of cue presentation included in the 'decision time'?? "Cues were presented at a variable interval 200-700ms after initiation and until animals left the well, 'Well Exit'. The time from cue onset to well exit was identified as the decision time (yellow)." Yet on the figure only the period after cue presentation is in yellow. This is critical because, given the duration of the cue, the animals are probably capable of deciding (to exit the well) before the cue turns off. Indeed, as shown in fig 2D, the animals can decide within about 500 ms. So to what extent is the 'cue response' actually a 'decision response'? When looking at figure 3A, there is clearly a pattern on the raster, a line going from top left to bottom right. If the trials are sorted chronologically, something is happening over time. If, as I suspect, trials are sorted by ascending response time, this raster is showing that what authors are calling a 'response to cues' is actually a response around action. Basically, if propranolol slows down reaction time, the spikes will be delayed from cue onset only because they remain locked to the action. Then the whole analysis and interpretation need to be reconsidered. But it might be for the best: as I mentioned earlier, recent work on LC activity has clearly emphasized its influence on motor rather than sensory processing (Poe et al, 2020).

Fig 2D-F: it is hard to believe that the increase in firing rate induced by propranolol in females is not significant. Presumably, because the range of the median firing rate is so high in the first place, distribution (2E) really indicates an increase in firing. Maybe some other test? e.g paired t.test, or standardized values (z.score) to get rid of variability in firing across neurons?

Along those lines, would it be worth looking for effects on specific populations (interneurons) which are sometimes characterized by thinner spikes and higher mean firing rates? Given the distribution of beta receptors RNA on interneurons, one would actually expect an effect of propranolol on the firing rate irrespective of task events. Or what is it that prevents the influence of propranolol on interneurons from changing the firing rate? In any case, one of the strengths of this study is the localization of beta receptors on specific neuronal populations in the cortex, so I think that the authors should really try to build on it and find something related to the neurophysiological effects. Otherwise, one cannot exclude the possibility that the behavioral effects are not related to the influence of the drug on these receptors in that region.

The conclusion that neuronal discrimination decreases because the proportion of neurons showing no effect increases is confusing (negative results, basically). It would be clearer if they were reporting the number of neurons that do show an effect, and presumably that this number shows a significant decrease.<br /> Figs 3F-I: a good proportion of neurons (at least 20%) show a significant encoding before cue onset. How is it possible? This raises the issue of noise level/ null hypothesis for this kind of repeated analysis. How did the author correct for multiple comparison issues?<br /> The description of the action-related activity is globally confusing. Again, how can the authors discriminate between activity related to planning vs action itself? What is significant and what is not, in males vs females? What is being measured here? For example, a very unclear statement on line 238: "Propranolol primarily disrupted active inhibition of irrelevant action selection in M2 activity, reducing the ability to maintain action plan representation in M2, delaying lever press responses (Figure 4L, 4M)." What is 'active inhibition? What is an irrelevant action plan? What is selection? All of that should be defined using objective behavioral criteria and tested formally.<br /> Also, the description of the classifier analysis should be more thorough. Referencing the toolbox is not sufficient to understand what has been done.<br /> Measuring Beta adrenoceptors is a great idea, and the results are interesting, especially the difference between neuron types. But again, how does that fit with neurophysiological results? Note, that since this is RNA measures, it should not be phrased as 'receptors' but 'receptors RNA' throughout. One possible interpretation of these anatomical results that cannot be reconciled with physiology is that protein expression at the membrane shows a distinct pattern.

In conclusion, I think that this is a very interesting study and that the results are potentially relevant for a wide audience. But the paper would clearly benefit from revisions. If the authors could clearly identify a significant relationship between the action of NA on beta receptors on specific cortical neurons, at a physiological and behavioral level, that would be a seminal study. At the moment, the evidence is not convincing enough but the data suggest that it is the case.

Reviewer #1 (Public Review):

The authors developed a new approach to enable the reconstruction of fine axonal morphologies based solely on extracellular action potential recordings from in vitro mammalian neurons using a high-density microelectrode array system with an integrated CMOS camera. They provide compelling evidence of fine resolution in mapping functional connections between neurons via very fine axons. The advantage of the approach is that it provides a label-free electrical visualization of axon conduction trajectories as well as the ability to access the AP waveforms. The work may have broad use in neurobiology, bioengineering, stem cell biology, as well as tissue engineering in functional characterization.

Reviewer #2 (Public Review):

This is a very interesting and compelling paper reporting a method for analyzing the features of action potential conduction in cortical and spinal neurons in vitro using high-density CMOS micro-electrode arrays. The authors report the performances of their detection algorithm allowing them to reconstruct the functional map of single-branching axons. In particular, they compare the functional conduction maps of cortical and spinal axons, and they show that spinal axons display larger spike signals in their distal part compared to cortical axons, but a lower number of branches. In addition, they reveal that spinal axons display a higher conduction velocity compared to cortical ones.

This study is particularly interesting as it constitutes a compelling methodological report of action potential propagation up to 5-8 mm in single axons in vitro.

Reviewer #2 (Public Review):

There are data to suggest that intratumour mutational heterogeneity (ITH; the proportion of all mutations that are found only within cancer subclones) is associated with worse therapeutic outcomes. Specifically, patients with more mutations (and thus neoantigens) mostly expressed by subclones (high ITH) have poorer responses to checkpoint immunotherapy. The authors set out to explore the mechanisms underlying this by studying 2 dimensions of neoantigen biology: firstly, distribution (clonal vs subclonal) and secondly, immunogenicity (weak vs strong binding to MHC class I). Using a panel of lung cancer cell lines modified to express individual or dual neoantigens in order to model clonal and subclonal expression, elegant studies show that clonal co-expression with a "strong" neoantigen can boost the immunogenicity of a "weak" neoantigen and result in tumour control. Mechanistically, this is related to engulfment of both neoantigens by cross presenting type 1 conventional dendritic cells and the associated enhanced activation state of this cell type. This is an interesting and potentially important finding that may be related to mechanisms of epitope spreading as immune responses diverge from targeting more to less immunogenic epitopes. Overall, the study is thought-provoking, informative in relation to how neoantigen immunogenicity is shaped and may have practical relevance.

Reviewer #1 (Public Review):

Zhou et al. have set up a study to examine how metabolism is regulated across the organism by taking a combined approach looking at gene expression in multiple tissues, as well as analysis of the blood. Specifically, they have created a tool for easily analyzing data from GTEx across 18 tissues in 310 people. In principle, this approach should be expandable to any dataset where multiple tissues of data were collected from the same individuals. While not necessary, it would also raise my interest to see the "Mouse(coming soon)" selection functional, given that the authors have good access to multi-tissue transcriptomics done in similarly large mouse cohorts.

Summary:

The authors have assembled a web tool that helps analyze multiple tissues' datasets together, with the aim of identifying how metabolic pathways and gene regulation are connected across tissues. This makes sense conceptually and the web tool is easy to use and runs reasonably quickly, considering the size of the data. I like the tool and I think the approach is necessary and surprisingly under-served; there is a lot of focus on multi-omics recently, but much less on doing a good job of integrating multi-tissue datasets even within a single omics layer.

What I am less convinced about is the "Research Article" aspect of this paper. Studying circadian rhythm in GTEx data seems risky to me, given the huge range in circadian clock in the sample collection. I also wonder (although this is not even remotely in my expertise) whether the circadian rhythm also gets rather desynchronized in people dying of natural causes - although I suppose this could be said for any gene expression pathway. Similarly for looking at secreted proteins in Figure 4 looking at muscle-hippocampus transcript levels for ADAMTS17 doesn't make sense to me - of all tissue pairs to make a vignette about to demonstrate the method, this is not an intuitive choice to me. The "within muscle" results look fine but panels C-E-G look like noise to me...especially panel C and G are almost certainly noise, since those are pathways with gene counts of 2 and 1 respectively.

I think this is an important effort and a good basis but a significant revision is necessary. This can devote more time and space to explaining the methodology and for ensuring that the results shown are actually significant. This could be done by checking a mix of negative controls (e.g. by shuffling gene labels and data) and a more comprehensive look at "positive" genes, so that it can be clearly shown that the genes shown in Fig 1 and 2 are not cherry-picked. For Figure 3, I suspect you would get almost an identical figure if instead of showing pan-tissue circadian clock correlations, you instead selected the electron transport chain, or the ribosome, or any other pathway that has genes that are expressed across all tissues. You show that colon and heart have relatively high connectivity to other tissues, but this may be common to other pathways as well.

Reviewer #2 (Public Review):

Summary:

Zhou et al. use publicly available GTEx data of 18 metabolic tissues from 310 individuals to explore gene expression correlation patterns within-tissue and across-tissues. They detect signatures of known metabolic signaling biology, such as ADIPOQ's role in fatty acid metabolism in adipose tissue. They also emphasize that their approach can help generate new hypotheses, such as the colon playing an important role in circadian clock maintenance. To aid researchers in querying their own genes of interest in metabolic tissues, they have developed an easy-to-use webtool (GD-CAT).

This study makes reasonable conclusions from its data, and the webtool would be useful to researchers focused on metabolic signaling. However, some misconceptions need to be corrected, as well as greater clarification of the methodology used.

Strengths:

GTEx is a very powerful resource for many areas of biomedicine, and this study represents a valid use of gene co-expression network methodology. The authors do a good job of providing examples confirming known signaling biology as well as the potential to discover promising signatures of novel biology for follow-up and future studies. The webtool, GD-CAT, is easy to use and allows researchers with genes and tissues of interest to perform the same analyses in the same GTEx data.

Weaknesses:

A key weakness of the paper is that this study does not involve genetic correlations, which is used in the title and throughout the manuscript, but rather gene co-expression networks. The authors do mention the classic limitation that correlation does not imply causation, but this caveat is even more important given that these are not genetic correlations. Given that the goal of their study aligns closely with multi-tissue WGCNA, which is not a new idea (e.g., Talukdar et al. 2016; https://doi.org/10.1016/j.cels.2016.02.002), it is surprising that the authors only use WGCNA for its robust correlation estimation (bicor), but not its latent factor/module estimation, which could potentially capture cross-tissue signaling patterns. It is possible that the biological signals of interest would be drowned out by all the other variation in the data but given that this is a conventional step in WGCNA, it is a weakness that the authors do not use it or discuss it.

Reviewer #3 (Public Review):

Summary:

A useful and potentially powerful analysis of gene expression correlations across major organ and tissue systems that exploits a subset of 310 humans from the GTEx collection (subjects for whom there are uniformly processed postmortem RNA-seq data for 18 tissues or organs). The analysis is complemented by a Shiny R application web service.

The need for more multisystems analysis of transcript correlation is very well motivated by the authors. Their work should be contrasted with more simple comparisons of correlation structure within different organs and tissues, rather than actual correlations across organs and tissues.

Strengths and Weaknesses:

The strengths and limitations of this work trace back to the nature of the GTEx data set itself. The authors refer to the correlations of transcripts as "gene" and "genetic" correlations throughout. In fact, they name their web service "Genetically-Derived Correlations Across Tissues". But all GTEx subjects had strong exposure to unique environments and all correlations will be driven by developmental and environmental factors, age, sex differences, and shared and unshared pre- and postmortem technical artifacts. In fact we know that the heritability of transcript levels is generally low, often well under 25%, even studies of animals with tight environmental control.

This criticism does not comment materially detract for the importance and utility of the correlations-whether genetic, GXE, or purely environmental-but it does mean that the authors should ideally restructure and reword text so as to NOT claim so much for "genetics". It may be possible to incorporate estimates of chip heritability of transcripts into this work if the genetic component of correlations is regarded as critical (all GTEx cases have genotypes).

Appraisal of Work on the Field:

There are two parts to this paper: 1. "case studies" of cross-tissue/organ correlations and 2. the creation of an R/Shiny application to make this type of analysis much more practical for any biologist. Both parts of the work are of high potential value, but neither is fully developed. My own opinion is that the R/Shiny component is the more important immediate contribution and that the "case studies" could be placed in the context of a more complete primer. Or Alternatively, the case studies could be their own independent contributions with more validation.

Reviewer #3 (Public Review):

In this paper, the authors analyze a large previously published deep mutational scanning data set using a reference-free regression approach. They extract the contributions of single locus and epistatic effects to the functionality of the sequence (no, weak or strong transcription activation of two response elements). They find that pairwise epistasis plays a crucial and dominant role at creating functional sequences and at connecting the functional sequence space.

I enjoyed reading the paper and the topic (role of epistasis at creating and connecting functional sequences; development of measures of epistasis) is very exciting to me. However, I found it difficult to judge the strength of the paper both because it is written in a rather dense and yet potentially redundant fashion (see comment 1) and because I was left with a number of questions upon reading. I will focus on conceptual questions in the following comments, since I am not able to judge the statistical approach in detail.

1/ Regarding the biological result (importance of pairwise epistasis) I was wondering how potentially redundant the consecutive sections of the paper are. In which situation would the authors expect that pairwise epistasis does *not* play a crucial role for mutational steps, trajectories, or space connectedness, if it is dominant in the genotype-phenotype landscape? I would also appreciate an explanation of how much new biological results this paper delivers as compared with the paper in which the data were published (which I, unfortunately, cannot access at the moment of writing this report).

2a/ Regarding the regression approach: I very much appreciate a reference-free approach to the estimation of epistasis. However, I would enjoy an explanation of how the results would have been (potentially) different if a reference-based approach was used, and how it compares with other reference-free approaches to estimating epistasis (e.g., linear regression or the gamma statistics of Ferretti et al. 2015).

2b/ When comparing the outcomes with and without epistasis, I understood that the authors compare the estimated "full model" with the outcome if epistatic effects were ignored - but without a new estimation of main effects if epistasis is ignored. Wouldn't that be a more fair comparison?

2c/ Where do the authors see the applicability of their approach to data beyond those analyzed in the present study? What are the requirements to use it? Does it only work for combinatorially complete landscapes? I did not have a chance to look at the code - how easily could other researchers apply the approach to their data?

Reviewer #1 (Public Review):

Metzger et al develop a rigorous method filling an important unmet need in protein evolution - analysis of protein genetic architecture and evolution using data from combinatorially complete 20^N variant libraries. Addressing this need has become increasingly valuable, as experimental methods for generating these datasets expand in scope and scale. Their model incorporates several key features - (1) it reports the effects of mutations relative to the average across all variants, rather than a particular genotype, making it useful for examining genetic architectures and evolution in a less biased way, (2) it infers contributions from both "specific" and "non-specific" epistasis, which is essential for some experimental measurements, and perhaps most importantly (3) it does this for all possible 20 states at each site, in contrast to the binary analyses in prior work. These features are not individually novel but integrating them into a single analysis framework is novel and will be incredibly valuable to the protein evolution community. Using a previously published dataset generated by two of the authors, they conclude that (1) changes in function are largely attributable to pairwise but not higher-order interactions, and (2) epistasis potentiates, rather than constrains, evolutionary paths. These findings are well-supported by the data, though the authors' claim that higher-order epistasis cannot account for the variation they see could be better supported by additional analyses or discussion (as noted in recommendations for authors). Overall, this work has important implications for predicting the relationship between genotype and phenotype, which is of considerable interest to protein biochemistry, evolutionary biology, and numerous other fields.

