Reviewer #2 (Public Review):

Summary:<br /> The authors utilized (permeabilized) fibers from muscle samples obtained from brown and black bears, squirrels, and Garden dormice, to provide interesting and valuable data regarding changes in myosin conformational states and energetics during hibernation and different types of activity in summer and winter. Assuming that myosin structure is similar between species then its role as a regulator of metabolism would be similar and not different, yet the data reveal some interesting and perplexing differences between the selected hibernating species.

Strengths:<br /> The experiments on the permeabilized fibers are complementary, sophisticated, and well-performed, providing new information regarding the characteristics of skeletal muscle fibers between selected hibernating mammalian species under different conditions (summer, interarousal, and winter).

The studies involve complementary assessments of muscle fiber biochemistry, sarcomeric structure using X-ray diffraction, and proteomic analyses of posttranslational modifications.

Weaknesses:<br /> It would be helpful to put these findings on permeabilized fibers into context with the other anatomical/metabolic differences between the species to determine the relative contribution of myosin energetics (with these other contributors) to overall metabolism in these different species, including factors such as fat volume/distribution.

Reviewer #2 (Public Review):

Summary

In this work, the authors seek to test a version of an old idea, which is that our perception of the world and our understanding of the objects in it are deeply influenced by the nature of our bodies and the kinds of behaviours and actions that those objects afford. The studies presented here muster three kinds of evidence for a discontinuity in the encoding of objects, with a mental "border" between objects roughly of human body scale or smaller, which tend to relate to similar kinds of actions that are yet distinct from the kinds of actions implied by human-or-larger scale objects. This is demonstrated through observers' judgments of the kinds of actions different objects afford; through similar questioning of AI large-language models (LLMs); and through a neuroimaging study examining how brain regions implicated in object understanding make distinctions between kinds of objects at human and larger-than-human scales.

Strengths 

The authors address questions of longstanding interest in the cognitive neurosciences -- namely how we encode and interact with the many diverse kinds of objects we see and use in daily life. A key strength of the work lies in the application of multiple approaches. Examining the correlations among kinds of objects, with respect to their suitability for different action kinds, is novel, as are the complementary tests of judgments made by LLMs. The authors include a clever manipulation in which participants are asked to judge action-object pairs, having first adopted the imagined size of either a cat or an elephant, showing that the discontinuity in similarity judgments effectively moved to a new boundary closer to the imagined scale than the veridical human scale. The dynamic nature of the discontinuity hints that action affordances may be computed dynamically, "on the fly", during actual action behaviours with objects in the real world.

Weaknesses 

A limitation of the tests of LLMs may be that it is not always known what kinds of training material was used to build these models, leading to a possible "black box" problem. Further, presuming that those models are largely trained on previous human-written material, it may not necessarily be theoretically telling that the "judgments" of these models about action-object pairs shows human-like discontinuities. Indeed, verbal descriptions of actions are very likely to mainly refer to typical human behaviour, and so the finding that these models demonstrate an affordance discontinuity may simply reflect those statistics, rather than providing independent evidence for affordance boundaries.

The relatively small sample size of the brain imaging experiment, and some design features (such as the task participants performed, and the relatively narrow range of objects tested) provide some limits on the extent to which it can be taken as support for the authors' claims.

Reviewer #2 (Public Review):

Summary:<br /> This paper reports that mechanical stress from egg accumulation is a biological stimulus that drives the formation of extruded vesicles from the neurons of C. elegans ALMR touch neurons. Using powerful genetic experiments only readily available in the C. elegans system, the authors manipulate oocyte production, fertilization, embryo accumulation, and egg-laying behavior, providing convincing evidence that exopher production is driven by stretch-dependent feedback of fertilized, intact eggs in the adult uterus. Shifting the timing of egg production and egg laying alters the onset of observed exophers. Pharmacological manipulation of egg laying has the predicted effects, with animals retaining fewer eggs having fewer exophers and animals with increased egg accumulation having more. The authors show that egg production and accumulation have dramatic consequences for the viscera, and moving the ALMR process away from eggs prevents the formation of exophers. This effect is not unique to ALMR but is also observed in other touch neurons, with a clear bias toward neurons whose cell bodies are adjacent to the filled uterus. Embryos lacking an intact eggshell with reduced rigidity have impaired exopher production. Acute injection into the uterus to mimic the stretch that accompanies egg production causes a similar induction of exopher release. Together these results are consistent with a model where stretch caused by fertilized embryo accumulation, and not chemical signals from the eggs themselves or egg release, underlies ALMR exopher production seen in adult animals.

Strengths:<br /> Overall, the experiments are very convincing, using a battery of RNAi and mutant approaches to distinguish direct from indirect effects. Indeed, these experiments provide a model generally for how one would methodically test different models for exopher production. The paper is well-written and easy to understand. I had been skeptical of the origin and purpose of exophers, concerned they were an artefact of imaging conditions, caused by deranged calcium activity under stressful conditions, or as evidence for impaired animal health overall. As this study addresses how and when they form in the animal using otherwise physiologically meaningful manipulations, the stage is now set to address at a cellular level how exophers like these are made and what their functions are.

Weaknesses:<br /> Not many. The experiments are about as good as could be done. Some of the n's on the more difficult-to-work strains or experiments are comparatively low, but this is not a significant concern because of the number of different, complementary approaches used. The microinjection experiment in Figure 7 is very interesting, there are missing details that would confirm whether this is a sound experiment.

Reviewer #2 (Public Review):

This study describes a deep mutational scan across CDKN2A using suppression of cell proliferation in pancreatic adenocarcinoma cells as a readout for CDKN2A function. The results are also compared to in silico variant predictors currently utilized by the current diagnostic frameworks to gauge these predictors' performance. The authors also functionally classify CDKN2A somatic mutations in cancers across different tissues.

This study is a potentially important contribution to the field of cancer variant interpretation for CDKN2A, but is almost impossible to review because of the severe lack of details regarding the methods and incompleteness of the data provided with the paper. We do believe that the cell proliferation suppression assay is robust and works, but when it comes to the screening of the library of CDKN2A variants the lack of primary data and experimental detail prevents assessment of the scientific merit and experimental rigor.

Reviewer #2 (Public Review):

Clément Mazeaud et al. identified the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a proviral cellular protein that regulates Zika virus RNA replication by modulating the biogenesis of virus-induced replication organelles.

The absence of IGF2BP2 specifically dampens ZIKV replication without having a major impact on DENV replication. The authors show that ZIKV infection changes IGF2BP2 cellular distribution, which relocates to the perinuclear viral replication compartment. These assays were conducted by infecting cells with an MOI of 10 for 48 hours. Considering the ZIKV life cycle, it is noteworthy that at this time there may be a cytopathic effect. One point of concern arises regarding how the authors can ascertain that the observed change in localization is a consequence of the infection rather than of the cytopathic effect. To address this concern, shorter infection periods (e.g., 24 hours post-infection) or additional controls, such as assessing cellular proteins that do not change their localization or infecting with another flavivirus lacking the IGF2BP2 effect, could be incorporated into their experiments.

By performing co-immunoprecipitation assays on mock and infected cells that express HA-tagged IGF2BP2, the authors propose that the observed change in IGF2BP2 localization results from its recruitment to the replication compartment by the viral NS5 polymerase and associated with the viral RNA. Given that both IGF2BP2 and NS5 are RNA-binding proteins, it is plausible that their interaction is mediated indirectly through the RNA molecule. Notably, the authors do not address the treatment of lysates with RNAse before the IP assay, leaving open the possibility of this indirect interaction between IGF2BP2 and NS5.

In in vitro binding assays, the authors demonstrate that the RNA-recognition motifs of the IGF2BP2 protein specifically bind to the 3' nontranslated region (NTR) of the ZIKV genome, excluding binding to the 5' NTR. However, they cannot rule out the possibility of this host protein associating with other regions of the viral genome. Using a reporter ZIKV subgenomic replicon system in IGF2BP2 knock-down cells, they additionally demonstrate that IGF2BP2 enhances viral genome replication. Despite its proviral function, the authors note that the "overexpression of IGF2BP2 had no impact on total vRNA levels." However, the authors do not delve into a discussion of this latter statement.

In this study, the authors extend their findings by illustrating that ZIKV infection triggers a remodeling of IGF2BP2 ribonucleoprotein complex. They initially evaluate the impact of ZIKV infection on IGF2BP2's interaction with its endogenous mRNA ligands. Their results reveal that viral infection alters the binding of specific mRNA ligands, yet the physiological consequences of this loss of binding in the cell remain unexplored. Additionally, the authors demonstrate that ZIKV infection modifies the IGF2BP2 interactome. Through proteomic assays, they identified 62 altered partners of IGF2BP2 following ZIKV infection, with proteins associated with mRNA splicing and ribosome biogenesis being the most represented. In particular, the authors focused their research on the heightened interaction between IGF2BP2 and Atlastin 2, an ER-shaping protein reported to be involved in flavivirus vesicle packet formation. The validation of this interaction by Western blot assays prompted an analysis of the effect of ZIKV on organelle biogenesis using a newly described replication-independent vesicle packet induction system. Consequently, the authors demonstrate that IGF2BP2 plays a regulatory role in the biogenesis of ZIKV replication organelles.

Based on these findings and previously published data, the authors propose a model outlining the role of IGF2BP2 in ZIKV infectious cycle, detailing the changes in IGF2BP2 interactions with both cellular and viral proteins and RNAs that occur during viral infection.

The conclusions drawn in this paper are generally well substantiated by the data. However, it is worth noting that the majority of infections were conducted at a high MOI for 48 hours, spanning more than one infectious cycle. To enhance the robustness of their findings and mitigate potential cell stress, it would be valuable to observe these effects at shorter time intervals, such as 24 hours post-infection.<br /> Furthermore, the assertion regarding the association of IGF2BP2 with NS5 could be strengthened through additional immunoprecipitation (IP) assays. These assays, performed in the presence of RNAse treatment, would help exclude the possibility of an indirect interaction between IGF2BP2 and NS5 (both RNA-binding proteins) through viral RNA, thus providing more confidence in the observed association.

Reviewer #2 (Public Review):

The manuscript raises interesting observations about the potential evolution of release factors and tRNA to readdress the meaning of stop codons. The manuscript is divided into two parts: The first consists of revealing that the presence of a trp tRNA with an AS of 5bp in Condylostoma magnum is probably linked to contamination in the databases by sequences from bacteria. This is an interesting point which seems to be well supported by the data provided. It highlights the difficulty of identifying active tRNA genes from poorly annotated or incompletely assembled genomes. The second part criticises the fact that a mutation at position S67 of eRF1 is required to allow the UGA codon to be reassigned as a sense codon. As supporting evidence, they provide a phylogenetic study of the eRF1 factor showing that there are numerous ciliates in which this position is mutated, whereas the organism shows no trace of the reassignment of the UGA codon into a sense codon. While this criticism seems valid at first glance, it suffers from the lack of information on the level of translation of UGA codons in the organisms considered. It has been clearly shown that S67G or S67A mutations allow a strong increase in the reading of UGA codons by tRNAs, so this point is not in doubt. However, this has been demonstrated in model organisms, and we now need to determine whether other changes in the translational apparatus could accompany this mutation by modifying its impact on the UGA codon. This is a point partly raised at the end of the manuscript. Indeed, it is quite possible that in these organisms the UGA codon is both used to complete translation and is subject to a high level of readthrough. Actually, in the presence of a mutation at position 67 (or elsewhere), the reading of the UGA can be tolerated under specific stress conditions (nutrient deficiency, oxidative stress, etc.), so the presence of this mutation could allow translational control of the expression of certain genes. On the other hand, it seems obvious to me that there are other ways of reading through a stop codon without mutating eRF1 at position S67. So the absence of a mutation at this position is not really indicative of a level of reading of the UGA codon. Before writing such a strong assertion as that found on page 3, experiments should be carried out. The authors should therefore moderate their assertion.

To make a definitive conclusion, we would need to be able to measure the level of termination and readthrough in these organisms. So, from my point of view, all the arguments seem rather weak. Moreover, the authors themselves indicate that the conjunction between a Trp tRNA that is efficient at reading the UGA codon and an eRF1 factor that is not efficient at recognising this stop codon could be the key to reassignment.

Reviewer #2 (Public Review):

Summary:<br /> In the present manuscript, Zhang et al utilize single-nuclei RNA-Seq to investigate the heterogeneity of perirenal adipose tissue. The perirenal depot is interesting because it contains both brown and white adipocytes, a subset of which undergo functional "whitening" during early development. While adipocyte thermogenic transdifferentiation has been previously reported, there remains many unanswered questions regarding this phenomenon and the mechanisms by which it is regulated.

Strengths:<br /> The combination of UCP1-lineage tracing with the single nuclei analysis allowed the authors to identify four populations of adipocytes with differing thermogenic potential, including an "whitened" adipocyte (mPRAT-ad2) that retains the capacity to rapidly revert to a brown phenotype upon cold exposure. They also identify two populations of white adipocytes that do not undergo browning with acute cold exposure.

Anatomically distinct adipose depots display interesting functional differences, and this work contributes to our understanding of one of the few brown depots present in humans.

Weaknesses:<br /> The most interesting aspect of this work is the identification of a highly plastic mature adipocyte population with the capacity to switch between a white and brown phenotype. The authors attempt to identify the transcriptional signature of this ad2 subpopulation, however the limited sequencing depth of single nuclei somewhat lessens the impact of these findings. Furthermore, the lack of any form of mechanistic investigation into the regulation of mPRAT whitening limits the utility of this manuscript. However, the combination of well-executed lineage tracing with comprehensive cross-depot single-nuclei presented in this manuscript could still serve as a useful reference for the field.

Reviewer #2 (Public Review):

Summary:

The authors investigated the molecular evolution of members of the gasdermin (GSDM) family. By adding the evolutionary time axis of animals, they created a new molecular phylogenetic tree different from previous ones. The analyzed result verified that non-mammalian GSDMAs and mammalian GSDMAs have diverged into completely different and separate clades. Furthermore, by biochemical analyses, the authors demonstrated non-mammalian GSDMA proteins are cleaved by the host-encoded caspase-1. They also showed mammalian GSDMAs have lost the cleavage site recognized by caspase-1. Instead, the authors proposed that the newly appeared GSDMD is now cleaved by caspase-1.

Through this study, we have been able to understand the changes in the molecular evolution of GSDMs, and by presenting the cleavage of GSDMAs through biochemical experiments, we have become able to grasp the comprehensive picture of this family molecules. However, there are some parts where explanations are insufficient, so supplementary explanations and experiments seem to be necessary.

Strengths:

It has a strong impact in advancing ideas into the study of pyroptotic cell death and even inflammatory responses involving caspase-1.

Weaknesses:

Based on the position of mammalian GSDMA shown in the molecular phylogenetic tree (Figure 1), it may be difficult to completely agree with the authors' explanation of the evolution of GSDMA.

(1) Focusing on mammalian GSDMA, this group and mammalian GSDMD diverged into two clades, and before that, GSDMA/D groups and mammalian GSDMC separated into two, more before that, GSDMB, and further before that, non-mammalian GSDMA, when we checked Figure 1. In the molecular phylogenetic tree, it is impossible that GSDMA appears during evolution again. Mammalian GSDMAs are clearly paralogous molecules to non-mammalian GSDMAs in the figure. If they are bona fide orthologous, the mammalian GSDMA group should show a sub-clade in the non-mammalian GSDMA clade. It is better to describe the plausibility of the divergence in the molecular evolution of mammalian GSDMA in the Discussion section.

(2) Regarding (1), it is recommended that the authors reconsider the validity of estimates of divergence dates by focusing on mammalian species divergence. Because the validity of this estimation requires recheck of the molecular phylogenetic tree, including alignment.

(3) If GSDMB and/or GSDMC between non-mammalian GSDMA and mammalian GSDMD as shown in the molecular phylogenetic tree would be cleaved by caspase-1, the story of this study becomes clearer. The authors should try that possibility.

Reviewer #2 (Public Review):

Summary:

In this study, Rana and colleagues present interesting findings demonstrating potential beneficial effects of AMPA receptor modulator with ampakines in the context of neurogenic bladder following acute spinal cord injury. Neurogenic bladder dysfunction is characterized by urinary retention and/or incontinence, with limited treatments available. Based on recent observations showing that ampakines improved respiratory function in rats with SCI, the authors explored the use of ampakine CX1739 on bladder and external urethral sphincter (EUS) function and coordination early after mid-thoracic contusion injury. Using continuous flow cystometry and EUS myography the authors showed that ampakine treatment led to decreased peak pressures, threshold pressure, intercontraction interval and voided volume in SCI rats versus vehicle-treated controls. Although CX1739 did not alter EUS EMG burst duration, treatment did lead to EUS EMG bursting at lower bladder pressure compared to baseline. In a subset of rats that did not show regular cystometric voiding, CX1739 treatment diminished non-voiding contractions and improved coordinated EUS EMG bursting. Based on these findings the authors conclude that ampakines may have utility in recovery of bladder function following SCI.

Strengths and Weaknesses:

The experimental design is thoughtful and rigorous, providing evaluation of both the bladder and external urethral sphincter function in the absence and presence of ampakine treatment. The data in support of a role for CX1789 treatment in the context of neurogenic bladder are presented clearly, and the conclusions are adequately supported by the findings. The authors have addressed essentially all of the weaknesses related to translational significance, CX1789 half-life, and the use of female animals only in this study.

Reviewer #2 (Public Review):

Summary:<br /> This manuscript mainly studied the biological effect of tenascin XB (TNXB) on hemophilic arthropathy (HA) progression. Using bioinformatic and histopathological approaches, the authors identified the novel candidate gene TNXB for HA. Next, the authors showed that TNXB knockdown leads to chondrocyte apoptosis, matrix degeneration, and subchondral bone loss in vivo/vitro. Furthermore, AKT agonists promoted extracellular matrix synthesis and prevented apoptosis in TNXB knockdown chondrocytes.

Strengths:<br /> In general, this study significantly advances our understanding of HA pathogenesis. The authors utilize comprehensive experimental strategies to demonstrate the role of TNXB in cartilage degeneration associated with HA. The results are clearly presented, and the conclusions appear appropriate.

Weaknesses:<br /> Additional clarification is required regarding the gender of the F8-/- mouse in the study. Is the mouse male or female?

Reviewer #2 (Public Review):

The following review for a revised manuscript is updated where appropriate and otherwise unchanged for completeness.

Summary<br /> The paper concerns the phenomenon of continuous flash suppression (CFS), relevant to questions about the extent and nature of subconscious visual processing. Whereas standard CFS studies only measure the breakthrough threshold-the contrast at which an initially suppressed target stimulus with steadily increasing contrast becomes visible-the authors also measure the re-suppression threshold, the contrast at which a visible target with decreasing contrast becomes suppressed. Thus, the authors could calculate suppression depth, the ratio between the breakthrough and re-suppression thresholds. To measure both thresholds, the authors introduce the tracking-CFS method, a continuous-trial design that results in faster, better controlled, and lower-variance threshold estimates compared to the discrete trials standard in the literature. The study finds that suppression depths are similar for different image categories, providing an interesting contrast to previous results that breakthrough thresholds differ for different image categories. The new finding calls for a reassessment of interpretations based solely on the breakthrough threshold that subconscious visual processing is category-specific.

Strengths<br /> (1) The tCFS method quickly estimates breakthrough and re-suppression thresholds using continuous trials, which also better control for slowly varying factors such as adaptation and attention. Indeed, tCFS produces estimates with lower across-subject variance than the standard discrete-trial method (Fig. 2). The tCFS method is straightforward to adopt in future research on CFS and binocular rivalry.

(2) The CFS literature has lacked re-suppression threshold measurements. By measuring both breakthrough and re-suppression thresholds, this work calculated suppression depth (i.e., the difference between the two thresholds), which warrants different interpretations from the breakthrough threshold alone.

(3) The work found that different image categories show similar suppression depths, suggesting some aspects of CFS are not category-specific. This result enriches previous findings that breakthrough thresholds vary with image categories. Re-suppression thresholds vary symmetrically, such that their differences are constant.

Weakness<br /> The following concern remains from my initial review. Reviewer #3 raised a similar point in the last revision round, and I believe the authors do not fully address either comment. Thus, here I paraphrase my initial concern with reference to the authors' reply and discuss why it needs further elaboration.

I do not follow the authors' reasoning as to why the suppression depth is a better (or fuller, superior, more informative) indication of subconscious visual processing than the breakthrough threshold alone. To my previous round of comments, the authors replied that 'breakthrough provides only half of the needed information.' I do not understand this. One cannot infer the suppression depth from the breakthrough threshold alone, but *one cannot obtain the breakthrough threshold from the suppression depth alone*, either. The two measures are complementary. (To be sure, given *both* the suppression depth and the re-suppression threshold, one can trivially recover the breakthrough threshold. The discussion concerns the suppression depth *alone* and the breakthrough threshold *alone*.) I am fully open to being convinced that there is a good reason why the suppression depth may be more informative than the breakthrough threshold about a specific topic, e.g., inter-ocular suppression or subconscious visual processing. I only request that the authors make such an argument explicit. Preferably, this argument will precede claims that require it. For example, in the significance statement, the authors write, 'all images show equal suppression when both thresholds are measured. We *thus* find no evidence of differential unconscious processing and *conclude* reliance on breakthrough thresholds is misleading' (emphasis added). Just what supports the 'thus' and the 'conclude'? Similarly, at the end of the introduction, the authors write, '[...] suppression depth was constant for faces, objects, gratings and visual noise. *In other words*, we find no evidence to support differential unconscious processing among these particular, diverse categories of suppressed images' (emphasis added). I believe the statements before and after the period have not been shown to be equivalent. In the abstract, the authors revised, 'variations in bCFS thresholds alone are insufficient for inferring whether the barrier to achieving awareness exerted by interocular suppression is weaker for some categories of visual stimuli compared to others.' While I appreciate the added specificity, this claim still needs more support because the authors have not established that suppression depth is a better index than the breakthrough threshold of 'the barrier to achieving awareness exerted by interocular suppression.'

The authors' reply included a discussion of neural CRFs, which may explain why the bCFS thresholds differ across image categories. However, CRFs do not explain why the bCFS threshold does not implicate some component of subconscious processing. For example, the bCFS threshold may reflect the aspect of subconscious visual processing that corresponds to V1/V4 neural responses.

Reviewer #2 (Public Review):

As a report of the first structure of VMAT2, indeed the first structure of any vesicular monoamine transporter, this manuscript represents an important milestone in the field of neurotransmitter transport. VMAT2 belongs to a large family (the major facilitator superfamily, MFS) containing transporters from all living species. There is a wealth of information relating to the way that MFS transporters bind substrates, undergo conformational changes to transport them across the membrane and couple these events to the transmembrane movement of ions. VMAT2 couples the movement of protons out of synaptic vesicles to the vesicular uptake of biogenic amines (serotonin, dopamine and norepinephrine) from the cytoplasm. The new structure presented in this manuscript can be expected to contribute to an understanding of this proton/amine antiport process.

The structure contains a molecule of the inhibitor TBZ bound in a central cavity, with no access to either luminal or cytoplasmic compartments. The authors carefully analyze which residues interact with bound TBZ and measure TBZ binding to VMAT2 mutated at some of those residues. These measurements allow well-reasoned conclusions about the differences in inhibitor selectivity between VMAT1 and VMAT2 and differences in affinity between TBZ derivatives.

The structure also reveals polar networks within the protein and hydrophobic residues in positions that may allow them to open and close pathways between the central binding site and the cytoplasm or the vesicle lumen. The authors propose involvement of these networks and hydrophobic residues in coupling of transport to proton translocation and conformational changes. However, these proposals are quite speculative in the absence of supporting structures and experimentation that would test specific mechanistic details.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Lane and colleagues measured the abundance of SARS-CoV-2 on breath in 60 outpatients after the development of COVID-19 symptoms using a novel breath collection apparatus. They found that, overall, viral abundance remains high for approximately eight days following the development of symptoms, after which viral abundance on breath drops to a low level that may persist for approximately 20 days or more. They did not identify significant differences in viral shedding on breath by vaccination status or viral variant. They also noted substantial variation in the degree and duration of shedding across individuals.

Strengths:

The primary strengths of this study are (1) the focus on breath, rather than the more traditional nasal/oropharyngeal swabs, and (2) the fact that the data were collected at multiple time points for each infection. This allows the authors to characterize not only mean viral abundance across individuals but also how that abundance changes over time, allowing for a better understanding of the potential duration of infectiousness of SARS-CoV-2.

Weaknesses:

The sample size is moderate (60) and focuses only on outpatients. While these are minor weaknesses (as the authors note, the majority of SARS-CoV-2 transmission likely occurs among those with symptoms below the threshold of hospitalization), it would nevertheless be useful to have a fuller understanding of variation in viral shedding across clinical groups. Furthermore, the study lacks information on viral shedding prior to the development of symptoms, which may be a critical period for transmission. Since the samples were collected at home by study participants using a novel apparatus, it is difficult to assess the degree to which actual variation in viral abundance, user variability, and/or measurement variation is inherent to the apparatus.

Reviewer #2 (Public Review):

This study focuses on the association between weight at birth and area, volume and thickness of the cerebral cortex measured at timepoints throughout the lifespan. Overall, the study is well designed, supported by evidence from a large sample drawn from three geographically distinct cohorts with robust analytical and statistical methods.

The authors test the hypotheses: that higher birth weight is associated with greater cortical area in later life; that associations are robust across samples and age; and that associations are stable across the lifespan. Analyses are performed separately in three cohorts: ABCD, UKBB and LCBC and the pattern of associations compared by means of spatial correlations. They find that BW is positively associated with cortical area (and, as a consequence, cortical volume) across most of the cortex, with effect sizes greatest in frontal and temporal regions. These associations remain largely unchanged when accounting for age, sex, length of gestation and (in one cohort) ethnicity. Variations due to MRI scanner and site are accounted for statistically. Measures are taken to determine within sample replicability through split-half analyses.

The authors conclude that BW, as a marker of early development, is associated with brain characteristics throughout the lifespan.

Reviewer #2 (Public Review):

Previous studies have shown that two hair cell transcription factors, Pou4f3 and Gfi1 are both necessary for the survival of cochlear hair cells, and that Gfi1 is regulated by Pou4f3. The authors have previously also shown that mosaic inactivation of the RNA-binding protein RBM24 leads to outer hair cell death.

In the present study, the authors show that hair cells dies in Pou4f3 and Gfi1 mutant mice. They show that Gfi1 is regulated by Pou4f3. Both these observations have been published before. They then show that RBM24 is absent in Pou4f3 knockouts, but not Gfi1 knockouts. They ectopically activate RMB24 in the hair cells of Poui4f3 knockouts, but this does not rescue the hair cell death. Finally the authors validate three RMB24 enhancers that are active in young hair cells and which have been previously shown to bind Pou4f3.

The experiments are well-executed and the data are clear. The results support the conclusions of the paper. The authors have revised the paper slightly, mostly to modify the red/green staining in the figures, and to perform additional analyses of the RBM24 and Ikzf2 mutants, now shown in Supplementary Figure 3.

Much of the work in the paper has been reported before. The result that hair cell transcription factors operate in a network, with some transcription factors activating only a subset of hair cell genes, is an expected result. Since RBM24 is only one of many genes regulated directly by Pou4f3, it is not surprising that it cannot rescue the Pou4f3 knockout hair cell degeneration, and indeed the rationale for attempting such a rescue experiment is not provided by the authors.

The identification of new hair cell enhancers may be of use to investigators wishing to express genes in hair cells.

In sum, this work, although carefully performed, does not shed significant new light on our understanding of hair cell development or survival.

Reviewer #2 (Public Review):

Summary: This study is a superbly written and illustrated documentation of the external sensilla of the Drosophila larva. Serial electron microscopy and digital modeling is used to the fullest to provide a definitive and clear picture of the sensory organs, which is dearly needed in the field.

