DOI: 10.1101/2023.01.13.523942

Resource: RRID:ZFIN_ZDB-ALT-071016-1

Curator: @scibot

SciCrunch record: RRID:ZFIN_ZDB-ALT-071016-1

What is this?

Reviewer #1 (Public Review):

This study reports the results of a computational and EEG analysis of altruistic decision making. The authors intend to examine whether fundamentally different mechanisms operate to drive altruistic decision making in different contexts, which they here manipulate by examining choices in the realm of advantageous and disadvantageous inequality. The authors find that changes in self payoff are encoded in opposite manners in the two contexts, but that a similar evidence accumulation mechanism leading up to the time of response seems to operate equally in both. In addition, they find that individual differences in generosity are predicted more by differences in sensitivity to change in the other's payoff in the disadvantageous inequality condition, and by stronger phase coupling between sensors related to this delta-other signal and sensors related to the evidence accumulation signal.

This study makes a valuable contribution by combining a sophisticated suite of modelling and neurophysiological analyses to shed light on the decision parameter adjustments that inform altruistic decisions in different contexts. The conclusions regarding those adjustments appear well supported by the data. One aspect that could be clarified is that there is an apparent discrepancy between the cross-condition bound adjustments identified by the modelling and the absence of any corresponding neural evidence accumulation signal amplitude difference.

One of the stated overarching goals of this study is to determine whether the neural mechanisms and circuits for altruistic decisions are context-specific or general. The manuscript would benefit from greater clarity on this point, in particular defining what is meant by 'mechanisms' and what qualitative and quantitative criteria should be applied when identifying them as distinct versus common. As all decisions in this study are reported via the same manual actions it seems implausible that there would be no overlap at all in the circuits and mechanisms involved. In addition, the prior literature has demonstrated that even individual neurons can trace different computations depending on the circumstances. Therefore, it is necessary to clarify whether the authors are searching for context-dependence in the brain areas/signals that are recruited and/or in the computations that are performed within a brain area.

Reviewer #1 (Public Review):

In this study, the authors examine the function of Tomosyn, in dense core vesicle fusion using CRE-mediated deletion in neuronal cultures from mice expressing conditional alleles of tomosyn and tomosyn-2. Tomosyn is a large soluble SNARE protein, where earlier work in multiple species suggested that it functions as a competitive inhibitor of cognate SNARE interactions impairing fusion. The authors show that while loss of tomosyns did not affect dense core vesicle exocytosis, it reduced the expression of several key dense core cargos, including BDNF. Limited (if anything opposite) impact of tomosyn loss-of-function on intracellular vesicle trafficking or Golgi function.

The authors concluded that tomosyns regulate neuropeptide and neurotrophin secretion by regulating dense core vesicle cargo production but not exocytosis.

Reviewer #1 (Public Review):

These findings for the first time provide a comprehensive multiscale assessment of the arrhythmogenic potential of elite exercise training.

The authors trained canines using a treadmill over 16 weeks, and compared these animals (n=12) to sedentary animals (n=13). The authors found global evidence of electrophysiologic remodeling ECG indices and heart rate, as well as repolarization variability in trained animals relative to controls.

The authors also demonstrate a range of effects of ventricular cardiomyocyte ion channels and fibrosis. Finally, using an induction protocol, the authors show enhanced risk for ventricular fibrillation as well as spontaneous arrhythmias in trained dogs.

The authors conclude that structural and electrophysiologic remodeling of ventricles in elite trained athletes is associated with ventricular arrhythmogenesis.

First, this is a difficult study to achieve given the logistical challenges of managing a large animal set up as utilized in this study. Further protocols that involve in vivo and subsequently in vitro studies of tissues from large animals are challenging to accomplish. Finally, the multimodal assessments undertaken in this study to achieve these comprehensive objectives are an additional strength.

Weaknesses include the descriptive nature of the work and somewhat low level of rigor in presenting the observed data. The presentation of the data in the text could also be improved. Finally, some of the counterintuitive/conflicting findings e.g. enhanced HCN4 expression with reduced heart rate.

Reviewer #1 (Public Review):

This article is aimed at constructing a recurrent network model of the population dynamics observed in the monkey primary motor cortex before and during reaching. The authors approach the problem from a representational viewpoint, by (i) focusing on a simple center-out reaching task where each reach is predominantly characterised by its direction, and (ii) using the machinery of continuous attractor models to construct network dynamics capable of holding stable representations of that angle. Importantly, M1 activity in this task exhibits a number of peculiarities that have pushed the authors to develop important methodological innovations which, to me, give the paper most of its appeal. In particular, M1 neurons have dramatically different tuning to reach direction in the movement preparation and execution epochs, and that fact motivated the introduction of a continuous attractor model incorporating (i) two distinct maps of direction selectivity and (ii) distinct degrees of participation of each neuron in each map. I anticipate that such models will become highly relevant as neuroscientists increasingly appreciate the highly heterogeneous, and stable-yet-non-stationary nature of neural representations in the sensory and cognitive domains.

As far as modelling M1 is concerned, however, the paper could be considerably strengthened by a more thorough comparison between the proposed attractor model and the (few) other existing models of M1 (even if these comparisons are not favourable they will be informative nonetheless). For example, the model of Kao et al (2021) seems to capture all that the present model captures (orthogonality between preparatory and movement-related subspaces, rotational dynamics, tuned thalamic inputs mostly during preparation) but also does well at matching the temporal structure of single-neuron and population responses (shown e.g. through canonical correlation analysis). In particular, it is not clear to me how the symmetric structure of connectivity within each map would enable the production of temporally rich responses as observed in M1. If it doesn't, the model remains interesting, as feedforward connectivity between more than two maps (reflecting the encoding of many more kinematic variables) or other mechanisms (such as proprioceptive feedback) could well explain away the observed temporal complexity of neural responses. Investigating such alternative explanations would of course be beyond the scope of this paper, but it is arguably important for the readers to know where the model stands in the current literature.

Below is a summary of my view on the main strengths and weaknesses of the paper:

1. From a theoretical perspective, this is a great paper that makes an interesting use of the multi-map attractor model of Romani & Tsodyks (2010), motivated by the change in angular tuning configuration from the preparatory epoch to the movement execution epoch. Continuous attractor models of angular tuning are often criticised for being implausibly homogeneous/symmetrical; here, the authors address this limitation by incorporating an extra dimension to each map, namely the degree of participation of each neuron (the distribution of which is directly extracted from data). This extension of the classical ring model seems long overdue! Another nice thing is the direct use of data for constraining the model's coupling parameters; specifically, the authors adjust the model's parameters in such a way as to match the temporal evolution of a number of "order parameters" that are explicitly manifested (i.e. observable) in the population recordings.

I believe the main weakness of this continuous attractor approach is that it - perhaps unduly - binarises the configuration of angular tuning. Specifically, it assumes that while angular tuning switches at movement onset, it is otherwise constant within each epoch (preparation and execution). I commend the authors for carefully motivating this in Figure 2 (2e in particular), by showing that the circular variance of the distribution of preferred directions is higher across prep & move than within either prep or move. While this justifies a binary "two-map model" to first order, the analysis nevertheless shows that preferred directions do change, especially within the preparatory epoch. Perhaps the authors could do some bootstrapping to assess whether the observed dispersion of PDs within sub-periods of the delay epoch is within the noise floor imposed by the finite number of trials used to estimate tuning curves. If it is, then this considerably strengthens the model; otherwise, the authors should say that the binarisation reflects an approximation made for analytical tractability, and discuss any important implications.

2. While it is great to constrain the model parameters using the data, there is a glaring "issue" here which I believe is both a weakness and a strength of the approach. The model has a lot of freedom in the external inputs, which leads to relatively severe parameter degeneracies. The authors are entirely forthright about this: they even dedicate a whole section to explaining that depending on the way the cost function is set up, the fit can land the model in very different regimes, yielding very different conclusions. The problem is that I eventually could not decide what to make of the paper's main results about the inferred external inputs, and indeed what to make of the main claim of the abstract. It would be great if the authors could discuss these issues more thoroughly than they currently do, and in particular, argue more strongly about the reasons that might lead one to favour the solutions of Fig 6d/g over that of Fig 6a. On the other hand, I see the proposed model as an interesting playground that will probably enable a more thorough investigation of input degeneracies in RNN models. Several research groups are currently grappling with this; in particular, the authors of LFADS (Pandarinath et al, 2018) and other follow-up approaches (e.g. Schimel et al, 2022) make a big deal of being able to use data to simultaneously learn the dynamics of a neural circuit and infer any external inputs that drive those dynamics, but everyone knows that this is a generally ill-posed problem (see also discussion in Malonis et al 2021, which the authors cite). As far as I know, it is not yet clear what form of regularisation/prior might best improve identifiability. While Bachschmid-Romano et al. do not go very far in dissecting this problem, the model they propose is low-dimensional and more amenable to analytical calculations, such that it provided a valuable playground for future work on this topic.

3. As an addition to the motor control literature, this paper's main strengths lie in the model capturing orthogonality between preparatory and movement-related activity subspaces (Elsayed et al 2016), which few models do. However, one might argue that the model is in fact half hand-crafted for this purpose, and half-tuned to neural data, in such a way that it is almost bound to exhibit the phenomenon. Thus, some form of broader model cross-validation would be nice: what else does the model capture about the data that did not explicitly inspire/determine its construction? As a starting point, I would suggest that the authors apply the type of CCA-based analysis originally performed by Sussillo et al (2015), and compare qualitatively to both Sussillo et al. (2015) and Kao et al (2021). Also, as every recorded monkey M1 neuron can be characterized by its coordinates in the 4-dimensional space of angular tuning, it should be straightforward to identify the closest model neuron; it would be very compelling to show side-by-side comparisons of single-neuron response timecourses in model and monkey (i.e., extend the comparison of Fig S6 to the temporal domain).

4. The paper's clarity could be improved.

Reviewer #1 (Public Review):

DeRisi and colleagues used a new phage-display peptide platform, with 238,068 tiled 62-amino acid peptides covering all known P falciparum coding regions (and numerous other entities), to survey seroreactivity in 198 Ugandan children and adults from two cohorts. They find that breadth of responses to repeat-containing peptides was twofold higher in children living in the high versus moderate exposure setting, while no such differences were observed for peptides without repeats. Additionally, short motifs associated with seroreactivity were extensively shared among hundreds of antigens, with much of this driven by motifs shared with PfEMP1 antigens.

Malaria immunity is complex, and this new platform is a potentially valuable addition to the toolkit for understanding humoral responses. The two cohorts differed in fundamental ways: 1) high versus moderate exposure to infective bites; 2) samples drawn at the time of malaria for most donors in the high zone versus ~100 days after the last malaria episode in the moderate zone. The effect of acute malaria to boost short-term cross-reactive antibodies can confound the ability to draw inferences when comparing the two cohorts, and this should be further explored to understand its role in the patterns of seroreactivity observed.

Reviewer #1 (Public Review):

Osteoclasts, giant multinucleated bone-resorbing cells, are crucial regulators of bone homeostasis and pathology. An underestimated aspect of their biology is that they are very heterogeneous, with at least 2 sub-populations (inflammatory osteoclasts and tolerogenic osteoclasts) existing, and exerting different actions, especially in the context of inflammatory bone loss. In this report, Madel, Halper (co-first authors), and colleagues present an interesting report investigating this heterogeneity, and showing that the probiotic yeast S. boulardii (probably through β-glucans) may be useful in managing inflammation-mediated bone loss, including oestrogen deprivation-mediated osteoporosis, as the authors show in vivo using an OVX mouse model.

The authors first evaluate the differences in the transcriptional landscape of tolerogenic vs inflammatory osteoclasts with RNAseq, and then they evaluate the differences in miRNA expression between the two. Finding that some of the pathways/genes that vary are related to pattern recognition receptors (PRRs), specialized in recognizing non-self antigens including those arising from bacteria and yeasts, they wonder if the probiotic yeast S. boulardii could influence the balance between tolerogenic and inflammatory osteoclasts. Indeed, when the authors treated OVX mice, characterised by an increase in inflammatory osteoclasts and estrogen deprivation/inflammation-induced bone loss, with the probiotic, the bone loss is avoided and inflammatory osteoclasts are reduced. This challenges the classical way in which osteoclast-mediated bone loss is treated, since targeting specifically the inflammatory osteoclasts could allow the good osteoclasts to keep working and improving bone health and immunity, while only the bad osteoclasts are targeted. Current treatments are not able to distinguish between the two, which can cause a paradoxical degradation in bone health and atypical fractures. The report is therefore potentially very important for the field, and although quite focused on a specific strain, it can pave the way to treating bone diseases with probiotics, or specific molecules derived from them including beta-glucans.

Reviewer #1 (Public Review):

This well-done platform trial identifies that ivermectin has no impact on SARS-CoV-2 viral clearance rate relative to no study drug while casirivimab lead to more rapid clearance at 5 days. The figures are simple and appealing. The study design is appropriate and the analysis is sound. The conclusions are generally well supported by the analysis. Study novelty is somewhat limited by the fact that ivermectin has already been definitively assessed and is known to lack efficacy against SARS-CoV-2. Several issues warrant addressing:

1) Use of viral load clearance is not unique to this study and was part of multiple key trials studying paxlovid, remdesivir, molnupiravir, and monoclonal antibodies. The authors neglect to describe a substantial literature on viral load surrogate endpoints of therapeutic efficacy which exist for HIV, hepatitis B and C, Ebola, HSV-2, and CMV. For SARS-CoV-2, the story is more complicated as several drugs with proven efficacy were associated with a decrease in nasal viral loads whereas a trial of early remdesivir showed no reduction in viral load despite a 90% reduction in hospitalization. In addition, viral load kinetics have not been formally identified as a true surrogate endpoint. For maximal value, a reduction in viral load would be linked with a reduction in a hard clinical endpoint in the study (reduction in hospitalization and/or death, decreased symptom duration, etc...). This literature should be discussed and data on the secondary outcome, and reduction in hospitalization should be included to see if there is any relationship between viral load reduction and clinical outcomes.

2) The statement that oropharyngeal swabs are much better tolerated than nasal swabs is subjective. More detail needs to be paid to the relative yield of these approaches.

3) The stopping rules as they relate to previously modeled serial viral loads are not described in sufficient detail.

4) The lack of blinding limits any analysis of symptomatic outcomes.

5) It is unclear whether all 4 swabs from 2 tonsils are aggregated. Are the swabs placed in a single tube and analyzed?

6) In supplementary Figure 7, both models do well in most circumstances but fail in the relatively common event of non-monotonic viral kinetics (multiple peaks, rebound events). Given the importance of viral rebound during paxlovid use, an exploratory secondary analysis of this outcome would be welcome.

Reviewer #1 (Public Review):

In the study, Zhao et al. investigated loop conformational changes in the active site of L1 Metallo-beta-lactamase. Antibiotic resistance is on the rise and beta-lactamases are enzymes that cleave a lactam ring. Authors investigate class B3 MBLs since these could be used for designing drugs for treating antibacterial resistance. Authors find specific loops that act as gates to the shape and access to the active site of the enzymes. They study these loops via MD simulations, Markov state models, and CVAE-based deep learning to experimentally reveal how each residue affects activity as well as remodeling of the active site.

Strengths<br /> - The authors make a good case for why MD is important for this scaffold and protein class. The study performs MD simulations coupled with Markov State Models - this coupled with CVAE to understand the different states the protein exists in shapes the state-of-the-art study. Authors are able to isolate three different states that the protein exists in and pinpoint which interactions cause a reshaping of the active site.<br /> - Furthermore, they isolate the likely states that also correspond with lower free energy indicating why these states might be more populated. This study adds to the depth of their work.

Weaknesses<br /> - Overall, the impact of work on the currently used antibiotic classes is unclear since the total market presence of all antibiotics is discussed not the carbapenem-based antibiotics class. Statistics related to broad antibiotic class reduce the impact statement instead of improving it.<br /> - Finally in the experimental testing only a few variants at each position were tested, leading to limited learning of the impact of active site interactions.<br /> - Authors state from previous studies on TEM-1 that disruption of the salt bridge between the two loops would alter the binding site, thus reducing antibiotic resistance. The authors continue on to hypothesize that this would hold true for the structure in consideration for this paper as well. While a good hypothesis, this cannot be inferred until we see experimental evidence for the same or a sequence comparison discussing how similar TEM1 is to the L1 MBL in question.<br /> - The authors do not explain how different splits of this data in terms of splitting (80:20 vs 70:30 or others) and reducing interaction matrix lower than 22 x 22 residues can impact their results. Also, the effect of changing the distance shell (8A) for matrix generation is not described. This variation is unaccounted for and can enable authors to pressure test their method and learnings.

Reviewer #1 (Public Review):

Bornstein and colleagues address an important question regarding the molecular makeup of the different cellular compartments contributing to the muscle spindle. While work focusing on single components of the spindle in isolation - proprioceptors, gamma-motor neurons, and intrafusal muscle fibres - have been recently published, a comprehensive analysis of the transcriptome and proteome of the spindle was missing and it fills an important gap considering how local translation and protein synthesis can affect the development and function of such a specialised organ.

The authors combine bulk transcriptome and proteome analysis and identify new markers for neuronal, intrafusal, and capsule compartments that are validated in vivo and are shown to be useful for studying aspects of spindle differentiation during development. The methodology is sound and the conclusions in line with the results. I feel a bit more analysis regarding the specificity and developmental expression profiles of the identified markers would be a great addition. In particular:

- Are any of the proprioceptive sensory neurons markers specific for fibres innervating the muscle spindles or also found in Golgi tendon organs?

- On the same line are any of the gamma motor neurons markers found also in alpha?

- How early expression of ATP1A3 is found in neurons at the spindle or fibres starting to innervating the muscle? A couple of late embryonic timepoints would be great.

- Given that the approach used allows to obtain insights on whether local translation plays a major role into the differentiation of the spindle it would be interesting to assess whether the proprioceptor and gamma motor neuron markers identified are also found in the cell body or exclusively at the spindle.

Altogether, this is a novel and important work that will benefit scientists studying the neuromuscular and musculoskeletal systems by pushing the field toward an holistic understanding of the muscle spindle. These datasets in combination with the previous ones can be used to develop new genetic and viral strategies to study muscle spindle development and function in healthy and pathological states by analysing the roles and relative contributions of different components of this fascinating and still mysterious organ.

Reviewer #1 (Public Review):

The authors devised a new mRNA imaging approach, MASS, and showed that it can be applied to investigate the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans. The approach is potentially useful, but this manuscript will benefit by addressing the following questions:

Major comments:

1. In Figure 1-figure supplement 1, the authors claimed that MASS could verify the lamellipodia-localization of beta-actin mRNAs. However, the image showed the opposite of the authors' claim as the concentration of beta-actin mRNA was lower in lamellipodia than the rest of the cytosol. This result disagreed with ref. 17 (Katz, Z.B. et al., Genes and Development, 2012). Hence, the authors cannot make the statement that "MASS can be readily used to image RNA molecules in live cells without affecting RNA subcellular localization". To thoroughly test this notion, the authors should image beta-actin mRNA using MASS and the conventional MS2 system side by side and calculate the polarization index in the same way as shown in Katz, Z.B. et al., Genes and Development, 2012.

2. The experiments that validate this new RNA imaging method are not sufficient. The authors need to systematically compare MASS and the MS2 system, including their RNA signal intensity, signal-to-background ratio.

3. In line with this, does beta-actin mRNA display the same behavior as in (Figure 1C-F) when the mRNA was imaged with the MS2 system? The movies do not indicate the type of motility expected of mRNA. For instance, it seems that almost all of the GFP dots, which are presumably single beta-actin mRNAs, stayed stationary over a time course of tens of seconds (Movie 1). This seems to be very different from what has been observed before. It's not clear that the dots are real mRNAs molecules. This further stresses the importance for them to compare their new imaging system with the conventional MS2 application.

4. The authors claimed that a major advantage of MASS is that it has only 8xMS2 stem loops (350 nt) and overcomes "the previous obstacle of the requirement of inserting a long 1,300 nt 24xMS2". This statement lacks experimental support in this manuscript. The authors need to quantitatively compare the genomic tagging efficiency of 8xMS2 and 24xMS2.

