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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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.

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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):

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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):

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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #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 #2 (Public Review):

The authors tried to characterize the function of the SWI/SNF remodeler family, BAF, in spermatogenesis. The authors focused on ARID1A, a BAF-specific putative DNA binding subunit, based on gene expression profiles. The study has several serious issues with the data and interpretation. The conditional deletion mouse model of ARIDA using Stra8-cre showed inefficient deletion; spermatogenesis did not appear to be severely compromised in the mutants. Using this data, the authors claimed that meiotic arrest occurs in the mutants. This is obviously a misinterpretation. In the later parts, the authors performed next-gen analyses, including ATAC-seq and H3.3 CUT&RUN, using the isolated cells from the mutant mice. However, with this inefficient deletion, most cells isolated from the mutant mice appeared not to undergo Cre-mediated recombination. Therefore, these experiments do not tell any conclusion pertinent to the Arid1a mutation. Furthermore, many of the later parts of this study focus on the analysis of H3.3 CUT&RUN. However, Fig. S7 clearly suggests that the H3.3 CUT&RUN experiment in the wild-type simply failed. Thus, none of the analyses using the H3.3 CUT&RUN data can be interpreted. Overall, I found that the study does not have rigorous data, and the study is not interpretable. If the author wishes to study the function of ARID2 in spermatogenesis, they may need to try other cre-lines to have more robust phenotypes, and all analyses must be redone using a mouse model with efficient deletion of ARID2.

Reviewer #2 (Public Review):

The authors present the OpenApePose database constituting a collection of over 70000 ape images which will be important for many applications within primatology and the behavioural sciences. The authors have also rigorously tested the utility of this database in comparison to available Pose image databases for monkeys and humans to clearly demonstrate its solid potential. However, the variation in the database with regards to individuals, background, source/setting is not clearly articulated and would be beneficial information for those wishing to make use of this resource in the future. At present, there is also a lack of clarity as to how this image database can be extrapolated to aid video data analyses which would be highly beneficial as well.

I have two major concerns with regard to the manuscript as it currently stands which I think if addressed would aid the clarity and utility of this database for readers.

1. Human annotators are mentioned as doing the 16 landmarks manually for all images but there is no assessment of inter-observer reliability or the such. I think something to this end is currently missing, along with how many annotators there were. This will be essential for others to know who may want to use this database in the future.

Relevant to this comment, in your description of the database, a table or such could be included, providing the number of images from each source/setting per species and/or number of individuals. Something to give a brief overview of the variation beyond species. (subspecies would also be of benefit for example).

2. You mention around line 195 that you used a specific function for splitting up the dataset into training, validation, and test but there is no information given as to whether this was simply random or if an attempt to balance across species, individuals, background/source was made. I would actually think that a balanced approach would be more appropriate/useful here so whether or not this was done, and the reasoning behind that must be justified.

This is especially relevant given that in one test you report balancing across species (for the sample size subsampling procedure).

And another perhaps major concern that I think should also be addressed somewhere is the fact that this is an image database tested on images while the abstract and manuscript mention the importance of pose estimation for video datasets, yet the current manuscript does not provide any clear test of video datasets nor engage with the practicalities associated with using this image-based database for applications to video datasets. Somewhere this needs to be added to clarify its practical utility.

Reviewer #2 (Public Review):

In this manuscript, the authors conducted a straightforward molecular approach to link FMRP and MAP1B proteins functionally. Both proteins are connected since FMRP is a translational regulator of the MAP1B protein, a microtubule-stabilizing factor.

The results combined molecular genetics (FMRP knock-out mice) with acute inactivation of FRMP and MAP1B to conclusively support the notion that FMRP-dependent regulation of MAP1B is necessary for proper neuronal migration toward the olfactory bulb using the rostral migratory stream.

Overall, these results increase our knowledge of the molecular mechanism that controls how neurons migrate in the brain to reach their final destinations and confirms that cytoskeleton regulators are key players in this process.

Reviewer #2 (Public Review):

Kimchi et al. examined the role of cholinergic inputs to the amygdala in regulating reward-seeking behavior. To investigate this, the authors developed a head-fixed behavioral task where animals were trained to lick at random intervals, with some of these responses being reinforced ("windows of opportunity") as opposed to control epochs when no reward was delivered.

The authors conducted in vivo optogenetic stimulation of basal forebrain cholinergic neurons and discovered that a 2-second optical stimulation of these neurons encouraged licking behavior when followed by reward delivery. This was in comparison to time epochs where no reward was delivered or compared to control mice only expressing EYFP. However, it remained unclear how many trials were required for this effect to manifest.

Furthermore, they demonstrated that the stimulation of basal forebrain cholinergic neurons did not induce real-time place preference or affect locomotion. The reward-driven licking behavior was also mitigated by systemic cholinergic receptor antagonists.<br /> Next, the authors observed the bulk calcium dynamics from these neurons in a version of the task where an auditory cue predicted reward availability. They found strong calcium signals when mice were licking and when the tone was present, but also reported signals when mice were spontaneously licking.

By injecting a genetically encoded Acetylcholine (Ach) sensor directly into the Basolateral Amygdala (BLA), they showed that Ach signals were present when mice were engaged in licking, both during reward availability and for non-rewarded licks. Photostimulation of Ach terminals directly in the BLA increased licking behavior when a reward was available.

Finally, using in vivo and ex vivo physiology, they demonstrated that Ach signaling influences the electrophysiological dynamics in the BLA. This may help clarify some of the postsynaptic responses triggered by this neuromodulator.

Strengths of the paper:

1. The experiments were well-executed and sufficiently powered, with most statistics being correctly reported.<br /> 2. The paper is a technical tour de force, employing fiber photometry, in vivo and ex vivo electrophysiology, optogenetics, and behavioral approaches.<br /> 3. Robust effects were observed in most of the experiments.<br /> Weaknesses:<br /> 1. The experimental design varies slightly across each behavioral experiment, making it difficult to directly compare one effect to another.<br /> 2. The paper doesn't include data showing the precise location for the Ach recordings. As a result, it is unclear whether these signals are specific to the BLA, or whether they might also be coming from neighboring regions.

Reviewer #2 (Public Review):

Hage et al examine how the foraging behavior of marmoset monkeys in a laboratory setting systematically takes into account the reward value and anticipated effort cost associated with the acquisition and consumption of food. In an interesting comprehensive framework, the authors study how experimental and natural variation of these factors affect both the decisions and actions necessary to gather and accumulate food, as well as the actions necessary to consume the food.

The manuscript proposes a computational model of how the monkeys may guide all these aspects of behavior, by maximizing a food capture rate that trades off the food that can be gathered with the effort and duration of the underlying actions. They use this model to derive qualitative predictions for how monkeys should react to an increase in the effort associated with food consumption: Monkeys should work longer before deciding to consume the accumulated food, but should move more slowly. The model also predicts that monkeys should show a different reaction to an increase in reward value of the food, also working longer but moving faster. The authors test these predictions in an interesting experimental setup that requires monkeys to collect small increments of food rewards for successful eye movements to targets. The monkeys can decide freely when to interrupt work and consume the accumulated food, and the authors measure the speed of the eye movements involved in the food acquisition as well as the tongue movements involved in the food consumption.

By and large, the behavioral findings fall in line with the qualitative model predictions: When the effort involved in food consumption increases, monkeys collect more food before deciding to consume it, and they move slower both during food acquisition and food consumption. In a second test, the authors approximate the effects of reward value of the food at stake, by comparing monkey behavior during different days with natural variations in body weight. These quasi-experimental increases in the reward value of food also lead to longer work times before consumption, but to faster movements during food consumption. Finally, the authors show that these effects correlate with pupil size, with pupils dilating more for low-effort foraging actions with increased saccade speed and decreased work duration. The authors conclude that the effort associated with anticipated actions can lead to changes in global brain state that simultaneously affect decisions and action vigor.

The paper proposes an interesting model for how one unified action policy may simultaneously affect multiple types of decisions and movements involved in foraging. The methods employed to measure behavior and test these predictions are generally sound, and the paper is well written. While the model and paper in their present form can clearly inspire researchers to consider this integrated perspective, and trigger further research employing such a framework, there are some conceptual and methodical shortcomings that reduce the conclusiveness of the results and the usefulness of the proposed model.

(1) The model proposed in the paper takes a very specific functional form that is neither motivated by the previous literature nor particularly useful for indexing the behavioral tendencies of individual monkeys (or of the same monkey in different contexts). For example, while it is clear that the saccade effort cost will need to outgrow the increase in the utility of the accumulated food for the monkey to start feeding, it is unclear why this needs to be modeled with a fixed quadratic exponent on the number of saccades? Similarly, why do licks deplete the food stash with the specific rate hard-coded in the model? Finally, the proportion of successful saccades and lick events is assumed to be fixed, even though it very likely to be directly influenced by movement speed (speed-accuracy trade-off), which is also contained in the model. It would strongly increase the plausibility and potential impact of the model if the authors could clearly state where these hard-coded model terms come from. Ideally, they would formulate the model in more general terms and also consider other functional forms, as briefly suggested in the discussion. This latter point would be particularly important since not all model predictions were actually borne out in the data.

(2) The authors derive qualitative predictions, by simulating their model with apparently arbitrary parameters. They then test these qualitative predictions with conventional statistics (e.g., t-tests of whether monkeys lick more for high vs low effort trials). The reader wonders why the authors chose this route, instead of formulating their model with flexible parameters and then fitting these to data. This would allow them (and future researchers) to test their model not just qualitatively but also quantitatively, and to compare the plausibility of different functional forms. The authors certainly have enough data and power to do this, given the vast number of sessions the monkey completed.

(3) The effort manipulation chosen by the authors (distance of food tube) goes hand in hand with a greater need for precision since the monkey's tongue needs to hit an opening of similar size, but now located at a greater distance. This raises the question of whether the monkeys moved slower to enhance its chance of collecting the food (in line with a speed-accuracy trade off). The manuscript would benefit from an explicit test of this possibility, for example by reporting whether for each of the two conditions, the speed of tongue movements on a trial-by-trial basis predicts the probability of food collection? At the very least, the manuscript should explicitly discuss this issue and how it affects the certainty with which effects of tube distance can be linked to anticipated effort cost alone.

(4) The authors report most of the effects on the different measures (work duration, movement vigor, lick vigor, etc) in separate analyses. However, their model predicts that all of these measures result from the same action policy (maximization of the capture rate) and should therefore be related on a trial-by-trial basis. This is so far hardly tested in the presented analyses (with the exception of the pupil correlations in Figure 5). The model's assumed action policy would appear more plausible if the authors could demonstrate these trial-by-trial interrelations with some tests of association (e.g., correlations/regressions as already done for pupil measures in Figure 5) or possibly with dimensionality reduction of the multivariate data.

(5) The manuscript measures pupil dilation in a time period ranging from -250ms before to 250 ms after saccade onset. However, the pupil changes strongly during saccade execution relative to the preceding baseline, leaving doubts as to whether the aggregated measure blurs several interesting and potentially different effects. It would be more conclusive if the manuscript could report the analyses of pupil size separately for a period prior to saccade onset and during/after the saccade.