Strengths: Serial electron microscopy and digital modeling is used to the fullest to provide a comprehensive, definitive and clear picture of the sensory organs, which is dearly needed in the field.

Weaknesses: none detected.

Reviewer #2 (Public Review):

Summary:<br /> Etcheverry et al. present two computational frameworks for exploring the functional capabilities of gene regulatory networks (GRNs). The first is a framework based on intrinsically-motivated exploration, here used to reveal the set of steady states achievable by a given gene regulatory network as a function of initial conditions. The second is a behaviorist framework, here used to assess the robustness of steady states to dynamical perturbations experienced along typical trajectories to those steady states. In Figs. 1-5, the authors convincingly show how these frameworks can explore and quantify the diversity of behaviors that can be displayed by GRNs. In Figs. 6-9, the authors present applications of their framework to the analysis and control of GRNs, but the support presented for their case studies is often incomplete.

Strengths:<br /> Overall, the paper presents an important development for exploring and understanding GRNs/dynamical systems broadly, with solid evidence supporting the first half of their paper in a narratively clear way.

The behaviorist point of view for robustness is potentially of interest to a broad community, and to my knowledge introduces novel considerations for defining robustness in the GRN context.

Some specific weaknesses, mostly concerning incomplete analyses in the second half of the paper:

(1) The analysis presented in Fig. 6 is exciting but preliminary. Are there other appropriate methods for constructing energy landscapes from dynamical trajectories in gene regulatory networks? How do the results in this particular case study compare to other GRNs studied in the paper?

Additionally, it is unclear whether the analysis presented in Fig. 6C is appropriate. In particular, if the pseudopotential landscapes are constructed from statistics of visited states along trajectories to the steady state, then the trajectories derived from dynamical perturbations do not only reflect the underlying pseudo-landscape of the GRN. Instead, they also include contributions from the perturbations themselves.

(2) In Fig. 7, I'm not sure how much is possible to take away from the results as given here, as they depend sensitively on the cohort of 432 (GRN, Z) pairs used. The comparison against random networks is well-motivated. However, as the authors note, comparison between organismal categories is more difficult due to low sample size; for instance, the "plant" and "slime mold" categories each only have 1 associated GRN. Additionally, the "n/a" category is difficult to interpret.

(3) In Fig. 8, it is unclear whether the behavioral catalog generated is important to the intervention design problem of moving a system from one attractor basin to another. The authors note that evolutionary searches or SGD could also be used to solve the problem. Is the analysis somehow enabled by the behavioral catalog in a way that is complementary to those methods? If not, comparison against those methods (or others e.g. optimal control) would strengthen the paper.

(4) The analysis presented in Fig. 9 also is preliminary. The authors note that there exist many algorithms for choosing/identifying the parameter values of a dynamical system that give rise to a desired time-series. It would be a stronger result to compare their approach to more sophisticated methods, as opposed to random search and SGD. Other options from the recent literature include Bayesian techniques, sparse nonlinear regression techniques (e.g. SINDy), and evolutionary searches. The authors note that some methods require fine-tuning in order to be successful, but even so, it would be good to know the degree of fine-tuning which is necessary compared to their method.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, Jiang et al., explore the role of neurexins at glycinergic MNTB-LSO synapses. The authors utilize elegant and compelling ex vivo slice electrophysiology to assess how the genetic conditional deletion of Nrxns1-3 impacts inhibitory glycinergic synaptic transmission and found that TKO of neurexins reduced electrically and optically evoked IPSC amplitudes, slowed optically evoked IPSC kinetics and reduced presynaptic release probability. The authors use classic approaches including reduced [Ca2+] in ACSF and EGTA chelation to propose that changes in these evoked properties are likely driven by the loss of calcium channel coupling. Intriguingly, while evoked transmission was impaired, the authors reported that spontaneous IPSC frequency was increased, potentially due to an increased number of synapses in LSO. Overall, this manuscript provides important insight into the role of neurexins at the glycinergic MNTP-LSO synapse and further emphasizes the need for continued study of both the non-redundant and redundant roles of neurexins.

Strengths:<br /> This well-written manuscript seamlessly incorporates mouse genetics and elegant ex vivo electrophysiology to identify a role for neurexins in glycinergic transmission at MNTB-LSO synapses. Triple KO of all neurexins reduced the amplitude and timing of evoked glycinergic synaptic transmission. Further, spontaneous IPSC frequency was increased. The evoked synaptic phenotype is likely a result of reduced presynaptic calcium coupling while the spontaneous synaptic phenotype is likely due to increased synapse numbers. While neuroligin-4 has been identified at glycinergic synapses, this study, to the best of my knowledge, is the first to study Nrxn function at these synapses.

Weaknesses:<br /> The data are compelling and report an intriguing functional phenotype. The role of Neurexins redundantly controls calcium channel coupling has been previously reported. Mechanistic insight would significantly strengthen this study.<br /> The claim that triple KO of Nrxns from MNTB increases the number of synapses in LSO is not strongly supported.<br /> Despite the stated caveats of measuring electrically evoked currents and the more robust synaptic phenotypes observed using optically evoked transmission, the authors rely heavily on electrical stimulation for most measurements.<br /> The differential expression of individual neurexins might indicate that specific neurexins may dominantly regulate synaptic transmission, however, this possibility is not discussed in detail.

Reviewer #2 (Public Review):

Summary:<br /> The authors introduce a new 192-channel OPM system that can be configured using different helmets to fit individuals from 2 to 34 years old. To demonstrate the veracity of the system, they conduct a sensorimotor task aimed at mapping developmental changes in beta oscillations across this age range. Many past studies have mapped the trajectory of beta (and gamma) oscillations in the sensorimotor cortices, but these studies have focused on older children and adolescents (e.g., 9-15 years old) and used motor tasks. Thus, given the study goals, the choice of a somatosensory task was surprising and not justified. The authors recorded a final sample of 27 children (2-13 years old) and 24 adults (21-34 years) and performed a time-frequency analysis to identify oscillatory activity. This revealed strong beta oscillations (decreases from baseline) following the somatosensory stimulation, which the authors imaged to discern generators in the sensorimotor cortices. They then computed the power difference between 0.3-0.8 period and 1.0-1.5 s post-stimulation period and showed that the beta response became stronger with age (more negative relative to the stimulation period). Using these same time windows, they computed the beta burst probability and showed that this probability increased as a function of age. They also showed that the spectral composition of the bursts varied with age. Finally, they conducted a whole-brain connectivity analysis. The goals of the connectivity analysis were not as clear as prior studies of sensorimotor development have not conducted such analyses and typically such whole-brain connectivity analyses are performed on resting-state data, whereas here the authors performed the analysis on task-based data. In sum, the authors demonstrate that they can image beta oscillations in young children using OPM and discern developmental effects.

Strengths:<br /> Major strengths of the study include the novel OPM system and the unique participant population going down to 2-year-olds. The analyses are also innovative in many respects.

Weaknesses:<br /> Several weaknesses currently limit the impact of the study. First, the choice of a somatosensory stimulation task over a motor task was not justified. The authors discuss the developmental motor literature throughout the introduction, but then present data from a somatosensory task, which is confusing. Of note, there is considerable literature on the development of somatosensory responses so the study could be framed with that. Second, the primary somatosensory response actually occurs well before the time window of interest in all of the key analyses. There is an established literature showing mechanical stimulation activates the somatosensory cortex within the first 100 ms following stimulation, with the M50 being the most robust response. The authors focus on a beta decrease (desynchronization) from 0.3-0.8 s which is obviously much later, despite the primary somatosensory response being clear in some of their spectrograms (e.g., Figure 3 in older children and adults). This response appears to exhibit a robust developmental effect in these spectrograms so it is unclear why the authors did not examine it. This raises a second point; to my knowledge, the beta decrease following stimulation has not been widely studied and its function is unknown. The maps in Figure 3 suggest that the response is anterior to the somatosensory cortex and perhaps even anterior to the motor cortex. Since the goal of the study is to demonstrate the developmental trajectory of well-known neural responses using an OPM system, should the authors not focus on the best-understood responses (i.e., the primary somatosensory response that occurs from 0.0-0.3 s)?

Regarding the developmental effects, the authors appear to compute a modulation index that contrasts the peak beta window (.3 to .8) to a later 1.0-1.5 s window where a rebound is present in older adults. This is problematic for several reasons. First, it prevents the origin of the developmental effect from being discerned, as a difference in the beta decrease following stimulation is confounded with the beta rebound that occurs later. A developmental effect in either of these responses could be driving the effect. From Figure 3, it visually appears that the much later rebound response is driving the developmental effect and not the beta decrease that is the primary focus of the study. Second, these time windows are a concern because a different time window was used to derive the peak voxel used in these analyses. From the methods, it appears the image was derived using the .3-.8 window versus a baseline of 2.5-3.0 s. How do the authors know that the peak would be the same in this other time window (0.3-0.8 vs. 1.0-1.5)? Given the confound mentioned above, I would recommend that the authors contrast each of their windows (0.3-0.8 and 1.0-1.5) with the 2.5-3.0 window to compute independent modulation indices. This would enable them to identify which of the two windows (beta decrease from 0.3-0.8 s or the increase from 1.0-1.5 s) exhibited a developmental effect. Also, for clarity, the authors should write out the equation that they used to compute the modulation index. The direction of the difference (positive vs. negative) is not always clear.

Another complication of using a somatosensory task is that the literature on bursting is much more limited and it is unclear what the expectations would be. Overall, the burst probability appears to be relatively flat across the trial, except that there is a sharp decrease during the beta decrease (.3-.8 s). This matches the conventional trial-averaging analysis, which is good to see. However, how the bursting observed here relates to the motor literature and the PMBR versus beta ERD is unclear.

Another weakness is that all participants completed 42 trials, but 19% of the trials were excluded in children and 9% were excluded in adults. The number of trials is proportional to the signal-to-noise ratio. Thus, the developmental differences observed in response amplitude could reflect differences in the number of trials that went into the final analyses.

Finally, the discussion could be improved to focus on the somatosensory literature and how this contributes to that. Currently, the discussion includes very little from the somatosensory literature.

Reviewer #2 (Public Review):

Summary:<br /> The authors address an important outstanding question: what forces are the primary drivers of evolutionary rate covariation? Exploration of this topic is important because it is currently difficult to interpret the functional/mechanistic implications of evolutionary covariation. These analyses also speak to the predictive power (and limits) of evolutionary rate covariation. This study reinforces the existing paradigm that covariation is driven by a varied/mixed set of interaction-types that all fall under the umbrella explanation of 'co-functional interactions'.

Strengths:<br /> Very smart experimental design that leverages individual protein domains for increased resolution.

Weaknesses:<br /> Nuanced and sometimes inconclusive results that are difficult to capture in a short title/abstract statement.

EDIT: The authors have done a satisfactory job of honing their language to get the nuanced ideas across clearly. The added scholarship and theoretical discussion they added strengthen the impact of the manuscript. The revised edition addresses my concerns.

Reviewer #3 (Public Review):

The study focuses on in vivo and in vitro cellular responses intranasal instillation of glycoforms and mutants of SARS-CoV2 spike trimer or spike bearing VLP in mice. Collectively, the experiments suggest that SARS-CoV2 spike has pro-inflammatory roles through increase M1 macrophage associated cytokines and induction of neutrophil netosis/necrosis, a proinflammatory cell death pathway. These effects seem largely independent of hACE2 interaction and partly depend upon interactions with SIGLECs on macrophages and neutrophils. A strength of the study is that a number sophisticated methods are used, including intravital microscopy in the cramaster and liver as well as acute lung slice models, to look at uptake of the spike proteins and immune cell dynamics. The weakness is that some of the reagents maybe contaminated with uncharacterized glycoforms and some important controls, such as control spike protein and control VLP are unevenly applied or not included. The authors have revised the manuscript through some improvements in the writing, but the survey nature and suggestive level of evidence is still a weakness. The study calls attention to sources of proinflammatory activity in the SARS CoV2 spike that may involve some carbohydrate interactions.

Reviewer #2 (Public Review):

This manuscript reports the discovery and analysis of a large protein complex that controls mating type and sexual reproduction of the model ciliate Tetrahymena thermophila. In contrast to many organisms that have two mating types or two sexes, Tetrahymena is multi-sexual with 7 distinct mating types. Previous studies identified the mating type locus, which encodes two transmembrane proteins called MTA and MTB that determine the specificity of mating type interactions. In this study, mutants are generated in the MTA and MTB genes and mutant isolates are studied for mating properties. Cells missing either MTA or MTB failed to co-stimulate wild-type cells of different mating types. Moreover, a mixture of mutants lacking MTA or MTB also failed to stimulate. These observations support the conclusion that MTA and MTB may form a complex that directs mating-type identity. To address this, the proteins were epitope-tagged and subjected to IP-MS analysis. This revealed that MTA and MTB are in a physical complex, and also revealed a series of 6 other proteins (MRC1-6) that together with MTA/B form the mating type recognition complex (MTRC). All 8 proteins feature predicted transmembrane domains, three feature GFR domains, and two are predicted to function as calcium transporters. The authors went on to demonstrate that components of the MTRC are localized on the cell surface but not in the cilia. They also presented findings that support the conclusion that the mating type-specific region of the MTA and MTB genes can influence both self- and non-self-recognition in mating.

Taken together, the findings presented are interesting and extend our understanding of how organisms with more than two mating types/sexes may be specified. The identification of the six-protein MRC complex is quite intriguing. It would seem important that the function of at least one of these subunits be analyzed by gene deletion and phenotyping, similar to the findings presented here for the MTA and MTB mutants. A straightforward prediction might be that a deletion of any subunit of the MRC complex would result in a sterile phenotype. The manuscript was very well written and a pleasure to read.

Reviewer #2 (Public Review):

Summary:<br /> This work describes a new pharmacological targeting approach to inhibit selective functions of the ubiquitously expressed chemokine receptor CXCR4, a potential target of immunomodulatory or anti-cancer treatments. Overall, the results build a strong case for the potential of this new compound to target specific functions of CXCR4, particularly linked to tumorigenesis. However, a more thorough evaluation of the function of the compound as well as future studies in mammalian model systems are needed to better assess the promise of the compound.

Strengths:<br /> The work elegantly utilizes in silico drug modelling to propose new small molecule compounds with specific features. This way, the authors designed compound AGR1.137, which abolishes ligand-induced CXCR4 receptor nanoclustering and the subsequent directed cell migration without affecting ligand binding itself or some other ligand-induced signaling pathways. The authors have used a relatively broad set of experiments to validate and demonstrate the effects of the drug. Importantly, the authors also test AGR1.137 in vivo, using a zebrafish model of tumorigenesis and metastasis. A relatively strong inhibitory effect of the compound is reported.

Weaknesses:<br /> The data would be significantly strengthened by adding kinetics and titration of concentrations. This is particularly important as it is the first description of these particular compounds and would help to evaluate the potency and possible side effects of the drug.

The authors carry out single-molecule tracking experiments to analyze nanoclustering of CXCR4 upon ligand binding. This complex data is presented in a sub-optimal manner. Representative images of the data should be included together with more thorough analysis tools like autocorrelation function or mean square displacement to get a more conclusive view of receptor clustering and the effects of the compound.

In the in vivo tumorigenesis experiments, again more kinetics and different concentrations of the drug would generate more convincing data. Also, the individual data points should be visualized to allow full evaluation of the data, throughout the experiments.

Reviewer #2 (Public Review):

Summary:<br /> This study aims to demonstrate that E. coli can acquire rapid antibiotic resistance mutations in the absence of a DNA damage response. To investigate this, the authors employed a sophisticated experimental framework based on a modified Adaptive Laboratory Evolution (ALE) workflow. This workflow involves numerous steps culminating in the measurement of antibiotic resistance. The study presents evidence that a recA strain develops ampicillin resistance mutations more quickly than the wild-type, as shown by measuring the Minimum Inhibitory Concentration (MIC) and mutation frequency. Whole-genome sequencing of 15 recA- colonies resistant to ampicillin revealed predominantly inactivation of genes involved in the multi-drug efflux pump system, whereas, in the wild-type, mutations appear to enhance the activity of the chromosomal ampC cryptic promoter. By analyzing mutants involved in the SOS response, including a lexA3 mutant incapable of inducing the SOS response, the authors conclude that the rapid evolution of antibiotic resistance occurs in an SOS-independent manner when recA is absent.

Furthermore, RNA sequencing (RNA-seq) of the four experimental conditions suggests that genes related to antioxidative responses drive the swift evolution of antibiotic resistance in the recA- strain.

Weaknesses:<br /> However, a potential limitation of this study is the experimental design used to determine the 'rapid' evolution of antibiotic resistance. It may introduce a significant bottleneck in selecting ampicillin-resistant mutants early on. A recA mutant could be more susceptible to ampicillin than the wild-type, and only resistant mutants might survive after 8 hours, potentially leading to their enrichment in subsequent steps. To address this concern, it would be critical to perform a survival analysis at various time points (0h, 2h, 4h, 6h, and 8h) during ampicillin treatment for both recA and wild-type strains, ensuring there is no difference in viability.

The observation that promoter mutations are absent in recA strains could be explained by previous research indicating that amplification of the AmpC genes is a mechanism for E. coli resistance to ampicillin, which does not occur in a recA-deficient background (PMID# 19474201).

The section describing Figure 3 is poorly articulated, and the conclusions drawn are apparent. The inability of a recA strain to induce the SOS response is well-documented (lines 210 and 278). The data suggest that merely blocking SOS induction is insufficient to cause 'rapid' evolution in their experimental conditions. To investigate whether SOS response can be induced independently of lexA cleavage by recA, alternative experiments, such as those using a sulA-GFP fusion, might be more informative.

In Figure 4E, the lack of increased SulA gene expression in the wild-type strain treated with ampicillin is unexpected, given that SulA is an SOS-regulated gene. The fact that polA (Pol I) is going down should be taken into account in the interpretation of Figures 2D and 2E.

The connection between compromised DNA repair, the accumulation of Reactive Oxygen Species (ROS) based on RNA-seq data, and accelerated evolution is merely speculative at this point and not experimentally established.

Reviewer #3 (Public Review):

Bae et al. described the key roles of pericytes in cavernous tissues in diabetic erectile dysfunction using both mouse and human single-cell transcriptomic analysis. Erectile dysfunction (ED) is caused by dysfunction of the cavernous tissue and affects a significant proportion of men aged 40-70. The most common treatment for ED is phosphodiesterase 5 inhibitors; however, these are less effective in patients with diabetic ED. Therefore, there is an unmet need for a better understanding of the cavernous microenvironment, cell-cell communications in patients with diabetic ED, and the development of new therapeutic treatments to improve the quality of life.

Pericytes are mesenchymal-derived mural cells that directly interact with capillary endothelial cells (ECs). They play a vital role in the pathogenesis of erectile function as their interactions with ECs are essential for penile erection. Loss of pericytes has been associated with diabetic retinopathy, cancer, and Alzheimer's disease and has been investigated in relation to the permeability of cavernous blood vessels and neurovascular regeneration in the authors' previous studies. This manuscript explores the mechanisms underlying the effect of diabetes on pericyte dysfunction in ED. Additionally, the cellular landscape of cavernous tissues and cell type-specific transcriptional changes were carefully examined using both mouse and human single-cell RNA sequencing in diabetic ED. The novelty of this work lies in the identification of a newly identified pericyte (PC)-specific marker, LBH, in mouse and human cavernous tissues, which distinguishes pericytes from smooth muscle cells. LBH not only serves as a cavernous pericyte marker, but its expression level is also reduced in diabetic conditions. The LBH-interacting proteins (Cryab and Vim) were further identified in mouse cavernous pericytes, indicating that these signaling interactions are critical for maintaining normal pericyte function. Overall, this study demonstrates the novel marker of pericytes and highlights the critical role of pericytes in diabetic ED.

Comments on revised version:

Bae and colleagues substantially improved the data quality and revised their manuscript "Pericytes contribute to pulmonary vascular remodeling via HIF2a signaling". While these revisions clarify some of the concerns raised, others remain. In my view, the following question must be addressed.

In my prior question on #3, I completely disagree with the statement that "identified cells with pericyte-like characteristics in the walls of large blood vessels". The staining that authors provided for LBH, was clearly stained for SMCs, not pericytes. Per Fig 2E, the authors are correct that LBH is colocalized with SMA+ cells( SMCs). However, the red signal from LBH clearly stains endothelial cells. In the rest of 2E and 2D, LBH is CD31- and their location suggests LBH stained for SMCs in the Aorta, Kidney vasculature, Dorsal vein, and Dorsal Artery.

Reviewer #2 (Public Review):

Summary: The authors seek to elucidate the early evolution of cnidarians through computer modeling of fluid flow in the oral region of very small, putative medusozoan polyps. They propose that the evolutionary advent of the free-swimming medusoid life stage was preceded by a sessile benthic life stage equipped with circular muscles that originally functioned to facilitate feeding and that later became co-opted for locomotion through jet propulsion.

Strengths: Assumptions of the modeling exercise laid out clearly; interpretations of the results of the model runs in terms of functional morphology plausible. An intriguing investigation that should stimulate further discussion and research.

Weaknesses: Speculation on the origin of the medusoid life stage in cnidarians heavily dependent on prior assumptions concerning the soft part anatomy and material properties of the skeleton of the modeled fossil organism that may be open to alternative interpretations. Logically, of course, the hypothesis that cnidarian medusae originated from benthic polyps must be evaluated along with the alternative hypotheses that the medusa came first and that the ancestral cnidarian exhibited both life stages.

Reviewer #2 (Public Review):

Summary and strengths:

1) The work provides significant insights because usually non-significant studies can be considered replicated by their null replications as well. The work discuss and provide data demonstrating that when analyzing studies with p > 0.05 for the result to be replicated, equivalence tests and bayes factor approaches are more suitable, since studies can be underpowered even if replications use larger samples than their original studies in general. Non-significant p-values are highly expected even with 80% of power for a true effect.

2) The evidence used features methods and analyses more rigorous than current state-of-the-art research on replicability.

Weaknesses:<br /> I am satisfied with the revisions made by the authors in response to my initial suggestions, as well as their subsequent responses to my observations throughout the reviewing process.

Reviewer #2 (Public Review):

Summary:<br /> In this study, the authors present a robust pipeline that integrates high-content phenotypic imaging of a targeted pool of 366 CRISPRi-screened genes with in situ sequencing of single cells, achieving a resolution for 1.3 million cells. The application of this pipeline on the U2OS cell line effectively screens for nuclear and actin morphology changes. One study's strength lies in the utilization of a barcode system, enabling efficient imaging and genotype determination for 85% of cells. The authors employ two distinct approaches to delineate phenotypic changes. In the first approach, cells are characterized by approximately 1,000 morphological features, with dimensionality reduction via PCA using 25 principal components and a novel image sampling method called VIEWED (Visual Interpretation of Embedding by Constrained Walkthrough Sampling). The second approach employs a deep learning technique, specifically the Beta-variational encoder, to identify morphological differences, offering a generative AI approach for visualizing interpreted distinctions learned through the algorithm. While the Beta-variational encoder is deemed simpler to use and interpret, the classical PCA approach demonstrates superiority due to its heightened sensitivity in identifying more genes with phenotypic changes. Both methods, however, successfully identify shared phenotypic gene hits, showing consistent replication across multiple individual guides for each gene hit. Key phenotypic clusters are identified and replicated similarly by both the conventional PCA feature approach and the Beta-variational encoder approach.

Strengths:<br /> - A novel barcode methodology for efficient genotyping via in situ sequencing, minimizing rounds of imaging and genotyping 85% of cells.<br /> - Use of a beta variational autoencoder, generative AI approach to facilitate detection of morphological change in cells, gene hits, and phenotypic gene clusters.

Weaknesses:<br /> Although the outcome is reproduced with 3 gRNA/gene, no biological replicate is presented and is as such limiting on convincing on reproducibility of the phenotypic detection approach.

The presented work is highly compelling as it employs an optical pooled CRISPRi screen, showcasing the capability to conduct pool screening beyond the typical frequency count of guides with the next-generation sequencing approach, effectively establishing a direct link between cell images and guide RNAs in the pool screen approach. This achievement, typically associated with arrayed screens, sets the study apart. Moreover, the study offers captivating images of individual cells that vividly portray convincing phenotypic changes. Additionally, the work effectively highlights the potency of generative AI in interpreting cell phenotypic changes detected by the algorithm. This aspect of the study is particularly relevant in the present time, as it introduces a potentially highly valuable methodology. Overall, the research provides a robust demonstration of innovative techniques and methodologies, contributing significantly to the field.

Reviewer #2 (Public Review):

Summary:<br /> This interesting study challenges a dogma regarding the link between bacterial metabolism decrease and tolerance to aminoglycosides (AG). The authors demonstrate that mutants well-known for being tolerant to AG, such as those of complexes I and II, are not so due to a decrease in the proton motive force (PMF) and thus antibiotic uptake, as previously reported in the literature.

Strengths:<br /> This is a complete study. These results are surprising and are based on various read-outs, such as ATP levels, pH measurement, membrane potential, and the uptake of fluorophore-labeled gentamicin. Utilizing a proteomic approach, the authors show instead that in tolerant mutants, there is a decrease in the levels of proteins associated with ribosomes (targets of AG), causing tolerance.

Weaknesses:<br /> The use of a single high concentration of aminoglycoside: my main comment on this study concerns the use of an AG concentration well above the MIC (50 µg/ml or 25 µg/ml for uptake experiments), which is 10 times higher than previously used concentrations (Kohanski, Taber) in study showing a link with PMF. This significant difference may explain the discrepancies in results. Indeed, a high concentration of AG can mask the effects of a metabolic disruption and lead to less specific uptake. However, this concentration highlights a second molecular level of tolerance. Adding experiments using lower concentrations (we propose 5 µg/ml to compare with the literature) would provide a more comprehensive understanding of AG tolerance mechanisms during a decrease in metabolism.

Another suggestion would be to test iron limitation (using an iron chelator as DIP), which has been shown to induce AG tolerance. Can the authors demonstrate if this iron limitation leads to a decrease in ribosomal proteins? This experiment would validate their hypothesis in the case of a positive result. Otherwise, it would help distinguish two types of molecular mechanisms for AG tolerance during a metabolic disruption: (i) PMF and uptake at low concentrations, (ii) ribosomal proteins at high concentrations.

Reviewer #2 (Public Review):

Summary:<br /> The authors tried to understand the mechanism of how a drug candidate, VLZ, works on a receptor, 5-HTR1A, by activating the SRC/MAPK pathway to promote the formation of platelets.

Strengths:<br /> The authors used both computational and experimental methods. This definitely saves time and funds to find a useful drug candidate and its therapeutic marker in the subfield of platelets reduction in cancer patients. The authors achieved the aim of explaining the mechanism of VLZ in improving thrombocytopenia by using two cell lines and two animal models.

Weaknesses:<br /> Only two cell lines, HEL and Meg-01 cells, were evaluated in this study. However, using more cell lines is really depending on the workflow and the grant situations of the current research team.

Reviewer #2 (Public Review):

Summary:<br /> The paper sets out to understand the mechanisms underlying the colonization and degradation of marine particles using a natural Vibrio isolate as a model. The data are measurements of motility and gene expression using microfluidic devices and RNA sequencing. The results reveal that degradation products of alginate do stimulate motility but not chemotaxis. The evidence for these claims is strong. The story of how particle degradation occurs through colonization and dispersal has modest support in the data. A quantitative description of these dynamics awaits future studies.

Strengths:<br /> The microfluidic and transcriptional measurements are the central strengths of the paper as they allow the delineation of phenotypes at the cellular and molecular levels in the presence of polymer and byproducts of polymer degradation.

Weaknesses:<br /> The explanation of the microfluidics measurements is somewhat confusing but I think this could be easily remedied. The quantitative interpretation of the dispersal data could also be improved and I'm not clear if the data support the claim made.