Reviewer #2 (Public Review):

The authors aimed to understand how epistasis influences the genetic architecture of the DNA-binding domain (DBD) of steroid hormone receptor. An ordinal regression model was developed in this study to analyze a published deep mutational scanning dataset that consists of all combinatorial amino acid variants across four positions (i.e. 160,000 variants). This published dataset measured the binding of each variant to the estrogen receptor response element (ERE, sequence: AGGTCA) as well as the steroid receptor response element (SRE, sequence: AGAACA). This model has major strengths of being reference free and able to account for global nonlinearity in the genotype-phenotype relationship. Thorough analyses of the modelling results have performed, which provided convincing results to support the importance of epistasis in promoting evolution of protein functions. This conclusion is impactful because many previous studies have shown that epistasis constrains evolution. However, the model in this study requires transformation of continuous functional data into categorical form, which would reduce precision in estimating the genetic architecture. Besides, generalizability of the findings in this study is unclear. These limitations, which are acknowledged by the authors, are minor and should not affect the conclusion of this study. The novelty of this study will likely stimulate new ideas in the field. The model will also likely be utilized by other groups in the community.

Reviewer #2 (Public Review):

This study aims to describe a physical interaction between the kinase DYRK1A and the Tuberous Sclerosis Complex proteins (TSC1, TSC2, TBC1D7). Furthermore, this study aims to demonstrate that DYRK1A, upon interaction with the TSC proteins regulates mTORC1 activity and cell size. Additionally, this study identifies T1462 on TSC2 as a phosphorylation target of DYRK1A. Finally, the authors demonstrate the role of DYRK1A on cell size using human, mouse, and Drosophila cells.

This study, as it stands, requires further experimentation to support the conclusions on the role of DYRK1A on TSC interaction and subsequently on mTORC1 regulation. Weaknesses include, 1) The lack of an additional assessment of cell growth/size (eg. protein content, proliferation), 2) the limited data on the requirement of DYRK1A for TSC complex stability and function, and 3) the limited perturbations on the mTORC1 pathway upon DYRK1A deletion/overexpression. Finally, this study would benefit from identifying under which nutrient conditions DYRK1A interacts with the TS complex to regulate mTORC1.

The interaction described here is highly impactful to the field of mTORC1-regulated cell growth and uncovers a previously unrecognized TSC-associated interacting protein. Further characterization of the role that DYRK1A plays in regulating mTORC1 activation and the upstream signals that stimulate this interaction will be extremely important for multiple diseases that exhibit mTORC1 hyper-activation.

Reviewer #1 (Public Review):

In this manuscript, Wang et al. demonstrate that knockdown of DYRK1A results in reduced cell size, which is mediated by mTORC1 activity. They found that DYRK1A interacts with TSC1/TSC2 proteins which leads to the phosphorylation of TSC2 at T1462. Phosphorylation of TSC2 at T1462 inhibits TSC2 activity leading to the activation of mTORC1. The authors complement their findings by demonstrating that overexpression of RHEB (positive regulator of mTORC1) rescues the phenotype of DYRK1A (mnb in flies) mutation in the NMJ.

The authors' findings on the regulation of cell size and mTORC1 activity by DYRK1A reflect the previous findings of Levy et al. (PMID: 33840455) that cortical deletion of Dyrk1a in mice causes decreased neuronal size associated with a decreased activity of mTORC1 that can be rescued by the inhibition of Pten or supplementation of IGF1.

The authors demonstrate that T1462 phospho-site at TSC2 is phosphorylated in response to the overexpression of WT but not kinase-dead DYRK1A. However, the authors do not provide any evidence that the regulation of mTORC1 is mediated via phosphorylation of this site. In addition, T1462 site is known to be phosphorylated by Akt. There is a possibility that Akt was co-purified with TSC1/TSC2 complex and DYRK1A promotes phosphorylation of TSC2 indirectly via the activation of AKT that can be tested by using AKT depleted cells.

RHEB is the most proximal regulator of mTORC1 and can activate mTORC1 even under amino acid starvation. The fact that RHEB overexpression rescues the cell size under DYRK1A depletion or mnb (DYRK1A in Drosophila) mutant phenotype does not prove that DYRK1A regulates the cell size via TSC1 as it would rescue any inhibitory effects upstream to mTORC1.

Reviewer #3 (Public Review):

The manuscript describes a combination of in vitro and in vivo results implicating Dyrk1a in the regulation of mTORC. Particular strengths of the data are this combination of cell and whole animal (drosophila) based studies. However, most of the experiments seem to lack a key additional experimental condition that could increase confidence in the authors' conclusions. Overall some tantalizing data is presented. However, there are several issues that should be clarified or otherwise addressed with additional data.

1. In Figure 1G, why not test overexpression levels of Dyrk1a via western rather than only looking at the RNA levels?

2. In Figure 2, while there is clearly TSC1 protein in the Dyrk1a and FLAG-Dyrk1a IPs that supports an interaction between the proteins, it would be good to see the reciprocal IP experiment wherein TSC1 or TSC2 are pulled down and then the blot probed for Dyrk1a.

3. Figures 3 A and D tested the effects of Dyrk1a knockdown using different methods in different cell lines. This is a reasonable approach to ascertain the generalizability of findings. However, each experiment is performed differently. For example, in 3A, the authors found no difference in baseline pS6, so they did a time course of treatment to induce phosphorylation and found differences depending on Dyrk1a expression. In 3D, they only show baseline effects from the CRISPr knockdown. Why not do the time course as well for consistency? Also, why the inconsistency in approaches wherein one shows baseline effects and the other does not? The authors could also consider the pharmacologic inhibition of Dyrk1a activity as well.

4. In Figure 4, RHEB overexpression increases cell size in both Dyrk1a wt and Dyrk1a shRNA treated cells, although the magnitude of the effect appears reduced in Dyrk1a shRNA cells. However, there is the possibility here that RHEB acts independently of Dyrk1a. Why not also do the experiment of Figure 1 wherein Dyrk1a is overexpressed and then knockdown RHEB in that context? If the hypothesis is supported, then RHEB knockdown should eliminate the cell size effect of Dyrk1a overexpression.

5. The discussion should incorporate relevant findings from other models, such as Arabidopsis. Barrada et al., Development (2019), 146 (3).

Reviewer #3 (Public Review):

This work provides a novel design of implantable and high-density EMG electrodes to study muscle physiology and neuromotor control at the level of individual motor units. Current methods of recording EMG using intramuscular fine-wire electrodes do not allow for isolation of motor units and are limited by the muscle size and the type of behavior used in the study. The authors of myomatrix arrays had set out to overcome these challenges in EMG recording and provided compelling evidence to support the usefulness of the new technology.

Strengths:

• They presented convincing examples of EMG recordings with high signal quality using this new technology from a wide array of animal species, muscles, and behavior.<br /> • The design included suture holes and pull-on tabs that facilitate implantation and ensure stable recordings over months.<br /> • Clear presentation of specifics of the fabrication and implantation, recording methods used, and data analysis

Weaknesses:

• The justification for the need to study the activity of isolated motor units is underdeveloped. The study could be strengthened by providing example recordings from studies that try to answer questions where isolation of motor unit activity is most critical. For example, there is immense value for understanding muscles with smaller innervation ratio which tend to have many motor neurons for fine control of eyes and hand muscles.

Reviewer #1 (Public Review):

The main objective of this paper is to report the development of a new intramuscular probe that the authors have named Myomatrix arrays. The goal of the Myomatrix probe is to significantly advance the current technological ability to record the motor output of the nervous system, namely fine-wire electromyography (EMG). Myomatrix arrays aim to provide large-scale recordings of multiple motor units in awake animals under dynamic conditions without undue movement artifacts and maintain long-term stability of chronically implanted probes. Animal motor behavior occurs through muscle contraction, and the ultimate neural output in vertebrates is at the scale of motor units, which are bundles of muscle fibers (muscle cells) that are innervated by a single motor neuron. The authors have combined multiple advanced manufacturing techniques, including lithography, to fabricate large and dense electrode arrays with mechanical features such as barbs and suture methods that would stabilize the probe's location within the muscle without creating undue wiring burden or tissue trauma. Importantly, the fabrication process they have developed allows for rapid iteration from design conception to a physical device, which allows for design optimization of the probes for specific muscle locations and organisms. The electrical output of these arrays are processed through a variety of means to try to identify single motor unit activity. At the simplest, the approach is to use thresholds to identify motor unit activity. Of intermediate data analysis complexity is the use of principal component analysis (PCA, a linear second-order regression technique) to disambiguate individual motor units from the wide field recordings of the arrays, which benefits from the density and numerous recording electrodes. At the highest complexity, they use spike sorting techniques that were developed for Neuropixels, a large-scale electrophysiology probe for cortical neural recordings. Specifically, they use an estimation code called kilosort, which ultimately relies on clustering techniques to separate the multi-electrode recordings into individual spike waveforms.

An account of the major strengths and weaknesses of the methods and results.<br /> The biggest strength of this work is the design and implementation of the hardware technology. It is undoubtedly a major leap forward in our ability to record the electrical activity of motor units. The myomatrix arrays trounce fine-wire EMGs when it comes to the quality of recordings, the number of simultaneous channels that can be recorded, their long-term stability, and resistance to movement artifacts.

The primary weakness of this work is its reliance on kilosort in circumstances where most of the channels end up picking up the signal from multiple motor units. As the authors quite convincingly show, this setting is a major weakness for fine-wire EMG. They argue that the myomatrix array succeeds in isolating individual motor unit waveforms even in that challenging setting through the application of kilosort.

Although the authors call the estimated signals as well-isolated waveforms, there is no independent evidence of the accuracy of the spike sorting algorithm. The additional step (spike sorting algorithms like kilosort) to estimate individual motor unit spikes is the part of the work in question. Although the estimation algorithms may be standard practice, the large number of heuristic parameters associated with the estimation procedure are currently tuned for cortical recordings to estimate neural spikes. Even within the limited context of Neuropixels, for which kilosort has been extensively tested, basic questions like issues of observability, linear or nonlinear, remain open. By observability, I mean in the mathematical sense of well-posedness or conditioning of the inverse problem of estimating single motor unit spikes given multi-channel recordings of the summation of multiple motor units. This disambiguation is not always possible. kilosort's validation relies on a forward simulation of the spike field generation, which is then truth-tested against the sorting algorithm. The empirical evidence is that kilosort does better than other algorithms for the test simulations that were performed in the context of cortical recordings using the Neuropixels probe. But this work has adopted kilosort without comparable truth-tests to build some confidence in the application of kilosort with myomatrix arrays? Furthermore, as the paper on the latest version of kilosort, namely v4, discusses, differences in the clustering algorithm is the likely reason for kilosort4 performing more robustly than kilosort2.5 (used in the myomatrix paper). Given such dependence on details of the implementation and the use of an older kilosort version in this paper, the evidence that the myomatrix arrays truly record individual motor units under all the types of data obtained is under question.

There is an older paper with a similar goal to use multi-channel recording to perform source-localization that the authors have failed to discuss. Given the striking similarity of goals and the divergence of approaches (the older paper uses a surface electrode array), it is important to know the relationship of the myomatrix array to the previous work. Like myomatrix arrays, the previous work also derives inspiration from cortical recordings, in that case it uses the approach of source localization in large-scale EEG recordings using skull caps, but applies it to surface EMG arrays. Ref: van den Doel, K., Ascher, U. M., & Pai, D. K. (2008). Computed myography: three-dimensional reconstruction of motor functions from surface EMG data. Inverse Problems, 24(6), 065010.

The incompleteness of the evidence that the myomatrix array truly measures individual motor units is limited to the setting where multiple motor units have similar magnitude of signal in most of the channels. In the simpler data setting where one motor dominates in some channel (this seems to occur with some regularity), the myomatrix array is a major advance in our ability to understand the motor output of the nervous system. The paper is a trove of innovations in manufacturing technique, array design, suture and other fixation devices for long-term signal stability, and customization for different muscle sizes, locations, and organisms. The technology presented here is likely to achieve rapid adoption in multiple groups that study motor behavior, and would probably lead to new insights into the spatiotemporal distribution of the motor output under more naturally behaving animals than is the current state of the field.

Reviewer #2 (Public Review):

Motoneurons constitute the final common pathway linking central impulse traffic to behavior, and neurophysiology faces an urgent need for methods to record their activity at high resolution and scale in intact animals during natural movement. In this consortium manuscript, Chung et al. introduce high-density electrode arrays on a flexible substrate that can be implanted into muscle, enabling the isolation of multiple motor units during movement. They then demonstrate these arrays can produce high-quality recordings in a wide range of species, muscles, and tasks. The methods are explained clearly, and the claims are justified by the data. While technical details on the arrays have been published previously, the main significance of this manuscript is the application of this new technology to different muscles and animal species during naturalistic behaviors. Overall, we feel the manuscript will be of significant interest to researchers in motor systems and muscle physiology, and we have no major concerns. A few minor suggestions for improving the manuscript follow.

The authors perhaps understate what has been achieved with classical methods. To further clarify the novelty of this study, they should survey previous approaches for recording from motor units during active movement. For example, Pflüger & Burrows (J. Exp. Biol. 1978) recorded from motor units in the tibial muscles of locusts during jumping, kicking, and swimming. In humans, Grimby (J. Physiol. 1984) recorded from motor units in toe extensors during walking, though these experiments were most successful in reinnervated units following a lesion. In addition, the authors might briefly mention previous approaches for recording directly from motoneurons in awake animals (e.g., Robinson, J. Neurophys. 1970; Hoffer et al., Science 1981).

For chronic preparations, additional data and discussion of the signal quality over time would be useful. Can units typically be discriminated for a day or two, a week or two, or longer? A related issue is whether the same units can be tracked over multiple sessions and days; this will be of particular significance for studies of adaptation and learning.

It appears both single-ended and differential amplification were used. The authors should clarify in the Methods which mode was used in each figure panel, and should discuss the advantages and disadvantages of each in terms of SNR, stability, and yield, along with any other practical considerations.

Is there likely to be a motor unit size bias based on muscle depth, pennation angle, etc.?

Can muscle fiber conduction velocity be estimated with the arrays?

The authors suggest their device may have applications in the diagnosis of motor pathologies. Currently, concentric needle EMG to record from multiple motor units is the standard clinical method, and they may wish to elaborate on how surgical implantation of the new array might provide additional information for diagnosis while minimizing risk to patients.

Reviewer #1 (Public Review):

The current manuscript provides a timely contribution to the ongoing discussion about the mechanism of the apical sodium/bile acid transporter (ASBT) transporters. Recent structures of the mammalian ASBT transporters exhibited a substrate binding mode with few interactions with the core domain (classically associated with substrate binding), prompting an unusual proposal for the transport mechanism. Early structures of ASBT homologues from bacteria also exhibit unusual substrate binding in which the core substrate binding domain is less engaged than expected. Due to the ongoing questions of how substrate binding and mechanism are linked in these transporters, the authors set out to deepen our understanding of a model ABST homolog from bacteria N. meningitidis (ABST-NM).

The premise of the current paper is that the bacterial ASBT homologs are probably not physiological bile acid transporters, and that structural elucidation of a natively transported substrate might provide better mechanistic information. In the current manuscript, the authors revisit the first BASS homologue to be structurally characterized, ABST-NM. Based on bacteriological assays in the literature, the authors identify the coenzyme A precursor pantoate as a more likely substrate for ABST-NM than taurocholate, the substrate in the original structure. A structure of ASBT-NM with pantoate exhibits interesting differences in structure. The structures are complemented with MD simulations, and the authors propose that the structures are consistent with a classical elevator transport mechanism.

