{"messages":[{"count":100,"status":"ok","interval":"2021-03-21:2021-04-28","cursor":"5","total":519}], "discussions":[{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225v2","doi":"10.1101\/2020.12.07.20245225"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Response to Public Reviews**\n\n**Response to Reviewer #1 (Public Review):**\n\nWe thank the reviewer for their kind comments and were glad to learn that the reviewer felt the manuscript significantly contributed to body of knowledge on COVID-19.\n\n**Response to Reviewer #2 (Public Review):**\n\nWe thank the reviewer for their dedication to a detailed review, and we greatly appreciated the constructive suggestions given that have helped strengthen the overall manuscript.\n\nWe agree with the reviewer that the loss of power for detecting a loss of sensitivity by the ID NOW PCR assay was hindered by the overall low population frequency of COVID-19 disease; this resulted in a low number of positive patients and ultimately led to early study termination as a result. Because of the reviewer\u2019s helpful observation, we have now re-estimated the power of\nthis study without reference to the observed results, but with consideration for the sample size and proportion of RT-PCR positive tests that were observed when the study was terminated. This new re-estimation suggests that the study retained 80% power to find a difference of 15% or more in sensitivity between ID NOW isothermal PCR and conventional RT-PCR; this analysis also\ndemonstrated over 95% power to find a difference in specificity of more than 5%. Indeed, the significant drop in population prevalence that led to a loss of power for detecting loss of sensitivity expectedly resulted in an increase in power for detecting loss of specificity. We have expanded the Methodssection of the paper to better expose these issues, and we have expanded\nour statement of strengths and limitations in the Discussion for the same reason.\n\nThe Methods section of the manuscript now reads as follows:\n\n\"The original study design called for enrolling 2000 symptomatic and 500 asymptomatic subjects, which would have provided, in the symptomatic population, power of 80 % for finding a difference (at \u03b1 = 0.05) of 5% in the sensitivity of ID NOW compared with the RTPCR reference standard; inclusion of at least 1350 negative patients would have provided 95% power (at \u03b1 =0.025) for finding a 5% difference in specificity. The study design assumed a population prevalence of 10%, and the study was terminated early when the population prevalence dropped to such a low level as to make the study unaffordable. We have re-estimated the power of this study without reference to the observed results\nbut considering the sample size and proportion of RT-PCR positive tests that were observed when the study was terminated. This re-estimation suggests that the study retained 80% power to find a difference of 15% or more in sensitivity between ID NOW and RTPCR, and well over 95% power to find a difference in specificity of more than 5%. Indeed, the significant drop in population prevalence that led to a loss of power for detecting loss of sensitivity resulted, as expected (Bujang and Adnan, 2016), in an increase in power for detecting loss of specificity.\n\nThe revised section of the Discussion regarding strengths and weaknesses now reads as follows:\n\n\"Our clinical study also suffered a significant loss of power to assess ID NOW sensitivity as a result of the low number of positive results, and the reduction of sample size caused by the decision to terminate the study as a result. The meta-analysis is also limited by the small number of studies meeting inclusion criteria, and the fact that positive cases are heavily concentrated in only a single study. Strengths of the clinical study include pretrial power analysis with sample size estimation, precise adherence to the ID NOW specimen acquisition protocol, and extremely high power for assessing assay specificity. Taken together with the focus on initial diagnosis of disease in the studies included in the meta-analysis, we believe the combination of trial and meta-analysis provides useful\ninformation for clinicians for whom point-of-care testing is helpful.\"\n\nWe thank the reviewer for noting the unique findings from the current cohort study in comparison to existing literature. The current cohort study was done with meticulous care to identify apparent \u201cfalse positives\u201d returned by ID NOW PCR assay. This is also reflected in some of the high-quality studies available in the literature. In the supplementary data, we have provided confusion matrices for all the studies included in the meta-analysis. We have identified four such cases out of 1,942 total ID NOW tests. Cell sizes of 0 (from our study) and 4 are two small to allow use of Chi-squared for assessment of heterogeneity, and unfortunately the total\nnumber of tests is too large to allow computation of Fisher\u2019s exact test; however, with such small numbers it is reasonable to treat them as samples drawn from Poisson distributions. The confidence interval around a Poisson estimate of 4 is 1.08987 \u2264 \u03bc \u2264 10.24159, and that around a Poisson estimate of 0 is 0.00000 \u2264 \u03bc \u2264 3.68888. The overlap is such that the estimates, 0 and 4,\nare consistent with having sampled the same distribution. While this does not allow us to conclude that no difference exists between our results and those of the other studies, it does not provide any evidence that there is a difference from the current cohort study and those previously published. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225v2","doi":"10.1101\/2020.12.07.20245225"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nTu et al. submit a manuscript that evaluates the performance of the Abbott ID NOW SARS-CoV-2 test in an ambulatory cohort relative to RT-PCR tests. They enrolled 785 symptomatic patients, 21 tested positive for SARS-CoV-2 by ID NOW and PCR (Hologic) while 2 tested positive only via PCR. They also tested 189 asymptomatic individuals, none of whom tested positive by either ID NOW or PCR. The positive agreement between ID NOW and PCR was 91.3%, and the negative percent agreement was 100%. The authors also provide a review and meta-analysis of ID NOW performance across at least a dozen other named studies which is thorough and interesting. The cohort assessed in this study is small and localized. The data is undermined by sample size, with the most glaring example being the 100% negative percent agreement, which doesn't compare with the known performance of the test in broader populations."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225v2","doi":"10.1101\/2020.12.07.20245225"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThe study presents relatively high and robust sensitivity of Abbott ID NOW for the detection of SARS-CoV-2 (COVID-19) in an ambulatory population, utilizing the RT-PCR methodology as a comparative correlation. The study was well designed and enrolled both symptomatic and asymptomatic populations to provide sufficient statistical power for the comparative analysis of the methodologies, as well as to represent accurately the patient populations. This is a useful and timely study that has a great impact in clinical setting for the rapid detection of COVID-19."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.07.20245225v2","doi":"10.1101\/2020.12.07.20245225"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThe authors evaluate the performance of the Abbott ID NOW SARS-CoV-2 test in a group of non-hospitalized individuals being tested for COVID-19 and compared that performance to an RT-PCR test. The authors also provide an interesting review and meta-analysis of ID NOW performance across the literature. The cohort assessed in this study, however, was small and localized, which currently undermines its comparison with the known performance of the test in broader applications.