Reviewer #1 (Public Review):

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

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

Reviewer #1 (Public Review):

This manuscript describes the identification of influential organisms on rice growth and an attempt of validation. The analysis of eDNA on rice pot and mimic field provides rice growth promoting organisms. This approach is novel for plant ecology field. However current results did not fully support whether eDNA analysis-based detection of influencing organism.

The strength of this manuscript is to attempt application of eDNA analysis-based plant growth differentiation. The weakness is too preliminary data and experimental set-up to make any conclusion. The trials of authors experiments are ideal. However, the process of data analysis did not meet certain levels. For example, eDNA analysis of different time points on rice growth stages resulted in two influential organisms for rice growth. Then they cultivate two species and applied rice seedlings. Without understanding of fitness and robustness, how we can know the effect of the two species on rice growth.

The authors did not check the fate of two species after introducing into rice. If this is true, it is difficult to link between the rice gene expression after treatments and the effectiveness of two species. I think the validation experiment in 2019 needs to be re-conducted.

Reviewer #1 (Public Review):

This study investigates the context-specificity of facial expressions in three species of macaques to test predictions for the 'social complexity hypothesis for communicative complexity'. This hypothesis has garnered much attention in recent years. A proper test of this hypothesis requires clear definitions of 'communicative complexity' and 'social complexity'. Importantly, these two facets of a society must not be derived from the same data because otherwise, any link between the two would be trivial. For instance, if social complexity is derived from the types of interactions individuals have, and different types of signals accompany these interactions, we would not learn anything from a correlation between social and communicative complexity, as both stem from the same data.

The authors of the present paper make a big step forward in operationalising communicative complexity. They used the Facial Action Coding System to code a large number of facial expressions in macaques. This system allows decomposing facial expressions into different action units, such as 'upper lid raiser', 'upper lip raiser' etc.; these units are closely linked to activating specific muscles or muscle groups. Based on these data, the authors calculated three measures derived from information theory: entropy, specificity and prediction error. These parts of the analysis will be useful for future studies.

The three species of macaque varied in these three dimensions. In terms of entropy, there were differences with regard to context (and if there are these context-specific differences, then why pool the data?). Barbary and Tonkean macaques showed lower specificity than rhesus macaques. Regarding predicting context from the facial signals, a random forest classifier yielded the highest prediction values for rhesus monkeys. These results align with an earlier study by Preuschoft and van Schaik (2000), who found that less despotic species have greater variability in facial expressions and usage.

Crucially, the three species under study are also known to vary in terms of their social tolerance. According to the highly influential framework proposed by Bernard Thierry, the members of the genus Macaca fall along a graded continuum from despotic (grade 1) to highly tolerant (grade 4). The three species chosen for the present study represent grade 1 (rhesus monkeys), grade 3 (Barbary macaques), and grade 4 (Tonkean macaques).

The authors of the present paper define social complexity as equivalent to social tolerance - but how is social tolerance defined? Thierry used aggression and conflict resolution patterns to classify the different macaque species, with the steepness of the rank hierarchy and the degree of nepotism (kin bias) being essential. However, aggression and conflict resolution are accompanied by facial gestures. Thus, the authors are looking at two sides of the same coin when investigating the link between social complexity (as defined by the authors) and communicative complexity. Therefore, I am not convinced that this study makes a significant advance in testing the social complexity for communicative complexity hypothesis. A further weakness is that - despite the careful analysis - only three species were considered; thus, the effective sample size is very small.

Reviewer #1 (Public Review):

This study aims to address the mechanism of eccDNA generation during spermatogenesis in mice. Previous efforts for cataloging eccDNA in mammalian germ cells have provided inconclusive results, particularly in the correlation between meiotic recombination and the generation of eccDNA. The authors employed an established approach (Circle-seq) to enrich and amplify eccDNA for sequencing analyses and reported that sperm eccDNA is not associated with miotic recombination hotspots. Rather, the authors reported that eccDNAs are widespread, and oligonucleosomal DNA fragments from sperm undergoing apoptosis, with the ligation of DNA ends by microhomology-mediated end-joining, would be a major source of eccDNA.

The strength of the study includes evaluating the eccDNA contents not only in sperm but also from earlier stages of cells in spermatogenesis. The differences in eccDNA size peaks between sperm and other progenitors, in particular, the unique peak in sperm around 360 bp, are intriguing. Results from sequencing data analysis were presented elegantly.

I also have critiques. First, the lack of eccDNA quality control step is a concern. Previous studies employed electron microscopy to ensure that DNA species are mostly circular before rolling-circle amplification. Phi29 polymerase is widely used for DNA amplification, including whole genome amplification of linear chromosomal DNA. Phi29 polymerase has a high processivity and strand displacement activity. When those activities occur within a molecule, it creates circular DNA from linear DNA in vitro. In vitro-created eccDNA from linear DNA would be randomly distributed in the genome, which may explain the low incidence of common eccDNA between replicates. Therefore, it will be crucial to show that DNA prior to amplification is dominantly circular. Electron microscopy would be challenging for the study because the relatively small number of cells were processed to enrich eccDNA. An alternative method for quality controls includes spiking samples with linear and circular exogenous DNA and measuring the ratios of circular/linear control DNA before and after column purification/exonuclease digestion. eccDNA isolation procedures can be validated by a very high circular/linear control DNA ratio.

Another critique is regarding the limitation of the study. It is important to remind the readers of the limitations of the study. As the authors mentioned, rolling circle amplification preferentially increases the copy numbers of smaller eccDNA. Therefore, the native composition of eccDNA is skewed. In addition, the candidate eccDNAs are identified by split reads or discordant read pairs. The details of the mapping process are unclear from the methods, but such a method would require reads with high mapping quality; the identification of eccDNA is expected to require sequencing reads that are mapped to genomic locations uniquely with high confidence, and reads mapped to more than one genomic location, such as highly similar repeat sequences or duplications, are eliminated. Such identification criteria would favor eccDNA formed by little or no homology at the junction sequences, and eliminate eccDNA formed by long homologies at the ends, such as eccDNA formed exclusively by satellite DNA. Therefore, it is not surprising that the authors found the dominance of microhomology-mediated eccDNA. It remains to be determined whether small eccDNA with microhomologies are the dominant species of eccDNA in the native composition. In this regard, it is noted that similar procedures of eccDNA enrichment (column purification, exonuclease digestion, and rolling circle amplification ) revealed variable sizes and characteristics of eccDNA in sperm (human from Henriksen et al. or mice from this study), dependent on the methods of sequencing (long-read or short-read sequencing). Considering these limitations, the last sentence of the introduction, "We conclude that germline eccDNAs are formed largely by microhomology mediated ligation of nucleosome protected fragments, and barely contribute to de novo genomic deletions at meiotic recombination hotspots" needs to be revised.

Small eccDNA (microDNA) data from various mouse tissues are available from the study by Dillion et al., (Cell Reports 2015). Authors are encouraged to examine whether the notable findings in this study (oligonucleosomal-sized eccDNA peaks and the association with apoptotic cell death) are unique to sperm or common in the eccDNA from other tissues.

Reviewer #1 (Public Review):

In their manuscript "Spindle assembly checkpoint-dependent mitotic delay is required for cell division in absence of centrosomes," Farrell and colleagues employ carefully chosen approaches to assay mitotic timeliness in the absence of centrosomes in mammalian culture cells, namely the mechanism behind it and its function. The authors acknowledge prior work well and present their data succinctly, clearly, and with a clear logical flow of experiments. The experiments are thoughtfully designed and presented, with appropriate controls in place.

The authors' model whereby centrosome separation and its early definition of poles mediates timely mitosis without relying on a SAC-dependent delay is compelling, and the authors' data are consistent with it. They show using two different MPS1 inhibitors that acentrosomal cell division fails, supporting their claims that a SAC-dependent delay is required in these instances. Furthermore, they show that reintroducing a time delay is sufficient to restore cell division, but inhibiting a different aspect of SAC function does not restore cell division. Next, the authors rule out polyploidy as a potential confounding factor for requiring a SAC-dependent delay, and instead demonstrate that inhibiting centrosome separation by Eg5 inhibition is sufficient to require this delay for mitotic progression. The authors' findings overall support their proposed model.

Probing what aspects of centrosomes protect against a requirement for SAC-dependent delays would strengthen the work and specifically the conclusion that centrosomes provide "two-ness". For example, the authors could examine division in a population of cells with only one centrosome. Seeing some restoration of mitotic progression in the absence of SAC-dependent delays would suggest that even one centrosome with uninhibited Eg5 is sufficient to negate SAC-dependent delays, and would limit models for what exactly centrosomes contribute. This would help disentangle the roles of actual centrosomes and their biochemical cues, Eg5-driven centrosome separation, and early definition of poles on mitotic progression in the absence of SAC-dependent delays. Making a high fraction of cells with one centrosome could be achieved by using centrinone for a shorter time.

Reviewer #1 (Public Review):

Based on a recent report of spontaneous and reversible remapping of spatial representations in the enthorhinal cortex (Low et al 2021), this study sets out to examine possible mechanisms by which a network can simultaneously represent a positional variable and an uncorrelated binary internal state. To this end, the authors analyse the geometry of activity in recurrent neural networks trained to simultaneously encode an estimate of position in a one-dimensional track and a transiently-cued binary variable. They find that network activity is organised along two separate ring manifolds. The key result is that these two manifolds are significantly more aligned than expected by chance, as previously found in neural recordings. Importantly, the authors show that this is not a direct consequence of the design of the model, and clarify scenarios by which weaker alignment could be achieved. The model is then extended to a two-dimensional track, and to more than two internal variables. The latter case is compared with experimental data that had not been previously analysed.

Strengths:<br /> - rigorous and careful analysis of activity in trained recurrent neural networks<br /> - particular care is taken to show that the obtained results are not a necessary consequence of the design of the model<br /> - the writing is very clear and pleasant to read<br /> - close comparison with experimental data<br /> - extensions beyond the situations studied in experiments (two-dimensional track, more than two internal states)

Weaknesses:<br /> - no major weaknesses<br /> - (minor) the comparison with previous models of remapping could be expanded

Altogether the conclusions claimed by the authors seem to be strongly supported and convincing.

Reviewer #1 (Public Review):

Meta-cognition, and difficulty judgments specifically, is an important part of daily decision-making. When facing two competing tasks, individuals often need to make quick judgments on which task they should approach (whether their goal is to complete an easy or a difficult task).

In the study, subjects face two perceptual tasks on the same screen. Each task is a cloud of dots with a dominating color (yellow or blue), with a varying degree of domination - so each cloud (as a representation of a task where the subject has to judge which color is dominant) can be seen an easy or a difficult task. Observing both, the subject has to decide which one is easier.

It is well-known that choices and response times in each separate task can be described by a drift-diffusion model, where the decision maker accumulates evidence toward one of the decisions ("blue" or "yellow") over time, making a choice when the accumulated evidence reaches a predetermined bound. However, we do not know what happens when an individual has to make two such judgments at the same time, without actually making a choice, but simply deciding which task would have stronger evidence toward one of the options (so would be easier to solve).

It is clear that the degree of color dominance ("color strength" in the study's terms) of both clouds should affect the decision on which task is easier, as well as the total decision time. Experiment 1 clearly shows that color strength has a simple cumulative effect on choice: cloud 1 is more likely to be chosen if it is easier and cloud 2 is harder. Response times, however, show a more complex interactive pattern: when cloud 2 is hard, easier cloud 1 produces faster decisions. When cloud 2 is easy, easier cloud 1 produces slower decisions.

The study explores several models that explain this effect. The best-fitting model (the Difference model is the paper's terminology) assumes that the decision-maker accumulates evidence in both clouds simultaneously and makes a difficulty judgment as soon as the difference between the values of these decision variables reaches a certain threshold. Another potential model that provides a slightly worse fit to the data is a two-step model. First, the decision maker evaluates the dominant color of each cloud, then judges the difficulty based on this information.

Importantly, the study explores an optimal model based on the Markov decision processes approach. This model shows a very similar qualitative pattern in RT predictions but is too complex to fit to the real data. It is hard to judge from the results of the study how the models identified above are specifically related to the optimal model - possibly, the fact that simple approaches such as the Difference model fit the data best could suggest the existence of some cognitive constraints that play a role in difficulty judgments.

The Difference model produces a well-defined qualitative prediction: if the dominant color of both clouds is known to the decision maker, the overall RT effect (hard-hard trials are slower than easy-easy trials) should disappear. Essentially, that turns the model into the second stage of the two-stage model, where the decision maker learns the dominant colors first. The data from Experiment 2 impressively confirms that prediction and provides a good demonstration of how the model can explain the data out-of-sample with a predicted change in context.

Overall, the study provides a very coherent and clean set of predictions and analyses that advance our understanding of meta-cognition. The field would benefit from further exploration of differences between the models presented and new competing predictions (for instance, exploring how the sequential presentation of stimuli or attentional behavior can impact such judgments). Finally, the study provides a solid foundation for future neuroimaging investigations.

Reviewer #1 (Public Review):

This paper performed a functional analysis of the poorly characterized pseudo-phosphatase Styxl2, one of the targets of the Jak/Stat pathway in muscle cells. The authors propose that Styxl2 is essential for de novo sarcomere assembly by regulating autophagic degradation of non-muscle myosin IIs (NM IIs). Although a previous study by Fero et al. (2014) has already reported that Styxl2 is essential for the integrity of sarcomeres, this study provides new mechanistic insights into the phenomenon. In vivo studies in this manuscript are compelling; however, I feel the contribution of autophagy in the degradation of NM IIs is still unclear.

Major concerns:

1) The contribution of autophagy in the degradation of Myh9 is still unclear to this reviewer. It has been reported that autophagy is dispensable for sarcomere assembly in mice (Cell Metab, 2009, PMID; 1994508). In Fig. 7A, the authors showed that overexpressed Styxl2 downregulated the amount of ectopically expressed Myh9 in an ATG5-dependent manner in C2C12 cells; however, the experiment is far from a physiological condition. Therefore, the authors should test ATG5 knockdown and the genetic interaction between Styxl2 and ATG5 in vivo. That is, 1) loss of ATG5 on sarcomere assembly in zebrafish, and 2) the genetic interaction between Styxl2 and ATG5; co-injection of Styxl2 mRNA and ATG5-MO into the zebrafish embryos.

2) As referenced, Yamamoto et al. reported that Myh9 is degraded by autophagy. Mechanistically, Nek9 acts as an autophagic adaptor that bridges Atg8 and Myh9 through interactions with both. Inconsistent with the model, the authors mentioned on page 12, lines 365-367, "A recent report showed that Myh9 could also undergo Nek9-mediated selective autophagy (Yamamoto et al., 2021), suggesting that Myh9 is ubiquitinated". I think it is not yet explored whether autophagic degradation of Myh9 requires its ubiquitination. Moreover, I cannot judge whether Myh9 is ubiquitinated in a Styxl2-dependent manner from the data in Fig. 7C. The author should test whether Nek9 is required for Myh9 degradation in muscles. If Nek plays a role in the Myh9 degradation, it would be better to remove Fig. 7C.

3) In Fig. 5F, the protein level of Styxl2 and Myh10 should be checked because the efficiency of Myh10-MO was not shown anywhere in this manuscript.

Reviewer #1 (Public Review):

C. elegans is a pre-eminent model for developmental genetics, and its invariant lineage makes it possible in theory to define molecular features such as gene expression comprehensively and at single cell resolution across the organism.

Previously published single-cell RNA-seq studies have mapped gene expression across the lineage through the 16-cell stage (Tintori et al 2017, Hashimshony et al 2016), and at later stages (Packer et al 2019, with good coverage starting at the 100-cell stage and some coverage at the ~50-cell stage). This left the critical period around gastrulation (~28-cell and ~50-cell) without comprehensive transcriptome data. This study covers this gap with a heroic effort involving the manual isolation and analysis of over 800 cells from embryos of known stage, combined with painstaking curation using known markers from small scale studies and larger imaging-based expression atlases. Importantly, the dataset overlaps at early and late stages with data from prior studies.

The data quality and overlap with Tintori and Packer datasets both appear high, but to make this inference required additional analysis from Supplemental Table 6 by this reviewer as it is not explored or described in the manuscript. Analyses demonstrating continuity with these datasets would greatly increase the value of the resource.

The authors show that specific lineages and stages preferentially express TFs with different classes of DNA binding domains. This extends previous work implicating homeodomains as preferentially involved in nervous system patterning and as enriched in neural and muscle progenitors in mid-stage embryos.

They also show that C. elegans homologs of Drosophila early embryonic regulators (which function based on spatial position in that system) tend to also be patterned in early C. elegans embryos, but with lineage-specific patterns. This conserved use of regulators would be fairly remarkable given the dramatically different developmental modes in these two species, although this observation is not backed up by quantitative analyses.

Finally, there is an argument that combinations of TFs expressed in lineage-specific patterns give rise to "stripe" patterns. This section is also not based on statistical analyses but suggests the possibility that lineage and positional regulation may be more convoluted than was previously thought.

Reviewer #1 (Public Review):

The authors of this well-described publication provided strong evidence that current DNA-based microbial genomics methodologies have an inherent constraint. These approaches cannot detect the source of sequenced DNA, and they fail to demonstrate the origin of sequenced DNA from live or non-viable bacteria. Moreover, scientists proved in people and mice that live bacteria for the most part remained within hair follicles rather than on the skin's surface. Overall, this study is of excellent quality and has broad implications beyond a particular subject.

Strengths:

The study is well-designed, and the experimental methods are well-described.<br /> The results are presented clearly and are supported by statistical analyses.<br /> The study's findings are novel and have important implications for understanding the skin microbiome and the biology of the skin.

Weakness:

RNA-based NGS could parallelly study the results of this DNA-based microbiome study. The bulk RNA-Seq can sequence thousands of transcripts from each viable bacterium and match them with the bacterial genome and transcriptome references. It is one of the best confirmatory methods for showing the diversity of viable cutaneous bacteria.

Reviewer #1 (Public Review):

Previous reports suggested an association between ceramide accumulation in skeletal muscle and disruption of insulin signaling and metabolic dysregulation. Mechanistically, however, how intracellular ceramide attenuates insulin action and reduces metabolism is not fully understood. It was suggested that insulin receptor (IR) signaling to PI3-K/AKT is inhibited by elevated intracellular ceramide. However, other studies failed to demonstrate an inhibitory effect of ceramide on PI3K/AKT. More recently, a study was published describing that intracellular localization of diacylglycerols and sphingolipids influences insulin sensitivity and mitochondrial function in human skeletal muscle (PMID: 29415895). In the present study, Diaz-Vegas and colleagues used an in vitro system to investigate this topic further and better understand how intracellular ceramide accumulation causes cellular insulin resistance and metabolic dysregulations in cultured myocytes.

The authors applied multiple methods to achieve this goal. Among these procedures are:

1) The overexpression of enzymes involved in mitochondrial ceramide synthesis and degradation;

2) Treatments of myocytes with different pharmacological tools to validate their findings;

3) Mitochondrial proteomics and lipidomics analyses.

The effects of these experimental conditions and treatment on intracellular lipids contents, mitochondrial functions, and insulin signaling in myocytes were then evaluated.

Findings:

The authors' findings indicate that incubation of myocytes with palmitate increases mitochondrial ceramide and reduces the insulin-stimulated GLUT4-HA translocation to the myocyte surface without affecting AKT activation. The elevation in mitochondrial ceramide lowers the coenzyme Q levels e depletes the electron transport chain (ETC) components, impairing mitochondrial respiration. Such mitochondrial dysfunction appears to attenuate the translocation of GLUT4-HA to the plasma membrane of the L6-myotubule. Also, mitochondrial proteomic analysis revealed an association of insulin sensitivity with mitochondrial ceramide and ETC expression levels in human muscle.

Based on these findings, the authors propose a mechanism whereby the building up of ceramide inside mitochondria depletes CoQ and compromise mitochondrial respiratory complexes, raising ROS. The resulting mitochondrial dysfunction causes insulin resistance in cultured myocytes. They postulate that CoQ depletion links ceramides with insulin resistance and define the respirasome as a critical connection between ceramides and mitochondrial dysfunction.

Relevance and critiques:

This original study provides direct evidence that mitochondrial ceramide accumulation depletes CoQ and downregulates multiple ETC components in myocytes. Consequently, elevation in the levels of reactive oxygen species (ROS) and mitochondrial dysfunctions occur. The authors proposed that such mitochondrial dysregulation attenuates insulin-stimulated GLUT4 translocation to the plasma membrane of L6-myotubules. Moreover, mitochondrial ceramide accumulation does not affect insulin action on AKT activation.

Overall, this is a well-done study, showing that in obesity, elevated mitochondrial ceramide suppresses mitochondrial function and attenuates insulin action on glucose transporter GLUT4 translocation into the myocyte surface. The main conclusion is supported by the results presented. The study also applied multiple methods and described several experiments designed to test the author's central hypothesis.

Importantly, these new findings shed light on possible cellular mechanisms whereby ectopic fat deposition in skeletal muscle drives insulin resistance and metabolism dysregulation. The results demonstrating that alterations in mitochondrial ceramide are sufficient to attenuate insulin-stimulated GLUT4 trafficking in cultured myocytes are very interesting. Well-done.

Comments for further discussion and suggestions:

Although the authors' results suggest that higher mitochondrial ceramide levels suppress cellular insulin sensitivity, they rely solely on a partial inhibition (i.e., 30%) of insulin-stimulated GLUT4-HA translocation in L6 myocytes. It would be critical to examine how much the increased mitochondrial ceramide would inhibit insulin-induced glucose uptake in myocytes using radiolabel deoxy-glucose.

Another important question to be addressed is whether glycogen synthesis is affected in myocytes under these experimental conditions. Results demonstrating reductions in insulin-stimulated glucose transport and glycogen synthesis in myocytes with dysfunctional mitochondria due to ceramide accumulation would further support the authors' claim.

In addition, it would be critical to assess whether the increased mitochondrial ceramide and consequent lowering of energy levels affect all exocytic pathways in L6 myoblasts or just the GLUT4 trafficking. Is the secretory pathway also disrupted under these conditions?

Reviewer #1 (Public Review):

In this manuscript, Elkind et al. use a deep learning segmentation algorithm trained on detecting putative cell nuclei in mouse brains to count cells in the Allen Mouse Brain Connectivity Atlas. The Allen Mouse Brain Connectivity Atlas is a dataset compromising hundreds of mice brains. The authors use this increased statistical power for detecting differences in volume, cell count, and cell density between strains (C57BL/6J and FVB.CD1) as well as sex differences.

Both volume, cell count, and cell density are regularly used in neuroanatomy to normalize or benchmark results so having a large available dataset for others to compare their data would be a useful resource. The trained segmentation algorithm might also find utility in assays where investigators for one reason or another can't dedicate an entire labeled channel to count cell nuclei.

Nevertheless, because of technical reasons, I find the current work problematic.

Major:

The authors make use of the "red" channel from the Allen Mouse Brain Connectivity Project (AMBCP). The AMBCP was acquired using two-photon tomography with the TissueCyte 1000 system (http://help.brain-map.org/download/attachments/2818171/Connectivity_Overview.pdf?version=2&modificationDate=1489022310670&api=v2). The sample is illuminated at 925 nm wavelength and the channel the authors describe as autofluorescence is collected through a 593/40 nm bandpass filter. The authors go on to describe their rationale for using this channel for quantifying cell nuclei:<br /> "We noticed that the red (background) channel of STPT images, taken for the purpose of atlas alignment, typically features dark, round-like objects resembling cell nuclei. We had observed this phenomenon in our own imaging of mouse brains but found little more than anecdotal mentions of it in the literature8,9,10,11".<br /> The authors here cite a Scientific Reports paper from 2021 with 11 citations, a Journal of Clinical Pathology paper from 2005 with 87 citations, and lastly a paper in Laboratory Investigation from 2016 with 41 citations. The authors completely fail to cite the work from Watt Webb's group (co-inventor of 2p microscopy) in PNAS from 2003 that entirely described the phenomena of native fluorescence by multiphoton-excitation (https://www.pnas.org/doi/10.1073/pnas.0832308100 ), citations so far: 1959 citations. This is either indicative of poor scholarship or an attempt to describe something as novel. Either way, the native fluorescence and second harmonic generation from multiphoton illumination are perfectly characterized by Webb and colleagues and they clearly show the differential effect on nucleosides, retinol, indoleamines, and collagen. This is also where the authors should have paid more attention to discrepancies in their own data when correlated to well-established cell nuclei markers (Murakami et al). The authors will note "black large spots" in the data at specific anatomical regions and structures, like the fornix and stria medullaris:<br /> https://connectivity.brain-map.org/projection/experiment/siv/263780729?imageId=263780960&imageType=TWO_PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=15702&y=18833&z=5

which is not reproduced in for example the Allen Reference Atlas H&E staining:<br /> http://atlas.brain-map.org/atlas?atlas=1&plate=100960284#atlas=1&plate=100960284&resolution=4.19&x=5507.4000244140625&y=5903.39990234375&zoom=-2

In connection here notice the poor signal in the 2p "autofluorescence" within the paraventricular nucleus:<br /> https://connectivity.brain-map.org/projection/experiment/siv/263780729?imageId=263780960&imageType=TWO_PHOTON,SEGMENTATION&initImage=TWO_PHOTON&x=15702&y=17833&z=6

and then compare it to the H&E staining:<br /> http://atlas.brain-map.org/atlas?atlas=1&plate=100960280#atlas=1&plate=100960276&resolution=1.50&x=5342.476283482143&y=5368.023856026786&zoom=0

These multiphoton-specific signals are especially pronounced in the pons and medulla which makes quantification especially dubious, which is even apparent simply from looking at Figure 1c in the manuscript. The authors here use the correlation on log-log coordinates between their data and that of Murakami et al to argue that the method has validity. However, the variance explained here is R^2 = 0.74 which is very poor given the log-log coordinates. A more valid metric would use linear coordinates and computing the ICC and interpret it according to established guidelines (e.g. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913118/).

In addition to the above concern, the authors argue that the large sample size of the AMBCP is what would enable them to find statistically significant small effect sizes that might have gone undetected in the literature. However, this argument falls flat once we examine some of the main findings the authors report. Although the authors do not directly report measures of dispersion we can estimate it from the figures and then arrive at the sample size needed to find the reported effect size. For example, the effect that describes ORBvl2/3 volume is larger in female mice compared to males would only require n=13 mice at the desired power of 0.8. Likewise, the sample size needed to detect the increased BST volume in male mice looks to be roughly n=16 mice at the desired power of 0.8. Both of these estimates are well within what is a reasonable sample size to expect in an ordinary study. This begs the question: why did the authors simply not verify some of their main findings in an independent sample obtained through traditional ways to quantify volume and cell density since it is well within reach? Such validation would strengthen the arguments of the paper.