The structural experiments are generally solid, although showing omit maps would bolster the identification of the substrate binding site. One shortcoming is that, although pantoate binding is observed, the authors do not show transport of this substrate, undercutting the argument that the pantoate structure represents binding of a "better" or more native substrate. Mechanistic proposals, like the proposed role of T112 in unlocking the transporter, would be much better supported by transport data.

Reviewer #2 (Public Review):

The manuscript starts with a demonstration of pantoate binding to ASBTnm using a thermostability assay and ITC, and follows with structure determinations of ASBTnm with or without pantoate. The structure of ASBTnm in the presence of pantoate pinpoints the binding site of pantoate to the "crossover" region formed by partially unwinded helices TMs 4 and 9. Binding of pantoate induces modest movements of side chain and backbone atoms at the crossover region that are consistent with providing coordination of the substrate. The structures also show movement of TM1 that opens the substrate binding site to the cytosol and mobility of loops between the TMs. MD simulations of the ASBT structure embedded in lipid bilayer suggests a stabilizing effect of the two sodium ions that are known to co-transport with the substrate. Binding study on pantoate analogs further demonstrates the specificity of pantoate as a substrate.

The weakness of the manuscript includes a lack of transport assay for pantoate and a lack of demonstration that the observed conformational changes in TM1 and the loops are relevant to the binding or transport of pantoate.

Overall, the structural, functional and computational studies are solid and rigorous, and the conclusions are well justified. In addition, the authors discussed the significance of the current study in a broader perspective relevant to recent structures of mammalian BASS members.

Reviewer #3 (Public Review):

The manuscript describes new ligand-bound structures within the larger bile acid sodium symporter family (BASS). This is the primary advance in the manuscript, together with molecular simulations describing how sodium and the bile acids sit in the structure when thermalized. What I think is fairly clear is that the ligands are more stable when the sodiums are present, with a marked reduction in RMSD over the course of repeated trajectories. This would be consistent with a transport model where sodium ions bind first, and then the bile acid binds, followed by a conformational change to another state where the ligands unbind.

While the authors mention that BASS transporters are thought to undergo an elevator transport mechanisms, this is not tested here. In my reading, all the crystal structures describe the same conformational state, and the simulations do not make an attempt to induce a transition on accessible simulation timescales. Instead, there is a morph between two states where different substrates are bound, which induces a conformational change that looks unrelated to the transport cycle.

Instead, the focus is on what kinds of substrates bind to this transporter, interrogating this with isothermal calorimetry together with mutations. With a Kd in the micromolar range, even the best binder, pantoate, actually isn't a particularly tight binder in the pharmaceutical sense. For a transporter, tight binding is not actually desirable, since the substrate needs to be able to leave after conformational change places it in a position accessible to the other side.

There is one really important point that readers and authors should be aware of. In Figure 2A, the names are not consistent with the chemical structure. "-ate" denotes when a carboxylic acid is in the deprotonated form, creating a charged carboxylate. What is drawn is pantoic acid, ketopantoic acid, and pantoethenic acid. Less importantly, the wedges and hashes for the methyl group are arguably not appropriate, since the carbon they are attached to is not a chiral center. For the crystallization, this makes no difference, since under near-neutral pKas the carboxylic acid will spontaneously deprotonate, and the carboxylate form will be the most common. However, if the structures in Figure 2A were used for classical molecular simulation, that would be a big problem, since now that would be modeling the much rarer neutral form rather than the charged state. I am reasonably sure based on Figure 5 that the MD correctly modeled the deprotonated form with a carboxylate, but that is inconsistent with Figure 2A. Otherwise, the structure and simulation analysis falls into the mainstream of modern structural biology work.

Reviewer #1 (Public Review):

The authors set out to use structural biology (cryo-EM), surface plasmon resonance, and complement convertase assays to understand the mechanism(s) by which ISG65 dampens the cytoxicity/cellular clearance to/of trypanosmes opsonised with C3b by the innate immune system.

The cryo-EM structure adds significantly to the author's previous crystallographic data because the latter was limited to the C3d sub-domain of C3b. Further, the in vitro convertase assay adds an additional functional dimension to this study.

The authors have achieved their aims and the results support their conclusions.

The role of complement in immunity to T. brucei (or lack thereof) has been a significant question in molecular parasitology for over 30 years. The identification of ISG65 as the C3 receptor and now this study providing mechanistic insights represents a major advance in the field.

Reviewer #2 (Public Review):

This is an excellent paper that uses structural work to determine the precise role of one of the few invariant proteins on the surface of the African trypanosome. This protein, ISG65, was recently determined to be a complement receptor and specifically a receptor of C3, whose binding to ISG65 led to resistance to complement-mediated lysis. But the molecular mechanism that underlies resistance was unknown.

Here, through cryoEM studies, the authors reveal the interaction interface (two actually) between ISG65 and C3, and based on this, make inferences regarding downstream events in the complement cascade. Specifically, they suggest that ISG65 preferably binds the converted C3b (rather than the soluble C3). Moreover, while conversion to a C3bB complex is not blocked, the ability to bind complement receptors 1 and 3 is likely blocked.

Of course, all this is work on proteins in isolation and the remaining question is - can this in fact happen on the membrane? The VSG-coated membrane is supposed to be incredibly dense (packed at the limits of physical density) and so it is unclear whether the interactions that are implied by the structural work can actually happen on the membrane of a live trypanosome. This is not necessarily a ding but it should be addressed in the manuscript perhaps as a caveat.

Reviewer #3 (Public Review):

The authors investigate the mechanisms by which ISG65 and C3 recognize and interact with each other. The major strength is the identification of eco-site by determining the cryoEM structure of the complex, which suggests new intervention strategies. This is a solid body of work that has an important impact on parasitology, immunology, and structural biology.

Reviewer #1 (Public Review):

The apicoplast, a non-photosynthetic vestigial chloroplast, is a key metabolic organelle for the synthesis of certain lipids in apicomplexan parasites. Although it is clear metabolite exchange between the parasite cytosol and the apicoplast must occur, very few transporters associated with the apicoplast have been identified. The current study combines data from previous studies with new data from biotin proximity labeling to identify new apicoplast resident proteins including two putative monocarboxylate transporters termed MCT1 and MCT2. The authors conduct a thorough molecular phylogenetic analysis of the newly identified apicoplast proteins and they provide compelling evidence that MCT1 and MCT2 are necessary for normal growth and plaque formation in vitro along with maintenance of the apicoplast itself. They also provide indirect evidence for a possible need for these transporters in isoprenoid biosynthesis and fatty acid biosynthesis within the apicoplast. Finally, mouse infection experiments suggest that MCT1 and MCT2 are required for normal virulence, with MCT2 completely lacking at the administered dose. Overall, this study is generally of high quality, includes extensive quantitative data, and significantly advances the field by identifying several novel apicoplast proteins together with establishing a critical role for two putative transporters in the parasite. The study, however, could be further strengthened by addressing the following aspects:

Main comments<br /> 1. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of IPP is only supported by indirect measurements (effects on host GFP uptake or trafficking, possibly due to effects on IPP dependent proteins such as rabs, and mitochondrial membrane potential, possibly due to effects on IPP dependent ubiquinone). This conclusion would be more strongly supported by directly measuring levels of IPP. If there are technical limitations that prevent direct measurement of IPP then the author should note such limitations and acknowledge in the discussion that the conclusion is based on indirect evidence.

2. The conclusion that condition depletion of AMT1 and/or AMT2 affects apicoplast synthesis of fatty acids is also poorly supported by the data. The authors do not distinguish between the lower fatty acid levels being due to reduced synthesis of fatty acids, reduced salvage of host fatty acids, or both. Indeed, the authors provide evidence that parasite endocytosis of GFP is dependent on AMT1 and AMT2. Host GFP likely enters the parasite within a membrane bound vesicle derived from the PVM. The PVM is known to harbor host-derived lipids. Hence, it is possible that some of the decrease in fatty acid levels could be due to reduced lipid salvage from the host. Experiments should be conducted to measure the synthesis and salvage of fatty acids (e.g., by metabolic flux analysis), or the authors should acknowledge that both could be affected.

Reviewer #2 (Public Review):

In this study Hui Dong et al. identified and characterized two transporters of the monocarboxylate family, which they called Apcimplexan monocarboxylate 1 and 2 (AMC1/2) that the authors suggest are involved in the trafficking of metabolites in the non-photosynthetic plastid (apicoplast) of Toxoplasma gondii (the parasitic agent of human toxoplasmosis) to maintain parasite survival. To do so they first identified novel apicoplast transporters by conducting proximity-dependent protein labeling (TurboID), using the sole known apicoplast transporter (TgAPT) as a bait. They chose two out of the three MFS transporters identified by their screen based and protein sequence similarity and confirmed apicoplast localisation. They generated inducible knock down parasite strains for both AMC1 and AMC2, and confirmed that both transporters are essential for parasite intracellular survival, replication, and for the proper activity of key apicoplast pathways requiring pyruvate as carbon sources (FASII and MEP/DOXP). Then they show that deletion of each protein induces a loss of the apicoplast, more marked for AMC2 and affects its morphology both at its four surrounding membranes level and accumulation of material in the apicoplast stroma. This study is very timely, as the apicoplast holds several important metabolic functions (FASII, IPP, LPA, Heme, Fe-S clusters...), which have been revealed and studied in depth but no further respective transporter have been identified thus far. hence, new studies that could reveal how the apicoplast can acquire and deliver all the key metabolites it deals with, will have strong impact for the parasitology community as well as for the plastid evolution communities. The current study is well initiated with appropriate approaches to identify two new putatively important apicoplast transporters, and showing how essential those are for parasite intracellular development and survival. However, in its current state, this is all the study provides at this point (i.e. essential apicoplast transporters disrupting apicoplast integrity, and indirectly its major functions, FASII and IPP, as any essential apicoplast protein disruption does). The study fails to deliver further message or function regarding AMC1 and 2, and thus validate their study. Currently, the manuscript just describes how AMC1/2 deletion impacts parasite survival without answering the key question about them: what do they transport? The authors yet have to perform key experiments that would reveal their metabolic function. I would thus recommend the authors work further and determine the function of AMC1 and 2.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of CRB3 in the formation of the primary cilium both in a mouse model and in human cells. They confirm in a conditional knock-out (KO) mouse model that Crb3 is necessary for the formation of the primary cilium in mammary and renal epithelial tissues and the new-born mice exhibit classical traits of ciliopathies. In the mouse mammary gland, the absence of Crb3 induces hyperplasia and tumorigenesis and in the human mammary tumor cells MCF10A the knock-down (KD) of CBR3 impairs ciliogenesis and the formation of a lumen in 3D-cultures with less apoptosis and spindle orientation defects during cell division.

To determine the subcellular localization of CRB3 the authors have expressed exogenously a GFP-CRB3 in MCF10A and found that this tagged protein localizes in cell-cell junctions and around pericentrin, a centrosome marker while endogenous CRB3 localizes at the basal body. To dissect the molecular role of CRB3 the authors have performed proteomic analyses after a pull-down assay with the exogenous tagged-CRB3 and found that CRB3 interacts with Rab11 and is present in the endosomal recycling pathway. CRB3 KD also decreases the interactions between components of the gamma-TuRC. In addition, the authors showed that CRB3 interacts with a tagged-Rab11 by its extracellular domain and that CRB3 promotes the interaction between Rab11 and CEP290 while CRB3 KD decreased the co-localization of GCP6 with Rab11 and gamma-Tub.

Finally, the authors showed that CRB3 depletion cannot activate the Hh pathway as opposed to the Wnt pathway.

Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provide some evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma-tubulin ring complex (gamma-TuRC) component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. Although the authors provide evidence that CRB3 promotes ciliogenesis via (indirect) physical association with Rab11 and gamma-TuRC, the precise mechanism by which CRB3 promotes ciliogenesis remains to be clarified. Specific comments are as follows:

1) For all cell-based assays using shRNA to knock down CRB3, it would be desirable to perform rescue experiments to ensure that the observed phenotype of CRB3 depleted cells is specific and not due to off-target effects of the shRNA.<br /> 2) Figure 3G: it is very difficult to see that the red stained structures are primary cilia.<br /> 3) Figure 5A: it is unfortunate the authors chose not to show the original dataset (Excel file) used for generating this figure; this makes it difficult to interpret the data. It is general policy of the journal to make source data accessible to the scientific community.<br /> 4) The authors have a tendency to overinterpret their data, and not all claims put forth by the authors are fully supported by the data provided.

Reviewer #1 (Public Review):

Here, Ensinck et al. investigated the composition of the yeast mRNA m6A methyltransferase complex required for meiosis. This complex was known to contain three proteins, but is much more complex in mammals, insects and plants. Through IP-MS analysis they identified three more proteins Kar4, Ygl036w and Dyn2. Of these Kar4 and Ygl036w are homologous to Mettl14 and Virma, respectively, and, like the previously described factors are essential for m6A deposition, mating and binding of the reader Pho92 to mRNA during meiosis by evidence acquired with appropriate methodology. Dyn2 is a novel factor not described for any m6A complex and is not essential for m6A deposition, mating and binding of the reader Pho92 to mRNA during meiosis.

In addition, detailed analysis of the Slz1 revealed homology to the mammalian factor m6A complex member ZC3H13 to comprise a conserved complex of five proteins, Mettl3, Mettl14, Mum2/WTAP, Virma and Slz/ZC3H13. When co-expressed in insects cells, they co-purify stoichiometrically and presence of Mum2 as a dimer is also indicated as shown for WTAP.

Complementary to these data they show that stability of the individual complex members is affected in mutants supporting that they are stabilized through complex formation.

Furthermore, the authors then show that kar4 has additional roles in mating that is separable from its role through the m6A complex in meiosis.

The authors employ appropriate methodology throughout to address their aims and present convincing evidence for their claims. The evidence presented here reinforces that the m6A complex is evolutionary highly conserved which has broad scope for its functional analysis in humans and model organisms.

Reviewer #2 (Public Review):

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited by the m6A methyltransferase complexes (MTC). While MTC in mammals/flies/plants consists of at least six subunits, yeast MTC was known to contain only three proteins. Ensinck, Maman, et al. revisited this question using a proteomic approach and uncovered three new yeast MTC components, Kar4/Ygl036w/Dyn2. By applying sequence and structure comparisons, they identified Kar4, Ygl036w, Slz1 as homologs of the mammalian METTL14, VIRMA. ZC3H13, respectively. While these proteins are essential for m6A deposition, the dynein light chain protein, Dyn2, is not involved in mRNA methylation. Interestingly, while mammalian and fly MTCs are configured as MAC (METTL3 and METTL14) and MACOM (other subunits) complexes, yeast MTC subunits appear to have different configurations. Finally, Kar4 has a different role as transcription regulator in mating, which is not mediated by other MTC members. These data establish fundamental framework for the yeast MTC and also provide novel insights for those studying m6A deposition.

Reviewer #1 (Public Review):

This manuscript compiles the colonization of shrubs during the Late Pleistocene in Northern America and Europe by comparing plant sedimentary ancient DNA (sedaDNA) records from different published lake sediment cores and also adds two new datasets from Island. The major findings of this work aim to illuminate the colonization patterns of woody shrubs (Salicaceae and Betulaceae) in these sediment archives to understand this process in the past and evaluate its importance under future deglaciation and warming of the Arctic.