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411v1","doi":"10.1101\/2021.01.27.428411"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nIn this manuscript, Xue et al. assessed many AAV vectors and demonstrated that Thioredoxin-interacting protein (TXNIP) saves RP cones by enhancing their lactate catabolism. The results of this study were based on cone counting, IHC and reporter. While the authors focus on the cellular metabolism in the Txnip-mediated rescue effect, it is unknown whether anti-oxidative stress plays a role as well."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411v1","doi":"10.1101\/2021.01.27.428411"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThe goal of this manuscript is to develop gene-agonistic approaches for promoting cone survival in retinal degenerative diseases. Based on their previous studies, the authors tested a total of 20 genes by subretinal delivery using an AAV vector which utilized a cone-specific promoter. Most of these genes augmented glucose utilization. Interestingly, only Txnip showed a positive result by prolonging cone survival (tested up to 50 days in rd1 retina). Txnip therapy also appears to be effective in rd10 and rho-\/- retina. Additional strength of this study is the use of Txnip C247S allele that blocks its association with thioredoxin. Furthermore, additional work on how Txnip may contribute to cone survival by better utilization of lactate for energy is well presented though the conclusion on \"heathier\" mitochondria require additional data. This manuscript is potentially of great interest. The data are extensive and biological implications of the study are clear. However, the broad conclusions with respect of Txnip therapy for RP (or even AMD) are less than justified based on the data. Two weaknesses are apparent: the first is related to the method of quantification using whole mount retina, and the second related to the duration of the study. Immunostainings of retinal sections (and even TEMs) are critical to elucidate the structure of surviving cone photoreceptors (specially in the absence of rods) and their relationship to other cells (e.g., RPE, bipolar cells, glia). Similarly, Prusky's OMR can't be equated to visual acuity. The authors need to show cone structure\/function at P50 and beyond (how long do the cones survive?) in rd1 and other models before claiming the potential benefit of Txnip for retinal and macular degeneration."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.27.428411v1","doi":"10.1101\/2021.01.27.428411"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis authors used AAV in mouse retinas to express several candidate genes that they thought might have favorable effects on cone metabolism and therefore make cones more robust to stress caused by genetic deficiencies. Txnip is the most effective at prolonging cones survival and a combo of HK and PFK is the most effective at shortening cone survival. The investigators evaluated effects of specific mutations in Txnip with known biochemical effects. Their general conclusion is that Txnip may be enhancing mitochondrial function and ATP production and it may allow cones to use alternative fuels more effectively. This is an interesting and informative set of findings and it is presented and discussed in the context of what currently is known about retina metabolism and its influences on photoreceptor survival.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740v1","doi":"10.1101\/2021.01.14.426740"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nIn this revised manuscript (Oon and Prehoda), the authors performed additional live-imaging experiments and recorded aPKC and actin dynamics simultaneously in larval neuroblasts. They also provide evidence that aPKC polarization is lost upon F-actin disruption by Latrunculin A treatment. These are great improvements. The pulsatile dynamics of actin and myosin II showed in the manuscript are compelling. Images presented in this manuscript are of high-quality and impressive.\n\nHowever, the pulsatile apical myosin network in delaminating neuroblasts in Drosophila embryos was reported previously (An Y. et al., Development, 2017). This important and relevant paper should be cited in the introduction of the current manuscript. Therefore, the finding on the pulsatile actomyosin in larval brain neuroblasts reported in this manuscript is not a total novel discovery. Another major concern is that Lat-A did not specifically disrupt actomyosin pulsatile movements, as it generally disrupts the F-actin network. So these experiments only strengthened the link between the F-actin network and Par polarity (which was already demonstrated in Kono et al., 2019; Oon 22 and Prehoda, 2019). Low doses of Cytochalasin D are known to disrupt myosin pulses still allowing the assembly of the actomyosin network (Mason et al., Nature Cell Biology 2014). The author should treat neuroblasts with low doses of CytoD to only disrupt actomyosin pulses, not the entire F-actin network, and examine the effect on Par polarity. It is also worthwhile to knockdown sqh to disrupt apical pulsatile actin dynamics. Besides, most of the concerns previously raised by the reviewer were not addressed in the revised manuscript."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740v1","doi":"10.1101\/2021.01.14.426740"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nPreviously, Oon and Prehoda showed apically directed movement of aPKC clusters during polarization of the neuroblast prior to asymmetric cell division. They found that these movements required F-actin, but the distribution of F-actin has only been reported for later stages of neuroblast polarization and division. Here, the authors report pulses of cortical F-actin during interphase, followed by an apically directed flow at the onset of mitosis, a strong apical accumulation of F-actin at metaphase and anaphase, followed by fragmentation and basally directed flow of the fragments. aPKC clusters are shown to colocalize with the F-actin networks as they flow apically. The F-actin networks are also shown have partial colocalization with non-muscle myosin II, suggesting a possible mechanism for their movement. Finally, the authors solidify the results of actin inhibitor studies from their 2019 study by showing that reported effects on aPKC localization are preceded by F-actin loss as would be expected but was not previously shown. Overall, the Research Advance extends the past study by more directly showing the involvement of F-actin and myosin in the apical localization mechanism of aPKC, and by describing F-actin and myosin dynamics prior to this transition. The following concerns should be addressed.\n\n1) The pulsatile nature of broad F-actin networks is evident during interphase, but these pulsations substantially subside upon entry into mitosis, and at this stage an apically directed flow of F-actin is the main behavior evident. This transition from pulses to flow is evident in both the movies and the kymographs of the F-actin probe. However, the authors state that the pulsations continue at the onset of mitosis and as the apical cap of aPKC matures. It is unclear whether the apical flow of aPKC and F-actin is associated with small-scale defined F-actin pulses, or small-scale random fluctuations of F-actin. The F-actin flow alone is an informative finding. The authors should consider revising their descriptions of these data (including in the manuscript title), or provide clearer examples of defined F-actin pulsations during the stage when aPKC polarizes.\n\n2) I checked the main text, methods, figures and figure legends, but could not find listings of sample sizes. Thus, the reproducibility of the findings has not been reported.