5. MASS has the same strategy as SunRISER (Guo, Y. & Lee, R.E.C., Cell Reports Methods, 2022). Both methods use Suntag to amplify signals of MS2- or PP7-tagged RNA. The authors need to elaborate the discussions and describe the similarities and differences of the two studies. In particular, the Guo paper needs to be properly referenced.

6. In Guo, Y. & Lee, R.E.C., Cell Reports Methods, 2022, they showed that 8XPP7 with 24XSunTag configuration led to fewer mRNA per cell (Figure 5B of the Cell Reports Methods paper). Does MASS, which has 8xMS2 with 24xSunTag, similarly lead to few mRNAs? The authors should compare the number of mRNAs detected by MASS and the conventional MS2, or by FISH.

Reviewer #1 (Public Review):

Auxin-induced degradation is a strong tool to deplete CHK-2 and PLK-2 in the C. elegans germ line. The authors strengthen their conclusions through multiple approaches, including rescuing mutant phenotypes and biochemical analyses of CHK-2 and PLK-2.

The authors overcame a technical limitation that would hinder in vitro analysis (low quantity of CHK-2) through the clever approach of preventing its degradation via the proteasome. In vitro phosphorylation assays and mass spectrometry analysis that establishes that CHK-2 is a substrate of PLK-2 nicely complement the genetic data.

The authors argue that the inactivation of CHK-2 by PLK-2 promotes crossover designation; however, the data only indicate that PLK-2 promotes proper timing of crossover designation.

It is not clear whether the loss of CHK-2 function with the S116A and T120A mutations is the direct result of the inability to phosphorylate these residues or whether it is caused by the apparent instability of these proteins, as their abundance was reduced in IPs compared to wild-type.

The mechanism of CHK-2 inactivation in the absence of PLK-2 remains unclear, though the authors were able to rule out multiple candidates that could have played this role.

Reviewer #1 (Public Review):

In this manuscript, Li et al identify sleep and circadian regulatory role for ecdysone signaling in cortex glia. Prior to this report, numerous studies have linked ecdysone to sleep regulation, though these have primarily focused on its function in neurons. The manuscript is of high interest for a number of reasons. First, it provides a systematic analysis of how NHRs impact sleep. Second, the identification of ecdysone as a critical regulator of both sleep and circadian neurons provides new avenues to study how glia regulate sleep. Finally, the link to lipid accumulation is interesting, but perhaps preliminary. The manuscript is well written, and the data are clearly presented with appropriate controls. Overall this is an exciting manuscript that opens up new directions for the field.

Reviewer #1 (Public Review):

The manuscript entitled "Endo-lysosomal assembly variations among Human Leukocyte 1 Antigen class I (HLA-I) allotypes" by Eli Olson and co-workers reports an interesting observation that HLA-I alleles known not to require the standard peptide-loading complex for assembly and egress from the endoplasmic reticulum, may assemble with peptide ligands within the endo/lysosomes and are more adept at antigen cross-presentation to the CD8+ subset of T cells.

The strengths of the work are (a) a novel hypothesis that HLA-I allotype variations caused by HLA-I gene polymorphisms control endo/lysosomal HLA-I assembly and antigen cross-presentation even though there is ample evidence for cross-presentation using the endo/lysosomal pathway; and (b) new evidence to support this hypothesis.

Weaknesses are (a) the use of qualitative serologic assays in which specificity of broadly reactive antibodies such as the anti-Bw6 antibody cannot be easily controlled; (b) poor resolution of co-localization micrographs and quantification based on such data; (c) evidence that endo/lysosomal pathway dominates in cross-presentation by B35.1 allotype is weak as the data suggest a significant role for the standard cytoplasmic pathway itself in this immunologic process; and (d) narrow focus on a single member of the B7 supertype that is prevalent at low frequency in the African American (AA: 0.05) and White American (WA: 0.07) populations. These weaknesses can be addressed by using (a) more quantitative biochemical assays; (b) high resolution microscopy; & (c) extending cellular biochemical studies to one or more additional allotypes in the B7 superfamily-e.g., B7.2 (AA: 0.08; WA: 0.155) itself &/or B35.3 (AA: 0.005; WA: 0.027) or B53.1 (AA: 0.133; WA: 0.004).

Reviewer #1 (Public Review):

This study used MEG to investigate the neural changes induced by two weeks of reading instruction in pre-literate children. The study addresses a topic of great importance, measuring neural changes resulting from learning to read. While there have been several previous studies investigating this question, this may be the first study to use a truly experimental approach (i.e., involving random assignment).

There are some weaknesses in the current presentation of results that limit the conclusions that can be drawn. First, there is no control region (e.g., the right FFA or object-selective LO) to show that learning to read specifically affected tuning in a region corresponding to the VWFA, as hypothesized. This is important also to exclude more general differences between conditions (e.g., increased attention to letters after two weeks of training to recognize letters). Second, the statistical pattern of results is closely linked to the specific time window of interest (135-235 ms after stimulus onset) but there is no evidence that this time window is selective for words in this age group. For comparison, the face-selective response in children of this age is only observed at around 250 ms after onset (Taylor et al., Clinical Neurophysiology 1999). Third, the power analysis is very optimistic, with an estimated effect size of d=4.65. Considering the between-subjects design, the relatively low SNR of data in young children, and the multiple comparisons that are inherent to neuroimaging data, the study may be underpowered to detect the likely subtle effects of the 2 weeks of training.

Reviewer #1 (Public Review):

This study comprehensively categorizes the olivocochlear efferents, using single nucleus RNA-sequencing and 3D reconstructions of individual fibers and their pre-synaptic contacts onto target neurons in the cochlea.

The major strengths of the methods and results are the gene expression studies, which reveal 5 clusters of olivocochlear neurons. Traditionally, efferents have been divided into two groups, medial olivocochlear neurons that terminate on outer hair cells, and the lateral olivocochlear neurons, that terminate on spiral ganglion neurons postsynaptic to the auditory hair cells. The analyses here revealed 3 main clusters, one large cluster of medial olivocochlear neurons, and two clusters of lateral olivocochlear neurons.

In a second major strength, the study shows changing patterns of physiologically relevant gene expression over development. The authors further showed changes in the neuropeptide expression in the lateral olivocochlear neurons days after acoustic trauma.

There are no significant weaknesses, barring the issue of a gap between gene and protein expression. This is mitigated by a close match with previous protein expression studies.

Thus, the authors have achieved their aims to characterize the phenotypes and arborization patterns of the cochlear efferents. They have confirmed and enlarged upon what has previously known about this important population.

Since these neurons have been difficult to characterize, and are important for auditory function, particularly in noise, the likely impact of the work on the field is high.

Reviewer #1 (Public Review):

In this study, the authors describe an elegant genetic screen for mutants that suppress defects of MCT1 deletions which are deficient in mitochondrial fatty acid synthesis. This screen identified many genes, including that for Sit4. In addition, genes for retrograde signaling factors (Rtg1, Rtg2 and Rtg3), proteins influencing proteasomal degradation (Rpn4, Ubc4) or ribosomal proteins (Rps17A, Rps29A) were found. From this mix of components, the authors selected Sit4 for further analysis. In the first part of the study, they analyzed the effect of Sit4 in context of MCT1 mutant suppression. This more specific part is very detailed and thorough, the experiments are well controlled and convincing. The second, more general part of the study focused on the effect of Sit4 on the level of the mitochondrial membrane potential. This part is of high general interest, but less well developed. Nevertheless, this study is very interesting as it shows for the first time that phosphate export from mitochondrial is of general relevance for the membrane potential even in wild type cells (as long as they live from fermentation), that the Sit4 phosphatase is critical for this process and that the modulation of Sit4 activity influences processes relying on the membrane potential, such as the import of proteins into mitochondria. However, some aspects should be further clarified.

1. It is not clear whether Sit4 is only relevant under fermentative conditions. Does Sit4 also influence the membrane potential in respiring cells? Fig. S2D shows the membrane potential in glucose and raffinose. Both carbon sources lead to fermentative growths. The authors should also test whether Sit4 levels influence the membrane potential when cells are grown under respirative conditions, such in ethanol, lactate or glycerol. Even if deletions of Sit4 affect respiration, mutants with altered activity can be easily analyzed.<br /> 2. The authors should give a name to the pathway shown in Fig. 4D. This would make it easier to follow the text in the results and the discussion. This pathway was proposed and characterized in the 90s by George Clark-Walker and others, but never carefully studied on a mechanistic level. Even if the flux through this pathway cannot be measured in this study, the regulatory role of Sit4 for this process is the most important aspect of this manuscript.<br /> 3. To further support their hypothesis, the authors should show that deletion of Pic1 or Atp1 wipes out the effect of a Sit4 deletion. In these petite-negative mutants, the phosphate export cycle cannot be carried out and thus, Sit4, should have no effect.<br /> 4. What is the relevance of Sit4 for the Hap complex which regulates OXPHOS gene expression in yeast? The supplemental table suggests that Hap4 is strongly influenced by Sit4. Is this downstream of the proposed role in phosphate metabolism or a parallel Sit4 activity? This is a crucial point that should be addressed experimentally.<br /> 5. The authors use the accumulation of Ilv2 precursors as proxy for mitochondrial protein import efficiency. Ilv2 was reported before as a protein which, if import into mitochondria is slow, is deviated into the nucleus in order to be degraded (Shakya,..., Hughes. 2021, Elife). Is it possible that the accumulation of the precursor is the result of a reduced degradation of pre-Ilv2 in the nucleus rather than an impaired mitochondrial import? Since a number of components of the ubiquitin-proteasome system were identified with Sit4 in the same screen, a role of Sit4 in proteasomal degradation seems possible. This should be tested.

Reviewer #1 (Public Review):

The authors optimize a live cell imaging method based on the detection of FAD/NAD(P)H adopted from the fast-growing field of live metabolic imaging. They build upon a method described by KreiB et al 2020 that used metabolic ratio and collagen fiber second harmonic generation imaging. They follow by combining metabolic imaging with morphologic measurements to train a machine-learning model that is able to identify cell types accurately. Upon visualization, authors detected structures hypothesized and then proven to resemble the "goblet cell associated antigen passages" previously studied in intestinal epithelia.

STRENGTHS<br /> - The manuscript is succinct, well written, and overall done rigorously.<br /> - The optimization of the method at multiple levels to the point of identifying both common and rare cell types is impressive.<br /> - Describes the elegant implementation of a sorely needed method in epithelial biology.<br /> - Provides an approach to studying the cholinergic response in epithelial cells, a poorly understood phenomenon despite broad clinical use for diagnosis and treatment.

WEAKNESSES<br /> A) For what is in large part a methods-development paper, the methods are not explained or shared in a manner that facilitates reproducibility. For example:<br /> A.1.) The training and validation datasets seem to come from the same sample (or the source is not clearly described). Therefore, it is not clear whether the "96% accuracy" refers to accuracy within the sample measured, or whether it can extrapolate to other samples.<br /> A.2.) It is unclear whether the model needs to be re-trained within each new sample measured, or if it's applicable to others. This has implications for method adoption by others. Either way is useful but needs to be clarified.<br /> A.3.) Code was only listed in a PDF file, which makes reproducing the analysis very cumbersome.

B) Whereas the optimization to improve cell type detection is very well described, the implementability of the approach could benefit from exploration (using the data already obtained) of the minimal set of measurements needed to identify cell types. For example, is the FAD/NAD(P)H ratio necessary? Or could just morphologic measurements achieve the same goal?

C) Whereas the conclusions are overall supported by the data, need small adjustments in some cases:<br /> C.1.) For example, P3L80: Claims autofluorescence imaging is more specific than "functional markers", however, this is done in the setting of a very specific intervention that massively affects a protein often used as a secretory cell marker (CCSP aka SCGB1A1), which is known to be secreted (and depleted) in secretory cells upon stimulation.<br /> C.2.) Relatedly, it is unclear how the method's accuracy would be affected in conditions that affect redox/metabolic state; the approach may be highly affected in inflammation and injury, for example.

D) The data used to describe "SAPs" is very cursory.<br /> D.1.) Unclear if FITC dextran uptake occurs in other cells too, or in secretory cells prior to methacholine stimulation, or induced nonspecifically due to epithelia manipulation. Secretory and goblet cells are very sensitive to stimulation and often considered minimal, for example, see the paper by Abdullah et al DOI:10.1007/978-1-61779-513-8_16 in which extreme care had to be applied to prevent any secretion at all.<br /> D.2.) A single image is provided for the SAP timeline (Figure 5C), which appears to be the same cell shown in the supplementary video.

IMPACT AND UTILITY<br /> This is well-done work with high potential for widespread adoption within the epithelial biology community, particularly if the methods and code are shared in better detail.

Reviewer #1 (Public Review):

This study used GWAS and RNAseq data of TCGA to show a link between telomere length and lung cancer. Authors identified novel susceptibility loci that are associated with lung adenocarcinoma risk. They showed that longer telomeres were associated with being a female nonsmoker and early-stage cancer with a signature of cell proliferation, genome stability, and telomerase activity.

Major comments:

1. It is not clear how are the signatures captured by PC2 specific for lung adenocarcinoma compared to other lung subtypes. In other words, why is the association between long telomeres specific to lung adenocarcinoma?

2. The manuscript is lacking specific comparisons of gene expression changes across lung cancer subtypes for identified genes such as telomerase etc since all the data is presented as associations embedded within PCs.

3. It is not clear how novel are the findings given that most of these observations have been made previously i.e. the genetic component of the association between telomere length and cancer.

Reviewer #1 (Public Review):

The authors set out to answer the standing mystery of an origin of a unique and complex system that is hagfish slime. They formulated a cogent scenario for the co-option of epidermal thread cells and mucous cells into slime and slime glands. Both histology and EM images back this up. It is a delight to see detailed and careful morphological analysis of both the cells and the secretion.

The weakness of the manuscript lies in: a) the absence of an alternative hypothesis (therefore the lacking sense of hypothesis testing); and b) oversimplification and insufficient description of results in transcriptomic and phylogenetic comparison. These are both key elements of the narrative. Because all the data "support" the only scenario considered in this paper, it could risk giving the impression of a just-so story. My reading of the results of their transcriptomic and phylogenetic analyses is more nuanced than explained in the paper. For example, the authors didn't explain in sufficient detail how the data summary in Fig. 5 "demonstrate" that the epidermal thread cells are "ancestral", and that the diversity of alpha and gamma thread biopolymer genes is a prerequisite to slime (without a functional analysis), or that the gene duplication events facilitated the origin of hagfish slime.

This work stands unique. I am not aware of any other study that attempted to explain the origin of this truly bewildering adaptation in hagfish with such a multifaceted approach.

Reviewer #1 (Public Review):

The manuscript aims to provide a comprehensive insight into the development of the tuberal hypothalamus of the chick by carefully analyzing the expression patterns of a plethora of proteins involved and perturbation of BMP signaling.

Strengths:<br /> This manuscript presents the results of an in-depth analysis aimed to unravel the expression of a variety of transcription factors, and the role of signaling molecules, in particular BMP, SHH and Notch, and, and the role of BMP for the development of the tubular hypothalamus. For this, the authors applied a variety of methods, including in-situ RNA hybridizations to chick embryos, fate mapping, explant cultures, and loss and gain-functions studies in embryos, complemented by carefully mining previously performed scRNA-Seq data. From the data they derive a model, which explains the dynamic changes of expression of signaling molecules and transcription factors from anterior to posterior during chick development. In addition, they show that fate specification and growth occur concomitantly.<br /> Overall, the data provide a plethora of information on expression patterns and consequences of BMP signaling perturbation, which will be valuable for scientists interested in the events taking place during the development of the chick tubular hypothalamus.

Weaknesses:<br /> The plethora of data presented makes it very difficult for a reader, who is not familiar with this system, to follow the major conclusions from each of the panels. This difficulty is enhanced by the lack of a concise, simple and focused summary at the end of most chapters, which, from my point of view, still contains too many details. Similarly, the discussion too often refers to details presented in the figures of the Results section, rather than giving a broader and focused summary and pointing out to novel conclusions.

I also suggest that the authors check the Materials and Methods section, which does not always contain the information required. For example, in the chapter on "Chicken HCR": I guess they used the HCR IHC kit from Molecular Instruments? What kind of "modification" of the Molecular Instruments protocol did they introduce?

Reviewer #1 (Public Review):

In this work, Zhang et al. test neutralizing antibody immunity elicited by a primary vaccination series and homologous boosting with the Sinovac-inactivated COVID-19 vaccine CoronaVac.

While the interpretation of the data is complicated by how some of the experiments were done, it seems that boosting with CoronaVac has only a marginal effect on Omicron (BA.1 subvariant) neutralization.

After primary vaccination comprising two doses, SARS-CoV-2 neutralization, as assessed by a live virus neutralization assay and pseudovirus neutralization assay, are low in absolute terms at peak response 1-month post-second dose (~GMT ~20), then wane. Boosting with a third dose of CoronaVac results in neutralization levels about an order of magnitude higher relative to 1-month post-primary vaccination. However, neutralizing capacity against Omicron (subvariant BA.1) is very limited even at the peak response from the boost, and the great majority of participant samples neutralize less than 50% of Omicron infection with relatively concentrated plasma (1:50).

Form an immunogenicity perspective, puts the utility of homologous boosting with CoronaVac into question given the current Omicron circulating subvariants.

While the strength of this study lies in the implications of the results, a weakness is how the pseudovirus results were done, and these are key to interpreting the data. For these, the authors did not fit a dose-response curve to a dilution series but rather used one plasma concentration (1:50 dilution) and measured percent inhibition. Not doing the measurement with multiple dilutions make the results less accurate and conclusions weaker.

Reviewer #1 (Public Review):

In this paper Lei et al analyzed the interaction between HIV-1 Gag and the viral RNA packaging signal Psi in living cells using the CLIP-seq method. The authors convincingly showed that NC alone is not sufficient to bind specifically to Psi sequence, while CANC does. They further showed that CANC mutants that are deficient in CA multimerization failed to bind specifically to the Psi sequence. The results indicate that correct assembly of Gag is required for specific binding of the protein to the Psi sequence.

Most of the data are convincing and support the conclusions. My only concern is that the authors analyzed the binding of some CA mutant proteins to the Psi sequence, but it is not so clear whether the specific binding of these mutants would lead to effective packaging of the RNA into virions. Measuring RNA/Gag ratios of some of the mutants in Figure 4 might help to address this concern.

Reviewer #1 (Public Review):

There is growing precedent for the utility of GWAS-type analyses in elucidating otherwise cryptic genotypic associations with specific Mtb phenotypes, most commonly drug resistance. This study represents the latest instalment of this type of approach, utilizing a large set of WGS data from clinical Mtb isolates and refining the search for DR-associated alleles by restricting the set to those predicted (or known) to be phenotypically DR. This revealed a number of potential candidate mutations, including some in nucleotide excision repair (uvrA, uvrB), in base excision repair (mutY), and homologous recombination (recF). In validating these leads functional assays, the authors present evidence supporting the impact of the identified mutations on antibiotic susceptibility in vitro and in macrophage and animal infection models. These results extend the number of candidate mutations associated with Mtb drug resistance, however the following must be considered:

(i) The GWAS analysis is the basis of this study, yet the description of the approach used and presentation of results obtained is occasionally obscure; for example, the authors report the use of known drug resistance phenotypes (where available) or inferences of drug-resistance from genotypic data to enhance the potential to identify other mutations that might be implicated in enabling the DR mutations, yet their list of known DR mutations seem to be predominantly rare or unusual mutations, not those commonly associated with clinical DR-TB. In addition, the distribution of the identified resistance-associated mutations across the different lineages need to be explained more clearly.

(ii) By combining target gene deletions with different complementation alleles, the authors provide compelling microbiological evidence supporting the inferred role of the mutY and uvrB mutations in enhanced survival under antibiotic treatment. The experimental work, however, is limited to assessments of competitive survival in various models, with/without antibiotic selection, or to mutant frequency analyses; there is no direct evidence provided in support of the proposed mechanism.

(iii) The low drug concentrations used (especially of rifampicin against M. smegmatis) suggest the identified mutations confer low-level resistance to multiple antimycobacterial agents - in turn implying tolerance rather than resistance. If correct, it would be interesting to know how broadly tolerant strains containing these mutations are; that is, whether susceptibility is decreased to a broad range of antibiotics with different mechanisms of action (including both cidal and static agents), and whether the extent of the decrease be determined quantitatively (for example, as change in MIC value).