Reviewer #2 (Public Review):

This manuscript links the distinctive stinging behavior of sea anemones in different ecological niches to varying inactivation properties of voltage-gated calcium channels that are conferred by the identity of auxiliary Cavbeta subunits. Previous work from the Bellono lab established that the burrowing anemone, Nematostella vectensis, expresses a CaV channel that is strongly inactivated at rest which requires a simultaneous delivery of prey extract and touch to elicit a stinging response, reflecting a precise stinging control adapted for predation. They show here that by contrast, the anemone Exaiptasia diaphana which inhabits exposed environments, indiscriminately stings for defense even in the absence of prey chemicals, and that this is enabled by the expression of a CaVbeta splice variant that confers weak inactivation. They further use the heterologous expression of CaV channels with wild type and chimeric anemone Cavbeta subunits to infer that the variable N-termini are important determinants of Cav channel inactivation properties.

1. The authors found that Exaiptasia nematocytes could be characterized by two distinct inactivation phenotypes: (1) nematocytes with low-voltage threshold inactivation similar to that of Nematostella (Vi1/2 = ~ -85mV); and (2) a distinct population with weak, high-voltage threshold inactivation (Vi1/2 = ~ -48mV). What were the relative fractions of low-voltage and high-voltage nematocytes? Do the low-voltage Exaiptasia nematocytes behave similarly to Nematostella nematocytes with respect to requiring both prey extract and touch to discharge?

2. The authors state in Fig 3 legend and in the results that Exaiptasia nematocyte voltage-gated Ca2+ currents have weak inactivation compared with Nematostella. This description is imprecise and inaccurate. Figure 3 in fact shows that Exaiptasia nematocyte voltage-gated Ca2+ currents display a faster rate of inactivation compared to Nematostella Ca2+ currents. A sub-population of Exaiptasia nematocytes does display less resting state (or steady-state) inactivation compared to Nematostella Ca2+ currents. The authors need to be more accurate and qualify what type of inactivation property they are talking about.

3. In a similar vein, the authors need to be more accurate when referring to 'rat beta' used in heterologous expression experiments. It should be made explicit throughout the manuscript that the rat beta isoform used is rat beta2a. Among the distinct beta isoforms, beta2a is unique in being palmitoylated at the N-terminus which confers a characteristic slow rate of inactivation and a right-shifted voltage-dependence of steady-state inactivation consistent with the data shown in Fig. 4D. Almost all other rat beta isoforms do not have these properties.

4. The profiling of the impact of different Cnidarian Cavbeta subunits on reconstituted Ca2+ channel current waveforms is nice (Fig 5 and Fig 5S1). The N-terminus sequence of EdCaVβ2 is different from palmitoylated rat beta2a, though both have similar properties in showing slow inactivation and a right-shifted voltage-dependence of steady-state inactivation. Does EdCaVβ2 target autonomously the plasma membrane when expressed in cells? If so, this would reconcile with what was previously known and provide a rational explanation for the observed functional impact of the distinct Cavbetas.

Reviewer #2 (Public Review):

This work presents a remarkably extensive set of experiments, assaying the interaction between methylation and expression across most CpG positions in the genome in two cell types. To this end, the authors use mSTARR-seq, a high-throughput method, which they have previously developed, where sequences are tested for their regulatory activity in two conditions (methylated and unmethylated) using a reporter gene. The authors use these data to study two aspects of DNA methylation: 1. Its effect on expression, and 2. Its interaction with the environment. Overall, they identify a small number of 600 bp windows that show regulatory potential, and a relatively large fraction of these show an effect of methylation on expression. In addition, the authors find regions exhibiting methylation-dependent responses to two environmental stimuli (interferon alpha and glucocorticoid dexamethasone).

The questions the authors address represent some of the most central in functional genomics, and the method utilized is currently the best method to do so. The scope of this study is very impressive and I am certain that these data will become an important resource for the community. The authors are also able to report several important findings, including that pre-existing DNA methylation patterns can influence the response to subsequent environmental exposures.

The main weaknesses of the study are: 1. The large number of regions tested seems to have come at the expense of the depth of coverage per region (1 DNA read per region per replicate). I have not been convinced that the study has sufficient statistical power to detect regulatory activity, and differential regulatory activity to the extent needed. This is likely reflected in the extremely low number of regions showing significant activity. 2. Due to the position of the tested sequence at the 3' end of the construct, the mSTARR-seq approach cannot detect the effect of methylation on promoter activity, which is perhaps the most central role of methylation in gene regulation, and where the link between methylation and expression is the strongest. This limitation is evident in Fig. 1C and Figure 1-figure supplement 5C, where even active promoters have activity lower than 1. Considering these two points, I suspect that most effects of methylation on expression have been missed.

Overall, the combination of an extensive resource addressing key questions in functional genomics, together with the findings regarding the relationship between methylation and environmental stimuli makes this a key study in the field of DNA methylation.

Reviewer #2 (Public Review):

Dietary restriction (DR) increases lifespan, an effect that has been consistently observed in several organisms, but we still lack a clear mechanism to explain this phenomenon. In this work, Hwangbo et al. revisited the role of the circadian clock in DR-mediated lifespan effects. They found that the increase in lifespan produced by DR is missing on a clock mutant, a clock dependency that is also observed at the level of nutrient-dependent egg laying. By conducting RNA-seq with an impressive temporal resolution, they showed that DR triggers an increment in the number of cycling genes expressed in the fat body, the fly functional analog of the mammalian liver. Interestingly, from these genes, a group of them are de novo daily expressed genes, meaning that their expression was not rhythmic under the control diet but appear rhythmically expressed under DR. Among those, genes encoding proteasome subunits are enriched. The authors finally showed that adult-specific knockdown of these genes in the fat body prevents the increase in lifespan under DR, further supporting a role of the proteasome in this process. Overall, the conclusions are mostly supported by the evidence presented, and the authors' discussion nicely frame their results with other research in the field.

Strengths:

- Many studies have limited their observations of DR on lifespan to a few dietary conditions which makes the reach of some previous conclusions somewhat limited. The dilution strategy that the authors used in this work provides a strong indication that the effect of DR on lifespan relies on clock expression regardless of the conditions used. Furthermore, the inclusion of the egg-laying assay is a good addition to support this hypothesis.<br /> - Because the strength of the rhythmicity statistics relies heavily on the number of data points collected, the temporal resolution used for the RNA-seq experiments (every 2 hrs per 48hrs) is remarkable. This allows exquisite dissection of the phase of rhythmic genes in different conditions. The dataset produced in this work might be of use to other groups interested in weighting the role of other represented gene clusters in DR.

Weaknesses:

I see only minor flaws in this work, that if addressed, might strengthen the authors' conclusions, particularly:

- The results of the lifespan assays are quite variable and in some instances contradictory (Fig. S8) across trials, possibly because there are other unaccounted variables we still do not understand. The fecundity assay, in contrast, seems to be a better readout (Fig. 2). Confirming at least the two genes picked for the study (Fig. 5) would be good support for the claim that the proteasome mediates the effects of DR.<br /> - According to the model, the acute effect of DR on gene expression is related to CLOCK protein function. However, I am not sure how this link was established. It is tempting to assume that CLOCK upstream is the reason for having an increase in rhythmic genes under DR, but the experiments did not test this. The tests conducted either assessed the role of clk or the effect of an impaired proteasome on DR-dependent extension of lifespan. Thus, it is difficult to assert the authors' claims on the link between CLK and the changes in cycling genes and to the proteasome upon DR.

Reviewer #2 (Public Review):

The authors sought to characterize normal placental aging to better understand how the molecular and cellular events that trigger the labor process. An understanding of these mechanisms would not only provide insight into term labor, but also potential triggers of preterm labor, a common pregnancy complication with no effective intervention. Using bulk transcriptomic analysis of mouse and human placenta at different gestational timepoints, the authors determined that stabilization of HIF-1 signaling accompanied by mitochondrial dysfunction and cellular senescence are molecular signatures of term placenta. They also used in vitro trophoblast (choriocarcinoma) and a uterine myocyte culture system to further validate their findings. Lastly, using chemically induced HIF-1 induction in vivo in mice, the authors showed that stabilization of HIF-1 protein in the placenta reduced the gestational length significantly.

The major strength of this study is the use of multiple model systems to address the question at hand. The consistency of findings between mouse and human placenta, and the validation of mechanisms in vitro and in vivo modeling are strong support for their conclusions. The rationale for studying the term placentas to understand the abnormal process of preterm birth is clearly explained. Although the idea that hypoxic stress and placental senescence are triggers for labor is not novel, the comprehensiveness of the approach and idea to study the normal aging process are appreciated.

There are some areas of the manuscript that lack clarity and weaknesses in the methodology worth noting. The rationale for focusing on senescence and HIF-1 is not clearly given that other pathways were more significantly altered in the WGCNA analysis. The placental gene expression data were from bulk transcriptomic analyses, yet the authors do not explicitly discuss the limitations of this approach. Although the reader can assume that the authors attribute the mRNA signature of aging to trophoblasts - of which, there are different types - clarification regarding their interpretation of the data and the relevant cell types would strengthen the paper. Additionally, while the inclusion of human placenta data is a major strength, the differences between mouse and human placental structure and cell types make highlighting the specific cells of interest even more important; although there are correlations between mouse and human placenta, there are also many differences, and the comparison is further limited when considering the whole placenta rather than specific cell populations.

Additional details regarding methods and the reasons for choosing certain readouts are needed. Trophoblasts are sensitive to oxygen tension which varies according to gestational age, and it is unclear if this variable was taken into consideration in this study. Many of the cellular processes examined are well characterized in the literature yet the rationale for choosing certain markers is unclear (e.g., Glb1 for senescence; the transcripts selected as representative of the senescence-associated secretory phenotype; mtDNA lesion rate).

Overall, the findings presented are a valuable contribution to the field. The authors provide a thoughtful discussion that places their findings in the context of current literature and poses interesting questions for future pursuit. Their efforts to be comprehensive in the characterization of placental aging is a major strength; few placental studies attempt to integrate mouse and human data to this extent, and the validation and presentation of a potential mechanism by which fetal trophoblasts signal to maternal uterine myocytes are exciting. Nevertheless, a clear discussion of the methodologic limitations of the study would strengthen the manuscript.

Reviewer #2 (Public Review):

The authors describe a computational study into the energetics of KcsA inactivation. Using enhanced sampling, a converged free energy landscape of the inactivation process is achieved in two modern molecular mechanics force fields. The obtained profiles confirm the literature finding of too rapid inactivation, in particular in simulations using the CHARMM force field. Interestingly, it is found that selectivity filter collapse does not gradually follow opening of the inner gate, but proceeds rather switch-like. A key role for residue L81 is proposed as opening gateway in this process.

The study is impressive and interesting. However, I have a number of concerns that the authors may wish to address in a revised version of the manuscript.

First, concerning a set of unbiased simulations spawned at different regions of the investigated free energy landscapes, the authors write: "These simulations have the expected stability based on their starting values".<br /> Fig 2.c shows a rather smooth downhill slope in the free energy curve towards the closed state for AMBER , so wouldn't the expected behavior in that case be that all unbiased trajectories end up in the closed state, or at least travel a substantial amount in that direction? However, that is not observed. This should be further investigated.

Second, "This suggests that stabilization of the partially open state by the removal of bound lipids can explain the increase in open probability" is an odd statement, as "stabilization of the partially open state" means almost the same as "increase in open probability".

The statement "both force fields yield inactivation barriers that are orders of magnitude lower than what is expected from electrophysiology experiments" seems inaccurate. Perhaps the authors mean "inactivation rates that are orders of magnitude lower" rather than barriers?