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740v1","doi":"10.1101\/2021.01.14.426740"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nOon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how\/why actin filament was required to localize\/polarize Par complex. Specifically, using spinning disc confocal microscopy with high temporal resolution, they found the directed actin movement toward the apical pole, which nicely correlates with concentration of aPKC. They also show that myosin II is involved in this pulsatile movement of actin filament. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems. Although descriptive in nature, I think this is an important observation and indicates a universal mechanism by which cells are polarized. I think this is a well executed study."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-23","createdOn":"2021-03-23","completedOn":"2021-03-23","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.14.426740v1","doi":"10.1101\/2021.01.14.426740"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nOon and Prehoda report pulsatile contraction of apical membrane in the process of Par protein polarization in Drosophila neuroblasts. This explains how\/why actin filament was required to localize\/polarize Par complex. This very much resembles the observation in C. elegans embryos, and nicely unifies observations across systems.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782v2","doi":"10.1101\/2020.05.28.120782"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nNICEdrug.ch integrates well-established previous methods\/pipelines from the same group and provides an easy-to-use platform for users to identify reactive sites, create repurposing and druggability reports, and reactive site-specific similarity searches between compounds. Case studies provided in the manuscript are quite strong and provide ideas to the reader regarding how this service can be useful (i.e., for which kinds of scientific aims\/purposes NICEdrug.ch can be utilized). On the other hand, there are a few critical issues related to the current state of the manuscript, which, in my opinion, should be addressed with a revision.\n\nMajor issues: \n\n1) Two of the most critical drawbacks are, first, the lack of quantitative assessment of the abilities of the service and its analysis pipeline. Use cases provide valuable information; however, it is not possible to assess the overall value of any computational tool\/service without large-scale quantitative analyses. One analysis of this kind has been done and explained under \"NICEdrug.ch validation against biochemical assays\" and \"Comparison of NICEdrug.ch predictions and biochemical assays\"; however, this is not sufficient as both the experimental setup and the evaluation of results are quite generic (e.g., how to evaluate an overall accuracy of 0.73 without comparing it to other computational methods that produce such predictions, as there are many of them in the literature). Also, similar quantitative and data-driven evaluations should be made for other sections of the study as well. \n\n2) The second critical issue is that, in the manuscript, the emphasis should be on NICEdrug.ch, since most of the underlying computational methods have already been published. However, the authors did not sufficiently focus on how the service can actually be used to conduct the analysis they mention in the use cases (in terms of usability). Via use cases, authors provide results and its biological discussion (which actually is done very well), but there is no information on how a potential user of NICEdrug.ch (who is not familiar with this system before and hoping to get an idea by reading this paper) can do similar types of analyses. I recommend authors to support the textual expressions with figures in terms of screenshots taken from the interface of NICEdrug.ch at different stages of doing the use case analyses being told in the manuscript. This will provide the reader with the ability to effectively use NICEdrug.ch. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782v2","doi":"10.1101\/2020.05.28.120782"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nThe authors developed a very interesting tool, named NICEdrug.ch, used it to identify drug metabolism and toxicity, and finally predicted druggability of disease-related enzymes and reposition drugs. Comprehensive integration effort based on publicly available datasets and several previous methods developed by the authors (e. g. BridgeIT, BNICE.ch, ATLAS of Biochemistry) results with a resource named NICEdrug.ch. The idea is interesting and addresses a very important problem in the field. The manuscript is clearly written, provides enough analysis of overall challenges and an overview of the most important results. Also, it presents figures that are remarkable. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.28.120782v2","doi":"10.1101\/2020.05.28.120782"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nIn this study, the authors proposed a new web service\/tool and its database, NICEdrug.ch, to be used in the fields of drug discovery and repurposing with the exploration of the metabolic fate of small molecules. The study is timely and will potentially have a high impact as metabolic evaluation of drugs\/compounds is a critical topic that is still understudied.\n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802v1","doi":"10.1101\/2021.01.22.427802"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nIn this manuscript, Lamers et al wanted to characterise the previously reported adaptation of SARS-CoV-2 to non-human (Vero) cells. Vero cells are commonly used by laboratories to grow experimental stocks of some viruses as these cells permit high titres of many viruses, they lack the ability to produce type I interferons (cytokines which could interfere with downstream assays), and their non-human nature means soluble factors in virus stocks are less likely to impact experiments in human cells. However, a number of reports have recently been published describing that growth of SARS-CoV-2 in Vero cells leads to loss of the SARS-CoV-2 Spike protein multibasic cleavage site (MBCS). This apparent adaptation to the Vero cell-line leads to a virus compromised in its ability to enter, and therefore replicate in, human cells, meaning that experimental results obtained in human cells using the Vero-adapted SARS-CoV-2 may not fully reflect the situation occurring with authentic SARS-CoV-2. It is therefore important for the research community to understand SARS-CoV-2 adaptation to laboratory cell-lines\/conditions and to have propagation methods that are suitable for maintaining the authenticity of clinical virus isolates.\n\nThe major finding of Lamers et al in this manuscript is that human cell-lines (e.g. Calu-3) and primary human organoid systems can be used to propagate clinical isolates of SARS-CoV-2 to high titres without the acquisition of 'laboratory adaptations'. To get to this finding, the authors carefully study the adaptation of a representative SARS-CoV-2 isolate in Vero cells, monitoring plaque size phenotypes and performing whole-genome deep sequencing to identify adaptive variants that appear in the viral Spike gene. These variants (including newly-described substitutions as well as deletions around the MBCS) are validated for their impact on viral infectivity in human and Vero cells using pseudovirus assays, fusion assays, and western blot assays, and their role in affecting the entry route of SARS-CoV-2 is dissected using pathway-specific inhibitors (such as camostat and E64D) and cell-lines with\/without TMPRSS2 (an important protease for Spike cleavage). Importantly, using these assays and tools, the authors can make solid and well-reasoned arguments as to why SARS-CoV-2 adapts to Vero cells, and thus why certain culture conditions and cell substrates lead to a loss of SARS-CoV-2 genetic stability. Using similar tools, this also allows the authors to carefully study whether any adaptations occur when SARS-CoV-2 stocks are passaged in human cell substrates (such as Calu-3 or primary human organoids), and study culture conditions in Veros (such as expression of TMPRSS2) that prevent changes in SARS-CoV-2.