Reviewer #1 (Public Review):

Alignment between high dimensional data which express their dynamics in a subspace is a challenge which has recently been addressed both with analytic-based solutions like the Procrustes transformation, and, most interestingly, via deep learning approaches based on adversarial networks. The authors have previously proposed an adversarial network approach for alignment which relied on first dimensionally-reducing the binned neural spikes using an autoencoder. Here, they use an alternative approach to align data without use of an initial dimensional-reduction step.

The results are fairly clear - the Cycle-GAN approach works better than their previous ADAN approach and one based on dimensionality reduction followed by the Procrustes transform. In general, a criticism of this entire field is to understand what alignment teaches us about the brain or how it specifically will be used in a BCI context.

There are a few issues with the paper.

1.) To increase the impact of their work, the investigators have now used it to align data in multiple types of tasks. There was an unanswered question about this related to neuroscience - does alignment in one task predict alignment for another?

2) Investigators use decoding as a way of comparing alignment performance. The description of the cycle GAN was not super detailed, and it wasn't clear whether there was any dynamic information stored in the network that might create questions of causality in actual use. It seems that input is simply the neural activity at a current time point rather than neural activity across the trial, which would alleviate this concern. However, they mention temporal alignment but never describe in detail whether all periods of spikes are properly modeled by the system or if only subsets of data (specific portions of task or non-task time) will work. Perhaps this is more a question of the Wiener filter, for which precise details are missing.

3) In general, precise details of the algorithms should have been provided.

4) Cross validation for day-0 alignment is not explained.

5) Details of statistical tests is not provided.

6) (minor) The idea that for neurons that have disappeared that the CycleGAN can "infer their response properties", seems an incorrect description. A proper description should be that it "hallucinates" their response properties?

Reviewer #1 (Public Review):

The study by Yang et al. reports a new mechanistic role of vinculin in inhibiting the Mef2c nuclear translation and sclerostin expression in osteocytes and promoting bone formation. The authors showed the reduction of vinculin in aged bone human bone samples. A 10kb DMP-1-Cre mouse model was generated that deleted vinculin in osteocytes. They found that vinculin deletion caused bone loss and decreased bone formation associated with increased sclerostin expression. This increase does not affect the protein level of transcription factor Met2c but interestingly enhances nuclear translocation. Vinculin is interested in Mef2c and appears to retain Mef2c in the cytosol. As expected, as a component of the mechanosensory focal adhesion complex, bone formation via tibial loading was decreased in vinculin deletion. Intriguingly, the bone loss associated with estrogen deficiency through ovariectomy was attenuated. Overall, the study unveiled an important role concerning a key player of focal adhesion and the study was well designed and executed. The paper would be strengthened by including a more thorough discussion including variables such as male vs. female, and cortical vs. trabecular bone as the vinculin deletion appeared to primarily affect trabecular bone while mechanical loading exerts anabolic effects on both bone types. The effect of estrogen deficiency effect is interesting and is worth some discussion.

Strengths:<br /> The paper shows a novel mechanism that vinculin retains Mef2c in the cytosol via protein interaction to prevent it from migrating to the nucleus and increases transcription of sclerostin, an inhibitory factor for Wnt/β-catenin signaling, a critical pathway for osteoblast activity and bone formation.<br /> They employed various in vivo and in vitro models as well as human tissue samples including generating conditional knockout of vinculin in osteocytes in vivo and vinculin gene knockdown in MLO-Y4 cells. They also used physiological/pathological relevant models, tibial loading, and ovariectomy to study the role of vinculin under mechanical loading and estrogen deficiency. The adopted standard techniques to study bone properties include microCT, bone formation, bone histomorphometry, histochemistry as well as biochemical assays such as immunoprecipitation, ChIP assays, etc.

The study is comprehensive and thorough and the noticeable uniqueness is that after observing the phenotypes from in vivo data, they further explored the underlying mechanisms using cell models. The experiments in general are well-designed and presented with adequate repeats and statistical analysis. The paper is also logically written and the figures were clearly labeled.

Minor weaknesses:<br /> More discussion is necessary concerning the potential difference in responses between male and female. Most of the studies were conducted in male mice except ovariectomy mice.<br /> It is interesting that the cKO of vinculin in osteocytes primarily affects trabecular bones with limited effect on cortical bones. However, sclerostin is increased in cortical bones. The promotion of bone formation by mechanical loading appears to affect both cortical and trabecular bones. If focal adhesion is a key mechanosensory complex, how to reconcile the different responses in the cKO model?<br /> The OVX response is interesting and it is worthwhile to elaborate more regarding the potential underlying mechanism and what's the relationship between estrogen and mechanical loading and if the action of estrogen on vinculin shares any similar mechanisms with mechanical loading, etc.

Reviewer #1 (Public Review):

This study represents in exciting collaboration between two young independent scientists in Uruguay and Japan. Trigo and Kawaguchi provide evidence for the presynaptic modulation of the opening-probability of calcium channels as a major mechanism of digital-analog coupling in immature cerebellar molecular layer interneurons (MLI). Applying a combination of electrophysiological methods including direct axonal whole-cell patch-clamp recordings and glutamate photolysis in acute brain slices and dissociated cultured neurons, the authors provide the following empirical findings: 1) Spontaneous and evoked EPSPs are reliably transmitted into the presynaptic compartment. The amplitude of the spontaneous EPSPs decayed with a length constant of 180 µm in the axon. 2) Physiologically relevant short and subthreshold (< 10 mV) depolarizations before action potentials ('pre-AP') increase the release probability and subsequently short-term depression at the MLI-Purkinje cell synapse without changing the duration of APs and just a minor reduction in amplitude of APs (< 10%). 3) The pre-AP subthreshold depolarizations subsequently increase the amplitude of AP-induced presynaptic calcium currents and GABAergic postsynaptic currents. 4) A short interval of only 3 ms duration between the pre-AP depolarization and the AP blocks the analog coupling. 5) A biophysical model of presynaptic calcium channel gating is proposed, which involves depolarization-induced intermediate gating steps that increase the probability of activating the channels during the AP.

A particular strength of this study is the large data set of technically very challenging direct recordings from small presynaptic terminals. The proposed mechanism provides an innovative explanation for the experimental findings. The most innovative experiments might be those with a 3-ms-gap between the pre-APs and APs. At this synapse, elevated residual intracellular calcium concentration was previously shown to mediate analog coding (https://doi.org/10.1523/JNEUROSCI.5127-10.2011). However, the elevated residual calcium cannot explain the surprising block of analog coding by a 3-ms-gap in the depolarization, because intracellular calcium signals decay with kinetics in the range of 100 ms. Both mechanisms (residual calcium and priming of calcium channels) are probably operating in parallel and future studies should resolve the exact interplay of both mechanisms. A potential weakness of the study is that the proposed priming of calcium channels is not shown explicitly to be able to explain the experimental data. Quantitive simulations of calcium channel gating states were only performed in steady-state but not in a time-dependent manner during pre-APs and APs.

Reviewer #1 (Public Review):

This manuscript investigates the mechanisms of 'summiting disease' using a previously characterised Drosophila model. The authors also show that E. muscae infiltrates the brain likey through a defective blood-brain barrier and populates regions of the brain in the medial protocerebrum. It likely releases metabolites into the haemolymph of summiting flies that has the ability to induce summiting in uninfected flies. They also show that a burst of locomotor activity precedes death. To understand the circuit basis of this, they perform a screen of more than a hundred neuronal lines and genes to identify an active DPN1>pars intercerebralis neurons> corpora allata>JH axis as being invovled in the summiting behaviour while not affecting death.

Reviewer #1 (Public Review):

The manuscript by Gochman and colleagues reports the discovery of a very strong sensitization of TRPV2 channels by the herbal compound cannabidiol (CBD) to activation by the synthetic agonist 2-aminoethoxydiphenyl borate (2-APB). Using patch-clamp electrophysiology the authors show that the ~100-fold enhancement by micromolar CBD of TRPV2 current responses to low concentrations of 2-APB reflects a robust increase in apparent affinity for the latter agonist. Cryo-EM structures of TRPV2 in lipid nanodiscs in the presence of both drugs report two-channel conformations. One conformation resembles previously solved structures whereas the second conformation reveals two distinct CBD binding sites per subunit, as well as changes in the conformation of the S4-S5 linker. Interestingly, although TRPV1 and TRPV3 are highly homologous to TRPV2 and both CBD binding sites are relatively conserved, the CBD-induced sensitization towards 2-APB is observable only for TRPV3 but not for TRPV1. Moreover, the simultaneous substitution of non-conserved residues in the CBD binding sites and the pore region of TRPV1 with the amino acids present in TRPV2 fails to confirm strong CBD-induced sensitization. The authors conclude that CBD-dependent sensitization of TRPV2 channels depends on structural features of the channel that are not restricted to the CBD binding site but involve multiple channel regions.

These are important findings that promote our understanding of the molecular mechanisms of TRPV family channels, and the data provide convincing evidence for the conclusions.

Reviewer #1 (Public Review):

The authors' objectives were to identify the features of uORFs that determine their effects on the translation of the main ORF found in the same transcript. The major strengths of the paper are the creative and powerful experimental platforms used to measure translation, the computational approaches used to identify the key features that determine the effect of uORFs on translation and the comparative analysis of two closely related species to understand how uORF activity evolves. The authors successfully and convincingly identify features associated with the regulatory effects of uORFs and have results suggesting that uORFs that would have strong repressive effects would be selected against. Although these insights regarding evolution are very interesting and may contribute to our understanding of regulatory evolution, at a level that is rarely explored, this section could benefit from additional analyses of existing data to fully support the conclusions. Another aspect that would need to be considered is the possible interaction between the uORFs and the main ORFs. Here, all experiments are performed with the same main ORFs (YFP) for practical and essential reasons, but it would be useful to know whether some uORF features would have effects whose sign and magnitude may depend on which main ORFs they associate with. Overall, there are several areas in which the authors' claims or conclusions are not fully justified and require either additional statistical analysis or new experimentation.

Reviewer #1 (Public Review):

The manuscript by Muthana et al. describes the effect of injection of an antibody specific for human CTLA4 conjugated to a cytotoxic molecule (Ipi-DM1) in knock-in mice expressing human CTLA4. The authors show that Ipi-DM1 administration causes a partial decrease (about 50% in absolute number) of mature B cells in blood and bone marrow 9-14 days after the beginning of treatment. Ipi-DM1 also results in a partial decrease in Foxp3+ Tregs (about 40% in absolute number) and a slight increase in activation of conventional T cells (Tconvs) in the blood at D9. Tconv depletion, CTLA4-Ig or anti-TNF mAb partially prevents the effect of ipi-DM1 on B cells. This work is interesting but has the following major limitations:

1- This work could have been of more interest if the Ipi-DM1 molecule would be used in the clinic. As this is not the case, the intimate mechanism of the effect of this molecule in mice is of reduced interest.<br /> 2- The fact that a partial deletion of Tregs is associated with activation of Tconvs and a decrease in B cells has been published several times and is therefore not new. According to the authors, their work would be the first to show that activation of Tconvs would lead to B cell depletion. However, this is shown in an indirect way and the mechanisms are not really elucidated. Indeed, this work shows a correlation between an increase in Tconv activation and a decrease in the number of B cells in the blood. The experiments to try to show a causal link are of 2 types: deletion of T cells (Fig 4) and blocking T cell activation with CTLA4-Ig (Fig 5) (neutralization of TNF addresses another question). Neither of these 2 experiments is totally convincing. Indeed, the absence of B cell depletion when T cells are deleted can be explained by other mechanisms than the preservation of B cell destruction by activated T cells. The phenomenon could be explained by B cell recirculation to lymphoid tissues or an effect of massive T cell death for example. The experiment shown in Fig. 5 with Belatacept is more convincing because this time the effect is targeted to activated T cells only. However, the prevention of B cell ablation is only partial. Again, since only blood is analyzed, other mechanisms could explain the B cell loss, such as their recirculation in lymphoid tissues.<br /> 3- It is disappointing that only the blood (and sometimes the bone marrow) was studied in this work. The interest of doing experiments in mice is to have access to many tissues such as the spleen, lymph nodes, colon, lung, and liver. To conclude that there is B cell deletion without showing lymphoid organs (where the majority of B cells reside) is insufficient. As discussed above, the drop in B cells in the blood could be due to their recirculation in lymphoid organs. In addition, there is no measurement of functional B cells activity. Do mice treated with Ipi-DM1 have a decreased ability to develop an antibody response following immunization?<br /> 4- Although it is difficult to study in vivo, there is not a single evidence of increased B cell death after injection of Ipi-DM1.<br /> 5- In most of the experiments, B cells are quantified with the B220 marker alone, but this marker, in some cases, can be expressed by other cells. It would have been preferable to use a marker more specific to B cells such as CD19 for example.

In conclusion, the concept that T cell activation can lead to B cell deletion is interesting but this study shows it only in an indirect and incomplete way.

Reviewer #1 (Public Review):

This study was designed to examine the bypass of Ras/Erk signaling defects that enable limited regeneration in a mouse model of hepatic regeneration. The authors show that this hepatocyte proliferation is marked by expression of CD133 by groups of cells. The CD133 appears to be located on intracellular vesicles associated with microtubules. These vesicles are loaded with mRNA. The authors conclude that the CD133 vesicles mediate an intercellular signaling pathway that supports cell proliferation. These are new observations that have broad significance to the fields of regeneration and cancer.

The primary observation is that the limited regeneration observed in livers with Ras/Erk signaling defects is associated with CD133 expression by groups of cells. The functional significance of CD133 was tested using Prom1 KO mice - the data presented are convincing.

The major weakness of the study is that some molecular mechanistic details are unclear - this is, in part, due to the extensive new biology that is described. Nevertheless, the data used to support some key points in this study are unclear:

a) What is the evidence that the observed CD133 groups of cells are not due to clonal growth. Is this conclusion based on the time course (the groups appear more rapidly than proliferation) or is this based on the GFP clonal analysis?

b) What is the evidence that the CD133 vesicles mediate intercellular communication. This is an exciting hypothesis, but what is the evidence that this happens? Is this inferred from IEG mRNA diversity? or some other data. Is there direct evidence of transfer - for example, the does the GFP clonal analysis show transfer of GFP that is not mediated by clonal proliferation? Moreover, since the hepatocytes are isogenic, what distinguishes the donor and recipient cells?

Increased clarity concerning what is hypothesis and what is directly supported by data - would improve the presentation of this study.

Reviewer #1 (Public Review):

This is an interesting manuscript that proposes a new approach to for accounting for viral diversity within hosts in phylogenetic analyses of pathogens. Concretely, the authors consider sites for which a minor allele exist as an additional base in the substitution model. For example, if at a particular site 60% of reads have an C and 40% have a G, then this site is assigned Cg, as opposed to an C which is typical of analysing consensus sequences. Because we typically model sequence evolution as a Markovian process, as is the case here, the data become naturally more informative, given that there are more states in the Markov chain when adding these bases. As a result, phylogenetic trees estimated using these data are better resolved than those from consensus sequences. The branches of the trees are probably also longer, which is why temporal signal becomes more apparent.

I commend the authors on their rigorous simulation study and careful empirical data analyses. However, I strongly suggest they consider whether treating minor alleles as an additional base is biologically realistic and whether this may have implication for other analyses, particularly when there is very high within-host diversity and the number of states in becomes very large.

Reviewer #1 (Public Review):<br /> <br /> Beta-hemoglobinopathies, such as sickle cell disease and beta-thalassemia, are common and debilitating genetic diseases caused by mutations in the adult beta-globin gene. Many in the field are pursuing various strategies to therapeutically upregulate fetal gamma-globin to treat these diseases. In this paper, the authors aimed to instead edit the promoter of the delta-globin gene to cause upregulation of delta-globin expression. Delta-globin is highly homologous to adult beta-globin and is pan-cellularly expressed in adult red blood cells, albeit at low levels due to the low activity of its promoter. Gene editing to activate the promoter of delta-globin could allow delta-globin expression to be elevated which could compensate for defective beta-globin in patients with beta-hemoglobinopathies. This is an underexplored and very interesting approach, and this study represents the first time that delta-globin upregulation has been attempted using gene editing in adult-like human erythroid immortalised and primary cells.

The first major finding from this study was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta-globin promoter was sufficient to cause upregulation of delta-globin expression at the mRNA and protein levels in immortalized HUDEP-2 cells. Modest upregulation was seen in pooled populations of HUDEP-2 cells (where ~25% of cells were HDR edited). Robust expression of delta-globin was seen in homozygously edited clonal populations of HUDEP-2 cells, with delta-globin constituting ~25% of total beta-like globin expression at the mRNA level in these cells. The results presented thus strongly support this finding.

The second major finding was that gene editing to insert KLF1, beta-DRF, and TFIIb sites into the delta globin promoter was sufficient to cause upregulation of delta-globin in primary human CD34+ cells. Despite HDR editing efficiencies of ~25% in these primary cells, and possibly due to only two donor cell populations being used, significant upregulation of delta-globin was not detected in pooled populations of edited primary CD34+ cells. Encouraging evidence of upregulation was seen in the clonal population of edited cells from the two donors. As such the results provide moderate support for this finding.

In combination, the HUDEP-2 cell and CD34+ cell data provide compelling evidence that gene editing of the delta-globin promoter is a promising line of enquiry for the treatment of beta-hemoglobinopathies.

This important study establishes and provides a proof-of-principle for this alternative therapeutic approach for those with beta-hemoglobinopathies. Future studies based on this work may enable delta-globin to be upregulated to therapeutically relevant levels in patient cells, including in cells from patients with beta-hemoglobinopathies. The therapeutic benefits of delta-globin upregulation will then be able to be assessed. This finding will be of interest to those in the globin switching and gene editing fields.

Reviewer #1 (Public Review):

In this interesting manuscript, Nasser et al explore long-term patterns of behavior and individuality in C. elegans following early-life nutritional stress. Using a rigorous, highly quantitative, high-throughput approach, they track patterns of motor behavior in many individual nematodes from L1 to young adulthood. Interestingly, they find that early-life food deprivation leads to decreased activity in young larvae and adults, but that activity between these times, during L2-L4, is largely unaffected. Further, they show that this "buffering" of stress requires dopamine signaling, as L2-L4 activity is significantly reduced by early-life starvation in cat-2 mutants. The paper also provides evidence that serotonin signaling has a role in modulating sensitivity to stress in L1 larvae and adults, but the size of these effects is modest. To evaluate patterns of individuality, the authors use principal components analysis to find that three temporal patterns of activity account for much of the variation in the data. While the paper refers to these as "individuality types," it may be more reasonable to think of these as "dimensions of individuality." Further, they provide evidence that stress may alter the strength and/or features of these dimensions. Though the circuit mechanisms underlying individuality and stress-induced changes in behavior remain unknown, this paper lays an important foundation for evaluating these questions. As the authors note, the behaviors studied here represent only a small fraction of the behavioral repertoire of this system. As such, the findings here are an interesting and very promising entry point for a deeper understanding of behavioral individuality, particularly because of the cellular/synaptic-level analysis that is possible in this system. This paper should be of interest to those studying C. elegans behavior and also more generally to those interested in behavioral plasticity and individuality.

Reviewer #1 (Public Review):

The DNA damage checkpoint is a cellular signalling pathway that responds to DNA damage and replication stress. This manuscript by Ho et al. systematically investigates an aspect of the checkpoint response in budding yeast that has been previously understudied, namely which proteins change subcellular and how these changed depend on the checkpoint kinases Mec1 and Rad53. By nice detective work the authors find a new mode of activation of Rad53, which is Mec1-independent, but rather depends on factors of so called retrograde signalling. Currently, we view checkpoint signalling as hierarchical, with Mec1 and Tel1 activating Rad53, despite both Mec1 and Rad53 having independent targets. This manuscript challenges that view by finding a Mec1 (and Tel1) independent mode of activation. It is very clear from survival and mass spectrometry data that in the absence of Mec1 this activation pathway and Rtg3 has a key role in activating Rad53. In the current form of the manuscript, it remains however difficult to assess what is the contribution of these factors on Rad53 activation in an otherwise WT background.

Reviewer #1 (Public Review):

In this paper, Reato, Steinfeld et al. investigate a question that has long puzzled neuroscientists: what features of ongoing brain activity predict trial-to-trial variability in responding to the same sensory stimuli? They record spiking activity in the auditory cortex of head-fixed mice as the animals performed a tone frequency discrimination task. They then measure both overall activity and the synchronization between neurons, and link this 'baseline state' (after removing slow drifts) of cortex to decision accuracy. They find that cortical state fluctuations only affect subsequent evoked responses and choice behavior after errors. This indicates that it's important to take into account the behavioral context when examining the effects of neural state on behavior.

Strengths of this work are the clear and beautiful presentation of the figures, and the careful consideration of the temporal properties of behavioral and neural signals. Indeed, slowly drifting signals are tricky as many authors have recently addressed (e.g. Ashwood, Gupta, Harris). The authors are well aware of the difficulties in correlating different signals with temporal and cross-correlation (such as in their 'epoch hypothesis'). To disentangle such slow trends from more short-lived state fluctuations, they remove the impact of the past 10 trials and continue their analyses with so-called 'innovations' (a term that is unusual, and may more simply be replaced with 'residuals').

I do wonder if this throws out the baby with the bathwater. If the concern is statistical confound, the 'session permutation' method (Harris) may be better suited. If the concern is that short-term state fluctuations are more behaviorally relevant (and obscured by slow drifts), then why are the results with raw signals in the supplement (Suppfig 8) so similar?

While the authors are correct that go-nogo tasks have drawbacks in dissociating sensitivity from response bias, they only cursorily review the literature on 2AFC tasks and cortical state. In particular, it would be good to discuss how the specific method - spikes, EEG (Waschke), widefield (Jacobs) and algorithm for quantifying synchronization may affect outcomes. How do these population-based measures of cortical state relate to those described extensively with slightly different signals, notably LFP or EEG in humans (e.g. work by Saskia Haegens, Niko Busch, reviewed in https://doi.org/10.1016/j.tics.2020.05.004)? This review also points out the importance of moving beyond simple measures of accuracy and using SDT, which would be an interesting improvement for this paper too.

Public Review:

Barreat and Katzourakis analyze the evolutionary history of eukaryotic viruses (and related mobile elements) in the Bamfordvirae kingdom, and discuss potential scenarios regarding the origin of different viral taxa in this group. This version of their manuscript includes a larger number of sequences to better represent diversity in these viral groups, and explored new evolutionary scenarios, including a "virophage-first" hypothesis now presented as the one best supported by phylogenetic analyses. The authors also present compelling analyses suggesting that the "nuclear escape" hypothesis in which these different viral groups separately "escaped" from nuclear (integrated) elements is not consistent with the current genomic and phylogenetic information available.

This work is thus an important step in our collective understanding of the ancient evolutionary history of eukaryotic viruses, and more generally of the constraints and main drivers of virus evolution.

Reviewer #1 (Public Review):

Owen D et al. investigated the protein partners and molecular functions of ZMYM2, a transcriptional repressor with key roles in cell identity and mutated in several human diseases, in human U2OS cells using mass spectrometry, siRNA knockdown, ChIP-seq and RNA-seq. They tried to identify chromatin bound complexes containing ZMYM2 and identified known and novel protein partners, including ADNP and the newly described partner TRIM28. Focusing mainly on these two proteins, they show that ZMYM2 physically interacts with ADNP or TRIM28, and co-occupies an overlapping set of genomic regions with ADNP and TRIM28. By generating a large set of knockdown and RNA-seq experiments, they show that ZMYM2 co-regulates a large number of genes with ADNP and TRIM28 in U2OS cells. Interestingly, ZMYM2-TRIM28 do not appear to repress genes directly at promoters, but the authors find that ZMYM2/TRIM28 repress LTR elements and suggest that this leads to gene deregulation at distance by affecting the chromatin environment within TADs.

A strength of the study is that, compared to previous studies of ZMYM2 protein partners, it investigates binding partners of ZMYM2 using the RIME method on chromatin. The RIME method makes it possible to identify low-affinity protein-protein interactions and proteins interactions occurring at chromatin, therefore revealing partners most relevant for gene regulation at chromatin. This allowed the identification of novel ZMYM2 partners not identified before, such as TRIM28.

The authors present solid interaction data with appropriate controls and generated an impressive amount of datasets (ChIP-seq for TRIM28 and ADNP, RNA-seq in ZMYM2, ADNP and TRIM28 knockdown cells) that are important to understand the molecular functions of ZMYM2. These datasets were generated with replicates and will be very useful for the scientific community. This study provides important novel insights into the molecular roles of ZMYM2 in human U2OS cells.

The authors could have been more precise in the manuscript title and abstract to emphasize that these findings apply to human cells, as indeed there is no demonstration yet that the findings presented here can be transposed to mouse cells.

The manuscript's main conceptual advance is that the authors propose a novel model of gene regulation whereby transcriptional repressors of transposable elements could regulate genes at distance by modulating the local chromatin environment within TADs. Additional experiments would be needed to strengthen this model. For example the authors could have performed TRIM28 ChIP in ZMYM2-kd cells to test if ZMYM2 favors the recruitment of TRIM28 to its genomic targets, as well as ChIP-seq of repressive chromatin marks (such as H3K9me3) in ZMYM2-kd cells to investigate if the loss of ZMYM2 leads to reduced H3K9me3 in ERVs and over large regions surrounding the ERVs.

Reviewer #1 (Public Review):

In the manuscript there is not much comparison between the crystal and cryoEM structures provided, and on inspection they appear to be very similar. The crystal structures also reveal parts of the CC domains in Las1, which is not present in the cryoEM structures. It is interesting the CC domains in Sc and Cj are quite different as illustrated in Figure 4B. They also seem to be somewhat disconnected from the rest of the complex (more so for Cj), even though that's not apparent in Figures 2-4. Despite this, it would be very useful to show the cryoEM densities when describing the catalytic site and C-terminal domain interactions, for example, as this can be very useful to increase confidence in the model and proposed mechanisms.

