AssayResult: 0.1

AssayResultAssertion: Abnormal

ReplicateCount: 19

StandardErrorMean: 0.1

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1. (Personal communication: A. Glazer)

AssayResult: 86.7

AssayResultAssertion: Normal

ReplicateCount: 28

StandardErrorMean: 8.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0.7

AssayResultAssertion: Abnormal

ReplicateCount: 17

StandardErrorMean: 0.6

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 115.6

AssayResultAssertion: Normal

ReplicateCount: 19

StandardErrorMean: 24.7

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

HGVS: NM_198056.2:c.1003T>C p.(Cys335Arg)

AssayGeneralClass: BAO:0010044 targeted transcriptional assay

AssayMaterialUsed: BTO:0000773 lymphoblastoid cell line derived from control individuals or individuals with germline TP53 variants

AssayDescription: Comparative transcriptomic analysis using RNA-Seq to compare EBV cell lines of wild type and pathogenic TP53 in the context of genotoxic stress induced by doxorubicin treatment. 10 biomarkers corresponding to p53 targets were measured to determine a functionality score.

AdditionalDocument: PMID: 23172776

AssayReadOutDescription: In the treated condition, the peak height of each of the 10  p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition. In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays.

AssayRange: An arbitrary functionality score was calculated from the induction score of the 10 p53 targets.

AssayNormalRange: N/A

AssayAbnormalRange: N/A

AssayIndeterminateRange: N/A

AssayNormalControl: wild type TP53

AssayAbnormalControl: LFS patient cells

ValidationControlPathogenic: 8 Individuals with dominant-negative TP53 missense variants, 10 Individuals with null TP53 variants, and 13 Individuals with other TP53 missense variants

ValidationControlBenign: 3 patients with wild type TP53

Replication: experiments were performed in triplicates.

StatisticalAnalysisDescription: Differentially expressed genes between doxorubicin-treated and untreated cells were arbitrarily defined using, as filters, a P<0.01 and fold-change cutoffs >2 or <2, for up and down regulation, respectively. The resultant signal information was analyzed using one-way analysis of variance (ANOVA, P= 0.001), assuming normality but not equal variances with a Benjamani–Hochberg correction for multiple comparisons using three groups: controls, null, and missense mutations.

SignificanceThreshold: P=0.001

Comment: statistical analysis and P value from previous publication.

Reviewer #1 (Public Review):

In this work, Panigrahi et. al. develop a powerful deep-learning-based cell segmentation platform (MiSiC) capable of accurately segmenting bacteria cells densely packed within both homogenous and heterogeneous cell populations. Notably, MiSiC can be easily implemented by a researcher without the need for high-computational power. The authors first demonstrate MiSiC's ability to accurately segment cells with a variety of shapes including rods, crescents and long filaments. They then demonstrate that MiSiC is able to segment and classify dividing and non-dividing Myxococcus cells present in a heterogenous population of E. coli and Myxococcus. Lastly, the authors outline a training workflow with which MiSiC can be trained to identify two different cell types present in a mixed population using Myxococcus and E. coli as examples.

While we believe that MiSiC is a very powerful and exciting tool that will have a large impact on the bacterial cell biological community, we feel explanations of how to use the algorithm should be more greatly emphasized. To help other scientists use MiSiC to its fullest potential, the range of applications should be clarified. Furthermore, any inherent biases in MiSiC should be discussed so that users can avoid them.

Major Concerns:

1) It is unclear to us how a MiSiC user should choose/tune the value for the noise variance parameter. What exactly should be considered when choosing the noise variance parameter? Some possibilities include input image size, cell size (in pixels), cell density, and variance in cell size. Is there a recommended range for the parameter? These questions along with our second minor correction can be addressed with a paragraph in the Discussion section.

2) Could the authors expand on using algorithms like watershed, conditional random fields, or snake segmentation to segment bacteria when there is not enough edge information to properly separate them? How accurate are these methods at segmenting the cells? Should other MiSiC parameters be tuned to increase the accuracy when implementing these methods?

3) Can the MiSiC's ability to accurately segment phase and brightfield images be quantitatively compared against each other and against fluorescent images for overall accuracy? A figure similar to Fig. 2C, with the three image modalities instead of species would nicely complement Fig. 2A. If the segmentation accuracy varies significantly between image modalities, a researcher might want to consider the segmentation accuracy when planning their experiments. If the accuracy does not vary significantly, that would be equally useful to know.

4) The ability of MiSiC to segment dense clusters of cells is an exciting advancement for cell segmentation algorithms. However, is there a minimum cell density required for robust segmentation with MiSiC? The algorithm should be applied to a set of sparsely populated images in a supplemental figure. Is the algorithm less accurate for sparse images (perhaps reflected by an increase in false-positive cell identifications)? Any possible biases related to cell density should be noted.

5) It is exciting to see the ability of MiSiC to segment single cells of M. xanthus and E. coli species in densely packed colonies (Fig. 4b). Although three morphological parameters after segmentation were compared with ground truth, the comparison was conducted at the ensemble level (Fig. 4c). Could the authors use the Mx-GFP and Ec-mCherry fluorescence as a ground truth at the single cell level to verify the results of segmentation? For example, for any Ec cells identified by MiSiC in Fig. 4b, provide an index of whether its fluorescence is red or green. This single-cell level comparison is most important for the community.

Reviewer #1 (Public Review):

In this paper, authors did a fine job of combining phylogenetics and molecular methods to demonstrate the parallel evolution across vRNA segments in two seasonal influenza A virus subtypes. They first estimated phylogenetic relationships between vRNA segments using Robinson-Foulds distance and identified the possibility of parallel evolution of RNA-RNA interactions driving the genomic assembly. This is indeed an interesting mechanism in addition to the traditional role for proteins for the same. Subsequently, they used molecular biology to validate such RNA-RNA driven interaction by demonstrating co-localization of vRNA segments in infected cells. They also showed that the parallel evolution between vRNA segments might vary across subtypes and virus lineages isolated from distinct host origins. Overall, I find this to be excellent work with major implications for genome evolution of infectious viruses; emergence of new strains with altered genome combination.

Comments:

I am wondering if leaving out sequences (not resolving well) in the phylogenic analysis interferes with the true picture of the proposed associations. What if they reflect the evolutionary intermediates, with important implications for the pathogen evolution which is lost in the analyses?

Lines 50-51: Can you please elaborate? I think this might be useful for the reader to better understand the context. Also, a brief description on functional association between different known fragments might instigate curiosity among the readers from the very beginning. At present, it largely caters to people already familiar with the biology of influenza virus.

Lines 95-96 Were these strains all swine-origin? More details on these lineages will be useful for the readers.

Lines 128-132: I think it will be nice to talk about these hypotheses well in advance, may be in the Introduction, with more functional details of viral segments.

Lines 134-136: Please rephrase this sentence to make it more direct and explain the why. E.g. "... parallel evolution between PB1 and HA is likely to be weaker than that of PB1 and PA" .

Lines 222-223: Please include a set of hypotheses to explain you results? Please add a perspective in the discussion on how this contribute might to the pandemic potential of H1N!?.

Lines 287-288: I am wondering how likely is this to be true for H1N1.

Reviewer #1 (Public Review):

In this paper, the authors tried to investigate complex roles of immune cells during acute myocardial infarction (AMI) by examining immune cells in blood samples from acute coronary syndrome (ACS) patients. They found an increase in the circulating levels of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the blood of ACS patients compared to healthy people, all of which were correlated with elevated levels of inflammatory markers in serum. Those findings were then further explored at a mechanistic level by using in vitro and in vivo experiments. Interestingly, the researchers also found that high cytomegalovirus (CMV) antibody titers could affect the immunoregulatory mechanisms in AMI patients. Taken together, the findings of the researchers could potentially contribute to the development of a more effective strategy to prevent cardiac deterioration and cardiovascular adverse events after AMI.

Strengths:

This paper contains novel insight regarding role of neutrophil and monocyte subset in pathophysiology of AMI. Although the increased level of CD10neg subsets of neutrophils in AMI patients has recently been reported (Marechal, P., et al. 2020. Neutrophil phenotypes in coronary artery disease. Journal of Clinical Medicine), the current paper aptly complemented the previous findings obtained by using its in vitro and in vivo mice model. This study also has robust methods to support their conclusion.

Weakness:

To further improve the strength of their conclusion, the experiments investigating the effects of immunoregulatory function of immature neutrophils and HLA-DRneg/low monocytes subsets would be advised.

Reviewer #1 (Public Review):

The manuscript by He et al. reveals a novel role for PKC-theta, following T cell receptor (TCR) stimulation, in regulating the nuclear translocation of several key activation-dependent transcription factors by regulating the assembly of key components of the nuclear pore complex (NPC). The authors make use of T cell lines and primary T cells to show that following TCR stimulation, PKC-theta phosphorylates RanGAP1 to promote its interaction with Ubc9 and increase the sumoylation of RanGAP1, which, in turn, enhances assembly of the RanBP2 subcomplex of the NPC that then promotes the nuclear import of AP-1, NFAT and NFB. These conclusions are well supported by a rigorous experimental approach, which included the use of PKC-theta deficient, sumoyltion-defective, kinase-dead, and constitutively active mutants, and RanGAP1-deficient cells.

Reviewer #1 (Public Review):

This paper uses a large breeding colony of guppies to measure genetic correlations between hormonal stress responses and behavior in an open-field test. Although we know a lot about the mechanisms of hormone-mediated behavior, we know less about variation in hormonal systems, particularly genetic variation. Understanding how hormones relate genetically to the behaviors they mediate is particularly important because it helps us understand how the entire hormone-behavior system evolves. A priori, we would expect genetic correlations between hormones and the behavior they underlie, such that selection on the hormone would lead to a response in the behavior and vice versa. However, evidence for this pattern is rare.

Here, the authors show that stress-induced levels of cortisol are repeatable and heritable. Interestingly, they also show that individuals show a lower stress response to later stress and slightly less variation, indicating a G X E interaction. There was a significant genetic correlation between the hormonal response and one of the behaviors measured in the open field test, and the hormone loaded positively in the first genetic principal component along with all the behaviors. This is evidence of an correlated suite of traits that would evolve together in response to selection.

This is an important study, because evidence of genetic variation in hormonal systems, not to mention genetic covariation with hormone-mediated traits, is rare. The results presented here provide insight into how a hormone-behavior complex might adapt to a changing environment. They are also relevant to ideas about the maintenance of variation in coping styles in natural populations.

Reviewer #1 (Public Review):

Using various voltage and concentration protocols in a heterologous expression system, the authors provide compelling evidence for strong block of GluR1 AMPA receptors by intracellular NASPM, and unlike spermine, the block is independent of auxiliary subunit expression. The authors also show that intracellular NASPM provides a more complete block than spermine of synaptic currents in GluR2-KO neurons.

Overall the manuscript contains high quality data that is clearly presented. It seems likely that this approach will be useful for assessing the contribution of CP-AMPARs in various scenarios. However, currently the authors have fallen short of providing a comprehensive analysis of the use of NASPM to differentiate between CP and CI AMPARs in intact systems containing multiple AMPAR subunits and auxiliary proteins.

Reviewer #1 (Public Review):

In "Asymptomatic Bordetella pertussis infections in a longitudinal cohort of young African infants and their mothers", the authors analyze longitudinal data from a cohort in Zambia of infant/mother pairs to investigate the evidence for subclinical and asymptomatic infections in both pairs as well as the use of IS481 qPCR cycle threshold (CT) values in providing evidence for pertussis infection. Overall, the manuscript lacks substantial statistical support or clear evidence of some of the patterns they are stating and would require a substantial revision to justify their conclusions. The majority of the manuscript relies on 8 infant/mother pairs where they have evidence of pertussis infection and rely on the dense sampling to investigate infection dynamics. However, this is a very small sample size and further, based on the results displayed in Figure 1, it is not obvious that the data has a very pattern that warrant their assertions.

Major comments:

The main results and conclusions are highly reliant on details from eight mother/infant pairs. However, Figure 1 does not show a clear picture of the fade-in/fade-out. The authors go into great detail describing each of these 8 pairs, however based on the figure and text there does not appear to be clear evidence of an underlying pattern. While there are some instances with a combination of higher/lower IS481 CT values, it does not appear to have a clear pattern. For example, what are possible explanations for time periods between samples with evidence of IS481 and those without (such as pair A, C, D, E, F and H)? There also does not appear to be a clear pattern of symptoms in any of these samples (aside from having fewer symptoms in the mothers than infants). Further, it is not obvious how similar these observed (such as a mixture of times of high or low values often preceded or followed by times when IS481 was not detected) is similar to different to the rest of the cohort (in contrast to those who have a definitive positive NP sample during a symptomatic visit). The main results are primarily a descriptive analysis of these 8 mother/infant pairs with little statistical analyses or additional support.

The authors do not provide evidence or detail about what is known about the variability in IS481 CT values, amongst individuals, or over time, or pre/post vaccination. Without this information, it is not clear how informative some of this variability is versus how much variability in these values is expected. I think particularly in Figure 1, how many of the individuals have periods between times when IS481 evidence was observed when it was not observed, is concerning that these data (at this granular a level) are measuring true infection dynamics. Adding in additional information about the distribution and patterns of these values for the other cohort members would also provide valuable insight into how Figure 1 should be interpreted in this context. As it stands, the authors do not provide sufficient interpretation and evidence for having relevant infection arcs.

It appears that Figure 2A was created using only 8 data points (from the infant data values). If so, this level of extrapolation from such few data points does not provide enough evidence to support to the results in the text (particularly about evidence for fade-in/fade-out population-level dynamics). Also, in Figure 2, it is not clear to me the added value of Figure 2C and the main goal of this figure.

The authors have created a measure called, evidence for infection (EFI), which is a summary measure of their IS481 CT values across the study. However, it is not clear why the authors are only considering an aggregated (sum) value which loses any temporality or relationship with symptoms/antibiotic use. For example, the values may have been high earlier in the study, but symptoms were unrelated to that evidence for infection - or visa versa. This seems to be an important factor - were these possible undiagnosed, asymptomatic, or mild symptomatic pertussis infections? It is not clear why the authors only focus on a sum value for EFI versus other measures (such as multiple values above or below certain thresholds, etc.) to provide additional support and evidence for their results.

It is not clear why the authors have emphasized the novelty and large proportion of asymptomatic infections observed in these data. For example, there have been household studies of pertussis (see https://academic.oup.com/cid/article-abstract/70/1/152/5525423?redirectedFrom=PDF which performed a systematic review that included this topic) that have also found such evidence. While cross-sectional surveys may be commonly used in practice, it is not clear that there is no other type of study that provides any evidence for asymptomatic infections. Further, it is not clear why the authors refer to widespread asymptomatic pertussis when a large proportion of individuals with evidence for pertussis infection had symptoms. Would it not be undiagnosed pertussis if it is associated with clinical symptomatology?

Reviewer #1 (Public Review):

Galdadas et al. applied a combinatorial approach of equilibrium and nonequilibrium molecular dynamics methods to study two important members of the Class A β-lactamase enzyme family in detail. Authors carefully chose two representative enzymes from this family, TEM-1 and KPC-2 in this study. Understanding of the nature of the communication pathways between allosteric ligand binding site and the active site has been the main focus of this study. Another very interesting finding of this study was the position of clinical variants that was precisely mapped along the allosteric communication pathway. This approach certainly has broad utility as it can be applied to study long-range communications in enzymes that are triggered by binding of a ligand (drug candidate) to an alternative/remote site, and also in cases where certain mutations occur far away from the active site but lead to drug resistance.