Reviewer #1 (Public Review):

In this manuscript, Wang et al focused on defining the importance of the ER proteostasis factor HSP47 in regulating the folding, assembly, trafficking, and activity of GABAA receptors. Previous mass spectrometry-based interactomics identified HSP47 as the most enriched GABAA interacting chaperone in HEK293T cells. Here, the authors expand this study, demonstrating that HSP47 interacts with GABAA subunits in mouse brain homogenates and in vitro, demonstrating that HSP47 binds the alpha1 GABAA subunit with high affinity. They went on to show that depletion of HSP47 reduces the surface expression and activity of GABAA receptors in primary hippocampal neurons. Alternatively, overexpression of HSP47 increased the trafficking of GABAA receptors in HEK293T cells. Interestingly, chemical or genetic disruption of critical disulfide bonds within the alpha1 subunit of GABAA decreased interactions with HSP47, while increasing interactions with the ATP-dependent ER chaperone BiP, suggesting that HSP47 binds folded GABAA subunits in the ER lumen and promoting receptor assembly. Consistent with this, the authors employed a FRET-based system and biochemical assays to demonstrate that HSP47 enhances the assembly of GABAA receptors. Further, they demonstrate that overexpression of HSP47 could enhance the trafficking and surface activity of the epilepsy-associated alpha1(A322D) GABAA mutant. Finally, the authors show that HSP47 promotes the assembly and activity of other Cys-loop receptors including nAchR.

Overall, this work expands our understanding of how membrane receptors including GABAA and nAchRs are folded and assembled. In particular, the demonstration that HSP47 works after canonical ATP-dependent chaperones such as BiP in promoting the assembly of GABAA receptors is intriguing, as it is beginning to demonstrate the sequence of events important for ER quality control of these membrane proteins. The use of multiple biochemical and genetic approaches to manipulate GABAA receptors and following the assembly, trafficking, and activity of these receptors is also a strength. However, one outstanding question is how does manipulation of HSP47 (with either overexpression or depletion) influence overall ER proteostasis and function. Can these effects be specifically attributed to HSP47 or does this reflect more global impairment of ER function induced by altered signaling through pathways such as the UPR? This is important because, while the authors do demonstrate direct interactions with HSP47, the direct importance of these interactions on the assembly and activity of Cys loop receptors remains somewhat unclear. Additional efforts addressing the specific impact of HSP47 manipulation on overall ER proteostasis should address this comment and allow for a more complete understanding of the role of HSP47 in regulating the assembly and trafficking of these proteins. Regardless, this is a solid manuscript that reviews new insights into membrane protein quality control and the importance of HSP47 in regulating ER proteostasis and function.

Reviewer #1 (Public Review):

This is an outstanding manuscript that takes a comprehensive approach to studying allosteric modulation at the M4R. I think it is an important addition to the literature and provides important insights into allosteric modulation. Overall the pharmacological approaches are very rigorous and are the types of analyses that need to be performed to move this field forward.

Reviewer #1 (Public Review):

The authors set out to extend modeling of bispecific engager pharmacology through explicit modelling of the search of T cells for tumour cells, the formation of an immunological synapse and the dissociation of the immunological synapse to enable serial killing. These features have not been included in prior models and their incorporation may improve the predictive value of the model.

The model provides a number of predictions that are of potential interest- that loss of CD19, the target antigen, to 1/20th of its initial expression will lead to escape and that the bone marrow is a site where the tumour cells may have the best opportunity to develop loss variants due to the limited pressure from T cells.

A limitation of the model is that adhesion is only treated as a 2D implementation of the blinatumomab mediated bridge between T cell and B cells- there is no distinct parameter related to the distinct adhesion systems that are critical for immunological synapse formation. For example, CD58 loss from tumours is correlated with escape, but it is not related to the target, CD19. While they begin to consider the immunological synapse, they don't incorporate adhesion as distinct from the engager, which is almost certainly important.

While the random search is a good first approximation, T cell behaviour is actually guided by stroma and extracellular matrix, which are non-isotropic. In a lymphoid tissue the stroma is optimised for a search that can be approximated as brownian, or more accurately, a correlated random walk, but in other tissues, particularly tumours, the Brownian search is not a good approximation and other models have been applied. It would be interesting to look at observations from bone marrow or other sites to determine the best approximating for the search related to BiTE targets.

Reviewer #1 (Public Review):

Sex determination and dosage compensation are two fundamental mechanisms in organisms with distinct sexes. These mechanisms vary greatly across the various model organisms in which they have been studied. Comparisons across more closely related members of the same genus have already proven productive in the past, to understand how these essential mechanisms evolve. In this study, the authors compare some aspects of the dosage compensation and sex determination mechanisms across two Caenorhabditis species that diverged ~15-30 MYA.

Previously, the authors have studied dosage compensation and sex determination extensively in C. elegans. Here, they first identify the homologs of some key factors in C. briggsae, a species that independently evolved hermaphroditism. The authors show that some of the key players in these processes play the same roles in C. briggsae as they do in C. elegans. Namely, they show that the nematode-specific SDC-2 protein plays a role in both dosage compensation and sex determination also in C. briggsae, they find the homologs of some of the SMC protein complex that performs dosage compensation also in C. elegans and they study the binding specificity on the X chromosome.

Overall, the work is thorough and compelling and is very clearly presented. The authors generate a number of genetic tools in C. briggsae and the careful genetic analyses together with a number of binding assays in vivo and in vitro, support the authors' main conclusions: that the main players and genetic regulatory hierarchy are conserved between these two nematodes, but the binding sites for the DCC on the X chromosome have diverged and the mode of binding has changed as well. Whereas in C. elegans the DCC binds sites in the X chromosome that contain multiple sequence motifs in a synergistic manner, in briggsae they seem to do so additively. This latter point is supported by the data, but it could be explored a bit more deeply using the available ChIP-seq data that the authors have generated. In addition, it would be interesting to discuss the possible implications of this difference.

One minor weakness of this work is that it could be better put in the context of other related comparisons of these mechanisms. For example, the comparison of sex determination pathway by Haag et al. in Genetics 2008, and the comparison of dosage compensation across Drosophila species (Ellison and Bachtrog, Plos Genetics, 2019), and possibly others. The other point that the authors could provide deeper insight into, is the rate of divergence of proteins like SDC-2 (which is thought to be the protein that contacts DNA), versus some other proteins in the DCC and in general other proteins not involved in sex determination or dosage compensation (this doesn't need to be limited to comparing elegans and briggsae as there are numerous Caenorhabditis genomes available). This would provide a more complete view of the evolution of these processes.

Reviewer #1 (Public Review):

Several new observations on the fascinating marine midge system are provided. The results are robust and have broad interest. First, multiple polymorphic chromosomal inversions are shown to be segregating in the study populations but these inversions are not strongly associated with ecotype differentiation. At least 4 QTL are detected that together explain a large part of the timing difference between co-located populations that emerge at full and new moon. Good candidate genes under these QTL are identified, with unusually high differentiation between populations. These are involved in the circadian clock (period) and in nervous system rewiring. The involvement of period suggests a link between the circadian and circalunar clocks.

The major context provided by the authors is the idea of 'magic traits' that influence multiple components of reproductive isolation and so are potentially important in population differentiation because associations between traits do not need to be built and maintained in the face of recombination. This idea is not described very clearly in the current MS. In particular, the authors suggest that magic traits are expected to have simple genetic basis, an expectation that they say should be re-evaluated on the basis of their results. However, there is no strong justification for this expectation. It tends to confuse the magic trait idea with the possibility of pleiotropy and with the advantage of recombination suppression where local adaptation is opposed by gene flow. The latter is relevant here because of the involvement of inversions, which might be expected to capture multiple alleles contributing to local adaptation but here apparently do not do so.

Reviewer #1 (Public Review):

The manuscript is of importance for vaccine design and understanding tissue microenvironmental influence on the functionality of monocytes and DCs to respiratory viruses such as Influenza A virus or SARS-CoV-2 virus. The methods used were mainly flow cytometry, ELISA, Luminex as well as TNFα secretion Assay Detection. The authors wanted to evaluate if and how the tissue microenvironment might impact DC and monocyte subset presence and functionality during Influenza A virus infection.

Strength:<br /> The study is summarizing a large cohort of human samples of blood, nasal swabs and nasopharyngeal aspirates. This is very uncommon as most of the time studies focus on the blood and serum of patients. Within the study, 3 monocyte and 3 DC subsets have been followed in healthy and Influenza A virus-infected persons. The study also includes functional data on the responsiveness of Influenza A virus-infected DC and monocyte populations. The authors achieved their aims in that they were able to show that the tissue microenvironment is important to understand subset specific migration and activation behavior in Influenza A virus infection and in addition that it matters with which kind of agent a person is infected. Thus, this study also impacts a better understanding of vaccine design for respiratory viruses.

Weakness:<br /> In the described study, the authors used a different nomenclature to introduce the DC subsets. This is confusing and the authors should stick to the nomenclature introduced by Guilliams et al., 2014 (doi.org/10.1038/nri3712) and commented in Ginhoux et al., 2022 (DOI: 10.1038/s41577-022-00675-7 ) or at least should introduce the alternative names (cDC1, cDC2, expression markers XCR1, CD172a/Sirpa). Further, Segura et al., 2013 (doi: 10.1084/jem.20121103) showed that all three DC subpopulations were able to perform cross-presentation when directly isolated. Overall, a more up-to-date introduction would be useful.<br /> As the data of this was already obtained in 2016-2018 it is clear that the FACS panel was not developed to study DC3. If possible, the authors might be able to speculate about the role of this subset in their data set. Moreover, there were other studies on SARS-CoV-2 infection and DC subset analyses in blood (line 87, and line 489) e.g. Winheim et al., (DOI: 10.1371/journal.ppat.1009742 ), which the authors should introduce and discuss in regard to their own data. Taken together, although the data are very important and very interesting, my overall impression of the manuscript is that in the era of RNA seq and scRNA seq analyses the study lacks a bit of comprehensiveness.

Reviewer #1 (Public Review):

In this manuscript, the authors propose a model to explain how oxysterols provide protection against bacteria and viruses by modulating a cell's "accessible cholesterol". The paper concentrates on one particular oxysterol, 25-hydroxycholesterol (25HC) which provides an example of a wider group of side-chain oxysterols. Previous studies have shown that 25HC can protect cells against microbial infection and have suggested that this is achieved by depleting "un-sequestered" or "accessible" cholesterol from the plasma membrane. Here, Heisler et al provide convincing evidence that this is achieved initially by activation of the enzyme acyl coenzyme A: cholesterol acyltransferase (ACAT also known as Sterol O-acyltransferase, SOAT, not to be confused with acetyl-coenzyme A acetyltransferase) to rapidly reduce plasma membrane accessible cholesterol by conversion of cholesterol to its cholesteryl ester. This is followed by 25HC-induced inhibition of the processing of SREBPs, the master transcription factors for genes of the cholesterol biosynthesis pathway and also the LDL receptor, to maintain cholesterol depletion.

The data presented throughout are solid, however, some of the structures drawn of the oxysterols in Figure 1 are not chemically correct. 24(S)HC is drawn as 24(R)HC and visa versa, also the oxysterol sulfate should have a bond between C-3 and the O of OSO3H. It would also help the reader if the vehicle for oxysterol additions was clarified.<br /> The data presented in Figures 2 and 3 show that inhibition of SREBP processing by 25HC is important for the long-term maintenance of depletion of plasma membrane accessible cholesterol, but I wonder if activation of LXR may also be important here. I appreciate that the data in Figure 2 points against LXR being involved in the rapid depletion of accessible cholesterol in HEK293 cells, but perhaps it is important for the long-term depletion of accessible cholesterol. Could there be some cell type specificity here?<br /> Something that always concerns me when the antimicrobial activity of 25HC is discussed is the fact that 25HC is usually a minor side-chain oxysterol compared to 24(S)HC and 27HC (and 22(R)HC in steroidogenic tissue), except for a short time after infection. Perhaps any long-term antimicrobial activity, and diminishment of accessible cholesterol, results from these other side-chain oxysterols. This may be worthy of some additional discussion.

In summary, the authors present a convincing model for the depletion of accessible cholesterol by oxysterols and their involvement in antimicrobial activities.

Reviewer #1 (Public Review):

This work provides a new multimodal blastocyst evaluation method utilising both blastocyst images and patient couple's clinical features (e.g., maternal age, hormone profiles, endometrium thickness, and semen quality) to predict live birth outcomes.<br /> The manuscript was reviewed using the checklist from the "Transparent reporting of a multivariable prediction model for individual prognosis or diagnosis (TRIPOD): The TRIPOD statement" (https://www.equator-network.org/reporting-guidelines/tripod-statement/ ). Generally, the authors have achieved their aims, and the results support their conclusions.

The major study strengths are as follows:

The study dataset consists of a huge amount (17,580) of blastocysts with known live birth outcomes, as well as blastocyst images, and data included the clinical features of couples.<br /> The authors developed a new artificial intelligence model consisting of a convolutional neural network to process blastocyst images and a multilayer perceptron to process patient couple's clinical features. This model demonstrated an AUC of 0.77 for live birth prediction, which is significantly higher than that achieved by the previously developed models. The conclusions of this paper are mainly well supported by the data.

Nevertheless, there are some weaknesses:

Regarding testosterone, the method of testosterone assessment is essential. The statistical significance of testosterone as a predictor could change when calculated free T or bioavailable testosterone is used.

According to the data presented in Supplementary Table 1, there are more than 15 statistically significant predictors of live birth. However, the value of predictive significance is presented only for 15 (Fig. 3).

Reviewer #1 (Public Review):

Chen and colleagues report the results of 3 experiments assessing how one or both eyes open under a patch influence resting EEG activity, contrast sensitivity, and binocular balance in normally sighted subjects. They found that keeping an eye open (as opposed to closed) under a patch enhances contrast sensitivity and evoked responses through the unpatched eye as well as interocular shifts in contrast sensitivity and binocular balance in favor of the patched eye once the patch is removed. These results suggest that the state of eye opening temporarily, but significantly, influences shifts in ocular dominance with relevance for treatment of binocular visual disorders such as amblyopia that are treated with periodic monocular occlusion.

Strengths:<br /> 1. Elegant simplicity in study design.<br /> 2. Well-designed and executed psychophysical assessments of contrast sensitivity and binocular balance. More than one assay for binocular balance is used.<br /> 3. Cross-modality relationships are analyzed and support the underlying hypothesis.

Weaknesses:<br /> 1. The investigators demonstrate an effect of eye open status under the patch on occipital oscillatory activity, but subsequent results cannot be directly attributed to these changes since occipital oscillation are not directly manipulated. Therefore, the extent to which the oscillatory activity in visual cortex mediates differential open versus closed eye effects on contrast sensitivity and binocular balance cannot be concluded based on these data alone.<br /> 2. Long-term effects produced or enabled through open or closed eye patching are not reported, limiting translational potential for visual disorders such as amblyopia.

Reviewer #1 (Public Review):

The contribution of disease-specific factors to the capacity of iPSCs for chondrogenic differentiation is unknown. A better understanding of the underlying mechanism will facilitate approaches to design more effective therapies and interventions to benefit cartilage regeneration.<br /> The authors adequately characterized the stemness of OA-derived iPSC clones compared with previously generated healthy iPSC (AC-iPSCs) based on accepted molecular markers, progenitor properties, and chondrogenic potential pointing to undifferentiated pluripotent phenotype. Clones from AC and OA-iPSCs were then successfully differentiated into mesenchymal progenitor intermediates and displayed similar phenotype characteristics. Immunophenotypic analyses were also performed and confirmed the expression of typical MSC markers in both population progenitors and the lack of hematopoietic and endothelial markers. In terms of multipotency, both iMSCs differentiated into OBs, adipocytes, and chondrocytes, although AC-iPSCs displayed enhanced chondrogenic potential compared with OA-iPSCs. This was confirmed in the chondrogenic differentiation assay using the pellet culture method and 3D-micromass culture wherein iPSCs derived from healthy chondrocytes displayed significantly higher chondrogenic potential compared with OA-iPSCs. The authors logically concluded that the reduced ECM generation by OA-iMSCs is likely due to retention (or memory) of OA phenotype of the original cell source.

RNA-seq analysis of the transcriptome of both AC and OA-iPSCs revealed significant differences between the two cell clones. Similarly, PC analysis suggested that the two populations are genomically distinct. Enrichment of GO terms and KEGG pathway analysis revealed metabolic pathways, epigenetic regulation, and chromatin organization are mostly enriched in AC-iPSCs. These findings suggested that metabolic and epigenetic pathways in AC cells support enhanced chondrogenic differentiation. It should be also noted that the profile of metabolic and epigenetic gene networks exhibited significant differences not only in the terminally differentiated cells but also in the undifferentiated state, further highlighting their distinct chondrogenic potential.

Altogether, using advanced pan-transcriptomic analyses, the authors convincingly demonstrate that distinct expression signature of epigenetic and metabolic marks was detected in healthy iPSCs different from OA-derived iPSCs.

The Implication of epigenetic pre-disposition in OA-iPSC is critically important for designing appropriate strategies to control chondrogenesis and potential cartilage regenerative approaches.

Reviewer #1 (Public Review):

Sorkac et al. devised a genetically encoded retrograde synaptic tracing method they call retro-Tango based on their previously developed anterograde synaptic tracing method trans-Tango. The development of genetically encoded trans-synaptic tracers has long been a difficult stumbling block in the field, and the development of trans-Tango a few years back was a breakthrough that was immediately, widely, and successfully applied. The recent development of the retrograde tracer method BActrace was also exciting for the field, but requires lexA driver lines and required by its design the test of candidate presynaptic neurons instead of an unbiased test for connectivity.

Retro-Tango now provides an unbiased retrograde tracer. They cleverly used the same reporter system as for trans-Tango by reversing the signaling modules to be placed in pre-synaptic neurons instead of post-synaptic neurons. Therefore, synaptic tracing leads to the labeling of pre-synaptic neurons under the regulation of the QUAS system. Using visual, olfactory as well sexually dimorphic circuits authors went about providing examples of specificity, efficiency, and usefulness of the retro-Tango method. The authors successfully demonstrated that many of the known pre-synaptic neurons can be successfully and specifically labelled using the retro-Tango method.

Most importantly, because it is based on the most used, very well tested and widely adopted trans-Tango method, retro-Tango promises to not just be a clever development, but a really widely and well-used technique as well. This is an outstanding contribution.

Reviewer #1 (Public Review):

The authors in this manuscript investigate the effect of co-substrate cycling on the metabolic flow. The main finding is that this cycling can limit the flux through a pathway. The authors examine implications of this effect in different simple configurations to highlight the potential impact on metabolic pathways. Overall, the manuscript follows logical steps and is accessible. Once the main point-reduction in flux of a pathway with limited pool of a cycled co-substrate-is established, some of the following steps become expected (e.g. the fraction of the flux in a branched pathway). Nevertheless, it is understandable that the authors have picked a few simple examples of the metabolic network motifs to highlight the implications. The results presented in the manuscript overall support the conclusions. One weakness is that some of the details of the assumptions (e.g. the choices of rates) are not explicitly spelt out in the manuscript. This work is impactful because it brings into light how cycling of some of the intermediates in a pathway can influence metabolic fluxes and dynamics. This is a factor in addition to (and separate from) reaction rates which are often considered as the main driver of metabolic fluxes.

Reviewer #1 (Public Review):

This study analyses associations between different blood groups and 1,312 hospital diagnosis codes, among >480,000 Danish patients who had their blood type determined in hospitals. While biological relationships between blood types and disease are of substantial interest, unfortunately, the analyses do not adjust for ethnicity (which is correlated with both blood types and many diseases). Thus it is unclear to which extent disease associations represent relationships with the blood types, as opposed to possible differences in disease incidence or severity between people with different ethnic backgrounds (which could also be due to socioeconomic differences as well as any other factors correlated with ethnicity).

Reviewer #1 (Public Review):

It has recently been shown that the HIV-1 protease can cleave and activate the inflammasome-forming sensor CARD8 upon treatment of infected cells with non-nucleoside reverse-transcriptase inhibitors (Wang et al., Science 2021). Here, Kulsuptrakul and colleagues show that the high susceptibility to proteolytic activation by the HIV-1 protease is a specific feature of human CARD8. They show that changes in human-specific F-F motif render the CARD8 protein of non-human primates largely resistant to cleavage. Interestingly, the protease of SIVcpz the direct precursor of pandemic HIV-1 strains are also capable of cleaving human but not chimpanzee CARD8. Thus, the authors propose that a human-specific CARD8 motif may contribute to the increased levels of inflammation and disease progression in HIV-infected humans compared to non-human primates that are naturally infected with SIV.