The description of the complex as a butterfly is engaging, and from a certain angle it can be made to look as such; this was also described previously in (Pillon et al., 2019, NSMB) for the same complex from a different organism (Ct). However, it is a bit misleading, because the complex is actually C2 symmetric. Under this symmetry, the 'body' would consist of two 'heads' one pointing up, one down facing towards the back, and one wing would have its back toward the viewer, the other the front. The structures presented here in Sc and Cj seem quite similar to the previous structure of the same complex in Ct, though the latter was only solved with cryoEM, and was also lacking the structure of the CC domain in Las1.

For the model suggested in Figure 8, perhaps in the 'weak activity' state, the LCT in Las1 could still be connected to Grc3, via the LCT, rather than disconnected as shown. This could facilitate faster assembly of the 'high activity' state. The complex is described as 'compact and stable', but from the structure and this image, it appears more dynamic, which would serve its purpose and the illustrated model better. The two copies of HEPN appear to have more connective area, meaning they are indeed more likely to remain assembled in the 'weak activity' state. On the other hand, HEPN in one protein appears to have less binding surface with PNK in Grc3, and even less so with the CTD (both PNK and CTD being from the other associated protein), meaning these bindings could release easily to form the 'weak activity' state.

There is also the potential to speculate that the GCT is bound to HEPN near the catalytic area in the 'weak activity' state. The reduced activity when the GCT residues are replaced by Alanine could then be explained by the complex not being able to assemble as quickly upon binding of the substrate, as it could if the GCT remained bound, rather than by a conformational change that it induces upon binding. The conformational change is also likely to be influenced by the combined binding of PNK and CTD in the assembled state, which also contact HEPN, rather than by GCT alone.

When comparing the structure of the HEPN domain in the lone Las1 protein to the structure of Las1-HEPN in the Las1-Grc3 complex, it is mentioned that 'large conformational changes are observed'. These could be described a bit better. The conformational change is ~3-4Å C-alpha RMSD across all ~150 residues in the domain (~90 residues forming a stable core that only changes by ~1Å). There is also a shift in the associated HEPN domain in Las1B domain compared to the bound HEPN in the Las1-Grc3 complex, as shown in Figure 7D: ~1Å shift and ~12degrees rotation. This does point to the conformation of HEPN changing upon complex formation, as does the relative positions of the HEPN domains in Las1A and Las1B. The conformational change and relative shift could indeed by key for the catalysis of the substrate as mentioned.

Overall, the structures presented should be very useful in further study of this system, even though the exact dynamics and how the substrate is bound are aspects that are perhaps not fully clear yet. The addition of the structures of the CC domain in two different organisms and the Las1 HEPN domain (not in complex with Grc3) as new structural information should allow for increasing our understanding of the overall complex and its mechanism.

Reviewer #1 (Public Review):

Membrane receptor guanylyl cyclases are important for many physiological processes but their structures in full-length and their mechanism are poorly understood. Caveney et al. determined the cryo-EM structure of a highly engineered GC-C in a complex with endogenous HSP90 and CDC37. The structural work is solid and the structural information will be useful for the membrane receptor guanylyl cyclases field and the HSP90 field. However, a detailed characterization of the protein sample is lacking. Moreover, the physiological significance of this structure is not fully exploited by supporting experiments and the mechanistic insight is currently limited.

1. The characterization of the protein sample is lacking. SDS-PAGE would be useful to identify potential proteolysis, leading to the dissociation of GC dimer. Further size-exclusion chromatography would be helpful to estimate the molecular weight of the complex and to determine if only GC-C monomer is purified.

2. The orientation distribution of the particles is not homogenous in Fig. S1D. It would be helpful to present the 3DFSC curve to evaluate the effect of preferred orientation on the reconstruction.

3. Description of protein expression details is lacking. Did the author use transient transfection, stable cell line or virus-mediated transduction?

4. HSP90 binds ATP and is often co-purified with endogenous ATP/ADP. Is there ATP or ADP present in the sample/cryo-EM maps? Is the conformation of NBD similar to ATP-bound HSP90? The author needs to include the description/figures about the nucleotide state of HSP90.

5. The catalytic domains of GC have to be dimerized to perform cyclase function. The presence of only one GC-PK monomer in the cryo-EM structure indicates the structure does not represent an active state of GC. These results suggest the GC expressed in this way is not functional. The authors need to explain why most of the GC protein is trapped in this inactive form.

6. The GC-C construct used here is a highly engineered "artificial" construct, which has not been fully characterized in this work. Does this construct have similar activity as the activated wt GC-C? Does the protein (this engineered construct) expressed in CHO cells show activity?

7. Are the residues on the interface between GC and HSP conserved in other members of membrane receptor guanylyl cyclases? Would mutations on this interface affect the activity of GC?

8. The authors propose that targeting HSP90 would tune the activity of GC. Is there any experimental data supporting this idea?

9. The model in Fig. S3 is largely speculative due to the lack of supporting functional data. In addition, it would be better to change the title to "structure of the protein kinase domain of guanylyl cyclase receptor in complex with HSP90 and cdc37" because the mechanistic insight is limited.

Reviewer #1 (Public Review):

This manuscript conducts a classic QTL analysis to identify the molecular basis of natural variation in disease resistance. This identifies a pair of glycosyltransferases that contribute to steroidal glycoalkaloid production. Specifically altering the final hexose structure of the compound. This is somewhat similar to the work in tomatine showing that the specific hexose structure mediates the final potential bioactivity. Using the resulting transgenic complementation lines that show that the gene leads to a strong resistance phenotype to one isolate of Alternaria solani and the Colorado potato beetle. This is solid work showing the identification of a new gene and compound influencing plant biotic interactions. While the experiments are solid, the introduction, discussion and associated claims don't accurately reflect my reading of what is known and said in the current literature.

The sentence on line 53-54 is misleading. It provides only three citations on specific links between specialized metabolism and disease resistance. However, there are actually at least 40 on specific links of camalexin and indolic phytoalexins to disease resistance. Similarly there are dozens of uncited papers on benzoxazinoids, indolic glucosinolates, aliphatic glucosinolates and tomatine to both non-host and host based resistance mechanisms. This even goes as far as showing how the pathogens resist an array of these compounds. The choices in the introduction make it appear that little is known about specialized metabolism to disease resistance but I would suggest that this is not an allusion supported by the literature. I would agree that given the breadth of specialized metabolism we have a lot of knowledge about a set of them but that there are hundreds to thousands of untested compounds but to indicate that little is known is unfair to the specialized metabolism community. This is especially true as the introduction and discussion give no image of the large body of literature on specialized metabolism to insect interactions even though this is a major component of this manuscript.

I would also agree that specialized metabolism is not a conscious target of breeding programs but the work on benzoxazinoids in maize and glucosinolates in the Brassica's has shown that these compounds have been influenced by breeding programs. Similarly work on de novo domestication of multiple crops is focused on the adjustment of specialized metabolism in these crops.

I would disagree with the hint on line 49-50 and again on lines 236-239 that specialized metabolism may have less pleiotropy. This is not supported by recent work on benzoxazinoids and glucosinolates showing that they have numerous regulatory links to the plant and can be highly pleiotropic. Even the earliest avenicin work in oat showed that the deficient lines had altered root development.

My main message from the above three paragraphs is to point out that there are a number of places in the manuscript where the current state of the specialized metabolite literature is not accurately portrayed. To properly place the manuscript in the broader context, I would suggest a more even handed introduction and discussion that takes into account the current state of the specialized metabolism literature.

Is it accurate to say complete resistance to A. solani if only a single isolate of the pathogen is used? Is there evidence that I am unaware of that there are no isolates of this pathogen with saponin resistance? There are pathogens with natural tomatine resistance and this is a common feature of plant pathogens that they have genetic variation in the resistance to specialized metabolism. For example, it should be noted that Botrytis BO5.10 is a tomatine sensitive isolate and the van Kan and Hahn groups have published on isolates that are resistant to saponins. I would suggest caveating across the manuscript that this is a single isolate and that it is possible that there may be isolates with natural resistance to the steroidal glycoalkaloid?

In Figure 4b, is the infection site about 3.5 mm in size such that 3.5 mm means absolutely no infection? If not, that would mean there is some outgrowth by Alternaria and the resistance isn't complete.

Reviewer #1 (Public Review):

In this article, Cacioppo et al., report on a previously unappreciated mechanism of the regulation of Aurora Kinase A (AURKA) protein levels that is orchestrated via coordinated action of alternative polyadenylation of AURKA mRNA and hsa-let-7a miRNA. Moreover, it is proposed that this mechanism may play a major role in neoplasia. In support of their model, the authors demonstrate that short-to-long 3'UTR AURKA mRNA isoform ratio is elevated in triple negative breast cancer patients where it correlates with poor prognosis. The authors further generated reporters suitable for single cell live imaging that express different 3'UTR variants, which revealed highly variable ratios of short and long 3'UTR AURKA isoforms across different cell lines. This was followed by actinomycin D chase and nascent chain immunoprecipitation assays in U2OS osteosarcoma cells to demonstrate that while short and long 3'UTR AURKA isoforms have comparable stability, short 3'UTR AURKA isoforms appear to exhibit higher ribosome association which is indicative of higher translation activity. Furthermore, using an additional reporter assay which takes advantage of trimethoprim-based stabilization of highly unstable E. Coli dihydrofolate reductase mutants Cacioppo et al., provide evidence that in contrast to the short 3'UTR AURKA mRNA isoform which appears to be constitutively translated throughout the cell cycle, long 3'UTR AURKA mRNA isoform is preferentially translated in the G2 phase. Further evidence is provided that suppression of long 3'UTR AURKA mRNA isoform is at least in part mediated by hsa-let-7a miRNA. Finally, the authors provide evidence that disrupting the expression of long 3'UTR AURKA mRNA isoform using CRISPR-based strategy, leads to overexpression of AURKA driven by the short 3'UTR isoform which is paralleled by an increase in cancer-related phenotypes.

Strengths: Overall it was thought that this study is of potentially broad interest inasmuch as it delineates a hitherto unappreciated mechanisms of regulation of AURKA protein levels, whereby AURKA is emerging as one of the major factors in neoplasia, including resistance to anti-cancer treatments. In general, it was thought that the author's conclusions were sufficiently supported by provided data. It was also thought that this study incorporates innovative methodology including single-cell expression sensors coupled with live cell microscopy and an assay to study translation in different phases of cell cycle without need for cell synchronization.

Weaknesses: Several relatively minor issues were observed regarding methodology and data interpretation. Namely, some inconsistencies between the models and/or cell lines that were used throughout the manuscript were noted. For instance, key experiments were performed almost exclusively in U2OS osteosarcoma cells, whereby triple negative breast cancer patient data were used to set the scientific foundation of the study. Considering potential differences in alternative polyadenylation between cell and tissue types, it was thought that investigation across the broader compendium of cell lines may be required for generalization of findings observed in U2OS cells. It was also found that the precise mechanisms underpinning the role of hsa-let-7a miRNA in regulation of AURKA protein levels remain largely obscure.

Reviewer #1 (Public Review):

In this manuscript, the authors aimed to compare, from testis tissues at different ages from mice in vivo and after culture, multiple aspects of Leydig cells. These aspects included mRNA levels, proliferation, apoptosis, steroid levels, protein levels, etc. A lot of work was put into this manuscript in terms of experiments, systems, and approaches. However, as written the manuscript is incredibly difficult to follow. The Introduction and Results sections contain rather loosely organized lists of information that were altogether confusing. At the end of reading these sections, it was unclear what advance was provided by this work. The technical aspects of this work may be of interest to labs working on the specific topics of in vitro spermatogenesis for fertility preservation but fail to appeal to a broader readership. This may be best exemplified by the statements at the end of both the Abstract and Discussion which state that more work needs to be done to improve this system.

Reviewer #1 (Public Review):

In this study the authors sought to address the issue of whether the Steller's sea cow -- a massive extinct sirenian ("sea cow") species that differs from its living relatives (manatees and dugongs) not only in body mass but also in having inhabited cold climates in the northern Pacific -- had hemoglobin adaptations that enhanced the species' thermoregulatory capacities relative to those of the extant species, which are restricted to relatively warm waters. To do so, the authors synthesized recombinant hemoglobin proteins of all the major sea cow lineages and used these data to assess differences in O2 binding, Hb solubility, responses to allosteric effectors, and thermal sensitivity. The work presented is very innovative and in my opinion convincingly demonstrates that the Steller's sea cow had remarkable hemoglobin adaptations that allowed for an extreme range extension into cool waters despite several physiological constraints that are inherent to the sirenian (and paenungulate, afrotherian, etc.) clade. I did not detect any obvious weaknesses of the paper, whereas the use of ancient DNA to resurrect 'extinct' hemoglobins, and the various analyses of these extinct hemoglobins alongside those of extant relatives is very exciting and are major strengths of the paper that make this study a very important advance for our understanding of Steller's sea cow's paleophysiology, as well as our understanding of the potential for extreme hemoglobin phenotypes that have not been documented among living species. Moving forward, these methods can be used to study aspects of the paleophysiology of other recently extinct mammals. I applaud the authors on an excellent and innovative study that significantly augments our understanding of the Steller's sea cow.

Reviewer #1 (Public Review):

This study utilizes scRNA-seq to generate a detailed map of transcriptional changes that occur in asynchronously replicating the Trypanosoma brucei insect (PCF) and mammalian (BSF) stages. The analyses were performed on both fresh and cryo-preserved parasites, and transcriptional changes in PCF compared to existing proteomic datasets at the same stage. This is the first study to comprehensively map cell cycle-related transcriptional changes in T. brucei BSF and to undertake a side-by-side analysis of the two major parasite developmental stages. The study identified >1,500 transcripts that exhibit dynamic changes during the cell cycle across the two stages, substantially increasing the number of cell cycle-regulated (CCR) genes compared to previous analyses. Analysis of the data revealed common as well as stage-specific CCR transcripts and identified transcripts with known/suspected functions in cell cycle regulation as well as hypothetical proteins. The findings also support and quantify previous observations suggesting that most transcript changes (83-86% of CCR transcripts) are not reflected by similar changes in corresponding proteins, and where there is a correlation, protein expression levels expectedly lag behind transcripts. Overall, the study provides the most comprehensive transcriptome atlas of the T. brucei cell cycle undertaken to date, highlighting a large number of genes and cellular processes that are linked to cell cycle progression, while further confirming the importance of post-transcriptional regulatory processes in these divergent eukaryotes. The work represents a significant technical advance, particularly in the validation of the use of cryo-preserved parasites for single-cell RNS-seq, and nicely integrates results from previous proteomics and gene-knockout studies.

Reviewer #1 (Public Review):

The nuclear receptor Nurr1 is a target of interest in neurodegenerative diseases like Parkinson's and Alzheimer's, but its mechanism of activation on NBRE-containing promoters and potential druggability is unknown. A heterodimer of Nurr1 with RXRa can be activated by a subset of ligands that bind to the RXRa ligand binding domain (LBD). Here, the authors provide evidence that transcriptional activation occurs through ligand-induced dissociation of the heterodimer, leading to an active Nurr1 monomer.

NMR spectroscopy and other biophysical, biochemical, and cell-based assays provide a strong foundation for the work. The manuscript is well-written and easy to follow, and for the most part, it thoughtfully addresses experimental results and data interpretation with reasonable caveats. However, a reliance on simple correlative analyses, including some with rather modest correlations (R2 values {less than or equal to} 0.5), may fail to account for some potentially interesting outlier ligands and oversimplify conclusions. Despite this possible oversimplification, this manuscript provides solid evidence of their discovery of an interesting mechanism by which a subset of RXRɑ ligands leads to transcriptional activation of Nurr1 at NBRE promoters--this is an exciting finding that could be potentially relevant in the development of neuroprotective therapies.

Reviewer #1 (Public Review):

In this manuscript, the authors describe a novel HA20-causing missense mutation, p.(Leu236Pro), in three patients from one family with periodic fevers, GI symptoms, urogenital ulcers, arthritis, and pustular rash. The patients had elevated levels of multiple proinflammatory cytokines, including IL-1b, IL-6, and TNFa. Patients had reduced A20 expression levels, and in silico analysis suggested that the p.Leu236Pro mutation destabilized the A20 protein. Using transfection assays, the authors determined that steady state protein expression of mutant A20 protein was lower, and that the half-life of the mutant protein was shorter. Treatment with MG132 increased the half life of mutant A20, suggesting that the mutant protein underwent degradation through the proteasome. Further experiments in the transfection system revealed that mutant A20 failed to suppress TNFα-induced NF-κB activity.

This paper will be of great interest to the field. HA20 is a novel disease (first described in 2016), and although the effects of frameshift/truncating mutations are quite evident, there is quite a lot of debate about the potential effects of missense mutations. It is not really clear which missense mutations cause disease and why, and clinicians who treat this disease are frequently faced with the dilemma of evaluating a patient with a rare missense variant of unknown significance. Thus, a paper that can explain the potential mechanisms by which missense mutations cause disease is highly relevant -- and this is an area of active investigation by several groups.

The strengths of this study include the thorough functional assessment of this novel mutation: the authors have collected quite a lot of data to show the effects of their mutation on protein stability and function. Another strength is the comparison with other similar mutations in the OTU and other domains. However, the data are not currently sufficient to support the conclusions of the authors about the effects of their mutation on protein folding. Similarly, the data do not sufficiently support the generalizability of this mechanism to other mutations in the OTU domain.

Reviewer #1 (Public Review):

Yamanaka et al.'s research investigates into the impact of volatile organic compounds (VOCs), particularly diacetyl, on gene expression changes. By inhibiting histone acetylase (HDACs) enzymes, the authors were able to observe changes in the transcriptome of various models, including cell lines, flies, and mice. The study reveals that HDAC inhibitors not only reduce cancer cell proliferation but also provide relief from neurodegeneration in fly Huntington's disease models. Although the findings are intriguing, the research falls short in providing a thorough analysis of the underlying mechanisms.

HDAC inhibitors have been previously shown to induce gene expression changes as well as control cell division and demonstrated to work on disease models. The authors demonstrate diacetyl as a prominent HDAC inhibitor. Though the demonstration of diacetyl is novel, several similar molecules have been used before.

Reviewer #1 (Public Review):

Asthma is a syndromic disease with multiple subtypes with different pathogenetic paths to a final wheezing phenotype. This limits the insights gleaned from genetic investigations of asthma. One of the most important phenotypes is early life onset wheezing, which persists. Here, the authors use data from multiple birth cohorts and by coupling latent class analysis of clinical phenotypic data with GWAS discovery, identify a novel locus close to annexin 1 (ANXA1) associated exclusively with early-onset persistent wheeze. The methodology is a major strength of the work and highlights the importance of acquiring and analysing phenotypic over simple use of doctor labels for complex diseases.

The authors went on to demonstrate a putative mechanism such that the risk allele (T) may confer a reduction in ANXA1 expression. Altered ANXA1 expression was additionally recapitulated in a murine model of house dust mite (HDM)-induced allergic airway disease. In this model, ANXA1 increased, rather than decreased, which may be attributable to its role in resolving inflammation. ANXA1-deficient mice had a more severe phenotype. This strengthens the evidence for causality in the novel link between ANXA1 and asthma and opens the door for further investigations. While novel for this link, the finding is well supported by prior knowledge about ANXA1-related pathways and inflammation. ANXA1 is known to participate in phospholipase A2-dependent reduction of inflammatory mediator production. Glucocorticoids increase ANXA1 levels. ANXA1 deficiency leads to airway hyperreactivity in mice. Overall, ANXA1 appears to be suitable as a therapeutic target and this may spur further investigations into the pathway.

Reviewer #1 (Public Review):

In their study, Osorio-Valeriano and colleagues seek to understand how bacterial-specific polymerizing proteins called bactofilins contribute to morphogenesis. They do this primarily in the stalked budding bacterium Hyphomonas neptunium, with supporting work in a spiral-shaped bacterium, Rhodospirillum rubrum. Overall the study incorporates bacterial genetics and physiology, imaging, and biochemistry to explore the function of bactofilins and cell wall hydrolases that are frequently encoded together within an operon. They demonstrate an important, but not essential, function for BacA in morphogenesis of H. neptunium. Using biochemistry and imaging, they show that BacA can polymerize and that its localization in cells is dynamic and cell-cycle regulated. The authors then focus on lmdC, which encodes a putative M23 endopeptidase upstream of bacA in H. neptunium, and find that is essential for viability. The purified LmdC C-terminal domain could cleave E. coli peptidoglycan in vitro suggesting that it is a DD-endopeptidase. LmdC interacts directly with BacA in vitro and co-localizes with BacA in cells. To expand their observations, the authors then explore a related endopeptidase/bactofilin pair in R. rubrum; those observations support a function for LmdC and BacA in R. rubrum morphogenesis as well.

An overall strength of this study is the breadth and completeness of approaches used to assess bactofilin and endopeptidase function in cells and in vitro. The authors establish a clear function for BacA in morphogenesis in two bacterial systems, and demonstrate a physical relationship between BacA and the cell wall hydrolase LmdC that may be broadly conserved. The eventual model the authors favor for BacA regulation of morphogenesis in H. neptunium is that it serves as a diffusion barrier and limits movement of morphogenetic machinery like the elongasome into the elongating stalk and/or bud. However, there is no data presented here to address that model and the role of LmdC in H. neptunium morphogenesis remains unclear.

The data presented illuminate aspects of bacterial morphogenesis and the physical and functional relationship between polymerizing proteins and cell wall enzymes in bacteria, a recurring theme in bacterial cell biology with a variety of underlying mechanisms. Bactofilins in particular are relatively recently discovered and any new insights into their functions and mechanisms of action are valuable. The findings presented here are likely to interest those studying bacterial morphogenesis, peptidoglycan, and cytoskeletal function.

Reviewer #1 (Public Review):

The manuscript provides analyses on a very complete dataset on weight and length growth, as well as several physiological markers related to growth, in bonobos. Moreover, there is a good overview of the presence of adolescent growth spurts in non-human primates, by reviewing published data, in comparison to their own dataset. They discuss the need to consider scaling laws when interpreting and comparing growth curves of different species and variables.

The manuscript is very well written, the sample is large, and the methods are well explained. It seems they have analyzed a very complete dataset. Also, the discussion and the references supporting the findings are complete.

The main weakness of this manuscript is that they do not provide a direct comparison with previous analyzed datasets in other species, using their own method (in part maybe because there is not available data, but just published figures).

On the other side, conclusions are well supported by the results, and the previous published datasets are discussed in the manuscript, although not in detail.

Reviewer #1 (Public Review):

For many years it has been understood that transposable elements (TEs) are an important source of natural variation. This is because, in addition to simple knockouts of genes, TEs carry regulatory sequences that can, and sometimes do, affect the expression of genes near the TEs. However, because TEs can be difficult to map to reference genomes, they have generally not been used for trait mapping. Instead, single nucleotide polymorphisms are widely used because they are easy to detect when using short reads. However, improvements in sequencing technology, as well as an increased appreciation of the importance of TEs to both linked to favorable alleles and are more likely to be causing the changes that make those alleles beneficial in a given environment. Further, because TE activity can occur after bottlenecks, they can provide polymorphisms in the absence of variation in point mutations.

In this manuscript, the authors carefully examine insertion polymorphisms in rice and demonstrate linkage to differences in expression. To do this, they used expression quantitative trait locus (eQTL) GWAS using TIPs as genetic markers to examine variation in 208 rice accessions. Because they chose to focus on genes that were expressed in at least 10% of the accessions, presumably because more rare variants would end up lacking statistical power. This is an understandable decision, but it says that recent insertions, such as the MITE elements detailed in a previous paper, would not be included. Importantly, although TIPs associated with differentially expressed genes are far less common than SNPs' traditional eQTLs, there were a significant number of eQTLs that showed linkage to TIPs but not to QTL.

The authors then show that of the eQTLs associated with both TIPs and SNPs, TIPs are more tightly linked to the eQTL, and are more likely to be associated with a reduction in expression, with variation in the effects of various TEs families supporting that hypothesis. Here and throughout, however, the distance of the TEs could be an important variable. It is also worth noting the relative numbers in order to assess the claim in the title of the paper. The total number of eQTL-TIPs is ten-fold less than the number of eQTL-SNPs, and, of the eQTLs that have both, there are a significant number of eQTL-TIPs that are not more tightly linked to the expression differences than the eQTL.

The authors show that eQTL-TIPs are more likely to be in the promoter-proximal region, but this may be due to insertion bias, which is well documented in DNA-type elements. Here and throughout the authors are careful to state that the data is consistent with the hypothesis that TEs are the cause of the change, but do not claim that the data demonstrate that they are.

Throughout the rest of the manuscript, the authors systematically build the case for a causal role for TEs by showing, for instance, that eQTL-TIPs show much stronger evidence for selection, with increased expression being more likely to be selected than decreased expression. The authors provide examples of genes most likely to have been affected by TE insertions.

Overall, the authors build a convincing case for TEs being an important source of regulatory information. I don't have any issues with the analysis, but I am concerned about the sweeping claims made in the title. Once you get rid of eQTLs that could be altered by either SNPs or TIPs and include only those insertions that show strong evidence of selection, the number of genes is reduced to only 30. And even in those cases, the observed linkage is just that, not definitive evidence for the involvement of TEs. Although clearly beyond the scope of this analysis, transgenic constructs with the TEs present or removed, or even segregating families, would have been far more convincing.

The fact that many of the eQTL-TIPs were relatively old is interesting because it suggests that selection in domesticated rice was on pre-existing variation rather than new insertions. This may strengthen the argument because those older insertions are less likely to be purged due to negative effects on gene expression. Given that the sequence of these TEs is likely to have diverged from others in the same family, it would have been interesting to see if selection in favor of a regulatory function had caused these particular insertions to move away from more typical examples of the family.

Reviewer #1 (Public Review):

With this work, the authors address a central question regarding the potential consequences of post-translational modifications for the pathogenesis of neurodegenerative diseases. Phosphorylation and mislocalization of the RNA binding protein TDP43 are characteristic of ~50% of frontotemporal lobar degeneration (FTLD), as well as >95% of amyotrophic lateral sclerosis (ALS). To determine if acetylation is a primary, disease-driving event, they generated a TDP-43 mutant harboring an acetylation-mimicking mutation (K145Q). Animals carrying the acetylation-mimic mutation (K145Q) displayed key pathological features of disease, including more cytoplasmic TDP43 and impaired TDP43 splicing activity, together with behavioral phenotypes reminiscent of FTLD.