Overall, the manuscript is well written, and the conclusions are mostly well supported by data.

Reviewer #1 (Public Review):

The authors aimed to survey a large transfusion database in Sweden to catalog associations between ABO/RhD blood group antigens and a wide variety of clinical phenotypes in a systematic, unbiased and comprehensive manor. They succeed at surveying over 1200 phenotypes in over 5 million people and identify 49 statistically significant associations for ABO blood group and point out a couple novel associations. Their statistical methods are appropriate and help eliminate potential false positive associations. The strengths of this study are the unbiased survey of a large database and the appropriate corrections for multiple observations which allow the authors to explore a large number of associations without loosing site of what is really a significant association.

This study sheds light on a topic of interest to many scientists. The ABO gene encodes a glycosyltransferase enzyme that has 4 major haplotypes in human populations and results in a specific pattern of posttranslational modification of plasma proteins and blood cells including erythrocytes. Proteins decorated with an H antigen can receive additional carbohydrate antigens from ABO transferase intracellularly. The common A allele transfers UDP-GalNAc while the B allele transfers UDP-Gal. The A2 allele is hypomorphic compared to the A allele and transfers lower amounts of UDP-GalNAc and the common O allele is a null resulting in no transferase activity.

The allele frequencies of these common alleles varies by ancestry and has geographic differences. Variation at ABO is unconstrained with many rare variants contributing to the four common haplotypes at ABO. Interestingly, geographically specific selective pressures may have led to allele frequency differences. For example. ~40-50% of individuals are homozygous for the null (type O) allele. These null haplotypes are more common in individuals of Latino or African ancestry while 'A' haplotypes are slightly more common in individuals of European origin and 'B' alleles are more common in individuals of Asian and African ancestry. Overall, O is more common than A or B alleles. An unbiased survey of phenotype frequencies by blood type allows for confirmation of previous associations and discovery of novel associations. In this largely European ancestry cohort, blood type A is the most common (45-47%) while blood type O is second most common at 38-39%.

Limitations of Phenome-wide Association Studies (PheWAS) like the one presented in this manuscript should be noted. Associations with complex phenotypes or those with small effect size will not be detected even in a large cohort such as the SCANDAT. This study is also biased toward associations with phenotypes more common in the Scandinavian population. This may present associations related to the population substructure and not a direct association with ABO. In genome-wide association studies this can be addressed through multiple methods but it is not clear how the authors correct for population structure in this study. Likewise, the insight into the mechanistic reasons for ABO associations is not a strength of this study and will await subsequent studies for many phenotypes. Mechanistic insight might be particularly interesting for the novel associations uncovered by this study.

Reviewer #1 (Public Review):

In this manuscript, the authors use single cell RNA sequencing to investigate cell-type specific eQTL within C. elegans. This relies on the well known ability to genotype individuals via their transcriptome allowing the authors to generate both phenotypes and genotypes from single cell transcriptomes. This identifies a blend of cis and trans-eQTL that are cell type specific and starts to provide numerical observations to the communities expectation of cell type specificity.

The use of simultaneous single cell sequencing on a diversity of individuals is a unique method that is absolutely essential to get around the vast scale issues that are presented when contemplating single cell eQTL within multicellular organisms. However, an unfortunate outcome of this approach that the cell-autonomy of the eQTL cannot be studied. Instead the cell types have to be considered completely independent of each other.

The authors conduct an analysis of eQTL per each cell type to get at specificity. This identifies a number of eQTL found in only a single cell type but these binary tests can have an ascertainment issue that may be over-estimating the cell type specificity. Optimally, this would be conducted by incorporating the different cell types as different environments within a single eQTL model but given the different sample sizes, this may not be feasible. Alternatively an investigation of how eQTLs specific to one cell type are or are not found by shifting the detection threshold in the other tissues could test this possibility.

Reviewer #1 (Public Review):

This manuscript describes the curation of a training dataset, CEM500K, of cellular electron microscope (EM) data including STEM, TEM of sections, electron tomography, serial section and array tomography SEM, block-face and focused-ion beam SEM. Using CEM500K to train an unsupervised deep learning algorithm, MoCoV2, the authors present segmentation results on a number of publically available benchmark datasets. They show that the standard Intersection-over-Union scores obtained with the CEM500K-trained MoCoV2 model, referred to as CEM500K-moco, equal or exceed the scores of benchmark segmentation results. They also demonstrate the robustness of CEM500K-moco's performance with respect to input image transformations, including rotation, Gaussian blur and noise, brightness, contrast and scale. The authors make the remarkable discovery that MoCoV2 spontaneously learned to use organelles as "landmarks" to identify important features in images, simulating human behavior to some degree.

Reviewer #1 (Public Review):

The submitted manuscript 'Distinct higher-order representations of natural sounds in human and ferret auditory cortex' by Landemard and colleagues seeks to investigate the neural representations of sound in the ferret auditory cortex. Specifically, they examine the stages of processing via manipulating the complexity and sound structure of stimuli. The authors create synthetic auditory stimuli that are statistically equivalent to natural sounds in their cochlear representation, temporal modulation structure, spectral modulation structure, and spectro-temporal modulation structure. The authors use functional ultrasound imaging (fUS) which allowed for the measurement of the hemodynamic signal at much finer spatial scales than fMRI, making it particularly suitable for the ferret. The authors then compare their results to work done in humans that has previously been published (e.g. Norman-Haignere and McDermott, 2018) and find that: 1. While human non-primary auditory cortex demonstrates a significant difference between natural speech/music sounds and their synthetic counterparts, the ferret non-primary auditory cortex does not. 2. For each sound manipulation in humans, the dissimilarity increases as the distance from the primary auditory cortex increases, whereas for ferrets it does not. 3. While ferrets behaviorally respond to con-specific vocalizations, the ferret auditory cortex does not demonstrate the same hierarchical processing stream as humans do.

Overall, I find the approach (especially the sound manipulations) excellent and the overall finding quite intriguing. My only concern, is that it is essentially a null-result. While this result will be useful to the literature, there is always the concern that a lack of finding could also be due to other factors.

Major points:

1) What if the stages in the ferret are wrong? The authors use 4 different manipulations thought to reflect key elements of sound structure and/or the relevant hierarchy of the processing stages of the auditory cortex, but it's possible that the dimensions in the ferret auditory cortex are along a different axis than spectro/temporal modulations. While I do not expect the authors to attempt every possible axis, it would be beneficial to discuss.

2) For the ferret vocalizations, it is possible that a greater N would allow for a clearer picture of whether or not the activation is greater than speech/music? While it is clear that any difference would be subtle and probably require a group analysis, this would help settle this result/issue (at least at the group level).

3) Relatedly, did the magnitude of this effect increase outside the auditory cortex?

4) It would be useful to have a measure of the noise floor for each plot and/or species for NSE analyses. This would make it easier to distinguish whether, for instance, in 2A-D, an NSE of 0.1 (human primary) vs. an NSE of 0.042 (ferret primary) should be interpreted as a bit more than double, or both close to the noise floor (which is what I presume).

Reviewer #1 (Public Review):

In this study, the authors set out to address the interesting question of how activating septal cholinergic neurons affects learning and memory of reward locations. The work provides compelling evidence showing that activation of septal cholinergic cells at reward zones suppresses sharp wave-ripples and impairs memory performance in freely behaving animals. The data are properly controlled and analyzed, and the results support the conclusions. The results shed new light on the functional significance of cholinergic projections in reward learning. Future follow-up studies designed to selectively activate cholinergic projections specifically at times when sharp wave-ripples occur will be interesting to determine the importance of cholinergic sharp wave-ripple suppression for these effects.

Reviewer #1 (Public Review):

Here, Houri-Ze'evi, et al. treated progeny of parents that had inherited small RNA response (silencing of an artificial, single-copy GL-expressed gfp with anti-gfp dsRNA) with 3 stresses – heat shock, hyperosmolarity, or starvation – starting at L1, and examined gfp silencing. All three treatments reduced silencing (visible as increased GFP fluorescence) in subsequent generations (F1-F3). The authors tested resetting of endogenous (endo-siRNA) and piRNAs using an endo-siRNA sensor target sequence and piRNA recognition sites, respectively. Again, all 3 stressors reset both in the same generation, but did not reset the effect transgenerationally, suggesting that exogenous RNAi resetting functions through a different mechanism than endogenous.

Next, they tested adults, which also led to resetting. However, only the F1 generation, not F2, is susceptible to resetting (how? Why?), revealing a critical period for resetting susceptibility. Reversal of the stress with RNAi treatment does not result in resetting, nor does simpy changing conditions. The authors then went on to examine mutants that might be defective in stress responses or in resetting; MAPK genes and skn-1 are required for resetting. Small RNA-seq from stressed worms and their progeny showed a decrease overall with stresses, and reveals some potential classes of genes, including targets of the mutator genes, and overlap with classic stress response pathways (dauer, IIS). Overall, this work presents some interesting phenomena and moves towards explaining how it might work through the identification of a critical period and some genes that are required.

In this version, the authors have added more information regarding the relationship between MAPK and SKN-1, and transcriptional targets. Most importantly, they have performed tissue-specific rescue of sek-1; in neurons, this rescues, but intestine did not.

These data add to prior work from the Rechavi lab and others in the field, which together address the interplay of small RNAs, response to stress, and transgenerational inheritance.

Reviewer #1:

Single molecule localization microscopy (SMLM) has become an important method for understanding the subcellular distribution of fluorescently labelled biomolecules at length scales of a few tens of nanometers. A critical challenge has been to find out, whether and to what extent biomolecular clustering occurs. While methods have been published which address the problem of identifying biomolecular clusters in SMLM images, they still suffer from many user-defined parameters, which - if selected inappropriately - influence the obtained results substantially. The StormGraph-3D method proposed here addresses these issues, based on a comprehensive mathematical framework which reduces the number of user-defined input parameters. The method was evaluated using comprehensive simulations of data, which show its robustness compared to alternative approaches.

The methods part of the paper would benefit, however, from more realistic data of single molecule blinking behavior, and the evaluation of the consequences on the performance of the method. As the authors acknowledge, overcounting due to blinking has challenged data analysis previously, and gave rise to artifactual localization clusters that do not represent the underlying protein distribution. It would be of particular interest, which results in the method yielded for a random biomolecular distribution.

Reviewer #1:

The authors of Working Memory Gates Visual Input to Primate Prefrontal Neurons studied how working memory influences information transmitting from V4 to frontal eye field via extracellular recording and electrical stimulation on behaving primate. They found that V4 neurons target FEF neurons with both visual and motor properties, and its synaptic efficacy of V4 to FEF was enhanced by working memory. These findings are interesting and important to our understanding about how our brain acts during daily WM-related activity.

1) In classical working memory tasks, the task periods usually consist of fixation, cue, delay and then a response period. The neural activity during the delay period is typically considered to be a working memory-related signal. However, in the current study, the authors didn't point out whether only delay period activity was included in analysis when they compared synaptic efficacy between stimulation and non-stimulation trials, in Figure 4a. Because the differences of neuronal response during fixation period cannot be viewed as relevant to information held in working memory, it may be better if only neuronal activity in the delay period was included in their analysis.

2) Did the 96 visual-recipient FEF neurons exhibit working memory-related activity in their memory guided saccade task? The example neuron in Figure 3a didn't show significant difference between In and Out trials during the delay period. If the visual-recipient neuron didn't present working memory related activity, how could the authors say enhanced synaptic efficacy from V4 to FEF was caused by working memory?

3) Did the two example neurons in Figure 4c show adjusted values (subtracting the same measure during non-stimulated trials)? The authors mentioned in Method that Figure 4 showed adjusted values, but it may not be applicable for raster plot in Figure 4c. It may be helpful that using adjusted values show stimulation effects on evoked spike counts during memory In and Out trials.

4) Did the authors find some FEF cells showing elevated firing during delay period in outside-RF trials compared with baseline firing? These elevated firing was not caused by RF cue, may underlying working memory signal.

5) The sample size should be indicated in Figure 3b Venn diagram.

6) It's better to indicate electrical stimulus protocol in Figure 1.

Reviewer #1:

The present study sought to better characterize how listeners deal with competing speech streams from multiple talkers, that is, whether unattended speech in a multitalker environment competes for exclusively lower-level acoustic/phonetic resources or whether it competes for higher-level linguistic processing resources as well. The authors recorded MEG data and used hierarchical frequency tagging in an unattended speech stream presented to one ear while listeners were instructed to attend to stories presented in the other ear. The study found that when the irrelevant speech contained structured (linguistic) content, an increase in power at the phrasal level (1 Hz) was observed, but not at the word level (2 Hz) or the sentence level (0.5 Hz). This suggests that some syntactic information in the unattended speech stream is represented in cortical activity, and that there may be a disconnect between lexical (word level) processing and syntactic processing. Source analyses of the difference between conditions indicated activity in left inferior frontal and left posterior parietal cortices. Analysis of the source activity underlying the linear transformation of the stimulus and response revealed activation in the left inferior frontal (and nearby) cortex. Implications for the underlying mechanisms (whether attentional shift or parallel processing) are discussed. The results have important implications for the debate on the type and amount of representation that occurs to unattended speech streams.

The authors utilize clever tools which arguably provided a unique means to address the main research question, i.e., they used hierarchical frequency tagging for the distractor speech, which allowed them to assess linguistic representations at different levels (syllable-, word-, phrase-, and sentence-level). This technique enabled the authors to make claims about what level of language hierarchy the stimuli are being processed, depending on the observed frequency modulation in neural activity. These stimuli were presented during MEG recording, which let the authors assess changes in neurophysiological processing in near real time--essential for research on spoken language. Source analyses of these data provided information on the potential neural mechanisms involved in this processing. The authors also assessed a temporal response function (TRF) based on the speech envelope to determine the brain regions involved at these different levels for linguistic analysis of the distractor speech.

Critiques:

Speech manipulation:

In general, it is unclear what predictions to make regarding the frequency tagging of the unattended distractor speech. On the one hand, the imposed artificial rhythmicity (necessary for the frequency tagging approach) may make it easier for listeners to ignore the speech stream, and thus seeing an effect at higher-level frequency tags may be of greater note, although not entirely plausible. On the other hand, having the syllables presented at a consistent rate may make it easier for listeners to parse words and phrasal units because they know precisely when in time a word/phrase/sentence boundary is going to occur, allowing listeners to check on the irrelevant speech stream at predictable times. For both the frequency tagging and TRF electrophysiological results, the task-irrelevant structured speech enhancement could be interpreted as an infiltration of this information in the neural signal (as the authors suggest), but because the behavioral results are not different this latter interpretation is not easily supported. This pattern of results is difficult to interpret.

Behavioral Results:

Importantly, no behavioral difference in accuracy was observed between the two irrelevant speech conditions (structured vs. non-structured), which makes it difficult to interpret what impact the structured irrelevant speech had on attentive listening. If the structured speech truly "infiltrates" or "competes" for linguistic processing resources, the reader would assume a decrease in task accuracy in the structured condition. This behavioral pattern has been observed in other studies. This calls into questions the face validity of the stimuli and task being used.

Attention:

In this study activation of posterior parietal cortex was found, that could be indicative of a strong attentional manipulation, and that the task was in fact quite attentionally demanding in order for subjects to perform. This may align with the lack of behavioral difference between structured and non-structured irrelevant stimuli. Perhaps subjects attempted to divide their attention which may have been possible between speech that was natural and speech that was rather artificial. The current results may align with a recent proposal that inferior frontal activity may be distinguished by language selective and domain general patterns.