Strengths of the study are that the authors convincingly show that a single human-specific amino acid change in CARD8 determines its susceptibility to cleavage by the HIV-1 protease and that the results shown are well controlled and presented. It is also interesting that SIVcpz can cleave human CARD8 and activate an inflammatory response. The major weakness is that it remains unclear whether HIV-1 of SIVcpz may induce CARD8-dependent inflammatory responses in primary CD4+ T cells or macrophages. The most relevant setting in the study was the infection of THP-1 cells with the T cell line-adapted X4-tropic HIV-1 LAI molecular clone. However, the effects on cell death were modest (Figure 3A) and on IL-1ß secretion was not dose-dependent (Figure 3B). Altogether, stronger effects were observed with VSV-G-pseudotyped HIV-1 and only those were used in subsequent experiments involving human CARD8 cleavage mutants (Figure 4). Additional evidence that primary HIV-1 molecular clones and/or SIVcpz may indeed induce CARD8-dependent inflammatory responses in primary viral target cells would greatly increase the significance of the study. In the absence of such data, conclusions about the potential role of CARD8 sensing of the viral protease for the pathogenesis of AIDS should be cautioned throughout.

Reviewer #1 (Public Review):

In this study, Moulana et al. measured the binding affinity of four antibodies, Ly-CoV016, REGN10987, Ly-CoV555, and S309, against ~30,000 RBD variants that represent the possible intermediates between the ancestral SARS-CoV-2 RBD and Omicron BA.1 RBD. Mutational pathway to antibody escape and the importance of epistasis in antibody binding were examined. By comparing the data here with a previous study by the same authors (ref 23), the authors also conclude that escape mutations with deleterious effects on ACE2 affinity can be compensated by Q498R and N501Y. Overall, the results are clearly presented and provide important insights into antibody escape.

Reviewer #1 (Public Review):

The therapeutic effects of NSC transplantation is limited by the low survival and differentiation rates of NSCs due to the harsh environment in the brain after ischemic stroke. To solve this technical challenge, the authors employed NSCs derived from human induced pluripotent stem cells (iPSCs) together with NSC-derived exosomes extracted from NSCs to treat cerebral ischemia induced by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice. In the current study, the authors attempt to demonstrate that NSC-derived exosomes could act as a supportive adjuvant for NSC transplantation after stroke. They showed that NSC-derived exosomes significantly reduced the inflammatory response, alleviated oxidative stress after NSC transplantation, and facilitated NSCs differentiation in vivo. The combination of NSCs with exosomes ameliorated the injury of brain tissue including cerebral infarct, neuronal death and glial scarring, and promoted the motor function recovery. To explore the underlying mechanisms, they analyzed the miRNA profiles of NSC-derived exosomes and the potential downstream genes. Overall, the study provided solid rationale supporting the application of exosomes during stem cell-based therapy. The data quality is good and convincingly supports the major claims. The impact is high for the NSC-transplantation for treating various neurological diseases.

Reviewer #1 (Public Review):

RNA-based self-replication systems might have been concentrated and compartmentalized with peptides by forming droplet-like complexes before the emergence of cellular organisms enveloped by lipid membranes. This report clearly shows that the physical properties of such droplets (phenotypes) can be affected by the activities of a ligase ribozyme in the droplets. This suggests that sequences of such ribozymes (genotypes) might have been selected not only for their direct activities (e.g., elongation of RNA) but also for their indirect effects on the droplet phenotypes (e.g., more viscous/solid droplets formed by the elongated RNAs) on the ancient earth. However, the exact requirements (e.g., average/maximum length of the ligated RNAs + double strand formation) for such phenotypic changes are not assessed in this report. It is not demonstrated whether the droplet property changes caused by the ribozyme activity can be advantageous for the survival of the RNA-based system (e.g., for the ribozyme activity itself). Follow-up studies would be desired to clarify these points and the true values of this report.

Reviewer #1 (Public Review):

After giving a very accessible introduction to cellular processes during brain development, the authors present the computational model used in this study. It combines the kinematics of cell proliferation with the mechanic of brain tissue growth and is essentially equal to their model presented in Zarzor et al (2021), but extended for the outer subventricular zone (OSVZ), see for example Figs. 2 in the present manuscript and in Zarzor et al (2021). This zone, which is specific to humans, provides a second zone of cell proliferation. The division rate in the OSVZ is smaller and at most equal to that in the ventricular zone.

The authors present two main findings: The distance between sulci in the cortex is decreased whereas the cell density in the ventricular zone is increased in presence of the OSVZ. Furthermore, the "folding evolution", which is the ratio between the outer perimeter at time t and the initial perimeter increases in presence of the OSVZ. The strongest effect is seen, when division rates in both proliferating zones are equal. The authors compare the cases of varying and constant cortical stiffness, which they had also done in Zarzor et al (2021). Finally, they consider the feedback of cortical folding on OSVZ thickness.

The computational model provides a sound description of how cell proliferation and migration combined with tissue mechanics yield cortical folding patterns. However, only a few parameter values are varied in a limited range. Also, it remains unclear to me, how important the specific functional dependencies of, for example, the cell division rate on the radial coordinate are. This point seems of particular importance because the effect of the presence of the OSVZ on the folding patterns seems rather minute, see Fig. 5. The authors do not propose experiments that could be used to test their description and results. Finally, the analysis is restricted to 2 dimensions.

Reviewer #1 (Public Review):

In this work, Roche et al. study a 13-year long time series of microbiome samples from wild baboons from Kenya. The data used in this work challenge a previous finding from the same authors that temporal dynamics in microbiome changes are largely individualized. Using a multinomial logistic-normal modeling approach, the authors detect that co-variance in temporal dynamics in microbial pair-wise associations among individuals occurs more frequently between relatives. Furthermore, the authors identify that microbial phylogenetic proximity is associated with consistent co-abundance changes over time and that their metric of universal microbial relationships is robust across hosts and is detected even in human longitudinal data. The authors conduct a thorough statistical revision of publicly available results, highlighting this time (e.g. compared to Björk et al, doi: 10.1038/s41559-022-01773-4) the consistently shared microbial properties between individuals, rather that the individual microbial signatures highlighted in their previous work.

Strengths:<br /> This work is foundational in its compelling effort to generate a rigorous method to evaluate co-abundance dynamics in longitudinal microbiome data. The approach taken will likely inspire developments that will sharpen the capacity to extract co-varying microbial features, taking into account seasonality, diet, age, relatedness, and more. To the best of my understanding, their hierarchical model integrated into the Gaussian process to analyze microbial dynamics is reasonably robust and they clearly explain the implementation. Furthermore, this work introduces and defines the concept of a universality score for microbial taxon pairs.<br /> Overall, the work presented is clear and convincing and provides tools for the community to benefit from both methods and results. Furthermore, conceptually, this work stresses the value of consistent and shared microbial dynamics in groups, which enriches our understanding of host-associated microbial ecology, otherwise understood to be largely dependent on external fluctuations.

Weakness:<br /> It is not entirely clear the extent to which the presented results revise, refute, or support the previously published analysis performed by the authors on the same dataset (doi: 10.1038/s41559-022-01773-4), which was more focused on individuality.

Reviewer #1 (Public Review):

The synaptonemal complex (SC) is a meiosis-specific tripartite chromosome structure for chromosome synapsis and regulates crossover formation essential for proper chromosome segregation during meiosis. In this interesting paper, the authors studied the dynamic behavior of two components of SC central regions, SYP-2 and SYP-3, in both oocytes and spermatocytes in C. elegans and also the effect of the dosage of the proteins on meiotic recombination and found sex-dimorphic and/or SYP-2/3 dosage sensitive dynamics of the SC components and proteins involved in crossover formation on the meiotic chromosomes, suggesting the intimate relationship between SC central regions and meiotic recombination.

Reviewer #1 (Public Review):

The authors set out to achieve two overarching objectives: 1) to demonstrate that BAFF is a bona fide senescence-associated factor and 2) to expand the field's understanding of senescence outcomes across cell types and models. By inducing senescence in a multitude of ways and across cell culture and animal models, the authors demonstrate that BAFF is robustly upregulated in response to senescence induction. Beyond mere association, by knocking down, overexpressing, or ectopically adding BAFF, the authors demonstrate that various senescence-associated phenotypes can be altered, suggesting that it is an effector of the senescent state. Moreover, by comparing transcriptomic and proteomic profiles in two very different types of cells-diploid WI-38 human fibroblasts and cancerous THP-1 monocytes-the authors identify two parallel trajectories, one involving p53 and one involving NF-kB.

Although trajectory differences may stem from cell type differences, it is possible that the cancerous vs non-cancerous status of the cell lines used may be a more important variable in this case. One question the reader may be left with is: would the two trajectories be different if non-cancerous monocytes with intact p53 were profiled?

Regardless, this study establishes a precedent for characterizing senescence responses in additional cell types of either healthy or diseased origin. Though a number of technical and statistical issues exist in the current version of the manuscript (i.e. use of only a single reference gene for RT-qPCR and inconsistent fold change thresholding in RNA-seq analyses), the results appear robust enough to remain statistically significant after modification. Moreover, many analyses are carried out at both the transcriptomic and proteomic levels with consistent results, highlighting the robustness of their observations.

Ultimately, the results strongly suggest that BAFF plays a senomorphic role in senescence, modulating downstream senescence-associated phenotypes, and may be an interesting candidate for senomorphic therapy.

Reviewer #1 (Public Review):

Cryo-EM structures of respiratory complex I have in recent years have a large impact on our understanding of its mechanism, regulation, assembly, and evolution. However, the coupling mechanism of complex I is still not clear, and controversies exist about whether certain conformations are part of the catalytic cycle or arise from the deactivation of the enzyme. Padavannil and colleagues now add to the story with the first structures of insect complex I, from the model organism Drosophila melanogaster. One of the rationales for choosing this organism is that it lacks the active-to-deactive (A-to-D) transition that prevents the enzyme from going in reverse, which should make the interpretation of any different conformations more straightforward.

The authors showed that the A-D transition seen in mammals and fungi was indeed not present in Drosophila complex I and they determined the cryo-EM structure. In contrast to especially mammalian complex I, which is often found in an "open" and a "closed" state, there was only a single conformation. Drosophila complex I has lost two accessory subunits compared to the mammalian complex, and several other subunits have lost or gained elements, with possible implications for the assembly, stability, or regulation of the complex. The interface of the two peripheral and membrane arms was poorly resolved. A focused classification on this region yielded distinct structures, differing in the angle of the two arms and in the presence or absence of an alpha helix at the N terminus of subunit NDUFS4 (the "lock helix"), a region that is not present in mammalian or yeast complex I. The authors observe a transition between two states named "closed" and "locked open" and speculate that the transition constitutes a deactivation mechanism in insect complex I.

The conclusions of the paper are for the most part solid and supported by the data. Only the interpretation of the significance of the "lock helix" is not convincing: without any evidence, it is assumed to be a regulatory element responsible for an off-pathway deactive state. The nomenclature "closed" and "locked open" is unfortunate, as most of the structural features that differ between the states are reversed compared to the mammalian closed and open states: the disorder of several loops in the quinone binding regions and the presence of absence of a π bulge in helix 4 of the ND6 subunit. Thus, the "locked open" state, which the authors assign as an off-pathway resting state, shares the features of the mammalian closed state, which in all catalysis models is considered an "active" state. An especially important feature in the closed state is the alpha-helical conformation of ND6-helix 4, which has been shown to support a water wire connection from the Q site to the membrane arm, suggesting a role in proton transfer. Conversely, all structures considered as possible D states in mammalian or yeast complex I are open and show disordered loops and a π bulge. These features as shared by the "closed" state of Drosophila, which is however assumed to be on-pathway.

Reviewer #1 (Public Review):

Most previous studies about burn injuries only considered systemic inflammation with analyses of blood specimens from patients. The current study is unique in the fact that it utilizes skin samples. The authors used single-cell analyses by flow cytometry and RNA-seq to characterize in detail the different T-cell populations. The differences are striking. Burned skins have higher degrees of CD69-negative T cells, which indicates that these are recruited from probably blood circulation. They are also substantially more responsive to stimulation by producing higher amounts of immunologic molecules, such as IFNG and TNFA. The results are compelling because they indicate that following burn injury, T cells infiltrate the lesions to potentially protect the damaged tissue from secondary infections.

However, there is an important aspect missing. What does induce T-cell infiltration into the burned skins? A potential explanation is that resident myeloid cells directly or indirectly promote chemokine-mediated recruitment of T cells.

Another important consideration is the impact on other leukocyte populations. While the study is well focused on T cells, the immune system consists of a complex network of cells and molecules that interact with each other. The study does not address myeloid cells and innate lymphoid cells, which could also play important roles and display altered functions in burned injuries.

Nevertheless, the study provides important information about the "activation" statuses of several T cell populations following burned injuries and could help guide the development of better treatments.

Reviewer #1 (Public Review):

The authors studied Eurasian perch in an experimental setup facilitated by a nuclear cooling plant to provide a natural laboratory. The heated area of the ecosystem raised in temperature by 8 degrees centigrade, while a reference area remained unheated. The authors provide a thorough and convincing description that the two areas are segregated such that individuals could not escape from one area to another prior to 2004, and such use data only until 2003 to test their hypotheses. The authors used both length-at-catch and age-increment data in a series of Bayesian mixed effects models to estimate the growth rate and length-at-age. They find that in the warmed area, both younger, smaller fish and older adults grew faster, contrary to the prediction of the temperature-size rule as well as many predictions and observations from other systems that fish reach smaller terminal body sizes in warmer environments due to increased metabolic demands. The authors furthermore combine the estimated body sizes with a mortality rate to determine the size-spectrum slope for both areas and determine the increased growth and increased mortality combine to essentially leave the size-spectrum slope observed in the ecosystem unchanged.

This is a thorough and interesting paper presented clearly and succinctly. These authors present a strong and thorough analysis of how temperature affects growth when all other ecosystem factors remain unchanged in a population. The dataset is a powerful one to support this type of analysis, and the statistical analysis methods the authors used appear to be robust and thorough. The diagnostics and visualizations are complete and inspire confidence in the convergence and accuracy of the modeling approach. The use of the size spectrum exponent to roll up individual-level changes across the population into a single metric was useful and interesting.

The estimates of the von Bertalanffy growth parameters in the results and discussion are less convincing than the growth increment and length-at-age estimates which seem much more robust. The presentation of estimates of the von Bertalanffy growth parameters in Figure S6 exhibit the high negative correlation between the k and L infinity parameters that are typical whenever multiple VBGF models are fit to subsets of data. It is difficult to determine which changes in parameters correspond to actual differences in early vs late life stage growth when, in any given year, if k is estimated low, L infinity will skew high simply due to the model structure. An example of this can be seen in 1995-1997 where L infinity is quite high but k is estimated quite low concurrently - in this case, it seems more reasonable to conclude the likelihood surface is quite flat between different parameter values than that fish suddenly reached a larger asymptotic size in these three years than all of the rest. The data in this case so strongly show larger growth in the heated area even without the VBGF results, and it would be more credible to base the discussion and results of this paper on the growth rate or observed length-at-age (e.g. Figure S4) estimates which are so clear.

Reviewer #1 (Public Review):

Caetano and colleagues describe the changes caused by periodontal inflammation in terms of tissue structure and provide additional evidence to understand the involvement of fibroblasts in altering the immune microenvironment.

While interesting and a concise study, the authors should improve their work on two major points:

1. To improve the resolution, the authors introduced a method that addresses improving the resolution by combining more information from the neighbour structure and the existing database. This raises the question of whether the lack of previous gingival tissue spatial transcriptome sequencing results weakens the reliability of this method. Does it miss the identification of some gingival tissue-specific cells? Is the failure to match two populations of fibroblasts between single-cell sequencing and spatial transcriptome sequencing of gingival tissue fibroblasts related to this?

2. Although the authors did the identification of the captured tissues, the results seem to require more analysis. Take Figure 5A as an example, there is a clear overlap between endothelial cells and basal cells. In addition, it is suggested that the authors indicate the specific location of the 10 clusters of cells in Figures 1D and 2C.

Reviewer #1 (Public Review):

In this manuscript, the authors employed an adult-trained variational autoencoder deep learning model on a relatively large sample (over 700) of human fetal-neonatal resting fMRI data to enhance the individual non-linear compression of functional activity patterns of baby brains. This approach showed better performance in the reconstruction of functional fluctuation maps, age prediction accuracy, and age prediction generalizability in fetal and neonatal fMRI data compared with conventional linear models such as spatial independent component analysis. This method also revealed distinct baby brain functional networks spanning primary and high-level systems.

This is an inspired attempt to represent non-linear changes in fetal-neonatal brain fMRI data. Considering the high noises and inconsistent functional spatial distributions in baby fMRI images, stable and sensitive feature extraction approaches are urgently needed in the field of early brain studies. This work is well designed and well written in general.

Reviewer #1 (Public Review):

By performing immunopeptidomics of macrophages infected with virulent M. tuberculosis, the authors were able to appropriately address whether Mtb proteins are able to enter the MHC-I antigen processing pathway. Their interrogation provides convincing evidence that substrates of Mtb's type VII secretion systems (T7SS) are a significant contributor to the Mtb-derived peptides presented on MHC-I. Compelling data are provided to demonstrate that ESX-1 activity is required for the MHC-1 presentation of these newly identified peptides.

Strength:

Employing a virulent strain of Mtb for infection of human monocyte-derived macrophages to identify Mtb proteins that access the MHC-I antigen processing pathways and the associated mechanisms.

Weakness:

The immunogenicity of at least some of the identified peptides should have been evaluated.

Reviewer #1 (Public Review):

The accessory protein Orf3a from severe acute respiratory syndrome coronavirus (SARS-CoV-1 or SARS-CoV-2) was initially suggested to be a viroporin and function as a cation channel. In this study, Miller et.al performed a comprehensive structural and functional investigation of SARS-CoV-2 Orf3a utilizing a multidisciplinary approach, including extensive electrophysiological analysis using different systems and determination of multiple single-particle EM structures of the protein under different conditions. Their findings demonstrated that Orf3a has no channel function and is unlikely to be a viroporin. In addition, they tried, but failed to record any channel activity of Orf3a claimed in other studies. They demonstrated that large single-channel currents measured from vesicle-reconstituted Orf3a are due to transient membrane leakiness caused by high protein/lipid ratio and/or channel contamination. Furthermore, they found that SARS-CoV-2 Orf3a, but not SARS-CoV-1 Orf3a, interacts with VPS39, a host HOPS protein involved in autophagosome/late endosome fusion with the lysosome. They proposed that the interaction between SARS-CoV-2 Orf3a and VPS39 may function to assist with SARS-CoV-2 exit and host intracellular immune evasion. This is a meticulously executed research work. I appreciate the tremendous effort the authors spent in the study to clarify some misconceptions related to the role and function of Orfsa from coronavirus.

Reviewer #1 (Public Review):

The idea that because the hippocampal code generates responses that match the most needed variable for each task (time or distance) makes it a predictive code is not fully proved with the analyses provided in the manuscript. For example, in the elapsed time task, there are also place cells and in the fixed-distance travel there are also cells that encode other features. This, rather than a predictive code, can be a regular sample of the environment with an overrepresentation of the more salient variable that animals need to get in order to collect rewards. In addition, the analysis provided in the manuscript are rather simple, and better controls could be provided. Improving the analytical quantification of the results is necessary to support the main claim.

- What is the relationship of each type of cell with the speed of the animal?<br /> - What is the relationship with the n of trial that the animal has run (first 10 trials, last 10 trials..)?<br /> - What is the average firing rate of each neuron? Is there any relationship between intrinsic firing rate and the type of coding that the cell develops in each task?<br /> - What is the relation of the units of each type with LFP features (theta phase, ripple recruitment)?