Reviewer #2 (Public Review):

Summary

The authors proposed a toolset Photo-SynthSeg to the software FreeSurfer which performs 3D reconstruction and high-resolution 3D segmentation on a stack of coronal dissection photographs of brain tissues. To prove the performance of the toolset, three experiments were conducted, including volumetric comparison of brain tissues on AD and HC groups from MADRC, quantitative evaluation of segmentation on UW-ADRC and quantitative evaluation of 3D reconstruction on HCP digitally sliced MRI data.

Strengths

To guarantee successful workflow of the toolset, the authors clearly mentioned the prerequisites of dissection photograph acquisition, such as fiducials or rulers in the photos and tissue placement of brain slices with more than one connected component. The quantitative evaluation of segmentation and reconstruction on synthetic and real data demonstrates the accuracy of the methodology. Also, the successful application of this toolset on two brain banks with different slice thicknesses, tissue processing and photograph settings demonstrates its robustness. By working with tools of the SynthSeg pipeline, Photo-SynthSeg could further support volumetric cortex parcellation. The toolset also benefits from its adaptability of different 3D references, such as surface scan, ex vivo MRI and even probabilistic atlas, suiting the needs for different brain banks.

Weaknesses

Certain weaknesses are already covered in the manuscript. Cortical tissue segmentation could be further improved. The quantitative evaluation of 3D reconstruction is quite optimistic due to random affine transformations. Manual edits of slice segmentation task are still required and take a couple of minutes per photograph. Finally, the current toolset only accepts coronal brain slices and should adapt to axial or sagittal slices in future work.

Reviewer #2 (Public Review):

Summary:

This manuscript by Xu et al., is an interesting study aiming to identify novel features of macaque cortical development. This study serves as a valuable atlas of single cell data during macaque neurogenesis, which extends the developmental stages previously explored. Overall, the authors have achieved their aim of collecting a comprehensive dataset of macaque cortical neurogenesis and have identified a few unknown features of macaque development.

Strengths:

The authors have accumulated a robust dataset of developmental time points and have applied a variety of informatic approaches to interrogate this dataset. One interesting finding in this study is the expression of previously unknown receptors on macaque oRG cells. Another novel aspect of this paper is the temporal dissection of neocortical development across species. The identification that the regulome looks quite different, despite similar expression of transcription factors in discrete cell types, is intriguing.

Reviewer #2 (Public Review):

Summary:<br /> This paper focuses on an interesting question that has puzzled psychologists for decades, that is, why do people demonstrate a mix of uncertainty approach and avoidance behavior, given the fact that reducing uncertainty could always gain information and seems beneficial? This paper designed a novel task to demonstrate behavioral signatures of uncertainty approaching and avoidance during the exploration phase within the same task at both a within-subject and between-subject level. On the algorithmic level, this paper compared four different implementations of uncertainty-guided exploration and found that the model sensitive to relative uncertainty provides the best fit for human behavior compared to its counterparts using expected information gain or past exposure. This paper then links people's uncertainty attitude with accuracy and finds that uncertainty avoidance during exploration does not impair task performance, implying that uncertainty avoidance may be the output of a resource-rational decision-making process. To examine this account, this paper uses reaction time as an independent proxy of costly deliberation and shows that people deliberate shorter when engaging in repetitive choice, which presumably saves cognitive resources. Finally, the paper shows that people's tendency to engage in repetitive choice correlates with their tendency to avoid uncertainty, which supports the argument that avoiding uncertainty could be a strategy developed under the constraint of limited cognitive resources.

Strengths:<br /> One of the highlights of this paper, as mentioned in the previous paragraph, is that the authors can establish the existence of the uncertainty approach and avoidance behavior within the same task whereas previous work usually focuses on one of them. This dissociation allows the authors to examine what situational factor is related to the emergence of the act of avoiding uncertainty, and extract parameters describing participants' attitude towards uncertainty during baseline as well as during situations where uncertainty avoidance is more common. Besides documenting the existence of uncertainty avoidance behavior, this paper also tried to explain this behavior by proposing under the resource rational framework and has carefully quantified different aspects (e.g., accuracy; choice speed) of participants' behavior as well as examined their relationships. Though more experiments are needed to fully understand human uncertainty avoidance behavior, this paper has provided both empirical and theoretical contributions toward a mechanistic understanding of how people balance approaching and avoiding uncertainty.

Weaknesses:<br /> I have a couple of concerns related to this paper. First, there seems to exist an anti-correlation between total uncertainty and absolute relative uncertainty (Figure 5 panel C, \delta uncertainty is restricted to a small range when total uncertainty is high). It seems to be a natural product of the exploration process since the high total uncertainty phase is usually the period where the participant knows little about either option, leading to a less distinguishable relative uncertainty. However, it remains unknown whether the documented uncertainty avoidance still applies when extrapolating to larger absolute relative uncertainty. It would be great if the experiment allows for a manipulation of uncertainty in the middle of the experiment (e.g., introducing a new deck/informing that one deck has been updated). Relatedly, the current 'threshold' of uncertainty avoidance behavior, if I understand correctly, is found by empirically fitting participants' data. This brings the question: can we predict when people will demonstrate uncertainty avoidance behavior before collecting any data? Or, is it possible that by measuring some metrics related to cognitive cost sensitivity, we could predict the proportion of choices that participants will show uncertainty-avoidant behavior? Finally, regarding the analysis of different behavior patterns in the game, it seems that the authors try to link repetitive behavior, uncertainty attitude, and accuracy together by testing the correlation between the two of them. I wonder whether other multivariate statistical methods e.g., mediation analysis, will be better suited for this purpose.

Reviewer #2 (Public Review):

The authors demonstrate convincingly the potential of single mesodermal cells, removed from zebrafish embryos, to show cell-autonomous oscillatory signaling dynamics and differentiation. Their main conclusion is that a cell-autonomous timer operates in these cells and that additional external signals are integrated to tune cellular dynamics. Combined, this is underlying the precision required for proper embryonic segmentation, in vivo. I think this work stands out for its very thorough, quantitative, single-cell real-time imaging approach, both in vitro and also in vivo. A very significant progress and investment in method development, at the level of the imaging setup and also image analysis, was required to achieve this highly demanding task. This work provides new insight into the biology underlying embryo axis segmentation.<br /> The work is very well presented and accessible. I think most of the conclusions are well supported. Here a my comments and suggestions:

1) The authors state that "We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding remarkable agreement."

I think this statement needs to be better placed in context. In absolute terms, the period of oscillations and the timing of differentiation are actually very different in vitro, compared to in vitro. While oscillations have a period of ~30 minutes in vivo, oscillations take twice as long in vitro. Likewise, while the last oscillation is seen after 143 minutes in vivo, the timing of differentiation is very significantly prolonged, i.e.more than doubled, to 373min in vitro (Supplementary Figure 1-9). I understand what the authors mean with 'remarkable agreement', but this statement is at the risk of being misleading. I think the in vitro to in vivo differences (in absolute time scales) needs to be stated more explicitly. In fact, the drastic change in absolute timescales, while preserving the relative ones,i.e. the number of oscillations a cell is showing before onset of differentiation remains relatively invariant, is a remarkable finding that I think merits more consideration (see below).

2) One timer vs. many timers<br /> The authors show that the oscillation clock slowing down and the timing of differentiation, i.e. the time it takes to activate the gene mesp, are in principle dissociable processes. In physiological conditions, these are however linked. We are hence dealing with several processes, each controlled in time (and hereby space). Rather than suggesting the presence of 'a timer', I think the presence of multiple timing mechanisms would reflect the phenomenology better. I would hence suggest separating the questions more consistently, for instance into the following three:<br /> a. what underlies the slowing down of oscillations?<br /> b. what controls the timing of onset of differentiation?<br /> c. and finally, how are these processes linked?

Currently, these are discussed somewhat interchangeably, for instance here: "Other models posit that the slowing of Her oscillations arise due to an increase of time-delays in the negative feedback loop of the core clock circuit (Yabe, Uriu, and Takada 2023; Ay et al. 2014), suggesting that factors influencing the duration of pre-mRNA splicing, translation, or nuclear transport may be relevant. Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock."(page 14). In the first part, the slowing down of oscillations is discussed and then the authors conclude on 'the timer', which however is the one timing differentiation, not the slowing down. I think this could be somewhat misleading.

3) From this and previous studies, we learn/know that without clock oscillations, the onset of differentiation still occurs. For instance in clock mutant embryos (mouse, zebrafish), mesp onset is still occurring, albeit slightly delayed and not in a periodic but smooth progression. This timing of differentiation can occur without a clock and it is this timer the authors refer to "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14). This 'timer' is related to what has been previously termed 'the wavefront' in the classic Clock and Wavefront model from 1976, i.e. a "timing gradient' and smooth progression of cellular change. The experimental evidence showing it is cell-autonomous by the time it has been laid down,, using single cell measurements, is an important finding, and I would suggest to connect it more clearly to the concept of a wavefront, as per model from 1976.

4) Regarding question a., clearly, the timer for the slowing down of oscillations is operating in single cells, an important finding of this study. It is remarkable to note in this context that while the overall, absolute timescale of slowing down is entirely changed by going from in vivo to in vitro, the relative slowing down of oscillations, per cycle, is very much comparable, both in vivo and in vivo. To me, while this study does not address the nature of this timer directly, the findings imply that the cell-autonomous timer that controls slowing down is, in fact, linked to the oscillations themselves. We have previously discussed such a timer, i.e. a 'self-referential oscillator' mechanism (in mouse embryos, see Lauschke et al., 2013) and it seems the new exciting findings shown here in zebrafish provide important additional evidence in this direction. I would suggest commenting on this potential conceptual link, especially for those readers interested to see general patterns.

5) Regarding question c., i.e. how the two timing mechanisms are functionally linked, I think concluding that "Whatever the identity, our results indicate the timer ought to exert control over differentiation independent of the clock." (page 14), might be a bit of an oversimplification. It is correct that the timer of differentiation is operating without a clock, however, physiologically, the link to the clock (and hence the dependence of the timescale of clock slowing down), is also evident. As the author states, without clock input, the precision of when and where differentiation occurs is impacted. I would hence emphasize the need to answer question c., more clearly, not to give the impression that the timing of differentiation does not integrate the clock, which above statement could be interpreted to say.

6) A very interesting finding presented here is that in some rare examples, the arrest of oscillations and onset of differentiation (i.e. mesp) can become dissociated. Again, this shows we deal here with interacting, but independent modules. Just as a comment, there is an interesting medaka mutant, called doppelkorn (Elmasri et al. 2004), which shows a reminiscent phenotype "the Medaka dpk mutant shows an expansion of the her7 expression domain, with apparently normal mesp expression levels in the anterior PSM.". The authors might want to refer to this potential in vivo analogue to their single cell phenotype.

7) One strength of the presented in vitro system is that it enables precise control and experimental perturbations. A very informative set of experiments would be to test the dependence of the cell-autonomous timing mechanisms (plural) seen in isolated cells on ongoing signalling cues, for instance via Fgf and Wnt signaling. The inhibition of these pathways with well-characterised inhibitors, in single cells, would provide important additional insight into the nature of the timing mechanisms, their dependence on signaling and potentially even into how these timers are functionally interdependent.

Reviewer #3 (Public Review):

Summary:

1. Grandits and colleagues were trying to develop a new tool to accelerate pharmacological studies by using neural networks to emulate the human ventricular cardiomyocyte action potential (AP). The AP is a complex electrical signal that governs the heartbeat, and it is important to accurately model the effects of drugs on the AP to assess their safety and efficacy. Traditional biophysical simulations of the AP are computationally expensive and time-consuming. The authors hypothesized that neural network emulators could be trained to predict the AP with high accuracy and that these emulators could also be used to quickly and accurately predict the effects of drugs on the AP.

Strengths:

2. One of the study's major strengths is that the authors use a large and high-quality dataset to train their neural network emulator. The dataset includes a wide range of APs, including normal and abnormal APs exhibiting EADs. This ensures that the emulator is robust and can be used to predict the AP for a variety of different conditions.

Another major strength of the study is that the authors demonstrate that their neural network emulator can be used to accelerate pharmacological studies. For example, they use the emulator to predict the effects of a set of known arrhythmogenic drugs on the AP. The emulator is able to predict the effects of these drugs, even though it had not been trained on these drugs specifically.

Weaknesses:

One weakness of the study is that it is important to validate neural network emulators against experimental data to ensure that they are accurate and reliable. The authors do this to some extent, but further validation would be beneficial. In particular for the inverse problem, where the estimation of pharmacological parameters very challenging and led to particularly large inaccuracies.

Additional context:

4. The work by Grandits et al. has the potential to revolutionize the way that pharmacological studies are conducted. Neural network emulation has the promise to reduce the time and cost of drug development and to improve the safety and efficacy of new drugs. The methods and data presented in the paper are useful to the community because they provide a starting point for other researchers to develop and improve neural network emulators for the human ventricular cardiomyocyte AP. The authors have made their code and data publicly available, which will facilitate further research in this area.

5. It is important to note that neural network emulation is still a relatively new approach, and there are some challenges that need to be addressed before it can be widely adopted in the pharmaceutical industry. For example, neural network emulators need to be trained on large and high-quality datasets. Additionally, it is important to validate neural network emulators against experimental data to ensure that they are accurate and reliable. Despite these challenges, the potential benefits of neural network emulation for pharmacological studies are significant. As neural network emulation technology continues to develop, it is likely to become a valuable tool for drug discovery and development.

Reviewer #2 (Public Review):

Summary:

The malaria parasite Plasmodium develops into oocysts and sporozoites inside Anopheles mosquitoes, in a process called sporogony. Sporozoites invade the insect salivary glands in order to be transmitted during a blood meal. An important question regarding malaria transmission is whether all mosquitoes harboring Plasmodium parasites are equally infectious. In this paper, the authors investigated the progression of P. falciparum sporozoite development in Anopheles mosquitoes, using a sensitive qPCR method to quantify sporozoites and an artificial skin system to probe for parasite expelling. They assessed the association between oocyst burden, salivary gland infection intensity, and sporozoites expelled.

The data show that higher sporozoite loads are associated with earlier colonization of salivary glands and a higher prevalence of sporozoite-positive salivary glands and that higher salivary gland sporozoite burdens are associated with higher numbers of expelled sporozoites. Intriguingly, there is no clear association between salivary gland burdens and the prevalence of expelling, suggesting that most infections reach a sufficient threshold to allow parasite expelling during a mosquito bite. This important observation suggests that low-density gametocyte carriers, although less likely to infect mosquitoes, could nevertheless contribute to malaria transmission.

Strengths:

The paper is well written and the work is well conducted. The authors used two experimental models, one using cultured P. falciparum gametocytes and An. stephensi mosquitoes, and the other one using natural gametocyte infections in a field setup with An. coluzzii mosquitoes. Both studies gave similar results, reinforcing the validity of the observations. Parasite quantification relies on a robust and sensitive qPCR method, and parasite expelling was assessed using an innovative experimental setup based on artificial skin.

Weaknesses:

There is no clear association between the prevalence of sporozoite expelling and the parasite burden. However, high total sporozoite burdens are associated with earlier and more efficient colonization of the salivary glands, and higher salivary gland burdens are associated with higher numbers of expelled sporozoites. While these observations suggest that highly infected mosquitoes could transmit/expel parasites earlier, this is not directly addressed in the study. In addition, whether all expelled sporozoites are equally infectious is unknown. The central question, i.e. whether all infected mosquitoes are equally infectious, therefore remains open.

Reviewer #2 (Public Review):

Dipasree Hajra et al demonstrated that Salmonella was able to modulate the expression of Sirtuins (Sirt1 and Sirt3) and regulate the metabolic switch in both host and Salmonella, promoting its pathogenesis. The authors found Salmonella infection induced high levels of Sirt1 and Sirt3 in macrophages, which were skewed toward the M2 phenotype allowing Salmonella to hyper-proliferate. Mechanistically, Sirt1 and Sirt3 regulated the acetylation of HIF-1alpha and PDHA1, therefore mediating Salmonella-induced host metabolic shift in the infected macrophages. Interestingly, Sirt1 and Sirt3-driven host metabolic switch also had an effect on the metabolic profile of Salmonella. Counterintuitively, inhibition of Sirt1/3 led to increased pathogen burdens in an in vivo mouse model. Overall, this is a well-designed study. There are a few comments below that would further strengthen the current study.

Major comments:<br /> In the in vivo study (lines 436-446) - the authors noticed increased pathogen burden in the EX-527 or the 3TYP-treated mice cohorts but decreased pathogen burden within the F4/80+ macrophage population. What are the other cell types that have increased pathogen burden in splenocytes from EX-527 or the 3TYP treated? Can this be further explored and explained?

While the authors indicated that IL-6 cytokine storm and elevated ROS production could result in bacterial dissemination in vivo, one could also argue that Sirt1/3 inhibitors might have an impact on gut function and/or gut microbiota (PMID: 22115311). Did Sirt1/3 inhibitors also lead to increased pathogen burdens in the gut? If so, the potential effect of these in vivo treatments on gut microbiota/colonization resistance should be discussed.

Minor comment:<br /> Sirt1 has been shown to be degraded during Salmonella infection (PMID: 28192515), which is different from the current study. An explanation should be provided for this.

Reviewer #2 (Public Review):

Summary:<br /> The authors present a report of a large Pseudomonas aeruginosa hospital outbreak affecting more than 80 patients with first sampling dates in 2011 that stretched over more than 10 years and was only identified through genomic surveillance in 2020. The outbreak strain was assigned to the sequence type 621, an ST that has been associated with carpabapenem resistance across the globe. Ongoing transmission coincided with both increasing resistance without acquisition of carbapenemase genes as well as the convergence of mutations towards a host-adapted lifestyle.

Strengths:<br /> The convincing genomic analyses indicate spread throughout the hospital since the beginning of the century and provide important benchmark findings for future comparison.

The sampling was based on all organisms sent to the Multidrug-resistant Organism Repository and Surveillance Network across the U.S. Military Health System.

Using sequencing data from patient and environmental samples for phylogenetic and transmission analyses as well as determining recurring mutations in outbreak isolates allows for insights into the evolution of potentially harmful pathogens with the ultimate aim of reducing their spread in hospitals.

Weaknesses:<br /> The epidemiological information was limited and the sampling methodology was inconsistent, thus complicating the inference of exact transmission routes. Epidemiological data relevant to this analysis include information on the reason for sampling, patient admission and discharge data, and underlying frequency of sampling and sampling results in relation to patient turnover.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Hu and colleagues investigate telomerase-independent survival in Saccharomyces cerevisiae strains engineered to have different chromosome numbers. The authors describe the molecular patterns of survival that change with fewer chromosomes and that differ from the well-described canonical Type I and Type II, including chromosome circularization and other atypical outcomes. They then take advantage of the strain with 3 chromosomes to examine the effect of deleting all the subtelomeric elements, called X and Y'. For most of the tested phenotypes, they find no significant effect of the absence of X- and Y'-element, and show that they are not essential for survivor formation. They speculate that X- and Y'-elements are remnants of ancient telomere maintenance mechanisms.

Strengths:<br /> This work advances our understanding of the telomerase-independent strategies available to the cell by altering the structure of the genome and of the subtelomeres, a feat that was enabled by the set of strains they engineered previously. By using strains with non-standard genome structures, several alternative survival mechanisms are uncovered, revealing the diversity and plasticity of telomere maintenance mechanisms. Overall, the conclusions are well supported by the data, with adequate sample sizes for investigating survivors. The assessment of the genetic requirements for survivors in strains with different chromosome numbers greatly improved the quality of this work. The molecular analyses based on Southern blots are also very well-conducted.

Weaknesses:<br /> The authors discovered alternative telomerase-independent survival strategies beyond the well-described type I and II (including circularization, type X and atypical, as they called them) at play in the context of reduced number of chromosomes. Their work provides a molecular and a partial genetic characterization of these survival pathways. A more thorough analysis of the frequency of each type of survivors and their genetic requirements would have advanced our understanding or the diversity of survival strategies in the absence of telomerase. However, as noted by the authors, the quantification of the rate of emergence of survivors (and their subtypes) is very difficult to achieve. This comment is therefore not meant as a criticism but rather as a perspective on exciting future research avenues.

Reviewer #3 (Public Review):

The authors have done a fine job of updating the manuscript and it is substantially improved. In particular, the paragraphs towards the end of the Introduction and Discussion are vastly improved. The last paragraph of the Introduction now clearly explicates the hypotheses (save one minor point of confusion). The limitations section of the Discussion is also very helpful and fair. However, there are still areas where claims need to be tempered.

Major criticisms<br /> • The results still do not lead to the conclusion that the angular gyrus is causally involved in insight-driven memory configuration. Although the authors do state that other regions such as the hippocampus may have contributed to the pattern of results, there is still no evidence of target engagement or a link between target engagement and the behavioral results. Thus, while the results support that cTBS to the angular gyrus affects insight-driven memory configuration, it is a strong overstep to say that the angular gyrus is causally involved in insight-driven memory reconfiguration. In particular, this applies to both the title and the last line of the Abstract. In relation to this, have the authors conducted any target engagement analyses? It seems like a good starting point would be to identify the censor closest to the stimulation site in each individual, Hjorth transforms the signal of that sensor by subtracting the average of the surrounding sensors to increase signal localization, and then measure the effects of stimulation on theta power. Presumably, we would expect that cTBS would decrease theta power relative to sham stimulation. Although this isn't the only type of analysis that could at least partially confirm target engagement, there needs to be some sort of formal analysis to maintain the claims of the title and last line of the Abstract.<br /> • The authors removed the mentions of "inhibitory stimulation" from the manuscript to their credit, but a rigorous and fair treatment of the effects of cTBS is still lacking, and it is still unclear why cTBS to the angular gyrus would cause an inhibitory effect in the first place. The authors state that

"Previous evidence has demonstrated the inhibitory effect of cTBS on the targeted brain region under stimulation (Huang et al., 2005; Jannati et al., 2023). Nonetheless, the effects of cTBS appear to vary based on the targeted region, with cTBS to parietal regions demonstrating the capability to enhance hippocampal connectivity (Hermiller et al., 2019, 2020)."

The inhibitory effects of motor cortex cTBS s on corticospinal excitability in nine subjects from the Huang paper and the Jannati review (not a primary source) do not constitute sufficient evidence to hypothesize an inhibitory effect on insight-driven memory reconfiguration. The second sentence provides much more sufficient evidence that parietal stimulation should have some sort of a facilitatory effect, but this is simply glossed over without an explanation of why cTBS to the parietal cortex should inhibit insight-driven memory reconfiguration. Pilot data showing such inhibitory effects or a body of evidence showing inhibitory effects of angular gyrus stimulation on closely-related areas of cognition would have given reason to believe this. However, without these, an a priori assumption that parietal cTBS would be inhibitory seems highly debatable and paints the results as provisional, rather than confirmatory"

Reviewer #3 (Public Review):

The findings of Bo Yu and colleagues titled "Identification of fallopian tube microbiota and its association with ovarian cancer: a prospective study of intraoperative swab collections from 187 patients" describes the identification of the fallopian tube microbiome and relationship with ovarian cancer. The studies are highly rigorous obtaining specimens from the fallopian tube, ovarian surfaces, paracolic gutter of patients of known or suspected ovarian cancer or benign tumor patients. The investigators took great care to insure there was no or limited contamination including test the surgical suite air, as the test locations are from low abundance microbiota. The findings provide evidence that the microbiota in the fallopian tube, especially in ovarian cancer has similarities to gut microbial communities. This is a potentially novel observation.

The studies investigate the microbiome of >1000 swabs from 81 ovarian cancer and 106 non-cancer patients. The sites collected are low biomass microbiota making the study particularly challenging. The studies provide descriptive evidence that the ovarian cancer fallopian tube microbiota contain species that are similar to the gut microbiota. In contrast the fallopian tube microbiota of non-cancer patients that exhibit more similarity to the uterine/cervical microbiota. This may be a relevant observation but is highly descriptive with limited insights on the functional relevance.

The data indicate the presence of low biomass FT microbiota. The findings support the existence of FT microbiota in ovarian cancer that appears to be related to gut microbial species. While interesting, there is no insights on how and why these microbial species are found in the FT. The studies only identify the species but there is no transcriptomic analysis to provide an indication on whether the bacteria are activating DNA damage pathways. This is an interesting observation that requires more insights to address how these bacteria reach the fallopian tube and a related question is whether these bacteria are found in the peritoneum.

An additional concern is whether these data can be used to develop biomarkers of disease and early detection of disease.

Reviewer #2 (Public Review):

Summary:

The study provides valuable and compelling evidence that while activation of the mTOR cascade confers some similarities in alterations in cell size, mTOR pathway activation, cortical lamination, baseline firing properties, and synaptic activity, there are distinctions that could account for clinical differences in seizure and epilepsy phenotypes in patients harboring these mutations. These findings could have important implications going forward as we design clinical therapeutic strategies to modulate mTOR activity in these individuals to treat seizures.

This study presents a valuable finding on the role that distinct mTOR pathway genes play in altered cell shape, cortical laminar migration, and cellular excitability in the mouse medial prefrontal cortex (mPFC). The evidence supporting the claims of the authors is solid, although analysis of the role of the mTORC2 pathway and consideration of distinct metabolic states i.e., amino acid levels would have strengthened the study. The work will be of interest to neuroscientists working on human epilepsy. These genes have each been assayed in previous independent studies and thus the direct comparison is what provides the innovation and interest.

The manuscript by Nguyen and colleagues attempts to define both the common and differential roles of mTOR pathway genes, both by gene knockout (KO) and activation, on cortical neuronal size, cortical lamination, and excitability. They focused on 5 genes that have been linked to human malformations of cortical development (MCD) and epilepsy: RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO. The RhebY35L, mTORS2215Y are known and pathogenic human gain-of-function variants. Each of these genes is known to modulate the activity of mTORC1 and either KO or activation will lead to abnormal and persistent hyperactivation of mTOR activity. Using in utero electroporation they transfected plasmids containing these gene constructs into fetal mouse brains at E15.5 and then assessed neuronal shape and size, laminar positioning, spontaneous activity, synaptic activity, and expression of a novel voltage-gated potassium channel (HCN4) at varying time postnatally e.g., P7-9 (neonates) and P28-43 (young adults).

The study clearly achieves its stated aims i.e., that disruption of each of five distinct mTOR pathway genes, Rheb, mTOR, Depdc5, Pten, and Tsc1, individually impacts pyramidal neuron development and electrophysiological function in the mouse mPFC. The data from each of the 5 genes provides strong support to the notion that mTOR pathway gene mutations yield the unifying clinical parcellation of mTORopathies, likely as a consequence of mTOR pathway activation. The data also provide interesting evidence that subtle or even overt differences in clinical phenotypes between RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO in humans could be due to effects of these genes either on mTOR or on yet to be defined alternative pathways. Assuredly follow-up studies to examine how Rheb, mTOR, Dedpdc5, Pten, and Tsc1 engage with other protein binding partners or other pathways will be warranted in future studies.

Strengths:

The investigators demonstrate that gene KO or activation leads to common changes in cell size (enlargement) though with different effects across each gene subtype suggesting distinct genetic effects despite a common effect on mTOR signaling. The major effect was seen in forebrain neurons expressing mTORS2215Y. They also report gene-specific effects of each mTOR pathway gene on cortical lamination. For example, while RhebY35L, mTORS2215Y, Dedpdc5KO, and Tsc1KO significantly disrupted laminar positioning of neurons in layer 2/3, PtenKO had minimal effects on laminar positioning. This finding is intriguing since it means that simply activating mTOR during fetal brain development will not necessarily alter cortical lamination and that an increase in cell size by itself doesn't disrupt laminar fidelity. To verify that the expression of plasmids led to mTORC1 hyperactivation, phosphorylated levels of S6 (i.e., p-S6), a downstream substrate of mTORC1, were assayed by immunohistochemistry in P28-43 mice. Expression of the RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO plasmids all led to significantly increased p-S6 staining intensity, supporting that the expression of each of these plasmids leads to increased mTORC1 signaling.