The strength of evidence is solid as methods (sedimentary DNA) and data analyses broadly support the claims because the authors use an established metabarcoding approach with PCR replicates (supporting the replicability of PCR and thereby proving the occurrence of Salicaeae and Betulaceae in the samples) and quantitative estimation of plant DNA with qPCR (which defines the number of cycles used for each PCR amplification to prevent overamplification). However, the extraction methods need more explanation and the bioinformatic pipeline is not well-known and needs also some further description in the main text (not only referring to other publications).

The authors compare their own data with previously published data to indicate the different timing of shrubification in the selected sites and show that Salicaceae occurs always like a pioneer shrub after deglaciation, followed by Betaluaceae with a various time lag. The successive colonization of Salicaceae followed by Betulaceae is explained by its differences in environmental tolerance, the time lag of colonization in the compared records is e.g. explained by varying distance to source areas.

However, there are some weaknesses in the strength of evidence because full sedaDNA plant DNA assessment, quality of the sedaDNA data (relative abundance and richness of sedaDNA plant composition) and results from Blank controls (for sedaDNA) are not fully provided. I think it is important to show how the plant metabarcoding in general worked out, because it is known that e.g. poor richness can be indicative of less preserved DNA and a full plant assessment (shown in the supplement) would be more comprehensive and would likely attract a larger readership.

Further, it would allow us to see the relative abundance in changes of plants and would make it easier to understand if the families Salicaeae and Betulaceae are a major component of the community signal. Further, the possibility to reach higher taxonomic resolution with sedaDNA compared to pollen or to facilitate a continuous record (which is different from macrofossils) is not discussed in the manuscript but should be added. Also, the taxonomic resolution within these families in the discussed datasets would be of interest, also on the sequence type level if tax. assignments are similar.

Another important aspect is how the abundance/occurrence of Salicaceae is discussed. Many studies on sedaDNA confirm an overrepresentation of this family due to better preservation in the sediment, far-distance transport along rivers, or preferences of primers during amplification etc. As this family is the major objective of this study, such discussion should be added to the manuscript and data should be presented accordingly.

I also miss more clarity about how the authors defined the source areas (refugia) of the shrubs. If these source areas are described in other literature I suggest to show them in a map or so. Further, it should be also discussed and explained more in detail which specific environmental preferences these families have, this is too short in the introduction and too unspecific. Also, it would be beneficial to show relative abundances rather than just highlighted areas in the Figures and it would allow us to see if Salicaeae will be replaced by Betulaceae after colonizing or if both families persist together, which might be important to understand future development of shrubs in these areas.

The author started a discussion about shrubification in the future, but a more defined evaluation and discussion of how to use such paleo datasets to predict future shrubification and its consequences for the Arctic would give more significance to the work.

Reviewer #2 (Public Review):

Harding et al have analysed 75 sedaDNA samples from Store Vidarvatn in Iceland. They have also revised the age-depth model of earlier pollen, macrofossil, and sedaDNA studies from Torfdalsvatn (Iceland), and they review sedaDNA studies for first detection of Betulaceae and Salicaceae in Iceland and surrounding areas. Their Store Vidarvatn data are potentially very interesting, with 53 taxa detected in 73 of the samples, but only results on two taxa are presented. Their revised age-depth model cast new light on earlier studies from Torfdalsvatn, which allows a more precise comparison to the other studies. The main result from both sedaDNA and the review is that Salicaceae arrives before Betulaceae in Iceland and the surrounding area. This is a well-known fact from pollen, macrofossil, and sedaDNA studies (Fredskild 1991 Nordic J Bot, Birks & Birks QSR 2014, Alsos et al. 2009, 2016, 2022) and as expected as the northernmost Salix reach the Polar Desert zone (zone A, 1-3{degree sign}C July temperature) whereas the northernmost Betula rarely goes beyond the Southern Tundra (zone D, 8-9{degree sign}C July temperature, Walker et al. 2005 J. Veg. Sci., Elven et al. 2011 http://panarcticflora.org/ ).

My major concern is their conclusion that lag in shrubification may be expected based on the observations that there is a time gap between deglaciation and the arrival of Salicaceae and between the arrival of Salicaceae and Betulaceae. A "lag" in biological terms is defined as the time from when a site becomes environmentally suitable for a species until the species establish at the site (Alexander et al. 2018 Glob. Change Biol.). The climate requirement for Salicaceae highly depends on species. In the three northernmost zones (A-C), it appears as a dwarf shrub, and it only appears as a shrub in the Southern Tundra (D) and Shrub Tundra (E) zone, and further south it is commonly trees. Thus, Salicaceae cannot be used to distinguish between the shrub tundra and more northern other zones, and therefore cannot be used as an indicator for arctic shrubification. Betulaceae, on the other hand, rarely reach zone C, and are common in zone D and further south. Thus, if we assume that the first Betulaceae to arrive in Iceland is Betula nana, this is a good indicator of the expansion of shrub tundra. Thus, if they could estimate when the climate became suitable for B. nana, they would have a good indicator of colonisation lags, which can provide some valuable information about time lags in shrub expansion (especially to islands). They could use either independent proxy or information from the other species recorded in sedaDNA to reconstruct minimum July temperature (see e.g. Parducci et al. 2012a+b Science, Alsos et al. 2020 QSR).

The study gives a nice summary of current knowledge and the new sedaDNA data generated are valuable for anyone interested in the post-glacial colonisation of Iceland. Unfortunately, neither raw nor final data are given. Providing the raw data would allow re-analysing with a more extensive reference library, and providing final data used in their publication will for sure interest many botanists and palaeoecologist, especially as 73 samples provide high time resolution compared to most other sedaDNA studies.

Reviewer #1 (Public Review):

The current study tests the hypothesis that inhibition of ryanodine receptor 2 (RyR2) in failing arrhythmogenic hearts reduces sarcoplasmic Ca leak, ventricular arrhythmias and improves contractile function. A guinea pig model of nonischemic heart failure (HF) was used and randomized to receive dantrolene (DS) or placebo in early or chronic HF. The authors show that DS treatment prevented ventricular arrhythmias and sudden cardiac death by decreasing dispersion of repolarization. The authors conclude that inhibition of RyR2 hyperactivity with DS mitigates the vicious cycle of sarcoplasmic Ca leak-induced increases in diastolic Ca and reactive oxygen species-mediated RyR2 oxidation. Moreover, the consequent increase in sarcoplasmic Ca2+ load improves contractile function.

In general, the study is well designed and the findings are likely to be of interest to the field. The characterization of the phenotypes is comprehensive, however, the study appears relatively weak in terms of the proposed mechanisms. Only in vivo functional analyses were presented with no in vitro analyses. The rationale for only using the male animals remains unclear. Data presented in Supplemental Figure 1 lacks the HF with DS group. As presented, the manuscript appears relatively descriptive in nature.

Reviewer #2 (Public Review):

Joshi et al. investigated the use of dantrolene, an RyR stabilizing drug, in improving contractile function and slowing pathological progression of pressure-overload heart failure. In a guinea pig model, they found that dantrolene treatment reduced cytosolic Ca2+ levels, improved contractility, reduced the incidence of arrhythmias, reduced fibrosis, and slowed the progression of heart failure. Importantly, delaying treatment until 3 weeks after aortic banding (when heart failure was already established) also resulted in improvements in function and decreased arrhythmogenesis. While some of the mechanistic details remain to be worked out, the data suggest that improving intracellular Ca2+ handling can break the vicious cycle of sympathetic activation, ROS production, and further deterioration of cardiac function.

The functional ECG and echo data are convincing, and very clearly demonstrate the positive effects of dantrolene in heart failure. This is important because dantrolene is already FDA-approved to treat malignant hyperthermia and muscle spasms, so repurposing this drug as a heart failure therapeutic might have a straightforward path to clinical implementation. This also highlights the non-specific nature of dantrolene to interact with RyR1, and therefore, potential side effects. However, this does not detract from the main proof-of-concept demonstrated here.

The guinea pig model employed here is also a strength, as the guinea pig has intracellular Ca2+ handling and ionic currents that are much more similar to human (vs. a murine model, for example).

One weakness is the exclusion of female animals from the study. The authors report more heterogeneity in the progression of HF in the female guinea pig model, however it will be very important to determine effects of dantrolene in the female heart, as there are considerable known sex differences in intracellular Ca2+ handling and contractility. Therefore, it is possible that dantrolene could have sex-dependent effects.

The title and parts of the discussion of the manuscript focus on 'repolarization reserve'. This term is often used in the realm of safety pharmacology, and 'reserve' refers to the fact that blocking a single K+ channel (for example) may not impact action potential duration because there may be enough other K+ currents to ensure proper repolarization. The repolarization reserve refers to this overall balance of depolarizing and repolarizing currents and potential redundancies to ensure proper repolarization. Although the present study clearly demonstrates QT shortening with dantrolene (thus, there must be a change in the balance of depolarizing and repolarizing currents), the study does not definitively demonstrate changes in any membrane currents. While this may seem like a minor point of terminology, it may mislead readers as to the main focus of the study, which is not at all on ionic currents, but on functional outcomes.

Reviewer #1 (Public Review):

This study investigates the structuring of long calls in orangutans. The authors demonstrate long calls are structured around full pulses, repeated following a regular tempo (isochronic rhythm). These full pulses are themselves structured around different sub-pulses, themselves repeated following an isochronic rhythm. The authors argue this patterning is evidence for self-embedded, recursive structuring in orangutang long calls.

The analyses conducted are robust and compelling and they support the rhythmicity the authors argue is present in the long calls. Furthermore, the authors went above and beyond and confirmed acoustically the sub-categories identified were accurate.

However, I believe the manuscript would benefit from a formal analysis of the specific recursive patterning occurring in the long call. Indeed, as of now, it is difficult for the reader to identify what the authors argue to be recursion and distinguish it from simple repetitions of motifs, which is essential. Although the authors already discuss briefly why linear patterning is unlikely, the reader would benefit from expanding on this discussion section and clarifying the argument here (a lay terminology might help). I believe an illustration here might help.<br /> In the same logic, I believe a tree similar to the trees used in linguistics to illustrate hierarchical structuring would help the reader understand the recursive patterning in place here. This would also help get the "big picture", as Fig 1A is depicting a frustratingly small portion of the long call.

Notwithstanding these comments, this paper would provide crucial evidence for recursion in the vocal *production* of a non-human ape species. The implication it would have would represent a key shift in the field of language evolution. The study is very elegant and well-constructed. The paper is extremely well written, and the point of view adopted is original, well-argued and compelling.

Reviewer #2 (Public Review):

I am not qualified to judge the narrow claim that certain units of the long calls are isochronous at various levels of the pulse hierarchy. I will assume that the modelling was done properly. I can however say that the broad claims that (i) this constitutes evidence for recursion in non-human primates, (ii) this sheds light on the evolution of recursion and/or language in humans are, when not made trivially true by a semantic shift, unsupported by the narrow claims. In addition, this paper contains errors in the interpretation of previous literature.

The main difficulty when making claims about recursion is to understand precisely what is meant by "recursion" (arguably a broader problem with the literature that the authors engage with). The authors offer some characterization of the concept which is vague enough that it can include anything from "celestial and planetary movement to the splitting of tree branches and river deltas, and the morphology of bacteria colonies". With this appropriately broad understanding, the authors are able to show "recursion" in orangutans' long calls. But they are, in fact, able to find it everywhere. The sound of a plucked guitar string, which is a sum of self-similar periodic patterns, count as recursive under their definition as well.

One can only pick one's definition of recursion, within the context of the question of interest: evolution of language in humans. One must try to name a property which is somewhat specific to human language, and not a ubiquitous feature of the universe we live in, like self-similarity. Only after having carved out a sufficiently distinctive feature of human language, can we start the work of trying to find it in a related species and tracing its evolutionary history. When linguists speak of recursion, they speak of in principle unbounded nested structure (as in e.g. "the doctor's mother's mother's mother's mother ..."). The author seems to acknowledge this in the first line of the introduction: "the capacity to *iterate* a signal within a self-similar signal" (emphasis added). In formal language theory, which provides a formal and precise definition of one notion of recursivity appropriate for human language, unbounded iteration makes a critical difference: bounded "nested structures" are regular (can be parsed and generated using finite-state machines), unbounded ones are (often) context-free (require more sophisticated automaton). The hierarchy of pulses and sub-pulses only has a fixed amount of layers, moreover the same in all productions; it does not "iterate".

Another point is that the authors don't show that the constraints that govern the shape of orangutans long calls are due to cognitive processes. Any oscillating system will, by definition, exhibit isochrony. For instance, human trills produce isochronouns or near isochronous pulses. No cognitive process is needed to explain this; this is merely the physics of the articulators. Do we know that the rhythm of the pulses and sub-pulses in orangutans is dictated by cognition as opposed to the physics of the articulators?

Even granting the authors' unjustified conclusion that wild orangutans have "recursive" structures and that these are the result of cognition, the conclusions drawn by the authors are too often fantastic leaps of induction. Here is a cherry-picked list of some of the far-fetched conclusions:

- "our findings indicate that ancient vocal patterns organized across nested structural strata were likely present in ancestral hominids". Does finding "vocal patterns organized across nested structural strata" in wild orangutans suggest that the same were present in ancestral hominids?<br /> - "given that isochrony universally governs music and that recursion is a feature of music, findings (sic.) suggest a possible evolutionary link between great ape loud calls and vocal music". Isochrony is also a feature of the noise produced by cicadas. Does this suggest an evolutionary link between vocal music and the noise of cicadas?

Finally, some passages also reveal quite glaring misunderstandings of the cited literature. For instance:

- "Therefore, the search for recursion can be made in the absence of meaning-base operations, such as Merge, and more generally, semantics and syntax". It is precisely Chomsky's (disputable) opinion that the main operation that govern syntax, Merge, has nothing to do with semantics. The latter is dealt within a putative conceptual-intentional performance system (in Chomsky's terminology), which is governed by different operations.<br /> - "Namely, experimental stimuli have consisted of artificial recursive signal sequences organized along a single temporal scale (though not structurally linear), similarly with how Merge and syntax operate". The minimalist view advocated by Chomsky assumes that mapping a hierarchichal structure to a linear order (a process called linearizarion) is part of the articulatory-perceptual system. This system is likewise not governed by Merge and is not part of "syntax" as conceived by the Chomskyan minimalists.

Reviewer #1 (Public Review):

This work introduces a new method of imaging the reaction forces generated by small crawling organisms and applies this method to understanding locomotion of Drosophila larva, an important model organism. The force and displacement data generated by this method are a qualitative improvement on what was previously available for studying the larva, improving simultaneously the spatial, temporal, and force resolution, in many cases by an order of magnitude. The resulting images and movies are quite impressive.

As it shows the novel application of recent technological innovations, the work would benefit from more detail in the explanation of the new technologies, of the rationales underlying the choice of technology and certain idiosyncratic experimental details, and of the limitations of the various techniques. In the methods, the authors need to be sure to provide sufficient detail that the work can be understood and replicated. The description of the results and the theory of motion developed here focus only on forces generated when the larva pushes against the substrate and ignores the equally strong adhesive forces pulling the larva onto the substrate.