Reviewer #1 (Public Review):

In this manuscript, the authors describe a one-step genome editing method to replace endogenous EB1 with their previously-developed light-sensitive variant, in order to examine the effect of acute and local optogenetic inactivation of EB1 in human neurons. They then attempt to assess the effects of EB1 inactivation on microtubule growth, F-actin dynamics, and growth cone advance and turning. They also perform these experiments in neurons that are lacking EB3, in order to determine whether EB1 can function in a direct and specific way without possible EB3 redundancy.

First, the experiments depicting the methodology are rigorous and compelling. Most previous studies of +TIP function use knockout or knockdown studies in which the proteins are inactivated over many hours or days in non-human systems. This is the first study to acutely and locally inactivate a +TIP in human neurons. While this group previously published the effects of replacing endogenous EB1 with the light-sensitive variant, the novelty in this current study is that they use a one-step gene editing replacement method (using CRISPR/Cas9) along with using human neurons derived from iPSCs. After proving their new experimental system works, the authors next seek to test the effect that acutely inactivating EB1 (alongside chronic EB3 knockdown) has on microtubule dynamics, and they observe a marked reduction in MT growth and MT length. They then seek to investigate whether F-actin dynamics are immediately affected by EB1 inactivation. While measured F-actin flow rates are not significantly affected, which leads the authors to conclude that EB1 inactivation does not have any immediate effect, the included figures and movies show a different phenotype, which is not discussed. Finally, they examine the effect of EB1 inactivation on growth cone advance and growth cone turning, and find that both are affected. However, the lack of certain controls in these final experiments (specifically for Figures 3, 4, and 5) reduces the strength of their findings.

Thus, the first part of this paper describing the new methodology is very compelling and should be of interest to a wide readership, while the second part describing the functional analysis is mostly solid, with very high-quality imaging data. However, additional analysis and controls would be needed to increase confidence in their conclusions.

1) Analysis of F-actin dynamics is not thorough and their claim is not completely supported by the data. Figure 3 only depicts F-actin dynamics data from growth cones of π-EB1 EB3-/- i3Neurons and does not control growth cones (to compare dark and light conditions). While their conclusion is that F-actin dynamics are not affected, there do appear to be immediate changes in the F-actin images, other than flow rates. For example, the F-actin bundles do not appear to emanate straight out with the light condition, compared to the dark condition. There also appears to be more F-actin intensity in the transition domain of the growth cone, compared to the dark condition. If the reason is due to the effects of four minutes of blue light exposure, this would be made clear by doing this experiment with control growth cones as well.

2) Analysis of the effect of EB1 inactivation on growth cone advance and growth cone turning. Figure 4C, showing the neurite unable to cross the blue light barrier, is potentially quite compelling data, but it would be even more convincing if there were also data showing that the blue light barrier has no effect on a control neurite. Given that a number of previous recent studies have shown a detrimental effect of blue light on neurons, it seems important to include these negative controls in this current study.

3) This concern also holds true for the final experiment, in which the authors examine whether localized blue light would lead to growth cone turning. The authors report difficulty with performing this technically challenging experiment of accurately targeting the light to only a localized region of the growth cone. Thus, the majority of the growth cones (72%) were completely retracted, and so only a small subset of growth cones showed turning. However, this data would be more compelling if there were also a control condition of blue light with neurons that are not expressing the light-inactivated EB1. Another useful control would be to examine whether precise region-of-interest blue light leads to localized loss of EGFP-Zdk1-EB1C on MT plus-ends within the growth cone, or if the loss extends throughout the growth cone. Either outcome would be helpful to potential readers.

Reviewer #1 (Public Review):

This study by Noonan et al. explores the role of TGFb signaling in melanoma. TGFb signaling in melanoma and in the tumor microenvironment is complex, acting as both a tumor suppressor and tumor promoter, as well as an immune suppressor. The authors identified a human TGFb-responsive genomic regulatory element that is activated in TGFb-treated melanoma cell lines. This human genomic regulatory element also functions in a zebrafish melanoma model (TIE:EGFP) in specific regions of advanced melanoma. The enhancer region is bound by SMAD2/3, JunB, and ATF3. The proposed model that TIE:EGFP+ melanoma cells are preferentially phagocytosed by macrophages suggests there is some signal specific to this subset of melanoma cells. How this subset of melanoma cells is phagocytosed by macrophages is still poorly understood and will require further investigation. In addition, the authors found that SATB2 overexpression drives the early onset of the TIE:EGFP reporter in melanoma. This novel zebrafish TGFb reporter line has provided unique insights into the dynamic in vivo interactions between melanoma cells and the microenvironment, as well as immune cells. This study will be of interest to researchers looking for novel signaling mechanisms of melanoma progression.

Reviewer #1 (Public Review):

The authors initiated the study motivated by the lack of knowledge about the molecular events downstream of the polarity effector Emx2 in the mammalian inner ear, hypothesizing that some of those molecular players will be found by sequencing cells that normally express Emx2 in ears from Emx2-mutant mice.

The hypothesis is sound, the technologies used are standard and well-established, and the presented data is of high quality. The results largely support the authors' conclusions. However, the authors have not formally demonstrated that Stk32A is a transcriptional target of Emx2. It is clear that it is positioned downstream of the events triggered by Emx2, and that it can reverse Emx2 activity, but the data do not support the claim that the kinase is under direct transcriptional control of Emx2.

The revelation that Stk32A has two separate functions in planar polarity is significant.

The results will have a significant impact in the field because it provides one of the more persuasive molecular links between Emx2 and the polarization machinery.

Reviewer #1 (Public Review):

This is a nicely written, very compelling manuscript, comprehensive in scope, that reaches new molecular and mechanistic conclusions on metal transport by Nramp on the basis of extensive crystallographic, molecular dynamics, and metal binding/transport assays. The higher resolution of the structures reported here provides new insights into metal (both Mn and Cd) coordination chemistry along the transport pathway which was generally missing (or incomplete) from previous structural analysis of this well-studied model bacterial system. The findings are strongly topical and likely applicable to other Nramps that are present in higher eukaryotes.

The new crystallography coupled with the molecular dynamics provides support for the overall transport pathway model. The conclusions are by and large strongly supported by the data. The figures are absolutely outstanding, and readily accessible even to the non-specialist. The authors identify a lower affinity "external" site which may function as an Mn transfer site that kinetically enhances Mn-binding to the cognate "orthosteric" site essential for transport across the membrane.

Minor weaknesses are the ITC experiments in general. The authors use these experiments to estimate binding affinities of the external and orthosteric sites in a variety of conformations. Although these data are extensive (there are many titrations here), the robustness of the fits to these data is not apparent from what is provided. Clearly the stoichiometry, and thus the binding model (one site vs. two independent sites) was assumed prior to the data fitting; the uncertainties in K are then quite large.

Reviewer #1 (Public Review):

Han and Eckstein asked human participants to follow the gaze of a person and to judge the presence/absence of a target person in videos. The videos contained a gazer and an additional person as gaze goal in present conditions. In absent conditions, this person was digitally removed from the video. The results show that participants use peripheral information about the most likely gaze goal to predictively execute a saccade towards the gaze goal before the gazer's head is oriented towards the goal. At the same time, foveal information about the head velocity of the gazer is processed, leading to more reverse saccades to the gazer when the head velocity of the gazer is low and/or when the head accelerates before the first saccade to the goal. Further control experiments show that the reverse saccades are effective in reducing the error of the following saccade because additional foveal information of the gazer's head direction is sampled. Predictive saccades are also observed when participants are not instructed to follow the gaze.

Strengths:

The study uses very clever experimental manipulations and analysis methods to understand when and where information is sampled for saccade programming. This is especially challenging because natural videos are used to investigate gaze control in an ecologically highly relevant scenario. Compared to previous studies on the sampling of information, in which mostly artificial and static targets were used, this is a large conceptual and methodological step forward and advances the state-of-the-art. The complex stimulus material is analysed using advanced AI techniques and traditional human annotations. Overall, the study contains a complex and rich data set that is created and analysed with innovative methods and it will certainly stimulate further research.

Weaknesses:

While the study uses clever and sophisticated manipulations to dissect the influence of different types of information on eye movement control, these manipulations inevitably lead to a few limitations of ecological validity, which might contribute to the findings:

1. Role of expectations: It seems that whenever there was a second person present in the video, it was always the gaze goal. This might influence the gaze dynamics of participants because participants can anticipate that the gazer will look towards the second person. This expectation might allow participants to infer the gaze goal with peripheral vision and reduce the necessity to rely on foveal information about the head direction of the gazer. Some or all of the differences between the present/absent conditions might actually reflect the effect of this expectation.

2. Absent videos: Absent videos were created by digitally removing the target/distractor person from the video. This is definitely useful to maximize the visual similarity of absent and present videos, but it also might lead to absent videos that do not contain a meaningful gaze goal in the scene. This can be seen in Figure 1e, where the gazer seems to look towards something that is outside of the video frame. This absence of a potential gaze goal might delay saccades and render them more variable, especially in terms of amplitude.

Reviewer #1 (Public Review):

This paper addresses the question of Prdm9-dependent hotspots and Prdm9 alleles evolution. Two properties underlie this question: the erosion of hotspots by biased gene conversion and the high mutation rate of the Prdm9 zinc finger domain. Here the authors include an additional recently observed property of Prdm9: its role in DSB repair, by enhancing DSB repair efficiency when binding on both homologs (symmetric sites). The status of symmetric binding depends on Prdm9 level and affinity, possibly other factors. The authors present a model for simulating Prdm9 and hotspots co-evolution based on several assumptions (Number of DSB independent of Prdm9, two types of hotspots, strong or weak; hotspots compete; at least one symmetric DSB is required on the smallest autosome). Although the in vivo context is obviously more complex, these assumptions are reasonable (except for the number of Prdm9 bound sites) as they qualitatively recapitulate or get close to what is known about the requirement for fertility. The model leads to several important conclusions and predictions that Prdm9 limits the number of sites used since such conditions are predicted to allow for a weaker contribution of asymmetric sites.

The presentation of the model is clear, but the results are difficult to follow and require many readings to follow the text and the associated figures.

A few specific points also require clarification:<br /> Competition: It seems that in the context defined Prdm9 is limiting (since most Prdm9 can be bound to all weak sites); in addition, it is not clear how the competition for DSB activity between Prdm9 sites is taken into account.

The number of Prdm9-bound sites in vivo is not known, thus several values must be tested.

It would be interesting to discuss the model prediction in the context of several observations published on hybrids with variable Prdm9 gene dosage.

Reviewer #1 (Public Review):

It is well established that valuation and value-based decision making is context-dependent. This manuscript presents the results of six behavioral experiments specifically designed to disentangle two prominent functional forms of value normalization during reward learning: divisive normalization and range normalization. The behavioral and modeling results are clear and convincing, showing that key features of choice behavior in the current setting are incompatible with divisive normalization but are well predicted by a non-linear transformation of range-normalized values.

Overall, this is an excellent study with important implications for reinforcement learning and decision-making research. The manuscript could be strengthened by examining individual variability in value normalization, as outlined below.

There is a lot of individual variation in the choice data that may potentially be explained by individual differences in normalization strategies. It would be important to examine whether there are any subgroups of subjects whose behavior is better explained by a divisive vs. range normalization process. Alternatively, it may be possible to compute an index that captures how much a given subject displays behavior compatible with divisive vs. range normalization. Seeing the distribution of such an index could provide insights into individual differences in normalization strategies.

One possibility currently not considered by the authors is that both forms of value normalization are at work at the same time. It would be interesting to see the results from a hybrid model.

Reviewer #1 (Public Review):

This paper establishes a strong case for the post-translational modification of C/EBPalpha to play a strong role in its effects, in this case, to promote macrophage differentiation in collaboration with PU.1. The cellular system being used for most of the experiments here takes advantage of the dual roles of PU.1 in B cells, which normally do not express C/EBP family factors, and in myeloid cells, which normally do express C/EBP family factors. The authors and others have previously shown that PU.1 and C/EBPalpha are very powerful collaborators, both needed to establish a macrophage identity. Thus, the title of the paper provocatively implies that the C/EBP modification that keeps it from being methylated on Arg35 works by increasing the re-distribution of PU.1 from B cells to myeloid gene sites in combination with C/EBP. Indeed, the authors show proximity ligation data to show that PU.1-C/EBPalpha juxtaposition is more frequent in the nucleus if C/EBPalpha cannot be Arg-methylated. The paper also shows careful and thorough characterization of the B to myeloid lineage conversion gene expression changes and the mapping of the Arg residues in C/EBPalpha that are most important to keep demethylation. Similarly, the paper provides strong evidence that it is Carm1, and not another protein arginine methyltransferase, that is responsible for the regulatory modification. This is a valuable and well-characterized demonstration of a mechanism that should be considered more generally as a regulator of transcription factor action.

Some weaknesses:

1. The mechanism proposed by the authors is that C/EBPalpha relocates PU.1 to macrophage sites and that C/EBPalpha R35A binds and relocates PU.1 more efficiently than wildtype, and this seems likely and appealing. However, it is not as strongly supported by data within the paper itself as the other points in the paper are. There is a puzzling gap in the data: no direct evidence is shown that C/EBPalpha is really relocating PU.1 from B cell to macrophage regulatory elements at all. Despite the figure titles (Fig. 4 and Fig. S4), there is no ChIP-seq data to show PU.1 binding sites before and after interaction with either wildtype or R35A mutant C/EBPalpha, just accessibility data. There is also a question of whether such a redistribution would occur fast enough to account for the impressive speed of the R35A mutant's other effects. These questions seem fairly straightforward to address. If relevant data could be added, it would greatly increase the impact and generality of the paper.

2. Also, there is evidence presented that the mutant C/EBPalpha still binds PU.1 at least as well as wildtype in co-immune precipitation and that the bands co-immune precipitated by the mutant may be about twofold stronger. However, this important interaction experiment is not done under quantitative titration conditions that would give confidence about the magnitude of the differences seen.

3. Finally, the effect of the mutation is assumed to be only on the interface for interaction between C/EBPalpha and PU.1 (or other co-factors). However, C/EBPalpha is such a short-lived protein that any modification that slightly increased its half-life could increase its potency. It seems important to present some quantitative protein staining evidence to clarify whether the steady-state level of C/EBPalpha in C/EBPalpha R35A-expressing cells is really unchanged from C/EBPalpha wild-type-expressing cells.

In summary, the authors have demonstrated an exciting and precise mechanism for modulating the effects of C/EBPalpha, but more direct evidence would be needed before concluding that this mechanism operates primarily by exposing a stronger interaction interface to speed up the relocation of PU.1 from B cell sites to macrophage sites.

Reviewer #1 (Public Review):

The manuscript seeks to address a major limitation in the study of SFRS1, a critical and well-studied alternative splicing factor. Specifically, this protein is insoluble at high concentrations due to liquid phase separation driven by the RS domain. This work tests the hypothesis that short RS repeat peptides might compete with intermolecular interactions that drive phase separation, thus solubilizing the protein sufficiently for biochemical and structural studies. The data convincingly show that short peptides with RS, ER, or DR repeats can render recombinant SRSF1 soluble. Moreover, the authors present well-resolved and assignable NMR spectra of SRSF1 dissolved with RS8 as a co-solute. Finally, the authors use paramagnetic relaxation enhancement experiments to map interactions between RS8 and SFRS1, which suggests that the interactions are driven by a combination of ionic interactions between arginine and acidic side chains, and pi-stacking interactions with surface-exposed hydrophobic residues.

The second aspect of this study seeks to identify features of proteins that make them more likely to undergo phase separations. Specifically, the authors use a bioinformatics approach to correlate the presence of RS repeats with the identity of proteins in three available databases of phase-separated material. In addition, the authors use molecular modeling and software tools to predict additional RRM domains that might prone to phase separation by looking specifically for those that are enriched in acidic amino acids with neighboring hydrophobics. These analyses are less convincing -the fold enrichment is small, the sample size decreases by a large amount with increasing repeat length, and it is not clear that the statistical tests performed correctly for multiple hypothesis testing. Moreover, the predictions of the model derived from the computational analyses have not been explicitly tested.

All told, the NMR and PRE data are convincing and the use of short RS, ER, and DR peptides as a co-solute for insoluble SR-domain proteins is novel and clever, but the computational analyses are incomplete and potentially over-interpreted.

Reviewer #1 (Public Review):

Lemerle et al utilize elegant imaging and molecular biology approaches to convincingly demonstrate the presence of Bin1 and caveolae containing rings capable of tubulation in developing muscle. The data is of fundamental potential significance as it advances our understanding of t-tubule biogenesis, which represents a major knowledge gap in muscle biology. The paper will be of broad interest to skeletal and cardiac muscle biologists and physiologists. The paper is well written, with a comprehensive yet concise introduction, clearly presented results, and an appropriate discussion. The imaging is spectacular, and the use of CLEM provides compelling validation of the protein constituents of ring structures identified via EM. When combined with time-lapse imaging, the combination of approaches provides powerful nanoscale structural information alongside temporal dynamics and live-cell confirmation of tubulating ability by Bin1-Cav3 containing rings. The data indicate that Bin1 is sufficient to generate circular structures that are subsequently decorated by caveolae which facilitate tubule formation at the membrane, and they support the requirement of both Bin1 and Cav3 for efficient tubule initiation and elongation. The authors also utilize myotubes from patients with cav3 mutations to explore whether altered ring formation may contribute to muscle pathology - however, this section requires additional controls and validation to confer pathological insight. Further, additional quantification of imaging data across the study is required to increase the rigor and strength of the conclusions of this work.

Reviewer #1 (Public Review):

The authors have tried to correlate changes in the cellular environment by means of altering temperature, the expression of key cellular factors involved in the viral replication cycle, and small molecules known to affect key viral protein-protein interactions with some physical properties of the liquid condensates of viral origin. The ideas and experiments are extremely interesting as they provide a framework to study viral replication and assembly from a thermodynamic point of view in live cells.

The major strengths of this article are the extremely thoughtful and detailed experimental approach; although this data collection and analysis are most likely extremely time-consuming, the techniques used here are so simple that the main goal and idea of the article become elegant. A second major strength is that in other to understand some of the physicochemical properties of the viral liquid inclusion, they used stimuli that have been very well studied, and thus one can really focus on a relatively easy interpretation of most of the data presented here.

There are three major weaknesses in this article. The way it is written, especially at the beginning, is extremely confusing. First, I would suggest authors should check and review extensively for improvements to the use of English. In particular, the abstract and introduction are extremely hard to understand. Second, in the abstract and introduction, the authors use terms such as "hardening", "perturbing the type/strength of interactions", "stabilization", and "material properties", for just citing some terms. It is clear that the authors do know exactly what they are referring to, but the definitions come so late in the text that it all becomes confusing. The second major weakness is that there is a lack of deep discussion of the physical meaning of some of the measured parameters like "C dense vs inclusion", and "nuclear density and supersaturation". There is a need to explain further the physical consequences of all the graphs. Most of them are discussed in a very superficial manner. The third major weakness is a lack of analysis of phase separations. Some of their data suggest phase transition and/or phase separation, thus, a more in-deep analysis is required. For example, could they calculate the change of entropy and enthalpy of some of these processes? Could they find some boundaries for these transitions between the "hard" (whatever that means) and the liquid?

The authors have achieved almost all their goals, with the caveat of the third weakness I mentioned before. Their work presented in this article is of significant interest and can become extremely important if a more detailed analysis of the thermodynamics parameters is assessed and a better description of the physical phenomenon is provided.

Reviewer #1 (Public Review):

The manuscript by Shaikh and Sunagar addresses the question of the origin of spider venom proteins. It has been known for many years that an important component of spider venoms is a diverse group of small proteins known as disulfide-rich peptides (DRPs). However, it has not been clear whether this group of proteins has a common origin or evolved convergently in different lineages. The authors collected sequences of the genes encoding these proteins from publicly available genomes of spiders from a range of families. They aligned the sequences using the structural cysteines as guides and carried out a phylogenetic analysis of the different sequences, ultimately classifying the different proteins into over 50 super-families. One thing that is not clear from the text or from the references cited (I am not an expert on spider venom) is how many of these superfamilies were known before and how many are novel. There is also no clear indication of what criteria were used to define a subset of sequences as a superfamily. Nonetheless, the authors show that all these superfamilies have a single common ancestor, predating the divergence of araneomorphs and mygalomorphs and that the DRPs underwent independent diversification in each of these two lineages.