Whole-cell current- and voltage-clamp recordings were performed in P25-P51 mice in acute brain slice preparations. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to decreased depolarization-induced excitability, but only RhebY35L, mTORS2215Y, and Tsc1KO expression led to depolarized resting membrane potentials. Interestingly, expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to the abnormal presence of HCN4 channels with variations in functional expression suggesting a common cellular mechanism that could confer excitability. Treatment with rapamycin, an mTOR inhibitor, reversed the expression changes in HCN4. Expression of RhebY35L, mTORS2215Y, Dedpdc5KO, PtenKO, and Tsc1KO led to different impacts on sEPSC properties. Effects of treatment with the selective HCN channel blocker zatebradine on hyperpolarization-induced inward currents in mTORS2215Y neurons confirmed the identity of ΔI as Ih.

Overall the data presented provides a convincing and compelling direct comparison of the roles that select mTOR pathway genes play on brain development and network excitability. It is critical to directly compare these gene effects in mouse models because although these genes are part of the mTOR pathway and clearly cause augmentation of mTOR activation, there are mechanistic differences in how these gees modify mTOR and how they interact with other proteins and phenotypic differences in humans harboring mutations in these same genes.

Reviewer #2 (Public Review):

Summary: The authors have previously demonstrated that the E3 ligase PDLIM2 inhibits NF-kB and STAT3 and is epigenetically repressed in human lung cancers (Sun et al. Nat. Comm. 2019 10: 5324); therefore, PDLIM2 is a tumor suppressor in lung cancer. In this manuscript, they follow up on their previous findings and show that expression of PDLIM2 is downregulated in human lung cancers by both genetic deletion and promoter methylation. They further describe a novel approach to restore the expression of PDLIM2 in mouse lung tumors by systemically administering PDLIM2 plasmids encapsulated in nanoparticles (termed "nanoPDLIM2"). The nanoPDLIM2 approach was shown to exhibit efficacy with low toxicity in a urethane-induced mouse lung cancer model. The authors further demonstrated synergy of nanoPDLIM2 with chemotherapy and PD-1 blockade immunotherapy. The combination therapy of nanoPDLIM2, chemotherapy and immunotherapy proved most effective with complete tumor remission in 60% of mice. Mechanistically, nanoPDLIM2 upregulated MHC-I expression, enhanced CD4/CD8 T cell activation and tumor infiltration, and suppressed MDR1 induction and nuclear expression of STAT3, RelA and prosurvival genes in tumors. Overall, this study is important because it reinforces the critical roles of PDLIM2 in suppressing lung cancer, and also identifies a potential approach to restoring PDLIM2 expression in lung tumors. The experiments were well executed; the data are convincing and support the conclusions made by the authors.

Reviewer #2 (Public Review):

Summary:<br /> Liao and colleagues generated tagged SMAD1 and SMAD5 mouse models and identified genome occupancy of these two factors in the uterus of these mice using the CUT&RUN assay. The authors used integrative bioinformatic approaches to identify putative SMAD1/5 direct downstream target genes and to catalog the SMAD1/5 and PGR genome co-localization pattern. The role of SMAD1/5 on stromal decidualization was assayed in vitro on primary human endometrial stromal cells. The new mouse models offer opportunities to further dissect SMAD1 and SMAD5 functions without the limitation from SMAD antibodies, which is significant. The CUT&RUN data further support the usefulness of these mouse models for this purpose.

Strengths:<br /> The strength of this study is the novelty of new mouse models and the valuable cistromic data derived from these mice. Overall the present manuscript is an excellent resource paper for the field of reproductive biology.

Weaknesses:<br /> The weakness of the present version of the manuscript includes the self-limited data analysis approaches such as the proximal promoter based bioinformatic filter and an outdated method on inferring the cell type composition. Evidence was provided for potential associations between SMAD1/5 and other major transcription factors. However, causal effects of SMAD1/5 on the genome occupancy of other major uterine transcription factors were discussed but not experimentally examined in the present manuscript, which is understandable.

For data in Figure 2B, the current manuscript fails to elaborate the common and distinct features between clusters 1 and 3 as well as the biological significance of having two separate clusters for SMAD1. In addition, Figure S1A shows overlapping genome occupancy between SMAD1 and SMAD5, which is not clearly demonstrated in Figure 2B.

For data in Figure 5A, the result description does not provide adequate information to guide readers to full understanding of the data. The biological meaning behind the three PR clusters is not stated nor speculated. Moreover, Figure 5A and Figure S1B are inherently connected but fail to be adequately described in the main text.

Reviewer #2 (Public Review):

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

Strengths:<br /> These findings rely on state-of-the-art methods in the field of Statistical Genomics and help to address the issue of a low number of GWASs in non-European populations, ultimately contributing to reducing health inequalities across the globe.

Weaknesses:<br /> There is no replication cohort, as acknowledged by the authors (page 29, line 587), and no experimental validation to assess the biological effect of putative causal variants/genes. Thus, the study provides good evidence of association, rather than causation, between the genetic variants and COVID-19. Lastly, I consider it crucial to report the results for the SCOURGE Latin American GWAS, in addition to its meta-analysis with HGI results, since HGI data has a different phenotype scheme (Hospitalized COVID vs Population) compared to SCOURGE (Hospitalized COVID vs Non-hospitalized COVID).

Reviewer #2 (Public Review):

DNA gyrase is an essential enzyme in bacteria that regulates DNA topology and has the unique property to introduce negative supercoils into DNA. This enzyme contains 2 subunits GyrA and GyrB, which forms an A2B2 heterotetramer that associates with DNA and hydrolyzes ATP. The molecular structure of the A2B2 assembly is composed of 3 dimeric interfaces, called gates, which allow the cleavage and transport of DNA double stranded molecules through the gates, in order to perform DNA topology simplification.<br /> The article by Germe et al. questions the existence and possible mechanism for subunit exchange in the bacterial DNA gyrase complex.

The complexes are purified as a dimer of GyrA and a fusion of GyrB and GyrA (GyrBA), encoded by different plasmids, to allow the introduction of targeted mutations on one side only of the complex. The conclusion drawn by the authors is that subunit exchange does happen in vitro, favored by DNA binding and wrapping. They propose that the accumulation of gyrase in higher-order oligomers can favor rapid subunit exchange between two active gyrase complexes brought into proximity. This study is nicely illustrated with diagrams that explain the possible mechanism.

The authors are also debating the conclusions of a previous article by Gubaev, Weidlich et al 2016 (https://doi.org/10.1093/nar/gkw740). Gubaev et al. originally used this strategy of complex reconstitution to propose a nicking-closing mechanism for the introduction of negative supercoils by DNA gyrase, an alternative mechanism that precludes DNA strand passage, previously established in the field. Germe et al. propose that the detected negative supercoiling activity in this earlier study may be due to the subunit swapping of the recombinant protein with the endogenous enzyme.

Strengths

The mix of gyrase subunits is plausible, this mechanism has been suggested by Ideka et al, 2004 and also for the human Top2 isoforms with the formation of Top2a/Top2b hybrids being identified in HeLa cells (doi: 10.1073/pnas.93.16.8288).<br /> Germe et al have used extensive and solid biochemical experiments, together with thorough experimental controls, involving :<br /> - the purification of gyrase subunits including mutants with domain deletion, subunit fusion or point mutations.<br /> - DNA relaxation, cleavage and supercoiling assays<br /> - biophysical characterization in solution (size exclusion chromatography, mass photometry, mass spectrometry)

Together the combination of experimental approaches provides convincing evidence for subunit swapping in gyrase in vitro, despite the technical limitations of standard biochemistry applied to such a complex macromolecule.

Weaknesses

The conclusions of this study could be strengthened by in vivo data to identify subunit swapping in the bacteria. Indeed, if shown in vivo, together with this biochemical evidence, this mechanism could have a substantial impact on our understanding of bacterial physiology and resistance to drugs. These in vivo perspectives are beyond the scope of the present in vitro investigation but are however explained by the authors.

Reviewer #2 (Public Review):

Summary:<br /> This study examined the possible affect of spike-wave discharges (SWDs) on the response to visual or somatosensory stimulation using fMRI and EEG. This is a significant topic because SWDs often are called seizures and because there is non-responsiveness at this time, it would be logical that responses to sensory stimulation are reduced. On the other hand, in rodents with SWDs, sensory stimulation (a noise, for example) often terminates the SWD/seizure.

In humans, these periods of SWDs are due to thalamocortical oscillations. A certain percentage of the normal population can have SWDs in response to photic stimulation at specific frequencies. Other individuals develop SWDs without stimulation. They disrupt consciousness. Individuals have an absent look, or "absence", which is called absence epilepsy.

The authors use a rat model to study the responses to stimulation of the visual or somatosensory systems during and in between SWDs. They report that the response to stimulation is reduced during the SWDs. While some data show this nicely, the authors also report on lines 396-8 "When comparing statistical responses between both states, significant changes (p<0.05, cluster-) were noticed in somatosensory auditory frontal..., with these regions being less activated in interictal state (see also Figure 4). That statement is at odds with their conclusion. I do not see that this issue was addressed.

They also conclude that stimulation slows the pathways activated by the stimulus. I do not see any data proving this. It would require repeated assessments of the pathways in time. This issue was not addressed.

The authors also study the hemodynamic response function (HRF) and it is not clear what conclusions can be made from the data. This is still an issue. No conclusions appear to be possible to make.

Finally, the authors use a model to analyze the data. This model is novel and while that is a strength, its validation is unclear. The authors did not add any validation of their model.

Strengths:<br /> Use of fMRI and EEG to study SWDs in rats.

Weaknesses:<br /> Several aspects of the Methods and Results were improved but some are still are unclear.

Reviewer #2 (Public Review):

Summary:<br /> This study builds upon previous work that demonstrated that brain injury results in leakage of albumin across the blood brain barrier, resulting in activation of TGF-beta in astrocytes. Consequently, this leads to decreased glutamate uptake, reduced buffering of extracellular potassium and hyperexcitability. This study asks whether such a process can play a physiological role in cortical plasticity. They first show that stimulation of a forelimb for 30 minutes in a rat results in leakage of the blood brain barrier and extravasation of albumin on the contralateral but not ipsilateral cortex. The authors propose that the leakage is dependent upon neuronal excitability and is associated with an enhancement of excitatory transmission. Inhibiting the transport of albumin or the activation of TGF-beta prevents the enhancement of excitatory transmission. In addition, gene expression associated with TGF-beta activation, synaptic plasticity and extracellular matrix are enhanced on the "stimulated" hemisphere. That this may translate to humans is demonstrated by a break down in the blood brain barrier following activation of brain areas through a motor task.

Strengths:<br /> This study is novel and the results are potentially important as they demonstrate an unexpected break down of the blood brain barrier with physiological activity and this may serve a physiological purpose, affecting synaptic plasticity.

The strengths of the study are:<br /> 1) The use of an in vivo model with multiple methods to investigate the blood brain barrier response to a forelimb stimulation.<br /> 2) The determination of a potential functional role for the observed leakage of the blood brain barrier from both a genetic and electrophysiological view point<br /> 3) The demonstration that inhibiting different points in the putative pathway from activation of the cortex to transport of albumin and activation of the TGF-beta pathway, the effect on synaptic enhancement could be prevented.<br /> 4) Preliminary experiments demonstrating a similar observation of activity dependent break down of the blood brain barrier in humans.

Weaknesses:<br /> The authors adequately addressed most of my points. A few remain:<br /> 1) Although the reviewers have addressed the possible effects of anaesthesia on neuro-vascular coupling. They have not mentioned or addressed the possible effects of ketamine (an NMDA receptor antagonist) on synaptic plasticity. Indeed, the low percentage of SEP increase following potentiation (10-20%) could perhaps be explained by partial block of NMDA receptors by ketamine.<br /> 2) The experimental paradigms remain unclear to me. Now, it appears that drugs are applied for 50 minutes and that the stimulation occurs during the "washout period". The more conventional approach would be to have the drug application during the stimulation period to determine if the drugs occlude or enhance the effects of stimulation and then washout the drugs. The problem is that drugs variably washout at different rates depending upon their lipid solubility.<br /> 3) It is still not clear to what extent the experimenters and those doing the analysis were blinded to group. If one or both were blind to group, then please put this in the methods.

Reviewer #2 (Public Review):

Summary:<br /> The authors are studying the behavioral response to pathogen exposure. They and others have previously describe the role that the G-protein coupled receptors in the nervous system plays in detecting pathogens, and initiating behavioral patterns (e.g. avoidance/learned avoidance) that minimize contact. The authors study this problem in C. elegans, which is amenable to genetic and cellular manipulations and allow the authors to define cellular and signaling mechanisms. This paper extends the original idea to now implicate signaling and transcriptional pathways within a particular neuron (ASJ) and the gut in mediating avoidance behaviour.

Strengths:<br /> The work is rigorous and elegant and the data are convincing. The authors make superb use of mutant strains in C. elegans, as well tissue specific gene inactivation and expression and genetic methods of cell ablation. to demonstrate how a gene, NPR15 controls behavioral changes in pathogen infection. The results suggest that ASJ neurons and the gut mediate such effects. I expect the paper will constitute an important contribution to our understanding of how the nervous system coordinates immune and behavioral responses to infection.

Reviewer #2 (Public Review):

This paper describes the results of a set of complementary and convergent experiments aimed at describing roles for the non-selective cation channels NALCN and TRPC6 in mediating subthreshold inward depolarizing currents and action potential generation in VTA DA neurons under normal physiological conditions. In general, the authors have responded satisfactorily to reviewer comments, and the revised manuscript is improved. The manuscript could still benefit from additional revision, including the following:

1. From the previous review, we mentioned that " 'The HCN' as written in line 69 is a bit misleading, as HCN channels in the heart and brain are different members of a family of channels, although as written in the text, it seems that they are identical." This is still the case (now line 73).

2. The authors state in line 112 that "most of the experiments were also repeated in female mice" - this is true in the case of most electrophysiological experiments, although not behavioral experiments. Authors should amend the statement in line 112 and clarify in the Discussion section which findings are generalizable between sexes; e.g.:<br /> a. Discussion of HCN contribution to VTA DA activity (beginning line 453) should clarify male mice.<br /> b. Similarly, any discussion of behavioral findings should clarify male mice.

3. The authors' statement in lines 179-183 ("In contrast, fewer GABAergic neuronal markers (Glutamic acid decarboxylase, GAD1/2 and vesicular GABA transporter, VGAT) co-expressed with the DA neurons, which is consistent with previous studies that VTA DA neurons co-expressing GABAergic neuronal markers mainly project to the lateral habenula") is a little confusing - as stated, it seems that the authors are confirming DA/GABA coexpression in VTA-LHb neurons, which is not the case.

4. Additional information could be included in the Methods section description of Western Blotting procedures - e.g., what thickness of tissue and what size gauge were used to dissect VTA for these experiments?

Reviewer #2 (Public Review):

The manuscript investigates the function of basal forebrain cholinergic axons in mouse primary visual cortex (V1) during locomotion using two-photon calcium imaging in head-fixed mice. Cholinergic modulation has previously been proposed to mediate the effects of locomotion on V1 responses. The manuscript concludes that the activity of basal forebrain cholinergic axons in visual cortex provides a signal which is more correlated with binary locomotion state than locomotion velocity of the animal. Cholinergic axons did not seem to respond to grating stimuli or visuomotor prediction error. Optogenetic stimulation of these axons increased the amplitude of responses to visual stimuli and decreased the response latency of layer 5 excitatory neurons, but not layer 2/3 neurons. Moreover, optogenetic or chemogenetic stimulation of cholinergic inputs reduced pairwise correlation of neuronal responses. These results provide insight into the role of cholinergic modulation to visual cortex and demonstrate that it affects different layers of visual cortex in a distinct manner. The experiments are well executed and the data appear to be of high quality. However, further analyses are required to fully support some of the study's conclusions.

The manuscript concludes that cholinergic axons convey a binary locomotion signal and are not tuned to running speed. Getting head-fixed animals to run at the speeds typical of freely moving animals can require training, which was not undertaken in this study. Consequently, the typically low running velocity of mice is a potential limitation of this study.

The analyses of the effects of locomotion and stimulation of cholinergic inputs present grand averages of responses across all neurons, and therefore may mask heterogeneity across layer 2/3 and layer 5 neurons.

Reviewer #2 (Public Review):

The authors phototag DA and GABA neurons in the VTA in mice performing a t-maze task, and report choice-specific responses in the delay period of a memory-guided task, more so that in a variant task w/o a memory component. Overall, I found the results convincing. While showing responses that are choice selective in DA neurons is not entirely novel (e.g. Morris et al NN 2006, Parker et al NN 2016), the fact that this feature is stronger when there is a memory requirement is an interesting and a novel observation.

Reviewer #2 (Public Review):

In this manuscript, Yao et al. present a series of experiments aiming at generating a cellular atlas of the human hippocampus across aging, and how it may be affected by injury, in particular, stroke. Although the aim of the study is interesting and relevant for a larger audience, due to the ongoing controversy around the existence of adult hippocampal neurogenesis in humans, a number or technical weaknesses result in a poor support for many of the conclusions made from the results of these experiments.<br /> In particular, a recent meta analysis of five previous studies applying similar techniques to human samples has identified different aspects of sample size as main determinants of the statistical power needed to make significant conclusions. Some of this aspects are the number of nuclei sequenced and subject stratification. These two aspects are of concern in Yao's study. First, the number of sequenced nuclei is lower than the calculated numbers of nuclei required for detecting rare cell types. However, Yao et al. report succeeding in detecting rare populations, including several types of neural stem cells in different proliferation states, which have been demonstrated to be extremely scarce by previous studies. It would be very interesting to read how the authors interpret these differences. Secondly, the number of donors included in some of the groups is extremely low (n=1) and the miscellaneous information provided about the donors is practically inexistent. As individual factors such as chronic conditions, medication, lifestyle parameters, etc... are considered determinant for the variability of adult hippocampal neurogenesis levels across individuals, this represents a series limitation of the current study. Overall, several technical weaknesses severely limit the relevance of this study and the ability of the authors to achieve their experimental aims.

After a first review round, the manuscript is still lacking a clear discussion of its several technical limitations, which will help the audience to grasp the relevance of the findings. In particular, detailed information about individual patients health status and relevant lifestyle parameters that may have affected it is lacking. The authors make the point themselves that the discrepancies among studies might be caused by health state differences across hippocampi, which subsequently lead to different degrees of hippocampal neurogenesis.". So, even in the authors own interpretation this is a serious limitation to the manuscript, that however out of the authors control, impacts on the quality of their findings.

Reviewer #2 (Public Review):

Summary:

This study aims to test auditory confounds during transcranial ultrasound stimulation (TUS) protocols that rely on audible frequencies. In several experiments, the authors show that a commonly observed suppression of motor-evoked potentials (MEP) during TUS can be explained by acoustic stimulation. For instance, not only target TUS, but also stimulation of a control site and acoustic stimulation led to suppressed MEP.

The authors have convincingly addressed all of my comments and provided useful additional details. I believe that this is a strong study that will impact the field. Thanks also for making the sound stimuli open-source.

Reviewer #2 (Public Review):

The authors describe what they assert to be a very unusual trigeminal nuclear complex in the brainstem of elephants, and based on this, follow with many speculations about how the trigeminal nuclear complex, as identified by them, might be organized in terms of the sensory capacity of the elephant trunk.

The identification of the trigeminal nuclear complex/inferior olivary nuclear complex in the elephant brainstem is the central pillar of this manuscript from which everything else follows, and if this is incorrect, then the entire manuscript fails, and all the associated speculations become completely unsupported.

The authors note that what they identify as the trigeminal nuclear complex has been identified as the inferior olivary nuclear complex by other authors, citing Shoshani et al. (2006; 10.1016/j.brainresbull.2006.03.016) and Maseko et al (2013; 10.1159/000352004), but fail to cite either Verhaart and Kramer (1958; PMID 13841799) or Verhaart (1962; 10.1515/9783112519882-001). These four studies are in agreement, but the current study differs.

Let's assume for the moment that the four previous studies are all incorrect and the current study is correct. This would mean that the entire architecture and organization of the elephant brainstem is significantly rearranged in comparison to ALL other mammals, including humans, previously studied (e.g. Kappers et al. 1965, The Comparative Anatomy of the Nervous System of Vertebrates, Including Man, Volume 1 pp. 668-695) and the closely related manatee (10.1002/ar.20573). This rearrangement necessitates that the trigeminal nuclei would have had to "migrate" and shorten rostrocaudally, specifically and only, from the lateral aspect of the brainstem where these nuclei extend from the pons through to the cervical spinal cord (e.g. the Paxinos and Watson rat brain atlases), the to the spatially restricted ventromedial region of specifically and only the rostral medulla oblongata. According to the current paper, the inferior olivary complex of the elephant is very small and located lateral to their trigeminal nuclear complex, and the region from where the trigeminal nuclei are located by others appears to be just "lateral nuclei" with no suggestion of what might be there instead.

Such an extraordinary rearrangement of brainstem nuclei would require a major transformation in the manner in which the mutations, patterning, and expression of genes and associated molecules during development occur. Such a major change is likely to lead to lethal phenotypes, making such a transformation extremely unlikely. Variations in mammalian brainstem anatomy are most commonly associated with quantitative changes rather than qualitative changes (10.1016/B978-0-12-804042-3.00045-2).

The impetus for the identification of the unusual brainstem trigeminal nuclei in the current study rests upon a previous study from the same laboratory (10.1016/j.cub.2021.12.051) that estimated that the number of axons contained in the infraorbital branch of the trigeminal nerve that innervate the sensory surfaces of the trunk is approximately 400 000. Is this number unusual? In a much smaller mammal with a highly specialized trigeminal system, the platypus, the number of axons innervating the sensory surface of the platypus bill skin comes to 1 344 000 (10.1159/000113185). Yet, there is no complex rearrangement of the brainstem trigeminal nuclei in the brain of the developing or adult platypus (Ashwell, 2013, Neurobiology of Monotremes), despite the brainstem trigeminal nuclei being very large in the platypus (10.1159/000067195). Even in other large-brained mammals, such as large whales that do not have a trunk, the number of axons in the trigeminal nerve ranges between 400,000 and 500,000 (10.1007/978-3-319-47829-6_988-1). The lack of comparative support for the argument forwarded in the previous and current study from this laboratory, and that the comparative data indicates that the brainstem nuclei do not change in the manner suggested in the elephant, argues against the identification of the trigeminal nuclei as outlined in the current study. Moreover, the comparative studies undermine the prior claim of the authors, informing the current study, that "the elephant trigeminal ganglion ... point to a high degree of tactile specialization in elephants" (10.1016/j.cub.2021.12.051). While clearly, the elephant has tactile sensitivity in the trunk, it is questionable as to whether what has been observed in elephants is indeed "truly extraordinary".

But let's look more specifically at the justification outlined in the current study to support their identification of the unusually located trigeminal sensory nuclei of the brainstem.

(1) Intense cytochrome oxidase reactivity.<br /> (2) Large size of the putative trunk module.<br /> (3) Elongation of the putative trunk module.<br /> (4) The arrangement of these putative modules corresponds to elephant head anatomy.<br /> (5) Myelin stripes within the putative trunk module that apparently match trunk folds.<br /> (6) Location apparently matches other mammals.<br /> (7) Repetitive modular organization apparently similar to other mammals.<br /> (8) The inferior olive described by other authors lacks the lamellated appearance of this structure in other mammals.

Let's examine these justifications more closely.

(1) Cytochrome oxidase histochemistry is typically used as an indicative marker of neuronal energy metabolism. The authors indicate, based on the "truly extraordinary" somatosensory capacities of the elephant trunk, that any nuclei processing this tactile information should be highly metabolically active, and thus should react intensely when stained for cytochrome oxidase. We are told in the methods section that the protocols used are described by Purkart et al (2022) and Kaufmann et al (2022). In neither of these cited papers is there any description, nor mention, of the cytochrome oxidase histochemistry methodology, thus we have no idea of how this histochemical staining was done. To obtain the best results for cytochrome oxidase histochemistry, the tissue is either processed very rapidly after buffer perfusion to remove blood or in recently perfusion-fixed tissue (e.g., 10.1016/0165-0270(93)90122-8). Given: (1) the presumably long post-mortem interval between death and fixation - "it often takes days to dissect elephants"; (2) subsequent fixation of the brains in 4% paraformaldehyde for "several weeks"; (3) The intense cytochrome oxidase reactivity in the inferior olivary complex of the laboratory rat (Gonzalez-Lima, 1998, Cytochrome oxidase in neuronal metabolism and Alzheimer's diseases); and (4) The lack of any comparative images from other stained portions of the elephant brainstem; it is difficult to support the justification as forwarded by the authors. The histochemical staining observed is likely background reactivity from the use of diaminobenzidine in the staining protocol. Thus, this first justification is unsupported.

Justifications (2), (3), and (4) are sequelae from justification (1). In this sense, they do not count as justifications, but rather unsupported extensions.

(4) and (5) These are interesting justifications, as the paper has clear internal contradictions, and (5) is a sequelae of (4). The reader is led to the concept that the myelin tracts divide the nuclei into sub-modules that match the folding of the skin on the elephant trunk. One would then readily presume that these myelin tracts are in the incoming sensory axons from the trigeminal nerve. However, the authors note that this is not the case: "Our observations on trunk module myelin stripes are at odds with this view of myelin. Specifically, myelin stripes show no tapering (which we would expect if axons divert off into the tissue). More than that, there is no correlation between myelin stripe thickness (which presumably correlates with axon numbers) and trigeminal module neuron numbers. Thus, there are numerous myelinated axons, where we observe few or no trigeminal neurons. These observations are incompatible with the idea that myelin stripes form an axonal 'supply' system or that their prime function is to connect neurons. What do myelin stripe axons do, if they do not connect neurons? We suggest that myelin stripes serve to separate rather than connect neurons." So, we are left with the observation that the myelin stripes do not pass afferent trigeminal sensory information from the "truly extraordinary" trunk skin somatic sensory system, and rather function as units that separate neurons - but to what end? It appears that the myelin stripes are more likely to be efferent axonal bundles leaving the nuclei (to form the olivocerebellar tract). This justification is unsupported.

(6) The authors indicate that the location of these nuclei matches that of the trigeminal nuclei in other mammals. This is not supported in any way. In ALL other mammals in which the trigeminal nuclei of the brainstem have been reported they are found in the lateral aspect of the brainstem, bordered laterally by the spinal trigeminal tract. This is most readily seen and accessible in the Paxinos and Watson rat brain atlases. The authors indicate that the trigeminal nuclei are medial to the facial nerve nucleus, but in every other species, the trigeminal sensory nuclei are found lateral to the facial nerve nucleus. This is most salient when examining a close relative, the manatee (10.1002/ar.20573), where the location of the inferior olive and the trigeminal nuclei matches that described by Maseko et al (2013) for the African elephant. This justification is not supported.

(7) The dual to quadruple repetition of rostrocaudal modules within the putative trigeminal nucleus as identified by the authors relies on the fact that in the neurotypical mammal, there are several trigeminal sensory nuclei arranged in a column running from the pons to the cervical spinal cord, these include (nomenclature from Paxinos and Watson in roughly rostral to caudal order) the Pr5VL, Pr5DM, Sp5O, Sp5I, and Sp5C. However, these nuclei are all located far from the midline and lateral to the facial nerve nucleus, unlike what the authors describe in the elephants. These rostrocaudal modules are expanded upon in Figure 2, and it is apparent from what is shown that the authors are attributing other brainstem nuclei to the putative trigeminal nuclei to confirm their conclusion. For example, what they identify as the inferior olive in Figure 2D is likely the lateral reticular nucleus as identified by Maseko et al (2013). This justification is not supported.