The substrate applies upward, downward, and horizontal forces on the larva, but only upward and downward forces are measured, and only upward forces are considered in the discussions of "Ground Reactive Forces." An apparent weakness of the WARP technique for the study of locomotion is that it only measures forces perpendicular to the substrate surface ("vertical forces" in Meek et al.), while locomotion requires the generation of forces parallel to the substrate ("horizontal forces"). It should be clarified that only vertical forces are studied and that no direct information is provided about the forces that actually move the larva forward (or about the forces which impede this motion and are also generated by the substrate). Along with this clarification, it would be helpful to include a discussion of other techniques, especially micropillar arrays and traction force microscopy, that directly measure horizontal forces and of why these techniques are inappropriate for the motions studied here.

The larvae studied are about 1 mm long and 0.1 mm in cross-section. Their volumes are therefore on order 0.01 microliter, their masses about 0.01 mg, and their weights in the range of 0.1 micronewton. This contrasts with the force reported for a single protpodium of 1 - 7 micronewtons. This is not to say that the force measurements are incorrect. Larvae crawl easily on an inverted surface, showing gravitational forces are smaller than other forces binding the larva to the substrate. The forces measured in this work are also of the same magnitude as the horizontal forces reported by Khare et al. (ref 32) using micropillar arrays.

I suspect that the forces adhering the larva to the substrate are due to the surface tension of a water layer. This would be consistent with the ring of upward stress around the perimeter of the larva visible in S4D, E and in video SV3. The authors remark that upward deflection of the substrate may be due to the Poisson's ratio of the elastomer, but the calibration figure S5 shows that these upward deflections and forces are much smaller than the applied downward force. In any case, there must be a downward force on the larva to balance the measured upward forces and this force must be due to interaction with the substrate. It should be verified that the sum of downward minus upward forces on the gel equals the larva's weight (given the weight is neglible compared to the forces involved, this implies that the upward and downward forces should sum to 0)

Much of the discussion and the model imply that the sites where the larva exerts downward force on the gel are the sites where horizontal propulsion is generated. This assumption should be justified. Can the authors rule out that the larva 'pulls' itself forward using surface tension instead of 'pushing' itself forward using protopodia?

More detail should be provided about the methods, their limitations, and the rationale behind certain experimental choices.

Three techniques are introduced here to study how a crawling larva interacts with the substrate: standard brightfield microscopy of a larva crawling in an agarose capillary, ERISM imaging of an immobilized larva, and WARP imaging of a crawling larva. The authors should make clear why each technique was chosen for a particular study - e.g. could the measurements using brightfield microscopy also be accomplished using WARP? They should also clarify how these techniques relate to and possibly improve on existing techniques for measuring forces organisms exert on a substrate, particularly micropillar arrays and Traction Force Microscopy.

As written, "(ERISM) (19) and a variant, Wavelength Alternating Resonance Pressure microscopy (WARP) (20) enable optical mapping of GRFs in the nanonewton range with micrometre and millisecond precision..." (lines 53-55) may generate confusion. ERISM as described in this work has a much lower temporal resolution (requires the animal to be still for 5 seconds - lines 474-5); In this work, WARP does not appear to have nanonewton precision (judging by noise on calibration figures) and it is not clear that it has millisecond precision (the camera used and its frame rate should be specified in the methods).

It would be helpful to have a discussion of the limits of the techniques presented and tradeoffs that might be involved in overcoming them. For instance, what is the field of view of the WARP microscope, and could it be increased by choosing a lower power objective? What would be required to allow WARP microscopy to measure horizontal forces? Can a crawling larva be imaged over many strides by recentering it in the field of view, or are there only particular regions of the elastomer where a measurement may be made?

Reviewer #2 (Public Review):

With a much higher spatiotemporal resolution of ground dynamics than any previous study, the authors uncover new "rules" of locomotory motor sequences during peristalsis and turning behaviors. These new motor sequences will interest the broad neuroscience community that is interested in the mechanisms of locomotion in this highly tractable model. The authors uncover new and intricate patterns of denticle movements and planting that seem to solve the problem of net motion under conditions of force-balance. Simply put, the denticulated "feet" or tail of the Drosophila larva are able to form transient and dynamic anchors that allow other movements to occur.

The biology and dynamics are well-described. The physics is elementary and becomes distracting when occasionally overblown. For example, one doesn't need to invoke Newton's third law, per se, to understand why anchors are needed so that peristalsis can generate forward displacements. This is intuitively obvious. Another distracting allusion to "physics" is correlating deformation areas with displaced volume, finding that "volume is a consequence of mass in a 2nd order polynomial relationship". I have no idea what this "physics" means or what relevance this relationship has to the biology of locomotion.

The ERISM and WARP methods are state-of-the-art, but aside from generally estimating force magnitudes, the detailed force maps are not used. The most important new information is the highly accurate and detailed maps of displacement itself, not their estimates of applied force using finite element calculations. In fact, comparing displacements to stress maps, they are pretty similar (e.g., Fig 4), suggesting that all experiments are performed in a largely linear regime. It should also be noted that the stress maps are assumed to be normal stresses (perpendicular to the plane), not the horizontal stresses that are the ones that actually balance forces in the plane of animal locomotion.

But none of this matters. The real achievements are the new locomotory dynamics uncovered with these amazing displacement measurements. I'm only asking the authors to be precise and down-to-earth about the nature of their measurements.

It would be good to highlight the strength of the paper -- the discovery of new locomotion dynamics with high-resolution microscopy -- by describing it in simple qualitative language. One key discovery is the broad but shallow anchoring of the posterior body when the anterior body undertakes a "head sweep". Another discovery is the tripod indentation at the tail at the beginning of peristalsis cycles.

As far as I know, these anchoring behaviors are new. It is intuitively obvious that anchoring has to occur, but this paper describes the detailed dynamics of anchoring for the first time. Anchoring behavior now has to be included in the motor sequence for Drosophila larva locomotion in any comprehensive biomechanical or neural model.

Reviewer #1 (Public Review):

This study reports a long-term, multisite study of tropical herbivory on Piper plants. The results are clear that lack of water leads to lower plant survival and altered herbivory. The results varied substantially among sites. The caveats are that ecosystem processes beyond water availability are not investigated although they are brought into play in the title and in the paper, that herbivory beyond leaf damage was not reported (there might be none, the reader needs to be shown the evidence for this), that herbivore diversity is defined by leaf damage (authors need to give evidence that this is a valid inference), that the plots were isolated from herbivores beyond their borders, that the effects of extreme climate events were isolated to Peru, that intraspecific variation in the host plants needs to be explained and interpreted in more detail, the results as reported are extremely complicated, the discussion is overly long and diffuse.

Reviewer #2 (Public Review):

This is an important and large experimental study examining the effects of plant species richness, plant genotypic richness, and soil water availability on herbivory patterns on Piper species in tropical forests.

A major strength is the size of the study and the fact that it tackled so many potentially important factors simultaneously. The authors examined both interspecific plant diversity and intraspecific plant diversity. They crossed that with a water availability treatment. And they repeated the experiment across five geographically separated sites.

The authors find that both water availability and plant diversity, intraspecific and interspecific, influence herbivore diversity and herbivory, but that the effects differ in important ways across sites. I found the study to be solid and the results to be very convincing. The results will help the field grapple with the importance of environmental change and biodiversity loss and how they structure communities and alter species interactions.

Reviewer #1 (Public Review):

In principle a very interesting story, in which the authors attempt to shed light on an intriguing and poorly understood phenomenon; the link between damage repair and cell cycle re-entry once a cell has suffered from DNA damage. The issue is highly relevant to our understanding of how genome stability is maintained or compromised when our genome is damaged. The authors present the intriguing conclusion that this is based on a timer, implying that the outcome of a damaging insult is somewhat of a lottery; if a cell can fix the damage within the allocated time provided by the "timer" it will maintain stability, if not then stability is compromised. If this conclusion can be supported by solid data, the paper would make a very important contribution to the field.

However, the story in its present form suffers from a number of major gaps that will need to be addressed before we can conclude that MASTL is the "timer" that is proposed here. The primary concern being that altered MASTL regulation seems to be doing much more than simply acting as a timer in control of recovery after DNA damage. There is data presented to suggest that MASTL directly controls checkpoint activation, which is very different from acting as a timer. The authors conclude on page 8 "E6AP promoted DNA damage checkpoint signaling by counteracting MASTL", but in the abstract the conclusion is "E6AP depletion promoted cell cycle recovery from the DNA damage checkpoint, in a MASTL-dependent manner". These 2 conclusions are definitely not in alignment. Do E6AP/MASTL control checkpoint signaling or do they control recovery, which is it?

Also, there is data presented that suggest that MASTL does more than just controlling mitotic entry after DNA damage, while the conclusions of the paper are entirely based on the assumption that MASTL merely acts as a driver of mitotic entry, with E6AP in control of its levels. This issue will need to be resolved.

and finally, the authors have shown some very compelling data on the phosphorylation of E6AP by ATM/ATR, and its role in the DNA damage response. But the time resolution of these effects in relation to arrest and recovery have not been addressed.

Revised manuscript:<br /> I think the authors did a good job in revising the paper, and provide compelling support for a timer function in the checkpoint. I do think they still have missed one important point how MASTL could act as a timer to control recovery. The data clearly show that MASTL somehow controls ATM/ATR activity, whilst their final model (fig.9) places MASTL upstream of CDK activity, without mentioning its feedback on ATM/ATR. I think there are 2 possible explanations for the timer function of MASTL they have discovered here, both may be relevant. The first is enhanced CDK activation by direct control of CDK phosphorylation through MASTL/B55/PP2A. The second is through MASTL-mediated shut-down of ATM/ATR activation (mechanism to be determined) which is also reported here. Their final model and discussion do not display sufficient appreciation for this latter option, and I would argue that the HU-recovery experiment shown in Fig.5B is actually in strong support of the second explanation, rather than the first.

Reviewer #2 (Public Review):

This manuscript describes a role for the ATM-E6AP-MASTL pathway in DNA damage checkpoint recovery. However, the data in the first version of the manuscript strongly suggest that E6AP is involved in checkpoint activation, which raises doubts about the exact function of this pathway. Additional minor issues were raised regarding the quality of some of the data. Although some minor points were addressed in the revised manuscript, the major issue whether the E6AP-MASTL pathway mediates checkpoint activation or checkpoint recovery was not experimentally addressed. Instead, the authors state that "the expression level of MASTL is not upregulated during the activation stages of the DNA damage checkpoint". However, their data show otherwise: MASTL upregulation coinciding with RPA phosphorylation and p-ATM/ATR signal.

I am therefore not convinced the revised manuscript sufficiently addressed the comments to fully support the conclusions.

Reviewer #1 (Public Review):

Membrane receptor guanylyl cyclases are important for many physiological processes but their structures in full-length and their mechanism are poorly understood. Caveney et al. determined the cryo-EM structure of a highly engineered GC-C in a complex with endogenous HSP90 and CDC37. The structural work is solid and the structural information will be useful for the membrane receptor guanylyl cyclases field and the HSP90 field.

Reviewer #2 (Public Review):

Caveney et al have overexpressed an engineered construct of the human membrane receptor guanyl cyclase GC-C in hamster cells and co-purified it with the endogenous HSP90 and CDC37. They have then determined the structure of the resultant complex by single particle cryoEM reconstruction at sufficient resolution to dock existing structures of HSP90 and CDC37, plus an AlphaFold model of the pseudo-kinase domain of the guanylyl cyclase. The novelty of the work stems from the observation that the pseudo-kinase domain of GC-C associates with CDC37 and HSP90 similarly to how the bona fide protein kinases CDK4, CRAF and BRAF have been previously shown to interact.

Reviewer #3 (Public Review):

A detailed understanding of how membrane receptor guanylyl cyclases (mGC) are regulated has been hampered by the absence of structural information on the cytoplasmic regions of these signaling proteins. The study by Caveney et al. reports the 3.9Å cryo-EM structure of the human mGC cyclase, GC-C, bound to the Hsp90-Cdc37 chaperone complex. This structure represents a first view of the intracellular functional domains of any mGC and answers without doubt that Hsp90-Cdc37 recognizes mGCs via their pseudokinase (PK) domain. This is the primary breakthrough of this study. Additionally, the new structural data reveals that the manner in which Hsp90-Cdc37 recognizes the GC-C PK domain C-lobe is akin to how kinase domains of soluble kinases docks to the chaperone complex. This is the second major finding of this study, which provides a concrete framework to understand, more broadly, how Hsp90-Cdc37 recruits a large number of other diverse client proteins containing kinase or pseudokinase domains. Finally, the Hsp90-Cdc37-GC-C structure offer clues as to how GC-C may be regulated by phosphorylation and/or ubiquitinylation by serving as a platform for recruitment of PP5 and/or E3 ligases.

Reviewer #1 (Public Review):

The authors apply a new approach to monitor brain-wide changes in sensory-evoked hemodynamic activity after focal stroke in fully conscious rats. Using functional ultrasound (fUS), they report immediate and lasting (up to 5 days) depression of sensory-evoked responses in somatosensory thalamic and cortical regions.

Strengths: This a technically challenging and proof-of-concept study that employs new methods to study brain-wide changes in sensory-evoked neural activity, inferred from changes in cerebral blood flow. Despite the minor typos/grammatical errors and small sample size, the authors provide compelling images and rigorous analysis to support their conclusions. Overall, this was a very technically difficult study that was well executed. I believe that it will pave the way for more extensive studies using this methodological approach. Therefore I support this study and my recommendations to improve it are relatively minor in nature and should be simple for the authors to address.

Weaknesses: The primary weakness of this paper is the small sample sizes. Drawing conclusions based on the small sham control group (n=2) or 5-day stroke recovery group (n=2), is rather tenuous. One way to alleviate some uncertainty with regard to the conclusions would be to state in the discussion that the findings (ie. loss of thalamocortical function after stroke) are perfectly consistent with previous studies that examined thalamocortical function after stroke. The authors missed some of these supporting studies in their reference list (see PMID: 28643802, 1400649). A second issue that can easily be resolved is their analysis of the 69 brain regions. This seems like a very important part of the study and one of the primary advantages of employing efUS. As presented, I had difficulty seeing the data. I think it would be worthwhile to expand Fig 3 (especially 3C) into a full-page figure with an accompanying table in the Supplementary info section describing the % change in CBF for each brain region.

Other Recommendations for the authors:.<br /> - Since there is variability in spreading depolarizations, was there any trend in the relationship between # SD's and ischemic volume? I know there are few data points but a scatterplot might be of interest.<br /> - For statistical comparisons of 'response curves' in Fig 3 and 4, what exactly was the primary dependent measure: changes in peak amplitude (%) or area under the curve?<br /> - There are several typos and minor grammatical errors in the manuscript. Some editing is recommended.

Reviewer #2 (Public Review):

Brunner et al. present a new and promising application of functional ultrasound (fUS) imaging to follow the evolution of perfusion and haemodynamics upon thrombotic stroke in awake rats. The authors leveraged a chemically induced occlusion of the rat Medial Cerebral Artery (MCA) with ferric chloride in awake rats, while imaging with fUS cerebral perfusion with high spatio and temporal resolution (100µm x 110µm x 300µm x 0.8s). The authors also measured evoked haemodynamic response at different timepoints following whisker stimulation.