In addition, the assertion "The CHARMM force field, on the other hand, results in landscapes in agreement with the fact that one of the dominant states in activating conditions is the partially open state, as revealed by a combination of ssNMR+MD." seems to hold for the AMBER force field without PG lipids rather than for CHARMM?

Together with the higher barrier towards the inactivated state as well as covering most known x-ray structures along the inactivation pathway, this would seem to point all in the direction that the studied AMBER force field provides a more faithful picture of the inactivation pathway than CHARMM. I, therefore, find the somewhat inconclusive summary as presented in Fig. 5 a bit uninformative, as it suggests that both mechanisms might be equally likely.

Overall, the study would benefit from a follow-up step to become more conclusive. This could be either in the form of the suggested L81 mutation or changing the simulation conditions to inactivating conditions such as low salt, in which case the inactivated state would be expected to become a minimum, which would provide an additional reference point for validation. Either of these would narrow down the spectrum of possible mechanisms.

Reviewer #2 (Public Review):

In this paper, the authors seek to identify genes that contribute to gut inflammation by capitalizing on deep phenotyping data in a mouse genetic reference population fed a high-fat or chow diet and then integrating it with human genetic data on gut inflammatory diseases, such as inflammatory bowel disease (IBD) and Ulcerative Colitis (UC). To achieve this the authors performed genome-wide gene expression in the colon of 52 BXD strains of mice fed either a high-fat or chow diet. From this analysis, they observed significant variation in gene expression related to inflammation among the 52 BXD strains and differential gene expression of inflammatory genes fed a high-fat diet. Overlaying this data with existing mouse and human data of inflammatory gut disease identified a significant enrichment. Using the 52 BXD strains the authors were able to identify specific subsets of strains that were susceptible and resistant to gut inflammation and analysis of gene expression within the colon of these strains was enriched with mouse and human IBD. Furthermore, analysis of cytokine levels of IL-10 and IL-15 were analyzed and found to be increased in resistant BXD strains and increased in susceptible BXD strains.

Using the colon genome-wide gene expression data from the 52 BXD strains, the authors performed gene co-expression analysis and were able to find distinct modules (clusters) of genes that correlated with mouse UC and human IBD datasets. Using the two modules, termed HFD_M28 and HFD_M9 that correlated with mouse UC and human IBD, the authors performed biological interrogation along with transcription factor binding motif analysis to identify possible transcriptional regulators of the module. Next, they performed module QTL analysis to identify potential genetic regulators of the two modules and identified a genome-wide significant QTL for the HFD_M28 on mouse chromosome 16. This QTL contained 552 protein-coding genes and through a deduction method, 27 genes were prioritized. These 27 genes were then integrated with human genetic data on IBD and two candidate genes, EPHA6 and MUC4 were prioritized.

Overall, this paper provides a framework and elegant use of data from a mouse genetic reference population coupled with human data to identify two strong candidate genes that contribute to human IBD and UC diseases. In the future, it will be interesting to perform targeted studies with EPHA6 and MUC4 and understand their role in gut inflammatory diseases.

Reviewer #2 (Public Review):

Jamge et al. set out to delineate the relationship between histone variants, histone modifications and chromatin states in Arabidopsis seedlings and leaves. A strength of the study is its use of multiple types of data: the authors present mass-spec, immunoblotting and ChIP-seq from histone variants and histone modifications. They confirm the association between certain marks and variants, in particular for H2A, and nicely describe the loss of constitutive heterochromatin in the ddm1 mutant.

Overall, this study nicely illustrates that, in Arabidopsis, histone variants (and H2A variants in particular) display specificity in modifications and genomic locations, and correlate with some chromatin sub-states. This encourages future work in epigenomics to consider histone variants with as much attention as histone modifications.

Reviewer #2 (Public Review):

A key strength is the quantitative approaches all add rigor to what is being attempted. The approach with very different cell culture lines will in principle help identify constitutive genes that vary in a particular and predictable way. To my knowledge, one other study that should be cited posed a similar pan-tissue question using mass spectrometry proteomics instead of gene expression, and also identified a caveolae component (cavin-1, PTRF) that exhibited a trend with stiffness across all sampled tissues. The study focused instead on a nuclear lamina protein that was also perturbed in vitro and shown to follow the expected mechanical trend (Swift et al 2013).

Reviewer #2 (Public Review):

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

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

Reviewer #2 (Public Review):

The phenotypic instability of in vitro-induced Treg cells (iTregs) has been discussed for a long time, mainly in the context of the epigenetic landscape of Treg-signature genes; e.g. Treg-specifically CpG-hypomethylated Foxp3 CNS2 enhancer region. However, it has been insufficiently understood the upstream molecular mechanisms, the particularity of intracellular signaling of natural Treg cells, and how they connect to stable/unstable suppressive function.

Huiyun Lv et al. addressed the issue of phenotypic instability of in vitro-induced regulatory T cells (iTregs), which is a different point from the physiological natural Treg cells and an obstacle to the therapeutic use of iTreg cells. The authors focused on the difference between iTreg and nTreg cells from the perspective of their control of store-operated calcium entry (SOCE)-mediated cellular signaling, and they clearly showed that the sustained SOCE signaling in iTreg and nTreg cells led to phenotypic instability. Moreover, the authors pointed the correlation between the incomplete conversion of chromatin configuration and the NFAT-mediated control of effector-type gene expression profile in iTreg cells. These findings potentially cultivate our understanding of the cellular identity of regulatory T cells and may shed light on the therapeutic use of Treg cells in many clinical contexts.

The authors demonstrated the biological contribution of Ca2+ signaling with the variable methods, which ensure the reliability of the results and the claims of the authors. iTreg cells sustained SOCE-signaling upon stimulation while natural Treg cells had lower strength and shorter duration of SOCE-signaling. The result was consistent with the previously-proposed concept; a certain range of optimal strength and duration of TCR-signaling shape the Treg generation and maintenance, and it provides us with further in-depth mechanistic understanding.

In the later section, authors found the incomplete installment of Treg-type open chromatin landscape in some effector/helper function-related gene loci in iTreg cells. These findings propose the significance to focus on not only the "Treg"-associated gene loci but also "Teffector-ness"-associated regions to determine the Treg conversion at epigenetic level.

Limitations and weaknesses;<br /> (1) Some concerns about data processing and statistic analysis.<br /> The authors did not provide sufficient information on statistical data analysis; e.g. lack of detailed descriptions about<br /> -the precise numbers of technical/biological replicates of each experiment<br /> -the method of how the authors analyze data of multiple comparisons... Student t-test alone is generally insufficient to compare multiple groups; e.g. figure 1.<br /> These inappropriate data handlings are ruining the evidence level of the precious findings.

(2) Untransparent data production; e.g. the method of Motif enrichment analysis was not provided.<br /> Thus, we should wait for the author's correction to fully evaluate the significance and reliability of the present study.

(3) Lack of evidence in human cells.<br /> I wonder whether human PBMC-derived iTreg cells are similarly regulated.

(4) NFAT regulation did not explain all of the differences between iTregs and nTregs, as the authors mentioned as a limitation.<br /> Also, it is still an open question whether NFAT can directly modulate the chromatin configuration on the effector-type gene loci, or whether NFAT exploits pre-existing open chromatin due to the incomplete conversion of Treg-type chromatin landscape in iTreg cells. The authors did not fully demonstrate that the distinct pattern of chromatin regional accessibility found in iTreg cells is the direct cause of an effector-type gene expression.

Reviewer #2 (Public Review):

An important paper that confirms the validity of the initial findings of Chretien et al regarding the hot temperatures at which the mitochondrion is operating. There are certain gaps in the literature covered in its list of cited references and, as a consequence, in the argumentation of the paper - but these can be easily fixed.

Reviewer #2 (Public Review):

In this study, Dureux and colleagues show that marmosets are sensitive to the Frith and Happe social illusion. This result is particularly interesting from an evolutionary perspective as rhesus macaques are insensitive to this social illusion.

Although marmosets show sensitivity to the illusion of social interaction between two geometric shapes, behavioural and neuronal evidence also show differences between humans and marmosets.

Reviewer #2 (Public Review):

Associative learning assigns valence to sensory cues paired with reward or punishment. Brain regions such as the amygdala in mammals and the mushroom body in insects have been identified as primary sites where valence assignment takes place. However, little is known about the neural mechanisms that translate valence-specific activity in these brain regions into appropriate behavioral actions. This study identifies a small set of upwind neurons (UpWiNs) in the Drosophila brain that receive direct inputs from two mushroom body output neurons (MBONs) representing opposite valences. Through a series of behavioral, imaging, and electrophysiological experiments, the authors show that UpWiNs are differentially regulated by the two MBONs, i.e., inhibited by the glutamatergic MBON-α1(encoding negative valence) while activated by the cholinergic MBON-α3 (encoding positive valence). They also show that UpWiNs control the wind-directed behavior of flies. Activation of UpWiNs is sufficient to drive flies to orient and move upwind, and inhibition of UpWiNs reduces flies' upwind movement toward the source of reward-predicting odors (CS+). These results, together with existing knowledge about the function of the mushroom body in memory processing, suggest an appealing model in which reward learning decreases and increases the responses of MBON-α1 and MBON-α3 to the CS+ odor, respectively, and these changes cause UpWiNs to respond more strongly to the CS+ odor and drive upwind locomotion. Interestingly, in the final part of the results, the authors reveal a wind-independent function of UpWiNs: increasing the probability that flies will revisit the site where UpWiNs were activated. Thus, UpWiNs guide learned reward-seeking behavior with and without airflow. Although the mushroom body has been extensively studied for its role in learning and memory, the downstream neural circuits that read the information from the mushroom body to guide memory-driven behaviors remain poorly characterized. This study provides an important piece of the puzzle for this knowledge gap.

Strength

1. Memory studies have predominantly relied on binary choice (go or no-go) assays as measures of memory performance. While these assays are convenient and efficient, they fall short of providing a comprehensive understanding of underlying behavioral structures. In an effort to overcome this limitation, the current study used video recording and tracking software to delve deeper into memory-guided behavior. This innovative approach allowed the authors to uncover novel neurons and examine their contribution to behavior with a level of detail not possible with binary choice assays.

2. This study used electron microscopy-based Drosophila hemibrain connectome data to reveal the synaptic connection between UpWiNs and MBON-α1 and MBON-α3. Using this method, the study shows that a single UpWiN receives direct input from both MBON-α1 and MBON- α3, which is confirmed by a functional imaging experiment. The connectome dataset also reveals several neurons downstream of UpWiNs, opening avenues for further research into the neural mechanisms linking memory and behavior.

Weakness

1. The authors repeatedly state in the manuscript that MBON-α1 and MBON-α3 convey appetitive or aversive memories, respectively. This assertion may not be entirely accurate. Evidence from sugar reward conditioning experiments suggests that MBON-α3 is potentiated and required for sugar reward memory retrieval. Therefore, the compartmentalization for appetitive and aversive memories appears not as obvious at the level of MBONs.