Lack of word level response:

A major concern is that the results do not seem to replicate from an earlier study with the same structured stimuli, i.e., the effects were seen for sentence and word level frequency tagging. As the authors discuss, it seems difficult to understand how a phrasal level of effect could be obtained without word-level processing, and so a response at the word level is expected.

Familiarization phase:

The study included a phase of familiarization with the stimuli, to get participants to understand the artificial speech. However it would seem that it is much easier for listeners to report back on structured rather than unstructured stimuli. This is relevant to understanding any potential differences between the two conditions. It is unclear if any quantification was made of performance/understanding at this phase. If there is no difference in the familiarization phase, this might explain why there was no difference in behavior during the actual task between the two conditions. Or, if there is a difference at the familiarization phase (i.e. structured sequences are more easily repeated back than non-structured sequences), this might help explain the neural data result at 1 Hz, given that some higher level of processing must have occurred for the structured speech (such as "chunking" into words/phrasal units).

Joint Public Review:

Eisele et al. evaluated the direct action of erythropoietin (EPO) on hematopoietic stem and progenitor cells that included hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). They used cellular barcoding to enable in vivo tracking of cellular output and then used scRNA-seq to corroborate their findings. They observed the transiently promoted output of Myeloid-Erythroid (ME)-biased and Myeloid-B-cell (MB)-biased clones. Single-cell RNA sequencing analysis revealed that EPO acted mostly on MPP1 and MPP2. Based on these data, the authors concluded that EPO acts directly on MPPs and transiently modulates their output. Although the conceptual advance brought by this study is incremental as similar findings have been presented by previous studies, the integration and use of both barcoding and scRNA-seq adds strength to the conclusions reached in the present study.

Reviewer #1 (Public Review):

In this work, the authors set out to better understand the mechanisms by which the nematode C. elegans responds to bacterial pathogens.

Using behavioral assays and genetic manipulations, the authors find that C. elegans can rapidly learn to avoid the pathogen E. faecalis (E.f.). While recent studies from other groups have shown that small RNAs (sRNAs) produced by some pathogenic bacteria can trigger aversive learning, the authors find that this seems not to be the case for E. faecalis. Instead, they provide evidence that E. faecalis causes abdominal distention, and that this may provide the trigger for learning. Because the evidence for this is largely correlative, alternative explanations may still be possible. Further, the authors identify two TRPM-class ion channels whose function appears to be necessary for learned avoidance of E.f. The authors propose that one or both of these may mediate detection of abdominal distention, an interesting idea that merits further study. While the paper's title indicates that these channels "mediate" this function, this remains speculative.

The authors also find that wild-type C. elegans prefer olfactory stimuli from E.f. to those of their regular diet, E. coli, but that this pattern is reversed after exposure to E.f. This plasticity involves the function of the chemosensory neurons ASE, AWC, and AWB, as well as the cyclic-nucleotide-gated channel TAX-2/TAX-4. This finding provides important insight into the nature of the changes in neural circuit function that are triggered by pathogen exposure, leading to pathogen avoidance.

The paper also examines a role for the neuropeptide receptor npr-1 in learned E.f. avoidance. Animals lacking npr-1 function are known to strongly avoid high (ambient) oxygen concentrations, and instead prefer the lower-oxygen environment of a bacterial lawn. The authors find that this oxygen avoidance overcomes any avoidance of E.f.; thus, npr-1 mutants do not avoid E.f. when tested with ambient oxygen, but they do avoid it in a low-oxygen environment. This indicates that npr-1 is not required for pathogen avoidance per se. Although the authors suggest that npr-1 may be a target of the learning process, this is not well justified by the data and it may be more likely that oxygen avoidance and pathogen avoidance are separate processes.

Together, these findings demonstrate that the mechanisms underlying learned pathogen avoidance in C. elegans differ substantially depending on the nature of the pathogen, and that worms likely use a combination of strategies to deal with these threats in the wild.

Reviewer #1 (Public Review):

In this manuscript the authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. This traffic is mediated by Ccl19/Cccr7 and is NfkB independent but Traf activation dependent. This type of trafficking is anti-inflammatory with promotion of early survival.

Specifically, authors work shows the traffic of DC-biased myeloid progenitors through direct transit from BM to bone lymphatic capillaries. This type of trafficking is highly activated in endotoxic inflammation. Giving LPS to mice results in massive mobilization of myeloid progenitors from the BM to lymph and retention in LN takes place. This happens rapidly and before the appearance of these cells in PB. This type pf LPS challenge induces Ccr7 expression on GMPs as well as secretion of CcL9 in the LN. Importantly, loss of CcL9 or neutralizing Ccr7 inhibits GMP/MDP migration to the LN and inflammation induce mortality.

The studies are well performed and the data supports the conclusions. The role of this signaling axis in the recruitment of GMPs/MDPs has not been investigated in this detail.

Reviewer #1 (Public Review):

The manuscript by Tindle et al describes generation of adult lung organoids (ALO) from human lung biopsies and their use to study the changes in gene expression as a result of SARS-CoV-2 infection. The main advantage of the use of organoids is the ability to generate many cell types that make up the lung. In this particular case the authors report the presence of AT1, AT2 cells, Basal cells, Goblet cells, Ciliated cells and Club cells. The authors were able to cultivate the cells at the air-liquid interface and to establish cultures of predominately proximal and predominately distal airway cells. The main finding is that proximal cells are more prone to viral infection, while distal cells are governing the exuberant inflammatory response, with both cells required for the exuberant response to occur. A useful information provided by the paper is the analysis gene signatures of various cellular models, in comparison to the infected human lung.

Reviewer #1 (Public Review):

This manuscript by Taylor et al. carefully investigates (1) ParB-ParA and (2) ParB-ParB interactions in the F Plasmid SopABC system using microfluidics, TIRF microscopy and magnetic tweezers.

(1) The work shows that the activation of ParA ATP hydrolysis requires a dimer of ParA to bind to two protomers of ParB. Surprisingly, ParB can bind to ParA either using the two protomers of a single dimer or two protomers from distinct dimers. The former occurs in the absence of ligands, the latter upon addition of either CTP or parS DNA, thus presumably corresponding to the state of ParB found in the cells near a parS site. The authors suggest that this is crucial for the precise timing of ParA-ParB anchoring and release.

(2) Magnetic tweezer experiments demonstrate nucleotide-dependent compaction of DNA by ParB. This compaction is strictly parS-sequence dependent and robust even at elevated DNA extension force (5 pN) and at relatively low ParB concentrations. This implies ParB dimer-ParB dimer interactions exclusively on parS DNA.

The conclusions are generally well supported by the data. Few control experiments are suggested.

Reviewer #1 (Public Review):

The paper "Insights from a Pan India Sero-Epidemiological survey (Phenome-India cohort) for SARS-CoV2" reports a longitudinal survey of about 10000 subjects from laboratories of the CSIR (India) who consented to be tested for antibodies to SARS-CoV-2, across August and September 2020. The methodology is a standard one, using the Roche kit to test for antibodies to the nucleocapsid antigen with a followup to detect neutralising antibodies using the GENScript Kit. A questionnaire for all participants asked about age, gender, pre-existing conditions and blood group, among other questions.

The principal results of the study were:

1) An overall seropositivity of 10.14% [95% CI: 9.6 - 10.7] but a large variation across locations

2) Virtually all of the seropositive exhibited neutralising activity

3) Seropositivity correlated with population density in different locations

4) A weak correlation was seen to changes in the test positivity across locations

5) A large asymptomatic fraction (~75%) who did not recall symptoms

6) Of those symptomatic, most reported mild flu-like symptoms with fever

7) A correlation with blood group, with seropositivity highest for AB, follow by B, O and A

8) A vegetarian diet correlated with reduced seropositivity

9) Antibody levels remained constant for 3 months across a sub-sample white neutralising activity was lost in ~30% of this subsample. Over a longer period, in a still smaller subsample of those tested at 3 months, anti-nucleocapsid antibody levels declines while neutralising antibody levels remained roughly constant

10) There is a reasonable agreement with the results of the second Indian serosurvey which obtained a seroprevalence of about 7% India-wise, although excluding urban hotspots.

The deficiencies of this study are:

1) This is a very specific cohort, largely urban, with - presumably - relatively higher levels of education. It is hard to see how this might translate into a general statement about the population

2) The presentation of Figure 1 was quite confusing, especially the colour coding

3) It is surprising that the state of Maharashtra shows only intermediate to low levels of seropositivity, given that the impact of the pandemic was largest there and especially in the city of Pune. There have been alternative serosurveys for Pune which found much higher levels of seropositivity from about the same period.

4) The statement "Seropositivity of 10% or more was associated with reductions in TPR which may mean declining transmission": For a disease with R of about 2, this would actually be somewhat early in the epidemic, so you wouldn't expect to see this in an indicator such as TPR. TPR is also strongly correlated with amounts of testing which isn't accounted for.

5) The correlation with vegetarianism is unusual - you might have argued that this could potentially protect against disease but that it might protect against infection is hard to credit. Much of South Asia is not particularly vegetarian but has seen significantly less impact

6) On the same point above, it is possible that social stratification associated with diet - direct employees being more likely to be vegetarian than contract workers - might be a confounder here, since outsourced staff seem to be at higher risk.

7) There may be correlations to places of residence that again act as confounders. If direct employees are provided official accommodation, they may simply have had less exposure, being more protected.

8) The correlations with blood group don't seem to match what is known from elsewhere

9) The statement that "declining cases may reflect persisting humeral immunity among sub-communities with higher exposure" is unsupported. What sub-communities?

Reviewer #1 (Public Review):

In the manuscript "Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart", the authors isolated epicardial stromal cells (EpiSC) and cardiac interstitial/stromal cells (termed active CSCs) from the same I/R heart and identified transcriptionally distinct subpopulation of EpiSCs via 10x genomics technology. They also performed transcriptome profile comparison between EpiSCs and aCSCs. This manuscript shows rigorous scientific investigation. Their isolation protocol is supported by their previous publication. Method section documented in detail of step-wise QC process of bioinformatics analysis. In summary, the analysis identified 11 clusters of EpiSC, some of which overlap with the well-established epicardial marker WT1 with confirmed in situ anatomical localization. When compared to aCSC, the two groups showed clear different function/states as expected. In the lineage tracing model, RNA velocity predicts cell hierarchy, cell-cell communication between populations, as well as cell cycle activity. Overall this manuscript provides a significant degree of information that can be helpful to the field.

Reviewer #1 (Public Review):

The work by Lutes et al. addresses how thymocytes undergo positive selection during their differentiation into mature T cells. The authors make use of several in vitro and in vivo model systems to the test whether developing thymocytes at the critical preselection CD4+CD8+ stage, expressing T cell receptors (TCRs) with different levels of putative self-reactivity, undergo different or similar differentiation events, in terms of migration, thymic epithelial cell engagement and temporal kinetics, and gene expression changes.

The authors selected three TCR-transgenes, which have increasing levels of self-reactivity, TG6, F5 and OT1, respectively, to test their hypothesis, that TCR signals during positive selection are not only sensed differently but lead to different outcomes that then define the functional status of mature T cells. The author's conclusions that thymocytes with low self-reactivity differentiate with distinct kinetics (migration, engagement and temporal) and express a different suite of genes than thymocytes that experience high self-reactivity is well supported by several elegant approaches, and convincing findings.

The authors clearly established that low to high TCR signaling outcomes affect the timing of positive selection, which is beautifully illustrated in Figures 3-6, and extend that work to non-TCR transgenic mice as well. Lastly, their findings from RNA-seq analyses shed light into the different genetic programs experienced by high-reactivity fast differentiating CD8 T cells as compared to low-reactivity slower differentiating cells, which appear to retain the expression a unique set of ion channels during later stages of their differentiation process.

However, what the expression of these ion channels means in terms of either supporting the slow progression or perhaps responsible for the slow progression is not directly addressed, and likely beyond the scope. Nevertheless, the authors posit as to the potential role(s) for the differently expressed gene subsets. Overall, the work is crisply executed, and the findings reveal new aspects as to how positive selection can be achieved by thymocytes expressing very different TCR reactivities.

Reviewer #1 (Public Review):

Nielsen and colleagues describe a large new multi-ome database containing combinations of absolute mRNA quantities, proteome and amino acid concentrations in a set of 14 yeast populations grown in various conditions in chemostats. Apart from being a valuable resource for colleagues, analysis of the data confirms the results of several previous seminal studies.

For example, the authors confirm the relatively high correlation between transcript and corresponding protein abundance. Moreover, it is shown that for most genes, changes in transcript abundance related to manipulated changes in growth rate largely reflected the availability of RNA polymerase II. Interestingly, this was not the case for genes involved in central carbon metabolism, suggesting that these are regulated separately, likely to maintain the cells' ATP levels. Similarly, manipulation of growth through the use of different nitrogen sources led to changes in transcription that correlated with certain amino-acid-derived metabolites (including nucleotides), but not with RNAPolII levels. Genes involved in central carbon metabolism are again an exception to this rule.

Reviewer #1 (Public Review):

This paper describes the development of a suite of viral vectors that allow expression (either on or off) of genes of interest depending on both Cre and Flp expression. They demonstrate that their system can solve the problem encountered with the other approach and use it for mapping axonal projections of the glutamatergic, LepR-expressing neurons and the consequences of chronic activation of these neurons on food intake and energy expenditure. The results are significant and clearly presented. The failure of the other system (INTRSECT) for their application is not clearly understood, but authors say that it may be due to low expression of Cre or Flp in these neurons; however, Supp Fig. 1 shows that it Lepr-Cre and Slc17a6-FLPo were sufficient to activate a transgenic reporter (Supp Fig. 1). The authors reveal that they probably could have used Nr5a1-Cre mice manipulate the activity of these VMH neurons. Nevertheless, it is worthwhile having multiple methods to attack a specific problem because of unforeseen complications with particular methods.

Reviewer #1 (Public Review):

The zebrafish has a rich history of enabling innovative microscopy techniques, and is also a well established system to model inflammation and infection by human pathogens. Consistent with this, Miskolci et al use zebrafish to test a novel imaging approach that has great potential to significantly impact the field of immunometabolism. Fluorescence lifetime is a label-free, non-invasive imaging approach to detect metabolic changes in situ, at the level of the single cell. In this report, Miskolci et al use fluorescence lifetime imaging of NAD(P)H and FAD to detect metabolic changes in zebrafish macrophages (with temporal and spatial resolution) in response to inflammatory and infectious cues.

Miskolci et al (eLife 2019) have previously characterized inflammatory and wound healing responses to distinct caudal fin injuries (tail wound, infection and tail wound, thermal injury). In this report, authors use these different injury models to show that fluorescence lifetime imaging can detect variations in macrophage metabolism. Although many interesting results are presented and future directions are proposed, the study in its current state is descriptive and lacks validation across different modalities. As a result, the reliability of fluorescence lifetime imaging in zebrafish macrophages is not yet convincing. Moreover, any metabolomic changes in macrophages are not functionally linked to zebrafish phenotypes (eg inflammation, bacterial burden, caudal fin regeneration).

Reviewer #1 (Public Review):

The manuscript presents a very nice and very detailed approach to illustrate the anatomical hierarchies and also some differences of signal transmission in the olfactory vs. thermosensory-/hygrosensory systems.