\n\nThe data in this manuscript are thorough and well-presented. Importantly, the conclusions are strongly supported by the data, particularly the overall take-home message that human cell substrates can be used to efficiently propagate SARS-CoV-2 isolates without introducing cell culture adaptations. However, beyond this simple message, the manuscript also provides new mechanistic insights into the reasons for such viral adaptations in the Vero cell system, and identifies previously undescribed adaptations in the MBCS region that will be valuable for other researchers to take note of. The authors also describe a methodological workflow to produce SARS-CoV-2 in human cells that highlights a buffer-exchange step to remove potentially interfering human cytokines\/debris, and which will be useful for other researchers.\n\nOverall, the manuscript makes a clear and important contribution to the SARS-CoV-2 field and will be of interest to active researchers who are studying this virus experimentally."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802v1","doi":"10.1101\/2021.01.22.427802"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThis manuscript, which follows on from a recent eLife paper documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2, shows that growing SARS-CoV-2 on relevant epithelial cell lines or differentiated stem cell-derived culture systems prevents the emergence of MBCS mutations than impact on properties of S that contribute to cell tropism and the viral entry mechanism.\n\nThe paper builds on the authors previous work and that of others, and in some respects the results are not surprising. Nevertheless, the paper sets out a number of important findings. 1) That SARS-CoV-2 grown in Vero cells rapidly acquire MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations that are not apparent in consensus sequence reads, 3) that factors such as the addition of fetal calf serum can influence the selection of mutant phenotypes and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the production of SARS-CoV-2, and potentially other viruses, avoiding the pitfalls that can arise from growing viruses in permissive transformed cell lines.\n\nThe data and manuscript are clearly presented, and my concerns are minimal. Overall, the paper will make a useful addition to the SARS-CoV-2 literature and will be of value to researchers working not just of SARS-CoV-2 but on many other viruses.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427802v1","doi":"10.1101\/2021.01.22.427802"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis manuscript follows up on work documenting the relevance of the multi-basic cleavage site (MBCS) in the spike (S) protein of SARS-CoV-2 for determining cell tropism and mode of cell entry. The paper describes a number of important findings: 1) That SARS-CoV-2 grown in Vero cells rapidly acquires MBCS mutations, where as virus grown in airway epithelial cells or Vero-TMPRSSR2 cells do not; 2) that deep sequencing is necessary to see mutations emerging that are not apparent in consensus sequence reads; 3) that factors such as fetal calf serum can influence the selection of mutant phenotypes, and 4) that cultures derived from differentiated stem cells can provide reproducible systems for virus culture. Together, the work sets out clear guidelines for the propagation of SARS-CoV-2 to avoid adaptations to laboratory cell-lines\/conditions and maintain the authenticity of clinical isolates. The work has relevance to other viruses and the use of permissive transformed cell lines.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993v1","doi":"10.1101\/2021.02.12.430993"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nIn this manuscript, B\u00f6hm et. al. aim to understand how precise kinetochore assembly is tied to cell cycle progression in budding yeast. In this work, the authors identify CDK phosphorylation sites concentrated in the N-terminus of Ame1, a protein of the COMA complex, and set out to characterize the role these phosphorylation sites may play protein function at the kinetochore. Although phospho-null Ame1 does not affect cell viability, expressing an Ame1 mutant that lacks the phosphorylated domain results in cell death. Interestingly, overexpression of the phospho-null Ame1 mutant accumulates to a higher level than the wild type protein leading the authors to hypothesize that these phosphorylation sites function as phosphodegrons in the Ame1 protein. Through molecular modeling and genetic analysis, the authors determine that Ame1 is a substrate of the SCF E3 ubiquitin ligase and is likely recognized by the Cdc4 F box protein. The authors go on to convincingly show that phosphorylation of what is referred to as the \"CDC4 phosphodegron domain\" is phosphorylated in a step-wise manner that is cell cycle dependent and that the phosphorylated Ame1 protein specifically is degraded in mitosis. In addition to Ame1 phosphorylation, the authors show that Ame1 degradation depends on whether Ame1 is bound to the Mtw1c (binding prevents degradation), which only happens at a fully assembled kinetochore. Based on these observations, the authors propose a model in which the phosphodegron motif functions to degrade any molecules of the COMA complex that are not incorporated into the kinetochore and in this way prevents kinetochore assembly at ectopic regions of the chromosome. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993v1","doi":"10.1101\/2021.02.12.430993"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nB\u00f6hm et al. investigated the phosphorylation of the Ctf19CCAN component Ame1CENP-U by Cdk1 which forms a phosphodegron motif recognized by the E3 ubiquitin ligase complex SCF-Cdc4. They identify phosphorylation sites on Ame1 and demonstrate that phosphorylation of Ame1 leads to its degradation by the SCF with Cdc4 in a cell-cycle dependent manner. They also demonstrate that the outer kinetochore component Mtw1c shields Ame1 from Cdk1 phosphorylation in vitro. Finally, they propose a model in which at least one component, Ame1, is present in excess at S-phase in yeast to incorporate into high levels of sub-complexes for efficient inner kinetochore formation on newly duplicated centromere DNA. Then, in mitosis, phosphodegrons serve to mediate the degradation of excess Ame1 (and presumably other CCAN components) and in so doing protect against the formation of ectopic outer kinetochores. \n\nThis manuscript puts forth well-designed and thorough experiments characterizing the phosphorylation of Ame1 and its regulation by the SCF-Cdc4 complex. The writing is clear and the figures are generally easy to understand. The authors succeed in asking pertinent questions, designing experiments to answer them, and considering potential alternative explanations or confounding factors. As a whole this creates a generally convincing study regarding the phospho-regulation of Ame1. However, I also have some important concerns: \n\n1) The authors begin the manuscript by mapping phosphorylation sites across Ctf19CCAN components but then largely narrow their experimental focus to Ame1 and to a lesser extent its binding partner Okp1. Without mutation of other components, the Ame1 mutant phenotypes are either absent or very mild. This would seem to implicate that, if this is an important process, that other targets for