2. This study did not conclusively establish the importance of the MBON-α1/α3 to UpWiN pathways in memory-driven behavior. In the experiments shown in Figure 5, flies were trained to associate the activation of reward-related DANs with a specific odor (CS+). After conditioning, UpWiNs were observed to show enhanced responses to the CS+ odor. However, the results should be interpreted with caution because the driver line used to activate DANs (R58E02-LexAp65) labels not only DANs projecting to the MBON-α1 compartment, but all DANs in the protocerebral anterior medial (PAM) cluster. Thus, it remains unclear to what extent the observed enhanced responses are influenced by changes in inhibitory inputs from MBON-α1. While UpWiNs have been shown to play a critical role in the expression of sugar reward memory (Figure 7), it should be noted that UpWiNs receive inputs from multiple upstream neurons, making it difficult to accurately assess the contribution of MBON-α1/α3 to UpWiN pathways in UpWiN recruitment. Further research is needed to fully address this issue.

3. UpWind neurons (UpWiNs) were so named because their activation promotes upwind locomotion. However, when activated in the absence of airflow, flies show increased locomotor speed and an increased probability of revisiting the same location (Figure 7 and Figure 7-figure supplement 1). The revisiting behavior can be observed during the activation of UpWiNs, which is distinct from the local search behavior that typically begins after a reward stimulus is turned off (e.g., Gr64f-GAL4 results in Figure 7-figure supplement 1). Because revisiting a location can also be a consequence of repeated turns, it seems more accurate to describe UpWiNs as controlling the speed and likelihood of turns and promoting upwind movement by integrating with neurons that sense the direction of airflow.

Reviewer #2 (Public Review):

Van der Grinten and De Ruyter van Steveninck et al. present a design for simulating cortical-visual-prosthesis phosphenes that emphasizes features important for optimizing the use of such prostheses. The characteristics of simulated individual phosphenes were shown to agree well with data published from the use of cortical visual prostheses in humans. By ensuring that functions used to generate the simulations were differentiable, the authors permitted and demonstrated integration of the simulations into deep-learning algorithms. In concept, such algorithms could thereby identify parameters for translating images or videos into stimulation sequences that would be most effective for artificial vision. There are, however, limitations to the simulation that will limit its applicability to current prostheses.

The verification of how phosphenes are simulated for individual electrodes is very compelling. Visual-prosthesis simulations often do ignore the physiologic foundation underlying the generation of phosphenes. The authors' simulation takes into account how stimulation parameters contribute to phosphene appearance and show how that relationship can fit data from actual implanted volunteers. This provides an excellent foundation for determining optimal stimulation parameters with reasonable confidence in how parameter selections will affect individual-electrode phosphenes.

Issues with the applicability and reliability of the simulation are detailed below:

1) The utility of this simulation design, as described, unfortunately breaks down beyond the scope of individual electrodes. To model the simultaneous activation of multiple electrodes, the authors' design linearly adds individual-electrode phosphenes together. This produces relatively clean collections of dots that one could think of as pixels in a crude digital display. Modeling phosphenes in such a way assumes that each electrode and the network it activates operate independently of other electrodes and their neuronal targets. Unfortunately, as the authors acknowledge and as noted in the studies they used to fit and verify individual-electrode phosphene characteristics, simultaneous stimulation of multiple electrodes often obscures features of individual-electrode phosphenes and can produce unexpected phosphene patterns. This simulation does not reflect these nonlinearities in how electrode activations combine. Nonlinearities in electrode combinations can be as subtle the phosphenes becoming brighter while still remaining distinct, or as problematic as generating only a single small phosphene that is indistinguishable from the activation of a subset of the electrodes activated, or that of a single electrode.

If a visual prosthesis happens to generate some phosphenes that can be elicited independently, a simulator of this type could perhaps be used by processing stimulation from independent groups of electrodes and adding their phosphenes together in the visual field.

2) Verification of how the simulation renders individual phosphenes based on stimulation parameters is an important step in confirming agreement between the simulation and the function of implanted devices. That verification was well demonstrated. The end use a visual-prosthesis simulation, however, would likely not be optimizing just the appearance of phosphenes, but predicting and optimizing functional performance in visual tasks. Investigating whether this simulator can suggest visual-task performance, either with sighted volunteers or a decoder model, that is similar to published task performance from visual-prosthesis implantees would be a necessary step for true validation.

3) A feature of this simulation is being able to convert stimulation of V1 to phosphenes in the visual field. If used, this feature would likely only be able to simulate a subset of phosphenes generated by a prosthesis. Much of V1 is buried within the calcarine sulcus, and electrode placement within the calcarine sulcus is not currently feasible. As a result, stimulation of visual cortex typically involves combinations of the limited portions of V1 that lie outside the sulcus and higher visual areas, such as V2.

Reviewer #2 (Public Review):

The authors utilized publicly available datasets to investigate age-related DNA methylation changes in six immune cell types. They identified 350 differentially methylated sites that were changing in the same directions among all cell types, while most of the differentially methylated sites were cell type-specific during aging. Further analyses of enriched pathways and motifs indicate that these DNA methylation changes may be induced by the fluctuations in oxygen availability.

Analyzing cell type-specific DNA methylation data and comparing cross-sectional and longitudinal datasets, the authors are able to identify age-associated DNA methylation sites that may be regulated by a common mechanism in aging. However, sex differences should be considered, and the proposed mechanism could spur future studies to test it.

Reviewer #2 (Public Review):

Insects have long been known to use cuticular hydrocarbons for communication. While the general pathways for hydrocarbon synthesis have been worked out, their specificity and in particular the specificity of the different enzymes involved is surprisingly little understood. Here, the authors convincingly demonstrate that a single fatty acid synthase gene is responsible for a shift in the positions of methyl groups across the entire alkane spectrum of a wasp, and that the wasps males recognize females specifically based on these methyl group positions. The strength of the study is the combination of gene expression manipulations with behavioural observations evaluating the effect of the associated changes in the cuticular hydrocarbon profiles. The authors make sure that the behavioural effect is indeed due to the chemical changes by not only testing life animals, but also dead animals and corpses with manipulated cuticular hydrocarbons.

I find the evidence that the hydrocarbon changes do not affect survival and desiccation resistance less convincing (due to the limited set of conditions and relatively small sample size), but the data presented are certainly congruent with the idea that the methyl alkane changes do not have large effects on desiccation.

Reviewer #2 (Public Review):

Keshav Thapa et al. investigated the role of melanocortin 1 receptor (MC1-R) in cholesterol and bile acid metabolism in the liver. First, they observed that MC1-R is present in the mouse liver and that its expression is reduced in response to a cholesterol-rich diet. To determine the role of MC1-R in the liver, they generated hepatocyte-specific MC1-R KO mice (L-Mc1r-/-). These animals exhibited a significant increase in liver weight, lipid accumulation, triglycerides and cholesterol levels, and fibrosis in comparison with control mice. By performing liquid chromatography-mass spectrometry, the authors also found that L-Mc1r-/- mice also have fewer bile acids in the plasma and faeces, but not in the liver. In accordance with these findings, mRNA/protein expression of different genes involved in these processes were altered in L-Mc1r-/- animals.

Secondly, in an attempt to evaluate the underlying mechanisms, they measured the expression of MC1-R in HepG2 cells under different treatments (i.e., palmitic acid, LDL, and atorvastatin). Moreover, they stimulated these cells with the endogenous MC1-R agonist - MSH, where they show that this molecule decreases the free cholesterol content, whereas increasing LDL and HDL uptake, as well as recapitulates some previously observed phenotypes in the proportions of bile acids. These effects were also encountered when using a selective agonist for MC1-R (i.e., LD211), further supporting the specific role of MC1-R. Finally, some experiments indicated that -MSH evokes not one single, but multiple intracellular signalling cascades for which MC1-R activation effects might take place.

Overall, this work provides novel and interesting findings on the role of MC1-R in cholesterol and bile acid metabolism in the liver, which undoubtedly will have some crucial implications for future research. Nevertheless, some experimental details should be better explained for the correct interpretation of the data. Besides, discrepant results exist regarding the molecular mechanisms behind MC1-R action that requires additional experimentation to support the conclusions drawn.

Reviewer #2 (Public Review):

Src is a well-studied non-receptor protein tyrosine kinase (PTK) with broad impacts on many signal transduction pathways. In this manuscript titled, "A Back-Door Insights into the modulation of Src kinase activity by the polyamine spermidine" Rossini et al investigated the mechanism of spermidine, a natural polyamine, in regulating Src tyrosine kinase activity and complex formation with IDO1, a known Src substrate. These data show a direct binding, and an allosteric binding site in the SH2 domain of Src, for spermidine. Interestingly, the manuscript also shows spermidine bound to Src promotes binding to IDO1, as well as its phosphorylation.

Overall, the molecular glue-like property of spermidine is an interesting finding. That Src substrate binding and phosphorylation for Src substrate is regulated by natural metabolites like spermidine is also a new and interesting finding. These discoveries further strengthen the idea to develop potential allosteric modulators for Src/PTK-mediated pathways.

Reviewer #2 (Public Review):

The manuscript has several areas of strength; it functionally explores a mutant that is detected in a portion of pancreatic cancers; it conducts mechanistic investigation and it uses human cell lines to validate the findings based on mouse models. Some areas for improvement are described below.

1) TGF-b is known to act as a tumor suppressor early in carcinogenesis, and as a tumor promoter later. The authors should extend their analysis of mouse models to determine whether the effect of SF3B1K700E is specific to promoting initiation (e.g. more, early acinar ductal metaplasia) or faster progression of PanINs following their formation. Another way to address this could be acinar cultures, to determine whether an increased propensity to ADM exists.

2) Given that the effect of SF3B1K700E expression is more prominent in KC mice, rather than in KPC mice, the authors should explain the rationale for using the latter for RNA sequencing.

3) Given that this mutation is found in about 3% of human pancreatic cancer, it would be interesting to know whether these tumors have any unique feature, and specifically any characteristic that could be harnessed therapeutically.

4) It would be interesting to know whether this mutation mutually exclusive to other mutations affecting response to TGF-b. Further, while the data might not be widely available, it would be interesting to know whether in human patients the mutation occurs in precursor lesions (PanIN might be difficult to assess, but IPMN might be doable) or at later stages.

Reviewer #2 (Public Review):

A new study by Kimble et al. examines the role of extensive resection in DNA double-strand break repair. Formation of ssDNA at DNA breaks is initiated by Mre11-Rad50-Xrs2 and followed by Exo1 or Sgs1/Dna2, which form longer ssDNA. This ssDNA is used to load recombination and DNA damage checkpoint proteins. Some studies suggested that very short ssDNA by MRX complex is sufficient for DSB repair. Here, the authors look carefully at the role of extensive resection in DSB repair by gene conversion. To address this question they have constructed a large number of new recombination assays. They find that sgs1 exo1 mutants that lack extensive resection are capable of DSB repair when recombining loci are present on a single DNA molecule and within 50 kb from each other. When the template for DSB repair is further away on the same molecule or present on a different chromosome, the repair is reduced by 5-10 folds in the absence of extensive resection. The authors present data suggesting that this defect relates to slower repair kinetics between more distant homologous sequences and the need for a Mec1-mediated DNA damage checkpoint that requires extensive resection. The role of the checkpoint response is likely not limited to simple cell cycle arrest but may also be necessary for the mobility of a broken molecule. Partial suppression of the sgs1 exo1 repair defect is accomplished by activating the checkpoint using an artificial system colocalizing checkpoint proteins on a separate chromosome. Altogether the manuscript addresses an important question, is well-written, and presents interesting data.