The authors first provide a complete description of the Drosophila olfactory system, from first, second and third-order neurons in the lateral horn. Using a generally applicable analysis methods, they extract information flow and layered organisation between olfactory input and descending interneurons. Among the results is the interesting finding that downstream of the mushroom body and lateral horn, output neurons converge to presumably regulate behavior. In an additional set of analyses, Schlegel et al. describe and quantify inter- vs. intraindividual stereotypy of neurons and motifs. They actually compare neurons from three hemispheres of two brains and show an astounding degree of similarity across brains. This is somewhat reassuring and helpful to the field of Drosophila connectomics.

While the many details and data make the manuscript a somewhat strenuous read, and the sheer flood of data could be a bit overwhelming, the data and findings are impressive and important.

1) The work is very complementary to the data presented by Li et al. on the mushroom body.

2) The structure and the step-by-step approach to showing increasingly complex circuitry and by defining different layers of the circuitry is very helpful for the reader to get an impression of the complexity of this brain.

3) Of significant importance and of use for the community are, in addition to the data, the described methods tools for data analysis.

4) Using this type of analysis, the authors test hypotheses and prevailing assumptions in the field. For instance, they find that in early layers of the olfactory system neurons tend to connect to the next higher layer, whereas neurons in higher layers interconnect or even connect back to earlier layers. This is a very interesting finding that might have important implications regarding top-down feedback and recurrent loops in olfactory processing.

5) Analysis of connectivity in the antennal lobe suggests that the system is highly lateralized. This finding also has important implications and helps to explain why flies might be able to discern left from right odor sources.

6) The manuscript shows many examples of what other scientists/readers of the manuscript could extract from the raw anatomical data. This will be very useful for the community beyond the data that is actually already shown in the manuscript.

7) The authors also compare their findings to the connectome/motifs identified for the larval olfactory system. There are many similarities as expected.

Reviewer #1 (Public Review):

Claudi et al. present a new tool for visualizing brain maps. In the era of new technologies to clear and analyze brains of model organisms, new tools are becoming increasingly important for researchers to interact with this data. Here, the authors report on a new tool for just this: exploring, visualizing, and rendering this high dimensional (and large) data. This tool will be of great interest to researchers who need to visualize multiple brains within several key model organisms.

The authors provide a nice overview of the tool, and the reader can quickly see its utility. What I would like to ask the authors to add is more information about computational resources and computing time for rendering; i.e. in the paper, they state "Brainrender uses vedo as the rendering engine (Musy et al., 2019), a state-of-the-art tool that enables fast, high quality rendering with minimal hardware requirements (e.g.: no dedicated GPU is needed)" - but would performance be improved with a GPU, runtimes, etc?

I would also be happy to see the limitations and directions expanded. For example, napari is a powerful n-dimensional viewer, how does performance compare (i.e. any plans for a napari plug in, or ImageJ plug in, or is this not compatible with this software's vision?). How does brain render compare (run time, computing wise) to Blender, for example, or another rendering tool standard in fields outside of neuroscience?

The methods are short (maybe check for all open source code citations are included, as needed), but they have excellent docs elsewhere; it would be nice to have minimal code examples in the methods though, i.e. "it's as easy as pip install brainrender" ... or such.

Lastly, I congratulate the authors on a clear paper, excellent documentation (https://docs.brainrender.info/), and I believe this is a very nice contribution to the community.

Reviewer #1 (Public Review):

The manuscript by Schrieber et al., explores whether inbreeding affects floral attractiveness to pollinators with additional factors of sex and origin in play, in male and female plants of Silene latifolia. The authors use a combination of spatial sampling, floral volatiles, flower color, and floral rewards coupled with the response of a specialized pollinator to these traits. Their results show that females are more affected by inbreeding and in general inbreeding negatively impacts the "composite nature" of floral traits. The manuscript is well written, the experiments are detailed and quite elaborate. For example., the methodology for flower color estimation is the most detailed effort in this area that I can remember. All the experiments in the manuscript show meticulous planning, with extensive data collection addressing minute details, including the statistics used. However, I do have some concerns that need to be addressed.

Core strengths: Detailed experimental design, elaborate data collection methods, well-defined methodology that is easy to follow. There is a logical flow for the experiments, and no details are missing in most of the experiemnts.

Weaknesses: A recent study has addressed some of the questions detailed in the manuscript. So, introduction needs to be tweaked to reflect this.

Some details and controls are missing in floral scent estimation. Flower age, a pesticide treatment of plants that could affect chemistry..needs to be better refined. While the study is laser-focused on floral traits, as the authors are aware inbreeding affects the total phenotype of the plants including fitness and defense traits. For example, there are quite a few studies that have shown how inbreeding affects the plant defense phenotype. This could be addressed in the introduction and discussion.

Reviewer #1 (Public Review):

In this study, Zhang et al. systematically analyze the effect of xanthohumol (XN) and TXN, a xanthohumol derivative, in a model of high-fat diet (HFD) feeding to mice, inducing several pathologies related to the metabolic syndrome. They authors convincingly show that XN and TXN attenuate HFD-induced weight gain, hepatic steatosis and lipid accumulation in adipose tissues. Furthermore, they newly show that XN and TXN bind to the PPARgamma ligand-binding domain pocket and that this inhibitory effect on PPARgamma is at least in part responsible for the observe beneficial effects.

Reviewer #1 (Public Review):

This manuscript describes a set of biochemical studies on the substrate and reaction specificity of PARP1, an important drug target and component of DNA damage response. The focus of the work is on the specific role of HPF1, and how PARP1's numerous activities are altered by complexation with it and with a variety of substrates. There are many important findings described in this paper, which will be of great interest to the researchers studying PARP1 and issues related to NAD+ metabolism. Perhaps the most significant finding is that HPF1 binding to PARP1 causes a shift from primarily PARylation activity to that of hydrolytic activity, yielding a large pool of free ADPR. The paper is very well written. Addressing the following issues would provide clarity.

1) The kcat enhancement from employing nucleosome substrates is exceedingly small, and probably will not ever be clearly correlated to a specific structural feature. However, more concerning is a possible uncontrolled variable when examining the nucleosome substrates. Specifically, the nucleosome substrates which yield a distinctly higher kcat (Table 1) are the larger, trivalent nucleosomes. It seems prudent to show that simply adding more potential binding sites, or perhaps just adding more protein itself is not causing these small increases in kcat (relative to DNA alone).

2) Concerning the assignment of E284 of HPF1 as the catalytic base in the deprotonation of the Ser hydroxyl, I'm wondering if there might be a dynamical explanation for its role instead. E284A causes a significant decrease in the KD for HPF1 binding, and an elimination of the observed PARylation activity, suggesting that it may play an allosteric role. Also, we see from Table 2 that H303Q also produces a large reduction in the activity and large reduction in the KD; the standard error on the H303Q binding data is very large, but does suggest that some observations were quite low (similar to E284A). Additionally, H303Q almost eliminates enzymatic activity as well. Overall, this set of data gives me pause about certainty of the assignment of E284 as the catalytic base, as there may be a more complex origin of the loss of enzymatic activity.

3) It may be that the reason that there is no apparent PARylation at the standard carboxylate residue sites (in the presence of HPF1) is that they are forming transient ester bonds with the anomeric carbon, which are labile to hydrolysis. I feel that a better development of the treadmilling effect would enhance the paper (e.g., mutation of the orthodox carboxylate nucleophiles and examination of changes in HPF1-induced hydrolytic activity). I'm not sure that it can be quantitatively shown that the shorter PAR chains in the presence of HPF1 account for the pool of free ADPR.

Reviewer #1 (Public Review):

This is an interesting manuscript which does a lot - both building and validating an epigenetic clock for the Amboseli baboons, and then looking to see which factors predict deviations in epigenetic age relative to chronological age. This is an important study, and perhaps the first of its kind from a free-ranging primate population. I believe it will be influential and well-cited.

In particular, it is extremely thorough in the data and analyses that it presents. It is also clearly structured and easy to follow, despite covering some dense material.

In sum, this manuscript is a high-quality and important manuscript that I believe will be influential.

Reviewer #1 (Public Review):

This study indicated that transient receptor potential channel subfamily melastatin 4 (TRPM4), a Ca2+ and voltage activated non-selective monovalent cation channel, might contribute to pressure overload-induced cardiac hypertrophy, although not through direct mechanical stretch-related activation. TRPM4 could possibly activate several Calmodulin (CaM)-related downstream signaling pathways, resulting in cardiac hypertrophy. However, the important question of what is mechanistic link of mechanical stretch and activation of TRPM4 ion channel is left unanswered.

Strength: The experiments are well designed with reliable data presented. The utilization of TAC mice model presented in this study was backed with proper reasoning with appropriate proof-of-concept results, especially concerning the 2-day TAC protocol.

Weakness: Trpm4cKO mice have been previously studied in another cardiac hypertrophy model by using angiotensin II, which lessened the novelty value in the findings of this study. Furthermore, the data presented in this paper were inadequate to fully answer their research questions and further in vivo and in vitro studies are needed to confirm the mechanism that can explain the phenomenon seen in the results.

Reviewer #1 (Public Review):

The authors employed population receptive field (pRF) mapping to characterize responses to visual stimuli in early visual cortical areas V1-V3 and to compare the similarity of pRF properties in pairs of monozygotic versus dizygotic twins. They find closer correspondence of the anatomical location and spatial extent of the visual areas, pRF locations (polar angle and eccentricity) in the retinotopic cortical maps of visual space, and spatial selectivity of responses (pRFs size) in monozygotic twins, relative to dizygotic twins, indicating heritability of these structural and functional properties of early visual cortex.

The pRF mapping procedures used in this study are appropriate and standard in the field, and the statistical analysis and data presentation are thorough and rigorous. Given the many previous demonstrations of heritability in multiple aspects of visual perception and physiological responses to visual stimuli, it would be very surprising if any of the properties studied by the authors did not exhibit some amount of heritability. This paper therefore adds to the list of known heritable properties of the visual system but does not contribute theoretical or conceptual advances or challenge any existing frameworks.

The fact that pRF eccentricity was more correlated and showed less heritability than pRF polar angle is interesting but was not interpreted or followed up in any meaningful way. Overall, the analyses are basic (% overlap of retinotopic maps and the three main pRF parameters) and descriptive.

Reviewer #1 (Public Review):

Using well-designed surveys, the authors collected mosquito samples and human data along with environmental variables to estimate parasite prevalence (PR) and the entomological inoculation rate (EIR) in three regions of Malawi. They developed advanced geostatistical models to estimate PR and EIR and illustrated the spatial-temporal variation. The online interactivity web-based application showing the spatial-temporal pattern of PR and EIR as well as hot spots in map is particularly useful for visual understandings. These estimations then allow to unveil the time-lagged relationship between PR and EIR. Their data and research approach add very useful information for improving vector-born disease control strategies. Certainly, the data and findings are very useful for malaria control in Malawi.

Their conclusion seems largely supported by their statistical models and data. However, some outstanding research questions remain. In addition, some statistical issues need to be justified and clarified.

1) While the spatial-temporal pattern of PR and EIR is illustrated, what are the mechanisms underlying those spatial-temporal variation? Specifically, I think environmental factors and spatial distribution of human population certainly play important roles. Indeed, environmental factors were included in their geostatistical models to estimate PR and EIR. However, the authors made no attempt to provide explanation and discussion for these results (results shown as tables in their appendix).

2) Furthermore, if environmental factors are left out of focus, what is the additional value of using modelled PfSR and PfEIR for evaluation instead of empirical (observed) PfSR and PfEIR? What is the scientific motivation and justification of using modelled PfSR and PfEIR instead of empirical ones to make the spatial-temporal map and further statistical analyses and then to draw their conclusion on the relationship between modelled PfSR and PfEIR? Statistically, if the same environmental variable is used to fit PfSR and PfEIR, then there is potential spurious correlation (statistical artifact) between the modelled PfSR and PfEIR. The authors need to demonstrated this is NOT the case in their results and analyses.

3) With A, B, C three regions separated by the national park in the middle (large spatial missing data), is the assumption of isotropic Gaussian process reasonable in their geostatistical model? Sites between A and B have very large distances, but there is no observation data in between. Alternatively, the authors can model the three regions separately?

4) For hotspot detection, it is unclear whether the hotspots are decided: (1) when the point estimates of PfEIR and PfPR exceed the threshold; or, (2) when the lower 95% confidence bound of the estimates exceed the threshold? If it is the case (1), please justify. Statistically, case (2) is more appropriate. The uncertainty associated with estimates needs to be carefully addressed throughout the manuscript. In any case, please elaborate how the exceedance probability is obtained. My similar concern also appears in other analyses, for example the confidence interval shown in Figure 4.

Reviewer #1 (Public Review):

This work described a novel approach, host-associated microbe PCR (hamPCR), to both quantify microbial load compared to the host and describe interkingdom microbial community composition with the same amplicon library preparation. The authors used the host single (low-copy) genes as PCR targets to set the host reference for microbial amplicons. To handle the problem that in many cases, the host DNA is excessive compared to the microbiome DNA, the authors adjusted the host-to-microbe amplicon ratio before sequencing. To prove the concept, hamPCR was tested with the synthetic communities, was compared to the shotgun metagenomics results, was applied in the biological systems involving the interkingdom microbial communities (oomycetes and bacteria), or diverse hosts, or crop hosts with large genomes. Substantial data from diverse biological systems confirmed the hamPCR approach is accurate, versatile, easy-to-setup, low-in-cost, improving the sample capacity and revealing the invisible phenomena using regular microbial amplicon sequencing approaches.

Since the amplification of host genes would be the key step for this hamPCR approach, the authors might also include more strategy discussions about the selection of single (low copy) genes for a specific host and the primer design for the host genes to guarantee the hamPCR usage in the biological systems other than those mentioned in the manuscript.

Reviewer #1:

The question is interesting, and the paradigm in principle well suited to answer it. Unfortunately, a number of shortcomings hinder a clear interpretation of the results. I think that the paper, notably the EEG analyses, need to be revised substantially, which might affect the results. Therefore I will just list the main points which need to be addressed and not go in more detail.

The behavioral effect of adaptation on duration perception appears very unspecific, namely it occurs in all but the spatially neutral condition. The authors conclude that the inversely directed motion did not have an effect because it did not survive the Bonferroni correction, yet they report a p-value of 0.02 and Cohen's d of 0.58, suggesting a medium effect. In order to prove the absence of an effect, I suggest to report Bayes factors, and only interpret the effect as absent if the Bayes factor is conclusive towards the H0.

In my view, if there was an effect of inversely directed motion, this poses a question as to the successful demonstration of specific adaptation effects in the behavior, which needs to be taken into account in the interpretation.

The EEG analyses and displayed results show some important shortcomings, which hinder a clear interpretation at this stage. Just to list a few main points:

-As apparent from Figures 3-5, the time-frequency plots show a lot of stripes and pixels, when one would expect rather smooth transitions over frequency and time. This suggests that the parameters for the time-frequency transformation might not be appropriate.

-The analyses compare time windows that differ in many respects, for instance the 15 s long adaptation phase versus short-lived stimulus-evoked activity at reference onset. Interpreting these differences as specific to the duration distortion effects does not seem justified, due to the diverging inputs presented during those time windows.

-Important aspects of the paradigm are not taken into account in the EEG analyses, for instance the fact that participants perform a saccade between the offset of adaptation and the onset of the reference. The saccade-related signatures in the EEG have to be accounted or controlled for, especially for effects occurring after adaptation offset.