This is a well-written and well-illustrated manuscript, with clear and convincing findings. The observations are significant and emphasize the importance of post-translational modifications to TDP-43 function and disease phenotypes. In addition, the TDP43(K145Q) mice may prove to be a valuable model for studying TDP-43-related mechanisms of neurodegeneration and therapeutic strategies.

However, as it stands it is challenging to determine if any or all of the phenotypes are a direct consequence of interrupted RNA binding by TDP-43, rather than acetylation per se. Furthermore, all the results are obtained using an acetylation-mimic mutation that may simply be disrupting a key residue involved in RNA binding by TDP43, instead of mirroring acetylation itself, which in theory is a reversible modification. Lastly, it remains unknown why TDP43(K145Q) mice developed features of FTLD, but not ALS, despite the fact that TDP-43 acetylation was found in ALS tissue and not FTLD.

Reviewer #1 (Public Review):

This manuscript reports the unique finding that specific ligands and receptors in the natriuretic peptide signaling pathway act during early embryogenesis to discriminate between neural crest (NC) and cranial placode (CP) fates. This is a significant finding for two reasons: 1) the developmental role of this pathway has not been studied in any detail; and 2) how cells located in the border between the neural ectoderm and non-neural ectoderm decide on NC versus CP fates is of broad interest and being actively pursued by a number of laboratories. The authors present logical and experimentally convincing support for their conclusions. They report the expression patterns by in situ hybridization and qPCR of the various ligands and receptors of the natriuretic peptide signaling pathway, clearly demonstrating that several of these molecules are expressed in the right place at the right time to influence NC and/or CP formation. They establish that Npr3 is a target of Pax3 and Zic1, two transcription factors previously shown to be required for NC and CP formation, further illustrating that it is part of the appropriate regulatory network. Next, the authors use morpholino knock down of Npr3 to show that the resulting embryos have deficient expression of two NC genes and two CP genes. The controls used for the knock-down are the correct ones and were confirmed by treatment with a high-affinity and selective Npr3 antagonist. The function of Npr3 was further explored by discriminating between its known two functions - clearance of natriuretic peptides and inhibition of adenylyl cyclase - by expressing either WT or mutant versions of human NPR3 in Npr3 morphant embryos. That WT rescued both NC and CP genes but the mutant version only rescued NC genes leads to the appropriate conclusion that Npr3 regulates NC and CP fates via different mechanisms. This conclusion was confirmed by treating Npr3 morphants with a specific adenylyl cyclase inhibitor, which restored CP gene expression, and treating CP promoting explants with an adenylyl cyclase activator, which repressed CP gene expression. Using similar knock-down approaches the authors convincingly demonstrate that Npr2 does not participate in NC/CP formation, but Npr1 does; again, the knock-down results were confirmed by treating embryos with a specific Npr1 antagonist. Finally, the authors complete the story by determining by equally well-controlled knock-down experiments which of the three natriuretic peptides participate in this process. In short, the many different experiments strongly support the conclusions, and the experiments are well controlled and include large numbers of embryos to provide exceptional rigor.

Reviewer #1 (Public Review):

Rapan et al. analyzed the cytoarchitectonic of the prefrontal cortex based on observer-independent analysis, confirming previous parcellations based on cyto-, myelo-, and immunoarchitectonic approaches, but also defining novel subdivisions of areas 10, 9, 8B, and 46 and identified the receptor density "fingerprint" of each area and subdivision. Furthermore, they analyzed the functional connectivity of the prefrontal cortex with caudal frontal, cingulate, parietal, and occipital areas to identify specific features for the various prefrontal subdivisions. Altogether, this study corroborates previous parcellations of the prefrontal cortex, adds new cortical subdivisions, and provides a neurochemical description of the prefrontal areas useful for comparative considerations and for guiding functional and clinical studies.

Strengths:<br /> - This study provides a detailed cytoarchitectonic map of the prefrontal cortex enriched with receptor density and functional connectivity data.<br /> - The authors shared the data via repositories and applied their map to a macaque MRI atlas to further facilitate data sharing.

Weaknesses:<br /> - The temporal cortex should be included in the functional connectivity analysis as it is known from anatomical studies that most prefrontal areas display rich connectivity with temporal areas. The aim of creating a comprehensive view of the frontal cortex makes the manuscript data-rich but cursory in discussing the relevant anatomical and functional literature.

Reviewer #1 (Public Review):

The authors have approached the study of the mechanism of maturation of retroviruses lattice, where Gag polyprotein is the main component. The Gag polyprotein is common to all retroviruses and makes up most of the observed lattice underlying the virion membrane. Within the lattice, 95% of the monomers are Gag, and 5% are Gag-Pol, which has the 6 domains of Gag followed by protease, reverse transcriptase and integrase domains (coming from Pol) embedded within the same polyprotein. For the maturation and infectivity of HIV retrovirus, the Gag proteins within the immature lattice must be cleaved by the protease formed from a dimer of Gag-Pol. Importantly, the lattice covers only 1/3 to 2/3 of the available space on the membrane. The incompleteness of the lattice results in a periphery of Gag monomers with unfulfilled intermolecular contacts. Recently, the structure of the immature lattice has been partially resolved using sub-tomogram averaging cryotomography (cryET) and it has been shown that the incompleteness of the lattice provides more accessible targets for the protease (Tan A. et al. 2021). Based on these, the authors have wondered: does the incompleteness of the lattice allow for dynamic rearrangements that ensure that protease domains embedded within the lattice can find one another to dimerize and activate? To answer this, they started from experimental cryoET data and used reaction-diffusion simulations of assembled Gag lattices with varying energies and kinetic rates to test how lattice structure and stability can support the dimerization of the Gag-Pols. They found that although they represent only 5% of the monomers that assemble into the lattice, the stochastic assembly ensure that at least a pair of them are adjacent within the lattice. They next showed that if the molecules are distant from one another, they would need to detach, diffuse, and reattach stochastically at the site of another Gag-Pol molecule.

I consider the work very interesting, which could contribute to a very important aspect of retroviruses maturation such as their infectivity. However, the observations made by the authors do not necessarily answer their initial question which seemed to be focused on studying the possible role of the incompleteness of the lattice on the protease activation rather than the mechanism of Pol activation itself. Maybe this is only a nuance to be polished in the writing.

The weakness of the work comes from both the fact their entire study has been done by computational methods and the exclusion in their computational approaches of well-known cellular components with a role in retrovirus maturation, which might obey to the fact of keeping their models into the simplest possible since handling atomistic models is already a heavy task. Maybe complementary molecular or structural studies would strengthen their results.

Reviewer #1 (Public Review):

The paper by Mohebi, Collins, and Berke describes the interactions between cholinergic interneurons and dopamine (DA) release in the core of the nucleus accumbens (NAc) in rats. The cholinergic triggering of DA release has been a debated issue in recent years, and this study provides data supporting cholinergic-dependent DA release.

The authors first show that optogenetic activation of cholinergic interneurons (CINs) induces DA release in the NAc, increasing with pulse width, frequency, and train pulse duration. They next show using simultaneous imaging of CIN calcium activity and DA release using RdLight that both are correlated in their response to sensory stimuli and entry to reward port in freely moving rats. They show that while CIN activity and DA release show ramping activity before entry to the center and food ports, such ramping is not seen in the spiking activity of DA cells. lastly, the authors show that blocking nicotinic receptors in the NAc by injection of DHBE impairs task performance, with similar (albeit weaker) effects as the DA antagonist flupenthixol. The uncoupling between DA release and DA cell firing, under certain conditions, has been shown by the authors in a previous paper (Mohebi et al, 2019). Here, the authors add the CINs calcium activity during the same task, showing that the dynamics of CIN activity resemble that of DA release. The results presented show correlations between CIN activity and DA release during behavior, however, the role of CINs in controlling DA release is not tested directly. The data presented in the paper are clear and it is well written. However, there are a few issues that need to be addressed, including some key experiments that could directly test the functional role of CIN-induced DA release.

Reviewer #1 (Public Review):

The authors compared the neural mechanisms of calling song in five Xenopus species. Two (X. laevis and X. petersii) were previously shown to produce fictive calls. This paper developed the techniques to evoke fictive calls for three additional species: X. cliivi, X. amieti, and X. tropicalis. The authors compared fast and low components of the calls and determined that the fast components in all species required bilateral coordination in the parabrachial nucleus (PBN), but the slow components were produced in the nucleus ambiguous (presumably with bilateral control, but that was not tested.

The abstract does not adequately summarize the content of the paper. There is no mention of stimulation, or bilateral connectivity, which is a large part of the paper. The names of all five species should appear in the abstract, not just X. laevis.

The conclusion that the "fast and slow CPGs identified in male X. laevis are conserved across species." is contradicted by the last paragraph, which states, "Fast trill-like CPGs are likely present only in fast clickers..." This inherent contradiction needs to be resolved.

The abstract also over-emphasizes the testosterone results. It states, "the development of fast CPGs [central pattern generators] depends on testosterone in a species-specific manner: testosterone facilitates the development of fast CPGs in a species with a courtship call containing fast clicks, but not in a species with a courtship call made entirely of slow clicks." The use of the word "development" implies embryology. Here, adults were treated and looked at 13 weeks later. There is no data presented about development. The effects of T could be simply to upregulate certain receptors of a circuit that was already present.

The concluding sentence of the abstract is, "The results suggest that species-specific calls of the genus Xenopus have evolved by utilizing conserved fast or slow CPGs that are broadly tuned to generate fast or slow trains of clicks, the development of which appear to be regulated by a strategic expression of testosterone receptors in the brain of each species." However, testosterone treatment was only applied to X. laevis females. The conclusion is based on plasma levels of testosterone in X. tropicalis. The conclusion that there is differential expression of testosterone receptors in the brain of each species is completely speculative and not supported by the data presented here.

Reviewer #1 (Public Review):

By the in vitro DNA damage response (DDR) assay with a defined DNA substrate using Xenopus extracts and in vitro binding assays with purified proteins, the authors nicely showed the role of APE1 (APEX1) in ATRIP recruitment for DDR activation, particularly a non-enzymatic (structural) role of APE1 in the binding to both ssDNAs and ATRIP. The results described in the paper are very convincing to support the authors' claim. However, these studies lack the quantification with proper statistics (and/or mentioning the reproducibility of the results). And, given the important discovery of APE1 in the DDR activation in vitro, it would be nice to demonstrate the role of APE1(APEX1) in ATR activation in vivo using siRNA-mediated knockdown of mammalian cells or yeast cells.

Reviewer #1 (Public Review):

In their study, Aman et al. utilized single cell transcriptome analysis to investigate wild-type and mutant zebrafish skin tissues during the post-embryonic growth period. They identified new epidermal cell types, such as ameloblasts, and shed light on the effects of TH on skin morphogenesis. Additionally, they revealed the important role of the hypodermis in supporting pigment cells and adult stripe formation. Overall, I find their figures to be of high quality, their analyses to be appropriate and compelling, and their major claims to be well-supported by additional experiments. Therefore, this study will be an important contribution to the field of vertebrate skin research. Although I have no major concerns, I would like to offer a few minor comments for the authors to consider.

1) The discovery of ameloblasts in the zebrafish skin is a fascinating finding that could potentially provide a new research model for understanding the development and regeneration of vertebrate teeth. It would be beneficial if the authors could provide further elaboration on this aspect and discuss how the zebrafish scale model could be utilized by researchers to better understand the morphogenesis of vertebrate teeth and/or hair.

2) While the overexpression-rescue experiments (i.e., fgf20a and pdafaa) provide crucial evidence to support the author's conclusions, it is important to note that overexpression driven by the heat-shock promoter is not spatially regulated. Therefore, it should be acknowledged that the rescue effects may not be cell-autonomous, as suggested in the current version.

3) Figure 7D. The authors used the ET37:EGFP lines to visualize hypodermis. Based on the absence of EGFP signal in the deep dermis of bnc2 mutants, the authors concluded that the hypodermis may be missing, suggesting the importance of the hypodermis in pigment cell formation. However, since the EGFP evidence is indirect, it is crucial to confirm the absence of the hypodermis structure with histology.

4) As the dataset is expected to be a valuable asset to the field, please provide Excel tables summarizing the key genes and their corresponding expression levels for each major cluster that has been identified.

Reviewer #1 (Public Review):

T2D in youth has been reported to reduce bone mass due to impaired bone anabolism, but the underlying mechanisms are not fully understood. The authors study the relationship between T2DM (Type 2 Diabetes Mellitus) and "skeletal fragility." Specifically, they look at glucose metabolism defects in osteoblasts during T2DM and their impacts on osteoblast activity. The results are novel as they elucidate the effects of low-dose STZ models of T2DM on osteoblast function and the function of osteoblasts from those mice in terms of glycolysis, glucose uptake, and function. Additionally, it covers recovery of glucose metabolic effects through overexpression of Hif1a or Pfkfb3 (targeted to osteoblasts) and metformin treatment. The role of Hif1a and Pfkfb3 in osteoblasts with regard to the rescue of T2DM bone effects is critical to the novelty of the paper and may benefit from being included and emphasized in the title and/or abstract. The study of osteoblasts and their glucose metabolism has been studied but not extensively at the mechanism level. The approach of using a mouse model is good for youth-onset T2D. It would be helpful if the author could include a bit more in the abstract about the critical role of Hif1a and Pfkfb3 in osteoblasts in recovery from T2DM treatment's bone effects in vivo.

Reviewer #1 (Public Review):

In their manuscript, Wang et al. investigate the changes occurring at the CNS borders upon neonatal bacterial meningitis. Both the dural meninges and the leptomeninges display changes. Using single nuc RNAseq and imaging approaches, they show that fibroblasts, endothelial cells and macrophages get inflamed, with an increase vascular leakage. Mechanistically, TLR4 KO but not CCR2 KO or liposome treatment (to deplete leptomeningeal macrophages) was able to rescue the vascular impairment. This is an interesting study that provides useful datasets for the community. However, we recommend several additions regarding data analysis (definitions, single cell, imaging) as well as additional studies (bacterial load, protein validation).

Reviewer #1 (Public Review):

This work puts forward a comprehensive characterisation of colorectal cancer (CCCRC), by classifying it into 4 subtypes with distinct TME features. It uses 10 public databases: 8 microarray datasets for the training of molecular classification and 2 RNAseq for validation (CRC-RNAseq) to identify the 4 subtypes using unsupervised machine learning (consensus clustering). These 4 subtypes were found to be somewhat distinct in terms of immune response and the possibilities for effective treatments. They found that one subtype may be more sensitive to chemotherapy, two to WNT pathway inhibitor SB216763 and Hedgehog pathway inhibitor vismodegib, and one to ICB treatment. They show an association with patient outcome in terms of PFS, validated in the validation cohort. They used histology to correspond the subtypes to known pathological types, as well as investigating their T cell makeup. They also investigated the genetic tumour evolution that may occur between the subtypes. A single-sample gene classifier was put forward as a way of identifying the class of cancer.

The evidence for the main results of the work is convincing, but a few areas need to be clarified and extended.

In the determination of the 4 subtypes (C1-C4) the methodology is clear, and the definition of the training and validation data are clear and well presented. The techniques used are well suited to the problem. The performance of the classification as a predictor of prognosis is presented as KM curves of PFS and OS for the training and validation sets. The training data shows a significant log-rank p-value in both PFS and OS. The validation data shows a significant effect in PFS.

What follows is quite an exhaustive process of finding differences between the cohorts using a multitude of techniques and datasets, including genomics, epigenetics, transcriptomics, and proteomics. These sections are mainly descriptive and do add understanding to the classification, especially with regard to the T-cell populations that are invasive.

Improvements could be made to the latter sections of the main paper. The basis for the potential clinical responses of the subtypes is arrived at via a "pre-clinical model" based on 81 genes. It would benefit from clarification on what genes were used in model training and details of the final model. Similarly the description of the "Single-sample gene classifier" could be enhanced similarly with a better description of which genes are in the final classifier.

Reviewer #1 (Public Review):

This paper investigates the metabolic basis of a node, posterior cingulate cortex (PCC), in the default node network (DMN). They employed sophisticated MRI-PET methods to measure both BOLD and CMRglc changes (both magnitude and dynamics) during attention-demanding and working memory tasks. They found uncoupling of BOLD and CMRglc in PCC with these different tasks. The implications of these findings are poorly interpreted, with a conclusion that is purely based on other work independent of this study. Various suggestions could allow them to place some speculations in line with a stronger interpretation of their results.

This is one of several papers in recent years investigating the metabolic underpinnings of activated (or task-positive) and deactivated (or task-negative) cortical areas in the human brain. In this study, they used BOLD fMRI and glucose PET scan to examine the metabolic distinction of the default node network (DMN), which is known to be deactivated during attention-demanding tasks, with different types of cognitively demanding tasks. Unlike the BOLD response in posteromedial DMN which is consistently negative, they found that CMRglc of the posteromedial DMN (a task-negative network) is dependent on the metabolic demands of adjacent task-positive networks like the dorsal attention network (DAN) and frontoparietal network (FPN). With attention-demanding tasks (like Tetris) the BOLD and CMRglc are both downregulated in DMN (specifically the posterior cingulate cortex, PCC, a task-negative node of DMN), but working memory induces CMRglc increase in PCC and which is decoupled from the negative BOLD response in PCC.

1. These complicated results are the main findings, and to provide a biological basis to these data they rather surprisingly, but without their own experimental evidence, conclude that the negative BOLD and negative CMRglc in PCC during attention-demanding tasks is due to decreased glutamate signaling (which was not measured in this study) and the negative BOLD and positive CMRglc in PCC during working memory is due to increased GABAergic activity (which was not measured in this study). It is rather surprising that without measurement, a conclusion is made which would at best be considered a hypothesis to be tested. Thus, independent of these hypothesized mechanisms, they need to summarize their results based on their own measurements in this study (see 3 for a hint).

2. It is mentioned that the FDG-PET scans allow quantitative CMRglc, both in terms of units of glucose use but also with high time resolution. Based on the method described, it isn't clear how this is possible. Important details of either prior work or their own work have been excluded that show how the time course of CMRglc (regardless of whether it's absolute or relative) can be compared with the BOLD time course. Furthermore, it is extremely difficult to conceive that quantitative CMRglc can be estimated without additional measurements (e.g., blood samples, etc). Significant methodological details have to be provided, which even should make their way to results given the importance of their BOLD-CMRglc coupling and decoupling in the same region.

3. It is surmised that the glutamatergic/GABAergic involvement of these metabolic differences in PCC is from another study, but what mechanism causes the BOLD signal to decrease in both stimuli? This is where the authors have to divulge the biophysical basis of the BOLD response. At the most basic level, the BOLD signal change (dS) can be positive or negative depending on the degree of coupling with changed blood flow (dCBF) and oxidative metabolism (dCMRO2) from resting condition. Unfortunately, neither CBF nor CMRO2 was measured in this study. In the absence of these additional measurements, the authors should at least discuss the basis of the BOLD response with regard to CBF and CMRO2. If we assume that both attention-demanding and working memory tasks decreased BOLD response in PCC in the same way, we have identical dCBF/dCMRO2 in PCC with both tasks, i.e., their results seem to suggest an alteration in aerobic glycolysis with different tasks. With attention-demanding tasks, CMRglc decreases similarly to CMRO2 decreases in PCC, whereas with working memory tasks, CMRglc increases differently from CMRO2 decreases. This suggests PCC may the oxygen to glucose index (OGI=CMRO2/CMRglc) would rise in PCC attention-demanding tasks, but fall in PCC with working memory tasks. This is obviously an implication rather than a conclusion as CBF or CMRO2 were not measured.

4. Given the missing attention that gives rise to the BOLD contrast mechanism, it is almost necessary to discuss the biophysical basis of BOLD contrast and specifically how metabolic changes have been linked to both increases and decreases in neuronal activity in the past. Although this type of work has largely been conducted in animal models, it seems that this topic needs to be discussed as well.

Reviewer #1 (Public Review):

The authors assessed the association between exposures and obesity by environment-wide and epigenome-wide association studies. The strength of this study is that exposures, body mass index, and waist-hip ratio were measured three times from adolescence to early adulthood, and the associations were repeatedly evaluated. A weakness of this study is that a loose significance threshold was used for the epigenome-wide association study and only a small number of study subjects were measured in early adulthood. Since this is an observational study, the confounding effect should be considered when interpreting the exposures associated with obesity reported in this study.

Reviewer #1 (Public Review):

This manuscript presents an exciting set of experiments on the mechanisms through which PSD proteins induce actin bundle formation. The study builds on a previous observation from the Zhang laboratory that phase condensates of six PSD proteins lead to the formation of actin bundles. Here, deep mechanistic analyses determine the necessity of upper vs. lower level PSD proteins for actin bundle formation, identify the domains and interactions of these proteins that are necessary and sufficient to induce actin bundles, and provide a first assessment in neurons of potential roles of the newly discovered mechanisms. The authors find that a patch of arginines in the Homer EVH1 domain plays a central role. Strikingly, no adaptors are needed for PSD condensates to induce actin bundles. This work is important for the understanding of roles and mechanisms of interactions between postsynaptic receptor scaffolds and cytoskeletal elements in dendritic spines. The mechanisms that are uncovered are likely mediators of structural and functional synaptic plasticity.

Overall, the data are rigorously acquired and convincing, the presentation of the findings is logical and clear, and the manuscript is well-written. In my view, a few adjustments in data presentation (quantitative assessment of in vitro experiments, statistical analyses) and additional analyses of existing data (on the localization and roles of transfected Homer proteins in neurons) will improve the paper, but new experiments are not necessary.

Reviewer #1 (Public Review):

This study presents a valuable comparison of fibre orientation estimates from three different modalities: diffusion MRI, scattered light imaging, and x-ray scattering. The comparison is interesting as each modality is sensitive to different aspects of tissue microstructure - water anisotropy, micron-scale structural coherence, and myelin lamella respectively. Where scattered light and x-ray imaging can be only applied ex vivo, diffusion MRI has in vivo applications but suffers from being an indirect estimate of the microstructure of interest. By acquiring all modalities in both a vervet monkey and human brain sample, the authors provide quantitative, pixel/voxel-wise comparisons of fibre orientation estimates within the same tissue samples. The authors show convincing agreement in fibre orientations from all three methods, giving confidence in the fidelity of the methods for neuroanatomical investigations. Differences are also observed: SLI is shown to have less reliable estimates of fibre inclination, and the CSD analysis presented overestimates the number of crossing fibre populations when compared to the microscopy methods, particularly in single fibre regions such as the corpus callosum, a known artefact in some diffusion analyses.

In the current PDF, it is very difficult to see fibre orientations in figures due to low resolution, limiting the reader's ability to assess the results. Higher-resolution images would provide more information and easier comparisons.

The methods are generally clear though some additional information is needed: 1) to specify the resolution that the orientations are compared in each figure and how data was up-/down-sampled for these comparisons respectively. For example, each SAXS pixel contains many SLI pixels. It is currently unclear whether the mean SLI orientation from a neighbourhood is equivalent to the SLI compared, or whether a comparison was made for each SLI pixel. Similarly, for the dMRI-microscopy comparisons. 2) I also could not follow why two SLI methods are presented in the methods: SLI scatterometry relating to Figure 2, and angular SLI relating to all other results. Further clarification is needed. 3) Since the quality of the data co-registration can strongly impact pixel/voxel-wise comparisons, quantification of the registration accuracy or overlays demonstrating the quality of the co-registration would be valuable.

A primary weakness of the work as a diffusion MRI validation study is that though diffusion MRI supports many different models to extract fibre orientations with different outputs, here only a single model is compared to the microscopy data, which may affect the generalisability of the results. Further, it only compares the primary orientations from the diffusion MRI and does not consider each fibre population's magnitude (density of fibres) or the orientation dispersion, both of which can influence downstream analyses.

The paper could be strengthened by a more detailed discussion on the differences between the imaging modalities - e.g. in terms of imaging resolution, signal-generating mechanisms, and sensitivity to specific aspects of the tissue microstructure - and how these differences may limit their application to specific neuroanatomical investigations, or ability to validate one another. For example, the microscopy sections are 80 microns thick whilst the diffusion voxel is 200 microns. I expect this could contribute to the difference in the number of fibre populations per voxel.

The hypothesis that dMRI signal contributions from extra-axonal water result in additional fibre populations could be investigated by running CSD on both low and high-b-value data (for example using the openly available MGH dataset, Fan 2016) where fewer secondary fibre populations should be observed at high b-value.

Reviewer #1 (Public Review):

The study tackles the topic of male harm (sexual selection favoring male reproductive strategies that incur a reduction of female fitness) from an interesting angle. The authors put emphasis on using wild-collected populations and studying them within their normal thermal range of reproductive conditions. Where previous studies have used temperature variation as a proxy for stressful environmental change, this approach should instead clarify what can be the role of male harm on female fitness in natural conditions. A minor caveat regarding this point is the fact the polygamy treatment also has a heavily male-biased sex ratio (3:1). The authors argue that this sex ratio is within the range of normal variation in that species, but it is likely that the average is still (1:1) in natural populations and using a male-biased sex ratio could magnify the intensity of male harm. This does not undermine the conclusions regarding the temperature sensitivity of sexual conflict but should be acknowledged.

The authors find that varying temperature within a range found in natural conditions affects the reproductive interactions between males and females, particularly through male-harm mechanisms. Male harm, measured as a reduction in lifetime reproductive success (LRS) from monogamy to polygamy settings is present at 20C, stronger at 24, and absent or undetectable at 28C. Female senescence is always faster in the polygamy mating systems as compared to monogamy, but the effect appears strongest at 20C. Mating behaviors of males and females in these different settings are used to attempt to uncover underlying mechanisms of the sensitivity of male harm to temperature.<br /> A weakness of the manuscript in its current form is the lack of clarity about the experimental design, which makes understanding the results a long and involved procedure, even for someone who is familiar with the field. If the authors consider revising the manuscript, I suggest giving a better overview of the experimental design(s) earlier in the manuscript, perhaps supported by a diagram or flowchart. I also suggest structuring the results better to aid the reader (e.g., make clearer distinctions between results that come from the different experiments). Finally, some additional figures and statistical tests corrected for multiple testing would help get a better feel of some aspects of the dataset.

I believe that the conclusions are generally justified and the results overall convincing. Overall, this is an impressive study with a lot of dimensions to it. Its complexity is a challenge and may require additional effort from the authors to make it easier to access. The core of the question is answered by LRS measures, but the authors have also provided a wealth of behavioral data as well as other fitness components. The manuscript could be greatly improved by putting more effort into linking the different metrics together to track down potential mechanisms for the observed variation in male-harm-induced reduction in female LRS. The discussion would also benefit from considering the female side of the sexual conflict coevolution arms race.