(8) In primates and related species, there is a distinct banded appearance of the inferior olive, but what has been termed the inferior olive in the elephant by other authors does not have this appearance, rather, and specifically, the largest nuclear mass in the region (termed the principal nucleus of the inferior olive by Maseko et al, 2013, but Pr5, the principal trigeminal nucleus in the current paper) overshadows the partial banded appearance of the remaining nuclei in the region (but also drawn by the authors of the current paper). Thus, what is at debate here is whether the principal nucleus of the inferior olive can take on a nuclear shape rather than evince a banded appearance. The authors of this paper use this variance as justification that this cluster of nuclei could not possibly be the inferior olive. Such a "semi-nuclear/banded" arrangement of the inferior olive is seen in, for example, giraffe (10.1016/j.jchemneu.2007.05.003), domestic dog, polar bear, and most specifically the manatee (a close relative of the elephant) (brainmuseum.org; 10.1002/ar.20573). This justification is not supported.

Thus, all the justifications forwarded by the authors are unsupported. Based on methodological concerns, prior comparative mammalian neuroanatomy, and prior studies in the elephant and closely related species, the authors fail to support their notion that what was previously termed the inferior olive in the elephant is actually the trigeminal sensory nuclei. Given this failure, the justifications provided above that are sequelae also fail. In this sense, the entire manuscript and all the sequelae are not supported.

What the authors have not done is to trace the pathway of the large trigeminal nerve in the elephant brainstem, as was done by Maseko et al (2013), which clearly shows the internal pathways of this nerve, from the branch that leads to the fifth mesencephalic nucleus adjacent to the periventricular grey matter, through to the spinal trigeminal tract that extends from the pons to the spinal cord in a manner very similar to all other mammals. Nor have they shown how the supposed trigeminal information reaches the putative trigeminal nuclei in the ventromedial rostral medulla oblongata. These are but two examples of many specific lines of evidence that would be required to support their conclusions. Clearly, tract tracing methods, such as cholera toxin tracing of peripheral nerves cannot be done in elephants, thus the neuroanatomy must be done properly and with attention to detail to support the major changes indicated by the authors.

So what are these "bumps" in the elephant brainstem?

Four previous authors indicate that these bumps are the inferior olivary nuclear complex. Can this be supported?

The inferior olivary nuclear complex acts "as a relay station between the spinal cord (n.b. trigeminal input does reach the spinal cord via the spinal trigeminal tract) and the cerebellum, integrating motor and sensory information to provide feedback and training to cerebellar neurons" (https://www.ncbi.nlm.nih.gov/books/NBK542242/). The inferior olivary nuclear complex is located dorsal and medial to the pyramidal tracts (which were not labelled in the current study by the authors but are clearly present in Fig. 1C and 2A) in the ventromedial aspect of the rostral medulla oblongata. This is precisely where previous authors have identified the inferior olivary nuclear complex and what the current authors assign to their putative trigeminal nuclei. The neurons of the inferior olivary nuclei project, via the olivocerebellar tract to the cerebellum to terminate in the climbing fibres of the cerebellar cortex.

Elephants have the largest (relative and absolute) cerebellum of all mammals (10.1002/ar.22425), this cerebellum contains 257 x109 neurons (10.3389/fnana.2014.00046; three times more than the entire human brain, 10.3389/neuro.09.031.2009). Each of these neurons appears to be more structurally complex than the homologous neurons in other mammals (10.1159/000345565; 10.1007/s00429-010-0288-3). In the African elephant, the neurons of the inferior olivary nuclear complex are described by Maseko et al (2013) as being both calbindin and calretinin immunoreactive. Climbing fibres in the cerebellar cortex of the African elephant are clearly calretinin immunopositive and also are likely to contain calbindin (10.1159/000345565). Given this, would it be surprising that the inferior olivary nuclear complex of the elephant is enlarged enough to create a very distinct bump in exactly the same place where these nuclei are identified in other mammals?

What about the myelin stripes? These are most likely to be the origin of the olivocerebellar tract and probably only have a coincidental relationship with the trunk. Thus, given what we know, the inferior olivary nuclear complex as described in other studies, and the putative trigeminal nuclear complex as described in the current study, is the elephant inferior olivary nuclear complex. It is not what the authors believe it to be, and they do not provide any evidence that discounts the previous studies. The authors are quite simply put, wrong. All the speculations that flow from this major neuroanatomical error are therefore science fiction rather than useful additions to the scientific literature.

What do the authors actually have?<br /> The authors have interesting data, based on their Golgi staining and analysis, of the inferior olivary nuclear complex in the elephant.

Reviewer #2 (Public Review):

Summary:<br /> The authors used a short hairpin RNA technique strategy to elucidate the functional activity of neurons in the retrotrapezoid nucleus (RTN), a critical brainstem region for central chemoreception. Dysfunction in this area is associated with the neuropathology of congenital central hypoventilation syndrome (CCHS). The subsequent examination of these rats aimed to shed light on the intricate aspects of RTN and its implications for central chemoreception and disorders like CCHS in adults. They found that using the short hairpin RNA (shRNA) targeting Phox2b mRNA, a reduction of Phox2b expression was observed in Nmb neurons. In addition, Phox2b knockdown did not affect breathing in room air or under hypoxia, but the hypercapnia ventilatory response was significantly impaired. They concluded that Phox2b in the adult brain has an important role in CO2 chemoreception. They thought that their findings provided new evidence for mechanisms related to CCHS neuropathology. The conclusions of this paper are well supported by data, but careful discussion seems to be required for comparison with the results of various previous studies performed by different genetic strategies for the RTN neurons.

Strengths:<br /> The most exciting aspect of this work is the modelling of the Phox2b knockdown in one element of the central neuronal circuit mediating respiratory reflexes, that is in the RTN. To date, mutations in the PHOX2B gene are commonly associated with most patients diagnosed with CCHS, a disease characterized by hypoventilation and absence of chemoreflexes, in the neonatal period, which in severe cases can lead to respiratory arrest during sleep. In the present study, the authors demonstrated that the role of Phox2b extends beyond the developmental period, and its reduction in CCHS may contribute to the respiratory impairment observed in this disorder.

Weaknesses:<br /> Whereas the most exciting part of this work is the knockdown of the Phox2b in the RTN in adult rodents, the weakness of this study is the lack of a clear physiological, developmental, and anatomical distinction between this approach and similar studies already reported elsewhere (Ruffault et al., 2015, DOI: 10.7554/eLife.07051; Ramanantsoa et al., 2011, DOI: 10.1523/JNEUROSCI.1721-11.2011; Huang et al., 2017, DOI: 10.1016/j.neuron.2012.06.027; Hernandez-Miranda et al., 2018, DOI: 10.1073/pnas.1813520115; Ferreira et al., 2022 DOI: 10.7554/eLife.73130; Takakura et al., 2008 DOI: 10.1113/jphysiol.2008.153163; Basting et al., 2015 DOI: 10.1523/JNEUROSCI.2923-14.2015; Marina et al., 2010 DOI: 10.1523/JNEUROSCI.3141-10.2010). In addition, several conclusions presented in this work are not directly supported by the provided data.

Reviewer #2 (Public Review):

Summary: The study by Cullen et al presents intriguing data regarding the contribution of mTOR complex 1 (mTORC1) versus mTORC2 or both in Pten-null induced macrocephaly and epileptiform activity. The role of mTORC2 in mTORopathies, and in particular Pten loss-off-function (LOF)-induced pathology and seizures, is understudied and controversial. In addition, recent data provided evidence against the role of mTORC1 in PtenLOF-induced seizures. To address these controversies and the contribution off these mTOR complexes in PtenLOF-induced pathology and seizures, the authors injected a AAV9-Cre into the cortex of conditional single, double and triple transgenic mice at postnatal day 0 to remove Pten, Pten+Raptor or Rictor, and Pten+raptor+rictor. Raptor and Rictor are essentially binding partners of mTORC1 and mTORC2, respectively. One major finding is that despite preventing the mild macrocephaly and increased cell size, Raptor knockout (KO, decrease mTORC1 activity) did not prevent the occurrence of seizures and the rate of SWD event, and aggravated seizure duration. Similarly, Rictor KO (decreased mTORC2 activity) partially prevented the mild macrocephaly and increased cell size but did not prevent the occurrence of seizures and did not affect seizure duration. However, Rictor KO reduced the rate of SWD events. Finally, the pathology and seizure/SWD activity were fully prevented in the double KO. These data suggest the contribution of both increased mTORC1 and mTORC2 in the pathology and epileptic activity of Pten LOF mice, emphasizing the importance of blocking both complexes for seizure treatment. Whether these data apply to other mTORopathies due to Tsc1, Tsc2, mTOR, AKT or other gene variants remain to be examined.

Strengths: The strengths are as follow: 1) they address an important and controversial question that has clinical application, 2) the study uses a reliable and relatively easy method to KO specific genes in cortical neurons, based on AAV9 injections in pups. 2) they perform careful video-EEG analyses correlated with some aspects of cellular pathology.

Weaknesses: the study has nevertheless a few weaknesses: 1) the conclusions are perhaps a bit overstated. The data do not show that increased mTORC1 or mTORC2 are sufficient to cause epilepsy. But the data clearly show that both increased mTORC1 and mTORC2 activity contribute to the pathology and seizure activity and as such are necessary for seizures to occur. 2) the data related to the EEG would benefit from having more mice. Adding more mice would have help determine whether there is a decrease in seizure activity with the Rictor or Raptor KO. 3) it would have been interesting to examine the impact of mTORC2 and mTORC1 overexpression related to point #1 above.

The authors properly addressed my comments. Number 3 above was only a suggestion that could be a follow-up in another study.

Reviewer #2 (Public Review):

Summary:<br /> The idea that various clinical conditions may be associated, at least partially, with a disrupted corollary discharge mechanism has been present for a long time.

In this paper, the authors draw a link between sensory overload, a characteristic of autism spectrum disorder, and a disturbance in the corollary discharge mechanism. The authors substantiate their hypothesis with strong evidence from both the motor and perceptual domains. As a result, they broaden the clinical relevance of the corollary discharge mechanism to encompass autism spectrum disorder.

The authors write:<br /> "Imagine a scenario in which you're watching a video of a fast-moving car on a bumpy road. As the car hits a pothole, your eyes naturally make quick, involuntary saccades to keep the car in your visual field. Without a functional efference copy system, your brain would have difficulty accurately determining the current position of your eye in space, which in turn affects its ability to anticipate where the car should appear after each eye movement."

I appreciate the use of examples to clarify the concept of efference copy. However, I believe this example is more related to a gain-field mechanism, informing the system about the position of the eye with respect to the head, rather than an example of efference copy per se.

Without an efference copy mechanism, the brain would have trouble accurately determining where the eyes will be in space after an eye movement, and it will have trouble predicting the sensory consequences of the eye movement. However it can be argued that the gain-field mechanism would be sufficient to inform the brain about the current position of the eyes with respect to the head.

The authors write:<br /> "In the double-step paradigm, two consecutive saccades are made to briefly displayed targets 21, 22. The first saccade occurs without visual references, relying on internal updating to determine the eye's position."

Maybe I have missed something, but in the double-step paradigm the first saccade can occur without the help of visual references if no visual feedback is present, that is, when saccades are performed in total darkness. Was this the case for this experiment? I could not find details about room conditions in the methods. Please provide further details.

In case saccades were not performed in total darkness, then the first saccade can be based on the remembered location of the first target presented, which can be derived from the retinotopic trace of the first stimuli, as well as the contribution from the surroundings, that is: the remembered relative location of the first target with respect to the screen border along the horizontal meridian (i.e. allocentric cues).

A similar logic could be applied to the second saccade. If the second saccade were based only on the retinotopic trace, without updating, then it would go up and 45 deg to the right, based on the example shown in Figure 1. With appropriate updating, the second saccade would go straight up. However, if saccades were not performed in total darkness, then the location of the second target could also be derived from its relationship with the surroundings (for example, the remembered distance from screen borders, i.e. allocentric cues).

If saccades were not performed in total darkness, the results shown in Figures 2 and 3 could then be related to i) differences in motor updating between AQ score groups; ii) differences in the use of allocentric cues between AQ score groups; iii) a combination of i) and ii). I believe this is a point worth mentioning in the discussion."

The authors write:<br /> "According to theories of saccadic suppression, an efference copy is necessary to predict the occurrence of a saccade."

I would also refer to alternative accounts, where saccadic suppression appears to arise as early as the retina, due to the interaction between the visual shift introduced by the eye movement, and the retinal signal associated with the probe used to measure saccadic suppression. This could potentially account for the scaling of saccadic suppression magnitude with saccade amplitude.

Idrees, S., Baumann, M.P., Franke, F., Münch, T.A. and Hafed, Z.M., 2020. Perceptual saccadic suppression starts in the retina. Nature communications, 11(1), p.1977.

Reviewer #2 (Public Review):

The authors repeated a previous behavioural study on the effects of overnight fasting on avoidance and extinction learning in healthy female participants in the 3T MRI scanner. Previous behavioural findings were replicated only in part. Fasting related changes of fMRI signals were less than expected.

This paper is not without interest. Anxiety disorders are very frequent, and there is still a need to better understand ways to improve extinction and reduced avoidance. The authors follow up on previous observations of their group using overnight fasting. The findings, however, were largely negative, and it is difficult to tell how robust the observed positive findings are. The paradigm did not work as well as expected in the MR scanner.

Introduction/main hypothesis: The reviewer does not understand why a smaller reward prediction error should result in faster extinction learning? The opposite should be the case. Plus, how much of a reward prediction error is expected in the CS- condition in extinction training? Here the US omission is expected. The reviewer may miss a key concept of the study.

Results: A major part of the behavioural data of a previous pure behavioural study was not reproduced (avoidance learning), plus many of the MRI findings did not show a difference between the fasting and re-feed groups. Given the large amount of comparisons it makes one wonder how robust the presented findings are. The advances to the field are therefore limited.

Reviewer #2 (Public Review):

This short manuscript by Zhu et al. describes an investigation into the role of gamma protocadherins in synaptic connectivity in the mouse cerebral cortex. First, the authors conduct a single-cell RNA-seq survey of postnatal day 11 mouse cortical neurons, using an adapted 10X Genomics method to capture the 5' sequences that are necessary to identify individual gamma protocadherin isoforms (all 22 transcripts share the same three 3' "constant" exons, so standard 3'-biased methods can't distinguish them). This method adaptation is an advance for examining individual clustered protocadherin transcripts, and it is helpful to publish the method in this manuscript. The results largely confirm what was known from other approaches, which is that a few of the 19 A and B subtype gamma protocadherins are expressed in an apparently stochastic and combinatorial fashion in each cortical neuron, while the 3 C subtype genes are expressed by most neurons. Second, using elegant paired electrophysiological recordings, the authors show that in gamma protocadherin knockout cortical slices, the likelihood of two neurons on layers 2/3 being synaptically connected is increased. That suggests that gamma protocadherins generally inhibit synaptic connectivity in the cortex; again, this has been reported previously using morphological assays, but it is helpful to see it confirmed here with physiology. Finally, the authors use an impressive sequential in utero electroporation method to provide evidence that the degree of isoform matching between two neurons negatively regulates their reciprocal synaptic connectivity. These are difficult experiments to do, and while some caveats remain (e.g., lack of demonstration of protein levels in electroporated neurons, lack of resolution of the differences between the present results and those of other papers, a focus on C4 rather than C3 or C5 when considering the highly expressed C-type isoforms), the main result is consistent. Strengths of this manuscript include the impressive methodology and improved demonstration of the previously-reported finding that gamma protocadherins work via homophilic matching to put a brake on synapse formation in the cortex. Because of the unique organization and expression pattern of the gamma protocadherins, it is not likely that these results will be broadly applicable to the general understanding of the role of cell adhesion molecules in synapse development. However, the methodology, which is now better described, should be applicable more broadly and the improved demonstration of the role of gamma protocadherin's negative role in cortical synaptogenesis is helpful in confirming earlier studies. There are several differences between the results here and those of other papers on the cortex, as well as those examining other neuronal populations such as spinal cord. The present findings do not resolve them, but adopting genetic approaches rather than overexpression in the future should help.

Reviewer #2 (Public Review):

Summary:

We often have prior expectations about how the sensory world will change, but it remains an open question as to how these expectations are integrated into perceptual decisions. In particular, scientists have debated whether prior knowledge principally changes the decisions we make about the perceptual world, or directly alters our perceptual encoding of incoming sensory evidence.

The authors aimed to shed light on this conundrum by using a novel psychophysical task while measuring EEG signals that have previously been linked to either the sensory encoding or response selection phase of perceptual choice. The results convincingly demonstrate that both features of perceptual decision making are modulated by prior expectations - but that these biases in neural process emerge over different time courses (i.e., decisional signals are shaped early in learning, but biases in sensory processing are slower to emerge).

Another interesting observation unearthed in the study - though not strictly linked to this perceptual/decisional puzzle - is that neural signatures of focused attention are exaggerated on trials where participants are given neutral (i.e. uninformative) cues. This is consistent with the idea that observers are more attentive to incoming sensory evidence when they cannot rely on their expectations.

In general, I think the study makes a strong contribution to the literature, and does an excellent job of separating 'perceiving' from 'responding'. More perhaps could have been done though to separate 'perceiving' and 'responding' from 'deciding' (see below).

Strengths:

The work is executed expertly and focuses cleverly on two features of the EEG signals that can be closely connected to specific loci of the perceptual decision making process - the SSVEP which connects closely to sensory (visual) encoding, and Mu-Beta lateralisation which connects closely to movement preparation. This is a very appropriate design choice given the authors' research question.

Another advantage of the design is the use of an unusually long training regime (i.e., for humans) - which makes it possible to probe the emergence of different expectation biases in the brain over different timecourses, and in a way that may be more comparable to work with nonhuman animals (who are routinely trained for much longer than humans).

Weaknesses:

In my view, the principal shortcoming of this study is that the experimental task confounds expectations about stimulus identity with expectations about to-be-performed responses. That is, cues in the task don't just tell participants what they will (probably) see, but what they (probably) should do.

In many respects, this feature of the paradigm might seem inevitable, as if specific stimuli are not connected to specific responses, it is not possible to observe motor preparation of this kind (e.g., de Lange, Rahnev, Donner & Lau, 2013 - JoN).

However, the theoretical models that the authors focus on (e.g., drift diffusion models) are models of decision (i.e., commitment to a proposition about the world) as much as they are models of choice (i.e., commitment to action). Expectation researchers interested in these models are often interested in asking whether predictions influence perceptual processing, perceptual decision and/or response selection stages (e.g., Feuerriegel, Blom & Hoogendorn, 2021 - Cortex), and other researchers have shown that parameters like drift bias and start point bias can be shifted in paradigms where observers cannot possibly prepare a response (e.g., Thomas, Yon, de Lange & Press, 2020 - Psych Sci).

The present paradigm used by Walsh et al makes it possible to disentangle sensory processing from later decisional processes, but it blurs together the processes of deciding about the stimulus and choosing/initiating the response. This ultimately limits the insights we can draw from this study - as it remains unclear whether rapid changes in motor preparation we see reflect rapid acquisition of new decision criterion or simple cue-action learning. I think this would be important for comprehensively testing the models the authors target - and a good avenue for future work.

In revising the manuscript after an initial round of revisions, the authors have done a good job of acknowledging these complexities - and I don't think that any of these outstanding scientific puzzles detract from the value of the paper as a whole.

Reviewer #2 (Public Review):

Summary:<br /> This study examined the longitudinal brain-behaviour link between attentional neural filtering and listening behaviour among a sample of aging individuals. The results based on the latent change score modeling showed that neither attentional neural filtering at T1 nor its T1-T2 change predicted individual two-year listening performance change. The findings suggest that neural filtering and listening behaviour may follow independent developmental trajectories. This study focuses on an interesting topic and has the potential to contribute a better understanding of the neurobiological mechanisms of successful communication across the lifespan.

Strengths:<br /> Although research suggests that speech comprehension is neurally supported by an attention-guided filter mechanism, the evidence on their causal association is limited. This study addresses this gap by testing longitudinal stability of neural filtering as a neural mechanism upholding listening performance, potentially shedding lights on translational efforts aiming at the preservation of speech comprehension abilities among aging individuals.

The latent change score modeling approach is appropriately used as a tool to examine key developmental questions and distinguish the complex processes underlying lifespan development in brain and behaviour with longitudinal data.

Weaknesses:<br /> Although the paper does have strengths in principle, the weaknesses of the paper are that the findings are merely based on a single listening task. Since both neural and behavioral indicators are derived from the same task, the results may be applicable only to this specific task, and it is difficult to extrapolate them to cognitive and listening abilities measured by the other tasks. Therefore, more listening tasks are required to comprehensively measure speech comprehension and neural markers.

The age span of the sample is relatively large. Although no longitudinal change from T1 to T2 was found at the group-level, from the cross-sectional and longitudinal change results (see Figure 3), individuals of different age groups showed different development pattern. Particularly, individuals over the age of 70 show a clear downward trend in both neural filtering index and accuracy. Therefore, different results may be found based on different age groups, especially older groups. However, due to sample limitations, this study was unable to examine whether age has a moderating effect on this brain-behaviour link.

In the Dichotic listening task, valid and invalid cues were manipulated. According to the task description, the former could invoke selective attention, whereas the latter could invoke divided attention. It is possible that under the two conditions, the neural filtering index may reflect different underlying cognitive processes, and thus may differ in its predictive effect on behavioral performance. The author could perform a more in-depth data analysis on indicators under different conditions.

Reviewer #2 (Public Review):

Summary: This paper aims to achieve a better understanding of how the antigenic or genetic compositions of the dominant influenza A viruses in circulation at a given time are related to key features of seasonal influenza epidemics in the US. To this end, the authors analyse an extensive dataset with a range of statistical, data science and machine learning methods. They find that the key drivers of influenza A epidemiological dynamics are interference between influenza A subtypes and genetic divergence, relative to the previous one or two seasons, in a broader range of antigenically related sites than previously thought.

Strengths: A thorough investigation of a large and complex dataset.

Weaknesses: The dataset covers a 21 year period which is substantial by epidemiological standards, but quite small from a statistical or machine learning perspective. In particular, it was not possible to follow the usual process and test predictive performance of the random forest model with an independent dataset.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors introduced ADSE, a SELEX-based protocol to explore the mechanism of emergency of species. They used DNA hybridization (to the bait pool, "resources") as the driving force for selection and quantitatively investigated the factors that may contribute to the survival during generation evolve (progress of SELEX cycle), revealing that besides individual-resource binding, the inter- and intra-individual interactions were also important features along with mutualism and parasitism.

Strengths:<br /> The design of using pure biochemical affinity assay to study eco-evolution is interesting, providing an important viewpoint to partly explain the molecular mechanism of evolution.

Reviewer #3 (Public Review):

The main findings of this study are as follows: (1) The authors defined "metabolism-type" and "kinase-type" in unclassified sporadic PCC patients through the single-cell transcriptomics-based differentially expressed genes and functional enrichment analyses. (2) They identified the limitation of Pheochromocytoma of the Adrenal gland Scaled Score (PASS) system and suggested the combination of molecular diagnostic methods like scRNA-seq with pathological tools like PASS in aiding the clinical evaluation of PCCs. (3) Analysis of the PCC microenvironment revealed a lack of immune cell infiltration in both metabolism-type and kinase-type PCCs, while only the kinase-type PCC patient exhibited the low expression of HLA-Ⅰ that potentially regulated by RET, providing clues for the combined therapy with kinase inhibitors and immunotherapy in kinase-type PCC patients.

The main strength of this manuscript is that it involves scRNA-seq analysis of an extremely rare tumor type-PCCs, which presents a single-cell transcriptomics-based molecular classification and microenvironment characterization of PCCs and further provides clues for potential therapeutic strategies to treat PCCs. The authors also validated the scRNA-seq analysis results (such as the expression levels of marker genes and the distribution of immune cells in the PCC microenvironment) with immunocytochemistry and multispectral immunofluorescent staining. In summary, the findings in this manuscript are quite interesting and significant, which will potentially be valuable for the molecular classification of PCCs.

Reviewer #2 (Public Review):

The manuscript of "IQCH regulates spermatogenesis by interacting with CaM to promote RNA-binding proteins' expression" from Ruan et al. identified a homozygous variant affect the splicing of IQCH in two infertile men from a Chinese family. The authors also generated a Iqch knockout mouse model to confirm the abnormal sperm phenotypes associated with IQCH deficiency. Further molecular biological assays supported the important role and mechanism of IQCH in spermatogenesis. This manuscript is informative for the clinical and basic research of male infertility.

Reviewer #2 (Public Review):

The authors provide a compelling data to demonstrate that the Notch-related transcription factor RBP-J can influence the number of circulating and recruited monocytes. The authors first delete the Rbpj gene in the myeloid lineage (Lyz2) and show that, as a proportion, only Ly6Clo monocytes are increased in the blood. The authors then attempted to identify why these cells were increased in proportion but ruled out proliferation or reduced apoptosis. Next, they investigated the gene signature of Rbpj null monocytes using RNA-sequencing and identified elevated Ccr2 as a defining feature. Crossing the Rbpj null mice to Ccr2 null mice showed reduced numbers of Ly6Clo monocytes compared with Rbpj null alone. Finally, the authors identify that an increased burden of blood Ly6Clo monocytes is correlated with increased lung recruitment and expansion of lung interstitial macrophages.

The main conclusion of the authors, that there is a 'cell intrinsic requirement of RBP-J for controlling blood Ly6CloCCR2hi monocytes' is strongly supported by the data. However, other claims and aspects of the study require clarification and further analysis of the data generated.

Strengths<br /> The paper is well written and structured logically. The major strength of this study is the multiple technically challenging methods used to reinforce the main finding (e.g. parabiosis, adoptive transfer). The finding reinforces the fact that we still know little about how immune cell subsets are maintained in situ, and this study opens the way for novel future work. Importantly, the authors have generated an RNA-sequencing dataset that will prove invaluable for identifying the mechanism - they have promised public access to this data via GEO - it is expected this will be made accessible upon publication.

Weaknesses - The main weakness of the study, is that although the main result is solidly supported, as written it is mostly descriptive in nature. For instance, there is no given mechanism by which RBP-J increases Ly6Clo monocytes. The authors conclude this is dependent on CCR2, however CCR2 deletion has a global effect on monocyte numbers and importantly in this study, it does not remove the Ly6Clo bias of cell proportions, if anything it seems to enhance the difference between the ly6C low and high populations in Rbpj null mice (figure 5C). This oversight in data interpretation likely occurred because: i) this experiment is missing a potentially important control (Lyz2cre/cre Ccr2RFP/RFP or RBP-J variations), and ii) lack of statistical comparisons between Ly6Clow and high subsets (e.g. two-way ANOVA design). In general, there seemed to be a focus on the Ly6C low cells, where the mechanism may be more identifiable in their precursors - likely the Ly6C high monocytes. Furthermore, the lack of this mechanism and data comparison may also be important, because it is possible that RBP-J signalling merely maintains the expression of Ly6C, rather than controls non-classical monocyte differentiation. In this case the comparison made for the sequencing data would be between Ly6C low non classical monocytes and 'artificially' Ly6C low classical monocytes. The basis of a population based on one marker is currently a widespread flaw in the field.