-Some of the effects (for instance the decoding analysis, or the linear mixed models testing for additive but not interactive effects) show differences in EEG activity related to visual processing of the stimuli, but might not specifically relate to the duration distortions. In my view, more trivial differences in processing the visual inputs should be accounted for (see also the point above), and clearly separated from specific timing effects.

Reviewer #1 (Public Review):

In this project, the authors set out to create an easy to use piece of software with the following properties: The software should be capable of creating immersive (closed loop) virtual environments across display hardware and display geometries. The software should permit easy distribution of formal experiment descriptions with minimal changes required to adapt a particular experimental workflow to the hardware present in any given lab while maintaining world-coordinates and physical properties (e.g. luminance levels and refresh rates) of visual stimuli. The software should provide equal or superior performance for generating complex visual cues and/or immersive visual environments in comparison with existing options. The software should be automatically integrated with many other potential data streams produced by 2-photon imaging, electrophysiology, behavioral measurements, markerless pose estimation processing, behavioral sensors, etc.

To accomplish these goals, the authors created two major software libraries. The first is a package for the Bonsai visual programming language called "Bonsai.Shaders" that brings traditionally low-level, imperative OpenGL programming into Bonsai's reactive framework. This library allows shader programs running on the GPU to seamlessly interact, using drag and drop visual programming, with the multitude of other processing and IO elements already present in numerous Bonsai packages. The creation of this library alone is quite a feat given the complexities of mapping the procedural, imperative, and stateful design of OpenGL libraries to Bonsai's event driven, reactive architecture. However, this library is not mentioned in the manuscript despite its power for tasks far beyond the creation of visual stimuli (e.g. GPU-based coprocessing) and, unlike BonVision itself, is largely undocumented. I don't think that this library should take center stage in this manuscript, but I do think its use in the creation of BonVision as well as some documentation on its operators would be very useful for understanding BonVision itself.

Following the creation of Bonsai.Shaders, the authors used it to create BonVision which is an abstraction on top of the Shaders library that allows plug and play creation of visual stimuli and immersive visual environments that react to input from the outside world. Impressively, this library was implemented almost entirely using the Bonsai visual programming language itself, showcasing its power as a domain-specific language. However, this fact was not mentioned in the manuscript and I feel it is a worthwhile point to make. The design of BonVision, combined with the functional nature of Bonsai, enforces hard boundaries between the experimental design of visual stimuli and (1) the behavioral input hardware used to drive them, (2) the dimensionality of the stimuli (i.e. 2D textures via 3D objects), (3) the specific geometry of 3D displays (e.g. dual monitors, versus spherical projection, versus head mounted stereo vision hardware), and (4) automated hardware calibration routines. Because of these boundaries, experiments designed using BonVision become easy to share across labs even if they have very different experimental setups. Since Bonsai has integrated and standardized mechanisms for sharing entire workflows (via copy paste of XML descriptions or upload of workflows to publicly accessible Nuget package servers), this feature is immediately usable by labs in the real world.

After creating these pieces of software, the authors benchmarked them against other widely used alternatives. IonVisoin met or exceeded frame rate and rendering latency performance measures when compared to other single purpose libraries. BonVision is able to do this while maintaining its generality by taking advantage of advanced JIT compilation features provided by the .NET runtime and using bindings to low-level graphics libraries that were written with performance in mind. The authors go on to show the real-world utility of BonVision's performance by mapping the visual receptive fields of LFP in mouse superior colliculus and spiking in V1. The fact that they were able to obtain receptive fields indicates that visual stimuli had sufficient temporal precision. However, I do not follow the logic as to why this is because the receptive fields seem to have been created using post-hoc aligned stimulus-ephys data, that was created by measuring the physical onset times of each frame using a photodiode (line 389). Wouldn't this preclude any need for accurate stimulus timing presentation?

Finally the authors use BonVision to perform one human psychophysical and several animal VR experiments to prove the functionality of the package in real-world scenarios. This includes an object size discrimination task with humans that relies on non-local cues to determine the efficacy of the cube map projection approach to 3D spaces (Fig 5D). Although the results seem reasonable to me (a non-expert in this domain), I feel it would be useful for the authors to compare this psychophysical discrimination curve to other comparable results. The animal experiments prove the utility of BonVision for common rodent VR tasks.

In summary, the professionalism of the code base, the functional nature of Bonsai workflows, the removal of overhead via advanced JIT compilation techniques, the abstraction of shader programming to high-level drag and drop workflows, integration with a multitude of input and output hardware, integrated and standardized calibration routines, and integrated package management and workflow sharing capabilities make Bonsai/BonVision serious competitors to widely-used, closed-source visual programming tools for experiment control such as LabView and Simulink. BonVision showcases the power of the Bonsai language and package management ecosystem while providing superior design to alternatives in terms of ease of integration with data sources and facilitation of sharing standardized experiments. The authors exceeded the apparent aims of the project and I believe BonVision will become a widely used tool that has major benefits for improving experiment reproducibility across laboratories.

Reviewer #1 (Public Review):

In this study, Boroumand et al investigate abundance and metabolic phenotype of Ly6Chi and Ly6Clo monocytes in the bone marrow (BM) following feeding a HFD for 3, 8 and 18 weeks compared with a control diet. The authors suggest that upon accumulation of white adipocytes in the BM (8 weeks of feeding), monocytes are skewed towards the Ly6Chi subset, which have been shown to give rise to many macrophage subsets in obese tissues. The authors further demonstrate metabolic changes in Ly6Clo monocytes which may contribute towards this phenotype. Finally, through a series of in vitro and ex vivo cultures, the authors suggest that the increase in Ly6Chi monocytes is due to conversion of Ly6Clo monocytes into Ly6Chi monocytes as a result of the increased prevalence of white adipocytes in the bone marrow.

Overall the findings of this work are interesting to the field and in the future it will be interesting to determine how these changes in the bone marrow relate to the different subsets of recruited macrophages present in obese tissues. For example, whether these monocytes preferentially generate CD9+Trem2+ Lipid associated macrophages recently described in obese adipose tissue (Jaitin et al, Cell, 2019) or if they are equally capable of generating monocyte-derived tissue resident macrophages in obese tissues.

The main strength of this paper is in the identification of the changes in the monocyte subsets abundance early after feeding a HFD and in uncovering the metabolic changes in and between these two monocyte subsets in obese mice. One concern regarding the data as a whole is that, while the authors have nicely indicated the number of samples/mice in each figure, there is no mention of how many times each experiment was performed. Including this would greatly aid in an understanding of the reproducibility of the results. Additionally, the inclusion of the different gating strategies used particularly for the first figures would be advantageous to fully appreciate the findings being presented. This is particularly relevant for the identification of Ly6Chi and Ly6Clo BM monocytes.

The conclusions made regarding the role of white adipocytes in skewing the monocyte subsets and particularly regarding the conversion of Ly6Clo monocytes to Ly6Chi are however less convincing. The authors use a culture strategy where they grow BM monocytes in vitro for 5 days. They then culture these 'monocytes' for a further 18 hours with conditioned media from BM adipocytes from control or HFD fed mice. They show that culture with 8 & 18 week conditioned media results in the increased abundance of Ly6Chi monocytes. The authors later claim this is not through proliferation of the existing Ly6Chi monocytes but conversion from Ly6Clo monocytes. However, the alternate explanation could be that there are some progenitors remaining in these cultures that can give rise to Ly6Chi monocytes following exposure to the conditioned media. To further validate these claims, it would be beneficial to sort Ly6Chi monocytes and culture them with the conditioned media to demonstrate the numbers do not increase. Moreover, it is important to demonstrate that there are no progenitors left in these cultures when the conditioned media is added. Indeed, later in the manuscript, when Ly6Clo monocytes are sorted and cultured with media from EWAT or BAT, it would be important to confirm that the sorted cells are a pure population of Ly6Clo monocytes with no contamination from progenitors that are also Ly6Clo that could give rise to Ly6Chi monocytes without going through the Ly6Clo monocyte stage.

In a similar vein, the authors suggest no conversion of Ly6Chi monocytes to Ly6Clo monocytes, but that Ly6Clo monocytes would convert into Ly6Chi monocytes (fig. 7). As this is a rather controversial claim, additional data in support of this conclusion would be beneficial. For example, after 18 hours of culture it is possible that if the authors are sorting Ly6Chi monocytes on the basis of Ly6Chi expression, that the antibody staining may be maintained for 18 hours. Similarly, after culture, it is possible that the cells are less healthy and hence non-specific binding should also be ruled out. Alternatively, qPCR for gene expression associated with Ly6Chi and Ly6Clo monocytes could be utilised to further substantiate the claims. For example, Spn expression for Ly6Clo monocytes, Ly6c2 expression for Ly6Chi monocytes.

Thus overall, this manuscript nicely demonstrates changes in the BM monocyte subsets and their metabolism, however some additional controls are required to further validate the claim that Ly6Chi monocytes are increased due to Ly6Clo monocyte conversion to Ly6Chi monocytes.

DOI: 10.7554/eLife.64267

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What is this?

DOI: 10.7554/eLife.64267

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What is this?

DOI: 10.7554/eLife.64267

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What is this?

Reviewer #1 (Public Review):

In this manuscript, Holt and colleagues investigate how the mechanoreceptor PIEZO1 mediates keratinocyte cell migration and re-epithelialization during wound healing. The authors utilized epidermal-specific Piezo1 knockout mice (Piezo1cKO) and epidermal-specific Piezo1 gain of function mice (Piezo1GoF) to investigate the contribution of keratinocyte Piezo1 to wound healing in vivo. Piezo1cKO mice exhibited faster wound closure, whereas Piezo1GoF mice exhibited slower wound closure compared to controls, suggesting that the presence of epidermal Piezo1 affects the speed of wound healing. To determine if these effects observed in vivo were due to changes in keratinocyte re-epithelization, the authors utilized an in vitro model of wound healing by inducing scratches to mimic "wounds" in keratinocyte monolayers. Similar to the in vivo findings, Piezo1cKO keratinocytes exhibited enhanced wound closure compared to controls. In a separate line of experiments, the authors found that enrichment of Piezo1 at the wound edge induces localized cellular retraction that slows keratinocyte re-epithelization and wound closure. Overall, major strengths are that the topic is of significant interest, Piezo channels and their function is of broad topical interest, and the manuscript is well written. Wound healing is a major health concern and understanding the mechanisms underlying how wounds heal could generate improved therapeutics for faster healing. The key weaknesses are that there are missing controls and missing cohorts (Piezo1GoF or Piezo1cKO) in several of the experimental data sets, and there is a concern about the wide variation in controls for some experiments.

Reviewer #1 (Public Review):

Slavetinsky et al., describe the development of monoclonal antibodies targeting the S. aureus MprF lipid flippase, which is responsible for membrane incorporation of the phospholipid lysyl-phosphatidylglycerol (LysPG). Incorporation renders the cell more positively charged and has been associated with increased virulence and resistance of MRSA to antibiotics and host antimicrobial peptides. MprF is a bifunctional protein; the N-terminal region translocates lipids (flippase), and the C-terminal region synthesizes LysPG. Overall, this is an interesting approach with significant potential.

Strengths:

Several epitopes on MprF (three outer loops) were targeted through the synthesis of peptides, which provided a number of antibodies that inhibit the flippase function. The authors identified one specific antibody (M-C7.1) that was shown to target a loop whose previous location was debatable; thus, these finding indicate the loop can be accessible from the outside of the cell. Antibody binding sensitized MRSA to host peptides and antibiotics (e.g., daptomycin). The antibody was shown to inhibit flippase function and also decreased bacterial survival in phagocytes. Overall, the antibody could be used as an anti-virulence agent, diminishing the severity of S. aureus-associated disease. The emergence of antibiotic resistance and difficult to treat S. aureus infections requires orthogonal therapeutic approaches; as such, the findings of this study could have significant impact.

Weaknesses:

A major emphasis of the study is that the antibody sensitizes S. aureus to host defenses. This reviewer would like to see dose-responses/titrations of the antibody vs the different CAMPs, using standard susceptibility testing methodology. In addition, during the preliminary ELISAs, have the authors established whether the mprF mutant has lower surface adhesion to maxisorp immuno plates? This would be an important control. When studying M-C7.1 mechanism of action, it is unclear why the data is being normalized to L-1 and why unbound cytochrome C is being quantified. It could be more intuitive to assess bound cytochrome C; can the raw data be included rather than normalized data? A control with delta-mprF alone would also be useful for these experiments. When assessing survival in phagocytes, Figure 5 would benefit from a delta-mprF control to compare M-C7.1 efficacy. This figure also requires statistical analysis. Overall, the conclusions of the study could be further strengthened from additional pre-clinical assessment of the antibody.

Reviewer #1 (Public Review):

This manuscript from Eric Snyder's laboratory details cell lineage states that are controlled by NKX2-1 and oncogenic MAPK signaling in BRAFV600E-driven lung cancers. The work builds on previous works from Snyder's group that showed NKX2-1 suppresses a latent gastric differentiation program in KRASG12D-driven lung cancers. Switching the model from KRAS to BRAF, now the Snyder laboratory demonstrates multiple similarities between the oncogenic drivers and details key differences that have significant impact on our understanding of lung cancer etiology and possibly treatment. The depth of data analysis and breadth of methodology used represent a real tour de force in cancer modeling. The insights highlight the complex interplay between mitogenic signaling and developmentally-related pathways during cancer progression. The insights gleaned from the study have some potential in influence treatment strategies. As such, this study will appeal to a broad audience. The stated conclusions from the work are entirely sound and wholly supported by the data presented.

The authors demonstrate that: Simultaneous activation of BRAFV600E expression and deletion of NKX2-1 suppresses the efficiency of tumor initiation (tumor number goes down). In contrast, genetic deletion of NKX2-1 after tumors have established does not impact tumor maintenance but instead is compatible with tumor progression. Modeling the effects of MAPK pathway inhibition (BRAFi+MEKi), the authors demonstrate that BRAF/p53 (BP) tumors enter a state of quiescence. However, BP tumors with NKX2-1 deletion (BPN) fail to enter the quiescent state. Mechanistically, this is due to activation of a WNT-dependent activation of CyclinD2 that acts with CDK4/6 to suppress RB. Further treatment with CDK4/6 inhibitors can drive cells into quiescence but does not lead to durable tumor growth inhibition as tumors rebound after treatment cessation. Consistent with their previous work in KRAS-driven lung cancers, deletion of NKX2-1 reveals a latent gastric cell differentiation program driven by relocalization of FOX factors toward gastric specific genes. Interestingly, MAPKi in BPN tumors further drives these cells toward a chief-like or tuft-like cell state that is also due to WNT-dependent signaling, and FOXA1/2-dependent effects at specific genes normally restricted to tuft and chief cells.

Reviewer #1 (Public Review):

The data in the paper are mostly convincing, but might be somewhat over-interpreted: statistical analysis of the Tables is required. Yes, long slender bloodstream forms can definitely differentiate to pro cyclic forms and infect Tsetse. However, they take longer to differentiate than stumpy forms do, and even though morphologically stumpy forms are not an obligatory intermediate, expression of at least one stumpy-form mRNA (and presumably, others in the pathway) is definitely required. This should be stated in the Abstract. The conclusion that there is no cell-cycle arrest at all is not really supported by the data.