Reviewer #2 (Public Review):

This represents an important study that demonstrates a high degree of heterogeneity within trailblazer cells in clusters that participate in collective migration. Solid methods highlight this heterogeneity and show that in TNBC cancers, trailblazer cells are defined by vimentin (and not Keratin 14) and are dependent on both TGFbeta and EGFR signaling. Additional, single cell studies would further support this work.

Strengths:

The paper highlights that collective migration, and the nature of trailblazer cells can be highly heterogeneous. This is important as it suggests that the ability to move between states may supersede a singular phenotype.

The paper uses animal models and organoids and in several areas attempts to correlate findings to human tissues.

The experiments are logically described.

Reviewer #2 (Public Review):

The paper by Pomper and coworkers is an elegant neurophysiological study, generally sound from a methodological point of view, which presents extremely relevant data of considerable interest for a broad audience of neuroscientists. Indeed, they shed new light on the mirror mechanism in the primate brain, trying to approach its study with a novel paradigm that successfully controls for some important factors that are known to impact mirror neuron response, particularly the target object. In this work, a rotating device is used to present the very same object to the monkey or the experimenter, in different trials, and neurons are recorded while the monkey (motor response) or the experimenter (visual response) performed a different action (twist, shift, lift) cued by a colored LED.

The results show that there is a small set of neurons with congruent visual and motor selectivity for the observed actions, in line with classical mirror neuron studies, whereas many more cells showed temporally unstable matched or even completely non-matched tuning for the observed and executed actions. Importantly, the population codes allow to accurately decode both executed and observed actions and, to some extent, even to cross-decode observed actions based on the coding principles of the executed ones.

In my view, however, the original hypothesis that an observer understands the actions of others by the activation of his/her motor representations of the observed actions constitutes circular reasoning that cannot be challenged or falsified, as the author may want to claim. Indeed, 1) there is no causal evidence in the paper favoring or ruling out this hypothesis (and there couldn't be), 2) there is no independent definition (neither in this paper nor in the literature) of what "action understanding" should mean (or how it should be measured). Instead, the findings provide important and compelling evidence to the recently proposed hypothesis that observed actions are remapped onto (rather than matched with) motor substrates, and this recruitment may primarily serve, as coherently hypothesized by the authors, to select behavioral responses to others (at least in monkeys).

1) One of the main problems of this manuscript is, in my view, a theoretical one. The authors follow a misleading, though very influential, proposal, advanced since the discovery of mirror neurons: if there are (mirror) neurons in the brain of a subject with an action tuning that is matched between observation and execution contexts, then the subject "understands" the observed action. This is clearly circular reasoning because the "understanding" hypothesis uniquely derives from the neuron firing features, which are what the hypothesis should explain. In fact, there is no independent, operational definition of the term "understanding". Not surprisingly there is no causal evidence about the role of mirror neurons in the monkey, and the human studies that have claimed to provide causal evidence of "action understanding" ended up using, practically, operational definitions of "recognition", "match-to-sample", "categorization", etc. Thus, "action understanding" is a theoretical flaw, and there is no way "to challenge" a theoretical flaw with any methodologically sound experiment, especially when the flaw consists of circular reasoning. It cannot be falsified, by definition: it must simply be abandoned.<br /> On these bases, I strongly encourage the authors to rework the manuscript, from the title to the discussion, by removing any useless attempt to falsify or challenge a circular concept and, instead, constructively shed new light on how mirror neurons may work and which may be their functional role.

2) An important point to be stressed, strictly related to the previous one, concerns the definition of "mirror neuron". I premise that I am perfectly fine with the definition used by the authors, which is in line with the very permissive one adopted in most studies of the last 20 years in this field. However, it does not at all fulfill the very restrictive original criteria of the study in which "action understanding" concept was proposed (see Gallese et al. 1996 Brain): no response to object, no response to pantomimed action or tool actions, activation during execution in the dark and during the observation of another's action. If the idea (which I strongly disagree with) was to simply challenge a (very restrictive) definition of mirroring (a very out-of-date one, indeed, and different from the additional implication of "action understanding"), the original definition of this concept should be at least rigorously applied. In the absence of additional control conditions, only the example neuron in Figure 2A could be considered a mirror neuron according to Gallese et al. 1996. Permissive criteria implies that more "non-mirror" neurons are accepted as "mirror": simply because they are permissively named "mirror", does not imply they are mirroring anything as initially hypothesized (Example neuron in Fig 2B, for example, could be related to mouth, rather than hand, movements, since it responds strongly and similarly around the reward delivery also during the observation task, when the monkey should be otherwise still). Clearly, these concerns impact all the action preference analyses. To practically clarify what I mean, it should be sufficient to note that 74% (reported in this study) is the highest percentage ever reported so far in a study of neurons with "mirror" properties in F5 (see Kilner and Lemon 2013, Curr Biol) and it is similar to the 68% recently reported by these same authors (Pomper et al. 2020 J Neurophysiol) with very similar criteria. Clearly, there is a bias in the classification criteria relative to the original studies: again, no surprise if by rendering most of the recorded neurons "mirror by definition" then they don't "mirror" so much. I suggest keeping the authors' definition but removing the pervasive idea to challenge the (misleading) concept of understanding.

3) It would be useful to provide more information on the task. Panel B in Figure 1 is the unique information concerning the type of actions performed by the monkey and the experimenter. Although I am quite convinced of the generally low visuomotor congruence, there are no kinematics data nor any other evidence of the statement "the experimental monkey was asked to pay attention to the same actions carried out by a human actor". First, although the objects were the same, the same object cannot be grasped or manipulated in the same way by a human and a macaque, even just because of the considerable difference in the size of their hands; this certainly changes the way in which monkeys' and experimenter's hands interact with the same object, and this is a quantifiable (but not quantified) source of visuomotor difference between observed and executed actions and a potential source of reduced congruency. Second, there is little information about monkey's oculomotor behavior in the two conditions, which is known to affect mirror neuron activity when exploratory eye movements are allowed (Maranesi et al. 2013 Eur J Neurosci), potentially influencing the present findings: a {plus minus}7 (vertical) and {plus minus}5 (horizontal) window at 49 cm implies that the monkey could explore a space larger than 10 cm horizontally and 14 cm vertically, which is fine, but certainly leaves considerable freedom to perform different exploratory eye movements, potentially different among observed actions and hence capable to account for different "attention" paid by the monkey to different conditions and hence a source of neural variability, in addition to action tuning.

4) Information about error trials and their relationship with action planning. The monkey cannot really "make errors" because, despite the cue, each object can be handled in a unique way. The monkey may not pay attention to the cue and adjust the movement based on what the object permits once grasped, depending on online object feedback. From the behavioral events and the times reported in Table 1, I initially thought that "shift" action was certainly planned in advance, whereas "lift" and "twist" could in principle be obtained by online adjustments based on object feedback; nonetheless, from the Methods section it appears that these times are not at all informative because they seem to depend on an explicit constraint imposed by the experimenters (in a totally unpredictable way). Indeed, it is stated that "to motivate the monkey even more to use the LED in the execution task, another timeout was active in 30% (rarely up to 100%) of trials for the time period between touch of object to start moving the object: 0.15 (rarely 0.1) for a twist and shift, 0.35 (rarely 0.3s) for a lift". This is totally confusing to me; I don't understand 1) why the monkey needed to be motivated, 2) how can the authors be sure/evaluate that the monkeys were actually "motivated" in this way, and 3) what kind of motor errors the monkey could actually do if any. If there is any doubt that the monkeys did actually select and plan the action in advance based on the cue, there is no way to study whether the activity during action execution truly reflects the planned action goal or a variety of other undetermined factors, that may potentially change during the trials. Please clarify.

5) Classification analysis. There seems to be no statistical criterion to establish where and when the decoding is significantly higher than chance: the classifier performance should be formally analyzed statistically. I would expect that, in this way, both the exe-obs and the obs-exe decoding may be significant. Together with the considerations of the previous point 2 about the permissive inclusion criteria for mirror neurons, this is a remarkable (even quite unexpected) result, which would prove somehow contrary to what the authors claim in the title of the paper. The fact that in any classification the "within task" performance is significantly better than the "between task" performance does not appear in any way surprising, considering both the inclusive selection criteria for "mirror neurons" and the unavoidably huge different sources of input (e.g. proprioceptive, tactile, top-down, etc. afferences) between execution and observation. So, please add a statistical criterion to establish and show in the figures when and where the classifications are significantly above chance.

6) "As the concept of a mirror mechanism posits that the observation performance can be led back to an activation of a motor representation, we restricted this analytical step to a comparison of the exe-obs and the obs-obs discrimination performance". I don't understand the rationale of this choice. The so-called "concept" of mirror mechanism in classical terms posits that mirror neurons have a motor nature and hence their functioning during observation should follow the same principle as during action execution. But this logical consideration has never been demonstrated directly (it is indeed costated by several papers), and when motor neurons are concerned (e.g. pyramidal tract neurons, see Kraskov et al. 2009) their behavior during action observation is by far more complex (e.g. suppression vs facilitation) than that hypothesized for classical "mirror neurons". Furthermore, when across-task decoding for execution and observation code has been used, both in neurophysiological (e.g. Livi et al. 2019, PNAS) and neuroimaging (Fiave et al. 2018 Neuroimage) data, the visual-to-motor direction typical produce better performance than the opposite one. Thus, I don't see any good reason not to show also (if not even just) the obs-exe results. Furthermore, I wonder whether it is considered the possible impact of a rescaling in the single neuron firing rate across contexts, as the observation response is typically less strong than the execution response in basically all brain areas hosting neurons with mirror properties, and this should not impact on the matching if the tuning for the three actions remains the same (e.g. see Lanzilotto et al. 2020 PNAS). The analysis shown in Figures 4 and 5 is, for the rest, elegant and very convincing - somehow surprising to me, as the total number of "congruent" neurons (7.5%) is even greater than in the original study by Gallese et al. (5.4%).

7) The discussion may need quite deep revision depending on the authors' responses and changes following the comments; for sure it should consider more extensively the numerous recent papers on mirror neurons that are relevant to frame this work and are not even mentioned.

Reviewer #2 (Public Review):

This manuscript pulls together a series of integrated genetic and metabolomic data sets to examine the molecular basis for biguanide action in C. elegans. Biguanides such as Metformin are important anti-diabetic drugs as well as being explored as a therapeutic mechanism for increasing human longevity. Understanding the molecular basis of biguanide action is of general interest to those in the ageing and age-related health fields as well as to those studying metabolism and obesity. The work here has been carried out in C. elegans but the work can be picked up by those working in mammalian systems. More could be done to highlight the conserved aspects of the mechanisms involved to assist with this translatability.

The methodology used is in general standard in the field and experiments are reported in detail. The successful use of metabolomics in C. elegans and its associated protocols is helpful as more labs expand to do this type of work.

Strengths: In general all the experiments presented are logical and well executed with the conclusions supported by the data. I am convinced that: 1) Metformin and Phenformin extend C. elegans lifespan (although that has previously been shown), 2) biguanides induce changes in ether lipids, 3) genes required for ether lipid biogenesis are required for the lifespan incurred with biguanide treatment and, in the case of fard-1 oe, can also promote longevity when levels are increased, 4) ether lipid biogenesis is also needed for other specific key longevity processes to extend lifespan, and 5) that some key ageing regulators (skn-1, aak-2 and daf-16) are required for fard-1 oe to extend lifespan.