As the fUS setup of the authors is limited to 2D imaging, Brunner and colleagues focused on a single coronal slice where they identified the primary Somatosensory Barrel Field of the Cortex (S1BF), directly perfused by the MCA and relay nuclei of the Thalamus: the Posterior (Po) and the Ventroposterior Medial (VPM) nuclei of the Thalamus. All these regions are involved in the sensory processing of whisker stimulation. By investigating these regions the authors present the hyper-acute effect of the stroke with these main results:

- MCA occlusion results in a fast and important loss of perfusion in the ipsilesional cortex.<br /> - Thrombolysis is followed by Spreading Depolarisation measured in the Retrosplenial cortex.<br /> - Stroke-induced hypo-perfusion is associated with a significant drop in ipsilesional cortical response to whisker stimulation, and a milder one in ipsilesional subcortical relays.<br /> - Contralesional hemisphere is almost not affected by stroke with the exception of the cortex which presents a mildly reduced response to the stimulation.

In addition, the authors demonstrate that their protocol allows to follow up stroke evolution up to five days post-induction. They further show that fUS can estimate the size of the infarcted volume with brilliance mode (B-mode), confirming the presence of the identified lesional tissue with post-mortem cresyl violet staining.

Upon measuring functional response to whisker stimulation 5 days after stroke induction, the authors report that:<br /> - The ipsilesional cortex presents no response to the stimulation<br /> - The ipsilesional thalamic relays are less activated than hyper acutely<br /> - The contralesional cortex and subcortical regions are also less activated 5d after the stroke.

These observations mainly validate the new method as a way to chronically image the longitudinal sequelae of stroke in awake animals. However, the potentially more intriguing results the authors describe in terms of functional reorganization of functional activity following stroke appear to be preliminary, and underpowered ( N = 5 animals were imaged to describe hyper-acute session, and N = 2 in a five day follow-up). While highly preliminary, the research model proposed by the author (where the loss of the infarcted cortex induces reduces activity in connected regions, whether by cortico-thalamic or cortico-cortical loss of excitatory drive), is interesting. This hypothesis would require a greatly expanded, sufficiently powered study to be validated (or disproven).

Reviewer #3 (Public Review):

The authors set out to demonstrate the utility of functional ultrasound for evaluating changes in brain hemodynamics elicited acutely and subacutely by the middle cerebral artery occlusion model of ischemic stroke in awake rats.

Functional ultrasound affords a distinct set of tradeoffs relative to competing imaging modalities. Acclimatization of rats for awake imaging has proven difficult with most, and the high quality of presented data in awake rats is a major achievement. The major weakness of the approach is in its being restricted to single-slice acquisitions, which also complicates the registration of acquisition across multiple imaging sessions within the same animal. Establishing that awake imaging represents an advancement in relation to studies under anesthesia hinges upon the establishment of the level of stress experienced by the animals in the course of imaging, i.e., requires providing data on the assessment of stress over the course of these long imaging sessions. This is particularly significant given how significant a stressor physical restraint has been established to be in rodent models of stress. Furthermore, assessment of the robustness of these measurements is of particular significance for supporting the wide applicability of this approach to preclinical studies of brain injury: the individual animal data (effect sizes, activation areas, kinetics) should thus be displayed and the statistical analysis expanded. Both within-subject, within/across sessions, and across-subjects variability should be evaluated. Thoughtful comments on the relationship between power doppler signal and cerebral blood volume are important to include and facilitate comparisons to studies recording other blood volume-weighted signals. Finally, the contextualization of the observations with respect to other studies examining acute and subacute changes in brain hemodynamics post focal ischemic stroke in rats is needed. It is also quite helpful, for establishing the robustness of the approach, when the statistical parametric maps are shown in full (i.e. unmasked).

Reviewer #1 (Public Review):

Many labs worldwide now use the blind source deconvolution technique to identify the firing patterns of multiple motor units simultaneously in human subjects. This technique has had a truly transformative effect on our understanding of the structure of motor output in both normal subjects and, increasingly, in persons with neurological disorders. The key advance presented here is that the software provides real-time identification of these firing patterns.

The main strengths are the clarity of the presentation and the great potential that real-time decoding will provide. Figures are especially effective and statistical analyses are excellent.

The main limitation of the work is that only male subjects were included in the validation of the software. The reason given - that yield of number of motor units identified is generally larger in males than females - is reasonable in the sense that this is the first systematic test of this real-time approach. At a minimum, however, the authors should clearly commit to future work with female subjects and emphasize the importance of considering sex differences.

A second weakness is that the Introduction does a poor job of establishing the potential importance of the real-time approach.

Reviewer #2 (Public Review):

Rossato et al present I-spin live, a software package to perform real-time blind-source separation-based sorting of motor unit activity. The core contribution of this manuscript is the development and validation of a software package to perform motor unit sorting, apply the resulting motor unit filters in real-time during muscle contractions, and provide real-time visual feedback of the motor unit activity.

I have a few concerns with the work as presented:

- I found it challenging to specifically understand the technical contributions of this manuscript. The authors do not appear to be claiming anything novel algorithmically (with respect to spike sorting) or methodologically (with respect to manual editing of spikes before the use of the algorithms in real-time). My takeaway is that the key contributions are C1) development of an open-source implementation of the Negro algorithm, C2) validating it for real-time application (evaluating its sorting efficacy, and closed-loop performance, etc), and developing a software package to run in closed-loop with visual feedback. I will comment on each of these items separately below. It would be great if the authors could more explicitly lay out the key contributions of this manuscript in the text.

- Related to the above, much of the validation of the algorithms in this manuscript has a "trust me" feel - the authors note that the Negro et al. algorithm has already been validated, so very few details or presentations of primary data showing the algorithm's performance are shown. Similarly, the efficacy of the decomposition approach is evaluated using manual editing of the sorting output as a reference, which is a subjective process, and users would greatly benefit from explicit guidance. There are very few details of manual editing shown in this manuscript (I believe the authors reference the Hug et al. 2021 paper for these details), and little discussion of the core challenges and variability of that process, even though it seems to be a critical step in the proposed workflow. So this is very hard to evaluate and would be challenging for readers to replicate.

- I found the User Guide in the Github package to be easy to follow. Importantly, it seems heavily tied to the specific hardware (Quattrocento). I understand it may be difficult to make the full software package work with different hardware, but it seems important to at least make an offline analysis of recorded data possible for this package to be useful more broadly.

- While this may be a powerful platform, it is also very possible that without more details and careful guidance for users on potential pitfalls, many non-experts in sorting could use this as a platform for somewhat sloppy science.

- The authors mention that data is included with the Github software package. I could not find any included data, or instructions on how to run the software offline on example data.

- Given the centrality of the real-time visual feedback to their system, the authors should show some examples of the actual display etc. so readers can understand what the system in action actually looks like (I believe there is no presentation of the actual system in the manuscript, just in the User Guide). Similarly, it would be helpful to have a schematic figure outlining the full workflow that a user goes through when using this system.

- The authors note all data was collected with male subjects because more motor units can be decomposed from male subjects relative to females. But what is the long-term outlook for the field if studies avoid female subjects because their motor units may be harder to decompose? This should at least be discussed - it is an important challenge for the field to solve, and it is unacceptable if new methods just avoid this problem and are only tested on male subjects.

Specific comments on the core contributions of this paper:

C1. Development of an open-source implementation of the Negro algorithm<br /> This seems an important contribution and useful for the community. There are very few figures showing any primary data, the efficacy of sorting, raw traces showing the waveforms that are identified, cluster shapes, etc. I realize the high-level algorithm has been outlined elsewhere, but the implementation in this package, and its efficacy, is a core component of the system and the claims being made in this paper. Much more presentation of data is needed to evaluate this.

Similarly, more information on the offline manual editing process (e.g. showing before/after examples with primary data) would be important to gain confidence in the method. The current paper shows application to both surface EMG and intramuscular EMG, but I could not find IM EMG examples in the Hug paper (apologies if I missed them). Surface and IM data are very, very different, so one would imagine the considerations when working with them should also be different.

All descriptions of math/algorithms are presented in text, without any actual math, variable definitions, etc. This presentation makes it difficult to understand what is done. I would strongly recommend writing out equations and defining variables where possible.

More details on how the level of sparseness is controlled during optimization would be helpful. And how this sparseness penalty is weighed against other optimization costs.

Overall the paper is not very rigorous about the accuracy of motor unit identification. For example, the authors note that SIL of 0.9 is generally used for offline evaluation (why is this acceptable?), but it was lowered to 0.8 for particular muscles in this study. But overall, it is unclear how sorting accuracy/inaccuracy affects performance in the target applications of this work.

C2. For real-time experiments, variability/jitter is important to characterize. Fig. 4 seems to be presenting mean computational times, etc, but no presentation of variability is shown. It would be helpful to depict data distributions somehow, rather than just mean values.

There is some description about the difference between units identified during baseline contractions, and how they might be misidentified during online contractions ("Accuracy of the real-time identification..."). This should be described in more detail.

Fig. 6: Given that a key challenge in sorting should be that collisions occur during large contractions, much more primary data should be presented/visualized to show how the accuracy of sorting changes during larger contractions in online experiments.

Fig.7: In presenting the accuracy of biofeedback, it is very hard to gain any intuition for performance by just looking at RMSE values. Showing the online decoded and edited trajectories would help readers understand the magnitude of errors.

Reviewer #3 (Public Review):

In this manuscript, Rossato and colleagues present a method for real-time decoding of EMG into putative single motor units. Their manuscript details a variety of decision points in their code and data collection pipeline that lead to a final result of recording on the order of ~10 putative motor units per muscle in human males. Overall the manuscript is highly restricted in its potential utility but may be of interest to aficionados. For those outside the field of human or nonhuman primate EMG, these methods will be of limited interest.

Notes<br /> 1. Artificial data should be used with this method to provide ground truth performance evaluations. Without it, the study assumptions are unchallenged and could be seriously flawed.

2. From the point of view of a motor control neuroscientist studying movement in animals other than humans or non-human primates, the title was misleadingly hopeful. The use case presented in this study requires human participants to perform isometric contractions, facilitating spatially redundant recordings across the muscle for the algorithm to work. It is unclear whether these methods will be of utility to use cases under more physiological conditions (ie. dynamic movement).

3. The text states that "EMG signals recorded with an array of electrodes can be considered and instantaneous mixture of the original motor unit spike trains and their delayed versions." While this may be a true statement, it is not a complete statement, since motor units at distal sites may be shared, not shared, or novel. It was not clear to me whether the diversity of these scenarios would affect the performance of the software or introduce artifacts. In other words, if at site 1 you can pick up the bulk signal of units 1,2,3,4; at site two you pick up the signals of units 2,3,4,5 and site three you pick up the signal of units 3,4,5,6, what does the algorithm assume is happening and what does it report and why?

4. I could not fully appreciate the performance gap solved by the current methods. What was not achievable before that is now achievable? The 125 ms speed of deconvolution? What was achievable before? Intro text around ln 85 states that 'most of the current implementations of this approach rely on offline processing, which restricts its ability to be used..." but no reference is provided here about what the non 'most' of can achieve.

5. Relatedly, it would have been nice to see a proof of concept using real-time feedback for some kind of biofeedback signal. If that is the objective here, why not show us this? I found the actual readout metrics of performance rather esoteric. They may be of interest to very close experts so I will defer to them for input.

6. I was disappointed to see that only male participants are used because of some vague statement that 'it is widely known in the field' that more motor units can be resolved in males, without thorough referencing. It seems that the objective of the algorithm is the speed of analysis, not the number of units, which makes the elimination of female participants not justified.

7. Human curation is often used in spike sorting, but the description of criteria used in this step or how the human curation choices are documented is missing.

8. The authors might try to add text to be more circumspect about the contributions of this method. I would recommend emphasizing the conceptual advances over the specifics of the performance of the algorithm since processor speed and implementation of the ideas in a faster environment (Matlab can be slow) will change those outcomes in a trivial way. Yet, much of the results section is very focused on these metrics.<br /> Minor<br /> Ln 115, "inversing" is not a word. "inverse" is not a verb<br /> Ln 186, typo, bioadhesive<br /> MVC should be defined on first use. It is currently defined on 3rd use or so.<br /> The term rate is used in a variety of places without units. Eg line 465 but not limited to that

Reviewer #1 (Public Review):

The manuscript by Kadkova et al. describes an electrophysiological analysis of 3 neurodevelopmental disease-causing SNAP-25 mutations in hippocampal neuron autaptic cultures. The work expands on a prior study of these 3 mutations, along with several others in SNAP-25, that was performed in acutely dissociated hippocampal cultures by another group (Alten et al, 2021). Most of the physiology defects found are pretty similar for the 3 mutations the two research groups characterized, with differences largely found in the effects on the size of the readily releasable pool (RRP) of SVs. These differences could be due to technical differences in the approach but are also likely to reflect in part differences in autapses as a model that has been previously described. In addition to the physiological analysis in cultured neurons, the current work extends the analysis beyond the prior study by analyzing the effects of these SNAP-25 mutations in vitro liposome fusion assays with purified proteins, and some modeling of the effects on energy landscapes during priming and fusion.

The authors use lentiviral expression of wildtype or one of the 3 mutants in SNAP-25 autaptic neurons and assay neuronal survival and synaptic output. The authors also combine wildtype with each of the 3 mutants as well, given these diseases manifest as spontaneous mutations in only 1 of the SNAP-25 alleles, suggesting a dominant effect. The authors observe that the V48F and D166Y alleles (that are suggested to disrupt the Syt1-SNAP-25/SNARE interface) result in a very large increase in a spontaneous release that exceeds the Syt1 null mutant alone, suggesting an effect on spontaneous SV release beyond a lack of Syt1 regulation of SNARE-mediated fusion. In contrast, Syt1 nulls have a much more severe loss of evoked release, though both V48F and D166Y also have modest decreases in release. They find both mutants also cause a decrease in the RRP. Applying some modeling for these results, the authors suggest V48F and D166Y lower the energy barrier for fusion, creating enhanced spontaneous release rates and causing a decrease of the RRP. They also find evidence for reduced SV priming. In contrast, a SNAP-25 I167N disease mutation in the SNARE assembly interaction layer causes dramatic decreases in both evoked and spontaneous release, consistent with a disruption to SNARE assembly/stability. In vitro fusion assays with these mutant SNAP-25 alleles were also done and provided supportive evidence for these interpretations for all 3 alleles. The ability to control calcium, Syt1, PIP2, and Complexin levels in the in vitro assays provided additional information on defining the precise steps of the fusion process these mutations disrupt. Together, the study indicates the I167N mutation acts as a dominant-negative allele to block fusion, while the other two alleles have both loss- and gain-of function properties that cause more complex disruptions that decrease evoked release while dramatically enhancing spontaneous fusion.

Overall, these results build on prior work and shed light on how disruptions to the SNAP-25 t-SNARE alter the process of SV priming and fusion.

Reviewer #2 (Public Review):

Kádková, Murach, Pedersen, and colleagues studied how three disease-causing missense mutations in SNAP25 affect synaptic vesicle exocytosis. These mutations have previously been studied by Alten et al., 2021. The authors observed similar impairments in spontaneous and evoked release as Alten et al., 2021, but the measurement of readily releasable pool (RRP) size differed between the two studies. The authors found that the V48F and D166Y mutations affect the interaction with the Ca2+ sensor synaptotagmin-1 (Syt1), but do not entirely phenocopy Syt1 loss-of-function because they also exhibit a gain-of-function. Thus, these mutations affect multiple aspects of the energy landscape for vesicle priming and fusion. The I67N mutation specifically increases the fusion energy barrier without affecting upstream vesicle priming.