Reviewer #1 (Public Review):

The authors address an important and understudied problem: how precise temporal properties of synaptic transmission might impact the kinds of neuronal correlations that instruct development. The methods used to characterize and simulate retino-thalamo-cortical development are carefully carried out and yield convincing results. Based on these simulations, the authors argue that features such as slow NMDA receptor-mediated currents are able to prevent aberrant development which might otherwise result from rapid timescale correlations that lack meaningful information about visual topography.

Reviewer #1 (Public Review):

The authors generated a detailed single-cell RNAseq dataset for the microfilariae stage of the human nematode parasite Brugia malayi. This is an impressive and important achievement, given that it is difficult to obtain sufficient material from human parasites and the microfilariae are protected by a chitin sheath. The authors collected microfilariae from jirds and carefully worked out a protocol of digestion, dissociation and filtering, to obtain single-cell material for sequencing.

The single-cell resource was complemented with a dataset derived from FACS-sorted large secretory cells, allowing the identification of several specific proteins expressed in this unique microfilarial cell-type important for immune evasion.

The authors also generated new data for secretory cells of Caenorhabditis elegans and concluded that there is limited similarity between the composition of Brugia and C. elegans secretory cell types.

In a further set of experiments, the authors analysed gene expression changes in dissociated Brugia cells to the commonly used anthelminthic drug ivermectin. This revealed specific gene expression changes across various cell types, providing new insights into how the drug effects the parasite.

Finally, the authors developed a method to keep dissociated Brugia cells alive in culture for two days. This method will aid cellular studies of this parasite.

The authors may want to explore the new resource in more detail to reach more specific biological conclusions. For example, the authors mention that the large secretory cells are critical to parasite survival and immune evasion. With a more complete list of genes expressed in these cells the authors could try to reach more specific conclusions or predictions. Are there newly identified secreted factors that could contribute to immune evasion? It would be important to read in more detail about such proteins (including an analysis of the sequences and phylogenies), especially if the authors could identify new candidates as potential vaccine or diagnostic targets. Likewise, can the data be used to understand in more detail the mechanism of immune evasion or ivermectin action?

The authors searched for known secreted proteins, including antigens, vaccine targets, and diagnostic markers and mapped the expression of these to the single-cell atlas. It is not clear from the paper how comprehensive previous studies to identify secretory proteins were. With the new resource in hand, the authors could look at all secreted proteins (with a signal peptide) expressed in the ES and other cells. The paper would benefit from a more comprehensive overview of the classes of secretory proteins and their expression.

The authors show that an abundance of C2H2 transcription factors is localizing almost exclusively to the secretory cells. It would be useful to see a classification of these proteins and phylogenetic analysis relating them to C2H2 from C. elegans and other animals.

In general, a more detailed bioinformatic analysis of secretory products and more discussions of potential functions (e.g. serpins etc.) would make the paper more interesting and could stimulate more mechanistic thinking.

Reviewer #1 (Public Review):

The manuscript Role of cytoneme-like structures and extracellular vesicles in Trichomonas vaginalis parasite: parasite communication by Salas N et al is an interesting manuscript with novel findings, clear strategies, and fine design of experiments. Despite the quality of the manuscript, it must be improved in order to deliver the best message in the area of cellular biology and molecular parasitology.

Reviewer #1 (Public Review):

The authors of this study exerted a variety of laboratory experiment methods and in silico analysis of expression data, and showed the differentiated aspects of the protein functions of the product of the duplicated genes eS27 and eS27L as well as their redundant aspects. These proteins are components of the cellular machinery for translation, namely 'readout' of the genome, in eukaryotes. This study provides a valuable test case of examining why seemingly redundant genes that underwent gene duplication during evolution have been retained in the genomes of many present-day organisms.

Reviewer #1 (Public Review):

Tunneling nanotubes, contrary to exosomes, directly connect remote cells and have been shown to allow the transfer of material between cells, including cellular organelles and RNAs. However, whether sorting mechanisms exist that allow to specifically transfer subspecies of RNAs, especially of mRNA, has not been shown, and the transcriptional consequences of RNA transfer have not been addressed yet.

Using cocultures (or mix or single cultures as controls) of human MCF7 breast cancer cell line, and immortalized mouse embryo fibroblasts (MEFs), followed by separation of human and mouse cells by cell sorting, the authors performed deep sequencing of the human mRNAs detected in mouse cells. An accurate analysis of the transferred material shows that all donor cell mRNAs transfer in a manner that correlates with their expression level, with less than 1% of total mRNA being transferred in acceptor cells. These results show that the process of RNA transfer is nonselective and that the consequences on the cells receiving the RNAs should depend on the phenotype of the sending cells. These results are complemented by the last part of the manuscript where the authors convincingly show that the coculture of the two cell lines results in significant transcriptomic changes in acceptor MEF cells that could become CAF-like cells.

Reviewer #1 (Public Review):

Animals respond to their environment in a state-dependent manner. One of the best examples of this is the dramatic changes in behaviours in the female after mating. In flies, this includes an overall increase in food consumption, a well-documented increase in protein appetite, increased salt appetite, increased egglaying behaviour, and reduced sexual receptivity.

In this study, the authors argue that sugar is a macronutrient that should be essential to support the increased metabolic needs of the fly and the lipid demand of the eggs. They isolate sugar (instead of providing it in a choice assay) and document that indeed mated flies have an increased appetite for sugars.

They then go on to demonstrate that this increase is not need-based, but is anticipatory in nature and that it is not changes in sensitivity of the sugar-sensing neurons, but central brain circuitry that drives this behvioural change. Finally, they work out the circuitry demonstrating that it diverges from the well-described three-layer mating circuit (SPSN>SAG>pC1) that is active in virgins but inhibited by sex-peptide in mated females. They use EM datasets to identify the pCd2>Lgr3+ neurons as downstream of pC1 and develop genetic tools to monitor and manipulate neuronal activity in these neurons to show that the Lgr3+ neurons are active in the mated state because they receive inhibitory inputs from the pCd2s.

As LG3 neurons are known to be activated by the DILPs, which mediate satiety, their model proposes the state of mating (as signalled by central brain circuitry) is essentially a state of additional hunger.

Reviewer #1 (Public Review):

The human genetic variant Dantu increases the surface tension of red blood cells making it hard for malaria parasites to invade. This was shown beautifully by Kariuki et al in 2020 (doi.org/10.1038/s41586-020-2726-6) by analysing blood from children using in vitro assays with cultured malaria parasites. Now Kariuki et al show that parasite growth is indeed restricted in vivo by infecting Dantu adults under controlled conditions with cryopreserved Plasmodium falciparum sporozoites and analysing parasite growth by qPCR. The authors compare parasite growth, peak parasitaemia and if / when treatment was sought for malaria symptoms between non-Dantu (111) and Dantu heterozygous (27) and homozygous (3) participants. Dantu either completely prevented malaria parasite detection in the blood (for 21 days) or slowed down parasite growth considerably.

The authors present compelling in vivo evidence that Dantu conveys protection by preventing malaria parasites from establishing a blood-stage infection. Because the effect on parasite growth is crystal clear the link to uncomplicated malaria follows - no/less parasites leads to less participants experiencing malaria symptoms and seeking treatment. It should however be noted that the paper does not show that Dantu reduces symptomatology at identical parasite densities to non-Dantu. Its protective effect seems to be purely parasitological.

Given that all volunteers were exposed to malaria prior to being experimentally infected (in various transmission settings ranging from low to high) the authors state that they adjusted for factors like schizont antibody concentration in their multi-variate analysis. More details on the assumptions and which dependent / independent variables were included would benefit interpretation. It would be also good to see if Dantu individuals were spread homogeneously across all transmission settings - if e.g. they all had history of intense malaria exposure and thus strong pre-existing anti-malaria immunity this might account in part for reduced parasite growth when compared to non-Dantu from lower transmission settings. Being able to de-convolute the effect of pre-existing immunity from Dantu would strengthen the paper.

The authors also presents data on other red cell polymorphisms known to modulate malaria infection and improve outcome: G6PD, blood group O, alpha thalassaemia and ATP2B4. However, no statistically significant differences between non-carriers and hetero/homozygous individuals were observed. This is probably because these mutations exert their effect not directly on parasite growth but modulate disease symptoms when parasite burden is high - which cannot be investigated in controlled human malaria infection settings as ethical considerations mandate treatment of all volunteers at parasite densities >500 parasites/ ul or any parasitaemia with symptoms. Controlled infections need to be complemented with other methods to understand the protective impact of genetic polymorphisms.

Reviewer #1 (Public Review):

In this manuscript the authors describe a new method for perturbing chromatin in living cells by delivering a local temperature gradient. Employing this approach, the authors uncover interesting behaviors that underscore the variability in the mechanical response of subnuclear domains and structures. The combination of a new experimental tool that should be accessible to many users and new insights are compelling, although there is the need for some controls and a broader discussion of prior work.

Strengths:<br /> 1. There is a need for non-invasive methods for probing the mechanical properties of chromatin, and nuclei and the approach developed by the authors has strong potential to be of broad utility.<br /> 2. By and large the authors provide a reasonable characterization of the technical aspects of the method, for example how local temperatures rise and the propagation of the temperature gradient relative to the rastering of the IR laser.<br /> 3. The findings that different chromatin compartments respond in distinct manners, in ways perhaps that were not intuited previously (for example, the highest level of deformation for "medium dense" chromatin domains regions), is provocative and raises new ideas about how the chromatin polymer and diffusible nuclear constituent molecules in different domains together contribute to the mechanical response.<br /> 4. The method provides insights into the viscoelastic properties of different chromatin domains, particularly different time scales of behavior, that have been challenging to access with existing approaches.<br /> 5. The authors provide new measurements of the behavior of nucleoli, which leads to insights that will impact our view of the mechanical behavior of such organelles.

Weaknesses:<br /> 1. Direct or indirect effects of the temperature gradient on the integrity of the DNA needs to be addressed, as this could influence the response particularly given the observation that there is a ~15% of the response that is not reversible (see next point).<br /> 2. The authors do not probe the basis for the irreversibility of the chromatin response, which seems to perhaps differ between different chromatin regions. The underlying factors that underlie this need to be further explored.<br /> 3. The authors need to acknowledge the time scales of behaviors that can be revealed using the approach and how this influences their observations. For example, they observe the creep behavior on the 1 second timescale, which is an order of magnitude below observations of the behavior of whole nuclei (~15 seconds) for nuclei from mammalian to yeast that has been suggested to reflect chromatin flow.<br /> 4. There are numerous studies important for the premise and interpretation of this study that need to be considered/cited.

Reviewer #1 (Public Review):

In this work, authors seek to understand how the polycomb complex may coordinate gene expression changes that occur during sequential stages of neuronal maturation. The main strengths are 1) choice of cerebellar granule neurons which mature over a protracted period during normal cerebellar development and constitute a relatively homogeneous population of neurons, 2) use of a genetic in vivo mouse model where a histone demethylase is knocked out, combined with an in vitro culture model of maturing cerebellar granule neurons in which a histone methyltransferase is inhibited, 3) use of CUT & TAG in neuronal cultures to investigate how changes in the H3K27me3 repressor chromatin modification at promoters correlate with gene expression and chromatin accessibility changes. The authors propose a bidirectional effect of the same chromatin repressor modification that is responsible, at least in part, for the timely loss of expression of early genes and the appearance of genes expressed later in maturation. This is the major impact of the work for those interested in cerebellar development. A weakness in the work lies in its narrow focus, which is on promoter regions almost exclusively.

The work is primarily bioinformatics driven and lacks physiological significance of the gene expression changes, or how the culture timing correlates with temporal regulation and chromatin changes in vivo. However, the results do support the proposal that polycomb-associated enzymatic activities play sequential roles during successive stages of cerebellar maturation.

Reviewer #1 (Public Review):

The authors have studied the effect of temperature on the interspecific interaction strength of coastal marine fish communities, using eDNA samples. Their introduction describes the state of the art concerning the dynamics of interspecific interactions in ecological communities. This introduction is well written and highly information dense, summarizing all that the reader needs to know to further understand their study setup and execution.

The authors hypothesize that water temperature changes could have an effect on the interspecific interaction strength between marine fishes, and they studied this with a two year long, bi-weekly eDNA sampling campaign at 11 study sites in Japan with different temperature gradients. These 550 water samples were analysed for fish biodiversity through eDNA-metabarcoding using MiFish primers. By using the most abundant fish species as an internal spike in and quantifying the copy numbers from this species by qPCR, the authors were able estimate DNA copy numbers for the total dataset. From the 50 most frequently detected fish species in these samples they showed that temperature affected the interspecific interaction strength between some species. Their work provides a highly relevant approach to perform species-interaction strength analysis based on eDNA biodiversity assessments, and as such provides a research framework to study marine community dynamics by eDNA, which is highly relevant in the study of ecosystem dynamics. The models and analytical methods used are clearly described and made available, enabling application of these methods by anyone interested in applying it to their own site and species group of interest.

Strengths: The authors have a study setup that is suitable to measure the effects of temperature of the eDNA diversity, and have taken a large number of samples and all appropriate controls to be able to accurately measure and describe these dynamics. The applied internal spike in to enable relative eDNA copy number quantification is convincing.

Weaknesses: The authors aim to study the relationship between species interaction strength and ecosystem complexity, and how temperature will influence this. However, there is only limited ecological context discussed explaining their results, and a link with climate change scenario's is also limited. A further discussion of this would have strengthened the manuscript.

The authors were able to find a correlation between water temperature and interaction strengths observed. However, since water temperature is dependent on many environmental variables that are either directly or indirectly influencing ecosystem dynamics, it is hard to prove a direct correlation between the observed changes in community dynamics and the temperature alone.

Reviewer #1 (Public Review):

The authors use a combination of structural and MD simulation approaches to characterize phospholipid interactions with the pentameric ligand-gated ion channel, GLIC. By analyzing the MD simulation data using clusters of closed and open states derived previously, the authors also seek to compare lipid interactions between putative functional states. The ultimate goal of this work is to understand how lipids shape the structure and function of this channel.

The strengths of this article include the following:

1) The MD simulation data provide extensive sampling of lipid interactions in GLIC, and these interactions were characterized in putative closed and open states of the channel. The extensive sampling permits confident delineation of 5-6 phospholipid interaction sites per subunit. The agreement in phospholipid binding poses between structures and the all-atom MD simulations supports the utility of MD simulations to examine lipid interactions.

2) The study presents phospholipid binding sites/poses that agree with functionally-important lipid binding sites in other pLGICs, supporting the notion that these sites are conserved. For example, the authors identify interactions of POPC at an outer leaflet intersubunit site that is specific for the open state. This result is quite interesting as phospholipids or drugs that positively modulate other pLGICs are known to occupy this site. Also, the effect of mutating W217 in the inner leaflet intersubunit site suggests that this residue, which is highly conserved in pLGICs, is an important determinant of the strength of phospholipid interactions at this site. This residue has been shown to interact with phospholipids in other pLGICs and forms the binding site of potentiating neurosteroids in the GABA(A) receptor.

Weaknesses of this article include the following:

1) The authors describe in detail state-dependent lipid interactions from the MD simulations; however, the functional significance of these findings is unclear. GLIC function appears to be insensitive to lipids, although this understanding is based on experiments where GLIC proteoliposomes were fused to oocyte membranes, which may not be optimal to control the lipid environment. Without functional studies of GLIC in model membranes, the lipid dependence of GLIC function is not definitively known. Therefore, it is difficult to interpret the meaning of these state-dependent lipid interactions in GLIC.

2) It is unlikely that the bound phospholipids in the GLIC structures, which are co-purified from e. coli membranes, are POPC. Rather, these are most like PE or PG lipids. While it is difficult to accommodate mixed phospholipid membranes in all-atom MD simulations, the choice of POPC for this model, while practically convenient, seems suboptimal, especially since it is not known if PE or PG lipids modulate GLIC function. Nevertheless, it is striking that the overall binding poses of POPC from the simulations agree with those identified in the structures. It is possible that the identity of the phospholipid headgroup will have more of an impact on the strength of interactions with GLIC rather than the interaction poses (see next point).

3) The all-atom MD simulations provide limited insight into the strength of the POPC interactions at each site, which is important to interpret the significance of these interactions. It is unlikely that the system has equilibrated within the 1.7 microseconds of simulation for each replicate preventing a meaningful assessment of the lipid interaction times. Although the authors report exchange of up to 4 POPC interacting at certain residues in M4, this may not represent binding/unbinding events (depending on how binding/interaction is defined), since the 4 Å cutoff distance for lipid interactions is relatively small. This may instead be a result of small movements of POPC in and out of this cutoff. The ability to assess interaction times may have been strengthened if the authors performed a single extended replicate up to, for example, 10-20 microseconds instead of extending multiple replicates to 1.7 microseconds.

Reviewer #1 (Public Review):

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

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

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

Reviewer #1 (Public Review):

In this report, the authors use what they describe as a novel phenotypic survival screening method to uncover ATP-dependent kinases that may show synthetic lethality (when inhibited) with BRCA2 loss. Interestingly, they find that inhibiting ROCK kinases in BRCA2 deficient cells (but not BRCA1 deficient cells), triggers synthetic lethality. They further show that the synthetic lethality is independent of acute replication stress and is preceded by enhanced M-phase defects (anaphase bridges and abnormal mitotic figures). These data, therefore, suggest a new pathway (ROCK kinases) that may be targeted to induce synthetic lethality in BRCA2 deficient cells.

Reviewer #1 (Public Review):

The manuscript by Zheng et al. examined the disease-causing mechanisms of two missense mutations within the homeodomain (HD) of CRX protein. Both mutations were found in humans and can produce severe dominant retinopathy. The authors investigated the two CRX HD mutants via in vitro DNA-binding assay (Spec-seq), in vivo chromatin-binding assay (ChIP-seq), in vivo expression assay of downstream target genes (RNA-seq), and retinal histological and functional assays. They concluded that p.E80A increased the transactivation activity of CRX and resulted in precocious photoreceptor differentiation, whereas p.K88N significantly changed the binding specificity of CRX and led to defects in photoreceptor differentiation and maintenance. The authors performed a significant amount of analyses. The claims are sufficiently supported by the data. The results not only uncovered the underlying disease-causing mechanisms, but also can significantly improve our understanding of the interaction between HD-TF and DNA during development.

Minor concerns:<br /> 1. The E80A, K88N and R90W (previously reported by the same group) mutations are located very close to each other in the homeodomain (Figure 1A), but had distinct effects on the activity of CRX. Has the structure of the homeodomain (of CRX) been resolved? If so, could the authors discuss this phenomenon (mutations close to each other but have distinct effects) based on the HD-DNA structure? In addition, has this phenomenon been observed in other homeodomain TFs?<br /> 2. The authors should briefly summarize the effects/disease-causing-mechanisms of all the reported CRX mutations in the discussion part. The readers can then have a better overview of the topic.<br /> 3. CRX can also function as a pioneer factor (reported by the same group). Would these HD mutations distinctively affect chromatin accessibility (which then leads to ectopic binding on the genome)?<br /> 4. The discussion part can be shortened and simplified.

Reviewer #1 (Public Review):

Zhou et al. investigated the factors that regulate mitotic chromosome size scaling during the early embryo divisions in Xenopus laevis using imaging of intact whole embryos and of embryo extracts with different sources of nuclei. They find that chromosome volume decreases during embryogenesis, and scales with nuclear and spindle volume throughout a broad range of embryo stages (stages 3 to 9) and cell sizes. They show that extracts from stage 3 or stage 8 embryos demonstrate significant differences in chromosome length, mirroring changes to chromosome volume observed in vivo. Using extracts from eggs or stage 8 embryos, and nuclei from sperm or stage 8 embryos, the authors demonstrate that chromosome length is dictated by the chromosomes and not the maternal mitotic environment, and find that the major determining factor is the amount of condensin I loading on mitotic chromosomes, which they correlate to changes in DNA loop size and layering. Interestingly, they find that the prior state of nuclei prior to entry into mitosis dictates mitotic chromosome length. They attribute this phenomenon to the nuclear to-cytoplasmic ratio during the prior interphase and suggest that some factor is titrated on chromatin that sets condensin I loading in mitosis. Notably, they found that chromosome length does not scale with nuclear or spindle size in vitro. In another set of experiments, the authors found that artificially increasing the palmitoylation of importin resulted in decreased chromosome length. However, this scaling effect is not due to condensin I loading differences, but to some unidentified importin cargo that would get released as cell size decreases during development. Overall, the conclusions of this paper are well supported by data, but some aspects of data interpretation and analysis need to be clarified and extended. The approaches used here are quite impressive and creative and provide compelling evidence for factors that regulate chromosome scaling during development in a vertebrate organism.

Reviewer #1 (Public Review):

The impact of the COVID-19 pandemic on cancer screening, diagnosis, referrals, and management has been well documented in high-resourced countries; but such quantitative estimates are rarely available from low- and middle-income countries (LMIC). The authors chose two very high human development index (HDI) category LMICs (Argentina and Thailand), two high HDI category LMICs (Colombia and Sri Lanka), and two medium HDI category LMICs (Bangladesh and Morocco), and looked at available data for cervical, breast, and colorectal cancer screening. The authors demonstrate that the reduction in the test volumes during the pandemic (2020) versus the previous year (2019) was quite comparable to that observed in high-income countries. Additionally, some countries demonstrated resilient catch-up of programmatic performance within a short period of time after the disruptions.

Major strengths include the use of national-level data estimates from key focal points for the CancScreen-5 project, an international data repository of cancer screening programmatic data, the use of appropriately comparable monthly estimates in the pre-pandemic vs. pandemic year, and representation of illustrative case studies from six countries across the medium-to-very high HDI status among LMICs.

Weaknesses include inherent limitations of such real-world outcome/registry data, lack of data across the screening continuum, inability to explore granular-level country-specific factors affecting disruptions as well as catch-up of screening, and high variability of performance of screening tests (especially those with subjective interpretation such as VIA for cervical cancer or clinical breast exam) across the comparison periods such that screen positivity rates may have been affected in unpredictable ways.

The authors have achieved their aims since this descriptive epidemiology analysis provides key estimates from LMICs that have not been explored/evaluated in the literature.

This work will be useful for future studies conducted by health modellers on measuring the impact on late/advanced stage detection and excess case burden and mortality.

Reviewer #1 (Public Review):

This paper is based on the premise that ketamine exerts antidepressant effects that are rapid by increasing glutamatergic transmission. However, the authors note that how this effect occurs is unclear because ketamine antagonizes the NMDA receptor, a glutamatergic receptor.<br /> Others have suggested a compensatory change in the glutamatergic transmission and the authors suggest how this might occur. The authors should clarify if prior studies suggested a mechanism different from theirs and if so, which might be correct.

There are also other mechanisms, such as the block of NMDA receptors on interneurons and the disinhibition of principal cells. It is important to clarify if this has already been addressed in the literature. Also, if their cultures are primarily glutamatergic neurons or they include interneurons and glia.

The authors show calcineurin is reduced after ketamine exposure and this increases AMPA receptor GluA1 phosphorylation. They also show that Calcium permeable AMPA receptors )CP-AMPARs) increase.

They also use suggest that the CP-AMPARs and other changes lead to enhanced synaptic plasticity, which could lead to antidepressant effects.

Although a lot of work is done in cultured hippocampal neurons, 14 days in vitro, they show effects in vivo that are consistent with the data from cultures. For example, ketamine increases GluA1 phosphorylation. Also, blocking CPAMPARs in vivo reduces anxiety/depressive behaviors such as the open field and tail suspension tests.

Overall the study appears to be done well and the presentation, writing, and references are good. There are important concerns regarding statistics, behavior, and pharmacology and several minor concerns.

Major concerns<br /> 1. Statistics.<br /> What was the stat test if the control was always 1?<br /> Often the control group is 1.00 with no SD but in other tests, the control group is 1.000 with an SD.<br /> e.g., line 145: "(CTRL) (CTRL, 1.000 and ketamine, 1.598 {plus minus} 0.543, p = 145 0.0039), but not GluA2 (CTRL, 1.000 and ketamine, 1.121 {plus minus} 0.464, p = 0.6498"

Line 188:<br /> Here the control group has a SD:<br /> Line 188 CTRL, 1.000 {plus minus} 0.106 and ketamine, 0.942 {plus minus} 0.051, p = 0.0170

2. Behavior.<br /> It is not clear that the open field and tail suspension tests measure antidepressant actions. Why were more standard tests such as forced swim or sucrose preference, novelty-suppressed feeding, etc not used?

3. Pharmacology.<br /> The conclusions rest on the specificity of drugs.<br /> Is 5 uM FK506 specific?<br /> 20 μM 1-naphthyl acetyl spermine (NASPM)?<br /> 10 mg/kg IEM-1460?

Reviewer #1 (Public Review):

The essentiality of Rv1636 has previously been predicted in numerous genetic studies. Here, the authors provide evidence that Rv1636 is an essential protein in Mtb. The authors report that chromosomal deletion of the gene encoding Rv1636 is only possible when an additional copy of the wild type gene is provided at the L5 integration site in the chromosome. While this is a standard method of demonstrating gene/protein essentiality in this system, the manuscript only provides a PCR reaction with "no amplicon" as proof of a double crossover event in an engineered merodiploid strain (Fig 6C). The authors fail to provide definitive evidence for a double crossover mutation in the merodiploid using primers that amplify a double crossover-dependent amplicon or the authors should a provide a southern blot demonstrating evidence for a bona fide double crossover event. The authors suggest that silencing the gene encoding Rv1636 with a CRISPRi system decreases viability of Mtb when a silencing guide RNA is expressed following Atc addition and spot plated onto agar. These studies lack a "no Atc control" and it is unclear how Mtb colonies appear after 6-7 days in these studies given the slow growth of this bacterium.

A sub-point of the manuscript describes the genetic organization around the gene that encodes Rv1636 in various Mycobacterial spp. Figure 1 also highlights the putative transcriptional start sites for the gene encoding Rv1636. The putative transcriptional start site information is just a summary of work from other groups and this information adds little to the main goals of this manuscript.