this quality control mechanism must exist. As it stands now, the focused investigation does not make the most compelling case for the broad conclusions that are claimed. More extensive investigation of phosphoregulation of CCAN subunits beyond Ame1 would certainly help justify the claim that phosphoregulation is used to clear excess CCAN subunits and protect against ectopic kinetochore assembly. Is there another lead from their initial mass spec work that could provide some molecular evidence that this is a general process? Failing that, the discussion could at least provide some hint at how the model could be tested in future studies. \n\n2) The conclusion that the binding of the Mtw1 complex shields Ame1 phosphodegrons is arguably one of the most significant and interesting claims made in this paper. However, the evidence presented to support this claim seems to rely exclusively on in vitro data. Thus, this part is out of balance with other parts of the paper where some in vivo correlations are attempted\/made. \n\n3) The central model mentioned at the outset strongly predicts that the mitotic degradation of Ame1 doesn't impact its abundance at centromeres. That is not the only possibility, though, and some measurement (fluorescence of a tagged Ame1 or a ChIP on centromere DNA) of Ame1 at centromeres before and through mitosis would help instill confidence in the proposal. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993v1","doi":"10.1101\/2021.02.12.430993"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nKinetochores are huge protein assemblies on chromosomes which are used as attachment point for microtubules and allow microtubules to pull chromosomes into daughter cells during cell division. The proteins that form the kinetochore are well known, but the temporal regulation of the assembly of all these proteins into functional kinetochores is less understood. \n\nIn this paper the authors have identified phosphorylation sites in the 'CCAN' of budding yeast, the 'inner', i.e. chromatin-proximal, part of the kinetochore. They characterize in detail the function of phosphorylation of Ame1 (CENP-U in humans), which is part of CCAN. \nThe data support the idea that a cluster of phosphorylation sites in Ame1 is phosphorylated by mitotic CDK1 and serves as phospho-degron for the E3 ligase SCF\/Cdc4. \n\nThe authors show phosphorylation of these CDK1 consensus sites in vivo and their phosphorylation by CDK1\/Clb2 in vitro. Genetic experiments and molecular dynamics simulations support the idea that phosphorylation sites on Ame1 can serve as phospho-degron for SCF\/Cdc4. Even the non-phosphorylatable mutant of Ame1 is stabilized in an SCF mutant background, though, suggesting that this phospho-degron is not the only way in which SCF influences kinetochore protein levels. \n\nMutants in the characterized phosphorylation sites do not impair budding yeast growth. This suggests that the degron characterized in this paper may be important for fine-tuning, but is not essential for the proper execution of mitosis. The observations overall add to prior evidence that kinetochore assembly can be regulated by phosphorylation and\/or ubiquitination. \n\nInterestingly, the authors find that phosphorylation of Ame1 by CDK1 in vitro is impaired when Ame1 binds Mtw1, another kinetochore protein. The fact that Mtw1 seems to shield these sites from phosphorylation leads the authors to put forward an interesting model: they propose that cell cycle-dependent phosphorylation and SCF-dependent degradation of kinetochore subunits allows for excess subunits during kinetochore assembly in S-phase (which will speed up assembly) while depleting any excess subunits after assembly, when the kinetochore needs to be functional. \n\nThis is an interesting model. The in vivo evidence is still limited, though. For now, it remains unknown whether the phosphorylation status of kinetochore-bound and free Ame1 is indeed different, whether more soluble Ame1 exists in S-phase, whether too early degradation of Ame1 (or possibly other kinetochore proteins) indeed impairs kinetochore assembly, or whether a failure to remove the soluble pool after assembly leads to mitotic defects. It is an attractive proposal, though, that can now be further explored experimentally. \n\nIn addition to the specific characterization of Ame1 sites, the paper also includes comprehensive data on CCAN phosphorylation sites obtained by mass spectrometry which can serve as basis for future studies. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.12.430993v1","doi":"10.1101\/2021.02.12.430993"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nThis paper will be of interest to those in the fields of chromosome biology, mitotic regulation, and proteostasis. The authors put forward an interesting model of phosphodegron regulation of kinetochore assembly based on convincing genetic and biochemical data. The novel model will require some additional evidence before it can be considered well-supported, but the paper represents an advance in our knowledge of kinetochore regulation with experiments that are rigorous, well-designed and carefully conducted. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671v1","doi":"10.1101\/2021.02.10.430671"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nComputational models, provide a way to understand emergent network function, and at their best provide a canvas for experimentalists to probe hypotheses regarding function. In this manuscript, Bui and colleagues provide a set of iterative models to describe the locomotor development of larval zebrafish at key developmental stages. These include coil, double coil, and swimming behavior that leads to 'beat-and-glide' behavior. During development, the model steadily moves from gap junction mediated connectivity to more complex synaptic-based network models. In my opinion, this is a very interesting foundation that can be used as a catalyst for future research for experimentalists or to develop more involved models. Like any model it is possible to be critical of the assumptions made. But I expect that it will not be static and be revised over the years. It is important to realize that these sets of models are unique in that they strive to provide models for motor control of a single species across development. The zebrafish is an excellent example since genetic models are widely used, development is swift, and there is active research to understand the physiology of locomotion. \n\nStrengths and weaknesses:\n \nThe key strength of this manuscript is the detailing of a set of related models detailing the motor output of the larval zebrafish across key stages of development. The models should form a basis for future research. It also a first of its kind - I don't know of similar models focusing on development of locomotor function. The main weakness is the reliance on assumptions of model connectivity. But I suggest that if the model is treated as a basis for the community to refine and validate it will be incredibly useful. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671v1","doi":"10.1101\/2021.02.10.430671"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nThis study presents iteratively constructed network models of spinal locomotor circuits in developing zebrafish. These models are shown to generate different locomotor behavior of the developing zebrafish, in a manner that is supported by electrophysiological and anatomical data, and by appropriate sensitivity analyses. The broad conclusions of the study result in the hypothesis that the circuitry driving locomotor movements in zebrafish could switch from a pacemaker kernel located rostrally during coiling movements to network-based spinal circuits during swimming. The study provides a rigorous quantitative framework for assessing behaviorally relevant rhythm generation at different developmental regimes of the zebrafish. The study offers an overarching hypothesis, and specific testable predictions that could drive further experimentation and further refinement of the model presented here. The models and conclusions presented here point to important avenues for further investigation, and provide a quantitative framework to address constituent questions in a manner that is directly relatable to electrophysiological recordings and anatomical data. The study would benefit from additional sensitivity analyses, and from the recognition that biological systems manifest degeneracy and significant variability along every scale of analysis. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671v1","doi":"10.1101\/2021.02.10.430671"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThe manuscript is somewhat readable but the many acronyms for the cell types in model and biology make it difficult to follow. Is there a reason why the biological neuron names cannot be used in the model? The presentation of data in figures can be more powerful. In many cases, the data in figures and the supplemental videos show apparently different results. This can be an artifact of how the videos were made and if yes, these can be improved. Tail tip coordinates can be plotted to show the behaviors in much better detail. \n\nEspecially for beat and glide swimming, the points regarding burst firing, inhibition, etc. have not been robustly made. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.10.430671v1","doi":"10.1101\/2021.02.10.430671"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nIn this manuscript, Roussel et al., build models of spinal networks capable of generating coiling and swimming behaviors of embryonic and larval zebrafish. The models use details obtained from earlier experimental studies and insert novel network elements, thus providing testable ideas for rhythm generation. The study will be of high value to those interested in motor pattern generation in general and zebrafish spinal cord function in specific. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784v1","doi":"10.1101\/2021.01.15.426784"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This rebuttal was posted by the corresponding author to *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Reply to the reviewers\n\nReviewer #1 (Evidence, reproducibility and clarity (Required)):\n\n*The authors aimed to understand the control and the elimination of disseminated tumor cells by NK cells within the lung, their main question being how pulmonary NK cells are able to prevent tumor cells from colonization in the lung.*\n\n*To dissect this question, Hiroshi Ichise and colleagues took advantage of the ultra-sensitive bioluminescence whole body imaging system combined with intravital two-photon microscopy technology involving genetically-encoded biosensors tumor or NK cells to explore the behavior and functional competences of NK cells in an experimental lung metastasis model.*\n\n*First, the authors have monitored the fate of intravenously injected B16-Akaluc cells from 5 min to 10 days and observe that tumor cells decrease rapidly within the first 12-24 hours. In parallel, they performed asialoGM1+ and NK1.1+ cells depletion by injection of depleting anti-aGM1 and anti-NK1.1 antibodies in order to see the involvement of these populations on the elimination of the disseminated tumor cells. They conclude that a rapid decrease of the tumor cells is mediated by NK cells. Consisting with this first data, the authors observe also the same early NK cells mediated impact on two other syngenic mouse tumor cell lines : the BRAFV600E melanoma and the colon adenocarcinoma MC-38.*\n\n*In a second part, the authors dissected NK cell dynamic behaviors in the pulmonary capillaries by taking advantage of the NKp46iCRExrosa26dtTomato mice where NKp46+ cells are fluorescents and performed 2P intravital imaging to follow the in situ the NKp46+ cells behavior. They could nicely observe that NK cells arrive from the capillaries and patrol on the lung epithelial cells in a stall-crawl-jump manner. Moreover, they also show that the attachment to the pulmonary capillaries is mediated by LFA-1. In the presence of B16F10 tumor model, they observe that NK cells stay longer in the capillaries and increase their duration time of crawling indicating that NK cells stay in contact longer with tumor cells.*\n\n*The authors then explored the NK-mediated tumor killing in the lung by measuring tumor cell apoptosis using B16F10-SCAT3 cells (which leads to visualize caspase 3 activation) and Ca2+ influx in tumor cells expressing two Ca2+ sensors, GCaMP6s and R-GECO. They could observe casp3 activation but also Ca+ influx on tumor cells within few minutes after encountering NK cells. They also observe that evasion of NK cell surveillance is mediated by Nectin-5 and Nectin-2 expressed on tumor cells.*\n\n*Then, they focus on NK cell activation by looking at ERK activation. To do so, they have isolated NK cells from Tg mice expressing a FRET-based ERK biosensor and performed in vitro killing assay against B16-R-GECO tumor cells but also in vivo experiments. For the in vivo experiments, they have developed reporter mice whose NK cells express the FRET biosensor for ERK. They observe that ERK-dependent NK cell activation contributes to the elimination of disseminated tumor cells within the first few hours but not after 24hours. Indeed, theu observe that B16F10-Akaluc tumor cells are equally eliminated when injected 24h after a first injection of B16F10 or PBS in mice. The authors concluded that tumor cell acquire the capacity to evade NK cell surveillance after 24h rather than a hypothesis toward NK cells loose tumoricidal activity over time.*\n\n*Finally, the authors have explored their last result on the potential tumor cell evasion of the NK cell surveillance. They show that this NK cell evasion is mediated by the shedding of cell surface Necl-5. They next show that clivage of extracellular domain of Necl-5 was mediated by thrombin in vitro and that anti-coagulation factors such as Warfarin, Edoxaban or Dabigatran Etexilate promote tumor elimination as observed by the bioluminescence experiments. This loss prevents the NK cell signaling needed for effective killing of tumor targets.*\n\n*However, most of the results remain correlations and have not been formally demonstrated or miss controls.*\n\n*B16F10 is a well known and characterized NK cell target in a in vivo model so the first part is not really knew except the in situ behavior of NK cells within the lung capillaries. The new mecanism of thrombin-mediated shedding of Necl-5 causing evasion from NK Cell surveillance is really concentrated on the last figure (Fig N{degree sign}6) and some supplemental experiments are mandatory and needed to really confirm this affirmation.*\n\nResponse: We deeply appreciate the reviewer\u2019s effort to evaluate our work. The reviewer criticizes that the mechanism is well known except \u201c*the in situ behavior of NK cells within the lung capillaries.\u201d* Indeed, this is what we wish to emphasize in our work. Nobody has ever seen how NK cells kill metastatic tumor cells in the lung. There is a big GAP between in vitro tissue culture experiments and in vivo macroscopic counting of metastatic nodules. Most researchers do not even know when and where in the lung NK cells kill metastatic tumor cells. Live imaging is a powerful approach to address such questions.\n\n*Reviewer #1 (Significance (Required)):*\n\n*There are several points to address to improve the significance of these data.*\n\n*\\*\\*Major points\\*\\**\n\n*1) A global point : 3 mice\/group is to small to analyse and interprete data because of the heterogeneity of the mice. Mean +\/- SEM have to represented instead of SD.