The strength of the study includes careful and technically excellent dissection of the synaptic release process and a combination of electrophysiology, biophysics, and modeling approaches. This study gained a deeper mechanistic understanding of these mutations in vesicle exocytosis than the previous study but did not result in a paradigm shift in our understanding of SNAP25-associated encephalopathy because the same spontaneous and evoked release phenotypes were previously identified.

1) The authors discussed possible reasons for the different results of the RRP sizes between this study and Alten et al., 2021. One of them is how the hypertonic solution is applied. The authors thought that the long application of hypertonic solution in Alten et al., 2021 caused an overlapping release of RRP and upstream vesicle pools because Alten et al., 2021 measured 10-fold larger RRP size than what was measured in this study. However, Alten et al., 2021 measured RRP from IPSCs and a single inhibitory vesicle fusion causes larger charge transfer than an excitatory vesicle. The authors need to take this into consideration and 10-fold is likely an overestimate.

2) Statistical tests should be performed for protein expression levels (Fig 2A and Fig 10A) and in vitro fusion assays (Fig 8D,E and Fig 9 B,C).

Reviewer #1 (Public Review):

Valk and Engert et al. examined the potential relations between three different mental training modules, hippocampal structure and functional connectivity, and cortisol levels over a 9-month period. They found that among the three types of mental training: Presence (attention and introspective awareness), Affect (socio-emotional - compassion and prosocial motivation), and Perspective (socio-cognitive - metacognition and perspective taking) modules; Affect training most consistently related to changes in hippocampal structure and function - specifically, CA1-3 subfields of the hippocampus. Moreover, decreases in diurnal cortisol correlated to bilateral increases in volume, and decreases in diurnal and chronic cortisol left CA1-3 functional connectivity. Chronic cortisol levels also related to right CA4/DG volume and left subiculum function. The authors demonstrate that mindfulness training programs impact hippocampus and are a potential avenue for stress interventions, a potential avenue to improve health. The data contribute to the literature on plasticity of hippocampal subfields during adulthood, the impact of mental training interventions on the brain, and the link between CA1-3 and both short- and long-term stress changes. Additional clarification and extension of the methods is needed to strengthen the authors' conclusions.

The authors thoughtfully approached the study of hippocampal subfields, utilizing a method designed for T1w images that outperformed Freesurfer 5.3 and that produced comparable results to an earlier version of ASHS. However, given the use of normalized T1-weighted images to delineate hippocampal subfield volume, some caution may be warranted (Wisse et al. 2020). While the authors note the assessment of quality control processes, the difficulty in ensuring valid measurement is an ongoing conversation in the literature. This also extends to the impact of functional co-registration using segmentations. I appreciate the inclusion of Table 5 in documenting reasons for missing data across subjects. Providing additional details on the distribution of quality ratings across subfields would help contextualize the results and ensure there is equal quality of segmentations across subfields.

Given the consistent pattern of finding results with CA1-3, in contrast to other subfields, it would help to know if the effects of the different training modules on subfields differed from each other statistically (i.e., not just that one is significant, and one is not) to provide an additional context of the strength of results focused on Affect training and CA1-3 (for example, those shown in Figure 3).

Reviewer #2 (Public Review):

In this study, Valk, Engert et al. investigated effects of stress-reducing behavioral intervention on hippocampal structure and function across different conditions of mental training and in relation to diurnal and chronic cortisol levels. The authors provide convincing multimodal evidence of a link between hippocampal integrity and stress regulation, showing changes in both volume and intrinsic functional connectivity, as measured by resting-state fMRI, in hippocampal subfield CA1-3 after socio-affective training as compared to training in a socio-cognitive module. In particular, increased CA1-3 volume following socio-affective training overlapped with increased functional connectivity to medial prefrontal cortex, and reductions in cortisol. The conclusions of this paper are well supported by the data, although some aspects of the data analysis would benefit from being clarified and extended.

A main strength of the study is the rigorous design of the behavioral intervention, including test-retest cohorts, an active control group, and a previously established training paradigm, contributing to an overall high quality of included data. Similarly, systematic quality checking of hippocampal subfield segmentations contributes to a reliable foundation for structural and functional investigations.

Another strength of the study is the multimodal data, including both structural and functional markers of hippocampal integrity as well as both diurnal and chronic estimates of cortisol levels. However, the included analyses are not optimally suited for elucidating multivariate interrelationships between these measures. Instead, effects of training on structure and function, and their links to cortisol, are largely characterized separately from each other. This results in the overall interpretation of results, and conclusions, being dependent on a large number of separate associations. Adopting multivariate approaches would better target the question of whether there is cortisol-related structural and functional plasticity in the hippocampus after mental training aimed at reducing stress.

The authors emphasize a link between hippocampal subfield CA1-3 and stress regulation, and indeed, multiple lines of evidence converge to highlight a most consistent role of CA1-3. There are, however, some aspects of the results that limit the robustness of this conclusion. First, formal comparisons between subfields are incomplete, making it difficult to judge whether the CA1-3, to a greater degree than other subfields, display effects of training. Relatedly, it would be of interest to assess whether changes in CA1-3 make a significant contribution to explaining the link between hippocampal integrity and cortisol, as compared to structure and functional connectivity of the whole hippocampus. Second, both structural and functional effects (although functional to a greater degree), were most pronounced in the specific comparison of "Affect" and "Perspective" training conditions, possibly limiting the study's ability to inform general principles of hippocampal stress-regulation.

Reviewer #1 (Public Review):

In this manuscript, the authors use a large dataset of neuroscience publications to elucidate the nature of self-citation within the neuroscience literature. The authors initially present descriptive measures of self-citation across time and author characteristics; they then produce an inclusive model to tease apart the potential role of various article and author features in shaping self-citation behavior. This is a valuable area of study, and the authors approach it with an appropriate and well-structured dataset.

The study's descriptive analyses and figures are useful and will be of interest to the neuroscience community. However, with regard to the statistical comparisons and regression models, I believe that there are methodological flaws that may limit the validity of the presented results. These issues primarily affect the uncertainty of estimates and the statistical inference made on comparisons and model estimates - the fundamental direction and magnitude of the results are unlikely to change in most cases. I have included detailed statistical comments below for reference.

Conceptually, I think this study will be very effective at providing context and empirical evidence for a broader conversation around self-citation. And while I believe that there is room for a deeper quantitative dive into some finer-grained questions, this paper will be a valuable catalyst for new areas of inquiry around citation behavior - e.g., do authors change self-citation behavior when they move to more or less prestigious institutions? do self-citations in neuroscience benefit downstream citation accumulation? do journals' reference list policies increase or decrease self-citation? - that I hope that the authors (or others) consider exploring in future work.

Statistical comments:

(1) Throughout the paper, the nested nature of the data does not seem to be appropriately handled in the bootstrapping, permutation inference, and regression models. This is likely to lead to inappropriately narrow confidence bands and overly generous statistical inference.

(2) The discussion of the data structure used in the regression models is somewhat opaque, both in the main text and the supplement. From what I gather, these models likely have each citation included in the model at least once (perhaps twice, once for first-author status and one for last-author status), with citations nested within citing papers, cited papers, and authors. Without inclusion of random effects, the interpretation and inference of the estimates may be misleading.

(3) I am concerned that the use of the inverse hyperbolic sine transform is a bit too prescriptive, and may be producing poor fits to the true predictor-outcome relationships. For example, in a figure like Fig S8, it is hard to know to what extent the sharp drop and sign reversal are true reflections of the data, and to what extent they are artifacts of the transformed fit.

(4) It seems there are several points in the analysis where papers may have been dropped for missing data (e.g., missing author IDs and/or initials, missing affiliations, low-confidence gender assessment). It would be beneficial for the reader to know what % of the data was dropped for each analysis, and for comparisons across countries it would be important for the authors to make sure that there is not differential missing data that could affect the interpretation of the results (e.g., differences in self-citation being due to differences in Scopus ID coverage).

Reviewer #2 (Public Review):

The authors provide a comprehensive investigation of self-citation rates in the field of Neuroscience, filling a significant gap in existing research. They analyze a large dataset of over 150,000 articles and eight million citations from 63 journals published between 2000 and 2020. The study reveals several findings. First, they state that there is an increasing trend of self-citation rates among first authors compared to last authors, indicating potential strategic manipulation of citation metrics. Second, they find that the Americas show higher odds of self-citation rates compared to other continents, suggesting regional variations in citation practices. Third, they show that there are gender differences in early-career self-citation rates, with men exhibiting higher rates than women. Lastly, they find that self-citation rates vary across different subfields of Neuroscience, highlighting the influence of research specialization. They believe that these findings have implications for the perception of author influence, research focus, and career trajectories in Neuroscience.

Overall, this paper is well written, and the breadth of analysis conducted by authors, with various interactions between variables (eg. gender vs. seniority), shows that the authors have spent a lot of time thinking about different angles. The discussion section is also quite thorough. The authors should also be commended for their efforts in the provision of code for the public to evaluate their own self-citations. That said, here are some concerns and comments that, if addressed, could potentially enhance the paper:

1. There are concerns regarding the data used in this study, specifically its bias towards top journals in Neuroscience, which limits the generalizability of the findings to the broader field. More specifically, the top 63 journals in neuroscience are based on impact factor (IF), which raises a potential issue of selection bias. While the paper acknowledges this as a limitation, it lacks a clear justification for why authors made this choice. It is also unclear how the "top" journals were identified as whether it was based on the top 5% in terms of impact factor? Or 10%? Or some other metric? The authors also do not provide the (computed) impact factors of the journals in the supplementary.

By exclusively focusing on high impact journals, your analysis may not be representative of the broader landscape of self-citation patterns across the neuroscience literature, which is what the title of the article claims to do.

2. One other concern pertains to the possibility that a significant number of authors involved in the paper may not be neuroscientists. It is plausible that the paper is a product of interdisciplinary collaboration involving scientists from diverse disciplines. Neuroscientists amongst the authors should be identified.

3. When calculating self-citation rate, it is important to consider the number of papers the authors have published to date. One plausible explanation for the lower self-citation rates among first authors could be attributed to their relatively junior status and short publication record. As such, it would also be beneficial to assess self-citation rate as a percentage relative to the author's publication history. This number would be more accurate if we look at it as a percentage of their publication history. My suspicion is that first authors (who are more junior) might be more likely to self-cite than their senior counterparts. My suspicion was further raised by looking at Figures 2a and 3. Considering the nature of the self-citation metric employed in the study, it is expected that authors with a higher level of seniority would have a greater number of publications. Consequently, these senior authors' papers are more likely to be included in the pool of references cited within the paper, hence the higher rate.

While the authors acknowledge the importance of the number of past publications in their gender analysis, it is just as important to include the interplay of seniority in (1) their first and last author self-citation rates and (2) their geographic analysis.

4. Because your analysis is limited to high impact journals, it would be beneficial to see the distribution of the impact factors across the different countries. Otherwise, your analysis on geographic differences in self-citation rates is hard to interpret. Are these differences really differences in self-citation rates, or differences in journal impact factor? It would be useful to look at the representation of authors from different countries for different impact factors.

5. The presence of self-citations is not inherently problematic, and I appreciate the fact that authors omit any explicit judgment on this matter. That said, without appropriate context, self-citations are also not the best scholarly practice. In the analysis on gender differences in self-citations, it appears that authors imply an expectation of women's self-citation rates to align with those of men. While this is not explicitly stated, use of the word "disparity", and also presentation of self-citation as an example of self-promotion in discussion suggest such a perspective. Without knowing the context in which the self-citation was made, it is hard to ascertain whether women are less inclined to self-promote or that men are more inclined to engage in strategic self-citation practices.

Reviewer #3 (Public Review):

This paper analyses self-citation rates in the field of Neuroscience, comprising in this case, Neurology, Neuroscience and Psychiatry. Based on data from Scopus, the authors identify self-citations, that is, whether references from a paper by some authors cite work that is written by one of the same authors. They separately analyse this in terms of first-author self-citations and last-author self-citations. The analysis is well-executed and the analysis and results are written down clearly. There are some minor methodological clarifications needed, but more importantly, the interpretation of some of the results might prove more challenging. That is, it is not always clear what is being estimated, and more importantly, the extent to which self-citations are "problematic" remains unclear.

When are self-citations problematic? As the authors themselves also clarify, "self-citations may often be appropriate". Researchers cite their own previous work for perfectly good reasons, similar to reasons of why they would cite work by others. The "problem", in a sense, is that researchers cite their own work, just to increase the citation count, or to promote their own work and make it more visible. This self-promotional behaviour might be incentivised by certain research evaluation procedures (e.g. hiring, promoting) that overly emphasise citation performance. However, the true problem then might not be (self-)citation practices, but instead, the flawed research evaluation procedures that emphasis citation performance too much. So instead of problematising self-citation behaviour, and trying to address it, we might do better to address flawed research evaluation procedures. Of course, we should expect references to be relevant, and we should avoid self-promotional references, but addressing self-citations may just have minimal effects, and would not solve the more fundamental issue.

Some other challenges arise when taking a statistical perspective. For any given paper, we could browse through the references, and determine whether a particular reference would be warranted or not. For instance, we could note that there might be a reference included that is not at all relevant to the paper. Taking a broader perspective, the irrelevant reference might point to work by others, included just for reasons of prestige, so-called perfunctory citations. But it could of course also include self-citations. When we simply start counting all self-citations, we do not see what fraction of those self-citations would be warranted as references. The question then emerges, what level of self-citations should be counted as "high"? How should we determine that? If we observe differences in self-citation rates, what does it tell us?

For example, the authors find that the (any author) self-citation rate in Neuroscience is 10.7% versus 15.9% in Psychiatry. What does this difference mean? Are psychiatrists citing themselves more often than neuroscientists? First author men showed a self-citation rate of 5.12% versus a self-citation rate of 3.34% of women first authors. Do men engage in more problematic citation behaviour? Junior researchers (10-year career) show a self-citation rate of about 5% compared to a self-citation rate of about 10% for senior researchers (30-year career). Are senior researchers therefore engaging in more problematic citation behaviour? The answer is (most likely) "no", because senior authors have simply published more, and will therefore have more opportunities to refer to their own work. To be clear: the authors are aware of this, and also take this into account. In fact, these "raw" various self-citation rates may, as the authors themselves say, "give the illusion" of self-citation rates, but these are somehow "hidden" by, for instance, career seniority.

Again, the authors do consider this, and "control" for career length and number of publications, et cetera, in their regression model. Some of the previous observations then change in the regression model. Neuroscience doesn't seem to be self-citing more, there just seem to be junior researchers in that field compared to Psychiatry. Similarly, men and women don't seem to show an overall different self-citation behaviour (although the authors find an early-career difference), the men included in the study simply have longer careers and more publications.