Another sub-point of this manuscript is that Rv1636 may be secreted by Mtb in a SecA2 dependent manner. The authors demonstrate that Rv1636 is not present in the culture filtrate of Mtb lacking SecA2 (Fig 2). However, these data are difficult to interpret without a secreted protein "loading control" which is typical for these types of experiments. The authors also report the development of a luciferase-based detection method for quantifying protein secretion in Mtb and use this to support their conclusion. This is a new tool that could be useful in detecting secreted proteins in Mtb. However, this method is not rigorously validated in these studies and do not present controls for cell lysis for example. Additionally, the authors fuse a ~19 kDA luciferase subunit to the C-terminus of CFP10 as a reporter for Esx1-dependent secretion. It is known that this region of CFP10 is critical for interactions with secretory components of the Esx1 system fractionation and it unclear if the CFP10 fusion protein is actually secreted.

The authors explore the idea that Rv1636 may potentially function as a "sink" for cAMP and quantify the molar amounts cAMP, ATP, and Rv1636 in Mtb. These studies demonstrate that the molar amounts of Rv1636 exceeds the levels of cAMP (free or protein-bound) in the cytosol of the Mtb. The authors conclude that the excess of Rv1636 may potentially be a "sink" for unbound cAMP but do not test this idea experimentally in Mtb due to the very low levels of cAMP in this bacteria.

Instead, the authors continue exploring the idea that specific proteins can serve as a cAMP "sink" using M. smegmatis (Msm) since this bacterium produces more cAMP (~25x) in the cytosol compared to Mtb. The authors present data that over expression of Rv1636 in Msm increases the amount of protein-bound cAMP. It is presumed here that the protein-bound cAMP is bound to Rv1636. Alternatively, deleting the Rv1636 homolog in Msm (MSMEG_3811) results in an increase in the amount of "free cAMP". Again, it is presumed that deleting the cAMP binding protein MSMEG_3811 is responsible for the increase in the amount of "free cAMP" in the cell.

Lastly, the authors use two small molecule compounds that may bind Rv1636 and demonstrate some level of bacterial inhibition using a spot plating method. No evidence is provided to demonstrate that these compounds are specifically binding/inhibiting Rv1636. These studies are lacking rigorous demonstration of "on target" inhibition and add very little to the reliable conclusions in this paper.

Reviewer #1 (Public Review):

Villalobos-Cantor et al. describe a chemical/genetic strategy to enable cell-type-specific labeling of nascent proteins in living tissues (called POPPi). O-propargyl-puromycin (OPP) is a commonly used compound to label nascent proteins in cells and tissue, however, its application is limited in vivo because it can not be targeted to individual cell types, tissues, or organs. Using Drosophila as a genetically tractable in vivo model organism, Villalobos-Cantor et al. incubate live tissue with a puromycin analog called phenylacetyl-OPP (PhAc-OPP) in combination with cell-type expression of Penicillin G acylase (PGA), which converts PhAc-OPP to OPP. As PGA is under the control of the Gal4/UAS system, a vast library of tissue-specific Gal4 lines can in theory be used to conduct labeling experiments in vivo.

The major strength of the methods and results is the demonstration that labeling can occur in specific cell types of the dissected brain - neurons and glia. For example, protein synthesis in individual dopamine neurons in the brain can be visualized and distinguished from neighboring cells, a remarkable achievement and striking image. These results in dissected brains nicely demonstrate that PhAc-OPP can penetrate into brain tissue, diffuse to internal locations, pass through the cell membrane, and become converted to OPP and label nascent proteins. A major weakness of the methods and results is the lack of exploration of POPPi in tissues other than the brain, as well as in non-dissected living animals. For example, the authors do not test if PhAc-OPP delivery can occur by feeding animals, or if PhAc-OPP can penetrate into various dissected tissues. Results from these experiments would be of great importance to others interested in applying this technique in non-brain tissues, and would properly support the authors' claims in the title and abstract that this is a general method (not only for the brain).

Assuming that PhAc-OPP can penetrate various dissected tissues, this method would have a significant impact on tissue-specific measurements of protein synthesis and could be a valuable new molecular reporter for gene function analysis (e.g. tissue-specific gene knockout + POPPi). If PhAc-OPP could be ingested by flies, perfuse through the body, and label nascent proteins in a cell-type specific manner, then POPPi could be incredibly useful for tissue-specific proteome profiling (i.e. mass spectrometry) in an in vivo living animal (non-dissected), similar to the BioID system.

Reviewer #1 (Public Review):

This project aimed to understand if decision making impairments commonly observed in older adults arise from working memory (WM) or reinforcement learning (RL) deficits. Evidence in the paper suggests it is the former; they observe poorer task accuracy in older adults that is accompanied by a faster memory decay in older adults using a novel hierarchical instantiation of a previously validated computational model. There were no similar changes in RL in this model. These results are extended using Magnetic Resonance Spectroscopy (MRS) to measure glutamate and GABA levels in striatum, prefrontal and parietal regions. They found that impairments in working memory were linked to reductions of glutamate in PFC, particularly in the older adult group.

The task employed is elegant and has been studied extensively in different populations and is well-validated (though here a hierarchical Bayesian extension is developed and validated). The results however may not be definitive in some respects; the paper did not replicate previously observed RL deficits. It therefore, remains possible that this is due to the sensitivity of the task to this RL component in ageing and future work is needed to fully bridge the gap in the literature.

Although the study is well-executed, there is an obvious limitation in the use of a cross-sectional design to address this question. The authors acknowledge this limitation in the discussion but could go further to highlight the potential confound of cohort effects on gaming, RL and WM tasks more generally. Without within-person change data, the evidence can only be suggestive of potential age-related decline. For this reason, it may be more appropriate to use the terminology "age-related differences' rather than "age-related declines" given the study design.

Reviewer #1 (Public Review):

This study represents an important work in the field of (CAR)T-cell immunotherapy by analyzing the effect of different oxygen tension on the function and differentiation of T-cells (especially CD8+). Although it has been described that low oxygen levels can influence effector function/differentiation of T-cells, as nicely acknowledged by the authors in the introduction, a comprehensive analysis in the context of immunotherapy has been missing so far and this study adds significant findings that will be relevant for patient care in all fields applying (CAR)T-cell immunotherapy.

The strength of the evidence is generally solid although there are some discrepancies between the different ways to induce HIF-1α (i.e. low O2, pharmacological inhibition, shRNA knockdown) that need to be clearly stated and/or discussed.

1) The first section of the results determines the impact of low oxygen and pharmacological HIF-1α stabilization on CD8+ T-cell activation/differentiation. Low oxygen diminishes cell growth but induces T-cell activation and effector cytokines, while HIF-1a stabilization mimics the effects on activation without alterations in expansion. Unfortunately, it remains unclear why effects upon low O2 are more pronounced although pharmacological HIF-1a stabilization is more efficient.<br /> 2) As a next step, in vitro conditioned T-cells are transferred into a subcutaneous B16-OVA model. Although only the low O2 levels increase T-cell numbers in vivo after the transfer, the initial tumor burden was nicely decreased by both low O2 and HIF-1a stabilization. However, only the latter significantly improved survival and it remains unclear and uncommented why.<br /> 3) Next, the authors address whether pre-conditioning of human CART-cells to induce HIF-1α either by pharmacological stabilization or by silencing of VHL shows similar effects. Surprisingly, both ways of HIF-1a stabilization resulted in different effects concerning differential gene expression and cytotoxic capacity of CART-cells. Accordingly, pharmacologically pre-conditioned CART-cells did not have a significant impact on survival in an in vivo model, while the VHL-silenced ones did significantly improve animal survival. This discrepancy between the two modes of HIF-1a stabilization remains uncommented. Unfortunately, it also remains unclear why the pharmacological HIF-1a stabilization significantly improved the survival in animals of the B16-OVA model and not in the human CART-cell model.<br /> 4) After this, the researchers determine how the timing of hypoxic conditioning affects the (CAR)T-cells. Here it is convincingly shown that already a short period of hypoxic conditioning (1 day) with a subsequent expansion phase (additional 6 days) is sufficient to induce HIF-1a mediated alterations (e.g. metabolic changes, calcium flux, intracellular signaling). Although this section is coherent in itself, the switch between different times of hypoxic conditioning, expansion, and analysis is difficult to follow and might lead to confusion. The expression pattern of e.g. HIF-1a on day 1 and day 7 together with the nuclear amounts of NFAT and c-Myc might be misunderstood, like the other presented data as well.<br /> 5) Last, short-term hypoxic conditioning of CART cells is tested in a solid tumor mouse model. The previously identified conditioning protocol also increases CART-cell function against solid tumors (as shown by enhanced cytotoxicity, reduced tumor burden, and prolonged survival). Unfortunately, although both HER2-CART-cells and CD19-CART-cells are shown to have superior cytotoxicity in vitro after the pre-conditioning, only HER2-CART-cells are demonstrated to be superior upon low O2 conditioning in an in vivo adoptive transfer mouse model and CD19-CART-cells remain an open question.

Generally spoken, the limitations of the manuscript are:<br /> 1) The occurring discrepancies of determining effects caused by the different modes of Hif-1a stabilization which certainly are caused by the complex nature of Hif-1a regulatory network, and;<br /> 2) The limitation of detected effects primarily on CD8+ T cells while CART-cells products usually are a mixture of CD4+ and CD8+ ones.

Reviewer #1 (Public Review):

This manuscript described the role of ALKBH5, an evolutionarily conserved mRNA m6A demethylase as a key regulator of axon regeneration. The authors screened the function of m6A regulators during axon regeneration and found that ALKBH5 limits regenerative growth associated with DRG neurons, by enhancing the stability of Lpin2 mRNA via erasing a single m6A modification in the 3'UTR. The major strength of the manuscript is the convincing importance of ALKDH5 as an attenuator to initially suppress the axon regeneration in the CNS and in the PNS proven by in vivo model system. These findings further suggest the potential use of ALKDH5 inhibitors to enhance neural regeneration upon physical injury.

Reviewer #1 (Public Review):

Baggett C., Murphy K. R., and Sengun E. et al. investigated cell senescence as the basis of pro-arrhythmogenic changes associated with myocardial infarction in the aged heart using the rabbit as a model, with validation of senescence markers on human heart specimens. The study is interesting and addresses a relevant biological and health issue. The authors demonstrate that aged rabbits are prone to arrhythmogenesis associated with higher mortality within 72 h after induction of myocardial infarction. Analysis of scar morphology determined that fibrosis is not sufficient to explain age-associated arrhythmogenesis. Instead, the authors show that senescence, assessed by -galactosidase activity, expression of regulators of the senescence-associated secretory phenotype, and H2AX, is increased in myofibroblasts compared to endothelial cells in infarcted aged rabbit hearts. Accordingly, H2AX was detected in αSMA+ cells in human-aged hearts. The authors tested the influence of myofibroblasts on cardiomyocyte electrophysiology by exposing cardiomyocytes in vitro to conditioned media from fibroblasts in which senescence was induced by treatment with etoposide. Such treatment did not affect action potential duration, leading the authors to conclude that senescent fibroblasts are unlikely to influence cardiomyocytes through paracrine signaling. Instead, the authors propose a possible yuxtacrine effect. To test this, they performed immunofluorescence to infer potential myofibroblast-cardiomyocyte coupling by the presence of connexin 43 in the cell-cell interphase and tested the potential electrophysiological effects of coupling using a computational model.

The analysis of peri-procedure mortality, arrhythmogenesis, and senescence in young and aged rabbits subjected to myocardial infarction is valuable, represents a significant amount of work, and the results support the conclusions drawn. Stronger evidence that senescent myofibroblasts couple with cardiomyocytes in the aged heart is needed to support the proposed model.

The authors conclude a propensity of myofibroblast senescence based on the finding that 80% of αSMA+ cells are also positive for H2AX. Showing the immunofluorescence results on hearts 2 weeks after MI would help to more convincingly illustrate the result. From these immunofluorescence experiments, it is also concluded that most of the persistent senescent cells in the scar correspond to myofibroblasts. The results presented show a continued increase in the proportion of H2AX+ cells in aged hearts up to 12 weeks after myocardial infarction. According to results in Figures 4F and G, these cells do not correspond to either myofibroblasts or endothelial cells. Given that H2AX+ cells are significantly increased in the aged heart, could the results presented suggest that a different cell type might be more important for the aged heart's response to MI? Providing some insight into the identity of these cells would be helpful to better understand the results presented. For example, cardiomyocyte senescence could contribute to arrhythmic phenotypes.

The results presented show that treatment of cardiomyocytes with conditioned media from, and co-cultured with, senescent myofibroblasts did not change action potential duration in cardiomyocytes. This led to the conclusion that paracrine signalling is unlikely to contribute to a pro-arrhythmogenic phenotype. It is possible that cardiomyocytes do couple with myofibroblasts in the in vitro system used. In which case, the results presented would not favor the proposed model. Another important possibility to be considered is that myofibroblasts might not have produced senescence-associated secretory phenotype-mediators at concentrations high enough to alter action potential duration in the conditions tested. Experimental evidence of the levels of selected mediators of the senescence-associated secretory phenotype in conditioned media would help assess a potential paracrine effect.

The evidence of coupling, i.e., the presence of connexin-43 in the interphase between αSMA+ and cardiomyocytes needs to be strengthened. Perhaps analyzing Z-stack 3D reconstructions would help to better define adjacent cells and more precisely reveal the localization of connexin-43.

Reviewer #1 (Public Review):

This manuscript describes the differences in the plasma proteome and metabolome in healthy Tanzanian and healthy Dutch adults. The inflammatory plasma proteome was measured using the Olink 92 Inflammation panel, while the plasma metabolome was analyzed using a mass spectrometry-based untargeted approach. The plasma metabolome was measured only in the Tanzanian cohort. This study aimed to link the pro-inflammatory proteome of Tanzanian and Dutch healthy individuals with environmental factors and dietary lifestyles.

The correlation between the plasma proteome and food-derived metabolome profiles can shed light on the development of non-communicable diseases. This observation stresses the importance of dietary transition and lifestyle changes in expressing inflammation-related molecules. Moreover, this study describes the inflammatory proteome profile in healthy Tanzanian individuals covering a cohort with limited studies. The molecular differences in circulating biomolecules between healthy individuals living in East Africa and individuals living in Western Europe and the correlations with intrinsic and environmental features are novel.

This study lacks a robust and solid validation of some of the differentially regulated circulating proteins and correlations between food-derived metabolites and proteins in a selected cohort. The discovery-driven approach in this manuscript highlights potential findings that need to be supported by a validation phase. According to this reviewer, the lack of such validation impacts the robustness of the results and the hypotheses generated. Due to that, the manuscript should incorporate validation experiments.

Reviewer #1 (Public Review):

For PRLR, the question being asked is whether and how the intracellular domain (ICD) interacts with the cellular membrane or how the disordered ICD can relay and transmit information. The authors show that PI(4,5)P2 in the membrane localizes around the transmembrane domain (TMD) due to charge interactions and facilitates binding of the ICD to the membrane, even in the absence of the TMD. Furthermore, the ICD and PI(4,5)P2 form a co-structure with JAK2 which locks a disordered part of the ICD into an extended conformation, allowing for signal relay and, through multiple complex conformations, may enable switching signalling on and off.

Strengths:<br /> - NMR paired with MD is a powerful way to probe an interaction especially when peaks disappear and become difficult to probe by NMR.<br /> - Using NMR and MD to formulate hypotheses which are then tested by cell studies is quite informative. The combination of MD, NMR, and cell biology is a strength.<br /> - The authors are diligent in testing MD simulations on systems with and without PIP2.<br /> - The use of Pep1 and Pep2 to differentiate the KxK region that interacts with PIP2 is helpful.<br /> - The four utilized mutants help illustrate the co-dependence of the respective regions in the formation of the co-structure.

Weaknesses:

- In Figure 2G, there is a big change in CSP between 280 and 290, which the authors do not comment about.<br /> - The data in Figure 2 are summarized as indicating the formation of extended structure in the ICD upon binding. It is not clear to me what data show an extended structure.<br /> - No modelling or experiments were done with PIP3 despite conclusions and models which rely on the phosphorylation of PIP2 to PIP3. At the very least, these would be useful as negative controls.<br /> - Only R2 experiments were done when the authors mention investigating dynamics. R1 and -HetNOE dynamics would be useful for creating a complete picture.<br /> - Some of the exciting results are under-emphasized including Fig 3H and 3I.

Reviewer #1 (Public Review):

This study demonstrates that Chinmo promotes larval development as part of the metamorphic gene network (MGN), in part by regulating Br-C expression in some tissues (exemplified in the wing disc) and in a Br-C independent manner in other tissues such as the salivary gland. I have included below the following comments on the submitted version of this manuscript:

1. The authors have shown experimentally that Chinmo regulates Br-C expression in the wing disc but not the larval salivary gland. Based on this, they posit that Chinmo promotes larval development in a Br-C-dependent manner in imaginal tissues and a Br-C-independent manner in other larval tissues. This generalization of Chinmo's role in development would be more compelling if the relationship between Chinmo and Br-C were explored in other examples of imaginal/larval tissues.

2. Chinmo, Br-C, and E93 have all been shown to be EcR-regulated in larval tissues, including the brain and wing disc (as in Zhou et al. 2006, Dev Cell; Narbonne-Reveau and Maurange 2019, PLOS Biology; Uyeharu et al. 2017, ). It would be interesting (and I believe relevant to this study) to know whether the roles of these factors in their respective developmental stages are EcR-dependent and whether their regulation by EcR (or lack thereof) depends on whether the tissue is larval or imaginal.

3. In the chinmo qPCR analysis shown in Fig1A, whether animals were sex-matched or controlled was not indicated. Since Chinmo has a published role in regulating sexual identity (Ma et al. 2014, Dev Cell; Grmai et al. 2018, PLOS Genetics), and since growth/body size is known to be a sexually dimorphic trait (Rideout et al. 2015, PLOS Genetics), it seems important to establish whether the requirement of Chinmo for larval development and/or growth. I recommend either 1) controlling for sex by repeating qPCRs in Fig 1A in either males or females, or 2) reporting male/female chinmo levels at each stage side-by-side.

4. In Fig2E, the authors show that salivary gland secretion (sgs) genes are repressed in salivary glands lacking chinmo. Sgs genes are expressed during late larval stages as the animal prepares to pupate. Thus, based on the proposed model where Chinmo promotes larval development and represses the larval-to-pupal transition, one might expect that larval salivary glands lacking chinmo would express higher than normal levels of sgs genes. This expectation directly opposes the observed result - it would be helpful to speculate on this in the interpretation of results.

Reviewer #1 (Public Review):

The authors study single and pairs of MDCK cells adherent to an H-shaped geometry on a flat surface. In this pattern, the cells form strong peripheral stress fibers. To a lesser extent, these cells also exhibit stress fibers in the cell interior, which otherwise has a rather homogenous actin distribution. Using a combination of traction force microscopy, from which they infer the stress distribution by monolayer stress microscopy, and "contour analysis" the authors quantify the 'bulk' and the 'surface' stress in these cells. This analysis shows that single cells are mechanically polarized whereas pairs are not.

The authors then go on to optogenetically activate the actomyosin contractility of either one half of a single cell or one cell of a pair. Combining their stress measurements in these situations and using a finite element mechanical model, the authors convincingly show that the mechanical response in the non-activated part is active. By varying the aspect ratio of the adhesion patterns, they also find that the efficacy of active stress propagation depends on the mechanical and structural polarity of the cell. Furthermore, they provide evidence that their results on cell pairs generalize to tissues.

Strengths:

This study uses a nice combination of physical tools to address an important question in tissue mechanics. The data is compelling and fully supports the authors' conclusions.

Weaknesses:

There are no major weaknesses.

In summary, although the fact that mechanical stress propagation in tissues is an active process might not come as a surprise, the study makes substantial contributions to a quantitative contribution of this process. As such it is of fundamental significance in the field. It will be interesting to explore the consequences of this mechanism for mechanical stress propagation in the context of developmental processes. It will be also of great interest to study how this local process can be accounted for in large-scale theories.

Reviewer #1 (Public Review):

In this manuscript, the authors seek to define the transcriptional response to deafening in the songbird brain. They compare transcriptional changes in the song regions with changes in the non-singing-associated surrounds, compute a song degradation score against which they can compare gene expression, and they use single-cell sequencing data from these brain regions to map genes to cells. The study is impressively comprehensive for time points, replicates, brain regions, comparisons, and alternative strategies (e.g. the LMAN lesions). This dataset builds nicely upon studies that assessed gene expression changes upon singing and applies a broad and useful series of bioinformatics analyses to get the strongest evidence for function from the data.

I think this dataset will be of great interest to a broad range of researchers who study neuronal plasticity mechanisms.

Reviewer #1 (Public Review):

The network of neurons of the inferior olive has long been suggested as a timing machine that controls the precise timing of movements, correcting movement and participating in the prediction of movement time. These timing capabilities have been attributed to the unique feature of the neurons to generate subthreshold voltage oscillations that can be used as a timing machine. In this study, the effect of the inhibitory and excitatory synaptic inputs on the oscillatory behavior was examined, demonstrating their different effects as well as the effects of combing the two inputs.

Reviewer #1 (Public Review):

Overall, this manuscript by Liu et al. provides a largely convincing mechanism for both how Zfp467 regulates osteoblast differentiation and how PTH1R expression and function in osteoblast-lineage cells is regulated at the transcriptional level, finding that NF-kB (RelB/p50) regulates PTH1R expression downstream of Zfp467. PTH1R expression and activity in turn is enhanced in Zfp467-deficient osteoblasts. In turn, PTH signaling regulates Zfp467 expression through PKA activity. In particular, the new findings on mechanisms of regulating PTH1R expression and evidence that this in turn impacts osteoblast differentiation are felt to be of broad interest and importance. The approach used is felt to be largely sound. Areas of major concern are few and relate mostly to better fleshing out how the NF-kB pathway is impacted as a part of the molecular pathway implicated here and clarifying some confusion regarding uCT data that appears to be discussed but which this reviewer cannot locate in the figure.

Reviewer #1 (Public Review):

In this study, the authors sought to develop a measure of Staphylococcus aureus intracellular virulence levels in the lab (the InToxSa assay) that more closely mimics the activity seen in vivo. They then used untargeted approaches (GWAS, homoplasy) on a set of 387 Australasian clinical isolates to identify genes with mutants associated with reduced intracellular toxicity. The authors identified several mutated genes which reduced virulence in the strains chosen for the study, demonstrating that their approach can be used to uncover virulence-related genes in S. aureus.

The study is clearly written, with high-quality figures. The development of the InToxSa assay is carefully described and logical. InToxSa was shown to potentially be more sensitive than the tryptophan blue test in detecting reduced intracellular cytotoxicity phenotype. They also showed evidence for agrA mutants and other transposon mutants with reduced inToxSa cytotoxicity having increased bacterial cell numbers cells compared to wild-type (Fig 2, Fig5GH), which is critical to the argument that bacteremia selects for intracellular persistence as a way to escape the immune system. There was an interesting and thoughtful use of random forest to choose the most appropriate parameters of the kinetic model.

The GWAS studies used publicly deposited genome data and clearly showed lineage effects of reduced intracellular survival of CC239 and CC22, confirming previous results. GWAS also confirmed the well-known pervasive association of agr mutants with reduced toxicity. Using a well-described homoplasy test for convergent evolution to extract more power, several other potential genes associated with enhanced intracellular toxicity were discovered or rediscovered, perhaps most significantly, the ausA gene, with biosynthesizes aureusimines (pyrazinone secondary metabolites) posited to have a role in the phagosomal escape.

There are two main 'weaknesses'. The first is the limited power that comes from only using measuring the phenotype of 387 strains. Whether this is because of the expense/ difficulty of the inToxSa is not discussed, leaving open the question of how much this assay could be scaled up in the future. The second is that the main output of the assay is actually reduced intracellular toxicity (PI uptake AUC), which is inferred to be strongly linked to increased intracellular persistence. The linkage between the phenotypes comes primarily from microscopic studies on a limited number of strains. It may be true of all cases but the possibility exists that for some of the strains, reduced cytotoxicity may be associated with intracellular elimination, which would presumably be a negative outcome for systemic infection.

Overall, the authors achieved their aims in terms of assay development and showing the utility of the pipeline for mutation discovery. This is a waypoint in the larger aim of understanding mutational pathways that lead to increased persistence of systemic S. aureus. Obviously, a lot more data is needed. The InToxSa intracellular screening method is interesting and could be reused/adapted by the community. This research should also spark more interest in the role of ausA and aureusimines in virulence and some of the other genes discovered through the untargeted approach.

Reviewer #1 (Public Review):

In this paper, Krishnan et al. describe their findings on the genetic architecture of the heart mitochondrial proteome that influences cardiac hypertrophy. They analyzed common genetic variations contributing to mitochondrial and heart functions in a panel of inbred mouse strains called the Hybrid Mouse Diversity Panel (HMDP), by performing whole heart proteomics. The authors have published a number of papers on this panel, which appears to be a powerful system to study various genetic factors. They identified three trans-acting genetic loci, located on chromosome (chr) 7, chr13, and chr17, which control both mitochondrial proteins and heart hypertrophy. High-resolution regional mapping identified NDUFS4, LRPPRC, and COQ7 as the candidate genes for chr13, chr17, and chr7 loci, and variations of these genes were associated with heart mass in isoproterenol-induced heart failure and diet-induced obesity. Using co-expression protein networks using weighted gene co-expression network analysis (WGCNA), they show that the chr13 locus was highly enriched for complex-I proteins, the chr17 locus for mitochondrial ribonucleoprotein complex, and the chr7 locus for ubiquinone biosynthesis. They concluded that "common variations of certain mitochondrial proteins can act in trans to influence mitochondrial functions and contribute to heart hypertrophy, elucidating mechanisms that may underlie genetic susceptibility to heart failure in human populations."

Although these studies are interesting and provide novel findings in the genetics of cardiac hypertrophy, there are a number of technical and conceptual issues that need to be addressed.

Reviewer #1 (Public Review):

Mermithid nematodes are ecologically important parasitoids of arthropods, annelids and mollusks today. Their fossil record in amber reaches back into the Early Cretaceous, some 135 million years ago. Luo et al. more than triple this record by presenting, with ample illustrations, exceptionally well preserved new specimens from the beginning of the Late Cretaceous (99 Ma ago) of Myanmar. Their most important finding is that mermithids parasitized a number of insect clades in the Cretaceous that they are not known to infect today or in Cenozoic amber; further, the proportion of holometabolous insects among the hosts is found to be lower in the Cretaceous than in the Cenozoic. The strengths of the paper lie in the specimens, the illustrations of the specimens, and the documentation of when, where and how the specimens were acquired. Certain nomenclatural aspects of the paper require improvement. A potential weakness of the paper could be collection bias: it is not tested whether the collections used to show the shift toward holometabolous hosts from the mid-Cretaceous to the Cenozoic are representative of the fossil record as it is preserved and accessible today.