*\n\nResponse: For the sake of animal welfare, researchers are asked to use minimal number of mice. Moreover, only one mouse can be observed in each imaging session, which takes several hours. In most experiments we performed two independent experiments with three mice each. We believe, the number is appropriate for this type of experiment. In the case of small number of samples, we think SD is better than SEM.\n\n*2) The authors used the well known polyclonal anti-asialoGM1 Ab to deplete NK cells. AsialoGM1 is also expressed by ILC1, T, NKT and gd+T cells but also basophils (Trambley J et al., Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. JCI, 1999). The authors checked the involvement only for the basophils. They have to check the depletion of each of these populations specifically in the lung to assume that the depletion impact only the NK cells or they must change their conclusion on the entire manuscrit and say that not only NK cells is responsible and involved in the control of the disseminated tumor cells but maybe also ILC1, NKT and or gd+T cells.*\n\nResponse: We obtained similar observations by using BALB\/c nu\/nu mice, which lack T cells. Therefore, we can exclude the contribution of T cells at least in the acute phase (*3) Lines 133 to 136 : The authors say that they \u00ab did not observe any significant difference in the relative increase of the bioluminescence signal between the control and \u03b1AGM1-treated mice, implying that NK cells eliminate disseminated melanoma cells primarily in the acute phase (Response: After 24 hrs, the slope of increment of bioluminescence intensity (BLI) did not change significantly between\u03b1AGM1-treated mice and control mice. In both mice, the doubling times of melanoma cells are approximately one day.\n\n*4) Fig S3A-B : The authors say that basophils express aGM1 so they performed basophils involvement on the elimination of B16F10 tumor cells with depleting aCD200R3 mab. They also checked the involvement of neutrophils and monocytes. They observed that basophils, neutrophils and monocytes are not involved on the B16F10 elimination. But what is the hypohesis to assess the role of neutrophils and monocytes ? Moreover, they did not explore Basophil roles in the other models including MC-38, BRAFV600E and 4T1 tumor cells*.\n\nResponse: We depleted neutrophils and monocytes because antibody-mediated removal of leukocytes could have non-specifically increased the survival of tumor cells. As for expanding the number of experiments with different cell lines, we are afraid but it is too much burden, considering the period required for the experiments and animal welfare.\n\n*5a) Fig 1D : Missing control : the author must add the WT Balbc + a-AGM1 as contro*l.\n\nResponse: We have this data, which will be included in the revised paper.\n\n*5b) Lines 154 to 156 : the authors say that \u00ab T cell immunity does not contribute to tumor cell reduction \u00bb because tumor cells are eliminated in the nu\/nu mice as efficiently as in the WT Balbc mice. This is not correct because they are looking in a window that correspond to innate immunity activation (up to 24h) so they cannot talk about T cell immunity, the adpative response will come more later around 8 days after.*\n\nResponse: Yes, we are focusing on the early phase of the rejection of metastatic tumor cells. We will rephrase the sentences.\n\n*6) Line 159 : (refer to point #2) To affirm that NK cells is critical and involved in the elimination of the disseminated tumor, authors have to perform experiment in a model of NK cell deficiency. The most relevant nowaday is the NKp46ICRExrosa26DTA mice that are deficients in NK cells but also ILC1 cells. Indeed, the authors have used the NKp46iCre mice model for other questions.*\n\nResponse: As the reviewer stated, the contribution of NK cells in the rejection of metastatic tumors is very well known. We do not think we need to repeat the experiments by using other genetically modified mouse lines, which will take at least one year. We wish to emphasize again that the new findings of our paper are in the in vivo imaging.\n\n*7a) Fig 2F : IC missing*\n\nResponse: According to the reviewer's suggestion, we will perform control experiments with an isotype control.\n\n*7b) Lines 181-182 : Authors conclude that the effect of anti-LFA-1 on NK cells adhesion to the pulmonary endothelial cells is mediated primarily by LFA-1. It is not totally true because it is partially mediated as observed in the fig 2F. So authors should change their conclusion and precise that the involvement is partially mediated by LFA-1*.\n\nResponse: We will rephrase the result section in the revised paper.\n\n*8) Fig S5B-C-D and S7: The authors talk about tumor cell death. But they are analyzing Ca2+ influx in vitro so it is a little bit different from the cell death. I'm wondering how the cell death is mesured espacially in the fig S5D and S7?*\n\nResponse: Under microscopes, apoptosis can be easily recognized by the appearance of blebs. We will include videos in the revised paper.\n\n*9) Fig 4H and lines 232-233 : the authors conclude that \u00ab damage to tumor cells is dependent on the engagement of DNAM-1 on NK cells \u00bb. There is any experiment performed to affirm this point so the authors cannot maintain this conclusion. First, the authors only analyzed Ca2+ influx at a specific time point. So this result only show that Nectin-5 and\/or Nectin-2 expressed by B16F10 is involved in the Ca2+ influx following NK cell contact but there is any data on DNAM-1 contribution. So, the role on the NK cells and specifically DNAM-1+ NK cells have not been adressed here. To answer to that question, the author have to perform in vivo model of engrafted WT vs Necl-5\/2 ko B16F10 in a WT vs DNAM1 deficient NK cells mouse model to ascertain the contribution of Necl-5\/2-DNAM-1 on NK cells. Moreover, survival curve and bioluminescent experiments would be very appreciated.*\n\nResponse: We have shown the data with Necl-5\/Nectin-2-deficientB16F10 cells in Fig. S7. I understand the importance of the experiment with the DNAM-1-deficient mice. But the introduction of another knockout mouse line cannot be performed easily. Instead, we will tone down the conclusion on the requirement of signaling from Necl-5\/Nectin-2 to DNAM-1.\n\n*10) Lines 253-254 : the authors talk about tumor apoptosis but they are looking at Ca2+ influx. So, they should change their conclusion or show killing experiment.*\n\nResponse: In Figure S7, we have shown that the sustained Ca2+ influx is a useful surrogate marker for apoptosis. We will include this information explicitly in the revised paper.\n\n*11) Fig 6 : the authors conclude that the trombin dependent shedding of Necl-5 causes evasion of NK cells surveillance. Moreover, all experiments are correlations and do not implicate in the same experiment Necl-5, DNAM-1+ NK cells and trombin or anti-coagulation factors. So, as in the comment #9, to adress this point, the authors should inject WT vs Necl-5 deficient B16F10-Akaluc into WT vs NK cell depleted mice and monitor the bioluminescence of the tumor cells within 24h following injection of anti coagulation factors as in the fig 6H. Moreover, the monitoring of the survival curve and the number of the lung metastasis would be also very important and informative to really answer to this point.*\n\nResponse: We will try the requested experiments during revision.\n\n*\\*\\*Minor points\\*\\**\n\n*1) Fig 2E: The authors assess the involvement of LFA-1 and MAC-1 on the NK cells attachement to the the pulmonary endothelial cells. But there is other adhesion molecules that are known to be expressed by NK cells as for example CR4 (CD11c\/CD18). So, the attachement of NK cells could be also due to this molecule.*\n\nResponse: We agree. The text will be modified to suggest the involvement of other adhesion molecules.