Weaknesses: I was less convinced by the fat accumulation data and felt that the link between skn-1 gain of function and ether lipid genes was not clear and that the results were more correlative than mechanistic. If age-associated somatic depletion of fat is important for the lifespans seen here then this is interesting and important and identifying an epistatic, genetic link between the implicated genes and fat levels is desirable. Additionally, biguanides are reported to have major effects on the metabolism and growth of bacteria. As C. elegans grows on and eats E. coli, it is important that the biguanides in question do not alter the worm's food source. If bacterial growth is restricted or metabolically altered this would have a major impact on fat metabolism and the other outputs examined here (see Cabreiro et al 2013). Therefore the impact of these biguanide treatments on the C. elegans foods used here should be clearly addressed. Additionally, biguanide treatment is subject to dose dependence. Different concentrations of biguanide are used for different types of experiments to make correlative points e.g. growth inhibition at 160mM metformin, and metformin uptake measured in C. elegans treated with 50mM. It is not clear why, or whether this could impact the results. Can the authors be sure that these different doses do not alter metformin action and/or uptake either by the worms or the way the bacteria metabolise it? I appreciate that it is interesting and important to understand what biguanides are doing in the organism irrespective of whether this is a direct or indirect effect but knowing how the effects are achieved could be important for treatment strategies moving forwards.

Reviewer #2 (Public Review):

Khalil et al. aimed to gain insights into similarities and differences between circuits processing innate and learned threats. For this, they investigated a circuit that is well established to have a critical role in auditory associative threat learning, the projection from the medial geniculate nucleus (MGN) to the basolateral amygdala (BLA), and carried out a side-by-side comparison of its role in conditioned and innate threat.

Although the MGN is part of the main auditory stream, the neurons that project to BLA are multimodal. Khalil et al. took advantage of this to use visual looming stimuli to evoke innate threat. The authors showed that the MGN-BLA pathway processes both innate freezing responses to looming black circles and threat-conditioned freezing responses to tones. The disruption of the pathway impairs freezing in both cases, and the pathway is activated mostly in the presence of freezing. This suggests that the MGN-BLA processes threat independently of the sensory modality and of whether the threat is learnt or not. This further suggests that these different forms of threat may share similar mechanisms.

Nonetheless, the fact that MGN-BLA circuit disruptions were done during the conditioning phase of associative threat learning, and not during the recall phase only, complicates the side-by-side comparison: it could be argued that in this case what is disturbed is the processing of the unconditioned innately aversive stimulus in the task, the foot shock, instead of the learnt threat of the sound. Still, this would go in hand with one of the main conclusions of the study, which is that the MGN-BLA processes innate threats.

There are alternative interpretations of the results though, which are beyond the scope of the study: the circuit might be relevant for processing salient stimuli beyond threatening stimuli, for instance for positive valence stimuli as well; or this circuit might be relevant for processing the freezing response to threat in particular. To target the MGN-BLA circuit, the authors employ viral-vector mediated expression of proteins in mice. This way they delete, inhibit, or image either the activity of the neurons (or the axons) that project from MGN to BLA, or the BLA neurons themselves. They combine this with fiber-photometry and behavioural quantifications. Targeting these small and deep nuclei in the mouse brain bilaterally is challenging, which increases the value of the presented data. Conversely, it is important that the authors support more explicitly the specificity of their targeting methods and quantifications throughout the manuscript.

Overall, the main conclusions of this paper are mostly supported by data, but important methodological aspects need to be clarified, data analysis extended and the interpretation of results discussed further. The question of whether innate and learnt responses to stimuli share common mechanisms is timely. This study places the MGN-BLA pathway as a suitable model circuit to investigate this and paves the way for future work to dig into the implicated mechanisms.

Specific comments (strengths):<br /> a) The authors use two methods to interrupt the MGN-BLA pathway, a reversible one (chemogenetics) and an irreversible one (neuronal deletion via caspase 3 expression), obtaining consistent results that strengthen the evidence supporting their conclusions.<br /> b) The authors demonstrate the efficacy of their MGN-BLA pathway interruption methods with in vivo recordings.<br /> c) The approach of addressing the same behavioural output (freezing) in the two conditions (innate and learnt threat) helps the interpretability of results.

Specific comments (weaknesses):<br /> e) There are not enough analysis and method descriptions to demonstrate the specificity of the targeting approach, which is in some cases neither reflected in the pictures of the main figures. These include quantifications of the extension of expression/deletions in the brain and placement of viral-vector injections. In particular, these should show that i) protein expression does not extend beyond the BLA or MGN; ii) the MGN cells projecting to the striatum (right above the BLA) are not implicated, iii) that neurons in the visual thalamus are not affected by the manipulations. These are critical points that need to be addressed.<br /> f) There is a lack of digging into the mechanisms that could be enhanced with further analysis and discussion. For example, to start addressing this question, the authors administer blockers of beta-adrenergic receptors systemically. This reveals differences between MGN-BLA projecting neurons, BLA neurons, and innate and learnt threat, but the mechanistic implications are not clear and should be discussed. Also, the interpretation of the pathway's role in behaviour and its relation to neuronal activity could be deepened with further analysis.

Reviewer #2 (Public Review):

The authors of this study investigated the relationship between (under)confidence and the anxious-depressive symptom dimension in a longitudinal intervention design. The aim was to determine whether confidence bias improves in a state-like manner when symptoms improve. The primary focus was on patients receiving internet-based CBT (iCBT; n=649), while secondary aims compared these changes to patients receiving antidepressants (n=82) and a control group (n=88).

The results support the authors' conclusions, and the authors convincingly demonstrated a weak link between changes in confidence bias and anxious-depressive symptoms (not specific to the intervention arm)

The major strength and contribution of this study is the use of a longitudinal intervention design, allowing the investigation of how the well-established link between underconfidence and anxious-depressive symptoms changes after treatment. Furthermore, the large sample size of the iCBT group is commendable. The authors employed well-established measures of metacognition and clinical symptoms, used appropriate analyses, and thoroughly examined the specificity of the observed effects.

However, due to the small effect sizes, the antidepressant and control groups were underpowered, reducing comparability between interventions and the generalizability of the results. The lack of interaction effect with treatment makes it harder to interpret the observed differences in confidence, and practice effects could conceivably account for part of the difference. Finally, it was not completely clear to me why, in the exploratory analyses, the authors looked at the interaction of time and symptom change (and group), since time is already included in the symptom change index.

This longitudinal study informs the field of metacognition in mental health about the changeability of biases in confidence. It advances our understanding of the link between anxiety-depression and underconfidence consistently found in cross-sectional studies. The small effects, however, call the clinical relevance of the findings into question. I would have found it useful to read more in the discussion about the implications of the findings (e.g., why is it important to know that the confidence bias is state-dependent; given the effect size of the association between changes in confidence and symptoms, is the state-trait dichotomy the right framework for interpreting these results; suggestions for follow-up studies to better understand the association).

Reviewer #2 (Public Review):

This paper explores how minimal active matter simulations can model tissue rheology, with applications to the in vivo situation of zebrafish morphogenesis. The authors explore the idea of active noise, particle softness and size heterogeneity cooperating to give rise to surprising features of experimental tissue rheologies (in particular an increase and then a plateau in viscosity with fluid fraction). In general, the paper is interesting from a theoretical standpoint, by providing a bridge between concepts from jamming of particulate systems and experiments in developmental biology. The idea of exploring a free space picture in this context is also interesting.

However, I'm still unsure right now though of how much it can be applied to the specific system that the authors refer to - which could be fixed either by considering other experimental systems/models reported in the recent literature or by doing the following theoretical checks:

- Take your current simulations and smoothly change the ratio of polydispersity from 8 to 0 to see exactly how much dispersity is needed to explain viscosity plateauing, and at which point the transition occurs.

- Cellular self-propulsion does not seem to play a role in zebrafish blastoderm, see Ref. [14]. Active noise has been proposed to play key roles in other systems and you could check whether such active noise could replace self-propulsion in your model, see for example Kim & Campas, Nat Phys, 2021.

- Could you simulate realistic rheological deformations to see how much they match both your expectation and the data?

Reviewer #2 (Public Review):

The authors examine the use of metformin in the treatment of hepatic ischemia/reperfusion injury (HIRI) and suggest the mechanism of action is mediated in part by the gut microbiota and changes in hepatic ferroptosis. While the concept is intriguing, the experimental approaches are inadequate to support these conclusions.

The histological and imaging studies were considered a strength and reveal a significant impact of metformin post-HIRI.

Weaknesses largely stem from the experimental design. First, use of the iron chelator DFO would be strengthened using the ferroptosis inhibitor, liproxstatin. Second, the impact of metformin on the microbiota is profound resulting in changes in bile acid, lipid, and glucose homeostasis. Throughout the manuscript no comparisons are made with metformin alone which would better capture the metformin-specific effects. Lastly, the absence of proper controls including germ free mice, metformin treated mice, FMT treated mice, etc make it difficult to understand the outcomes and to properly reproduce the findings in other labs.

Overall, while the concept is interesting and has the potential to better understand the pleiotropic functions of metformin, the limitations with the experimental design and lack of key controls make it challenging to support the conclusions.

Reviewer #2 (Public Review):

This interesting research commendably revealed the association between sleep regularity and mortality. However, as authors acknowledged, the analysis can not accurately identify the cause and effect. In my opinion, the causality is important for this topic, cuz, sleep regularity and health (e.g. chronic disease) were long-term simultaneous status, especially given the participants are older. I suggest that the author could utilize MR analysis to find out for more evidence.

Reviewer #2 (Public Review):

In this paper the authors aim to investigate brain-wide activation patterns following administration of the anesthetics ketamine and isoflurane, and conduct comparative analysis of these patterns to understand shared and distinct mechanisms of these two anesthetics.

To this end, they perform Fos immunohistochemistry in perfused brain section to label active nuclei, use a custom pipeline to register images to the ABA framework and quantify Fos+ nuclei, and perform multiple complementary analyses to compare activation patterns across groups.

This is an interesting line of research and a tour de force in brain-wide Fos quantification. However, there are several issues with the analysis, and overall integration that dampen my enthusiasm for the article in its current form.

Major comments:

1- The authors report 987 brain regions in the introduction, but I cannot find any analysis that incorporates these or even which regions they are. Very little rationale is provided for the regions included in any of the analyses and numbers range from 53 in Figure 1, to 201 in Figure 3, to 63 in Figure 6. It would help if the authors could first survey Fos+ counts across all regions to identify a subset that is of interest (significantly changed by either condition compared to control) for follow up analysis.

2- Different data transformations are used for each analysis. One that is especially confusing is the 'normalization' of brain regions by % of total brain activation for each animal prior to PCA analysis in Figures 2 and 3. This would obscure any global differences in activation and make it unlikely to observe decreases in activation (which I think is likely here) that could be identified using the Fos+ counts after normalizing for region size (ie. Fos+ count / mm3) which is standard practice in such Fos-based activity mapping studies. While PCA can be powerful approach to identify global patterns, the purpose of the analysis in its current form is unclear. It would be more meaningful to show that regional activation patterns (measured as counts/mm3) are on separate PCs by group.