Reviewer #1 (Public Review):

This paper investigates the neural correlates of noise-induced hearing loss. The authors use an electrode array to capture neural responses across the inferior colliculus to speech and synthetic sounds in both normal-hearing gerbils, and gerbils with noise-induced hearing loss. They use dimensionality reduction to isolate a low-dimensional response subspace that captures most of the information about the speech signals, and find that this low-dimensional representation is altered considerably by hearing loss (evaluated with CCA). To probe the basis of these differences, the authors train an artificial neural network to predict the subspace responses to arbitrary stimuli, for instance to investigate the consequences of frequency-dependent amplification of sound with a hearing aid, or synthetic test stimuli. Using this approach, they find that the representation of sounds in quiet is largely restored by a hearing aid algorithm that amplifies high frequencies to render them audible. However, the representation of sounds in noise also differs between the IC of normal-hearing and hearing-impaired gerbils, and this difference is not eliminated by a hearing aid. Specifically, low-frequency maskers seem to distort the representation of high-frequency sounds (e.g. consonants in speech), even once the high-frequencies have been amplified to compensate for the hearing loss.

Overall, this is a strong paper. The topic is important, the methods are innovative, logical, and rigorous, and the whole thing is exceptionally clearly described. I greatly appreciate the care that clearly went into writing the paper. I have two major concerns. The first seems fairly critical to the paper's conclusions, but I hope can be addressed with some kind of control experiment. The second could potentially be thought of as more of a future direction, but it speaks to the specificity of the conclusions.

1. My main substantive concern is that the conclusions depend critically on believing the predictions of the DNN, and yet it is not clear we should expect it to generalize well to stimuli outside its training distribution. Current artificial neural networks typically work very well for stimuli like those they were trained on, but often do not generalize as well as one might like. The authors recorded responses to speech in quiet and in different noise levels, and show that the trained DNN (trained on these sounds and the associated responses) produces very accurate predictions on held-out sounds from this distribution. But the conclusions depend critically on the DNN predictions for sound processed by a hearing aid, and for synthetic sounds (pure tones, SAM noises) that are quite unlike the training data. The predictions look reasonable in places where we have some prior sense for what to expect (level-dependent frequency tuning to pure tones), which is reassuring, but I am not sure how to be confident that the predictions should be accurate for all of the conditions that are tested, in particular to the results with the simulated hearing aid. I am pretty sure that the predictions will be inaccurate for some types of stimuli (just based on the various pathologies that are known to occur with neural networks). I would hope that this would not be the case for the conditions tested by the authors, but it is hard to be sure, and this makes the conclusions seem a little more vulnerable than I would like.<br /> How do we know that the DNN generalizes beyond its training data well enough to render the conclusions airtight?

2. My second concern is the extent to which the results are specific to a) the IC, and b) noise. The authors assert that similar effects would not be present in the nerve, citing a Heinz paper, but I am not sure how clear this evidence is - it is not described in enough detail here to assess. It would be nice to show this, perhaps by repeating their analysis on a model of the nerve with and without simulated hearing loss. One can similarly wonder about the effects in the cortex, especially given the literature on noise invariance (Rabinowitz, Moore, Khalighinejad, Kell...), which would at least be worth discussing. It is similarly unclear whether the results are specific to additive noise. Would similar conclusions hold for any type of distortion? This could be easily addressed by an additional DNN analysis (e.g. with clipping, or segments of speech intermittently replaced by silence, or reverberation).

Reviewer #1 (Public Review):

This manuscript seeks a greater understanding of joint movements in recipients of total knee replacements who have symptoms of unstable prosthetic joints. The authors describe the results of a carefully conducted retrospective analysis of joint movements after total knee replacement (TKA) using a recently developed method based on videofluoroscopy. Kinematic data supplemented by electromyography measurements of muscle activation through normal gait. These measurements were conducted while walking on flat ground, down an incline, or down stairs. The kinematics and EMG data provide convincing evidence of altered knee kinematics when symptoms of joint instability occurred that were accompanied by subject-specific changes in patterns of muscle activation. The manuscript raises interesting questions about how patients adapt muscle activation patterns to limit discomfort prior to TKA and to what degree these same defensive strategies influence joint stability post-operatively.

Reviewer #1 (Public Review):

In this manuscript, Li et al characterize sex differences in the impact of macrophage RELMa in protection against diet-induced obesity [DIO]. This is a key area of interest as obesity studies in mice have generally focused exclusively on male animals, as they tend to gain more weight, faster than female mice. The authors use a combination of flow cytometry, adoptive transfer, and single-cell transcriptomics to characterize the mechanism of action for female-specific DIO protection. They identify a potential role for eosinophils in mediating female DIO protection downstream of RELMa production by macrophage. They also use the transcriptomic characterization of the stromal vascular fraction of the adipose tissue to evaluate molecular and cellular drivers of this sex-specific DIO protection.<br /> Although the authors provide solid evidence for many claims in the manuscript, there is generally not enough information about the studies' methods (especially on the computational/data analysis aspects) for a careful evaluation of the result's robustness at this stage.

Reviewer #1 (Public Review):

In vertebrates, ciliary motility is important for left-right body patterning, airway clearance, cerebrospinal fluid flow, and the locomotion of spermatozoa. The movement of cilia is powered by the action of dyneins tethered to axonemal doublet microtubules. The largest and most powerful axonemal dynein, OAD, is tethered by a pentameric docking complex (the OAD-DC). Here, Yamaguchi, Morikawa and Kikkawa show convincingly that the Calaxin and Armc4 subunits of the OAD-DC have discrete roles in docking OADs. Using zebrafish mutants, they show that loss of Armc4 causes complete loss of the OAD, whereas mutation of Calaxin causes only partial OAD loss. They demonstrate that Calaxin localization is dependent on Armc4 but independent of the OAD or calcium conditions. Using cryo-ET, they report a higher resolution structure of the wild-type zebrafish sperm axoneme than previously determined (Yamaguchi et al., 2018) and show that the OAD and OAD-DC structures resemble the cryo-EM structures of other organisms. Cryo-ET analysis of calaxin-/- axonemes reveals that without Calaxin, OADs have mostly normal conformations but make fewer connections with the OAD-DC and are less stably bound. The paper is well-written with appropriate methods and conclusions.

Reviewer #1 (Public Review):

This interesting manuscript by Nakajima-Takagi et al describes the roles of the PRC 1.1 member Pcgf1 in myeloid lineage commitment in hematopoiesis and in regulating myeloid differentiation and self-renewal during emergency myelopoiesis. The roles of Pcgf1 have been explored previously in the context of Runx1 depletion or in the context of myelofibrosis together with the JAK2V617F mutation, but this is the first report of the specific roles of Pcgf1 in HSCs and in myelopoiesis. The authors convincingly demonstrate that conditional deletion of Pcgf1 in hematopoietic cells causes a lineage switch in HSCs from lymphoid to myeloid fates and that a key mechanism for this lineage switch is regulation of the H2AK119ub1 chromatin mark, leading to de-repression of CEBPalpha, a key transcription factor that promotes myeloid cell fate. They also perform a single-cell RNAseq experiment and demonstrate an increase in the population of "self-renewing GMPs", and they attribute this increase to an upregulation in HoxA9 expression and beta-catenin activation. They also demonstrate that HoxA9 overexpression promotes beta-catenin activation, which has been observed in emergency myelopoiesis in other studies, though the mechanism for this is unclear. The authors also demonstrate that deletion of Pcgf1 in hematopoietic cells can also lead to deregulated myelopoiesis, leading to a lethal MPN in a subset of animals. They conclude that Pcgf1 plays a critical role to regulate emergency myelopoiesis, and to prevent the malignant transformation of myeloid progenitors.

Overall, the methods are highly rigorous and the results support the authors' conclusions. The only conclusion that would require further clarification is that Pcgf1 promotes emergency myelopoiesis. Emergency myelopoiesis typically starts with a proliferative burst of myeloid progenitors in response to a stress stimulus, followed by enhanced myeloid differentiation into mature functional myeloid cells. In this Pcgf1 KO mouse model, it is clear that there is an increase in the production of myeloid progenitors, and prolonged survival of myeloid progenitors in culture, but there is no demonstration that this results in the generation of mature functional myeloid cells. It appears that there may also be a differentiation block, likely due to the increase in "self-renewing progenitors", which is likely a consequence of HoxA9 upregulation, and possibly the beta-catenin activation in myeloid progenitors. Therefore, if there is also a differentiation block due to Pcgf1 deletion, the statement that emergency myelopoiesis is enhanced may be an oversimplification. What appears to occur is an expansion of a pool of self-renewing transformed or pre-transformed myeloid progenitors, and the relevance of this event to emergency myelopoiesis is not entirely clear. However, there is a clear significance of these findings and this new mouse model for studying the pathogenesis of myeloid malignancies, such as MPN, MDS, or AML, in which mutations in other components of PRC1.1 are frequently mutated, so this study is likely to have a significant impact in the field.

Reviewer #1 (Public Review):

This paper presents a thorough biochemical characterization of inferred ancestral versions of the Dicer helicase function. Probably the most significant finding is that the deepest ancestral protein reconstructed (AncD1D2) has significant double-stranded RNA-stimulated ATPase activity that was lost later, along the vertebrate lineage. These results strongly suggest that the previously known differences in ATPase activity between extant vertebrates and, for example, extant arthropods is due to loss of the ATPase activity over evolutionary time as opposed to gains in specific lineages. Based on their analysis, the authors also "restore" ATPase function in the vertebrate dicer, but they did so by making many (over 40) mutations in the vertebrate protein, and it is not clear which of these many mutations is required for the restoration of the activity. Thus, it is difficult to discern how the results of this experiment relate to the evolutionary history.

A criticism of the paper is the authors' tendency (probably unconscious) to ascribe a purposefulness to evolution. For example, in the introduction, "We speculate that the unique role of the RLR's in the interferon signaling pathway in vertebrates...created an incentive to jettison an active helicase in vertebrates." Although this sentence is clearly labelled as speculation and "incentive" is clearly a metaphor, the implication is that evolution somehow has forethought. (There are other instances of this notion in the paper, for example, in the last line of the abstract). The author's statement also implies that the developing interferon system somehow caused the loss of active helicase, but it seems equally plausible that the helicase function was lost before the interferon system co-opted it.

Reviewer #1 (Public Review):

VO2max is one of the most important gross criteria of peak performance ability and a plethora of studies focused on VO2max prediction. This manuscript provides huge and comprehensive data from male runners and male cyclists. The endurance-trained athletes performed cardiopulmonary exercise testing on a treadmill (n= 3330) or cycle ergometer (n=1094). In contrast to former studies, the authors used machine learning for algorithms and VO2max prediction. Models were derived and internally validated with multiple linear regression. The present study substantially expands current research.

Sadly, the manuscript has an important and relevant main shortcoming as the limitations of the study had not been addressed properly:<br /> - The authors paid no attention to the fact that their results are strongly influenced by the exercise protocol used. It is obvious e.g. that maximal performance attainable in protocols with 2-minute exercise steps will be higher compared to an identical protocol with 3- or 4-minute steps.<br /> - The exercise intensity was kept constant for only 2 minutes before the workload was increased (by 1km/h treadmill or by 20-30 W cycle ergometer). Due to the kinetics of lactate, VO2, etc., it is evident that the short 2-min intervals aggravate the correct determination of aerobic and anaerobic threshold. It is well-known that longer-lasting constant exercise steps (e.g. 4 minutes) are better when the focus is centered on threshold determinations.

The quality of this manuscript will be substantially improved when the authors could implement a comprehensive and blunt paragraph showing the limitations of their study.

Reviewer #1 (Public Review):

The authors provide evidence for chromatin, which in Drosophila muscle cells is peripherally localized in the nucleus, whereas the central region is depleted of chromatin, and is organised such that RNA polymerase II (RNAp) is surrounding dense regions of chromatin. The authors theoretically study the formation of these regions by describing chromatin as a multi-block copolymer, where the blocks correspond to active and inactive chromatin regions. These regions are assumed to phase separately and to have different solvability. The solvability of the active region is regulated by binding RNAp. The authors study the core-shell organization in a layered geometry by analyzing the various contributions to free energy. In this way, they in particular obtain the dependence of the shell-layer thickness, which is described as a polymer brush. From these results, they infer chromatin organization in spherical core-shell chromatin domains and compare these results to Brownian dynamics simulations.

The work is well done and even though it uses standard methods for studying block copolymers and polymer brushes obtains interesting information about local chromatin organization. These findings should be of great interest to researchers in the field of chromatin organization and in general to everybody interested in understanding the physical principles of biological organization.

The work has two main weaknesses: The experimental evidence for RNAp and chromatin micro-organization is weak as only one example is shown. It remains unclear whether the observed organization pattern is common or not. Also, no data is shown concerning the dependence of the extensions of the active and inactive phases on parameters, for example, solvent properties or transcriptional activity. Second, some parts could prove difficult for biologists to assess. For example, the expression for the brush-free energy should be explained in more detail and notions like that of 'mushrooms' need to be introduced. As a second example, biologists might benefit from a better explanation of the concept of a theta solvent and its relevance.

Reviewer #1 (Public Review):

Marchal-Duval et al studied the role of Prrx1 in lung fibroblasts. Prrx1 is a transcription factor expressed in lung fibroblasts but not in other cell types. The authors showed that Prrx1 gene expression was enhanced in IPF patients. Immunohistochemistry in IPF tissue suggested that Prrx1 was expressed in fibroblasts in fibroblastic foci. The authors then showed that Prrx1 expression was regulated by TGF-b1 stimulation or stiffness of substrate by in vitro experiments using primary human lung fibroblasts from either normal or IPF lungs. The authors also showed that Prrx1 regulated fibroblast proliferation and TGF-b signaling by regulating PPM1A and Tgfbr2 expression. Finally, the authors revealed that Prrx1 knockdown suppressed fibrosis in bleomycin-induced fibrosis or PCLS. This manuscript identified novel molecular roles of Prrx1 in fibroblast activation, which is expressed in not only lung fibroblasts but also in other injured or developing organs. To support the idea that Prrx1 plays a critical role in lung fibrosis, however, some discrepancies between in vitro and in vivo data need to be clarified.

1. Although the authors showed that Prrx1 knockdown in primary fibroblasts reduced Smad2/3 phosphorylation, the reduction of Acta2 or Col1a1 after Prrx1 knockdown and TGF-b1 stimulation was not impressive (Fig. S6), suggesting that the inhibition of TGF-b signaling by Prrx1 knockdown is only partial. In contrast, Prrx1 knockdown by ASO in bleomycin-induced fibrosis showed remarkable fibrosis suppression (Fig. 6, 7). Admittedly there are differences in models and nucleotides used, but this discrepancy needs to be addressed.

2. Fig.6 and 7 lack control groups, where mice are treated with PBS instead of bleomycin and treated with either control ASO or Prrx1 ASO.

3. In Fig. 6F, the hydroxyproline content is shown with ug collagen/ug protein. Total protein in the lung is influenced by infiltration of hematopoietic cells, which are the major population in injured lungs by cell count. Fibrosis should be ideally assessed as ug hydroxyproline/lung (or lobe).

4. Major proliferating populations in bleomycin-treated lungs are not mesenchymal cells but epithelial/endothelial/hematopoietic cells. Mki67+ cells (Fig. 7D) need to be identified by co-staining with mesenchymal markers if the authors claim that Prrx1 knockdown suppresses fibroblast proliferation in vivo.

5. Bleomycin-injured lungs or IPF tissue are patchy and mixed with normal and abnormal areas. Therefore, how areas of interest are chosen for histological quantifications (Fig. 6C, S14D) need to be described in the methods section.

Reviewer #1 (Public Review):

Here the authors investigate the mechanisms by which pulmonary endothelial cells (EC) contribute to alveolar repair post-H1N1-mediated acute lung injury and the molecular basis for the heterogeneity of this response among different EC subpopulations. Using single-cell transcriptomic analysis they identify the CREB family factor Atf3 differentially enriched in CAP1B cells, a subpopulation of EC previously known for its proliferative behavior in response to alveolar injury. They report a crucial role for Atf3 in injury repair but not during homeostasis. Using a combination of lineage tracing and loss function approach and an influenza mouse model in vivo, they show that Atf3 inactivation in ECs results in the inability of CAP1B ECs to initiate a proliferative response to repair the vascular compartment and ultimately regenerate the lung. Notably, the decreased number of Atf3 lineage-labeled EC capillaries was shown to correlate with the alveolar regions that failed to repair the post-H1N1 injury. They conclude that Atf3 is an essential factor for repair damaged capillaries in alveolar injury.

The study is carefully designed and the results provide novel important information about a previously undisclosed role of Atf3 in the regeneration of the lung vascular component. The work has many strengths and is supported by impressively coherent data from the analysis of mouse genetic models, single-cell transcriptomic, and phenotypic characterization.

Joint Public Review:

The manuscript "Monoallelically-expressed Noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression" reports the discovery of a family of ncRNAs they call SNULs for Single NUcleolus Localized RNA and examine their localization with respect to nucleoli and reports that the RNAs they are examining are monoallelically expressed in a mitotically stable manner similar to what happens in X inactivation.

These RNAs come from a screen which is not well described and the descriptions of the sequence analyses are unclear, so it is difficult to know exactly what they are analyzing in the manuscript. If these are RNAs with reasonable abundance, then they should be findable without the extensive PCR amplification they appear to have done for the PacBio sequencing (the methods section is not clear on exactly how many rounds of PCR were performed). Moreover, given the acknowledged sequence similarities of the SNULs with other RNAs, the possibility of chimaera formation during PCR amplification is high. They are clearly detecting RNAs associated with nucleoli but exactly what they are examining is unclear. It is possible that a clear determination of the genomic origin of these RNAs will be complicated by the repetitive sequences in the regions of the genome where they reside.

Note also that the idea of monoallelic expression from rRNA encoding loci is interesting, but has been established in 2009. Title: Allelic inactivation of rDNA loci. Genes Dev. 2009 Oct 15;23(20):2437-47. doi: 10.1101/gad.544509.

Reviewer #1 (Public Review):

Jordan and Keller investigated the possibility that sensorimotor prediction error (mismatch between expected and actual inputs) triggers locus coeruleus (LC) activation, which in turn drives plasticity of cortical neurons that detect the mismatch (e.g. layer 2/3 neurons in V1), thus updating the internal presentation (expected) to match more the sensory input. Using genetic tools to selectively label LC neurons in mice and in vivo imaging of LC axonal calcium responses in the V1 and motor cortex in awake mice in virtual reality training, they showed that LC axons responded selectively to a mismatch between the visual input and locomotion. The greater the mismatch (the faster the locomotion in relation to the visual input), the larger the LC response. This seemed to be a global response as LC responses were indistinguishable between sensory and motor cortical areas. They further showed that LC drove learning (updating the internal model) despite that LC optical stimulation failed to alter acute cellular responses. Responses in the visual cortex increased with locomotion, and this was suppressed following LC phasic stimulation during visuomotor coupled training (closed loop). In the last section, they showed that artificial optogenetic stimulation of LC permitted plasticity over minutes, which would normally take days in non-stimulated mice trained in the visuomotor coupling mode. These data enhance our understanding of LC functionality in vivo and support the framework that LC acts as a prediction error detector and supervises cortical plasticity to update internal representations.

The experiments are well-designed and carefully conducted. The conclusions of this work are in general well supported by the data.

Reviewer #1 (Public Review):

The authors evaluate a number of stochastic algorithms for the generation of wiring diagrams between neurons by comparing their results to tentative connectivity measured in cell cultures derived from embryonic rodent cortices. They find the best match for algorithms that include a term of homophily, i.e. preference for connections between pairs that connect to an overlapping set of neurons. The trend becomes stronger, the older the culture is (more days in vitro).

From there, they branch off to a set of related results: First, that connectivity states reached by the optimal algorithm along the way are similar to connectivity in younger cultures (fewer days in vitro). Second, that connectivity in a more densely packed network (higher plating density) differs only in terms of shorter-range connectivity and even higher clustering, while other topological parameters are conserved. Third, blocking inhibition results in more unstructured functional connectivity. Fourth, results can be replicated to some degree in cultures of human neurons, but it depends on the type of cell.

The culturing and recording methods are strong and impressive. The connectivity derivation methods use established algorithms but come with one important caveat, in that they are purely based on correlation, which can lead to the addition of non-structurally present edges. While this focus on "functional connectivity" is an established method, it is important to consider how this affects the main results. One main way in which functional connectivity is likely to differ from the structural one is the presence of edges between neurons sharing common innervation, as this is likely to synchronize their spiking. As they share innervation from the same set of neurons, this type of edge is placed in accordance with a homophilic principle. In other words, this is not merely an algorithmic inaccuracy, but a potential bias directly related to the main point of the manuscript. This is not invalidating the main point, which the authors clearly state to be about the correlational, functional connectivity (and using that is established in the field). But it becomes relevant when in conclusion the functional connectivity is implicitly or explicitly equated with the structural one. Specifically, considering a long-range connection to be more costly implies an actual, structural connection to be present. Speculating that the algorithm reveals developmental principles of network formation implies that it is the actual axons and synapses forming and developing. The term "wiring" also implies structural rather than functional connectivity. One should carefully consider what the distinction means for conclusions and interpretation of results.

The main finding is that out of 13 tested algorithms to model the measured functional connectivity, one based on homophilic attachment works best, recreating with a simple principle the distributions of various topological parameters.<br /> First, I want to clear up a potential misunderstanding caused by the naming the authors chose for the four groups of generative algorithms: While the ones labelled "clustering" are based on the clustering coefficient, they do not necessarily lead to a large value of that measure nor are they really based on the idea that connectivity is clustered. Instead, the "homophilic" ones are a form of maximizing the measure (but balanced by the distance term). To be clear, their naming is not wrong, nor needs to be changed, but it can lead to misunderstandings that I wanted to clear up. Also, this means that the principle of "homophilic wiring" is a confirmation of previous findings that neuronal connectivity features increased values of the clustering coefficient. What is novel is the valuable finding that the principle also leads to matching other topological network parameters.

The main finding is based on essentially fitting a network generation algorithm by minimizing an energy function. As such, we must consider the possibility of overfitting. Here the authors provide additional validation by using measures that were not considered in the fitting (Fig 5, to a lesser degree Fig 3e), increasing the strength of the results. Also, for a given generative algorithm, only 2 wiring parameters were optimized. However, with respect to this, I was left with the impression that a different set of them was optimized for every single in-vitro network (e.g. n=6 sets for the sparse PC networks; though this was not precisely explained, I base this on the presence of distributions of wiring parameters in Fig 6c). The results would be stronger if a single set could be found for a given type of cell culture, especially if we are supposed to consider the main finding to be a universal wiring principle. At least report and discuss their variability.

Next, the strength of the finding depends on the strengths of the alternatives considered. Here, the authors selected a reasonably high number of twelve alternatives. The "degree" family places connections between nodes that are already highly connected, implementing a form of rich-club principle, which has been repeatedly found in brain networks. However, I do not understand the motivation for the "clustering" family. As mentioned above, they do not serve to increase the measure of the clustering coefficient, as the pair is likely not part of the same cluster. As inspiration, "Collective dynamics of 'small-world' networks" is cited, but I do not see the relation to the algorithm or results presented in that study. A clearly explained motivation for the alternatives (and maybe for the individual algorithms, not just the larger families) would strengthen the result. 

Related to the interpretation of results, as they are presented in Fig3a, bottom left: What data points exactly go into each colored box? Specifically, into the purple box? What exactly is meant by "top performing networks across the main categories" mean? Compared with Supp Fig S4, it seems as if the authors do not select the best model out of a family and instead pool the various models that are part of the same family, albeit each with their optimized gamma and eta. Otherwise, the purple box at DIV14 in Fig3 would be identical to "degree average" at DIV14 in S4. If true, I find this problematic, as visually, the performance of one family is made to look weaker by including weak-performing models in it. I am sure one could formulate a weak-performing homophily-based rule that drives the red box up. If such pooling is done for the statistical tests in Supp Tables 3-7, this is outright misleading! (for some cases "degree average" seems not significantly worse than the homophily rules).

The next finding is related to the development of connectivity over the days in vitro. Here, the authors compare the connectivity states the network model goes through as the algorithm builds it up, to connectivity in-vitro in younger cultures. They find comparable trajectories for two global topological parameters. <br /> Here, once again it is a strength that the authors considered additional parameters outside the ones used in fitting. However, it should be noted that the values for "global efficiency" at DIV14 (the very network that was optimized!) are clearly below the biological values plotted, weakening the generality of the previous result. This is never discussed in the text.

The conclusion of the authors in this part derives from values of modularity decreasing over time in both model and data, and global efficiency increasing. The main impact of "time" in this context is the addition of more connections, and increasing edge density. And there is a known dependency between edge density and the bounds of global efficiency. I am not convinced the result is meaningful for the conclusion in this state. If one were to work backwards from the DIV14 model, randomly removing connections (with uniform probabilities): Would the resulting trajectory match DIV12, DIV10, and DIV7 equally well? If so, the trajectory resulting from the "matching" algorithm is not meaningful.

Further, the conclusion of the authors implies that connections in the cultures are formed as in the algorithm: one after another over time without pruning. This could be simply tested: How stable are individual connections in vitro over time (between DIV)? 

The next finding is that at higher densities, the connections formed by the neurons still have very comparable structures, only differing in clustering and range; and that the same generative algorithm is optimal for modelling them. I think in its current state, the correlation analysis in Fig. 4a supports this conclusion only partially: Most of these correlations are not surprising. Shortest path lengths feature heavily in the calculation of small worldness and efficiency (in one case admittedly the inverse). Also for example network density has known relations with other measures. The analysis would be stronger if that was taken into account, for example showing how correlations deviate from the ones expected in an Erdos-Renyi-type network of equal sizes.

Yet, overall the results are supported by the depicted data and model fits in Supp. Fig S7. With the caveat that some of the numerical values depicted seem off: <br /> What are the units for efficiency? Why do they take values up to 2000? Should be < 1 as in 4b. Also, what is "strength"? I assume it's supposed to be the value of STTC, but that's not supposed to be >1. Is it the sum over the edges? But at a total degree of around 40, this would imply an average STTC almost three times higher than what's reported in Fig 1i. Also, why is the degree around 40, but between 1000 and 1500 in Fig S2? <br /> Finally, it should be mentioned that "degree average" seems (from the boxplot) to work equally well.