But here's the key issue: what does it then mean to "control" for some variables? This doesn't make any sense, except in the light of causality. That is, we should control for some variable, such as seniority, because we are interested in some causal effect. The field may not "cause" the observed differences in self-citation behaviour, this is mediated by seniority. Or is it confounded by seniority? Are the overall gender differences also mediated by seniority? How would the selection of high-impact journals "bias" estimates of causal effects on self-citation? Can we interpret the coefficients as causal effects of that variable on self-citations? If so, would we try to interpret this as total causal effects, or direct causal effects? If they do not represent causal effects, how should they be interpreted then? In particular, how should it "inform author, editors, funding agencies and institutions", as the authors say? What should they be informed about?

The authors also "encourage authors to explore their trends in self-citation rates". It is laudable to be self-critical and review ones own practices. But how should authors interpret their self-citation rate? How useful is it to know whether it is 5%, 10% or 15%? What would be the "reasonable" self-citation rate? How should we go about constructing such a benchmark rate? Again, this would necessitate some causal answer. Instead of looking at the self-citation rate, it would presumably be much more informative to simply ask authors to check whether references are appropriate and relevant to the topic at hand.

In conclusion, the study shows some interesting and relevant differences in self-citation rates. As such, it is a welcome contribution to ongoing discussions of (self) citations. However, without a clear causal framework, it is challenging to interpret the observed differences.

Reviewer #1 (Public Review):

This study demonstrates that vitamin D-bound VDR increased the expression of SIRT1 and that vitamin D-bound VDR interacts with SIRT1 to cause auto-deacetylation on Lys610 and activation of SIRT1 catalytic activity. This is an important finding that is relevant to the actions of VDR on colorectal cancer. The data presented to support the presented conclusion is convincing.

A strength of the study is that it is focused on a narrow group of conclusions.

The major weakness of the study is that the site of SIRT1 regulatory lysine acetylation is defined by mutational analysis rather than by direct biochemical analysis. This issue is partially mitigated by previous reports of K610 acetylation using mass spec (https://www.phosphosite.org/proteinAction.action?id=5946&showAllSites=true). However, Fig. 4E is reassuring because it shows that the apparent acetylation of the K610 mutant SIRT1 appears to be lower than WT SIRT1

A second weakness of the study relates to the use of shRNA-mediated knockdown of VDR for some studies in which a previously reported cell line was employed. The analysis presented would be more compelling if similar data was obtained using more than one shRNA. Similarly, only a single siRNA for SIRT1 is presented in Table 1.

A third weakness of the study is that the conclusion that the VDR interaction with SIRT1 is the cause of auto-deacetylation rather than an associated event mediated by another mechanism would be more strongly supported by mutational analysis of SIRT1 and VDR residues required for the binding interaction. Will VDR increase SIRT1 activity when mutations are introduced to block the interaction? While the finding that catalytically inactive SIRT1 does not interact with VDR is helpful, this does not address the role of the binding surface.

A fourth weakness of the study is that it would be improved by testing the proposed hypothesis through in vitro reconstitution with purified proteins. Does VDR cause auto-deacetylation and activation of Sirt1 in vitro?

Reviewer #2 (Public Review):

The authors provide a comprehensive analysis of vitamin D-mediated signaling through VDR, SIRT1, and Ace H3K9. They specifically emphasize the significance of K610 in SIRT1 within this signaling pathway. The article effectively presents a convincing and straightforward argument. The experiments conducted are meticulously executed, and the statistical analysis is sound. The inclusion of complex biochemistry details adequately covers the topic at hand. These findings hold great relevance to both normal and pathological physiology across different cell lineages, making them of considerable interest.

Reviewer #1 (Public Review):

The objective of this investigation was to determine whether experimental pain could induce alterations in cortical inhibitory/facilitatory activity observed in TMS-evoked potentials (TEPs). Previous TMS investigations of pain perception had focused on motor evoked potentials (MEPs), which reflect a combination of cortical, spinal, and peripheral activity, as well as restricting the focus to M1. The main strength of this investigation is the combined use of TMS and EEG in the context of experimental pain. More specifically, Experiment 1 investigated whether acute pain altered cortical excitability, reflected in the modulation of TEPs. The main outcome of this study is that relative to non-painful warm stimuli, painful thermal stimuli led to an increase on the amplitude of the TEP N45, with a larger increase associated with higher pain ratings. Because it has been argued that a significant portion of TEPs could reflect auditory potentials elicited by the sound (click) of the TMS, Experiment 2 constituted a control study that aimed to disentangle the cortical response related to TMS and auditory activity. Finally, Experiment 3 aimed to disentangle the cortical response to TMS and reafferent feedback from muscular activity elicited by suprathreshold TMS applied over M1. The fact that the authors accompanied their main experiment with two control experiments strengthens the conclusion that the N45 TEP peak could be implicated in the perception of painful stimuli. Perhaps, the addition of a highly salient but non-painful stimulus (i.e. from another modality) would have further ruled out that the effects on the N45 are not predominantly related to intensity/saliency of the stimulus rather than to pain per se.

Reviewer #2 (Public Review):

The authors have used transcranial magnetic stimulation (TMS) and motor evoked potentials (MEPs) and TMS-electroencephalography (EEG) evoked potentials (TEPs) to determine how experimental heat pain could induce alterations in these metrics.
In Experiment 1 (n = 29), multiple sustained thermal stimuli were administered over the forearm, with the first, second, and third block of stimuli consisting of warm but non-painful (pre-pain block), painful heat (pain block) and warm but non-painful (post-pain block) temperatures respectively. Painful stimuli led to an increase in the amplitude of the fronto-central N45, with a larger increase associated with higher pain ratings. Experiments 2 and 3 studied the correlation between the increase in the N45 in pain and the effects of a sham stimulation protocol/higher stimulation intensity. They found that the centro-frontal N45 TEP was decreased in acute pain.

The study comes from a very strong group in the pain fields with long experience in psychophysics, experimental pain, neuromodulation, and EEG in pain. They are among the first to report on changes in cortical excitability as measured by TMS-EEG over M1.

While their results are in line with reductions seen in motor-evoked responses during pain and effort was made to address possible confounding factors (study 2 and 3), there are some points that need attention. In my view the most important are:<br /> 1. The method used to calculate the rest motor threshold, which is likely to have overestimated its true value : calculating highly abnormal RMT may lead to suprathreshold stimulations in all instances (Experiment 3) and may lead to somatosensory "contamination" due to re-afferent loops in both "supra" and "infra" (aka. less supra) conditions.<br /> 2. The low number of pulses used for TEPs (close to ⅓ of the usual and recommended), lack of measures to mask auditory noise.<br /> 3. A supra-stimulus heat stimulus not based on individual HPT, that oscillates during the experiment and that lead to large variations in pain intensity across participants is unfortunate. So is the lack of report on measures taken to correct for a fortuitous significance (multiple comparison correction) in such a huge number of serial paired tests.

Reviewer #3 (Public Review):

The present study aims to investigate whether pain influences cortical excitability. To this end, heat pain stimuli are applied to healthy human participants. Simultaneously, TMS pulses are applied to M1 and TMS-evoked potentials (TEPs) and pain ratings are assessed after each TMS pulse. TEPs are used as measures of cortical excitability. The results show that TEP amplitudes at 45 msec (N45) after TMS pulses are higher during painful stimulation than during non-painful warm stimulation. Control experiments indicate that auditory, somatosensory, or proprioceptive effects cannot explain this effect. Considering that the N45 might reflect GABAergic activity, the results suggest that pain changes GABAergic activity. The authors conclude that TEP indices of GABAergic transmission might be useful as biomarkers of pain sensitivity.

Pain-induced cortical excitability changes is an interesting, timely, and potentially clinically relevant topic. The paradigm and the analysis are sound, the results are mostly convincing, and the interpretation is adequate. The following clarifications and revisions might help to improve the manuscript further.

1. Non-painful control condition. In this condition, stimuli are applied at warmth detection threshold. At this intensity, by definition, some stimuli are not perceived as different from the baseline. Thus, this condition might not be perfectly suited to control for the effects of painful vs. non-painful stimulation. This potential confound should be critically discussed.<br /> 2. MEP differences between conditions. The results do not show differences in MEP amplitudes between conditions (BF 1.015). The analysis nevertheless relates MEP differences between conditions to pain ratings. It would be more appropriate to state that in this study, pain did not affect MEP and to remove the correlation analysis and its interpretation from the manuscript.<br /> 3. Confounds by pain ratings. The ISI between TMS pulses is 4 sec and includes verbal pain ratings. Considering this relatively short ISI, would it be possible that verbal pain ratings confound the TEP? Moreover, could the pain ratings confound TEP differences between conditions, e.g., by providing earlier ratings when the stimulus is painful? This should be carefully considered, and the authors might perform control analyses.<br /> 4. Confounds by time effects. Non-painful and painful conditions were performed in a fixed order. Potential confounds by time effects should be carefully considered.<br /> 5. Data availability. The authors should state how they make the data openly available.

Public Review:

In countries endemic for P vivax the need to administer a primaquine (PQ) course adequate to prevent relapse in G6PD deficient persons poses a real dilemma. On one hand PQ will cause haemolysis; on the other hand, without PQ the chance of relapse is very high. As a result, out of fear of severe haemolysis, PQ has been under-used.

In view of the above, the Authors have investigated in well-informed volunteers, who were kept under close medical supervision in hospital throughout the study, two different schedules of PQ administration: (1) escalating doses (to a total of 5-7 mg/kg); (2) single 45 mg dose (0.75 mg/kg).

It is shown convincingly that regimen (1) can be used successfully to deliver within 3 weeks, under hospital conditions, the dose of PQ required to prevent P vivax relapse.

As expected, with both regimens acute haemolytic anaemia (AHA) developed in all cases. With regimen (2), not surprisingly, the fall in Hb was less, although it was abrupt. With regimen (1) the average fall in Hb was about 4 G. Only in one subject the fall in Hb mandated termination of the study.

Since the data from the Chicago group some sixty years ago, there has been no paper reporting a systematic daily analysis of AHA in so many closely monitored subjects with G6PD deficiency. The individual patient data in the Supplementary material are most informative and more than precious.

Having said this, I do have some general comments.<br /> 1. Through their remarkable Part 1 study, the Authors clearly wish to set the stage for a revision of the currently recommended PQ regimen for G6PD deficient patients. They have shown that 5-7 mg/kg can be administered within 3 weeks, whereas the currently recommended regimen provides 6 mg/kg over no less than 8 weeks.<br /> 2. Part 2 aims to show that, as was known already, even a single PQ dose of 0.75 mg/kg causes a significant degree of haemolysis: G6PD deficiency-related haemolysis is characteristically markedly dose-dependent. Although they do not state it explicitly in these words (I think they should), the Authors want to make it clear that the currently recommended regimen does cause AHA.<br /> 3. Regulatory agencies like to classify a drug regimen as either SAFE or NOT-SAFE; they also like to decide who is 'at risk' and who is 'not at risk'. A wealth of data, including those in this manuscript, show that it is not correct to say that a G6PD deficient person when taking PQ is at risk of haemolysis: he or she will definitely have haemolysis. As for SAFETY, it will depend on the clinical situation when PQ is started and on the severity of the AHA that will develop.

The above three issues are all present in the discussion, but I think they ought to be stated more clearly.

Finally, by the Authors' own statement on page 15, the main limitation is the complexity of this approach. The authors suggest that blister packed PQ may help; but to me the real complexity is managing patients in the field versus the painstaking hospital care in the hands of experts, of which volunteers in this study have had the benefit. It is not surprising that a fall in Hb of 4 g/dl is well tolerated by most non-anaemic men; but patients with P vivax in the field may often have mild to moderate to severe anaemia; and certainly they will not have their Hb, retics and bilirubin checked every day. In crude approximation, we are talking of a fall in Hb of 4 G with regimen (1), as against a fall in Hb of 2 G with regimen (2), that is part of the currently recommended regimen: it stands to reason that, in terms of safety, the latter is generally preferable (even though some degree of fall in Hb will recur with each weekly dose). In my view, these difficult points should be discussed deliberately.

Reviewer #1 (Public Review):

Sun and colleagues investigated the cross-reactive antibodies between E.Coli and the host in severe alcoholic hepatitis (SAH). The study found that IgA and IgG were deposited in the liver of SAH patients. Complements C3d and C4d were also deposited in the SAH patient's liver. Moreover, they found that the Ig accumulated in the SAH liver, but not in the SAH serum, induced hepatocyte killing, suggesting that liver Ig is important. Then, they found that these Ig can recognize both human and E. Coli antigens. Very interestingly, SAH-derived Ig shows cross-reactivity to both human and E. Coli antigens, suggesting E, Coli-primed Ig in SAH may damage hepatocytes through host antigen recognition. These Ig are not observed in alcoholic cirrhosis patients. The liver RNA-seq data suggested that Ig was also produced in the liver, not only gut-derived Ig. This is a very interesting study showing the novel mechanism of SAH mediated by the Ig with the cross-reactivity with bacteria and host antigens, which is not observed in AC patients. Overall, the study design is reasonable and the data are consistent to support their central hypothesis. There are a few comments.

Specific comments:<br /> 1. Figures 1 and 2 show Ig deposition in the liver (it seems on hepatocytes). Not only Ig reaction to the specific antigen but also non-specific Fc receptor-mediated binding to hepatocytes could be contributed.<br /> 2. Similarly, in Figure 2G Ig-mediated hepatocyte killing, Fc receptor-mediated hepatocyte killing may be involved.<br /> 3. The study examined the possibility of liver resident B cell and plasma cell-mediated Ig. As the authors mentioned in the discussion, B cells may be translocated from the intestine to the liver. Or the resident B cells (not from the gut) are also involved.

Reviewer #2 (Public Review):

In this paper, Ahmadi et al demonstrated that antibodies produced locally in the liver by infiltrating B cells can enhance liver damage caused by fat accumulation. The main finding is that human samples extracted from severe alcoholic hepatitis showed antibody accumulation that may be related to an enhanced immune response to self-antigens, which could ultimately fuel liver damage - which was already present due to alcohol consumption. Their data are corroborated by arrays and gene ontology assays, and I strongly believe that these data could add to the future options we have to treat patients.

Reviewer #1 (Public Review):

The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions. This is well supported by a combination of immunofluorescence, RNA seq, CUT&RUN and ATAC-seq.<br /> The mouse model in this study is a conditional Stra8 Cre mouse. Loss of ARID1A in this mouse, caused up regulation of XY linked genes in prophase I spermatocytes and ingression of RNA pol II to the sex body, indicating a role for this chromatin remodeller in MSCI. Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes. ARID1A interacts with gene transcription start sites of sex-linked genes, and loss of ARID1A increased promoter accessibility of XY linked genes with concomitant gene up regulation.

This work suggests that ARID1A regulates chromatin composition of the sex body relative to the autosomes. In the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes regulated by ARID1A.

Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 is a protein with histone methyltransferase activity that catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the suggested role for ARID1A in genome instability in cancer cells (Nacarelli et al 2020: 10.1080/23723556.2019.1690923, Zhang et al. 2023: 10.1093/carcin/bgad011 and others). It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.

It is worth mentioning that authors show that there are Arid1a transcripts that escape the Cre system. This might mask the phenotype of the Arid1a knockout, given that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed (these mice produce sperm). ARID1A is present throughout prophase I and it might have pre-MSCI roles that impact earlier stages of Meiosis I and cell death might be happening in these earlier stages too.

Overall the research presented here is solid, adds new knowledge on how the sex chromatin is silenced during meiosis and has generated relevant databases for the field.

Reviewer #2 (Public Review):