Joint Public Review:

This is an elegant study that delves into germline initiation and ovule development at a resolution not previously reported. The topic is of general significance for developmental biologists, and particularly interesting for groups studying the basis for germline development. Using a multitude of assays, starting from 3D segmentation analysis, progressing to modelling, reporter line analysis and mutant characterization, the authors document cellular components of ovule primordium growth and uncover new aspects of spore mother cell (SMC) emergence, in which ovule geometry appears to play a relevant role. The authors concluded that anisotropic growth is one of important factors to drive overall development of ovules, especially in Phase I, and that the L1 dome and the basal domain, but not the SMC and neighboring L2 companion cells, are consecutive sites of cell proliferation, thus contributing to morphological changes of ovules in Phases I and II. In terms of novelty, this work identified growth principles conducive to ovule primordium growth, added a layer of complexity to the nucellar epidermis towards SMC specification, and provided a new concept of SMC development: SMC fate emergence and SMC singleness resolution, where cell geometry plays a very active role

The katanin mutant is an interesting choice since it has been reported previously to impact cell growth. As expected, in katanin mutants, the primordium became enlarged in size and was more isotropic (lower height/width ratio) in shape. A reduced anisotropy also induced aberrant enlargement of SMC companion L2 cells in katanin mutant ovules. From PCNA and CYCB1.1 expression patterns, which are S- and M-phase markers, respectively, the authors found that the SMC precursor and its companion cells showed a highly frequent S-phase pattern. Taken together with infrequent divisions, the SMC and its neighbors have properties distinct to other ovular cells in longer S-phase duration. In addition, SMC singleness was suggested to be determined partly by Katanin-dependent anisotropic condition.

The claims made through the work are well documented and supported. In terms of experimental clarity and composition, the authors describe very well how the samples were obtained/how they were named, the statistical analysis appears robust and well described, and several of the markers analyzed provide a comprehensive landscape of what is occurring in the ectopic cells.

Reviewer #1 (Public Review):

The neuroendocrine system of the maggot has been mapped in parts at both the light and electron microscopic levels in earlier studies. In this manuscript, Hückesfeld et al map the entire endocrine system all the way from its sensory input neurons to the interneurons and secretory neurons and the glands. This is invaluable for many reasons, including because information about external stimuli are likely integrated at the level of interneurons.

The authors use this connectome to model how and to what extent each sensory modality might influence the different neurosecretory cells. They use the CO2 sensing pathway to functionally validate their model in vivo using CaMPARI. Through this they validate a circuitry where CO2 sensing neurons in the trachea influence 4 types of neurosecretory cells via 4 interneuron pathways. Interestingly, they find that the CO2 sensory information is not necessarily what dominates the sensory input onto some these neurons.

DOI: 10.7554/eLife.54491

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What is this?

DOI: DOI:10.1016/j.cub.2020.08.103

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What is this?

DOI: 10.1126/sciadv.aaz3173

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What is this?

DOI: 10.7554/eLife.37001

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What is this?

DOI: 10.7554/eLife.37001

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What is this?

DOI: 10.1016/j.neuron.2018.10.045

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What is this?

DOI: 10.7554/eLife.42881

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What is this?

Dr. Tara C. Smith. (2021, January 23). A reminder: Especially among the elderly, some individuals will die shortly after receipt of the vaccine. What we need to understand is the background rate of such deaths. Are they higher then in the vaccinated population? We didn’t see that in the trials. Some data from @RtAVM. https://t.co/LJe9k1WJQC [Tweet]. @aetiology. https://twitter.com/aetiology/status/1352810672359428097

‘What Happens When the 1% Go Remote’. Bloomberg.Com, 16 December 2020. https://www.bloomberg.com/news/articles/2020-12-16/what-happens-when-the-1-move-to-miami-and-austin.

Reviewer #1 (Public Review):

In this manuscript Rao et al. describe an interesting relationship between KSR1 and the translation regulation of EPSTI1 (a regulator of EMT). They identified this relationship by polysome RNAseq of CRC cells in the context of KSR1 knockdown (KD) which they confirm by polysome QPCR. They then go on to show that KSR KD and add back influences EPSTI1 expression at the protein but not mRNA level and impacts cell viability, anchorage-independent growth, and possibly cell migration. They focus on the cell migration phenotype and show that it is associated with changes in EMT-related genes including E-cad and N-cad. Interestingly, add back of EPSTI1 can reverse the phenotype elicited by KSR1 deletion. Overall, this story is interesting and translation regulation by KSR1 has not been described previously. However, Rao et al. do not provide a mechanism for how KSR1 regulates the translation of EPSTI1, and it is unclear whether this occurs through eIF4E, as the authors suggest.

Reviewer #1 (Public Review):

The paper is investigating the mechanism of lineage switch in lung cancer. In about 10-15% of lung cancers treated with inhibitors of oncogenic receptors such as EGFR or KRAS, cancer cells emerge over time with newly acquired features of neuroendocrine differentiation. The authors proposed that it is a direct result of inhibition of MAPK pathway signaling so that reduced MAPK activity activates previously silent genes regulating neural crest differentiation. While this theory is of interest, the experiments presented herein are construed on the opposite sequence by way of introducing activated MAPK via oncogenic KRAS or EGFR to 3 neuroendocrine cell lines resulting in lower expression of neuronal transcription factors. The authors propose MAPK-activated ETS family TFs are responsible for the repression of NE lineage.

Several principal issues presented by the authors raise some concerns:

1) Despite presenting some evidence to the effect of suppression of NE transcription factors by overactivating MAPK signaling, the conversion of adenocarcinoma to NE (the opposite transition) is not being addressed in the paper. Therefore, it is rather illogical to investigate the process of transition that is not taking place in the real world.

2) The authors do not consider a possibility of multi-clonality of human cancers and clonal competition as a mechanism leading to acquired resistance and the emergence of NE clones that are not suppressible by the inhibitors of MAPK pathway (e.g. EGFR inhibitors, or KRAS/RAF/MEK inhibitors). Starting the experiments with clonal populations of long-term cultured cell lines may be an insurmountable difficulty to switch these cells between the epithelial and NE phenotypes which proved to be frustratingly non-productive in the hands of the authors. Taken out of context of tumor microenvironment, these phenotypic transitions may be co-regulated by a combination of cell-intrinsic and extrinsic factors.

3) Despite zeroing in on ETVs downstream of ERK1/2, the paper does not go as far as showing the direct effect of these TFs as repressors of NE differentiation (ASCL1, BRN2, NEUROD1 etc.).

4) The line of evidence that Dox-activated MAPK signaling via massive over expression of KRAS or EGFR induces dramatic increase in marks of transcriptionally active chromatin (such H3K27ac and others) is to be expected in this entirely artificial system. Indeed, the addition of doxycycline results in massive burst of proliferation and overexpression of ETV1 and ETV4, the canonical MAPK targets. Again, this switch appears unrelated with the opposite of epithelial-to-NE de-differentiation.

DOI: 10.7554/eLife.55771

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What is this?

DOI: 10.7554/eLife.55771

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What is this?

DOI: 10.7554/eLife.55771

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What is this?

DOI: 10.7554/eLife.53403

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What is this?

DOI: 10.7554/eLife.53403

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What is this?

DOI: 10.7554/eLife.53403

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What is this?

AssayResult: 100

AssayResultAssertion: Normal

Comment: See Table S3 for details

AssayResult: 20.5

AssayResultAssertion: Normal

Comment: See Table S3 for details

AssayResult: 6.4

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 6.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 7.1

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 7.7

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 3.5

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 3.1

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.4

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.2

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.1

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 3.5

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.9

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.4

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 6.7

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 10.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.9

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4.9

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 8.6

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 4

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 6

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.4

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 7.1

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.5

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 2.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 2

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 2.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 3.1

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.6

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 3.5

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 5.8

AssayResultAssertion: Abnormal

Comment: See Table S2 for details

AssayResult: 11.5

AssayResultAssertion: Normal

ControlType: Normal, wild type TP53 from control 3

Comment: See Table S2 for details

AssayResult: 10.8

AssayResultAssertion: Normal

ControlType: Normal, wild type TP53 from control 2

Comment: See Table S2 for details

AssayResult: 13.6

AssayResultAssertion: Normal

ControlType: Normal, wild type TP53 from control 1

Comment: See Table S2 for details

HGVS: NM_000546.5:c.-117G>T

DOI: 10.1016/j.neuron.2017.06.003

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Curator: @evieth

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What is this?

Reviewer #1 (Public Review):

SARM1 is an enzyme that is present in neurons and degrade NAD+. Previous studies have shown that disrupting SARM1 inhibits axon degeneration and thus it could be a target for treating neurodegenerative diseases. NAD+ is also an important metabolite that is required for many biological pathways. Thus, SARM1 activity must be carefully regulated. Recent studies have provided structural and biochemical insights about how SARM1 activity is auto-inhibited in basal states. The manuscript by Dr. Thompson and coworkers provide a nice new model regarding how SARM1 could be potentially activated. They provide strong in vitro data to support that phase transition, promoted by PEG molecules and citrate, could dramatically increase the activity of SARM1 TIR domain (which is the catalytic domain) in vitro. The authors also showed that in the worm, C. elegans, citrate promotes SARM1 puncta formation and axon degeneration, which is consistent with the in vitro data. They also generated multiple mutants of SARM1 TIR domain and showed many of the mutants have decreased phase transition and decreased activity in vitro. One of mutant, G601P, also showed decreased puncta formation when expressed in HEK 293T cells as SARM1 SAM-TIR domains E462A mutant (a catalytic mutant so that expression will not cause toxicity) fused with GFP.

The manuscript has many strengths, including the strong and very careful in vitro characterization of the purified SARM1 TIR domain, which provide a lot of useful information regarding the kinetic parameters, substrate specificity, and inhibition profiles. The worm data with citrate is consistent with the in vitro data, which is also a strength.

The impact of the finding lies in two aspects. First, it provides a new understanding about how SARM1 activity might be regulated in vivo by phase transition. This is especially true given most studies so far focuses on how it is inhibited at basal conditions. It also adds another example to the list of enzymes that are regulated by phase separation. Second, the finding that PEG and citrate strongly activate SARM1 in vitro also provides a much improved assay for the development of small molecule modulators of SARM1 for potential therapeutic applications.

There are two minor weaknesses associated with the studies of the manuscript. One is that all the in vitro studies used just the TIR domain of SARM1, not the full length SARM1. Another minor weakness is associated with the data in Figure 5. Most of the mutants have dramatically lower catalytic activities (>100-fold), but the precipitate formation is only modestly affected (2-fold). Although this does not affect the overall conclusion of the manuscript, it prevents the mutants from being more useful for mechanistic dissection.

Reviewer #1:

In this paper, the authors proposed a new approach by mounting a PDMS microwells of specific sizes on agar surface to confine swarming and planktonic SM3 cell, they found swarming bacteria exhibit a "single-swirl" motion pattern and concentrated planktonic bacteria exhibit"multi-swirls" motion pattern in the diameter range of 31-90 μm. The phase diagram shows that in smaller wells concentrated planktonic SM3 forms a single vortex and in larger wells swarming SM3 also breaks into mesoscale vortices.

After that, they conducted systematic experiments to explore parameters defining the divergence of motion patterns in confinement including cell density, cell length, cell speed and surfactant. They concluded that the single swirl pattern depends on cohesive cell-cell interaction mediated by biochemical factors removable through matrix dilution.

This paper gives a new method to discern swarmers from Planktonic Bacteria and carefully studies the factors that influence the formation of bacterial vortices under restriction. However, major revisions are required to improve the quality of this paper.

Major questions and comments:

1) When the authors put the PDMS chip mounting on the edge of the swarming colony, the PDMS chip is completely attached to agar or suspended in a bacterial solution. The distance between PDMS chip and agar surface should be quantified. It is better to have a schematic diagram of the experimental device.

2) Is the bacteria still expanding outward after a PDMS chip was mounted on agar surface? The effect of PDMS chips on the expansion of bacteria on the agar surface needs to be discussed.

3) "Diluted swarming SM3 show unique dynamic clustering patterns". In the diluted bacteria experiment, the authors found that the diluted swarming bacteria can form bacterial rafts and the concentrated planktonic SM3 disperse uniformly and move randomly. Hence, when bacteria expand and gradually fill up new empty microwells, is there a process of transition from raft to single vortex state?

4) In the experiment of altering the conditions of swarming SM3, the authors diluted the swarming cells in Lysogenic Broth (LB) by 20-fold, re-concentrated the cells by centrifugation and removed extra LB to recover the initial cell density. After these operations, they found the previous single swirl turned to multiple swirls and got a conclusion that matrix dilution can affect single swirl patterns. The authors think centrifugation may wash away some surrounding matrix or polymers on the surface of bacteria. Therefore, the steps of centrifugation need to be presented and the effect of centrifugation on the physiological behavior of bacteria should be discussed.

5) This article covers the PDMS chip directly on the agar surface and finds that swarm and planktonic bacteria have different spatial correlation scales in the restricted microwells. The authors have done a lot of experiments to prove the difference between clusters and planktonic bacteria and explain the reason for the single vortex. However, the conclusion is not clear. Therefore, the authors should focus more on the analysis of this new experimental phenomenon, such as critical length and vortex phase diagram, rather than just describing the experiments they did.

6) The authors mentioned the critical length for swarming SM3 is ~ 49 μm, whereas, for concentrated planktonic SM3, it is ~ 17 μm. Does this quoted data match what you get from their experimental method? I do not see any follow-up discussion and evidence.

7) As shown in Figure 1 and Movie_S1_mp4, the direction of the single vortex motion of bacteria is clockwise. However, the article simply ignores that the single vortexes of bacteria all present the same direction, and there is no analysis and reasonable explanation on the vortex direction. As shown in Movie_S5_mp4 on the numerical simulations of circularly confined SM3, simulated bacteria vortex counterclockwise in completely opposite directions. The influence of the microwell boundary on the direction of the vortex should be clearly explained at the level of bacterial movement and preferentially with theoretical simulation.

8) Swarming and concentrated planktonic Bacillus subtilis 3610 show the same motion pattern across different confinement sizes. However, the authors did not give definitive conclusions and evidence. As shown in Figure S1, bacillus subtilis 3610 show completely different cluster behavior. Therefore, the discussion of 3601WT may cause readers' confusion on the article. It may be better to put it in the supporting material.

Minor questions and comments

9) Figure 1C, 1D, 6A, 6B may be more convenient to have a scale bar.

Reviewer #1 (Public Review):

The authors have succeeded in their attempt to develop and characterize a rigorous preclinical model of prenatal methadone exposure secondary to pre-pregnancy prescription opioid use. The model is a technical advance in terms of the opioid exposure being consistent throughout pregnancy and the outcome measures of methadone impact are rigorous. Many aspects neurodevelopment and key physiological processes are assessed and key knowledge is provided about the effects of prenatal methadone exposure on physical development, sensorimotor behavior and neuronal properties.

Major strengths include the thoroughness and rigor of analyses and the multiple body systems study. In addition, scientific questions are approached using physical, biochemical and behavioral assessments to fully characterize the effects of prenatal methadone exposure.

The strengths of this paper outweighs the weaknesses. Weakness are minor and include an incomplete assessment or discussion of whether withdrawal in the postnatal period may explain the pathophysiology described and changes in circuitry. Similarly, white matter analyses are not included MRI assessments confining the results to gray matter brain regions.

Reviewer #1:

In this manuscript, the authors look at the influence of root stock genotype on a single scion genotype in Vitis. This includes a lovely highly replicated design including differential water availability. While the experimental design is very elegant, I'm less sure that using general PCs or ML is the best approach to grab the signal of interest.