Other specific weaknesses were identified (note these are in addition to the more important comments above):<br /> 1) The confirmation of knockout in supplemental figure 1A shows only a two third knockdown when this should be almost totally gone. The authors have confirmed this is perhaps poor primer design and cite a study which shows almost complete reduction in protein levels (though this could be made more clear).<br /> 2) Many figures (e.g. 1A) only show proportional data (%) when the addition of cell numbers would also be informative - for example, what if Ly6Chigh cells were decreasing, thus artificially increasing the proportion of Ly6Clo cells? Looking at figure 7B - where cell numbers are shown, it is clear that cell proportion differences often do not match number data - here RBP-J knockout also increases Ly6C high cells in number (but not %).<br /> 3) It was noted previously that many figures only have an n of 1 or 2 (e.g. 2B, 2C), the authors clarified that some of these displayed one dot to represent an experiment of multiple n.<br /> 4) There is incomplete analysis (e.g. Network analysis, comparison to subset-restricted gene expression) and interpretation of RNA-sequencing results (figure 4), additionally the difference between the genotypes in both monocyte subsets would provide a more complete picture and potentially reveal mechanisms<br /> 6) The experiments in figure 5 are missing a control (Lyz2cre/cre Ccr2RFP/RFP or the Rbpj+/+ versions) and may have been misinterpreted. For example if the control (RBP-J WT, CCR2 KO) was used then it would almost certainly show falling Ly6C low numbers compared to RBP-J WT CCR2 WT, but RBP-J KO CCR2 KO would still have more Ly6c low monocytes than RBP-J WT, CCR2 KO - meaning that the RBP-J function is independent of CCR2. I.e. Ly6c low numbers are mostly dependent on CCR2 but this is irrespective of RBP-J. Explained in another way, the normal ratio of Ly6C high to low is around 1.5 Ly6Chigh cells for every one Ly6Clow cell, this is flipped in the RBP-J knockout to 1 high to 1.25 low (the main finding of the paper), but when CCR2 is removed it actually becomes 1 high to 5 low (actual numbers 0.2% vs around 1%) - in which case all CCR2 removal is doing is lowering the number of monocytes and RBP-J's mechanism is independent of CCR2.<br /> 7) Figure 6 was difficult to interpret because of the lack of shown gating strategy. The authors state they copied the strategy from Schyns et al. however in order to review this correctly the authors should show a supplemental figure of their own gating.<br /> 8) Figure 7 has the same problem as figure 5, but this time has the correct control. CCR2 ablation has a global suppression of monocyte numbers however the increased ly6c low monocyte ratio is most likely still present in the DKO mice - the lower numbers reduce the clarity of the data. Additionally in Lung IM macrophages depletion of CCR2 in the DKO only had a partial effect in some cell types - so CCR2 is playing a role, but it is not fully dependent. A good comparison would be if they blocked PU.1 expression - the effect of RBP-J would also be muted but it doesn't mean anything in terms of mechanism. Statements about the origin of the cells may need to be removed due to lack of compelling evidence.<br /> 9) Even after being notified and acknowledging the study, the authors still have not referred to or cited a similar 2020 study in their manuscript. This study also investigated myeloid deletion of Rbpj (Zhang et al. 2020 - https://doi.org/10.1096/fj.201903086RR). Zhang et al identified that Ly6Clo alveolar macrophages were decreased in this model - it is intriguing to synthesise these two studies and hypothesise that the ly6c low monocytes steal the lung niche, but this was not discussed. The authors also indicated they looked at AM but saw no difference - perhaps they should look specifically at Ly6Clow AMs in their data to compare with this study?

Reviewer #2 (Public Review):

The study employs quantitative metabolomic and lipidomic analyses to scrutinize tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples from renal cell carcinoma (RCC) patients. The authors delve into the intricate world of renal cell carcinoma and its tumor microenvironment, shedding light on the factors that shape nutrient availability in both cancerous and adjacent normal tissues. The authors prove that non-cancer-driven tissue factors play a dominant role in shaping nutrient availability in RCC. This finding opens up new avenues for research, suggesting that the tumor microenvironment is profoundly influenced by factors beyond the presence of cancer cells. This study not only contributes valuable insights into RCC metabolism but also prompts a reevaluation of the factors governing nutrient availability in tumor microenvironments more broadly. Overall, it represents a significant step forward in our understanding of the intricate interplay between cancer and its surrounding milieu.

The study is overall well-constructed, including appropriate analysis. Likewise, the manuscript is written clearly and supported by high-quality figures. Since the authors exclusively employed samples from RCC patients and did not include kidney interstitial fluid and plasma samples from healthy individuals, we cannot accurately assess the true significance and applicability of the results until the role of cancer cells in reshaping KIF is understood. In essence, some metabolite levels in the tumor interstitial fluid did not show an increase or decrease compared to the adjacent normal kidney interstitial fluid. However, the levels of these metabolites in both TIF and KIF might be higher or lower than those in kidney interstitial fluid from healthy individuals, and the roles of these metabolites should not be overlooked. Similar concerns extend to plasma levels, emphasizing the importance of metabolites that synchronously change in RCC TIF, KIF, and plasma-whether elevated or reduced.

Reviewer #2 (Public Review):

In this manuscript, the authors uncover a variety of macromolecular Drosha complexes in NSCs and propose that they might exert specific functions in adult neurogenesis. This is an interesting and important area of research, the proteomics data are very useful, and the manuscript is well written and easy to understand. Overall, this manuscript has many strengths. The authors identify 165 proteins, several of them enriched in NSCs, and potentially specific for miRNA dependent or independent Drosha macromolecular complexes. Moreover, the authors convincingly show that Safb1 binds and post-transcriptionally destabilizes NFIB transcript in complex with Drosha, in vitro. With that said, most of the functional evidence are based on Safb1 overexpression in vitro, and in some cases with immortalized cell lines. This is a major limitation of the study. Further experiments should be done to convincingly demonstrate that Safb1 regulates cell fate determination in adult neural stem cells by enhancing Drosha cleavage of NFIB mRNA.

Reviewer #2 (Public Review):

The authors introduce MetaPathPredict, a method that infers the presence of functional units of gene sets, such as a set of genes coding enzymes for a common metabolic pathway, from a pool of genes or genetic sequences. MetaPathPredict employs a stacked ensemble of neural networks, each trained for a specific pathway, to consider mutual information between pathways.

In predicting the presence of metabolic pathways in incomplete genomes, MetaPathPredict outperforms alternative naive classifiers and single neural network methods. These results demonstrate the effectiveness of a stacked ensemble of neural networks in exploiting mutual information between metabolic pathways.

Reviewer #3 (Public Review):

Studying the late development of neural circuits in relation to developmental changes in behaviour is clearly of great interest, particularly during the period of adolescence when a number of developmental abnormalities can be revealed. This is however not an easy task, since there are many concurrent changes that occur simultaneously during this developmental making it difficult to establish causality rather than correlation.

The study focuses on behavioural and circuit changes that occur between juvenile and adulthood focusing in the prefrontal cortex and on its descending projections to the brainstem raphe nuclei. Because the pathway from the frontal cortex to serotonin raphe neurons has been involved in behavioural and stress control, exerting a top-down control on impulsive behavior, there is a good justification to focus on the development of this pathway during a period that is thought to correspond to adolescence.

The authors identified a behavioral change in foraging strategy, which they term persistence. They find that adults tend to be more persistent than juveniles in an exploration for reward. To analyse the maturation of the prefrontal to raphe circuit they use a genetic approach (the Rbp4 promoter which drives expression in layer 5 cortical neurons) recording the synaptic drive elicited by stimulation of the axons arriving into the raphe area. They find that this maturation starts very late in the late adolescent period. They then study the effects of ablation of the layer 5 Rbp4 neurons in adults and find that adult animals have a behavior that is more similar to that of the juveniles. They then conclude that cortical prefrontal projections to the raphe are involved in the control of this behavior.

The study is interesting in showing this new behavioural test quantifying developmental changes in exploratory behavior and indicating that some pathways derived mainly from the frontal cortex continue to mature late. However, there are a number of issues regarding the specificity of the genetic approach used. This makes it difficult to be convinced that the behaviour is related to changes in the cortico-raphe circuit.

Reviewer #2 (Public Review):

Using a single-cell omics approach combined with spatial proteomics and genetic fate mapping, Kayvanjoo et al found that fetal liver (FL) macrophages cluster into distinct yolk sac-derived subpopulations and that some of the HSCs in FL preferentially associate with one of the identified macrophage subpopulations. FLs lacking macrophages show a delay in erythropoiesis. The authors also try to identify a role of macrophages for HSCs function in FL, and claim that macrophages affect myeloid differentiation of HSCs. Experimental support for the function of macrophages on HSCs remains weak. Taken together, their data provide a precise map of FL macrophage subpopulations, which is novel and will serve the field well.

Reviewer #2 (Public Review):

This manuscript reports the results of an ancillary study of a prospective trial assessing the effects of androgen deprivation therapy (ADT) with Dagarelix (a GnRH antagonist) on body composition in patients with prostate cancer. An interesting relationship between FSH levels, that were suppressed by Dagarelix treatment, and body composition parameters (particularly fat body mass) was described after 12 months of therapy. Therefore, the authors conclude that FSH could be a promising marker to monitor the risk of sarcopenic obesity and cardiovascular complications in prostate cancer patients undergoing ADT. As acknowledged by the Authors the main limitation of the study is the limited sample of patients. However, since testosterone levels were not assessed it is not possible to firmly establish whether the changes in fat mass observed with treatment are directly or indirectly associated with a reduction in FSH (and therefore in the latter case mediated by testosterone). Moreover, it is not clear whether the effect of the change in FSH levels during the study and the body composition parameters achieved at 12 months was evaluated (instead of assessing the relationship between FSH changes and changes in body composition parameters). Finally, tests on bone muscle mass and strength were not performed, so the hypothesis that variation of FSH levels in prostate cancer patients in ADT may affect sarcopenia remains speculative.

Reviewer #2 (Public Review):

The Authors demonstrate compelling genetic evidence that the region that harbors rs6740960 plays a role in both normal craniofacial development risk for craniofacial disease. They show strong evidence that the conserved element harboring this variant is tested for LacZ reporter activity in the developing mouse that is has activity in relevant tissues. They perform several assays to demonstrate a physical link between this enhancer region to a specific target gene, PKDCC, in both cranial neural crest cells and differentiated chondrocytes. Removal of a single copy of the enhancer has little effect on PKDCC expression in CNCCs but strong impacts in chondryocytes. H1 derived cells that are heterozygous at the variant above show strong bias in H3K27ac signals in chondrocytes. The researchers then go on to recharacterize a PKDCC knockout mouse to show that it has craniofacial defects. They use modern micro-CT and analysis techniques to demonstrate subtle changes in jaw and skull structure in PKDCC heterozygous mice and confirm many of the phenotypes that were described by Kinoshita et al 2009. Overall these results point to dosage of PKDCC in craniofacial development with changes in skull shape and susceptibility to orofacial clefting. However the epigenomic differences presented in Figure 2B that serve as the foundation for the rest of the work do not agree with previously published work by this group (Prescot et al 2015). The researchers claim "enrichment of the coactivator p300 and of the active chromatin mark H3K27ac at this region is higher in the chimpanzee CNCCs as compared to human, suggesting that this non-coding element may have higher regulatory activity in the chimp. However this region was not identified in the top 1000 biased enhancer regions provided in the supplement of the Prescott et al 2015 paper. The authors do not indicate any statistical significance and largely rely on signal tracks that have not been corrected for input controls to make this conclusion. The in vivo assay for enhancer activity while excellent at demonstrating where an enhancer can be active is not well suited to quantitative comparisons. Furthermore the researchers claim that the mouse orthologous sequence is not active in the assay despite strong H3K27ac and other enhancer related signals in developing mouse craniofacial tissues as available from the Mouse Encode Project. This calls into questions whether this assay is informative at all if the native sequence which shows functionally conserved activity is not active in the mouse embryo. Lastly the authors only consider this region as a potential enhancer and not any other type of regulatory sequence. GENCODE gene annotations demonstrate a potential lncRNA (LINC02898 /ENST00000378711.2) that is directly adjacent to the region marked by this variant. This could be a promoter for an RNA that regulates PKDCC in cis. Inspection of gene expression data from a recent preprint Yankee et al 2022 as well as Prescot et al data available from the recount3 database indeed indicate RNA signal from both CNCCs and primary human tissue consistent with this annotation. The Mundlos lab has demonstrated similar regulatory mechanisms through lncRNA Maenli at the En1 locus that result in limb abnormalities.

Reviewer #2 (Public Review):

This study follows up on previous work from this group, and others, relating paternal diet to changes in sperm epigenetics, and offspring phenotypes. The authors focus on paternal diet (high-fat diet versus a control chow), sperm chromatin, and molecular changes in the placenta associated with offspring development.

The text is well written and the figures are generally well presented and clear. The sperm epigenetic analyses and analysis of the placenta epigenetics and gene expression are generally well performed. The study provides new insight into how paternally mediated intergenerational epigenetic inheritance could involve placenta-embryo signaling.

A major weakness is that the high-fat diet used was from a different manufacturer than the control (lower fat) diet. Therefore, it is difficult to judge whether the effects are due to a change in fat levels, or the many other molecules that are likely to differ in chow between different manufacturers. Other weaknesses include lack of methodological detail in parts, low n values for some experiments, and the need for more mechanistic data.

Whilst the authors may have achieved their aims, more data is needed to inform a potential mechanism.

This study adds to our understanding of how changes in paternal diet may alter sperm epigenetics and offspring development. The novelty is in the link to gene expression in the placenta associated with offspring development in utero.

Reviewer #2 (Public Review):

Summary:<br /> Verma et al. provide a short technical report showing that endogenously tagged dynein and dynactin molecules localize to growing microtubule plus-ends and also move processively along microtubules in cells. The data are convincing, and the imaging and movies very nicely demonstrate their claims. I don't have any large technical concerns about the work. It is perhaps not surprising that dynein-dynactin complexes behave this way in cells due to other reports on the topic, but the current data are among some of the nicest direct demonstrations of this phenomenon. It may be somewhat controversial since a separate group has reported that dynein does not move processively in mammalian cells (https://www.biorxiv.org/content/10.1101/2021.04.05.438428v3). Because of this, it might be nice for the authors to comment on this discrepancy in the field, although the aforementioned work is still in pre-print form.

Strengths:<br /> Using state-of-the-art methods to endogenously tag dynein/dynactin subunits and performing live-cell imaging is convincing and useful for the field.

Weaknesses:<br /> The claims are perhaps not surprising or novel given the extensive data already published in the field. However, there aren't many similar studies using endogenously tagged subunits to date.

Reviewer #2 (Public Review):

Summary:<br /> Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:<br /> One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:<br /> While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Benner et al. interrogate the transcriptional regulator OVO to identify its targets in the Drosophila germline. The authors perform ChIP-seq in the adult ovary and identify established as well as novel OVO binding motifs in potential transcriptional targets of OVO. Through additional bioinformatic analysis of existing ATAC-seq, CAGE-seq, and histone methylation data, the authors confirm previous reports that OVO is enriched at transcription start sites and suggest that OVO does not act as part of the core RNA polymerase complex. Benner et al. then perform bulk RNA-seq in OVO mutant and "wildtype" (GAL4 mediated expression of OVO under the control of the ovo promoter in OVO mutants) ovaries to identify genes that are differentially expressed in the presence of OVO. This analysis supports previous reports that OVO likely acts at transcription start sites as a transcriptional activator. While the authors propose that OVO activates the expression of genes that are important for egg integrity, maturation, and for embryonic development (nanos, gcl, pgc, bicoid), this hypothesis is based on correlation and is not supported by in vivo analysis of the respective OVO binding sites in some of the key genes. A temporal resolution for OVO's role during germline development and egg chamber maturation in the ovary is also missing. Together, this manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis but lacks important in vivo experimental evidence that would validate the high-quality datasets.

Strengths:

The manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis

Weaknesses:

1. The authors propose that OVO acts as a positive regulator of essential germline genes, such as those necessary for egg integrity/maturation and embryonic/germline development. Much of this hypothesis is based on GO term analysis (and supported by the authors' ChIP-seq data). However accurate interpretation of GO term enrichment is highly dependent on using the correct background gene set. What control gene set did the authors use to perform GO term analysis (the information was not in the materials and methods)? If a background gene set was not previously specified, it is essential to perform the analysis with the appropriate background gene set. For this analysis, the total set of genes that were identified in the authors' RNA-seq of OVO-positive ovaries would be an ideal control gene set for which to perform GO term analysis. Alternatively, the total set of genes identified in previous scRNA-seq analysis of ovaries (see Rust et al., 2020, Slaidina et al., 2021 among others) would also be an appropriate control gene set for which to perform GO term analysis. If indeed GO term analysis of the genes bound by OVO compared to all genes expressed in the ovary still produces an enrichment of genes essential for embryonic development and egg integrity, then this hypothesis can be considered.

2. The authors provide important bioinformatic analysis of new and existing datasets that suggest OVO binds to specific motifs in the promoter regions of certain germline genes. While the bioinformatic analysis of these data is thorough and appropriate, the authors do not perform any in vivo validation of these datasets to support their hypotheses. The authors should choose a few important potential OVO targets based on their analysis, such as gcl, nanos, or bicoid (as these genes have well-studied phenotypes in embryogenesis), and perform functional analysis of the OVO binding site in their promoter regions. This may include creating CRISPR lines that do not contain the OVO binding site in the target gene promoter, or reporter lines with and without the OVO binding site, to test if OVO binding is essential for the transcription/function of the candidate genes.

3. The authors perform de novo motif analysis to identify novel OVO binding motifs in their ChIP-seq dataset. Motif analysis can be significantly strengthened by comparing DNA sequences within peaks, to sequences that are just outside of peak regions, thereby generating motifs that are specific to peak regions compared to other regions of the promoter/genome. For example, taking the 200 nt sequence on either side of an OVO peak could be used as a negative control sequence set. What control sequence set did the authors use as for their de novo motif analysis? More detail on this is necessary in the materials and methods section. Re-analysis with an appropriate negative control sequence set is suggested if not previously performed.

4. The authors mention that OVO binding (based on their ChIP-seq data) is highly associated with increased gene expression (lines 433-434). How many of the 3,094 peaks (conservative OVO binding sites), and what percentage of those peaks, are associated with a significant increase in gene expression from the RNA-seq data? How many are associated with a decrease in gene expression? This information should be added to the results section.

5. The authors mention that a change in endogenous OVO expression cannot be determined from the RNA-seq data due to the expression of the OVO-B cDNA rescue construct. Can the authors see a change in endogenous OVO expression based on the presence/absence of OVO introns in their RNA-seq dataset? While intronic sequences are relatively rare in RNA-seq, even a 0.1% capture rate of intronic sequence is likely to be enough to determine the change in endogenous OVO expression in the rescue construct compared to the OVO null.

6. The authors conclude with a model of how OVO may participate in the activation of transcription in embryonic pole cells. However, the authors did not carry out any experiments with pole cells that would support/test such a model. It may be more useful to end with a model that describes OVO's role in oogenesis, which is the experimental focus of themanuscript.

Reviewer #2 (Public Review):

Summary:

The authors of this manuscript are interested in discovering and functionally characterizing genes that might cause obesity. To find such genes, they conducted a forward genetic screen in mice, selecting strains which displayed increased body weight and adiposity. They found a strain, with germ-line deficiency in the gene Spag7, which displayed significantly increased body weight, fat mass, and adipose depot sizes manifesting after the onset of adulthood (20 weeks). The mice also display decreased organ sizes, leading to decreased lean body mass. The increased adiposity was traced to decreased energy expenditure at both room temperature and thermoneutrality, correlating with decreased locomotor activity and muscle atrophy. Major metabolic abnormalities such as impaired glucose tolerance and insulin sensitivity also accompanied the phenotype. Unexpectedly, when the authors generated an inducible, whole body knockout mouse using a globally expressed Cre-ERT2 along with a globally floxed Spag7, and induced Spag7 knockout before the onset of obesity, none of the phenotypes seen in the original strain were recapitulated. The authors trace this discrepancy to the major effect of Spag7 being on placental development.

Strengths:

Strengths of the manuscript are its inherently unbiased approach, using a forward genetic screen to discover previously unknown genes linked to obesity phenotypes. Another strong aspect of the work was the generation of an independent, complementary, strain consisting of an inducible knockout model, in which the deficiency of the gene could be assessed in a more granular form. This approach enabled the discovery of Spag7 as a gene involved in the establishment of the mature placenta, which determines the metabolic fate of the offspring. Additional strengths include the extensive array of physiological parameters measured, which provided a deep understanding of the whole-body metabolic phenotype and pinpointed its likely origin to muscle energetic dysfunction.

Weaknesses:

Weaknesses that can be raised are the lack of molecular mechanistic understanding of the numerous phenotypic observations. For example, the specific role of Spag7 to promote placental development remains unclear. Also, the reason why placental developmental abnormalities lead to muscle dysfunction, and whether indeed the entire metabolic phenotype of the offspring can be attributed solely to decreased muscle energetics is not fully explored.

Overall, the authors achieved a remarkable success in identifying genes associated with development of obesity and metabolic disease, discovering the role of Spag7 in placental development, and highlighting the fundamental role of in-utero development in setting future metabolic state of the offspring.

Comments on revised version:

I have no further comments on my assessment of this interesting paper.

Reviewer #2 (Public Review):

Summary:<br /> In this work, the authors sought to 1) establish a method for measuring muscle fiber subcellular structure (myofibrils) using common, non-specialized laboratory techniques and equipment, and 2) use this method to provide evidence on whether loading-induced muscle fiber growth was the result of myofibril growth (of existing myofibrils) or myofbrillogenesis (creation of new myofibrils) in mice and humans. The latter is a fundamental question in the muscle field. The authors succeeded in their aims and provided useful methods for the muscle field and detailed insight into muscle fiber hypertrophy; specifically, that loading-induced muscle fiber hypertrophy may be driven mostly by myofibrillogenesis.

Strengths:<br /> 1) The usage of murine and human samples to provide evidence on myofibril hypertrophy vs myofibrillogenesis.<br /> 2) A nice historical perspective on myofibrillogenesis in skeletal muscle.<br /> 3) The description of a useful and tractable IHC imaging method for the muscle biology field supported by extensive validation against electron microscopy.<br /> 4) Fundamental information on how myofiber hypertrophy ensues.

Weaknesses:

- The usage of young growing mice (8-10 weeks) versus adult mice (>4 months) in the murine mechanical overload experiments, as well as no consideration for biological sex. The former point is partly curtailed by the adult human data that is provided (male only). Still, the usage of adult mice would be preferable for these experiments given that maturational growth may somehow affect the outcomes. For the latter point, it is not clear whether male or female mice were used.

Reviewer #2 (Public Review):

Making state-of-the-art (super-resolution) microscopy widely available has been the subject of many publications in recent years as correctly referenced in the manuscript. By advocating the ideas of open-microscopy and trying to replace expensive, scientific-grade components such as lasers, cameras, objectives, and stages with cost-effective alternatives, interested researchers nowadays have a number of different frameworks to choose from. In the iteration of the theme presented here, the authors used the existing modular UC2 framework, which consists of 3D printable building blocks, and combined a cheapish laser, detector and x,y,(z) stage with expensive filters/dichroics and an expensive high-end objective (>15k Euros).

The choice of using the UC2 framework has the advantage, that the individual building blocks can be 3D printed, although it should be mentioned that the authors used injection-moulded blocks that will have a limited availability if not offered commercially by a third party. The strength of the manuscript is the tight integration of the hardware and the software (namely the implementations of imSwitch as a GUI to control data acquisition, OS SMLM algorithms for fast sub-pixel localisation and access to Napari).

The presented experimental data is convincing, demonstrating (1) extended live cell imaging both using bright-field and fluorescence in the incubator, (2) single-particle tracking of quantum dots, and (3) and STORM measurements in cells stained against tubulin.

For the revised (current) version of the manuscript, the authors further polished the manuscript and, more importantly, added plenty of information on the GitHub page that should make it significantly easier for interested researchers to replicate the instrument.

Overall, this is compelling work that is helping to make super-resolved microscopy more accessible.

Reviewer #2 (Public Review):

Summary:<br /> The article by Shuai et al. describes a comprehensive collection of over 800 split-GAL4 and split-LexA drivers, covering approximately 300 cell types in Drosophila, aimed at advancing the understanding of associative learning. The mushroom body (MB) in the insect brain is central to associative learning, with Kenyon cells (KCs) as primary intrinsic neurons and dopaminergic neurons (DANs) and MB output neurons (MBONs) forming compartmental zones for memory storage and behavior modulation. This study focuses on characterizing sensory input as well as direct upstream connections to the MB both anatomically and, to some extent, behaviorally. Genetic access to specific, sparsely expressed cell types is crucial for investigating the impact of single cells on computational and functional aspects within the circuitry. As such, this new and extensive collection significantly extends the range of targeted cell types related to the MB and will be an outstanding resource to elucidate MB-related processes in the future.

Strengths:<br /> The work by Shuai et al. provides novel and essential resources to study MB-related processes and beyond. The resulting tools are publicly available and, together with the linked information, will be foundational for many future studies. The importance and impact of this tool development approach, along with previous ones, for the field cannot be overstated. One of many interesting aspects arises from the anatomical analysis of cell types that are less stereotypical across flies. These discoveries might open new avenues for future investigations into how such asymmetry and individuality arise from development and other factors, and how it impacts the computations performed by the circuitry that contains these elements.

Weaknesses:<br /> Providing such an array of tools leaves little to complain about. However, despite the comprehensive genetic access to diverse sensory pathways and MB-connected cell types, the manuscript could be improved by discussing its limitations. For example, the projection neurons from the visual system seem to be underrepresented in the tools produced (or almost absent). A discussion of these omissions could help prevent misunderstandings. Additionally, more details on the screening process, particularly the selection of candidate split halves and stable split-GAL4 lines, would provide valuable insights into the methodology and the collection's completeness.

Reviewer #2 (Public Review):

Summary:

The study investigates whether speech and music processing involve specific or shared brain networks. Using intracranial EEG recordings from 18 epilepsy patients, it examines neural responses to speech and music. The authors found that most neural activity is shared between speech and music processing, without specific regional brain selectivity. Furthermore, domain-selective responses to speech or music are limited to frequency-specific coherent oscillations. The findings challenge the notion of anatomically distinct regions for different cognitive functions in the auditory process.

Strengths:

1. This study uses a relatively large corpus of intracranial EEG data, which provides high spatiotemporal resolution neural recordings, allowing for more precise and dynamic analysis of brain responses. The use of continuous speech and music enhances ecological validity compared to artificial or segmented stimuli.

2. This study uses multiple frequency bands in addition to just high-frequency activity (HFA), which has been the focus of many existing studies in the literature. This allows for a more comprehensive analysis of neural processing across the entire spectrum. The heterogeneity across different frequency bands also indicates that different frequency components of the neural activity may reflect different underlying neural computations.

3. This study also adds empirical evidence towards distributed representation versus domain-specificity. It challenges the traditional view of highly specialized, anatomically distinct regions for different cognitive functions. Instead, the study suggests a more integrated and overlapping neural network for processing complex stimuli like speech and music.

Weaknesses:

While this study is overall convincing, there are still some weaknesses in the methods and analyses that limit the implication of the work.

The study's main approach, focusing primarily on the grand comparison of response amplitudes between speech and music, may overlook intricate details in neural coding. Speech and music are not entirely orthogonal with each other at different levels of analysis: at the high-level abstraction, these are two different categories of cognitive processes; at the low-level acoustics, they overlap a lot; at intermediate levels, they may also share similar features. The selected musical stimuli, incorporating both vocals and multiple instrumental sounds, raise questions about the specificity of neural activation. For instance, it's unclear if the vocal elements in music and speech engage identical neural circuits. Additionally, the study doesn't adequately address whether purely melodic elements in music correlate with intonations in speech at a neural level. A more granular analysis, dissecting stimuli into distinct features like pitch, phonetics, timbre, and linguistic elements, could unveil more nuanced shared, and unique neural processes between speech and music. Prior research indicates potential overlap in neural coding for certain intermediate features in speech and music (Sankaran et al. 2023), suggesting that a simple averaged response comparison might not fully capture the complexity of neural encoding. Further delineation of phonetic, melodic, linguistic, and other coding, along with an analysis of how different informational aspects (phonetic, linguistic, melodic, etc) are represented in shared neural activities, could enhance our understanding of these processes and strengthen the study's conclusions.

The paper's emphasis on shared and overlapping neural activity, as observed through sEEG electrodes, provides valuable insights. It is probably true that domain-specificity for speech and music does not exist at such a macro scale. However, it's important to consider that each electrode records from a large neuronal population, encompassing thousands of neurons. This broad recording scope might mask more granular, non-overlapping feature representations at the single neuron level. Thus, while the study suggests shared neural underpinnings for speech and music perception at a macroscopic level, it cannot definitively rule out the possibility of distinct, non-overlapping neural representations at the microscale of local neuronal circuits for features that are distinctly associated with speech and music. This distinction is crucial for fully understanding the neural mechanisms underlying speech and music perception that merit future endeavors with more advanced large-scale neuronal recordings.