The authors also looked at selective forces acting on the sequences using dN/dS analyses. They reach the conclusion that there are different modes of selection acting on different sequences based on their role - defensive or predatory venoms - building on previous work by the lead author on venom sequence evolution in diverse animals.

All in all, this is an admirable piece of molecular evolution work, providing new data on the evolution of spider venom proteins. There are some confusions in terminology that need to be cleared up, and somewhat more context needs to be given for non-specialists as detailed in the points below:<br /> 1) Common names of the main spider infraorders should be given.<br /> 2) Opisthothelae is not the common ancestor of Mygalomorphae and Araneamorphae, but the clade that encompasses those two clades. This incorrect statement appears in several places. Further on, it is stated that Opisthothelae is the common ancestor of all extant spiders. This is wrong both from a terminological point of view (a clade cannot be ancestral to another clade) and from a factual point of view, since there are extant spiders not included in Opisthothelae.<br /> 3) Several proteins and proteins families are mentioned without being introduced, e.g. knottin. Please provide short descriptions.

Reviewer #1 (Public Review):

In this manuscript, the authors present a study on the humanized mouse model of HIV, in which the major focus is inflammasome activation during acute infection and the potential for blocking this activation. They describe the mouse model as sufficient to study inflammasome activity after HIV infection and proceed to demonstrate potentially beneficial effects of a caspase-1 inhibitor, VX-765, given during acute infection resulting in mildly reduced viral load and increased CD4 T cell preservation.

The authors clearly demonstrate established HIV infection in huNSG mice through detection of plasma viremia, viral RNA/DNA in tissue and depletion of CD4 T cells. Furthermore, they show moderate but inconsistent increases in expression of inflammasome-related genes using qPCR across multiple tissues and timepoints. As expected, the authors found increased levels of inflammatory cytokines, particularly IL-18, during acute viral infection.

Systemic IL-18 levels are significantly reduced by VX-765, demonstrating clear in vivo capacity to impact inflammasome-related cytokines. Furthermore, there appears to be a statistically significant preservation of CD4 T cell populations in VX-765-treated animals, although this preservation is inconsistent across different tissues and may not be to a biologically relevant degree. Finally, VX-765 appears to significantly decrease plasma viral load at day 22 post-infection and potentially results in lower HIV DNA levels in the spleen. Finally, the manuscript demonstrates reduced caspase-1 activity after VX-765 treatment, but this finding is limited to CD11c+ and CD14+ cells with no impact found within CD4 and CD8 T cells, and the authors acknowledge that they may be unable to detect caspase-1 activity in CD4 and CD8 T cells after HIV infection.

Although the study is interesting, there are several important comments and potential caveats/limitations that must be addressed, including for the correlations between AIM2 and IFI6 with viral loads and CD4 T cells that appear strongly driven by measurements at day 0 post-infection; multiple cytokine measurements that appear to be below the manufacturer's described limit of detection for the assay described; a lack of measurable caspase activity in T cells; some inconsistency between DNA or RNA content and plasma viremia.

Reviewer #1 (Public Review):

This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 interacts with Gcn5 and Ada2 and, hence, could act directly to recruit these cofactors to the FRQ promoter. Importantly, they show that knock out of GCN5 eliminates rhythmic cell growth and FRQ expression (although surprisingly not FRQ mRNA abundance), as well as reducing H3ac levels and WCC binding at FRQ. They further show that TSA treatment can reverse the effects of histidine starvation on the circadian period in WT cells, and can partially restore rhythmic growth to histidine-starved cpc-3 cells, and that elimination of HDAC Hda1 increases H3ac at FRQ in WT cells. They provide some evidence that transcriptional activation of certain aa biosynthetic genes by CPC-1 is also rhythmic, although the evidence for this is not strong and it's unclear whether CPC-1 occupancy or its activation function would be periodic. They also did not address whether CPC-1 occupancy at FRQ is rhythmic.

This work is important in providing convincing evidence that CPC-1-mediated induction of transcription factor CPC-3 in starved Neurospora cells mediates CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter, which counteracts the function of histone deacetylase HDA1 to maintain high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ transcription. Although the work does not identify new regulatory circuits, such as rhythmic transcription of FRQ, the role of Gcn5, Hda1, and promoter histone acetylation in supporting transcriptional activation, and the general amino acid control response to amino acid starvation are all well-established mechanisms, the work is significant in showing how these pathways and mechanisms are integrated to maintain circadian rhythm in the face of amino acid limitation.

There is an abundance of convincing experimental evidence provided to support the key claims just summarized above. However, there are a few instances in which additional experiments might be required to resolve a discrepancy in the data or provide stronger evidence to support a claim.

Reviewer #1 (Public Review):

Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patient-derived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs that express human PLA2G6 and study expression levels in a human kidney cell line as well as in patent-derived NPCs. In the latter neuronal model, they show that expression of human PLA2G6 can rescue mitochondrial fragmentation associated with PLA2G6 loss-of-function. Lin et al then show that ICV (intracerebroventricular) and IV (intravenous) injection of a human PLA2G6-containing construct is able to partially rescue the rotarod phenotype in PLA2G6 transheterozygous PLA2G6 mutant mice between ~110 and 150 days. There is also an associated improvement in lifespan and body weight.

The strengths of this work are that the authors use a number of different model organism systems, including patient-derived neuronal cells, Drosophila models (INAD flies) and mouse models to study PLA2G6-associated neurodegeneration (PLAN) at the cellular level. They also screen drug compounds that are predicted to target endo-lysosomal trafficking and sphingolipid metabolic pathways to ameliorate PLAN, thus identifying potential new therapeutic strategies. The work in mice, showing that gene therapy with human PLA2G6 can rescue a behavioural phenotype and lifespan is the first proof-of-concept of such an advancement. This work will hopefully lead to further studies for optimisation toward clinical advancement.

The major weaknesses are that the pathogenic mechanisms shown in the patient-derived neuronal cells and mice do not extend as far as those previously shown in the fly model published by the authors. Of note, ceramide levels and retromer function are not studied, both key pathologies described in the previous fly models. In addition, the drug screening is limited by its testing in one fly behavioural assay and LAMP2 Western blot analysis on patient derived NPCs.

The results, in general, support the conclusions of the authors and represent well-performed work. However, the significance of elevated glucosylceramide levels is not clear in the present study. Although this was previously found to be elevated in INAD flies, it was ceramide levels that were thought to be the main toxic insult, with drugs aimed at reducing ceramide levels being shown to rescue INAD flies.

This work will no doubt be of significant interest to the field, confirming several previous findings in the Drosophila model of PLA2G6 (iPLA2-VIA) knockout. It also extends upon the fly work by identifying compounds that can be further studied for potential drug-re-purposing for the treatment of PLA2G6-associated disease. The gene therapy studies are also very interesting and a first proof-of-principle in PLAN using ICV and IV delivery in a mouse model.

Reviewer #1 (Public Review):

Targeting allosteric sites, including cryptic sites holds great potential for achieving drug design that distinguishes between isologous protein targets. Here, Meller et al seek to reveal the mechanisms by which blebbistatin, a selective allosteric inhibitor of myosins achieves selectivity between proteins with high structural and sequence similarity. Blebbistatin binds in a supposed cryptic pocket, and authors explore the hypothesis that this selectivity is modulated by dynamics of opening in the cryptic pocket. Studies use MD simulations to show that while cryptic pockets do not exist in experimental structures, they appear in simulations. Markov state models (MSM) generated from simulations are used to quantify probability of pocket opening. The same methods are used to show that ADP-bound myosins are more likely to open than ATP-bound state, consistent with higher blebbistatin binding affinity observed in the ADP-bound state. Myosin-II proteins are shown to have higher probability of opening than non-myosin-IIs, along with an observed correlation of probability with IC50. By docking blebbistatin into structures derived from MSMs, authors show a correlation between predicted binding affinity from docking and experimental binding. Binding was correctly predicted for a new isoform in a blind study, further establishing the utility of using conformational ensembles to predict sensitivity of blebbistatin binding to myosins.

Reviewer #1 (Public Review):

This study provides further detailed analysis of recently published Fly Atlas datasets supplemented with newly generated single cell RNA-seq data obtained from 6,000 testis cells. Using these data, the authors define 43 germline cell clusters and 22 somatic cell clusters. This work confirms and extends previous observations regarding changing gene expression programs through the course of germ cell and somatic cell differentiation.

This study makes several interesting observations that will be of interest to the field. For example, the authors find that spermatocytes exhibit sex chromosome specific changes in gene expression. In addition, comparisons between the single nucleus and single cell data reveal differences in active transcription versus global mRNA levels. For example, previous results showed that (1) several mRNAs remain high in spermatids long after they are actively transcribed in spermatocytes and (2) defined a set of post-meiotic transcripts. The analysis presented here shows that these patterns of mRNA expression are shared by hundreds of genes in the developing germline. Moreover, variable patterns between the sn- and sc-RNAseq datasets reveals considerable complexity in the post-transcriptional regulation of gene expression.

Overall, this paper represents a significant contribution to the field. These findings will be of broad interest to developmental biologists and will establish an important foundation for future studies. However, several points should be addressed.

In figure 1, I am struck by the widespread expression of vasa outside of the germ cell lineage. Do the authors have a technical or biological explanation for this observation? This point should be addressed in the paper with new experiments or further explanation in the text.

The proposed bifurcation of the cyst cells into head and tail populations is interesting and worth further exploration/validation. While the presented in situ hybridization for Nep4, geko, and shg hint at differences between these populations, double fluorescent in situs or the use of additional markers would help make this point clearer. Higher magnification images would also help in this regard.

Reviewer #1 (Public Review):

Wolfram syndrome 1 (WS1) is a rare genetic disorder characterized by diabetes mellitus, various neurological dysfunction, and blindness caused by optic atrophy. The primary impact of this paper is the characterization of vision loss, retinal dysfunction, and retinal ganglion cell (RGC) degeneration in the Wfs1exon8del murine model of WS1 combined with the generation of -omics datasets at RNA and protein levels. Based largely on a qualitative assessment of select targets, the authors propose mechanisms that could increase RGC susceptibility in WS1 pathology.

Strengths:

1. This study determines that Wfs1exon8del mice exhibit progressive disruption of RGC function similar to that reported in the Wfs1exon5del rat model.<br /> 2. This study performs an in-depth assessment of retinal anatomy, including in vivo OCT and FA, which provides analysis of both vascular and neural elements of pathology.<br /> 3. TEM and immunohistochemical assessment of optic nerve anatomy and RGC soma elucidate a timeline of degenerative events that begins with myelin thinning and axon pathology followed by axon and soma loss.<br /> 4. RNA sequencing and proteomic profiling provide a global assessment of potentially relevant pathways associated with retinal and optic nerve pathology induced by Wfs1 deletion.

Weaknesses:

1. Mechanisms are generally inferred from previous literature rather than demonstrated directly in this model.<br /> 2. Diverse phenotypes were noted in oligodendrocytes, astrocytes, Muller cells, and microglia. It is difficult to piece together the significance of these observations and their relationship to the RGC degeneration noted in earlier figures. There is a sense that the surface is skimmed for each of these.<br /> 3. ERG and f-VEP data do not rule out the possibility that the electrophysiological function of the retina is abnormal from birth.<br /> 4. Only positive correlations with existing literature are discussed.

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!- Comment : 30 to 1 ratio of unequal exchange to North to South Aid - structural unfairness baked into the system - the North knowingly benefits - Knowing this, it can be argued that North states are structurally corrupt and morally bankrupt knowingly imposing this mass suffering upon the south

Reviewer #1 (Public Review):

The current study proposed a drug discovery pipeline to accelerate the process of identifying drug candidates for LCA10 patients using cells from mouse retinal organoid for initial screening, human patient iPSC-derived retinal organoid for further testing, and then mouse mutants for in vivo validation. Reserpine was identified as the top candidate, possibly through modulating proteostasis and autophagy to promote cilium assembly. The study was with high translational value. However, the rationale using dissociated cells from mouse retinal organoid for initial drug screening needs to be justified. In addition, the consistency of phenotypic characteristics in human patient iPSC-derived retinal organoid needs to be reported. It was unclear if the rescued phenotypic changes were from the drug effects or a result of phenotypic variations in organoids.

Reviewer #1 (Public Review):

The manuscript by Webb et al., describes a proband with biallelic variants in the MCAT gene. The proband presents with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Subsequent studies in isolated lymphoblasts and fibroblast samples from the proband unveil combined OXPHOS deficiency. Although the manuscript is of interest and adds translational impact to the previous work of the authors, a few issues remain unresolved. The study would benefit from additional functional tests of the variant MCAT allele.

Reviewer #1 (Public Review):

Roncaioli et al. build upon their previous findings showing that the NAIP/NLRC4 inflammasome confers host resistance to oral Shigella flexneri infection. They investigate the role of additional programmed cell death pathways in the host response to Shigella infection in mice. They find that in the absence of the NAIP inflammasome, caspase-11 contributes but in a limited capacity due to OspC3 antagonism of caspase-11 activity. Furthermore, in the absence of both NAIP and caspase-11, TNF-mediated caspase-8 activation contributes. Thus, the authors conclude that there is a hierarchy of cell death pathways involving caspase-1, 11, and 8 that all contribute to restrict Shigella infection in mice. Overall, the manuscript is well-written, the studies are logically presented and well-designed, and the data largely support the authors' conclusions. The findings will be of great interest to the field. I have a few suggestions for improving the manuscript.

Reviewer #1 (Public Review):

Latshaw and colleagues show that interfering with the function of the tyramine receptor, AmTYR1, causes a precipitous decline in responses to olfactory stimuli, a decline consistent with AmTYR1's proposed involvement in the regulation of inhibitory networks within the antennal lobes (primary olfactory centres) of the honey bee brain. Interestingly, impacts on odour learning of disrupting AmTYR1 function are enhanced by repeatedly exposing bees to an odour without reinforcement ('familiarization'). The authors argue that disruption of AmTYR1 signalling increases the expression of latent inhibition without affecting appetitive conditioning. The results do not, in my view, support this claim. Nevertheless, the disruption of tyramine signalling in the bee brain clearly packs a powerful punch.

Strengths:<br /> Repeated presentation of an odour without reinforcement slows subsequent learning of an association between the odour in question and food reward (latent inhibition). This study's aim was to investigate the mechanisms that underlie individual differences in this trait.

The authors select honey bee lines showing high and low levels of latent inhibition and use QTL mapping to identify genes potentially responsible for this trait. Amtyr1 is identified, a gene that encodes a tyramine receptor the authors have shown elsewhere is expressed in the brain, including on presynaptic terminals of olfactory sensory neurons in the antennal lobes (primary olfactory centres of the brain). Using the tyramine receptor blocker, yohimbine, and Amtyr1 knockdown with dsiRNA, Latshaw et al. show that disruption of tyramine signalling via AmTYR1 receptors inhibits dramatically the magnitude of responses to odour signals at the level of the antennal lobes. Odour learning appears to remain largely intact unless, prior to conditioning, bees are exposed repeatedly to puffs of odour without reinforcement (a situation expected to induce latent inhibition). As a result of familiarization, learning not only of the familiarized odour, but also of novel odours declines dramatically. These findings are fascinating and consistent with the hypothesis that AmTYR1 is involved in regulating inhibitory networks within the AL.

Weaknesses:<br /> The authors argue that disruption of AmTYR1 signalling increases the expression of latent inhibition without affecting appetitive conditioning. The results, in my view, do not support this conclusion. Electrophysiological recordings from the AL show that blocking AmTYR1 function causes significant non-odour-specific suppression of responses at the level of the antennal lobes, a result that would be predicted if inhibiting AmTYR1 function increased lateral inhibition (as opposed to latent inhibition) globally in the antennal lobe.

Under these conditions, the consequences of odour familiarization would, I believe, be predicted by current models of inhibitory networks in antennal lobes. A schematic of olfactory circuits within the antennal lobes, and the location of AmTYR1 receptors within this network, would assist in enabling readers to navigate these complex networks and interpret the interesting findings presented in this study. While the stated aim was to investigate the mechanisms that underlie individual differences in latent inhibition, this goal seems to be lost along the way.

Reviewer #1 (Public Review):

In their paper "Spatiotemporal Ecological Chaos Enables Gradual Evolutionary Diversification Without Niches or Tradeoffs", Mahadevan, Pearce, and Fisher build on previous works to explore a compelling potential answer (what they term a "scenario") to an important open and fascinating question: what gives rise to micro strain-level diversity?

Naively, the ecological principle of competitive exclusion would suggest that closely related strains should not be able to co-exist. However, Fisher and collaborators have previously proposed an interesting and potentially novel and powerful solution to this paradox. If the species have (extremely) anti-correlated species-species interactions (i.e more A helps B but more B hurts A) then there is a reasonably large set of parameters under which you can have infinite diversity due to spatial-temporal chaos (STC). This is really an interesting and compelling picture.

The purpose of this paper is to explore a natural follow-up question: does the STC phase still support infinite diversity even when communities are assembled using evolution, or more accurately undergo evolution starting with a sufficiently large randomly assembled community? In other words, is STC still a reasonable explanation for strain-level diversity once evolution is considered? This is an extremely interesting question since evolution and ecology are so deeply intertwined at the time scales on which strains evolve. The work is especially impressive due to the extreme dearth of analytic and computational tools to really understand eco-evolutionary dynamics.

Reviewer #1 (Public Review):

This is one of the most careful analyses of sexual dimorphism in dinosaurs, based on a remarkable assemblage of 61 ornithomimosaur fossils from the Early Cretaceous of western France. The dimorphism is expressed in variations in the shaft curvature and the distal epiphysis width, analysed appropriately here and plausible because these are the kinds of morphological features that vary between males and females among birds and crocodilians, among others.

In the Introduction, it is right to highlight the shortage of convincing cases of demonstrated sexual dimorphism (SD) in dinosaurs. But note the points made by Hone, Saitta and others that SD can exist in many species today without major morphological differences, making it hard to demonstrate in fossils with such types of dimorphism. Also, some proposed statistical tests to ensure that SD has been convincingly demonstrated in fossils are so stringent they would be hard ever to pass (requiring enormous and constant morphological distinctiveness). In other words, we are conditioned not to find SD in dinosaurs, and yet may be massively under-reporting it because of preservation difficulties (of course) but also because of some overly rigorous demands for proof. These issues help argue that the current study is especially valuable because the data set is large (itself a rarity), and 3D bone shape analysis and proper statistical testing have been applied.

It's interesting the dinosaur example shows the same two dimorphic traits (femoral obliquity = bicondylar angle; width of distal epiphysis = bicondylar breadth) seen in mammals (MS, lines 117-123), where the femur angle may vary because of the need for broader hips in the female to accommodate the birth canal, and yet dinosaurs laid eggs. These are small dinosaurs, so perhaps their eggs were relatively large in proportion to body size. Perhaps the authors could comment on this. There is some discussion with regard to modern birds at MS lines 187-199.

Reviewer #1 (Public Review):

Here the authors sought to understand how BPGM/2,3-BPG levels are involved in adaptive responses to hypoxia and whether they are involved in fetal growth restriction. In the current state, I find the data to be confusing and lacking in mechanistic data to justify that increased BPGM is an adaptive response to hypoxia. While the authors find increased staining for the enzyme BPGM in SpA-TGCs after hypoxia, they did not assess 2,3-BPG in cord blood. This would show that increased enzymatic levels have a downstream impact. MRI experiments assessing placental and fetal haemoglobin-oxygenation, showed no differences. Human FGR samples, however, showed reduced 2,3-BPG in cord blood. Further evidence is required to show hypoxia increases BPGM as a compensatory mechanism to permit adequate 2,3-BPG and placental-fetal oxygenation levels as the authors claim. Additional experiments that demonstrate that BPGM is advantageous in the context of hypoxia would strengthen the authors arguments, and would provide a novel mechanism for adaptive responses to hypoxia in the placenta which is highly interesting.

Reviewer #1 (Public Review):

This manuscript describes a new method to perform online movement correction and extraction of calcium signals from a miniscope. The efficiency of the algorithm is tested by quantifying the accuracy of animal location decoding from hippocampal place cells. The online decoding happens with virtually no delay which is promising for closed-loop methods. It seems to be superior to online decoding without motion correction, which was the state of the art.

The strength of this technique is therefore that it achieves real-time processing.<br /> The weakness of the study is the lack of comparison of the decoding accuracy with what can be obtained with electrophysiological state of the art, which prevents really estimating how precise the technique is.