Further, the conclusion of the "matching" algorithm equally fitting both cases would be stronger if we were informed about the wiring parameters (η and γ) resulting in both cases. That way we could understand: Is it the same algorithm fitting both cases or very different variants of the same? It is especially crucial here, because the η and γ parameters determine the interplay between the distance- and topology-dependent terms, and this is the one case where a very different set of pairwise distances (due to higher density) are tested. Does it really generalize to these new conditions?

Conversely, the results relating to GABAa blocking show a case where the distances are comparable, but the topology of functional connectivity is very different. (Here again, the contrast between structural and functional connectivity could be made a bit clearer. How is correlational detection of connections affected by "bursty" activity?) The reduction in tentative inhibition following the application of the block is convincing.

The main finding is that despite of very different connectivities, the "matching" algorithm still holds best. This is adequately supported by applying the previous analyses to this case as well. <br /> The authors then interpret the differences between blocked and control by inspection of the η and γ parameters, finding that the relative impact of the distance-based term is likely reduced, as a lower (less negative) exponent would lead to more equal values for different distances. This is a good example of inspecting the internals of a generative algorithm to understand the modeled system and is confirmed by longer edge lengths in Supp Fig. S12C.

The authors further inspect the wiring probabilities used internally at each step of the algorithm and compare across conditions. They conclude from differences in the distribution of P_ij values that the GABAa-blocked network had a "more random" topology with "less specific" wiring. This is the opposite of the conclusion I would draw, given the depicted data. This may be partially because the authors do not clearly define their concept of "random" vs. "specific". I understand it to be the following: At each time step, one unconnected pair is randomly picked and connected, with probabilities proportional to P_ij, as in Akarca et al., 2021; "randomness" then refers to the entropy of that process. In that case, the "most random" or highest entropy case is given by uniform P_ij values, which would be depicted as a delta peak at 1 / n_pairs in the present plot. A flatter distribution would indicate more randomness if it was the distribution of P_ij over pairs of neurons (x-axis: pairs; y-axis P_ij). The conclusion should be clarified by the use of a mathematical definition and supported by data using that definition.

Next, the methods are repeated for various cultures of human neurons. I have no specific observations there.

In summary, while I think the most important methods are sound, and the main conclusions (reflected in the title of the paper) are supported, the analysis of more specific cases (everything from Fig 3e onwards, except for Fig 5) requires more work as in the current state their conclusions are not adequately supported.

Reviewer #1 (Public Review):

This work deals with courtship behaviour in mice. Authors try to identify the acoustic features that influence the attractivity level of male courtship songs to females. Courtship songs are made of sequences of short ultrasound syllables emitted at a rate of 7-10Hz. Authors manipulated these syllables by changing either the spectrotemporal content of each syllable or the intersyllable intervals. The authors found that it was only when sequences of syllables were irregular (with highly variable intersyllable intervals) that the female was less attracted to the song. The data, therefore, brings evidence that the acoustic features of syllables account less than the song's temporal regularity for the attractivity of courtship songs. The authors suggest that temporal regularity of syllable emission, building on breathing patterns, could reflect male fitness. They also suggest that temporal regularity could be an acoustic cue compressing the complex acoustic information carried by songs.

Strengths:

The study is well-written, very straightforward, and easy to follow. Behavioral tasks are well-designed and many tests, on a large enough set of animals have been done to support the conclusions. Results are clearly presented and provide enough details to see individual points. The discussion makes interesting connections between syllable rhythms and animals' fitness or brain rhythms.

Weaknesses:

Although the study is easy to understand and provides interesting results, the data analysis remains incomplete, and the interpretation of results is not cautious enough.

For instance, Fig. 2 shows a preference for song playback but we cannot determine if it is a general preference for a sound or a specific preference for male songs because only the difference between the presence of song or silence is tested. I acknowledge that the authors did not overstate their results, but the experimental design is incomplete and hard to interpret in that respect. For instance, the expression "preferential approach to song" is ambiguous.

There is no analysis of individual preference across tests and we might have the feeling that the effect shown mostly depends on the preference of only a few animals. Indeed, it seems that roughly one-third of animals showed a strong preference for the intact song while another third showed a strong preference for the modified song, whatever the modification. A few animals are therefore "swing voters". It would have been interesting, if not pertinent, to have a deeper analysis of the behavior of these later animals. Do they choose less (i.e. spend less time close to speakers) or do they swing from one corner to another? What about the animals which always chose the modified song? Are these animals that already showed a weak or strong preference for silence, therefore showing they were not comfortable with the songs played? There is no discussion of these aspects either.

Also, on page 11, it is written "female listeners perceptually compress the high sensory dimensionality of male songs by selectively monitoring a reduced subset of meaningful acoustic features in isolation." This statement or hypothesis is questionable. After all, if someone would change the inter-syllable intervals in human speech, that would become cryptic or at least annoying for the listener. Humans would definitely prefer normal speech. Is this because we compress acoustic features? Not really. It is likely that this modified speech just differs too much from the set of parameters typically encountered and therefore understood/interpreted while learning a language in childhood. Thus, the hypothesis here is rather to determine, for a given acoustic feature, if there is a range within which the perception of the message carried by the song (courtship) is maintained. Interpretation of "compressed acoustic features" with regards to animals' preference seems an overinterpretation. Same remark at the end of the conclusion.

Reviewer #1 (Public Review):

Tippett et al present whole cell and proteoliposome transport data showing unequivocally that purified recombinant SLC26A6 reconstituted in proteoliposomes mediates electroneutral chloride/bicarbonate exchange, as well as coupled chloride/oxalate exchange unassociated with detectable current. Both functions contrast with the uncoupled chloride conductance mediated by SLC26A9. The authors also present a novel cryo-EM structure of full-length human SLC26A6 chloride/anion exchanger. As part of the structure, they offer the first partial view of the STAS domain previously predicted to be unstructured. They further define a single Arg residue of the SLC26A6 transmembrane domain required for coupled exchange, mutation of which yields apparently uncoupled electrogenic chloride transport mechanistically resembling that of SLC26A9, although of lower magnitude. The authors further apply to proteoliposomes for the first time a still novel approach to the measurement of bicarbonate transport using a bicarbonate-selective Europium fluorophor. The evidence strongly supports the authors' claims and conclusions, with one exception.

The manuscript has numerous strengths.

As a structural biology contribution, the authors extend the range of SLC26 structures to SLC26A6, comparing it in considerable detail to the published SLC26A9 structure, and presenting for the first time the structure of a portion of the STAS IVS domain of SLC26A6 long considered unstructured.

The authors also apply a remarkably extensive range of creative technical approaches to assess the functional mechanisms of anion transport by SLC26A6, among them the first application of the novel, specific bicarbonate sensor Eu-L1+ to directly assess bicarbonate transport in reconstituted proteoliposomes. The authors also present the first (to this reviewer's knowledge) functional proteoliposome reconstitution of chloride-bicarbonate exchange mediated by an SLC26 protein. They define a residue in surrounding the anion binding pocket which explains part of the difference in anion exchange coupling between SLC26A6 and SLC26A9. In the setting of past conflicting results, the current work also contributes to the weight of previous evidence demonstrating that SLC26A6 mediates electroneutral rather than electrogenic Cl-/HCO3- exchange.

Each of these achievements constitutes a significant advance in our understanding.

The paper has only a few weaknesses. One is an incomplete explanation of the mechanistic determinants of anion exchange coupling in SLC26A6 vs. uncoupled anion transport by SLC26A9. A second minor weakness is the inconsistently repeated conclusion that SLC26A6 mediates strictly coupled chloride/oxalate exchange. The data presented do not measure the stoichiometry of Cl-/oxalate exchange. The AMCA proteoliposome assay documented extracellular oxalate-dependent proteoliposomal anion transport that was most simply interpreted as coupled exchange, whereas no stoichiometric coupled exchange was documented in the AMCA experiments as presented.

Overall, the manuscript represents an important advance in our understanding of the SLC26 protein family and of coupled vs uncoupled carrier-mediated anion transport.

Reviewer #1 (Public Review):

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

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. However, a major weakness of this study is that quantitative analysis of several key results is either lacking, not done appropriately, or is incompletely described. In addition, some of the cell-based experiments are lacking appropriate controls, and the claim that CRB3 directly binds to Rab11 is not supported by the data provided.

Reviewer #1 (Public Review):

In "Striatal ensemble activity in an innate behavior", Minkowicz et al. strive to characterize how the striatum, the primary input nucleus of the basal ganglia, represents grooming. Here, grooming is used as a paradigmatic habitual behavior. The pose dynamics of grooming are stereotyped: mice perform it spontaneously and prior work has shown that it is both represented and controlled by the striatum.

The manuscript presents a valuable contribution to the field by shedding light on how ensembles of neurons encode this innate behavior. Additionally, the use of supervised machine learning allowed the authors to collect and precisely align a large number of grooming repetitions, which enabled most of their downstream analysis.

I found the paper to be well-written and the conclusions are mostly well-supported. However, some of the data analysis was a bit opaque, and some more detail and reanalysis could substantially strengthen the authors' claims.

1) The authors identified grooming bouts using empirically defined thresholds and manual tweaking. Next, the boundaries of grooming were used for trial alignment and linear time warping. This is a completely sensible approach; however, in using only the boundaries of grooming episodes, the dynamics of grooming bouts are ignored. I am particularly concerned that pose dynamics of grooming bouts are most stereotyped at the boundaries (e.g. they always begin and end with specific paw movements). To play devil's advocate, if the striatum encodes pose dynamics and not boundaries and pose dynamics are noisy between the beginning and end of these bouts (either due to the dynamics of the behavior or how it was identified), then a "boundary-like" representation may emerge in the average. I strongly recommend re-running a subset of the analysis after accounting for variability in grooming dynamics. A simple thing to try would be to further cluster grooming bouts using 3D keypoint trajectories. Another would be to warp grooming bouts in a manner that accounts for keypoint trajectories (e.g. DTW or other recent time-warping variants).

2) The authors should consider if the correlation to grooming is due to (at least in part) a correlation with another aspect of movement, e.g. overall velocity, acceleration, height, or angular velocity. This should be straightforward to analyze with the current dataset. To start, I would simply take the velocity and acceleration of the mouse's centroid (head and body could be considered separately). Next, look at the correlation with DLS spiking. If a clear relationship emerges, then check to see how velocity (or another variable) maps onto grooming. It may be that DLS neurons appear to encode the boundaries of grooming when they (at least partially) encode other variables.

3) The ensemble analysis is potentially critical to our understanding of SPNs. Figure 4A suggests that ensembles encode grooming with a probabilistic code - ensembles appear to be engaged for a small number of grooming bouts in the session. First, a basic question is what is the probability a given ensemble is activated during grooming? Second, the more complex question is whether there is an explanation for why one ensemble is engaged for some trials and not others? Related to point 2, I wonder if another aspect of behavior - e.g. vigor, duration, or speed - determines this. I suggest some analysis to at least rule out some simple explanations.

Reviewer #1 (Public Review):

In this manuscript by Douglas et al, the investigative team seeks to identify Staphylococcus aureus genes (and associated polymorphisms) that confer altered susceptibility to human serum, with the hypothesis that such genes might contribute to the propensity of a strain to cause bacteremia, invasive disease, and/or death. Using an innovative GWAS-like approach applied to a bank of over 300 well-characterized clinical S. aureus isolates, the authors discover SNPs in seven different staphylococcal genes that confer increased survival in the setting of serum exposure. The authors then mainly focus on one gene, tcaA, and illustrate a potential mechanism whereby modification of peptidoglycan structure and WTA display leads to altered susceptibility to serum, serum-derived antimicrobial compounds, and antibiotics. One particularly significant finding is that the identified tcaA SNP is significantly associated with patient mortality, in that patients infected with the SNP bearing isolate are less likely to die from infection. It is therefore hypothesized that this SNP represents an adaptive mutation that promotes serum survival while decreasing virulence and host mortality. In a murine model of infection, the strain bearing the WT allele of tcaA is significantly more virulent than the tcaA mutant, suggesting that the role of tcaA in bacteremia is infection-phase dependent.

This manuscript has many strengths. The triangulation of genomic analysis, patient outcomes data, and in vitro and in vivo mechanistic testing adds to the significance of the findings in terms of human disease. Testing the impact of mutating tcaA in multiple staphylococcal lineages and backgrounds also increases the rigor of the study. The identification of bacterial loci that impact susceptibility to both host antimicrobial compounds and commonly used antibiotics is also a strength of this work, given the evolutionary and treatment implications for such genes.

One moderate weakness is that the impact of the identified SNP in tcaA is only tested in some of the assays, whereas the majority of the testing is performed with a whole gene knockout. Additionally, for some experiments, rigor is lacking in that statistical measures are not deployed to support the conclusions of biologically meaningful changes based on data with very modest differences between groups. In some cases this results in more speculative conclusions that will require further testing to validate. Finally, there are instances of inter-experiment variability that require further explanation. All in all, this is an exciting manuscript that will be of interest to the broader research communities focused on staphylococcal pathogenesis, bacterial evolution, and host-pathogen interactions, as well as to clinicians who care for patients with invasive staphylococcal infection.

Reviewer #1 (Public Review):

The manuscript by Hussein et al. uses cryoEM structure, microscale thermophoresis (MST), and molecular dynamics simulations (conventional and CpHMD) to unravel the Zn2+ and proton role in the function of the Cation Diffusion Facilitator YiiP. First, they generate mutants that abolish each of the three Zn2+ models to study the role of each of them separately, both structurally and functionally. Next, they used a Monte Carlo approach refining the CpHMD data with the MST points to establish the Zn2+ or proton binding state depending on the pH. That predicted a stoichiometry of one Zn2+ to 2 or 3 protons (1:3 under lower pH values). Finally, they proposed a mechanism that involves first the binding of Zn2+ to one low-affinity site and then, after the Zn2+ migrates to the highest affinity site in the transmembrane portion of the protein. The lack of Zn2+ in the low-affinity site might induce occlusion of the transporter.

The manuscript is well-written it is of interest to the field of Cation Facilitator Transporters. It is also an excellent example of a combination of different techniques to obtain relevant information on the mechanism of action of a transporter.

I have only a few comments that might need clarification from the authors:

- If the unbinding of Zn2+ to site B triggers the occlusion (and maybe the OF state) and the external pH does not affect that binding, how is it prevented from being always bound to Zn2+ and thus occluded also while it should be transporting protons (B to C panels in Figure 5)? Are there some other factors that I am missing?<br /> - I am not an expert on experiments, but the results for mutants that abolish site C are difficult to understand. For D287A/H263A, the SEC columns data suggest a population of higher oligomers. Still, for the D70A/D287A/H263A and D51A/D287A/H263A, they showed a native dimer. I understand your suggestion that the Fab induces the domain swap, but how do you explain the double mutant SEC column result? Please elaborate.<br /> - Since in the D287A mutant, you are disrupting the preferred tetrahedral coordination of Zn2+, but it still binds, do you observe any waters that compensate for the missing aspartate? Maybe in the MD simulations?

Reviewer #1 (Public Review):

Tomasi et al. performed a combination of bioinformatic, next-generation tRNA sequencing experiments to predict the set of tRNA modifications and their corresponding genes in the tRNAs of the pathogenic bacteria Mycobacterium tuberculosis. Long known to be important for translation accuracy and efficiency, tRNA modifications are now emerging as having regulatory roles. However, the basic knowledge of the position and nature of the modifications present in a given organism is very sparse beyond a handful of model organisms. Studies that can generate the tRNA modification maps in different organisms along the tree of life are good starting points for further studies. The focus here on a major human pathogen that is studied by a large community raises the general interest of the study. Finally, deletion of the gene mnmA responsible for the insertion of s2U at position 34 revealed defects in in growth in macrophage but in test tubes suggesting regulatory roles that will warrant further studies. The conclusions of the paper are mostly supported by the data but the partial nature of the bioinformatic analysis and absence of Mass-Spectrometry data make it incomplete. The authors do not take advantage of the Mass spec data that is published for Mycobacterium bovis (PMID: 27834374) to discuss what they find.

Important points to be considered:

1) The authors say they took a list of proteins involved in tRNA modifications from Modomics and added manually a few but we do not know the exact set of proteins that were used to search the M. mycobacterium genome.

2) The absence of mnmGE genes in TB suggested that the xcm5U derivatives are absent. These are present in M. bovis (PMID: 27834374). Are the MnmEG gene found in M. bovis? If yes, then the authors should perform a phylogenetic distribution analysis in the Mycobacterial clade to see when they disappeared. If they are not present in M. bovis then maybe a non-orthologous set of enzymes do the same reaction and then the authors really do not know what modification is present or not at U34 without LC-MS. The exact same argument can be given for the xmo5U derivatives that are also found in M.bovis but not predicted by the authors in M. tuberculosis.

3) Why is the Psi32 predicted by the authors because of the presence of the Rv3300c/Psu9 gene not detected by CMC-treated tRNA seq while the other Psi residues are? Members of this family can modify both rRNA and tRNA. So the presence of the gene does not guarantee the presence of the modification in tRNAs

4) What are tsaBED not essential but tsaC (called sua5 by the authors) essential?

Reviewer #1 (Public Review):

Muller glia function as retinal stem cells in the adult zebrafish retina. Following retinal injury, Muller glia are reprogramned (reactive Muller glia), and then divide to produce a progenitor that amplifies and differentiates into retinal neurons. Previous scRNAseq analysis used total retinal RNA from uninjured and injured retinas isolated at time points when Muller glia are quiescent, being reprogrammed, and proliferating to reveal genes and gene regulatory networks underlying these events (Hoang et al., 2020). The manuscript by Celotto et al., used double transgenic zebrafish that allow them to purify by FACS quiescent and reactive Muller glia, Muller glia-derived progenitors, and their differentiating progeny at different times post retinal damage. RNA from these cell populations was used in scRNAseq studies to identify the transcriptomes associated with these cell populations. Importantly, they report two quiescent and two reactive Muller glia populations. These results raise the interesting possibility that Muller glia are a heterogenous population whose members may exhibit different regenerative responses to retinal injury. However, without further experimentation, the validity and significance of this result remain unclear. In addition to putative Muller cell heterogeneity, Celotto et al., identified multiple progenitor classes, some of which are specified to regenerate specific retinal neuron types. Because of its focus on Muller glia and Muller glia-derived progenitors at mid to late stages of retina regeneration, this new scRNAseq data will be a useful resource to the research community for further interrogation of gene expression changes underlying retina regeneration.

Major concerns:

1) The identification of multiple populations of Muller glia, reactive Muller glia, and progenitors is interesting, but beyond a few in situ hybridization studies to validate injury-dependent gene inductions, there are no experiments that confirm that multiple cell populations exist in vivo, and no experiments examining the significance of these different populations in the regenerative process. It would be helpful to discuss how the peripheral to the central gradient of Muller cell maturation influences the scRNAseq-based cell clustering results.

2) While the reliance on transient GFP and mCherry expression may be sufficient, the final population used for the scRNAseq analysis is only partial in nature. Permanently marking the MG through a Cre-Lox system is more ideal. The authors mention the possibility of missing highly proliferative populations of MG/RPC through the dilution of fluorescent proteins; a transgenic system that allows for true lineage tracing may then capture more appropriate MG/RPC populations. The lack of gating for a pure GFP population also confounds this problem which the authors do mention in the discussions; this oversight was not explained.

3) Much time was taken to identify each cell cluster and to list the differentially expressed genes, but no functional significance for these genes was probed. While a lot of work has gone into the analysis shown, altering some of the MG/RPC trajectories through differentially expressed genes would go a long way to making this study more impactful.

4) The data presented in this paper has significant overlap with scRNAseq data presented by Hoang et al., 2020 in Science where Muller glia, reactive Muller glia, and Muller glia-derived progenitors were carefully analyzed. How does their data fit with the data presented here? The authors could have used that paper as a jumping-off point and offered more time points for comparison, especially as progenitors differentiate.

5) A major conclusion of the paper is that neurogenic progenitors in the injured retina differentiate into neurons with a similar order as that taking place during development. This analysis is based on two time points, and while the trends stay true to the authors' model, two time points are too few to make such a conclusion. In addition, because of the time points chosen for this analysis, many mature neuronal markers are lacking. Including additional time points so mature neuronal markers are detected in the dataset would enhance the trajectory proposed.

Reviewer #1 (Public Review):

Ciampa et al. investigated the role of the hypoxia-inducible factor 1 (HIF-1) pathway in placental aging. They performed transcriptomic analysis of prior data of placental gene expression over serial timepoints throughout gestation in a mouse model and identified increased expression of senescence and HIF-1 pathways and decreased expression of cell cycle and mitochondrial transcripts with advancing gestational age. These findings were confirmed by RT-PCR, Western blot, and mitochondrial assessment from mouse placental tissues from late gestation time points. Studies of human placental samples at similar late gestational ages showed similar trends in increased HIF-1 targets and decreased mitochondrial abundance with increasing gestation, but were not significantly significant due to the limited availability of uncomplicated preterm placenta samples. The authors demonstrated that stabilization of HIF-1 in vitro using primary trophoblasts and choriocarcinoma cell lines recapitulated the gene and mitochondrial dysfunction seen in the placental tissues and were consistent with senescence. Interestingly, cell-conditioned media from HIF-1 stabilized placenta cell lines induced myometrial cell contractions in vitro and correspondingly, induction of HIF-1 in pregnant mice was associated with preterm labor in vivo. These data support the role of the HIF-1 pathway in the process of placental senescence with increasing gestational age and highlight this pathway as a potentially important contributor to gestational length and a potential target for therapeutics to reduce preterm birth.

Overall, the conclusions of this study are mostly well supported by the data. The concept of placental aging has been controversial, with several prior studies with conflicting viewpoints on whether placental aging occurs at all, is a normal process during gestation, or rather only a pathologic phenomenon in abnormal pregnancies. This has been rather difficult to study given the difficulty of obtaining serial placental samples in late gestation. The authors used both a mouse model of serial placental sampling and human placental samples obtained at preterm, but non-pathologic deliveries, which is an impressive accomplishment as it provides insight into a previously poorly understood timepoint of pregnancy. The data clearly demonstrate changes in the HIF-1 pathway and cellular senescence at increasing gestational ages in the third trimester, which is consistent with the process of aging in other tissues.

Weaknesses of this study are that although the authors attribute alterations in HIF-1 pathways in advanced gestation to hypoxia, there are no experiments directly assessing whether the changes in HIF-1 pathways are due to hypoxia in either in vitro or in vivo experiments. HIF-1 has both oxygen-dependent and oxygen-independent regulation, so it is unclear which pathways contribute to placental HIF-1 activity during late gestation, especially since the third-trimester placenta is exposed to significantly higher oxygen levels compared to the early pregnancy environment. Additionally, the placenta is in close proximity to the maternal decidua, which consists of immune and stromal cells, which are also significantly affected by HIF-1. Although the in vitro experimental data in this study demonstrate that HIF-1 induction leads to a placenta senescence phenotype, it is unclear whether the in vivo treatment with HIF-1 induction acts directly on the placenta or rather on uterine myometrium or decidua, which could also contribute to the initiation of preterm labor.

Reviewer #1 (Public Review):

Summary

Favate et al. measure the relative levels of metabolites in 12 Escherichia coli strains isolated from different replicate populations after 50,000 generations of the Lenski long-term laboratory evolution experiment. They use untargeted LC/MS methods that include standards and report both positive and negative ionization mode measurements. They initially use principal component analysis (PCA) to broadly compare how the metabolomes of these strains are similar and different. Then, they describe several instances where the changes in metabolite abundance they see in specific pathways correlate with mutations that lead to changes in the expression of genes that encode enzymes in those pathways.

Strengths

The statistical analyses and presentation of the high-throughput data are excellent. The most compelling results are communicated in wonderful figures that integrate their measurements of metabolite levels in this study with results from a prior study they conducted looking at changes in gene expression levels in the same bacterial strains. These sections include the ones describing large increases in NAD(P) pools due to mutations in nadR, changes in the levels of arginine and related compounds due to mutations in argR, and changes in metabolites from glycolysis and the TCA cycle related to iclR and arcB.

Weaknesses

Showing that A-2 and especially A-3 are outliers in the PCA analysis is useful, but it may be hiding other interesting signals in the data. The other strains are remarkably colinear on these plots, hinting that if the outliers were removed, one main component would emerge along which they are situated. It also seems possible that this additional analysis step would allow the second dimension to better differentiate them in a way that is interesting with respect to their mutator status or mutations in key metabolic or regulatory genes.

There is a missed opportunity to connect some key results to what is known about LTEE mutations that reduce the activity of pykF (pyruvate kinase I). This gene is mutated in all 12 LTEE populations, and often these mutations are frameshifts or transposon insertions that should completely knock out its activity. At first glance, inactivating an enzyme for a step in glycolysis does not make sense when the nutrient source in the growth medium is glucose, even though PykF is only one of two isozymes E. coli encodes for this reaction. There has been speculation that inactivating pykF increases the concentration of phosphoenolpyruvate (PEP) in cells and that this can lead to increased rates of glucose import because PEP is used by the phosphotransferase system of E. coli to import glucose (see https://doi.org/10.1002/bies.20629). The current study has confirmed the higher PEP levels, which is consistent with this model.

In the introduction, the papers cited to show the importance of changes in metabolism for adaptation do not seem to fit the focus of this study very well. They stress production of toxins and secondary metabolites, which do not seem to be mechanisms that are at work in the LTEE. I can think of two areas of background that would be more relevant: (1) studies of how bacterial metabolism evolves in adaptive laboratory evolution (ALE) experiments to optimize metabolic fluxes toward biomass production (for example, https://doi.org/10.1038/nature01149 ), and (2) discussions of how cross-feeding, metabolic niche specialization, and metabolic interdependence evolve in microbial communities, including in other evolution experiments (for example, https://doi.org/10.1073/pnas.0708504105 and https://doi.org/10.1128/mBio.00036-12).

Impact and Significance

While there has been past speculation about the effects of LTEE mutations on metabolism, this study measures changes in the levels of metabolites in related metabolic pathways for the first time. Therefore, it provides useful information about how metabolism evolves, in general, and will also be a useful resource for those studying other aspects of the LTEE related to metabolism, such as contingency in the evolution of citrate utilization.