While classifying electrodes into 3 categories provides valuable insights, it may not fully capture the complexity of the neural response distribution to speech and music. A more nuanced and continuous approach could reveal subtler gradations in neural response, rather than imposing categorical boundaries. This could be done by computing continuous metrics, like unique variances explained by each category, or ratio-based statistics, etc. Incorporating such a continuum could enhance our understanding of the neural representation of speech and music, providing a more detailed and comprehensive picture of cortical processing.

Reviewer #2 (Public Review):

In this paper, Phan et al. investigate the properties of human HP1 paralogs, their interactions and abilities to undergo liquid-liquid phase separation. For this, they use a coarse-grained computational approach (validated with additional all-atom simulations) which allows to explore complex mixtures. Matching (wet-lab) experimental results, HP1 beta (HP1b) exhibits different properties from HP1 alpha and gamma (HP1a,g), in that it does not phase separate. Using domain switch experiments, the authors determine that the more negatively charged hinge in HP1b, compared to HP1a and HP1g, is mainly responsible for this effect. Exploring heterotypic complexes, mixtures between HP1 subtypes and DNA, the authors further show that HP1a can serve as a scaffold for HP1b to enter into condensed phases and that DNA can further stabilize phase separated compartments. Most interestingly, they show that a multicomponent mixture containing DNA, and HP1a and HP1b generates spatial separation between the HP1 paralogs: due to increased negative charge of DNA within the condensates, HP1b is pushed out and accumulates at the phase boundary. This represents an example how complex assemblies could form in the cell.

Overall, this is purely computational work, which however builds on extensive experimental results (including from the authors). The methods showcase how coarse-grained models can be employed to generate and test hypotheses how proteins can condense. Applied to HP1 proteins, the results from this tour-de-force study are consistent and convincing, within the experimental constraints. Moreover, the authors generate further models to test experimentally, in particular in light of multicomponent mixtures. Finally, the authors clearly discuss the computational methods, assumptions and limits of the methodology, which makes this a strong contribution to our understanding of biophysical basis of condensate formation in gene regulation.

Reviewer #2 (Public Review):

Summary:<br /> The authors were trying to identify and characterize the intrinsic factors that control the process of cell aging of bone marrow mesenchymal stromal cells (BMSCs), which is believed to be related to osteoporosis.

Strengths:<br /> The method is reasonable. The concept and methods used in this work can be easily extended to other systems and cells to study their aging process. It is also interesting to further examine if PCBP2 functions as an intrinsic aging factor in these other cell types.

The results are solid, supporting the claims and conclusions. The authors successfully identified and characterized PCBP2 as one of the intrinsic aging factors for BMSC cells.

Weaknesses:<br /> It is unclear if PCBP2 can also function as an intrinsic factor for BMSC cells in female individuals. More work may be needed to further dissect the mechanism of how PCBP2 impacts FGF2 expression. Could PCBP2 impact the FGF2 expression independent of ROS?

Additional context that would help readers interpret or understand the significance of the work:<br /> In the current work, the authors studied the aging process of BMSC cells, which are related to osteoporosis. Aging processes also impact many other cell types and their function, such as in muscle, skin, and the brain.

Reviewer #2 (Public Review):

Summary:<br /> The story of the co-evolution of TTX-bearing newts and their independently evolved TTX-resistant garter snake predators is a classic in evolutionary ecology/physiology. Over the years specific amino acid substitutions in the muscle-expressing (and other) sodium channels have been identified and the behavioral assays of snake crawling performance have indicated that the attainment of TTX-resistance comes at a cost in mobility. One previous study also examined how the amino acid mutations affected the biophysics of Nav channel properties. The present paper starts with this foundation and builds by adding in details and making causal connections across multiple snake populations with different degrees of resistance. The addition of muscle physiology bridges the gap between organismal performance and sodium channel biophysics. Moving in the other direction, examining molecular models of Nav channel structure and energetics allows a deep understanding of how amino acid substitutions affect channel properties. In the end, a clear picture is painted from molecular to organismal levels in two different parallel evolutions of TTX resistance.

Strengths:<br /> This study is a tour de force. It is clearly written, and nicely illustrated, and the methods and procedures are meticulously documented.

Weaknesses:<br /> One caveat is that the Nav channels used to test mutations in expression systems are rat channels engineered with TTX-resisting substitutions observed in snake populations. The ideal experiment would have been to use the snake channels. While the rat channels appear to be a good substitute for the snake channels and the authors have taken pains to show that the important amino acids are conserved between garter snakes and rats, the authors might explain why they did not use snake channels.

The noise analysis seems like a reasonable way to get at the question of single-channel conductance. But why did the authors not just measure single channel conductance in patches as opposed to this much more complex and roundabout method? It is recommended that the authors discuss how noise analysis deals with the problem of having the number of open channels changing rapidly during activation and fast inactivation. Is this a potential problem for deriving the total number of channels?

Reviewer #2 (Public Review):

Summary:<br /> In short, this paper uses a previously published method, ReplicaDock, to improve predictions from AlphaFold-multimer. The method generated about 25% more acceptable predictions than AFm, but more important is improving an Antibody-antigen set, where more than 50% of the models become improved.

When looking at the results in more detail, it is clear that for the models where the AFm models are good, the improvement is modest (or not at all). See, for instance, the blue dots in Figure 6. However, in the cases where AFm fails, the improvement is substantial (red dots in Figure 6), but no models reach a very high accuracy (Fnat ~0.5 compared to 0.8 for the good AFm models). So the paper could be summarized by claiming, "We apply ReplicaDock when AFm fails", instead of trying to sell the paper as an utterly novel pipeline. I must also say that I am surprised by the excellent performance of ReplicaDock - it seems to be a significant step ahead of other (not AlphaFold) docking methods, and from reading the original paper, that was unclear. Having a better benchmark of it alone (without AFm) would be very interesting.

These results also highlight several questions I try to describe in the weakness section below. In short, they boil down to the fact that the authors must show how good/bad ReplicaDock is at all targets (not only the ones where AFm fails. In addition, I have several more technical comments.

Strengths:<br /> Impressive increase in performance on AB-AG set (although a small set and no proteins).

Weaknesses:<br /> The presentation is a bit hard to follow. The authors mix several measures (Fnat, iRMS, RMSDbound, etc). In addition, it is not always clear what is shown. For instance, in Figure 1, is the RMSD calculated for a single chain or the entire protein? I would suggest that the author replace all these measures with two: TM-score when evaluating the quality of a single chain and DockQ when evaluating the results for docking. This would provide a clearer picture of the performance. This applies to most figures and tables. For instance, Figure 9 could be shown as a distribution of DockQ scores.

The improvements on the models where AFm is good are minimal (if at all), and it is unclear how global docking would perform on these targets, nor exactly why the plDDT<0.85 cutoff was chosen. To better understand the performance of ReplicaDock, the authors should therefore (i) run global and local docking on all targets and report the results, (ii) report the results if AlphaFold (not multimer) models of the chains were used as input to ReplicaDock (I would assume it is similar). These models can be downloaded from AlphaFoldDB.

Further, it would be interesting to see if ReplicaDock could be combined with AFsample (or any other model to generate structural diversity) to improve performance further.

The estimates of computing costs for the AFsample are incorrect (check what is presented in their paper). What are the computational costs for RepliaDock global docking?

It is unclear strictly what sequences were used as input to the modelling. The authors should use full-length UniProt sequences if they were not done.

The antibody-antigen dataset is small. It could easily be expanded to thousands of proteins. It would be interesting to know the performance of ReplicaDock on a more extensive set of Antibodies and nanobodies.

Using pLDDT on the interface region to identify good/bas models is likely suboptimal. It was acceptable (as a part of the score) for AlphaFold-2.0 (monomer), but AFm behaves differently. Here, AFm provides a direct score to evaluate the quality of the interaction (ipTM or Ranking Confidence). The authors should use these to separate good/bad models (for global/local docking), or at least show that these scores are less good than the one they used.

Reviewer #2 (Public Review):

This work probes the control of the hox operon in the cyanobacterium Synechocystis, where this operon directs the synthesis of a bidirectional hydrogenase that functions to produce hydrogen. In assessing the control of the hox system, the authors focused on the relative contributions of cyAbrB2, alongside SigE (and to a lesser extent, SigA and cyAbrB1) under both aerobic and microoxic conditions. In mapping the binding sites of these different proteins, they discovered that cyAbrB2 bound many sites throughout the chromosome repressed many of its target genes, and preferentially bound regions that were (relatively) rich in AT-residues. These characteristics led the authors to consider that cyAbrB2 may function as a nucleoid-associated protein (NAP) in Synechocystis, given its functional similarities with other NAPs like H-NS. They assessed the local chromosome conformation in both wild-type and cyabrB2 mutant strains at multiple sites within a 40 kb window on either side of the hox locus, using a region within the hox operon as bait. They concluded that cyAbrB2 functions as a nucleoid-associated protein that influences the activity of SigE through its modulation of chromosome architecture.

The authors approached their experiments carefully, and the data were generally very clearly presented and described.

Based on the data presented, the authors make a strong case for cyAbrB2 as a nucleoid-associated protein, given the multiple ways in which it seems to function similarly to the well-studied Escherichia coli H-NS protein. It would be helpful to provide some additional commentary within the discussion around the similarities and differences of cyAbrB2 to other nucleoid-associated proteins, and possible mechanisms of cyAbrB2 control (post-translational modification; protein-protein interactions; etc.). The manuscript would also be strengthened with the inclusion of biochemical experiments probing the binding of cyAbrB2, particularly focussing on its oligomerization and DNA polymerization/bridging potential.

Previous work had revealed a role for SigE in the control of hox cluster expression, which nicely justified its inclusion (and focus) in this study. However, the results of the SigA studies here suggested that SigA both strongly associated with the hox promoter, and its binding sites were shared more frequently than SigE with cyAbrB2. The focus on cyAbrB2 is also well-justified, given previous reports of its control of hox expression; however, it shares binding sites with an essential homologue cyAbrB1. Interestingly, while the B1 protein appears to bind similar sites, instead of repressing hox expression, it is known as an activator of this operon. It seems important to consider how cyAbrB1 activity might influence the results described here.

Reviewer #2 (Public Review):

Summary:<br /> The authors have previously engineered an antibody fusion protein targeting ZNRF3/RNF43 ubiquitin ligases, which enhances Wnt signaling specifically in hepatocytes. This is achieved using RSPO2RA (ZNRF3/RNF43 ligand with F105R/F109A mutations which abolish its binding to LGRs) and ASGR1 (hepatocyte-specific cell surface molecule). In the current study, they have identified two new ASGR1 and ASGR1/2 antibodies (8M24 and 8G8), which also enhance Wnt signaling when fused to RSPO2RA antibody. These also lead to the degradation of ASGR1, demonstrating that protein degradation and signaling enhancement can be dually targeted with a single molecule.

Strengths:<br /> The authors show crystal structures for binding of these antibodies to ASGR1/2, and hypothesize about why specificity is mediated through specific residues. They do not test these hypotheses.

The authors demonstrate a sub-picomolar affinity of these antibodies for ASGR1/2, which should be powerful clinically.

The authors demonstrate in hepatocyte cell lines that these function as mimetics, and that they do not function in HEK cells, which do not express ASGR1. They do not perform an exhaustive screen of all non-hepatocyte cells, nor do they test these molecules in vivo.

Surprisingly, these molecules also induced loss of ASGR1, which the authors hypothesize is due to ubiquitination and degradation, initiated by the E3 ligases recruited to ASGR1. They demonstrate that inhibition of either the proteasome or lysosome abrogates this effect and that it is dependent on E1 ubiquitin ligases. They do not demonstrate direct ubiquitination of ASGR1 by ZNRF3/RNF43.

Weaknesses:<br /> As co-listed with strengths above, the analysis is not always exhaustive but shows good preliminary findings for the field.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Lao et al. develop an open-source software (OpenNucleome) for GPU-accelerated molecular dynamics simulation of the human nucleus accounting for chromatin, nucleoli, nuclear speckles, etc. Using this, the authors investigate the steady-state organization and dynamics of many of the nuclear components.

Strengths:<br /> This is a comprehensive open-source tool to study several aspects of the nucleus, including chromatin organization, interactions with lamins and organization, and interactions with nuclear speckles and nucleoli. The model is built carefully, accounting for several important factors and optimizing the parameters iteratively to achieve experimentally known results. The authors have simulated the entire genome at 100kb resolution (which is a very good resolution to simulate and study the entire diploid genome) and predict several static quantities such as the radius of gyration and radial positions of all chromosomes, and time-dependent quantities like the mean-square displacement of important genomic regions.

Weaknesses:<br /> One weakness of the model is that it has several parameters. Some of them are constrained by the experiments. However, the role of every parameter is not clear in the manuscript.

Reviewer #2 (Public Review):

Summary:<br /> Pulfer A. et al. developed a deep learning-based apoptosis detection system named ADeS, which outperforms the currently available computational tools for in vitro automatic detection. Furthermore, ADeS can automatically identify apoptotic cells in vivo in intravital microscopy time-lapses, preventing manual labeling with potential biases. The authors trained and successfully evaluated ADeS in packed epithelial monolayers and T cells distributed in 3D collagen hydrogels. Moreover, in vivo, training and evaluation were performed on polymorphonucleated leukocytes in lymph nodes and spleen.

Strengths:<br /> Pulfer A. et colleagues convincingly presented their results, thoroughly evaluated ADeS for potential toxicity assay, and compared its performance with available state-of-the-art tools.

Weaknesses:<br /> The use of ADeS is still restricted to samples where cells are fluorescently labeled either in the cytoplasm or in the nucleus, which limits its use for in vitro toxicity assays that are performed on primary cells or organoids (e.g., iPSCs-derived systems) that are normally harder to transfect.

In conclusion, ADeS will be a useful tool to improve output quality and accelerate the evaluation of assays in several research areas with basic and applied aims.

Reviewer #2 (Public Review):

Summary<br /> This paper has three parts. The first part applied a coarse-grained model with proteome partition to calculate cell growth under respiration and fermentation modes. The second part considered single-cell variability and performed population average to acquire an ensemble metabolic profile for acetate fermentation. The third part used model and simulation to compare experimental data in literature and obtained substantial consistency.

Strengths and major contributions<br /> (i) The coarse-grained model considered specific metabolite groups and their inter-relations and acquired an analytical solution for this scenario. The "resolution" of this model is in between the Flux Balanced Analysis/whole-cell simulation and proteome partition analysis.

(ii) The author considered single-cell level metabolic heterogeneity and calculated the ensemble average with explicit calculation. The results are consistent with known fermentation and growth phenomena qualitatively and can be quantitatively compared to experimental results.

Weaknesses<br /> (i) If I am reading this paper correctly, the author's model predicts binary (or "digital") outcomes of single-cell metabolism, that is, after growth rate optimization, each cell will adopt either "respiration mode" or "fermentation mode" (as illustrated in Figure Appendix - Figure 1 C, D). Due to variability enzyme activity k_i^{cat} and critical growth rate λ_C, each cell under the same nutrient condition could have either respiration or fermentation, but the choice is binary.

The binary choice at the single-cell level is inconsistent with our current understanding of metabolism. If a cell only uses fermentation mode (as shown in Appendix - Figure 1C), it could generate enough energy but not be able to have enough metabolic fluxes to feed into the TCA cycle. That is, under pure fermentation mode, the cell cannot expand the pool of TCA cycle metabolites and hence cannot grow.

This caveat also appears in the model in Appendix (S25) that assumes J_E = r_E*J_{BM} where r_E is a constant. From my understanding, r_E can be different between respiration and fermentation modes (at least for real cells) and hence it is inappropriate to conclude that cells using fermentation, which generates enough energy, can also generate a balanced biomass.

(ii) The minor weakness of this model is that it assumes a priori that each cell chooses its metabolic strategy based on energy efficiency. This is an interesting assumption but there is no known biochemical pathway that directly executes this mechanism. In evolution, growth rate is more frequently considered for metabolic optimization. In Flux Balanced Analysis, one could have multiple objective functions including biomass synthesis, energy generation, entropy production, etc. Therefore, the author would need to justify this assumption and propose a reasonable biochemical mechanism for cells to sense and regulate their energy efficiency.

My feeling is that the mathematical structure of this model could be correct, but the single-cell interpretation for the ensemble averaging has issues. Each cell could potentially adopt partial respiration and partial fermentation at the same time and have temporal variability in its metabolic mode as well. With the modification of the optimization scheme, the author could have a revised model that avoids the caveat mentioned above.

Discussion and impact for the field<br /> Proteome partition models and Flux Balanced Analysis are both commonly used mathematical models that emphasize different parts of cellular physiology. This paper has ingredients for both, and I expect after revision it will bridge our understanding of the whole cell.

Reviewer #2 (Public Review):

In the current study, Latchoumane and collaborators focus on the Cav3.1 calcium channels in the mediodorsal thalamic nucleus as critical players in the regulation of brain-states and ethanol resistance in mice. By combining behavioural, electrophysiological, and genetic techniques, they report three main findings. First, KO Cav3.1 mice exhibit resistance to ethanol-induced sedation and sustained tonic firing in thalamocortical units. Second, knocked-down Cav3.1 mice reproduce the same behaviour when the mediodorsal, but not the ventrobasal, thalamic nucleus is targeted. Third, either optogenetic or electric stimulation of the mediodorsal thalamus reduces ethanol-induced sedation in control animals.

Overall, the study is well designed and performed, correctly controlled for confounds, and properly analysed. Nonetheless, it is important to address some aspects of the report. The results support the conclusions of the study. These results are likely to be relevant in the field of systems neuroscience, as they increase the molecular evidence showing how the thalamus regulates brain states.

Reviewer #2 (Public Review):

Summary:<br /> Animals exhibit different speeds of locomotion. In vertebrates, this is thought to be implemented by different groups of spinal interneurons and motor neurons. A fundamental assumption in the field has been that neural mechanisms that generate and sustain the rhythm at different locomotor speeds are the same. In this study, the authors challenge this view. Using rigorous in vivo electrophysiology during fictive locomotion combined with genetics, the authors provide a detailed analysis of cellular and synaptic properties of different subtypes of spinal V2a neurons that play a crucial role in rhythm generation. Importantly, they are able to show that speed-related subsets of V2a neurons have distinct cellular and synaptic properties and may utilize different mechanisms to implement different locomotor speeds.

Strengths:<br /> The authors fully utilize the zebrafish model system and solid electrophysiological analyses to study the active and passive properties of speed-related V2a subsets. Identification of the V2a subtype is based directly on their recruitment at different locomotor speeds and not on indirect markers like soma size, D-V position etc. Throughout the article, the authors have cleverly used standard electrophysiological tests and analysis to tease out different neuronal properties and link it to natural activity. For example, in Figures 2 and 4, the authors make comparisons of V2a spiking with current steps and during fictive swims showing spike rates measured with current steps are physiologically relevant and observed during natural recruitment. The experiments done are rigorous and well-controlled.

Weaknesses:<br /> The authors claim that a primary result of their study is that reciprocal inhibition is important for rhythmogenesis at fast speeds while recurrent inhibition is key at slow speeds. This is shown in Figure 6, however, the authors do not show any statistical tests for this claim. The authors also do not show any conclusive evidence that reciprocal inhibition is required for rhythmogenesis at fast speeds and vice versa for slow speeds. Additional experiments or modeling studies that conclusively show the necessity of these different inhibitory sources to the generation of different rhythms would be needed to strengthen this claim.

The authors do a great job of teasing out cellular and synaptic properties in the different V2a subsets, however, it is not clear if or how these match the final output. For example, V2aD neurons are tonic or bursting for fast and slow speeds respectively but it is not intuitive how these cellular properties would influence phasic excitation and inhibition these neurons receive.

It is not clear from the discussion why having different mechanisms of rhythm generation at different speeds could be an important circuit design. The authors use anguilliform and carangiform modes of swimming to denote fast and slow speeds but there are differences in these movements other than speed, like rostrocaudal coordination. The frequency and pattern of these movements are linked and warrant more discussion.

Reviewer #2 (Public Review):

Summary:<br /> In this work, Kashefi et al. investigate the planning of sequential reaching movements and how the additional information about future reaches affects planning and execution. This study, carried out with human subjects, extends a body of research in sequential movements to ask important questions: How many future reaches can you plan in advance? And how do those future plans interact with each other?

The authors designed several experiments to address these questions, finding that information about future targets makes reaches more efficient in both timing and path curvature. Further, with some clever target jump manipulations, the authors show that plans for a distant future reach can influence plans for a near future reach, suggesting that the planning for multiple future reaches is not independent. Lastly, the authors show that information about future targets is acquired parafoveally--that is, subjects tend to fixate mainly on the target they are about to reach to, acquiring future target information by paying attention to targets outside the fixation point.

The study opens up exciting questions about how this kind of multi-target planning is implemented in the brain. As the authors note in the manuscript, previous work in monkeys showed that preparatory neural activity for a future reaching movement can occur simultaneously with a current reaching movement, but that study was limited to the monkey only knowing about two future targets. It would be quite interesting to see how neural activity partitions preparatory activity for a third future target, given that this study shows that the third target's planning may interact with the second target's planning.

Strengths:<br /> A major strength of this study is that the experiments and analyses are designed to answer complementary questions, which together form a relatively complete picture of how subjects act on future target information. This complete description of a complex behavior will be a boon to future work in understanding the neural control of sequential, compound movements.

Weaknesses:<br /> I found no real glaring weaknesses with the paper, though I do wish that there had been some more discussion of what happens to planning with longer dwell times in target. In the later parts of the manuscript, the authors mention that the co-articulation result (where reaches are curved to make future target acquisition more efficient) was less evident for longer dwell times, likely because for longer dwell times, the subject needs to fully stop in target before moving to the next one. This result made me wonder if the future plan interaction effect (tested with the target jumps) would have been affected by dwell time. As far as I can tell, the target jump portion only dealt with the shorter dwell times, but if the authors had longer dwell time data for these experiments, I would appreciate seeing the results and interpretations.

Beyond this, the authors also mentioned in the results and discussion the idea of "neural resources" being assigned to replan movements, but it's not clear to me what this might actually mean concretely. I wonder if the authors have a toy model in mind for what this kind of resource reassignment could mean. I realize it would likely be quite speculative, but I would greatly appreciate a description or some sort of intuition if possible.

Reviewer #2 (Public Review):

Summary:<br /> Here Jeong et al., use a combination of theoretical and experimental approaches to define molecular contexts that support specific chromatin conformations. They seek to define features that are associated with TADs that are retained after cohesin depletion (the authors refer to these TADs as P-TADs). They were motivated by differences between single cell data, which suggest that some TADs can be maintained in the absence of cohesin, whereas ensemble HiC data suggest complete loss of TADs. By reananalyzing a number of HiC datasets from different cell types, the authors observe that in ensemble methods, a significant subset of TADs are retained. They observe that P-TADs are associated with mismatches in epigenetic state across TAD boundaries. They further observe that "physical boundaries" are associated with P-TAD maintenance. Their structure/simulation based approach appears to be a powerful means to generate 3D structures from ensemble HiC data, and provide chromosome conformations that mimic the data from single-cell based experiments. Their results also challenge current dogma in the field about epigenetic state being more related to compartment formation rather than TAD boundaries. Their analysis is particularly important because limited amounts of imaging data are presently available for defining chromosome structure at the single-molecule level, however, vast amounts of HiC and ChIP-seq data are available. By using HiC data to generate high quality simulated structural data, they overcome this limitation. Overall, this manuscript is important for understanding chromosome organization, particularly for contacts that do not require cohesin for their maintenance, and for understanding how different levels of chromosome organization may be interconnected. I cannot comment on the validity of the provided simulation methods and hope that another reviewer is qualified to do this.

Specific comments<br /> -It is unclear what defines a physical barrier. From reading the text and the methods, it is not entirely clear to me how the authors have designated sites of physical barriers. It may help to define this on pg 7, second to last paragraph, when the authors first describe instances of P-TAD maintenance in the absence of epigenetic mismatch.

-Figure 7 adds an interesting take to their approach. Here the authors use microC data to analyze promoter-enhancer/promoter-promoter contacts. These data are included as part of the discussion. I think this data could be incorporated into the main text, particularly because it provides a biological context where P-TADs would have a rather critical role.

-Figure 3a- the numbers here do not match the text (page 6, second to last paragraph). The numbers have been flipped for either chromosome 10 or chromosome 13 in the text or the figures.

In the revision, the authors have sufficiently addressed my specific concerns from above.

Reviewer #2 (Public Review):

The responses to the comments and changes in the manuscript are convincing, especially the secretion patterns of high and low secreting cells are interesting and reassuring. The only criticism I still have is that most observations are already published in the previous paper by the same authors.

Summary:<br /> In their manuscript titled "Stimulation-induced cytokine polyfunctionality as a dynamic concept," the authors investigate the dynamic nature of polyfunctional cytokine responses to established stimulants. The authors use their previously published single-cell encapsulation droplet-microfluidic platform to analyse the response of peripheral blood mononuclear cells (PBMCs) to different stimulants and measure the secretion dynamics of individual cytokines. This assay shows that polyfunctionality in cytokine responses is a complex but short-lived phenomenon that decreases with prolonged stimulation times. The study finds that polyfunctional cells predominantly display elevated cytokine concentrations with similar secretion patterns but higher secretion levels compared to their monocytokine-secreting counterparts. The method is promising to analyse the correlation between the secretion dynamics of different cytokines in primary samples and heterogeneous cell populations.

Strengths:<br /> This method provides single-cell-resolved and dynamic cytokine concentration information, which might be used to identify "fingerprints" of secretion patterns for selected cytokines. When extending the available data to more than one donor, this might be the basis for a diagnostic tool. The combination of established droplet microfluidics with an epi-fluorescence microscope-based readout makes it convincing that the method is transferable to other labs. Specifically, the dynamic analysis of cytokine concentrations is interesting, and the differences or similarities in secretion timepoints might be missed with end-point methods. The authors convincingly show that they detect up to three different cytokines in single cells.

Weaknesses:<br /> The conclusions of the study are based on samples from a single donor, which makes the conclusions on secretion patterns difficult to interpret. The choice of cytokines is explained, but the justification of the groupings of the antibodies into the two panels is missing. It would further be helpful to discuss how the single cell incubation might affect the secretion dynamics vs. the influence of co-culture of all cell types during the 24 h activation. The authors compare average secretion rates and levels. However, the right panel in Fig. 6 looks like there might be two different populations of mono- or polyfuntional cells that have two secretion rates. As the authors have single-cell data, I would find the separation into these populations more meaningful than comparing the mean values. In line with this comment, comparing the mean values for these cytokines instead of the mean of the populations with distinct secretion properties might actually show stronger differences than the authors report here.Is the plateau of the cytokine concentration caused by the fluorescence signal saturating the camera, saturation of the magnetic beads, exhaustion of the fluorescent antibodies, or constant cytokine concentrations? The high number of non-CSCs and the limited number of droplets decrease the statistical power of the method. The authors discuss their choice to use PBMCs and not solely T cells, but this aspect is missing in the discussion.

Reviewer #2 (Public Review):

Summary:<br /> Let-7 family miRNAs are largely redundant in function, and originate from multiple genomic loci ("clusters"). Erice et al demonstrate that two individual clusters (let7afd and let7bc2) in mice regulate the generation of IL-17 producing CD8 T cells in vitro and in vivo in a model of emphysema. These cells also express higher levels of the IL-17-inducing transcription factor RORgt, encoded by Rorc, which the authors demonstrate to be a direct target of let-7. Since multiple let-7 family miRNAs are downregulated in T cells and lung tissue in emphysema, these data support a model in which reduced let-7 allows increased IL-17 production by T cells, contributing to disease pathogenesis.