Although less critical, there is no demonstration of a closed-loop application. Real-time position decoding is technically nice, but the position can be obtained from tracking the animal so it is practically useless. It is also clear that decoding position on a linear track is easier than on a 2D arena, therefore it is difficult to estimate how much the efficiency of the method can be challenged in harder settings.

Thus despite its technical excellence, the impact of this method seems weak.

Reviewer #1 (Public Review):

In this study, the authors collected mandibular alveolar bone samples from control mice and the mice with apical periodontitis (AP) and performed single-cell RNA sequencing (scRNA-Seq) experiment. Using the data from cell subsets of the mandibular alveolar bone, the authors compared the expression profiles of the mice with AP with those from control mice. They also determined the relationship between MSCs and immune cells and confirmed the role of a subset of MSCs in inflammation. In addition, the authors demonstrated MSC differentiation potential to mature osteoblasts during AP inflammation. Using transgenic mouse models and samples derived from patients with chronic AP, they further confirmed the findings of scRNA-Seq data. Taken together, these results reveal the heterogeneity and interactions of alveolar bone cells during periodontitis inflammation. One of their key findings is to identify a subset of MSC cells and their differentiation in the inflamed tissues.

Reviewer #1 (Public Review):

This study intended to identify the metabolic at-risk profile within PLWH on ART, by integrating and analyzing the multiomics data from multi-omics including untargeted plasma metabolomic, lipidomic, and fecal 16s microbiome. The overall strength of the study is the long-term treatment (~15 years) of the study subjects with well-recovered CD4 cell count and viral suppression. The integration and analysis of multi-omics data using similarity network fusion and factor analysis, etc. to group or differentiate HIV patients are informative and useful. The weakness of the study is the lack of presentation of comparability between patients and healthy controls and the use of multiple regression analysis for controlling potential confounders.

Reviewer #1 (Public Review):

Complex I deficiency is associated with multiple diseases ranging from devastating inborn errors of metabolism to more common ailments associated with aging. It has long been known that Complex I transports electrons through a series of iron-sulfur clusters; however, the consequences of the likely dysfunction of these cofactors in models of Complex I deficiency have been surprisingly unexplored. The authors of this manuscript explore the contributions of iron to pathophysiology seen in a mouse model of Complex I deficiency, the Ndufs4 knockout model. Specifically, the authors hypothesize "that Complex I deficiencies may alter normal cellular or regional iron distribution which contributes to mitochondrial disease progression."

The authors begin by convincingly demonstrating that modulating iron availability affects clasping - a marker of neurodegeneration - as well as lifespan. Using either an iron-chelating agent or a low iron diet, the authors delay the onset of clasping (i.e., neurodegeneration) and extend lifespan in Ndufs4 knockout mice. As expected, a limited iron diet causes anemia, but does not affect overall weight in either wild type or Ndufs4 knockout mice, suggesting the diet is tolerated despite the hematological defects. To begin to understand the molecular mechanisms underlying these phenotypes, the authors quantify total iron levels across tissues, and surprisingly find tissue-specific effects of chelation and/or diet-induced modulation of total iron levels. These data suggest that a low iron diet rescues iron levels in the liver, kidney, and duodenum, but not in the brain, which is surprising given the rescue of the clasping phenotype. To follow up on this, the authors look at multiple metals whose uptake can be affected by altered iron homeostasis, and find select defects in other tissues, such as elevated zinc in the brains of knockout mice relative to wild-type controls and elevated manganese in Ndufs4 KO tissues. However, the effects of such metal imbalances were not further explored. The authors then show that the imbalance in liver free iron could cause oxidative stress and reprogram the cellular program of iron transport. Interestingly, these phenotypes are found in the liver but not the brain, consistent with the tissue-specific changes in metals found in previous experiments. Collectively, the data presented by the authors convincingly demonstrate that: 1. Complex I deficiency causes defects in metal homeostasis, albeit in a tissue-specific manner; 2. In tissues harboring elevated iron levels in Ndufs4 knockout mice, this leads to altered iron regulation pathways as well as elevated oxidative stress; and 3. That, despite the tissue specificity of the molecular changes in iron homeostasis, limiting iron uptake in Ndufs4 knockout mice rescues neurodegeneration and extends lifespan in mice. While these data support the authors' hypothesis that "Complex I deficiencies may alter... regional iron distribution which contributes to mitochondrial disease progression," the authors do not test the role of altered cellular distribution of iron within this model.

Overall, the data presented strongly suggest that modulating iron intake may ameliorate the pathophysiology associated with Complex I dysfunction. However, the specific tissues in which these benefits may occur, and the molecular mechanisms underlying this therapeutic benefit are not fully established in this study. Furthermore, the benefits of modulating iron levels as a potential therapeutic strategy for Complex I dysfunction would need to be balanced with potential complications, such as anemia.

Reviewer #1 (Public Review):

The manuscript by Seroussi et al. presents a comprehensive analysis of the expression and function of the entire, extensive family of Argonaute (AGO) proteins in C. elegans. Using genome editing methods, the authors fused tags to 19 endogenous argonaute genes to allow for visualization of their expression patterns in live worms and immunoprecipitation assays to detect RNA partners. Furthermore, they analyzed how the loss of specific AGOs impacts smRNA populations as well as fertility, developmental, and pathogen susceptibility phenotypes. The methods are rigorous and care was taken to maintain the functionality of tagged proteins. This study offers an extremely valuable resource in its comprehensive evaluation of all AGOs in an organism and the resulting datasets and reagents. Furthermore, the authors provide thoughtful analyses of their own data pointing out surprises and follow-up experiments to support their interpretations. A good example is the isolation of miRNAs in the ERGO-1 IP, which provide compelling evidence to indicate that this result is likely due to co-IP of ALG-1/2 on transcripts also bound by ERGO-1 rather than a new role for ERGO-1 in directly binding miRNAs. The authors are also to be commended on the clear and engaging figures, which are often difficult to produce from largely genomics data. Overall, this is a highly significant body of work that provides extensive datasets and reagents that will propel the smRNA field forward faster.

Reviewer #1 (Public Review):

This work identified a novel gene Belly roll (Bero) as a key protein that controls nociceptive escape behavior in Drosophila larvae using genome-wide association analysis. The authors show that constitutive deletion of Bero by CRISPR or RNAi knockdown of Bero expression shows enhanced escape rolling behavior induced by heat probe stimulation. They then showed that Bero is expressed specifically in dimm' pepti, Eh+, Ilp, and AbLK neurons. Next, they demonstrated that Bero RNAi knockdown specifically in ABLk neurons showed the mutant phenotype. Furthermore, they found that ABLK neurons exhibit spontaneous activity and, that Bero RNAi knockdown inhibited this spontaneous activity and increased the response latency to nociceptive stimulation in ABLK neurons.

Strong points of this study: Authors identify a novel gene as a key protein that negatively controls nociceptive escape behavior in Drosophila. The data were presented clearly and the approach to identifying the gene was unique.

Weakness of this study:<br /> I think if authors are able to link the variable bero expression levels in each GNP line and ABLK neural activity, the physiological function of Bero will be strong. I also appreciate the proposed model in the supplemental figure although it has not shown the evidence to link stress and Bero in this study.

Reviewer #1 (Public Review):

This study combines the biologging method with captive experiments and DNA metabarcoding to detail the hunting behavior of a bat species in the wild. Specifically, it shows that bats use two foraging strategies (echolocating small prey in the air and capturing large ground prey with passive listening) with different success rates and energetic gains. This result highlights that a species believed to be a specialist forager can, in fact, have mixed strategies depending on the condition and environment.

The detailed foraging behavior they show for such a small animal is impressive. A combination of several different methods, including captive experiments, is a major strength of the paper. I especially like the mastication sound analysis, although I don't know how new it is. However, I have a major concern about the presentation of this study. The manuscript is apparently written for a bat community, and it's hard to understand the significance of the results in the field of animal ecology.

Reviewer #1 (Public Review):

Recent studies indicate that osteoblasts formed during endochondral bone formation have arisen from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). In this study, through a single-cell transcriptomics approach, the authors found that HC descendent cells activate MMP14 and the PTH pathway as they transition to osteoblasts at postnatal and adult stages. HC-specific Mmp14 knockout mice had increased bone mass phenotype. The authors found that MMP14 cleaves the extracellular domain of PTH1R and inhibits PTH signaling. They also found that HC-derived osteoblasts contribute about 50% of osteogenesis promoted by the treatment with PTH 1-34 and this response was amplified in Mmp14 knockout mice. MMP14 controls PTH signaling through both HC- and non-HC-derived osteoblasts. The authors concluded that they have identified a novel mechanism of MMP14-mediated PTH signaling in the osteoblast lineage cells.

Reviewer #1 (Public Review):

King et al. provide an interesting reanalysis of existing fMRI data with a novel functional connectivity modeling approach. Three connectivity models accounting for the relationship between cortical and cerebellar regions are compared, each representing a hypothesis. Evidence is presented that - contrary to a prominent theoretical account in the literature - cortical connectivity converges on cerebellar regions, such that the cerebellum likely integrates information from the cortex (rather than forming parallel loops with the cortex). If true, this would have large implications for understanding the likely computational role of the cerebellum in influencing cortical functions. Further, this paper provides a unique and potentially groundbreaking set of methods for testing alternate connectivity hypotheses in the human brain. However, it appears that insufficient details were provided to properly evaluate these methods and their implications, as described below.

Strengths:<br /> • Use of a large task battery performed by every participant, increasing confidence in the generality of the results across a variety of cognitive functions.<br /> • Multiple regression was used to reduce the chance of confounding (false connections driven by a third region) in the functional connectivity estimates.<br /> • A focus on the function and connectivity of the cerebellum is important, given that it is clearly essential for a wide variety of cognitive processes but is studied much less often than the cortex.<br /> • The focus on clear connectivity-based hypotheses and clear descriptions of what would be expected in the results if different hypotheses were true.<br /> • Generalization of models to a completely held-out dataset further increases confidence in the generalizability of the models.

Concerns:<br /> • The main conclusion of the paper (including in the title) involves a directional inference, and yet it is notoriously difficult to make directional inferences with fMRI. The term "input" into the cerebellum is repeatedly used to describe the prediction of cerebellar activity based on cortical activity, and yet the cerebellum is known to form loops with the cortex. With the slow temporal resolution of fMRI it is typically unclear what is the "input" versus the "output" in the kinds of predictions used in the present study. Critically, this may mean that a cerebellar region could receive input from a single cortical region (i.e., the alternate hypothesis supposedly ruled out by the present study), then output to multiple cortical regions, likely resulting (using the fMRI-based approach used here) in a faulty inference that convergent signals from cortex drove the results. On pg. 4 it is stated: "We chose this direction of prediction, as the cerebellar BOLD signal overwhelmingly reflects mossy-fiber input, with minimal contribution from cerebellar output neurons, the Purkinje cells (Mathiesen et al., 2000; Thomsen et al., 2004)." First, it would be good to know how certain this is in 2022, given the older references and ongoing progress in understanding the relationship between neuronal activity and the BOLD signal (e.g., Drew 2019). Second, given that it's likely that activity in the mossy-fiber inputs has an impact on Purkinje cell outputs, and that some cortical activity supposedly reflects cerebellar output, it is possible that FC could also reflect the opposite direction (cerebellumcortex). It would seem important to consider these possibilities in the interpretation of the results.<br /> • It would be helpful to have more details included in the "Connectivity Models" sub-section of the Methods section. The GLM-based connectivity approach is highly non-standard, such that more details on the logic behind it and any validation of the approach would be helpful. More specifically, it would be helpful to have clarity on how this form of functional connectivity relates to more standard forms, such as Pearson correlation and perhaps less standard multiple regression (or partial correlation) approaches. If I understand this approach correctly, each cortical parcel's time series is modulated (up or down) using that parcel's task-evoked beta weights, then "normalized" by the standard deviation of that parcel's time series, with the resulting time series then used in a multiple regression model to explain variance in a given cerebellar voxel's time series. It would be helpful if each of these steps were better explained and justified. For example, it is unclear what modulation of the cortical parcel time series by task-related beta weights does to the functional connectivity estimates, and thus how they should be interpreted.<br /> • It appears that task-related functional connectivity is used in the present study, and yet the potential for task-evoked activations to distort such connectivity estimates does not appear to be accounted for (Norman-Haignere et al. 2012; Cole et al. 2019). For example, voxel A may respond to just the left hemifield of visual space while voxel B may respond to just the right hemifield of visual space, yet their correlation will be inflated due to task-evoked activity for any centrally presented visual stimuli. There are multiple methods for accounting for the confounding effect of task-evoked activations, none of which appear to be applied here. For example, the following publications include some options for reducing this confounding bias: (Cole et al. 2019; Norman-Haignere et al. 2012; Ito et al. 2020; Rissman, Gazzaley, and D'Esposito 2004; Al-Aidroos, Said, and Turk-Browne 2012). If this concern does not apply in the current context it would be important to explain/show why.<br /> • It is stated (pg. 21): "To reduce the influence of these noise correlations, we used a "crossed" approach to train the models: The cerebellar time series for the first session was predicted by the cortical time series from the second session, and vice-versa (see Figure 1). This procedure effectively negates the influence of noise processes, given that noise processes are uncorrelated across sessions." However, this does not appear to be strictly true, given that the task design (parts of which repeat across sessions) could interact with sources of noise. For example, task instruction cues (regardless of the specific task) likely increase arousal, which likely increases breathing and heart rates known to impact global fMRI BOLD signals. The current approach likely reduces the impact of noise relative to other approaches, but such strong certainty that noise processes are uncorrelated across sessions appears to be unwarranted.<br /> • It appears possible that the sparse cerebellar model does worse simply because there are fewer predictors than the alternate models. It would be helpful to verify that the methods used, such as cross-validation, rule out (or at least reduce the chance) that this result is a trivial consequence of just having a different number of predictors across the tested models. It appears that the "model recovery" simulations may rule this out, but it is unclear how these simulations were conducted. Additional details in the Methods section would be important for evaluating this portion of the study.

References:

Al-Aidroos, Naseem, Christopher P. Said, and Nicholas B. Turk-Browne. 2012. "Top-down Attention Switches Coupling between Low-Level and High-Level Areas of Human Visual Cortex." Proceedings of the National Academy of Sciences of the United States of America 109 (36): 14675-80.<br /> Cole, Michael W., Takuya Ito, Douglas Schultz, Ravi Mill, Richard Chen, and Carrisa Cocuzza. 2019. "Task Activations Produce Spurious but Systematic Inflation of Task Functional Connectivity Estimates." NeuroImage 189 (April): 1-18.<br /> Drew, Patrick J. 2019. "Vascular and Neural Basis of the BOLD Signal." Current Opinion in Neurobiology 58 (October): 61-69.<br /> Ito, Takuya, Scott L. Brincat, Markus Siegel, Ravi D. Mill, Biyu J. He, Earl K. Miller, Horacio G. Rotstein, and Michael W. Cole. 2020. "Task-Evoked Activity Quenches Neural Correlations and Variability in Large-Scale Brain Systems." PLoS Computational Biology. https://doi.org/10.1101/560730.<br /> Norman-Haignere, S. V., G. McCarthy, M. M. Chun, and N. B. Turk-Browne. 2012. "Category-Selective Background Connectivity in Ventral Visual Cortex." Cerebral Cortex 22 (2): 391-402.<br /> Rissman, Jesse, Adam Gazzaley, and Mark D'Esposito. 2004. "Measuring Functional Connectivity during Distinct Stages of a Cognitive Task." NeuroImage 23 (2): 752-63.

!- David Hume : book 1 and 2 - book 1 explains what persons are - book 2 explains that persons don't have selves

Reviewer #1 (Public Review):

The authors sought to define the molecular mechanism of activation of the thrombopoietin receptor (TpoR), a very important cytokine receptor that regulates megakaryocyte differentiation and platelet production. They conducted a thorough series of experiments combining mutagenesis experiments with sophistical biological assays and that also includes solid-state NMR structural measurements. This work builds on a body of previous studies of TpoR from this group and from others. They focused both on (1) the role and impact of W515 located in the juxtamembrane cytosolic domain and (2) the impact of introducing either Asn at sites in the transmembrane domain to induce various dimerization modes, or insertion of pairs of Ala residues to induce helical rotation to the TM domain. There is a lot of nice data in this paper, which is fairly intricate - a tough read, but that's because it's a complicated system. The writing is excellent.

This paper presents a model for receptor activation in which the inactive receptor is the monomeric form of the receptor in which the juxtamembrane domain, including W515, maintains a helical structure. Activation of the receptor triggers dimerization of the transmembrane domain and loss of helicity of the juxtamembrane segment, which facilitates optimal interactions of the kinase domains with their JACK2 domain phosphorylation substrates.

There is a lot to like in this careful work and the resulting manuscript. There is one major shortcoming in this manuscript, which concerns W515. It is known that mutation of W515 to any of 17 of the canonical amino acids, including Phe, is sufficient to trigger homodimerization and receptor activation. The authors present some evidence that the phenomenon behind this is that mutation of W515 to almost any other residues disrupts the helical secondary structure of the critical juxtamembrane segment, which promotes dimerization and receptor activation. What I find puzzling is why a Trp at site 515 promotes helix formation, but nearly all other amino acids at this site disrupt helix formation. This strongly suggests the side chain of W515 must be interacting with another domain of the protein in the inactive state, in a manner that is responsible for how Trp stabilizes the juxtamembrane helix which is a central feature that helps define that state. I think that for this paper, this dangling missing piece of their mechanistic model should be resolved.

Reviewer #1 (Public Review):

The manuscript by Xu et. al. does a very thorough characterization and molecular dissection of the role of SSH2 in spermatogenesis. Loss of SSh2 in germ cells results in germ cell arrest In step2-3 spermatids and eventually leads to germ cell loss by apoptosis. Molecular characterization of the mutant mice shows that the loss of SSH2 prevents the fusion of proacrosomal vesicles leading to the formation of a fragmented acrosome. The fragmentation of the acrosome is due to the impaired actin bundling and dephosphorylation of COFILIN. In short, this is a comprehensive body of work.

Reviewer #1 (Public Review):

This study demonstrates that MALPs (Marrow Adipogenic Lineage Progenitors), which were previously described by these authors and constitute approximately 0.5% of bone marrow mesenchymal cells, are major producers of Csf1 (M-CSF) in murine bone marrow. The initial discovery of Csf1 in MALPs occurred during review of scRNA-seq datasets. Here the authors show that deletion of Csf1 in MALPs with AdipoQ-Cre increased trabecular bone mass in long bones, but not vertebrae, and reduced the number of osteoclasts on trabecular bone surfaces. Cortical bone was not altered. Deletion of Csf1 with Adipo-Cre also prevented LPS-induces osteolysis and reduced numbers of hematopoietic progenitors and F4/80+ macrophages. Strengths of this study include use of two CKO lines (Adipo-Cre and Prx-Cre) to understand the relative contribution of MALPs to Csf1 levels in the bone marrow, examination of bone mass in both long bones and vertebrae at several ages, challenging bone responses in Csf1 CKOAdipoQ mice with LPS-induced osteolysis, and studying the effect of Csf1 deletion in MALPs on hematopoiesis and vasculature. Mechanical studies of bone strength were not included but would be necessary to determine if deletion of Csf1 in MALPs is sufficient to cause osteopetrosis. Additional information on other molecular changes Csf1 CKOAdipoQ mice would provide insights into how deletion of Csf1 in MALPs affects bone remodeling. Overall, this is a very important study that will have a high impact on the field because it is challenging the paradigm that osteoblasts and osteocytes are the major sources of M-CSF in the bone marrow.

Reviewer #1 (Public Review):

In the current study, the authors reanalyze a prior dataset testing effects of D2 antagonism on choices in a delay discounting task. While the prior report using standard analysis, showed no effects, the current study used a DDM to examine more carefully possible effects on different subcomponents of the decision process. This approach revealed contrasting effects of D2 blockade on the effect of reward size differences and bias. Effects were uncorrelated, suggesting separate mechanisms perhaps. The authors speculate that these opposing effects explain the variability in effects across studies, since they mean that effects would depend on which of these factors is more important in a particular design. Overall the study is novel and well-executed, and the explanation offers interesting insight into neural processes.