Is there evidence that the top 20 PCs of the metabolome or the top 100 PCs are an end point of gaining new information about the system. For example, if the top 20 PCs are all different descriptions of the water availability, then PC 21 might start to grab more information about the root-scion relationship. For example in this dataset, PC2-10 were largely about temporal block (line 314-316). In large genomic datasets like this, they have an immense amount of variation such that r2 is not a meaningful way to capture what is in a PC. I can understand the desire to minimize the statistical analysis but if the goal is to fully interrogate the dataset, the authors should provide an empirical reason for stopping at pre-ordained PCs. Or possibly better would be to grab the lsmeans for the main factors in the model to exclude factors of blocking and then run the PCs as that is the underlying interest in the experiment.

The focus on PCs or using ML on the full dataset also hinders the ability to get at the underlying root/scion and water availability connection. Given that phenology and blocking are the main sources of variance, using these approaches rather than a direct GLM or PC on lsmeans/BLUPs weakens the authors ability to use the power in their experimental design. PC and ML can only capture the largest components of variance while GLMS that account for these larger sources of variance can begin to dive into the underlying questions. There is a possibility that the authors did attempt these directed GLMS with no luck but that was not stated.

I think the use of PCs is maybe my biggest hindrance on the manuscript as the section on lines 409-430 which is the capstone of the paper but ends up being correlations of faceless PCs. Unfortunately this leaves the reader with the idea that phenology is simply too strong to obtain any information about the root/scion connection or the water availability connection.

Reviewer #1 (Public Review):

The authors find that plin2 transcript is induced in intestine of 6 dpf zebrafish larvae following a single feeding, while plin3 transcript is expressed in the fasted and fed states in the intestine. They use TALENS to knock-in EGFP and TagRFPt into the plin2 and plin3 loci, with the encoded gene products being the fusion proteins EGFP-plin2 and Plin3-TagRFPt. The EGFP-plin2 protein shows greater induction of fluorescence following a meal. The overall aim of these initial expression characterizations and development of lipid droplet reporter knock-ins is to be able to monitor the life cycle of these organelles in a living whole organism.

Higher resolution photomicrographs of lipid droplets with these knock-in lines concurrently stained with the the fluorescent lipid dyes BODIPY 558/568 C12 and BODIPY FL-C12 are presented with a time series following feeding in intestine; additional cell types beyond enterocytes (i.e., hepatocytes, adipocytes, and cells surrounding lateral line structures) are presented.

The authors have provided a technical advance to the field of lipid droplet biology. With the tractable revisions set out below, their tools set the stage for chemical and genetic screens for factors and compounds that modulate the normal life cycle of these dynamic organelles.

Reviewer #1 (Public Review):

In initial experiments, low levels of IL-33 were detected in Toxaplasma-infected mice. How do these levels compare with normal physiological levels? It would help the reader to understand the relative levels to expect.

The authors identify that most IL-33 is produced by stromal cells rather than hematopoietic cells. The frequency of tdTomato parasites appear to be much less than for the distribution of IL-33 producing cells. Does the parasite expression reflect 100% of parasites or are the number of IL-33-producing stromal cells stimulated in the infection much larger than the identifiable parasite number? That is, is the activation of the stromal cells a direct effect of the Toxaplasma infection or does it depend on intermediates to amplify the effect?

Although the data presented are interesting and the authors identify that both stromal cells and hematopoietic cells contribute to the protective effect of IL-33, it is somewhat confusing amongst the hematopoietic cells, which cells are really driving the response amongst those categorized as 'innate'. Within the hematopoietic compartment, a number of associations are delineated but the causal connections are less clear. The provision of exogenous cytokines indeed have the effect they show in their results, but it remains unclear to this reviewer, whether these effects directly act on the hematopoietic cells, or stromal cells which alone are not sufficient to contain the infection and thus develop a higher pathogen load confounding their contributions.

This work would be strengthened significantly by delineating more clearly the contributions of each compartment. Currently, the correlations are modelled on the responses in the omentum and it would be useful to understand if this reflects the broader response.

This work would benefit from a schematic to indicate how the authors believe the different cells are connected and which are the real drivers/where connections have been demonstrated in driving the immune response.

Reviewer #1 (Public Review):

1) I found the initial description of the overall structure confusing. At first the authors say the complex is a tetramer, which is not what was seen by the Conti lab and then follow that with a confusing discussion leading to the conclusion that the dimer with a rigid subunit and a flexible one is the functional unit. It rather feels like they arrive at this conclusion because that's what Conti's lab saw, rather than any other reason. Since the human complex is a tetramer, perhaps the tetrameric complex observed here is one possible form and that possibility should be considered more carefully. Please state whether there is any similarity in the arrangements between the human tetramer and the tetramer observed here. I found the figure 2 supp 1C was not easy to follow. Coloring each of the four protomers differently would make things clearer.

2) The authors previously determined the structure of yra1C domain bound to sub2 and several labs have shown this interaction activates Sub2 atpase activity. Are those interaction observed previously between Yra1 and Sub2 compatible with this new structure? If so, perhaps the authors could provide a model showing how Yra1 fits into this larger complex. Also, could Yra1 C domain and Gbp2 bind simultaneously to a single THO-Sub2 protomer or would one protomer bind Yra1 and perhaps another bind Gbp2? This is worth considering because this would strengthen the concept that TREX acts as a general platform engaging with multiple export factors to drive recruitment of multiple Mex67 molecules and eventual export of the Mex67:mRNP complex. In the human system, the SR proteins and Alyref have an overlapping binding site on Nxf1, suggesting they may not act together to recruit a single Nxf1, but rather they recruit different Nxf1 molecules perhaps to the same mRNP via a single multimeric THO platform.

Reviewer #1 (Public Review):

Overall this is a well-done study, but some additional controls and experiments are required, as discussed below. The authors have done a considerable amount of work, resulting in quite a lot of negative data, and so should be commended for persistence to eventually identify the link between neutrophils with IL-18, though type I IFN signaling.

Major Comments:

• A major conclusion of this manuscript is prolonged type I IFN production following vaginal HSV-2 infection, but the data presented herein did not actually demonstrate this. At 2 days post infection, IFN beta was higher (although not significantly) in HSV-2 infection, but much higher in HSV-1 infection compared to uninfected controls. At 5 days post infection the authors show mRNA data, but not protein data. If the authors are relying on prolonged type I IFN production, then they should demonstrate increased IFN beta during HSV-2 infection at multiple days after infection including 5dpi and 7dpi.

• Does the CNS viral load or kinetics of viral entry into the CNS differ in mice depleted of neutrophils, IFNAR cKO mice, or mice treated with anti- IL-18? Do neutrophils and/or IL-18 participate at all in neuronal protection from infection?

• In Figure 3 the authors show that neutrophil "infection" clusters 2 and 5 express high levels of ISGs. Only 4 of these ISGs are shown in the accompanying figures. Please list which ISGs were increased in neutrophils after both HSV-2 and HSV-1 infection, perhaps in a table. Were there any ISGs specifically higher after HSV-2 infection alone, any after HSV-1 infection alone?

• The authors claim that HSV-1 infection recruits non-pathogenic neutrophils compared to the pathogenic neutrophils recruited during HSV-2 infection. Can the authors please discuss if these differences in inflammation or transcriptional differences between the neutrophils in these two different infections could be due to differences in host response to these two viruses rather than differences in inflammation? Please elaborate on why HSV-1 used as opposed to a less inflammatory strain of HSV-2. Furthermore, does HSV-1 infection induce vaginal IL-18 production in a neutrophil-dependent fashion as well?

Reviewer #1 (Public Review):

In this paper, the authors investigated the role of the N-terminal acetyltransferase Naa10 in mouse development. In addition, they identified a new paralog, Naa12, and demonstrated that it has a redundant role with Naa10 in controlling mouse embryonic development. The results are very clear and should be of interests to those working on development and N-terminal acetylation.

I have several comments for the authors to consider:

1) It is important to show that N-terminal acetylation is lost in the double knockouts. Only with that, the authors can conclude that they have identified the "the complete machinery for the process of amino-terminal acetylation of proteins in mouse development."

2) Naa12 is new, so if not done yet, the sequence needs to be deposited into Genbank.

3) The presentation needs to be polished.

i) The title "Naa12 rescues embryonic lethality in Naa10-Deficient 1 Mice in the amino-terminal acetylation pathway" is misleading. When I saw the title, I got the impression that Naa10-dficient 1 mice show embryonic lethality. I would suggest to change it to indicate that Naa10 and Naa12 have redundant roles in embryonic development. Also, "Naa10-Deficient 1 Mice" needs to be changed to "Naa10-deficient mice."

ii) In the impact statement "Mice doubly deficient for Naa10 and Naa12 display embryonic lethality...", the word "doubly " is unnecessary.

iii) Too many acronyms, which make the reading a bit difficult. The terms NTA and Nt-acetylation could be avoided. iv) At the end of page 9, please cite the sequence alignment in Fig. S6

v) On page 12, "Naa12 may rescue loss of Naa10 in mice" could be more assertive.

vi) Overall, I feel that the authors could polish the manuscript so that the salient points could be conveyed more easily to readers.

The sensing components of the device being worn on the hand are ECG, acoustic, motion, and temperature sensors. It incorporates an electrocardiogram to measure heart activity using amplifiers and resistors to calculate the voltage versus time using electrodes placed on the skin. Assembling the sensors with EGaIn alloy to connect the sensor electronics and polyimine films allows the device to possess its’ flexibility and stretchability. Using polyimine allows for the breaking and reforming of bonds to allow self-healing, and polymerization and depolymerization to recycle the product.

Reviewer #1 (Public Review):

This is a highly interesting manuscript by Gonzalez-Calvo et al., describing the involvement of the CCP domain containing protein SUSD4 in the degradation of GluA4 receptors at cerebellar synapses. The novelty of this work lies in the specificity of this degradation pathway. In comparison, synaptic proteins involved in AMPA receptor endocytosis, such as GRIP1 and PICK1, play a role in multiple trafficking processes. In addition, CCP domain proteins play a role in synaptic pruning, which is closely related to LTD. We will return later to this point.

The paper will certainly enrich the field and further our understanding of cellular plasticity in the cerebellum. These are exciting findings that should be published. I have three relatively minor comments:

1) Figure 2E: it is surprising that the potentiation shown in WT mice is not longer lasting. Under the experimental conditions used here, plasticity seems to be biased towards depression. In the methods, the authors state that they use 2mM Calcium and 1mM Magnesium in their external saline. A recent study (Titley et al., J. Physiol. 597, 2019) has demonstrated that under realistic conditions (incl. an ion milieu of 1.2 mM Calcium and 1mM Magnesium), LTP results under most conditions - even those involving climbing fiber co-stimulation - while LTD only results from prolonged complex spike firing. Optimally, the authors would establish a real LTP control in their WT mice (using conditions as described in Titley et al or similar) and test for changes in the mutants. As LTP is not the focus of this paper and this might be out of the scope of this work, it should be acceptable to leave it as it is, but this caveat should at least be discussed.

2) Figure 3: The climbing fiber physiology is described in detail, but what is missing is a characterization of potential changes in the complex spike waveform, recorded in current-clamp mode. This should certainly be provided. This is important as it has been shown that changes in the complex spike waveform affect the probability for LTD induction (Mathy et al., Neuron 62, 2009). The CF-EPSC is a rather indirect measure.

3) Is synaptic pruning at parallel fiber synapses impaired in the SUSD4 mutants? The LTD deficit is quite obvious. In the light of the role of autophagy in pruning, and the molecular similarity between LTD and pruning, it would be of interest to see whether activity-dependent pruning at these synapses is altered. This aspect is somewhat addressed by the VGLuT1 measures shown in Figure 2, but should be discussed in more depth.

Reviewer #1 (Public Review):

Galbraith et al., using systems immunology approach document in a very detailed manner, provide the textbook example of innate and adaptive immune responses over time following an infection. Here, their clinical assessment is linked to SARS-CoV2 infection. While novelty aspects are not immense, this study is nonetheless well executed, detailed and thorough.

The authors perform association studies and propose that simple seroconversion test should be considered in determining the clinical treatment. While some would argue that is already practiced and perhaps expected, the authors have done an excellent job at detailed immune analyses which they coupled with statistically sound associations. Thus these findings are important to document, and should be considered as experimental ex vivo evidence of what clinical practice may have implicitly already considered.

Reviewer #1 (Public Review):

In this manuscript the authors present a new and improved open-source option for a home cage pellet dispensing device that carries with it the ability to offer continuous monitoring of feeding behavior as well home-cage operant testing. This device solves many issues in the way individuals typically go about studying animal feeding behavior including but not limited to testing at only certain times of the day for limited amounts of time and food restriction in a manner that optimizes cost, functionality, scalability, and customizability over traditional or commercial options. Of note, besides offering the ability to capture massive amounts of home cage feeding and operant data directly in the vivarium of animal housing facilities, a major strength of this approach is that the authors demonstrate that the same amount of learning that would typically require 16 days (one-hour testing sessions) can be accomplished overnight (and with interesting circadian effects on decision-making that are often overlooked). The authors demonstrate useability of this device across institutions in other labs and integration with optogenetics (as well as citing recent studies integrating the device with recording systems).

Reviewer #1 (Public Review):

The authors use smFRET and cross linking to constrain relative orientations of CC1-CC3 helices in STIM1 resting and active conformations. The data are excellent and especially because structures of full length STIM1 are currently lacking they paint an important picture of the structural basis for STIM1 activation. The number of smFRET pairs examined in the inactive state is fairly large and paints a good picture of the relative orientations of helices. In contrast, only a few pairs of sites were examined in activated STIM1 which paint a clear picture of CC1a1 dissociation from CC3, but the remaining postulated conformational changes during activation are inferred primarily from cross linking, and it would have been nice to probe those with smFRET as well. Nonetheless, the data yet provide very useful constraints on STIM1 conformational rearrangements that will be of great value to further structure-function studies.

Joint Public Review:

The manuscript by Tachinawa et al. presents a new method (named RhIP), to study incorporation of recombinant epitope-tagged histone dimers into permeabilized cell nuclei. Using RhIP, the authors demonstrate that both H3-H4 and H2A-H2B and their variants are incorporated in this setup. They proceed with investigating context-specific features of these events, providing evidence that ongoing replication and overall chromatin structure may influence histone dimer incorporation in RhIP. This argues for RhIP having the potential to reveal the mechanisms of chromatin assembly and disassembly genome-wide, and determine how cell cycle and chromatin structure influence these dynamics.

The system is capable of recapitulating major known chromatin assembly pathways and supports existing knowledge of histone dimer dynamics on chromatin. RhIP is also valuable in directly testing histone mutants or variants, as proven by authors.

H3.1 incorporation is shown to be exquisitely dependent on replication, demonstrating that replication itself, as well as replication-dependent chromatin assembly are successfully reconstituted with isolated nuclei, cytosolic extracts and recombinant histones.

The focus of the study is on the incorporation H2A variants, in particular H2A.Z. These data supports known notions about H2A.Z dynamics in chromatin, showing a preference for transcription start sites, and the dependence on the M6 region.

However, the major limitation of the current manuscript is that it remains unclear what properties are driving the observed RhIP effects. This is not fully elucidated and thus limits the ability of RhIP to enable the discovery of new mechanisms.

While replication-dependent mechanisms are well captured by RhIP, it is less clear if transcription and chromatin remodeling is functional in this system and thus if transcription-dependent nucleosome exchange processes are faithfully recapitulated. It is important to improve the comparison of RhIP with 'in vivo' (i.e. existing ChIP-seq datasets) localisation and explicitly develop hypotheses why in some cases the data matches the 'in vivo' situation and in others not. It would be helpful to improve the interpretation of the data to include all existing caveats to the assay setup.