3- Critical problem: The authors include a control group for each anesthetic (ketamine vs. saline, isofluorane vs. homecage) but most analyses do not make use of the control groups or directly compare Fos+ counts across the groups. Strictly speaking, they should have compared relative levels of induction by ketamine versus induction by isoflurane using ANOVAs. Instead, each type of induction was separate from the other. This does not account for increased variability in the ketamine versus isoflurane groups. There is no mention in the Statistics section or in Results section that any multiple comparison corrections were used. It appears that the authors only used Students t-test for each region and did not perform any corrections.

4- Figures 4 and 5 show brain regions 'significantly activated' following KET or ISO respectively, but again a subset of regions are shown and the stats seem to be t-tests with no multiple comparisons correction. It would help to show these two figures side by side, include the same regions, and keep the y axis ranges similar so the reader can easily compare the 'activation patterns' across the two treatments. Indeed, it looks like KET/Saline induced activation is an order or magnitude or two higher than ISO/Homecage. I would also recommend that this be the first data figure before any other analyses and maybe further analysis could be restricted to regions that are significantly changed in following KET or ISO here.

5- Analyses in Figure 6 and 7 are interesting but again the choice of regions to include is unclear and makes interpreting the results impossible. For example, in Figure 7 it is unclear why the list of regions in bar graphs showing Degree and Betweenness Centrality are not the same even within a single row?

Reviewer #2 (Public Review):

The authors attempt to show that event-related changes in the alpha band, namely a decrease in alpha power over parieto/occipital areas, explain the P300 during an auditory target detection task. The proposed mechanism by which this happens is a baseline-shift, where ongoing oscillations which have a non-zero mean undergo an event-related modulation in amplitude which then mimics a low frequency event-related potential. In this specific case, it is a negative-mean alpha-band oscillation that decreases in power post-stimulus and thus mimics a positivity over parieto-occipital areas, i.e. the P300. The authors lay out 4 criteria that should hold if indeed alpha modulation generates the P300, which they then go about providing evidence for.

Strengths:<br /> - The authors do go about showing evidence for each prediction rigorously, which is very clearly laid out. In particular, I found the 3rd section connecting resting-state alpha BSI to the P300 quite compelling.<br /> - The study is obviously very well-powered.<br /> - Very well-written and clearly laid out. Also, the EEG analysis is thorough overall, with sensible analysis choices made.<br /> - I also enjoyed the discussion of the literature, albeit with certain strands of P300 research missing.

Weaknesses:<br /> In general, if one were to be trying to show the potential overlap and confound of alpha-related baseline shift and the P300, as something for future researchers to consider in their experimental design and analysis choices, the four predictions hold well enough. However, if one were to assert that the P300 is "generated" via alpha baseline shift, even partially, then the predictions either do not hold, or if they do, they are not sufficient to support that hypothesis. This general issue is to be found throughout the review. I will briefly go through each of the predictions in turn:

1. The matching temporal course of alpha and P300 is not as clear as it could be. Really, for such a strong statement as the P300 being generated by alpha modulation, one would need to show a very tight link between the signals temporally. There are many neural and ocular signals which occur over the course of target detection paradigms: P300, alpha decrease, motor-related beta decrease, the LRP, the CNV, microsaccade rate suppression etc. To specifically go above and beyond this general set of signals and show a tighter link between alpha and P300 requires a deeper comparison. To start, it would be a good idea to show the signals overlapping on the same plot to really get an idea of temporal similarity. Also, with the P300-alpha correlation, how much of this correlation is down to EEG-related issues such as skull thickness, cortical folding, or cognitive issues such as task engagement? One could perhaps find another slow wave ERP, e.g. the Lateralised Readiness Potential, and see if there is a similar strength correlation. If there is not, that would make the P300 relationship stand out.

In Figure 3, it is clear that alpha binning does not account for even 50% of the variance of P300 amplitude. Again, if there is such a tight link between the two signals, one would expect the majority of P300 variance to be accounted for by alpha binning. As an aside, the alpha binning clearly creates the discrepancy in the baseline period, with all alpha hitting an amplitude baseline at approx. 500ms. I wonder if could you NOT, in fact, baseline your slow wave ERP signal, instead using an appropriate high pass filter (see "EEG is better left alone", Arnaud Delorme, 2023) and show that the alpha binning creates the difference in ERP at the baseline which then is reinterpreted as a P300 peak difference after baselining.

2. The topographies are somewhat similar in Figure 4, but not overwhelmingly so. There is a parieto-occipital focus in both, but to support the main thesis, I feel one would want to show an exact focus on the same electrode. Showing a general overlap in spatial distribution is not enough for the main thesis of the paper, referring to the point I make in the first paragraph re Weaknesses. Obviously, the low density montage here is a limitation. Nevertheless, one could use a CSD transform to get more focused topographies (see https://psychophysiology.cpmc.columbia.edu/software/csdtoolbox/), which apparently does still work for lower-density electrode setups (see Kayser and Tenke, 2006).

3. Very nice analysis in Figure 6, probably the most convincing result comparing BSI in steady state to P300, thus at least eliminating task-related confounds.

4. Also a good analysis here, wherein there seem to be similar correlation profiles across P300 and alpha modulation. One analysis that would really nail this down would be a mediation analysis (Baron and Kenny, 1986; https://davidakenny.net/cm/mediate.htm), where one could investigate if e.g. the relationship between P300 amplitude and CERAD score is either entirely or partially mediated by alpha amplitude. One could do this for each of the relationships. To show complete mediation of P300 relationship with a cog task via alpha would be quite strong.

One last point, from the methods it appears that the task was done with eyes closed? That is an extremely important point when considering the potential impact of alpha amplitude modulation on any other EEG component due to the well-known substantial increase in alpha amplitude with eyes closed versus open. I wonder, would we see any of these effects with eyes opened?

Overall, there is a mix here of strengths of claims throughout the paper. For example, the first paragraph of the discussion starts out with "In the current study, we provided comprehensive evidence for the hypothesis that the baseline-shift mechanism (BSM) is accountable for the generation of P300 via the modulation of alpha oscillations." and ends with "Therefore, P300, at least to a certain extent, is generated as a consequence of stimulus-triggered modulation of alpha oscillations with a non-zero mean." In the limitations section, it says the current study speaks for a partial rather than exhausting explanation of the P300's origin. I would agree with the first part of that statement, that it is only partial. I do not agree, however, that it speaks to the ORIGIN of the P300, unless by origin one simply means the set of signals that go to make up the ERP component at the scalp-level (as opposed to neural origin).

Again, I can only make these hopefully helpful criticisms and suggestions because the paper is very clearly written and well analysed. Also, the fact that alpha amplitude modulation potentially confounds with P300 amplitude via baseline shift is a valuable finding.

Reviewer #2 (Public Review):

How organism physiological state modulates establishment and perdurance of memories is a timely question that the authors aimed at addressing by studying the interplay between energy homeostasis and food-related conditioning in Drosophila. Specifically, they studied how starvation modulates the establishment of short-term vs long-term memories and clarified the role of the monoamine Octopamine in food-related conditioning, showing that it is not per se involved in formation of appetitive short-term memories but rather gates memory formation by suppressing LTM when energy levels are high. This work clarifies previously described phenotypes and provides insight about interconnections between energy levels, feeding and formation of short-term and long-term food-related memories. In the absence of population-specific manipulation of octopamine signaling, it however does not reach a circuit-level understanding of how these different processes are integrated.

Strengths<br /> - Previous studies have documented the impact of Octopamine on different aspects of food-related behaviors (regulation of energy homeostasis, feeding, sugar sensing, appetitive memory...), but we currently lack a clear understanding of how these different functions are interconnected. The authors have used a variety of experimental approaches to systematically test the impact of internal energy levels in establishment of appetitive memory and the role of Octopamine in this process.

- The authors have used a range of approaches, performed carefully controlled experiments and produced high quality data.

Weaknesses<br /> 1- In the tbh mutant flies, Tyramine -to- Octopamine conversion is inhibited, resulting not only in a lack of Octopamine, but also in elevated levels of Tyramine. If and how elevated levels of Tyramine contributes to the described phenotypes is unclear. In the current version of the manuscript, only one set of experiments (Figure 2) has been performed using Octopamine agonist. This is particularly important in light of recent published data showing that starvation modifies Tyramine levels.

2- Octopamine (and its precursor Tyramine) have been implicated in numerous processes, complicating the analysis of the phenotypes resulting from a general inhibition of tbh.

3- The manuscript explores various aspects of the impact of energy levels on food-related behaviors and the underlying sensing and effector mechanism, both in wild-type and tbh mutants, making it difficult to follow the flow of the results.

Reviewer #2 (Public Review):

This is a well-written paper using gene expression in tree sparrow as model traits to distinguish between genetic effects that either reinforce or reverse initial plastic response to environmental changes. Tree sparrow tissues (cardiac and flight muscle) collected in lowland and highland populations subject to hypoxia treatment were profiled for gene expression and compared in 1) highland birds; 2) lowland birds under normal conditions to test for differences in directions of changes between initial plastic response and subsequent colonized response.

The authors clarified several points and made revisions according to my comments. It is good to know that the highland and lowland samples were collected and processed at the same time and the previous publication reported part of the data. My concerns regarding the conclusions about reversal versus reinforcement remain even after the additional analyses. Further studies are needed to confirm these results.

Reviewer #2 (Public Review):

The authors used cutting-edge bio-telemetry technology to decipher the roles of wind speed and wave height on the take-off of albatrosses from the water surface. They revealed that each of these factors contributes to take-off in a unique way with interesting interactions of the two factors. The authors achieved their objectives and their results support their conclusions. This work will set new standards in integrating information about bird movement and environmental conditions experienced by the bird in a comprehensive, integrative and hypothesis-driven framework. The approach of the authors is highly advanced, providing heuristic insights for many additional systems where organisms are influenced by, and respond to small-scale environmental conditions.

Reviewer #2 (Public Review):

This work sheds new light on the growth trajectory of Bonobo and contributes heavily to the discussion of the exclusivity of certain aspects of growth in modern humans. These results are also interesting as long as they are based on the study of the largest sample ever considered in the study of the growth of this species by including morphometric measurements as well as endocrinological factors.

The authors approach the study of the presence of growth spurs (GS) in Bonobo on the basis that GS are exclusive to the growth in modern humans. This idea is fairly widespread, however studies on non-human primates have shown an acceleration of growth during adolescence in several species, these works are recalled, presented and discussed by the authors. The originality of this work lies in highlighting the importance of scaling in studies of growth trajectories. The absence of GS in Bonobo but also in other primate species may result from not considering the conjunction of weight and height in the analysis of growth, because the pronounced changes in the speed of the height are in relation to the speed of changes in weight and this is modified according to the size/age. The authors apply scaling corrections to their results and the GS become evident (or more obvious) in Bonobo. Thus, the exclusivity of GS in growth in modern humans may in fact result only by the application of analytical approach not very appropriate in non-human primates.

Reviewer #2 (Public Review):

Phage satellites are fascinating elements that have evolved to hijack phages for induction, packaging, and transfer, promoting their widespread dissemination in nature. It is remarkable how different satellites use conserved strategies of parasitism, utilising unrelated proteins that perform similar roles in their cognate elements. In the current manuscript, Dr. Seed and coworkers elucidated the mechanism used by one family of satellites, the PLEs, to produce small capsids, a process that inhibits phage reproduction while increasing PLE transmission. The work is presented beautifully, and the results are astonishing. The authors identified the gene responsible for generating the small capsids, characterised its role in the PLE transfer and phage inhibition, and determined the structure of the PLE-sized small capsids. It is a truly impressive piece of work.