Reviewer #1 (Public Review):

In this manuscript, Richardson et al. describe the repertoire characteristics of Ky mice that carry human immunoglobulin heavy (IgH) and light chain (Igk and l) genes. Immunophenotyping revealed no abnormalities in B cell subsets in the bone marrow, spleen, or lymph nodes of Ky mice (Fig. 1) and the light chain k/l ratio was similar to that observed in humans. Bulk RNA-seq showed some differences in VH, DH, and JH utilization in Ky mice compared to humans (Fig. 2). Ky sequences also had slightly shorter CDRH3 regions (Fig. 3) with a diversity index lying between that of mice and humans (Fig. 4). Use of a novel algorithm further substantiated similarities between Ky mice and human-derived sequences. The authors conclude that Ky mice are suitable to study human immune responses.

Richardson et al. have compiled a comprehensive dataset of Ig sequences from Ky mice to compare with human repertoires. The differences in gene segment utilization are potentially interesting but there is little discussion of the ramifications of these differences during immune responses. They briefly stated that previously published studies of immunizations in Ky mice support their conclusion that these mice respond like humans. However, no details were provided, and it is difficult to assess how similarly Ky mice respond to specific antigens compared to humans. Given the substantial amount of single-cell RNA-Seq data that is now available for responses against the SARS-CoV2 spike, this may be a good antigen to test in Ky mice. Finally, I defer to computational experts to speak to the novelty and validity of conclusions regarding diversity and structural variability in Ky mice compared to humans.

Reviewer #1 (Public Review):

This study will be of interest to those who want to understand how non-pharmaceutical interventions in response to epidemics in human populations (here using the example of SARS-CoV-2 in the Netherlands) might be designed to reduce negative societal impacts through subnational implementation. In most countries, including the Netherlands, non-pharmaceutical interventions such as movement restrictions and school closures were implemented simultaneously throughout entire jurisdictions. The authors of this paper investigated whether subnational heterogeneities in the prevalence of infection could be exploited to develop local level control measures that varied according to local changes in prevalence of infection, an innovation that could potentially reduce negative societal impacts of interventions while maintaining similar levels of epidemiological control. Using simulations from a carefully parameterised agent based model of SARS-CoV-2 transmission in the first wave in Netherlands , the authors generated convincing evidence to suggest that this would be, at least in theory, possible, though practical difficulties of implementing such a control policy were not explored.

Reviewer #1 (Public Review):

The data that is presented is quite clear, and expected given the prior in vitro work, as well as prior work in vivo with helminth infection and BCG vaccination. Overall, it is important to demonstrate that observations from in vitro experiments are relevant in vivo, however, there are concerns with the design of this study which limits its impact. In addition, the study confirms what is expected from prior work, but falls short of adding any new mechanistic insight.

In terms of the in vivo experimental design, it is unclear why the authors chose to administer BCG IP, when the vaccine is given SC (and then based on more recent data, IV could be arguably interesting and relevant). The focus on the peritoneum limits the potential application of these findings to address the important question of the effects of helminth infection on BCG vaccine responses. The ultimate in vivo experiment to be able to demonstrate a physiological relevance of the mechanisms explored here would be to see what the effect was on Mtb infection in the lung.

The authors do report different responses in the spleen and lymphnode, which is interesting, but lines 336-337 accurately point out that compartmentalized overexpression of IL-10 in the spleens but not the lymph nodes has been described in mice with chronic schistosomiasis. Mechanistic insight into this phenomenon was lacking, and the relevance to Mtb infection is still unknown.

Reviewer #1 (Public Review):

COVID-19 severity has been previously linked to a genetic region on chromosome 3 introgressed from Neandertals. The authors use several computational methods to, within this region, identify specific regions that putatively regulate gene expression, and to identify genes within these regions associated with COVID-19 severity. The use of several complementary computational approaches is a major strength of the paper as it bolsters confidence in the findings and narrows the search for significant genomic regions down to most likely candidates. They find 14 genes that exhibit expression regulated by the identified introgressed genomic regions. Among these are several chemokine receptors including two - CCR1 and CCR5 - whose upregulation is associated with severe COVID-19. The authors then use functional genomics to determine whether the identified regions do regulate gene expression.

In contrast to the robustness of the computational findings, the authors' MPRA results are less robust with respect to the significance of the paper to clinical severity of COVID-19. The MPRA shows that the computational methods were reasonably effective at identifying regulatory elements within the introgressed region (53%). The authors then focus on emVars where the H.n. allele differentially regulates expression and identify 4 putative emVars that may regulate expression of CCR1 and CCR5. However, the authors found in their MPRA that these emVars downregulate reporter gene expression, whereas the genes of interest CCR1 and CCR5 are upregulated during severe COVID.

This result highlights the principal weakness of using the MPRA in this context, as it assumes that reporter gene expression using a minimal promoter has identical regulatory determinants as expression of the gene of interest. Its strength is the high-throughput nature of the assay, but its weakness is the lack of specificity with respect to the question at hand. This lack of specificity mitigates the impact of the functional aspect of the work. The authors' computational findings certainly bolster previous work that H.n. introgressed alleles are associated with COVID-19 severity and that this association may be at least partly dependent on gene expression differences between the archaic and modern alleles. However, the specific question at hand, whether chemokine receptor expression is linked to the clinical phenotype, remains unaddressed.

Ultimately the authors results support the conclusions that the 4 emVars identified do regulate gene expression. However, the hypothesis that these specific regions are linked to COVID-19 severity is not supported. The authors' speculation as to why their results may differ from the observed upregulation during disease is intriguing, but lacks support.

Reviewer #1 (Public Review):

Implementation of host-directed therapies (HDTs) could alter the global trajectory of tuberculosis and several HDTs are under investigation. In this manuscript, sertraline (SRT) is proposed as an adjunctive therapy with the mechanism proposed to be due to possible effects on host immune cells. However, the mechanism by which SRT exerts its potentiating effects on Mtb growing in macrophages or in mice is unclear. Schump et al (2017) had previously demonstrated that SRT has a weak anti-tubercular effect in vitro but that this is further enhanced in macrophages simply because SRT acts as a weak base that accumulates in phagosomes. SRT, however, has immune effects as well, as demonstrated by its inhibition of IRF3 dependent gene expression by virtue of its inhibition of PI3K signaling (part of the TLR3 pathway)(Zhu et al., 2010). In this work, a variety of phenotypes are demonstrated for SRT but it is never conclusively demonstrated that the potentiating action of SRT can be ascribed to effects on type I interferon production. It should also be noted that while type I interferon production is generally accepted to promote Mtb growth and dissemination, type I interferons also have a positive role to play in immune regulation (see PMID 29666166). Comparing SRT to inhibitors such as BX795 and Isoliquiritigenin does not establish that the observed effects act through a common mechanism, especially in light of the fact that BX795 has a greater effect on inhibiting IRF3-dependent transcription than SRT but has weaker potentiating effects against Mtb in macrophages. BX795 also inhibits IRF3 transcription but through a different pathway. The role of cGAS/STING is also explored. The cGAS/STING pathway also results in IRF3-dependent signaling but this is through another upstream pathway where the cGAS/STING is activated by dsDNA and bacterial cyclic dinucleotides. Previous studies have shown that cGAS is protective in mice (Collins et al., 2015) which would suggest that inhibition of this pathway and possibly all pathways that mediate IRF3 signaling, would be detrimental to the host. The authors suggest that SRT enhances inflammasome activation but the data supporting this is not convincing and the control Isoliquiritigenin, a NLRP3 inflammasome inhibitor, potentially has other effects. In addition, NLRP3 inflammasomes appear to enhance Mtb growth and spread in host cells (see Beckwith et al. 2020) which would counter the argument that NLRP3 inflammasomes are central to SRT effects. Overall, studies to demonstrate that the activity of SRT can be directly to inhibition of SRT signaling would corroborate the hypothesis that SRT acts as an HDT.

Nevertheless, the fact that SRT has an effect and somehow potentiates the activity of drugs in macrophages and in mice is an important demonstration and a highlight of this work. The demonstration that SRT also acts on Mtb in different physiological states as well as a drug-tolerant clinical strain, is also an important advance.

Reviewer #1 (Public Review):

The purpose of this study was to determine whether heme oxygenase -2 deficiency translates to deficiencies in motor neuron function. This paper plays a plausible mechanism by which heme oxygenase-2 deficiency can lead to obstructive apneas. Indeed, this is among the first papers to comprehensively describe a signaling pathway in motor neurons and the consequences of its deficiency.

The major strengths of this paper include comprehensive pharmacological and genetic methods, and the combination of histology and functional electrophysiological measures of neuronal function. While it is not clear the mechanism by which heme oxygenase-2 deficiency might occur in motor neuron pools, or the relevance to human disease, the authors identify several targetable molecules in the hypoglossal motor neurons that are candidates for future study.

Furthermore, the work completed here may be relevant to other diseases in which motor neuron signal transmission is a key contributor.

Reviewer #1 (Public Review):

The manuscript discussed the combination use of pyrotinib, tamoxifen, and dalpiciclib against HER2+/HR+ breast cancer cells. Through a series of in vitro drug sensitivity studies and in vivo drug susceptibility studies, the authors revealed that pyrotinib combined with dalpiciclib exhibits better therapeutic efficacy than the combination use of pyrotinib with tamoxifen. Moreover, the authors found that CALML5 may serve as a biomarker in the treatment of HER2+/HR+ breast cancer.

The authors provide solid evidence for the following:<br /> 1. The combination use of pyrotinib with dalpiciclib exhibits better therapeutic efficacy than the combination use of pyrotinib with tamoxifen.<br /> 2. Nuclear ER distribution is increased upon anti-HER2 therapy and could be partially abrogated by the treatment of dalpiciclib.<br /> 3. CALML5 may serve as a putative risk biomarker in the treatment of HER2+/HR+ breast cancer.

The manuscript has significant strengths and several weaknesses. The strengths include the identification of the novel role of dalpiciclib in the treatment of HER2+/HR+ breast cancer. Moreover, the authors provide solid evidence that the combined use of dalpiciclib with pyrotinib significantly decreased the total and nuclear expression of ER. The main weakness of the manuscript is that the manuscript is difficult to read due to language inconsistency. In addition, some figure captions and figure legends should be carefully amended.

Reviewer #1 (Public Review):

Prostate cancer is the most common cancer and the second most common cause of cancer death in men. Hence, there continues to be a pressing need for new diagnostic and therapeutic approaches for this disease, as well as better prognostic biomarkers to guide treatment. In this manuscript, Lauer et al. show increased expression of PCA3 and decreased expression of PRUNE2 in prostate cancer compared with the adjacent normal prostate across all tumor grades and stages. And there was no association between the relative gene expression levels of PCA3 or PRUNE2 and time to disease recurrence. These findings suggest a role for PCA3 and PRUNE2 in prostate cancer. And most conclusions of this paper are supported by data.

Reviewer #1 (Public Review):

Sonobe et al provide compelling data on the translation initiation codons required for PR and PG production from the antisense C4G2 repeat expansion associated with C9orf72-ALS/FTD. The strengths of this study are the systematic approach by which the authors identify the start codons. They also provide data investigating if eIF2D is needed for DPR production, building upon previous findings. A major weakness of this work includes the lack of full characterization of their models as they relate to disease, including the need to define any potential toxicity associated with DPR production and any DPR aggregation. Additionally, the study would be strengthened by the quantification and further validation of some of the data presented. Overall, the findings within the study have the potential to be important in advancing our understandings of toxic dipeptide production from the G4C2 repeat expansion in C9orf72-ALS/FTD. This has implications clinically and increases our biological interpretation of this disease.

Reviewer #1 (Public Review):

The manuscript by Prem and colleagues uses neural progenitor cells (NPCs) from individuals with different types of autism (either idiopathic or 16p11.2 deletion) to determine whether cells from these individuals show similar or varying phenotypes. An equal number of control cell lines are also used for a total of 6 autism compared to 6 control lines. The results are surprising in that the NPCs from individuals with autism have common cell biology defects (in neurite outgrowth and cell migration) yet have no overlapping genetic defects between the groups. Using proteomics, the authors also show converging biology at the level of the phospho-proteome with mTOR signaling being affected (albeit in differing directions depending on the idiopathic case). Finally, the authors use a number of pharmacological approaches to show that the various cell biology phenotypes can be rescued or induced (in controls) through manipulation of mTOR signaling. In summary, it is a remarkable result that would seem to indicate that many forms of autism somehow alter mTOR signaling, at least in the NPC stage. I have only a handful of comments about the study:

A sample size of 3 idiopathic seems underpowered relative to the many types of genetic changes that can occur in ASD. Since the authors carried out WGS, it would be useful to know what potential causative variants were found in these 3 individuals and even if not overlapping if they might expect to be in a similar biological pathway. If the authors randomly selected 3 more idiopathic cell lines from individuals with autism, would these cell lines also have altered mTOR signaling? And could a line have the same cell biology defects without a change in mTOR signaling? The authors argue that the sample size could be the reason for lack of overlap of the proteomic changes (unlike the phosphor-proteomic overlaps), which makes the overlapping cell biology findings even more remarkable. Or is the phenotyping simply too crude to know if the phenotypes truly are the same?

Reviewer #1 (Public Review):

The authors used high-throughput nanodroplet-based microfluidics to measure the effect of individual species and the joint effects of species pairs and trios on the growth of six fluorescently labeled strains (E. coli and five soil isolates). In total, over 14,000 bacterial communities composed of subsets of a library of 61 soil and leaf isolates were quantified. They found that the effects of multiple species combine non-additively and are heavily dominated by the strongest single species effect.

Single species showed large variability in the ability to use different carbon sources and survive antibiotics. Interaction assays were carried out in minimal M9 media with 0.5% [w/v] glucose for 24 hours. Authors found that single species' effects on the focal strain is positive in ~1/3 of cases, and is negative in 60% of cases. When two species were added, 3/4 of cases became negative. The similarity of phenotypes (e.g. metabolic profile or phylogeny) between the focal and affecting species did not correlate strongly with the effect on focal species. Joint effects of two or more species are not additive, but rather dominated by the strongest single effect.

The paper presents an interesting example of how complexities of communities may be reduced. The assay combines different growth aspects (lag, rate, yield), and these different aspects should ideally be untangled. The mechanism of why joint effects are dominated by the strongest effect is not known. The generality of the findings awaits further studies (e.g. what will happen if environments are changed?) However, these future directions do not diminish the value of this study.

Reviewer #1 (Public Review):

By careful analyses of single particle images, the authors could identify two distinct structural forms of PSII, a stretched and a compact one. Furthermore, they could analyze a pair of two PSII complexes facing each other on their flat stroma-facing surfaces forming a protein sandwich, which can be made of both the stretched as well as the compact PSII forms. A crucial change in the positioning of the CP29 subunit in the two PSII configurations was identified that favors energy transfer between chlorophylls either to light-harvesting complex II or to the PSII-core antenna complexes. Further aspects like water channels to the oxygen-evolving complex, identification of a Na+ ion, and post-translational modifications were discovered. Overall, the manuscript provides a deep view into the structure of PSII unraveling novel aspects of its structural flexibility and fine details. The experiments and data analysis were conducted very carefully and thoroughly. The work could spark new discussions about the importance of PSII flexibility in photosynthetic membranes.

Reviewer #1 (Public Review):

The goal of the present study was to provide the spatial, morphological, and connectional properties of molecularly defined CeA neurons. The authors provide compelling, high-quality profiling of the spatial distribution of up to 29 molecular markers in the CeA and its surrounding. In addition, by combining their EASI-FISH pipeline with retrograde labeling, the authors offer a comprehensive view of the connectional and molecular diversity within the CeA. The spatial resolution and utility of the EASI-fish technique are showcased elegantly in the present study. The authors also develop a new method to combine EASI-FISH with retrograde labeling to provide information on the anatomical connectivity of molecularly defined cells. Overall, the authors do provide a novel resource for the field. However, the scRNAseq dataset presented at the beginning of the study contains ~1300 cells, which is a low number of cells. From this, the authors report that three of their sequencing clusters (c1, c4, and c12) do not have specific molecular markers and were seemingly excluded from spatial validation. As such, throughout the study, how to interpret the relationship between the sequencing clusters and the molecular clusters may require additional experiments or analyses.

Reviewer #1 (Public Review):

This paper shows that nuclear pore complex components are required for Kras/p53 driven liver tumors in zebrafish. The authors previously found that nonsense mutation in ahctf1 disrupted nuclear pore formation and caused cell death in highly proliferative cells in vivo. In the absence of this gene, there are multiple mitotic functions involving the nuclear pore that are defective, leading to p53 dependent cell death. Heterozygous fish are viable but have reduced kras/p53 liver tumor growth, and this is associated with multiple nuclear and mitotic defects that lead to cancer cell death/lack of growth. This therapeutic window suggests targetability of this pathway in cancer. I think the data are robust, rigorous, and clearly presented. I believe this in vivo work will encourage therapeutic targeting of NPCs in cancer.

Reviewer #1 (Public Review):

The study addresses an important question - how the composition of the microbiota influences the intestinal colonization of encapsulated vs unencapsulated B. theta, an important commensal organism. To answer the question, the authors develop a refurbished WITS with extended mathematical modeling to quantify B. theta population bottlenecks during intestinal colonization in the setting of different microbiota. Interestingly, they show that the colonization defect of an acapsular mutant is dependent on the composition of the microbiota, suggesting (but not proving) that interactions between gut bacteria, rather than with host immune mechanisms, explains why the mutant has a colonization defect. However, it is fairly difficult to evaluate some of the claims because experimental details are not easy to find and the number of animals is very small. Furthermore, some of the analyses and claims are compromised because the authors do not fully explain their data; for example, leaving out the zero values in Fig. 3 and not integrating the effect of bottlenecks into the resulting model, undermines the claim that the acapsular mutant has a longer in vivo lag phase.

Limitations:

1. The experiments do not allow clear separation of effects derived from the microbiota composition and those that occur secondary to host development without a microbiota or with a different microbiota. Furthermore, the measured bottlenecks are very similar in LCM and Oligo mice, even though these microbiotas differ in complexity. Oligo-MM12 was originally developed and described to confer resistance to Salmonella colonization, suggesting that it should tighten the bottleneck. Overall, an add-back experiment demonstrating that conventionalizing germ-free mice imparts a similar bottleneck to SPF would strengthen the conclusions.

2. It is often difficult to evaluate results because important parameters are not always given. Dose is a critical variable in bottleneck experiments, but it is not clear if total dose changes in Figure 2 or just the WITS dose? Total dose as well as n0 should be depicted in all figures.

3. This is in part a methods paper but the method is not described clearly in the results, with important bits only found in a very difficult supplement. Is there a difference between colonization probability (beta) and inoculum size at which tags start to disappear? Can there be some culture-based validation of "colonization probability" as explained in the mathematics? Can the authors contrast the advantages/disadvantages of this system with other methods (e.g. sequencing-based approaches)? It seems like the numerator in the colonization probability equation has a very limited range (from 0.18-1.8), potentially limiting the sensitivity of this approach.

4. Figure 3 and the associated model is confusing and does not support the idea that a longer lag-phase contributes to the fitness defect of acapsular B.theta in competitive colonization. Figure 3B clearly indicates that in competition acapsular B. theta experiences a restrictive bottleneck; i.e., in competition, less of the initial B. theta population is contributed by the acapsular inoculum. There is no need to appeal to lag-phase defects to explain the role of the capsule in vivo. The model in Figure 3D should depict the acapsular population with less cells after the bottleneck. In fact, the data in Figure 3E-F can be explained by the tighter bottleneck experienced by the acapsular mutant resulting in a smaller acapsular founding population. This idea can be seen in the data: the acapsular mutant shedding actually dips in the first 12-hours. This cannot be discerned in Figure 3E because mice with zero shedding were excluded from the analysis, leaving the data (and conclusion) of this experiment to be extrapolated from a single mouse.

5. The conclusions from Figure 4 rely on assumptions not well-supported by the data. In the high fat diet experiment, a lower dose of WITS is required to conclude that the diet has no effect. Furthermore, the authors conclude that Salmonella restricts the B. theta population by causing inflammation, but do not demonstrate inflammation at their timepoint or disprove that the Salmonella population could cause the same effect in the absence of inflammation (through non-inflammatory direct or indirect interactions).

6. Several of the experiments rely on very few mice/groups.