Reviewer #1:

The definition of individuality and its neurogenetic basis is a fundamental problem in ethology and neuroscience. Individuals might fall into discrete groups of personality types; alternatively, individuals might be better described by a broader spectrum of independent traits. An unbiased and quantitative analysis of behavioural traits that make up an individual's personality is a prerequisite of investigating the neuronal and genetic basis of individuality. Given the technical challenges in systematically measuring many behavioural traits across sufficiently large and genetically defined populations and over long time-scales, these questions remain unanswered. This manuscript represents a tour-de-force trying to shed more light in these directions. Werkhoven and colleagues aim at characterizing structure in correlations among a large set of quantitative behavioural measures obtained from the model organism Drosophila melanogaster. The authors performed a large number of high throughput behavioural experiments that cover behavioural paradigms ranging from locomotion to perceptual decision-making. Data were acquired from an inbred, hence isogenic fly line, an outbred line, and various neuronal circuit manipulations. In addition, gene expression data were obtained from individuals. In this way, the authors were able to capture hundreds of behavioural metrics from hundreds of flies, while keeping their individual identities over the course of 13 days. They developed a computational analysis pipeline that quantifies the correlation matrix computed from these metrics. In a 2-step procedure, they condense this matrix into a "distilled" matrix, the entries of which contain all remaining behavioural covariates that were not a priori expected by the authors.

A central claim in this paper is that any structure in this distilled matrix should reveal the principal axes along which individuality should be described. Based on these measurements and analyses flies could not be categorized into discrete types. Moreover, behavioral covariates appear rather sparse and derive from a high-dimensional behavioral space. This would mean that each individual fly is better described by a large combinatorial set of parameters. The same qualitative finding was made between inbred and outbred flies, leading the authors to a conclusion that larger genetic diversity does not change the principal organization of behaviour. The authors perform a set of neuronal-circuit manipulations and claim in conclusion that specific neuronal activity patterns underlie structure in behavioural correlations. Some correlations between gene expression and behavioral metrics were discovered, for example gene expression of metabolic pathways can predict some variability found in the behaviour of flies. The behavioural pipeline is sophisticated and presents a great leap forward in enabling researchers to capture a large set of behavioural measures from a large fly population, keeping the identity of individuals. The work is also presenting an innovative and interesting analysis pipeline.

Although we applaud these ambitious experimental paradigms and computational techniques used, we have several major reservations about this work. Reading through the manuscript multiple times, one is left confused whether the major finding is that no structure whatsoever can be found in these data and to what extent the remaining sparse correlations are of biological / ethological relevance. Another major concern arises from the high level of trial-trial variability that is found in the data, which seems to preclude identification of persistent idiosyncrasies in the behavioural traits of individuals and impedes the reproducibility of the data matrices in two repetitions of the main experiment. We feel that most of the authors' conclusions and claims are confounded by these caveats.

1) Distinguishing persistent idiosyncrasies from trial-to-trial variability and reproducibility of decathlon data

A major challenge in measuring personality traits or individuality is to distinguish between persistent idiosyncrasies and trial-to-trial variation; the latter could result from inherent stochastic properties of behaviors, environmental or measurement noise. To identify an idiosyncratic behavioral trait in an animal one needs to show that individuals exhibit a distinct distribution in a behavioral metric that cannot be explained by trial-to-trial variability. Such a distinction cannot be made if a behavioral metric is measured just once or during a short period, but requires repeated measures over longer time-scales from a sufficiently large population of animals. Unfortunately, in this study many measures have been taken during just one 1-2hs episode per individual of a decathlon. For other measures that were taken repeatedly (circadian assays, unsupervised video acquisition) no efforts have been undertaken by the authors to make the above distinction. Hence, the authors' conclusion that there are no "types" of flies seems premature. In Figure S1 we are surprised to see how low most behavioral measures auto-correlate when recorded on two subsequent days; most auto-correlations further drop to meaningless values when compared over time-periods that correspond to the different epochs of a decathlon. This indicates that trial-to-trial variability dominates the data. In our view it makes little sense to ask whether two behavioral metrics are correlated or not, if their autocorrelations measured over the same time-scale are already extremely low. Moreover, Fig S5B shows that the two decathlons generate largely different data matrices (correlation ~0.25), raising concerns that the results are not reproducible. We wonder whether any structure in behavioral correlations was masked by various sources of noise in this study.

Related to above, there should be error bars and number of flies for the plots in Fig S1. This figure undermines the starting point of the paper claiming persistent idiosyncratic behaviors.

2) Given the concerns above, it is not surprising that the outbred fly line delivers another set of covariates which lack otherwise any further structure. If experiments with >100 inbred flies cannot deliver reproducible results, it cannot be expected that a similarly sized population of outbred flies would. Perhaps the needed population size must be orders of magnitudes larger in this case.

3) Figure 3. It is intriguing to observe how the relationship between switchiness and clumpiness is perturbed upon temperature shifts. But, it seems rather uncorrelated at the restrictive temperature in the Iso line, with a slightly positive value. However, the switchiness-clumpiness correlation is not reproducible in both perturbation types at permissive temperatures. Note, that at both temperatures the Shi and Trp datasets show no - or very low correlations: the Trp lines produce correlations from approx. -0.2 (permissive T) to 0.1 (restrictive T); the Shi lines 0, 0.1 respectively. Fig 3D is very misleading in showing the best fits to the combined datasets. We are not convinced that there is a robust sign-inversion in any of these correlation. The authors' major conclusion that " thermogenetic manipulation and specific neuronal activity patterns underlie the structure of behavioral variation" is not supported by these data. The effect of temperature in the control line, although interesting, is a major caveat for interpreting the results from the Shi and Trp results.

4) The authors measure a large set of low- and high-level behavioral metrics, e.g. walking speed and choices in Y-mazes respectively. A fundamental problem is that many of these metrics potentially have common underlying but trivial causes, e.g. covariation between speeds measured in various conditions is expected. Therefore, the authors condense their original correlation matrix (Fig 1E) into a distilled matrix (1G) by making such judgements. In the present form, it is impossible to evaluate how systematic or arbitrarily these choices were. In many cases, where the same measure was recorded repeatedly (e.g. circadian bout length) or across different conditions (e.g. mean speed) it is obvious, but for other cases it is not obvious at all for the non-expert: for example, why are circadian-bout-length and LED-Y-maze-choice-number lumped into one block of expected behavioral covariates? The current manuscript lacks detailed explanations how the authors systematically created the distilled matrix. Can the sparseness of the distilled matrix be a consequence of too generous pre-allocations? See also point (6). The bulk of the analysis in this paper is done on the "distilled matrices" which are produced by removing correlations within previously defined groups of behavioral metrics. This is said to cleanly reveal unexpected correlations, leading to a main result of the paper, the correlations between "Switchiness" and "Clumpiness". However, if the a priori categories were defined differently, then in the extreme case this correlation would have been completely removed. How sensitive is this correlation to the choice of categories, especially given that many of the Switchiness and Clumpiness metrics are from similar assays (Fig. S8)?

5) For the second pipeline that uses t-SNE and watershed (Fig. 2 and S3C), a previous publication from some of the authors [1] appears to show low repeatability of this analysis.Thus, the repeatability and noise levels of the pipeline must be investigated further. These were 3x 1h recordings per decathlon. Related to comments (1-2), the authors need to show that the differences across flies (Fig 2C,D) are not expected from the level of trial-to-trial variability. Perhaps more data from individual flies need to be recorded?

6) 1G: To our understanding, within-block entries to the distilled matrix should indicate zero correlations, because these are correlations between PCA-projections. But we see many nonzero entries. Given the information provided in the methods it is unclear why this is the case; this requires further explanation.

In any case, within-block correlations are expected to be at least very low. Hence, we expect the distilled matrix to be relatively sparse given how it was calculated. Of interest are then the across-block correlations, the authors should make this point more clear to the readers.

7) Some of the author's claims are related to the spectral dimensionality reduction technique described in Fig. S9. However, none of the real data shown in the main paper figures look qualitatively similar to the toy data. Indeed, the histograms from the main figures are on a log scale, and are thus not comparable to the toy data results. Although the technique might be well suited for certain classes of data, one interpretation of the main paper figures seems to be that no structure is revealed whatsoever. More work should be done to exclude this as a possible interpretation, at least by generating toy data that look like the real Datasets; also with respect to point (6) above.

8) Throughout the paper, the authors use the term "independence" for orthogonal / uncorrelated datasets. Correlation/uncorrelation - dependence/independence are not interchangeable terms. To my understanding PCA decomposes into independent variables only under certain circumstances (multivariate normal distributed data). Have the authors tested for independence?

[1] Todd, J.G., Kain, J.S. and de Bivort, B.L., 2017. Systematic exploration of unsupervised methods for mapping behavior. Physical biology, 14(1), p.015002.

Reviewer #1:

Major points:

1) On the conceptual level, the authors claim that low-intensity amplitude-modulated transcranial focused ultrasound stimulation (AM-tFUS) inhibits local inhibitory interneurons and excites excitatory neurons at high intensity. However, the problem I have with this is that these cell types are highly interconnected within the local circuits, and changing the activity of the inhibitory cell type should have the opposite effect on the excitatory cell type. This has been documented in many experiments (Babl et al., Cell Reports, 2019; Royer et al., Europ.Journ. of Neurosc., 2010), and it unclear why the authors did not see similar effects. Furthermore, it is particularly troubling that the authors observe sustained suppression (five minutes) of the inhibitory neurons yet fail to see any effects on the excitatory neurons (Fig. 3B,D). This conceptual problem raises questions about the experimental setup, which I address below.

2) The authors performed electrophysiological recordings while delivering AM-tFUS with different intensities. To claim the differential effects on the excitatory and inhibitory interneurons, the authors first need to isolate single units in their recordings. However, the authors fail to cluster single units, as documented in the methods section (line 338). There could be several reasons why the authors failed to complete this step. I suggest the following ways to remedy this problem: A. The authors should use a silicone probe with a higher density of recording sites (the distance between the individual sites can be as small as 25 um in some NeuroNexus probes) than the one used in the MS, or use a Neuropixels probe so that the clustering algorithms have a chance to isolate single units. Using NeuroNexus probes with 100 um separation between the recording sites makes it impossible for different channels to "see" the same neuron and severely limits the spike sorting algorithms that separate units based on their unique spatio-temporal waveforms. B. After clustering, the authors should use autoccorellograms to verify that the single units do not violate the refractory period (Hill et al., Journal of Neuroscience, 2011). This is particularly important in areas, such as the hippocampus, which has a high density of neurons, and care should be taken to avoid multiunit recordings. C. The authors should perform one long recording session that comprises all experimental manipulations-the delivery of AM-tFUS, the sham control, and the rest period-to trace how the same units change their firing rate as a function of the experimental manipulations. This would also be very helpful in understanding how the firing rate change in one class of neurons is accompanied by changes in another class. D. Although this might be tricky, the authors could try to perform electrophysiological recordings by lowering the electrode perpendicular to the brain surface. This would allow them to record excitatory neurons and inhibitory interneurons that are connected to each other within the local circuit. This type of recording, would give the authors a greater chance of observing how changes in the firing of the inhibitory cell type affects the activity of the excitatory cell type and vice versa. This type of recording would also be highly desirable for understanding changes in oscillations of the local field potential (LFP) (see below).

3) The authors should report the sites that they have recorded by labelling the electrode with fluorescent dye or performing lesions at the recording sites.

4) When analyzing the effect of AM-tFUS on theta frequency oscillations, the authors should perform current source density (CSD) analysis to verify that the observed effects are local and do not originate from distant sources by volume conduction (Buzsaki et al., Nat. Rev. Neurosc. 2016). Performing electrophysiological recordings perpendicular to the brain surface, as I recommend in 2D, would be necessary for this. The CSD analysis would identify the location in the hippocampus where the change in theta power occurs.

5) The authors argue that temperature changes of 0.2 degrees were not sufficient to alter the firing rate of the neurons. However, the paper to which they refer (Darrow et al., Brain Stim, 2019) shows, in Fig. 7, that heating up brain tissue with a laser even at 0.2C can induce changes in somatosensory evoked LFPs. The authors should perform control experiments that are analogous to those in the cited paper to manipulate the temperature while recording the neurons in order to verify that the observed effects are not due to the changes in temperature.

Minor points:

1) The authors should not use label cells in Fig. 3 as they cannot claim that they recorded single units.

2) In Fig. 5C, Fig. S3B,C, and Fig. S4B,C, the authors should show the full scale of the values. Furthermore, the outliers in these plots (not seen in the figures) may drive the general trends, and removing them should be considered.

3) During AM-tFUS at intermediate power intensity (Fig. 4D,G), the authors observe a very dramatic change in LFP power in the 1-3 Hz frequency range. Although there is no clear underlying change in the firing of neurons at this intensity (Fig. 3E,F,G,H), the authors could speculate on what is happening in this case.

4) Fig. 5B shows a clear reduction of power in the theta frequency range after AM-tFUS in the dentate gyrus as well as in CA1 and CA3. This effect is also seen in Fig. 4G and Fig. S1,2. Although this effect does not reach the level of statistical significance, the authors should report the p-values.

5) Although the suppression of firing rates for a five-minute period after low-intensity AM-tFUS application is interesting, I am not sure if such prolonged after-stimulation effects have ever been documented using other modes of neuromodulation. Therefore, the authors should discuss this effect in line with previous work.

Reviewer #1 (Public Review):

In this paper, Judd et al performed intersectional viral-mediated genetics to resolve a projection map from Ntsr1-positive and inhibitory neurons in the anterior interposed nucleus. They show that, in contrast of what is currently thought, inhibitory neurons that project to the inferior olive in fact bifurcate to multiple brainstem and midbrain areas. This is a thorough and timely paper, with valuable information for cerebellar scientists with implications that will be of interest to the general neuroscience audience. As a direct consequence of the vast amount of information, this paper summarizes a lot of data using acronyms and summary schematics, which makes it at times difficult to follow the core story. A bigger concern is that the main conclusion arguing that inhibitory neurons make widespread extra-cerebellar projections relies on the assumption that the Cre-lines used in the study are able to specifically/exclusively mark to those inhibitory neurons – these details were not fully worked out in this study.

Reviewer #1 (Public Review):

The general thesis of the work, provided by the authors, is the demonstration that latrophilins 2 and 3 function as classical GPCRs at the synapse and that this activity is necessary for synapse formation at a specific synapse within the hippocampus. The topic is interesting and important for several reasons. First, the knowledge of GPCRs at synaptic connections is focused largely on neurotransmitter receptors in the literature – metabotropic GluR and AChR as well as neuromodulatory neurotransmitter receptors (NPY, Seratonin etc). The mechanism demonstrated in this work concerns the function of a GPCR receptor system that could confer specificity to synapse formation.

The effect sizes that are documented throughout this work are large, giving this reviewer confidence that the effects are robust and will be reproducible and, more importantly, are indeed a biological mechanism related to synapses.

The other major strength of the work is that the studies in neuronal cell culture are recapitulated in vivo providing additional confidence in the validity and importance of the work. Indeed, the concept of specificity requires this type of in vivo work as the identity of synapses in culture systems can not be readily determined.

A further strength is the rational and implementation of three mutant receptors that are used to dissect the signaling modalities of these receptors, validated for their effects on the protein and then used as rescue constructs in synaptogenesis assays.