Strengths:<br /> The inclusion of miRNA and pri-miRNA expression data from sorted human lung T cells as well as mouse T cells from an emphysema model is a strength.

The study includes complementary loss of function and gain of function experimental systems to test the effect of altered let-7 function, though it should be noted that these involved different let-7 family members and did not yield simple, complementary results for all experimental outcomes.

The most important finding is that deletion of just one let-7 cluster ("Let7bc2") is sufficient to exacerbate emphysema in the nCB and CS models.

Weaknesses:<br /> The functional analyses are unusually focused on IL-17 producing CD8 T cells, but it is not made clear whether these cells are an important player in emphysema pathogenesis in the nCB and CS models. The data shown reveal that they are far less numerous than IL-17-producing CD4 T cells. It is also notable that the Figure 1 expression data from human subjects used sorted CD4+ T cells. And as the author mentioned, prior work on let-7 showed that it regulated Th17 (CD4) responses.

Compared with Let7bc2 deletion, Let7afd deletion had a much larger effect on IL17 production by CD8 T cells in vitro, and it also had a larger effect on RORgt expression in untreated mice in vivo, especially in the lung. It would be valuable to more thoroughly characterize the let7afd mice. RORgt expression should be shown in the in vitro assays. In the results, the authors state that let7afdLOF mice "did not exhibit lung histopathology nor inflammatory changes" up to 6 months of age. Similarly, it is stated in the conclusion that "the let-7afdLOF mice ... did not exhibit changes in Tc17/Th17 subpopulations" in vivo. All these data should be shown, and if no baseline changes are apparent, then I also recommend challenging these mice with nCB and/or cigarette smoke.

This brings up the larger issue of redundancy among the let-7 family members and genomic clusters. This should be discussed, including some explanation of the relative expression of each mature family member in T cells, and how that maps to the clusters studied here (and those that were not investigated). It would also be helpful to explain the relationship between mouse Let7bc2 and human Let7a3b, since Let7bc2 is the primary focus of emphysema experiments in this manuscript.

This is especially important because the study of individual let-7 clusters is the core novelty of this body of work, as described in the first paragraph of the discussion. The regulation of let-7 expression has been reported before and its functional role has been investigated with a variety of tools.

Let7g overexpression caused a marked reduction in Rorgt expression in T cells at baseline and in the setting of nCB challenge, and it reduced the frequency of IL17+ producing CD8 T cells in the lung to baseline levels. Yet there was no change in the MLI measurement of histopathology. Is this a robust result? The responses in the experiment shown in Fig. 6C-D are quite muted compared to those shown in Figure 2. The latter also shows a larger number of replicates, and it is unclear whether the data in 6D include measurement from all of the mice tested (e.g. pooled from 2 small experiments) or only mice from one experiment.

Although RORgt is a great candidate to have direct effects on IL-17 expression, the mechanistic understanding of let-7 action on T cell differentiation and cytokine production is limited to this single target. As noted in the discussion, others have identified cytokine receptor targets that may play a role, but it is also likely others among the many targets of let-7 also contribute.

Reviewer #2 (Public Review):

This is a potentially interesting study addressing a possible scale-invariant log-normal characteristic of droplet size distribution in the phase separation behavior of biomolecular condensates. Some of the data presented are valuable and intriguing. However, as it stands, the validity and utility of this study are uncertain because there are serious deficiencies in the execution and presentation of the authors' results. Many of these shortcomings are fundamental, including a lack of clarity in the basic conceptual framework of the study, insufficient justification of the experimental setup, less-than-conclusive experimental evidence, and inadequate discussion of implications of the authors' findings to future experimental and theoretical studies of biomolecular condensates. Accordingly, this reviewer considers that the manuscript should undergo a major revision to address the following. In particular, the discussion should be significantly expanded by including references mentioned below as well as other references pertinent to the issues raised.

1. The theoretical analysis in this study is based on experimental data on condensed droplet size distributions for FUS and α-synuclein. The size data for FUS droplet is indirect as it relies on the assumption that FUS droplet diameter is proportional to fluorescence intensity of labeled FUS (page 10 of manuscript), with fluorescence data adopted from a previously published work by another group (Kar et al. & Pappu, ref.27). Because fluorescence of a droplet is expected to be dependent upon the condensed-phase concentration of FUS, this proportional relationship, even if it holds, must also be modulated by FUS concentration in the droplet. Moreover, why should fluorescence be proportional to diameter but not the cross-sectional area or volume of the FUS droplet, which would be more intuitive? These issues should be clarified. A new measure by microscopy is used to determine the size distribution of condensed α-synuclein; but no microscopy image is shown. It is of critical importance that such raw data (for example microscopy images) be presented for the completeness and reproducibility of the experiment because the entire study relies on the soundness of these experimental measurements.

2. Despite the authors' claim of a universal scaling relationship, the log-log scatter plots in Figure 1 (page 15 of the manuscript) exhibit significant deviations from linearity at low protein concentrations (ρ→0). Given this fact, is universal scaling really valid? Discussion of this behavior is conspicuously absent (except the statement that these data points are excluded in the fit). In any case, the possible origins of these deviations should be thoroughly discussed so that the regime of universal scaling can be properly delineated.

3. Droplet size distribution most likely depends on the time duration after the preparation of the sample. For α-synuclein, "liquid droplet size characterisation images were captured 10 minutes post-liquid droplet formation" (page 9 of the manuscript). Why 10 minutes? Have the authors tried imaging at different time points and, if so, do the distributions at different time points remain essentially the same? If they are different, what is the criterion for focusing only on a particular time point? Information related to these questions should be provided.

4. At least two well-known mechanisms can lead to the time-dependent distribution of liquid droplet sizes: (i) coalescence of droplets in spatial proximity to form a larger droplet, and (ii) Ostwald ripening, i.e., formation of larger droplets concomitant with the dissolution of smaller droplets without fusion of droplets. The implications of these mechanisms on the authors' droplet size distributions should be addressed. Indeed, maintaining a size distribution against these mechanisms in vivo often requires active suppression [Bressloff, Phys Rev E 101, 042804 (2020)] with possible involvement of chemical reactions [Kirschbaum & Zwicker, J R Soc Interface 18, 20210255 (2021)]. These considerations are central to the basic rationale of this study and therefore should be carefully tackled.

5. If coalescence and/or Ostwald ripening do occur, given sufficient time after sample preparation, the condensed phase may become a single large "droplet" or a single liquid layer. Does this occur in the authors' experiments?

6. It is unclear whether the authors aim to address the kinetic phenomenon of liquid droplet formation and evolution or equilibrium properties. The two types of phenomena appear to be conflated in the authors' narrative. Clarification is needed. If this work aims to address time-independent (or infinite-time) equilibrium properties, how are they expected to be related to droplet size distribution, which most likely is time-dependent?

7. The relationship between the potentially time-dependent droplet size distribution and equilibrium properties of ρt and ρc (transition and critical concentrations, respectively) should be better spelled out. An added illustrative figure will be helpful.

8. The authors comment that their findings appear to be inconsistent with Flory-Huggins theory because Flory-Huggins "characterizes droplet formation as a consequence of nucleation ..." (page 8 of the manuscript). Here, three issues need detailed clarification: (i) In what way does Flory-Huggins mandate nucleation? (ii) Why are the findings of apparent scale invariance inconsistent with nucleation? (iii) If liquid droplet formations do not arise from nucleation, what physical mechanism(s) is (are) envisioned by the authors to be underpinning the formation of condensed liquid droplets in protein phase separation?

9. Are any of the authors' findings related to finite-system effects of phase separation [see, e.g., Nilsson & Irbäck, Phys Rev E 101, 022413 (2020)]?

10. Since the authors are using their observation of an apparent scale-invariant droplet size distribution to evaluate phase separation theory, it is important to clarify whether their findings provide any constraint on the shape of coexistence curves (phase diagrams).

11. More specifically, do the authors' findings suggest that the phase diagrams predicted by Flory-Huggins are invalid? Or, are they suggesting that even if the phase diagrams predicted by Flory-Huggins are empirically correct (if verified by experimental testing), they are underpinned by a free energy function different from that of Flory-Huggins? It is important to answer this question to clarify the implications of the authors' findings on equilibrium phase behaviors and the falsifiability of the implications.

12. How about the implications of the authors' findings on other theories of protein phase separation that are based on interactions that are different from the short spatial range interactions treated by Flory-Huggins? For instance, it has been observed that whereas the Flory-Huggins-predicted phase diagrams always convex upward, phase diagrams for charged intrinsically disordered proteins with long spatial range Coulomb interactions exhibit a region that concave upward [Das et al., Phys Chem Chem Phys 20, 28558-28574 (2018)]. Can information be provided by the authors' findings regarding apparent scale-invariant droplet size distribution on the underlying interaction driving the protein molecules toward phase separation?

13. Table S1 (page 4) and Table S2 (page 7) are mentioned in the text but these tables are not in the submitted files.

14. The two systems studied (FUS and α-synuclein) have a single intrinsically disordered protein (IDP) component. It is not clear if the authors expect their claimed scaling relation to be applicable to systems with multiple IDP components and if so, why.

Reviewer #2 (Public Review):

Summary: In the mauscript entitled "The Intricate Relationship of G-Quadruplexes and Pathogenicity Islands: A Window into Bacterial Pathogenicity" Bo Lyu explored the interactions between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) in 89 bacterial genomes through rigorous computational approach. This paper handles an intriguing and complex topic in the field pathogenomics, it has the potential to contribute significantly to the understanding of G4-PAI interactions and bacterial pathogenicity.

Strengths: Chosen research area and summarizing the results through neat illustrations

Weaknesses: I did not find any significant ones.

Reviewer #3 (Public Review):

Smirnova et al. present a cryo-EM structure of human SIRT6 bound to a nucleosome as well as the results from molecular dynamics simulations. The results show that the combined conformational flexibilities of SIRT6 and the N-terminal tail of histone H3 limit the residues with access to the active site, partially explaining the substrate specificity of this sirtuin-class histone deacetylase. Two other groups have recently published cryo-EM structures of SIRT6:nucleosome complexes; this manuscript confirms and complements these previous findings, with the addition of some novel insights into the role of structural flexibility in substrate selection.

Reviewer #2 (Public Review):

Summary:<br /> The authors have conducted an exceptionally informative series of studies investigating the neural basis of interoception in transdiagnostic psychiatric symptoms. By comparing differential and overlapping neural activation during 'top-down' and 'bottom-up' interoceptive tasks, they reveal convergent activation largely localised to the ventral dysgranular subregion ('mid-insula'), which differs in extent between patients and controls, replicating and extending previous suggestions of this region as a central locus of disruption in psychiatric disorders. Their work also reveals different extents of divergent activation in the anterior insula during anticipation of interoceptive disruption. This substantially advances our previous knowledge of the anatomy of interoception and confirms theoretical predictions of the roles of different cytoarchitectural subregions of the insula in interoceptive dysfunction in mental health conditions.

Strengths:<br /> The work is exceptional in terms of breadth and depth, making use of multiple imaging and analysis techniques which are non-standard and go well beyond what is known today. The study is statistically well-powered and the tasks are well-validated in the literature. To my knowledge, these functions of the insula in interoception and mental health have never been compared directly before, so the results are novel and informative for both basic science and psychiatry. The work is strongly theory-driven, building on and directly testing results from influential theories and previous studies. It is likely that the results will strengthen our theoretical models of interoception and advance psychiatric studies of the insula.

Weaknesses:<br /> The study has three current limitations. (1) The interpretation of the resting-state data is not quite as clear-cut as the task-based data - as presented currently, changes could potentially represent fluctuations over time rather than following interoception specifically. In contrast, much stronger conclusions can be drawn from the authors' task-based data. (2) The transdiagnostic sample could be better characterised in terms of diagnostic information, and was almost entirely female; it is also unclear what the effect of psychotropic medications may have been on the results given the effects of (e.g.) serotonergic medication on the BOLD signal. (3) As the authors point out, there may have been task-specific preprocessing/analysis differences that influenced results, for example, due to physiological correction in one but not both tasks.

Reviewer #2 (Public Review):

Sasaki et al. investigated methods to entrain vasomotion in awake wild-type mice across multiple regions of the brain using a horizontally oscillating visual pattern which induces an optokinetic response (HOKR) eye movement. They found that spontaneous vasomotion could be detected in individual vessels of their wild-type mice through either a thinned cranial window or intact skull preparation using a widefield macro-zoom microscope. They showed that low-resolution autofluorescence signals coming from the brain parenchyma could be used to capture vasomotion activity using a macro-zoom microscope or optical fibre, as this signal correlates well with the intensity profile of fluorescently-labelled single vessels. They show that vasomotion can also be entrained across the cortical surface using an oscillating visual stimulus with a range of parameters (with varying temporal frequencies, amplitudes, or spatial cycles), and that the amplitude spectrum of the detected vasomotion frequency increases with repeated training sessions. The authors include some control experiments to rule out fluorescence fluctuations being due to artifacts of eye movement or screen luminance and attempt to demonstrate some functional benefit of vasomotion entraining as HOKR performance improves after repeat training. These data add in an interesting way to the current knowledge base on vasomotion, as the authors demonstrate the ability to entrain vasomotion across multiple brain areas and show some functional significance to vasomotion with regards to information processing as HOKR task performance correlates well with vascular oscillation amplitudes.

The aims of the paper are mostly well supported by the data, but some streamlining of the data presentation would improve overall clarity. The third aim to establish the functional significance of vasomotion in relation to plasticity in information processing could be better supported by the inclusion of some additional control experiments. Specifically:

1) The clarity and comprehensibility of the paper could be significantly enhanced by incorporating additional details in both the introduction and discussion sections. In the introduction, a succinct definition of the frequency range of vasomotion should be provided, as well as a better description of the horizontal optokinetic response (i.e. as they have in the results section in the first paragraph below the 'Entrainment of vasomotion with visual stimuli presentation' sub-heading). The discussion would benefit from the inclusion of a clear summary of the results presented at the start, and the inclusion of stronger justification (i.e. more citations) with regards to the speculation about vasomotion and neuronal plasticity (e.g. paragraph 5 includes no citations).

2) The novel methods for detecting vasomotion using low-resolution imaging techniques are discussed across the first four figures, but this gets a little bit confusing to follow as the authors jump back and forth between the different imaging and analysis techniques they have employed to capture vasomotion. The data presentation could be better streamlined - for instance by presenting only the methods most relevant for the functional dataset (in Figures 5-7), with the additional information regarding the various controls to establish the use of autofluorescence intensity imaging as a valid method for capturing vasomotion reduced to fewer figure panels, or moved to supplementary figures so as to not detract from the main novel findings contributed in this study.

3) The authors heavily rely on representative traces from individual vessels to illustrate their findings, particularly evident in Figures 1-4. While these traces offer a valuable visualization, augmenting their approach by presenting individual data points across the entire dataset, encompassing all animals and vessels, would significantly enhance the robustness of their claims. For instance, in Figures 1 and 2, where average basal and dilated traces are depicted for a representative vessel, supplementing these with graphs showcasing peak values across all measured vessels would enable the authors to convey a more holistic representation of their data. Or in Figure 3, where the amplitude spectrum is presented for individual Texas red fluorescence intensity changes in V1 across novice, trained, and expert mice, incorporating a summary graph featuring the amplitude spectrum value at 0.25Hz for each individual trace (across animals/imaging sessions), followed by statistical analysis, would fortify the strength of their assertions. Moreover, providing explicit details on sample sizes for each individual figure panel (where not a representative trace), including the number of animals or vessels/imaging sessions, would contribute to transparency and aid readers in assessing the generalisability of the findings.

4) In the experiments where mice are classed as "novice", "trained" or "expert", the inclusion of the specific range of the number of training sessions for each category would improve replicability.

5) The authors don't state whether mice were habituated to the imaging set-up prior to the first data collection, as head-fixation and restraint can be stress-inducing for animals, especially upon first exposure, which could impact their neurovascular coupling responses differentially in "novice" versus "trained" imaging sessions (e.g. see Han et al., 2020, DOI: https://doi.org/10.1523/JNEUROSCI.1553-20.2020). The stress associated with a tail vein injection prior to imaging could also partially explain why mice didn't learn very well if Texas Red was injected before the training session. If no habituation was conducted in these experiments, the study would benefit from the inclusion of some control experiments where "novice" responses were compared between habituated and non-habituated animals.

6) The experiments regarding the brain-wide vasomotion entrainment across the cortical surface would benefit from some additional information about how brain regions were identified (e.g. particularly how V1 and V2 were distinguished given how close together they are).

7) Whilst the authors show that HOKR task performance and vasomotion amplitude are increased with repeated training to provide some support to their aim of investigating the functional significance of vasomotion with regards to information processing plasticity, the inclusion of some additional control experiments would provide stronger evidence to address this aim. For instance, if vasomotion signalling is blocked or reduced (e.g. using optogenetics or in an AD mouse model where arteriole amyloid load restricts vasomotion capacity), does flocculus-dependent task performance (e.g. HOKR eye movements) still improve with repeated exposure to the external stimulus.

Reviewer #2 (Public Review):

Summary:<br /> In this manuscript, the authors attempted to study mechanisms of transcription inhibition in cells treated with IR. They observed that, unlike histone chaperone HIRA-dependent transcription inhibition during UV-induced damage, IR-induced transcription inhibition does not depend on HIRA. Through the CRISPR/Cas9 screen, they identified protein neddylation is important for transcription inhibition. By sequencing nascent RNA, they observed that down-regulated transcripts upon IR treatment are largely highly transcribed genes including histone genes and rDNA.

Strengths:<br /> The authors utilized comprehensive approaches to fill in the knowledge gap of IR-induced transcription inhibition.

Weaknesses:<br /> it is not clear that inhibition of histone genes by IR is due to a reduction of S phase progression.

Reviewer #2 (Public Review):

Summary:

There are two potential contributions made by this study, both of which are not fully supported by the data presented. First, that Twist-positive hepatocytes in the midlobular zone are derived from Twist2-expressing cells in embryonic livers via intermediate EpCAM-expressing cells. Second, that there is a population of hepatocytes with mesenchymal features that drive regeneration after various injuries. The concept that mid-lobular hepatocytes are more regenerative in adult injury settings has already been established and this paper further supports that body of knowledge.

Strengths:

There are copious scRNA-seq data that are supportive of the claims, but these analyses were not definitive.

Weaknesses:

1. There is not sufficient evidence to support the following assertion: "markers identified a mesenchymal-hepatocyte hybrid population (13.7% of total hepatocytes) that express signature genes of both lineages." Twist-Cre reporter mice mark hepatocytes and mesenchymal populations, but it is not clear whether or not this means that the hepatocyte population labeled by Twist is mesenchymal. It is very possible for hepatocytes to express mesenchymal genes without being a true hybrid population. There is not much evidence that zone 2 cells are a mix of hepatocyte and mesenchymal. The idea of a hybrid population needs to be defined. The definition probably needs to involve the concept that hybrid cells must have morphologic or functional features of mesenchymal cells, rather than just expressing some genes from each cell type.

Related to this, the authors claim that co-expression of Twist and EpCAM in E10.5 liver cells might support the existence of a hepatomesenchymal cell type. This is possible, but one should note that adult hepatocytes can express EpCAM, especially during ductular reactions, so it is not necessarily a mesenchymal marker per se.

2. The authors assert several times that Twist-Cre mice appear to have no effect on overall liver regeneration phenotypes. They use this to suggest a lack of an effect for heterozygous deletion of Twist by the Cre allele. It is still possible for these mice to have altered lineage tracing results. It is very difficult to rule this out. For example, Axin2-CreER mice did not have any overt liver function or regeneration phenotypes, but the lineage tracing results from these mice differed from other CreER mice.

3. The central problem with this study is that the authors use a Cre strain and not a CreER strain. With a Cre strain, there could be new labeling of Twist-positive cells at multiple later time points. Thus, it is very difficult to assert that the Tomato-positive population at later time points are really descendants of the originally labeled population. It is very difficult to interpret the results of Cre-based lineage tracing experiments.

With this technical limitation in mind, I do not think that there is enough evidence to support the assertion made on page 6: "These findings suggest that EpCAMlow progenitor cells give rise to hepatocytes and MCs." The authors use scRNA-seq trajectory analysis to come to the conclusion that mesenchymal cells give rise to hepatocytes between p1 and p14. Much more evidence is needed before the authors can arrive at this conclusion. It is much more likely that midlobular hepatocytes arise from other hepatocytes. To support their arguments, the authors would have to use a CreER line that exclusively labels mesenchymal cells in the liver, then lineage traces them until p14 to determine if they become hepatocytes. Without such an experiment, I do not think the current experiments are interpretable.

4. The injury experiments are again limited in their interpretability because they do not use CreER. It is very possible that Twist is turned on after CCl4 or surgical injury, and thus new hepatocytes might activate Tomato. It is unclear if previously Tomato-positive midzone hepatocytes were proliferating to increase the Tomato positive population. The authors use expression-based studies to argue against ectopic activation of Twist, but it is very difficult to exclude Cre activation using these types of studies.

Reviewer #2 (Public Review):

Summary:

Here, the authors show that neutral lipids play a role in spermatogenesis. Neutral lipids are components of lipid droplets, which are known to maintain lipid homeostasis, and to be involved in non-gonadal differentiation, survival, and energy. Lipid droplets are present in the testis in mice and Drosophila, but not much is known about the role of lipid droplets during spermatogenesis. The authors show that lipid droplets are present in early differentiating germ cells, and absent in spermatocytes. They further show a cell autonomous role for the lipase brummer in regulating lipid droplets and, in turn, spermatogenesis in the Drosophila testis. The data presented show that a relationship between lipid metabolism and spermatogenesis is congruous in mammals and flies, supporting Drosophila spermatogenesis as an effective model to uncover the role lipid droplets play in the testis.

Strengths and weaknesses:

The authors do a commendably thorough characterization of where lipid droplets are detected in normal testes: located in young somatic cells, and early differentiating germ cells. They use multiple control backgrounds in their analysis, including w[1118], Canton S, and Oregon R, which adds rigor to their interpretations. The authors employ markers that identify which lipid droplets are in somatic cells, and which are in germ cells. The authors use these markers to present measured distances of somatic and germ cell-derived lipid droplets from the hub. Because they can also measure the distance of somatic and germ cells with age-specific markers from the hub, these results allow the authors to correlate position of lipid droplets with the age of cells in which they are present. This analysis is clearly shown and well quantified.

The quantification of lipid droplet distance from the hub is applied well in comparing brummer mutant testes to wild type controls. The authors measure the number of lipid droplets of specific diameters, and the spatial distribution of lipid droplets as a function of distance from the hub. These measurements quantitatively support their findings that lipid droplets are present in an expanded population of cells further from the hub in brummer mutants. The authors further quantify lipid droplets in germline clones of specified ages; the quantitative analysis here is displayed clearly and supports a cell autonomous role for brummer in regulating lipid droplets in spermatocytes.

Data examining testis size and number of spermatids in brummer mutants clearly indicates the importance of regulating lipid droplets to spermatogenesis. The authors show beautiful images supported by rigorous quantification supporting their findings that brummer mutants have both smaller testes with fewer spermatids at both 29 and 25C. There is also significant data supporting defects in testis size, but not spermatid number, in 14-day-old brummer mutant animals compared to controls. Their analysis clearly shows an expanded region beyond the testis apex that includes younger germ cells, supporting a role for lipid droplets influencing germ cell differentiation during spermatogenesis.

The authors present a series of data exploring a cell autonomous role for brummer in the germline, including clonal analysis and tissue specific manipulations. The clonal data indicating increased lipid droplets in spermatocyte clones, and a higher proportion of brummer mutant GSCs at the hub are convincing and supported by quantitation. The authors also show a tissue specific rescue of the brummer testis size phenotype by knocking down mdy specifically in germ cells, which is also supported by statistically significant quantitation. The authors present data examining the number of spermatocyte and post-meiotic clones 14 days after clonal induction. Their finding is significant with a p-value of 0.0496, which they acknowledge is less robust than their other data reported in this study, and could be a result of a low sample size. They indicate that future studies might validate these results with additional samples.

The authors do a beautiful job of validating where they detect brummer-GFP by presenting their own pseudotime analysis of publicly available single cell RNA sequencing data. Their data is presented very clearly, and supports expression of brummer in older somatic and germline cells of the age when lipid droplets are normally not detected. The authors also present a thorough lipidomic analysis of animals lacking brummer to identify triglycerides as an important lipid droplet component regulating spermatogenesis.

Impact:

The authors present data supporting the broad significance of their findings across phyla. This data represents a key strength of this manuscript. The authors show that loss of a conserved triglyceride lipase impacts testis development and spermatogenesis, and that these impacts can be rescued by supplementing diet with medium-chain triglycerides. The authors point out that these findings represent a biological similarity between Drosophila and mice, supporting the relevance of the Drosophila testis as a model for understanding the role of lipid droplets in spermatogenesis. The connection buttresses the relevance of these findings and this model to a broad scientific community.

Reviewer #2 (Public Review):

This work investigates the mechanisms, patterns, and geographical distribution of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum. Rapid diagnostic tests (RDTs) detect P. falciparum histidine-rich protein 2 (PfHRP2) and its paralog PfHRP3 located in subtelomeric regions. However, laboratory and field isolates with deletions of pfhrp2 and pfhrp3 that can escape diagnosis by RDTs are spreading in some regions of Africa. They find that pfhrp2 deletions are less common and likely occur through chromosomal breakage with subsequent telomeric healing. Pfhrp3 deletions are more common and show three distinct patterns: loss of chromosome 13 from pfhrp3 to the telomere with evidence of telomere healing at breakpoint (Asia; Pattern 13-); duplication of a chromosome 5 segment containing pfhrp1 on chromosome 13 through non-allelic homologous recombination (NAHR) (Asia; Pattern 13-5++); and the most common pattern, duplication of a chromosome 11 segment on chromosome 13 through NAHR (Americas/Africa; Pattern 13-11++). The loss of these genes impacts the sensitivity of RDTs, and knowing these patterns and geographic distribution makes it possible to make better decisions for malaria control.

Reviewer #2 (Public Review):

The authors present an image-analysis pipeline for mother-machine data, i.e., for time-lapses of single bacterial cells growing for many generations in one-dimensional microfluidic channels. The pipeline is available as a plugin of the python-based image-analysis platform Napari. The tool comes with two different previously published methods to segment cells (classical image transformation and thresholding as well as UNet-based analysis), which compare qualitatively and quantitatively well with the results of widely accessible tools developed by others (BACNET, DelTA, Omnipose). The tool comes with a graphical user interface and example scripts, which should make it valuable for other mother-machine users, even if this has not been demonstrated yet.

The authors also add a practical overview of how to prepare and conduct mother-machine experiments, citing their previous work, referring to detailed instructions on their github page, and giving more advice on how to load cells using centrifugation.

Finally, the authors emphasize that machine-learning methods for image segmentation reproduce average quantities of training datasets, such as the length at birth or division. Therefore, differences in training can propagate to differences in measured average quantities. This result is not surprising but good to remember before interpreting absolute measurements of cell shape.

Reviewer #2 (Public Review):

This work by Knights et al., makes use of the Cam-CAN dataset to investigate functional compensation during a fluid processing task in older adults, in a fairly large sample of approximately 200 healthy adults ranging from 19 to 87. Using univariate methods, the authors identify two brain regions in which activity increases as a function of both age and performance and conduct further investigations to assess whether the activity of these regions provides information regarding task difficulty. The authors conclude that the cuneal cortex - a region of the brain previously implicated in visual attention - shows evidence of compensation in older adults.

The conclusions of the paper are well supported by the data, and the authors use appropriate statistical analyses. The use of multivariate methods over the last 20 years has demonstrated many effects that would have been missed using more traditional univariate analysis techniques. The data set is also of an appropriate size, and as the authors note, fluid processing is an extremely important domain in the field of cognition in aging, due to its steep decline over aging. However, it might have been nice to see an analysis of a more crystallised intelligence task included too, as a contrast since this is an area that does not demonstrate such a decline (and perhaps continues to improve over aging).