Reviewer #2 (Public Review):

This preprint by Pokrovsky and coworkers is a descriptive study reporting on non-breeding itinerant behaviour of an intrapalearctic migratory raptor, the rough-legged buzzard, and relating such non-breeding movements to snow cover across the European non-breeding range. The article is based on long-term GPS tracking data from a relatively large sample of individuals (n=43) that were equipped with state-of-the-art tracking devices in the Russian Arctic during 2013-2019. The results show that, upon breeding, buzzards migrated rapidly to southern non-breeding areas, located in open areas north of the Black and Caspian seas, where they perform continuous directional movements at a slower pace, initially moving SW (Oct to Jan) and then progressively moving NE (Feb to Apr) before embarking on rapid spring migration. It is suggested that such itinerant behaviour follows variation (expansion and retreat) of snow cover across the non-breeding range.

The results are definitely useful for researchers investigating the ecological drivers of bird movement patterns. The paper is generally well-written and the analytical framework is solid. However, there are significant weaknesses in the theoretical framework, unwarranted claiming of novelty, and interpretation of the data. Below are key points that the authors may wish to consider.

1) The authors underemphasize the fact that what they term 'fox-trot' migration is actually a well-known pattern for many other migratory species, both in the Nearctic and in the Afro-Palearctic migration systems. Such behaviour has previously been identified as 'itinerant', involving an alternation of stopovers and movements between different short-term non-breeding residency areas, and it seems that the pattern the authors report for this particular species is perfectly in line with such previous evidence. For instance, this is well-documented among migratory raptors, such as the Montagu's harrier, a lesser kestrel or black kite, that exploit Sahelian savannahs, where large spatio-temporal variation in greenness and hence resource availability occurs. And, besides the mentioned cuckoos and nightingales, there are studies of red-backed shrikes suggesting the same, as well as of tree swallows in the Nearctic. Therefore, the authors should avoid claiming novelty for this study and introducing unnecessary and confusing new terms in the literature (i.e. the 'fox-trot' migration patterns) when these are definitely not strictly needed as they have been previously observed and defined otherwise. Reference to all this previous body of literature is only hinted at and should be considerably expanded. The final sentence of the abstract, involving a general recommendation for future work, is definitely not warranted. Sentences such as 'We used the rough-legged buzzard as a model..." are also similarly unwarranted. This is simply a descriptive study reporting on such behaviour in yet another migratory species. The predictions paragraph is also overlong and could be considerably condensed.

2) The term 'migration' associated to so-called 'fox-trot' movements (see Fig. 1) is also highly confusing and possibly incorrect, as it is not in line with the commonly accepted definition of 'migration' (i.e. mass back and forth movements from the same areas). Apparently, the authors do not provide any evidence that the birds are moving back and forth from the same areas during the non-breeding period (i.e., there is no mention of site fidelity between early and late wintering areas, but judging from fall and spring migration distances it seems this is definitely not the case). 'Non-breeding itinerancy' is clearly a more appropriate term to describe this behaviour. More generally, the reference to 'winter migration', which is often mentioned in the manuscript, is not correct and should be amended.

3) The current title is unnecessarily general (it may recall rather a review or meta-analysis) and not adequately describing the content of the manuscript. It is not at all clear how the terms 'Conservation' and 'Anthropocene' are related to the content of the study (unless one believes that this is because any study of wildlife is aimed at its conservation, which is of course untrue, and that the study has been performed in the Anthropocene, which is the case for all wildlife studies carried out after 1950-1960). In order to be informative, the title should more tightly reflect the content of the article. A valid alternative would be 'Itinerant non-breeding behaviour of an intra-Palaearctic migratory raptor', far more adequate and informative. Although it might be worthwhile mentioning the association between movements and snow cover (or ecological conditions more generally) already in the title, perhaps that link is too indirect as currently reported in the manuscript. There are several possibilities to provide a more direct link between movements and snow cover, such as e.g. performing habitat selection analysis with respect to snow cover. Plotting temporal progression of snow cover (average) against movements (e.g. by showing monthly home ranges against snow cover) would help visualizing the association between snow cover and movement patterns.

4) The text, particularly the Introduction and (even more so) the Discussion, would benefit from profound reframing in light of the above comments. Any link to conservation is too weak and should be removed or considerably toned down. Moreover, the species is not of conservation interest (IUCN = Least Concern), as it has an extremely large range and population size, with largely fluctuating and non-declining populations (whose dynamics are related to Arctic small rodent cycles). Unless the authors are able to make prediction on how these movements will be affected by climate change (e.g. by using species distribution models or similar approaches), the link to the Anthropocene and to conservation is mostly unwarranted. In general, reference to 'winter' should be avoided and replaced with 'non-breeding season', which is a more general term.

Reviewer #2 (Public Review):

The manuscript by Wang et al. follows up on the group's previous publication on KLF1 (EKLF) K47R mice and reduced susceptibility to tumorigenesis and increased life span (Shyu et al., Adv Sci (Weinh). Sep 2022;9(25):e2201409. doi:10.1002/advs.202201409). In the current manuscript, the authors have described the dependence of these phenotypes on age, gender, genetic background, and hematopoietic translation of bone marrow mononuclear cells. Considering the current study is centered on the phenotypes described in the previous study, the novelty is diminished. Further, there are significant conceptual concerns in the study that make the inferences in the manuscript far less convincing. Major concerns are listed below:

1. The authors mention more than once in the manuscript that KLF1 is expressed in range of blood cells including hematopoietic stem cells, megakaryocytes, T cells and NK cells. In the case of megakaryocytes, studies from multiple labs have shown that while EKLF is expressed megakaryocyte-erythroid progenitors, EKLF is important for the bipotential lineage decision of these progenitors, and its high expression promotes erythropoiesis, while its expression is antagonized during megakaryopoiesis. In the case of HSCs, the authors reference to their previous publication for KLF1's expression in these cells- however, in this study nor in the current study, there is no western blot documented to convincingly show that KLF1 protein is expressed at detectable levels in these cells. For T cells, the authors have referenced a study which is based on ectopic expression of KLF1. For NK cells, the authors reference bioGPS: however, upon inspection, this is also questionable.

2. The current study rests on the premise that KLF1 is expressed in HSCs, NK cells and leukocytes, and the references cited are not sufficient to make this assumption, for the reasons mentioned in the first point. Therefore, the authors will have to show both KLF1 mRNA and protein levels in these cells, and also compare them to the expression levels seen in KLF1 wild type erythroid cells along with knockout erythroid cells as controls, for context and specificity.

3. To get to the mechanism driving the reduced susceptibility to tumorigenesis and increased life span phenotypes in EKLF K74R mice, the authors report some observations- However, how these observations are connected to the phenotypes is unclear.<br /> a. For example, in Figure S3, they report that the frequency of NK1.1+ cells is higher in the mutant mice. The significance of this in relation to EKLF expression in these cells and the tumorigenesis and life span related phenotypes are not described. Again, as mentioned in the second point, KLF1 protein levels are not shown in these cells.<br /> b. In Figure 4, the authors show mRNA levels of immune check point genes, PD-1 and PD-l1 are lower in EKLF K74R mice in PB, CD3+ T cells and B220+ B cells. Again, the questions remain on how these genes are regulated by EKLF, and whether and at what levels EKLF protein is expressed in T cells and B cells relative to erythroid cells. Further, while the study they reference for EKLF's role in T cells is based on ectopic expression of EKLF in CD4+ T cells, in the current study, CD3+ T cells are used. Also, there are no references for the status of EKLF in B cells. These details are not discussed in the manuscript.

4. The authors perform comparative proteomics in the leukocytes of EKLF K74R and WT mice as shown in Figure S5. What is the status of EKLF levels in the mutant lysate vs wild type lysates based on this analysis? More clarity needs to be provided on what cells were used for this analysis and how they were isolated since leukocytes is a very broad term.

5. In the discussion the authors make broad inferences that go beyond the data shown in the manuscript. They mention that the tumorigenesis resistance and long lifespan is most likely due to changes in transcription regulatory properties and changes in global gene expression profile of the mutant protein relative to WT leukocytes. And based on reduced mRNA levels of Pd-1 Pd-l1 genes in the CD3+ T cells and B220+ B cells from mutant mice, they "assert" that EKLF is an upstream regulator of these genes and regulates the transcriptomes of a diverse range of hematopoietic cells. The lack of a ChIP assay to show binding of WT EKLF on genes in these cells and whether this binding is reduced or abolished in the mutant cells, make the above statements unsubstantiated.

6. Where westerns are shown, the authors need to show the molecular weight ladder, and where qPCR data are shown for EKLF, it will be helpful to show the absolute levels and compare these levels to those in erythroid cells, along the corresponding EKLF knock out cells as controls.

7. Figure S1D does not have a figure legend. Therefore, it is unclear what the blot in this figure is showing. In the text of the manuscript where they reference this figure, they mention that the levels of the mutant EKLF vs WT EKLF does not change in peripheral blood, while in the figure they have labeled WBCs for the blot, and the mRNA levels shown do seem to decrease in the mutant compared to WT peripheral blood.

Reviewer #2 (Public Review):

The authors intended to identify a protein signature in extracellular vesicles of serum to distinguish pancreatic ductal adenocarcinoma from benign pancreatic diseases.

A major strength of the work presented is the valuable profiling of a significant number of patient samples, with a rich cohort of patients with pancreatic cancer, benign pancreatic diseases, and healthy controls. However, despite the strong cohorts presented, the numbers of patient samples for benign pancreatic diseases as well as controls were very limited.

Also, the method used to isolate vesicles, EVTrap, recognizes double bilayers, which means that it can detect cellular debris and apoptotic bodies, which are very common in the circulation of patients that are undergoing chemotherapy. It would be important to identify the patients that are therapy naïve and the ones that are not because of this possible bias. Additionally, the transmission electron microscopy data reflect this heterogeneity of the samples, also with little identification of double bilayered vesicles. It would be important to identify some extracellular vesicles markers in those preparations to strengthen the quality of the samples analysed. What is more, previously published work with this same methodology identifies around 2000 proteins per sample. It would be important to explain why in this study there seems to be a reduction in more than 50% of the amount of proteins identified in the vesicles.

One of the proteins that constantly surges on the analysis is KRT20. It would be important to proceed with the analysis by first filtering out possible contaminants of the proteomics, of which keratins are the most common ones. Finally, none of the 7-extracellular vesicle protein signatures has been validated by other techniques, such as western blot, in extracellular vesicles isolated by other, standard, methods, such as size exclusion chromatography.

A distinct technique for protein analysis was done but not a different method of isolation of these vesicles. This would strengthen the results and the origin of the proteins.

The conclusions that are reached do not fully meet the proposed aims of the identification of a protein signature in circulating extracellular vesicles that could improve early detection of the disease. The authors did not demonstrate the superiority of detection of these proteins in extracellular vesicles versus simply performing an ELISA, nor their superiority with respect to the current standard procedure for diagnosis.

The authors also suggest that profiling of circulating extracellular vesicles provides unique insights into systemic immune changes during pancreatic cancer development. How is this better than a regular hemogram is not clear.

Finally, it would be important to determine how this signature compares with many others described in the literature that have the exact same aim. Why and how would this one be better?