\n\n*2) Lines 190 to 197 : Authors should put this methodology part in the \u00ab material and method \u00bb in order to be more clear on the message they want to deliver.*\n\nResponse: We will modify the text according to the suggestion.\n\n*3) line 228 : There is any hypothesis or explanation regarding the use of Necl5\/Necl2 deficient B16F10. Why authors decided to go and explore this pathway ? Authors could add some transition sentence and explanation to help readers.*\n\nResponse: We will refer to previous papers suggesting the role of DNAM-1 and its ligands, Necl-5 and nectin-2.\n\n*4) The author could performed the same experiment as in Fig S7D and assessed ERK activation of DNAM+ vs - NK cells against WT vs Necl-5\/Necl-2KO R-GEKO B16F10 cells.*\n\nResponse: We will try the suggested experiments.\n\n*5) Line 283 : Thanks to reformulate the sentence. Check the firgures associated with the text.*\n\nResponse: We will correct this error. The figures will be Fig. 5E and 5F.\n\n*Reviewer #2 (Evidence, reproducibility and clarity (Required)):*\n\n*The authors use in vivo imaging techniques to investigate the killing of lung metastasis by NK cells. They demonstrate that the cleavage of CD155 may result in resistance of killing by NK cells and suggest that this could be an immune evasion mechanism of metastatic tumor cells.*\n\n*Overall, the subject is highly relevant, and the in vivo imaging is an interesting and highly relevant technique. However, the message, that tumor cells escape the killing by NK cells by cleavage of CD155 is interesting, but not yet fully supported by the data.*\n\n*\\*\\*Major comments:\\*\\**\n\n1. * Figure 6: To support their main claim the authors would need to transfect the tumor cells with a CD155 mutant, which cannot be cleaved by Thrombin and show that these tumor cells can no longer escape NK cell-mediated killing. This experiment is straight forward and feasible. Another important experiment along this line would be the use the CD155\/CD112 deficient tumor cells (Which the authors use in figure 4) in the experiments shown in figure 1. One would expect that tumor control by NK cells within the first 24h is absent when using these tumor cells.*\nResponse: We previously made five CD155 mutants, which could be resistant to thrombin-mediated cleavage, and re-expressed in CD155\/CD112 deficient tumor cells. However, none of the mutants was not killed by NK cells both in vivo and in vitro. It appears that the potential thrombin-cleavage site(s) reside in the recognition site by DNAM-1. We will include this observation in the discussion.\n\n\n* Figure 5: The demonstration that ERK is activated in this in vivo setting is novel. However, ERK activation is not DNAM-1 specific and the ERK inhibitor is significantly less effective that the depletion of NK cells. Therefore, the relevance of these data to the main message of the manuscript is unclear and the figure could be omitted.*\n\nResponse: We agree that the modest effect of MEKi implies that ERK activation is dispensable for NK activation. However, ERK activation is a useful marker of NK cell activation. The data shown here vividly show the timing of NK cell activation and following tumor cell killing. Because the in vivo dynamics of NK cell activation and tumor cell killing is the most important message of this work, we wish to show this data.\n\n\n* In general, the issue of NK cell exhaustion should be addressed in more detail. The experiments do not address serial killing activity of NK cells and more data is needed to show that it is not an exhaustion of NK cells but the cleavage of CD155 from the tumor cells that prevents further killing.*\n\nResponse: We believe, Fig. 5G clearly shows that NK cells are not exhausted 24 hours after tumor cell injection.\n\n\\*\\*Minor comments:\\*\\*\n\n\n* Figure 1C: The relevance of this experiment needs to be better explained.*\n\nResponse: We will rephrase the result section in the revised paper.\n\n\n* Figure 3A: What does SHG stand for?*\n\nResponse: It is shown in line 625, M&M section. We will show the statement that SHG stands for second harmonic generation channel in the figure legend.\n\n\n* Figure 3: Please add a statistical analysis for these experiments.*\n\nResponse: We will include P values in the revised paper.\n\n\n* Figure 4: The use of the caspase-3 and the calcium sensors may detect different cytotoxic mechanisms used by the NK cells. While caspase-3 can be activated by death receptor and perforin\/granzyme B mediated killing, the calcium sensor may report mostly on perforin mediated membrane damage. These killing mechanisms have different kinetics and are differentially used during serial killing by NK cells. This should be addressed (at least in the discussion).*\n\nResponse: We thank this invaluable comment. We will include this discussion.\n\n*Reviewer #2 (Significance (Required)):*\n\n*Investigating the in vivo cytotoxicity of NK cells against tumor cells by using live imaging technologies is highly relevant for the understanding of the dynamic relationship between tumor and killer cells. Therefore, the subject of this manuscript and the technologies used are very relevant, as in vivo killing activities do not always translate to the in vivo setting.*\n\nResponse: We thank the reviewer for the favorable comment.\n\n*Reviewer #3 (Evidence, reproducibility and clarity (Required)):*\n\n*\\*\\*Summary\\*\\**\n\n*Ichise et al., present a solid work describing the modality and time frame of action of NK control over seeding metastatic cells within the lung vasculature. Th authors use a variety of technique able to dissect how NK patrol lung vasculature, that they interact with cancer cells as they interact with the endothelial cells and they activate a ERK dependent activation leading to calcium influx in cancer cells leading to their death. The data support the notion that this NK control occur over an early time frame, 4h after cancer cells arrival and is mediated by Necl expression on cancer cells. After this time point cancer cells show a thrombin dependent loss of Necl expression on their surface and therefore become resistant to NK control.*\n\n*\\*\\*Comments:\\*\\**\n\n*The data presented are supporting the conclusions. This work utilizes a variety of elegant strategy combining reporter strategy with in vivo imaging to assess the phenomenon of interaction, ERK activation, Calcium Inflax and Apoptosis activation directly in the lung.*\n\n*In term of experiments, I found the work thorough and complete.*\n\n*The data a presented well overall and the statistics seems adequate.*\n\n*I only have few suggestions:*\n\n*Supplementary Figure S3, show the use of antiLy6G to deplete neutrophils in the lungs of C57BL\/6 mice injected with melanoma B16F10 cells. It was recently shown that this antibody is not efficient in depleting neutrophils in this background, but only lead neutrophils to internalise the Ly6G so they cannot be detected by FACS. As shown in Boivin et al 2020 http:\/\/doi.org\/10.1038\/s41467-020-16596-9) neutrophils depletion in C57BL\/6 mice can be achieved by using antiGr1 antibody. Therefore, if the authors aim to show this additional control, which I also agree is really good to have, I suggest performing the experiment accordingly to the best-known practice.*\n\nResponse: We will perform the suggested experiment.\n\n*Figure 1E: in the text the experiment is described as 4T1 Akaluc cells were inoculated into the foot pad of BALB\/c mice with either control antibody or \u03b1AGM1, but the legend states that mice subcutaneously injected with B16 Akaluc cells into footpad.*\n\n*As B16 melanoma cells are not in BALB\/c background, I assume the legend needs to be corrected as the cells should be 4T1, however I wonder if injecting 4T1 breast cancer cells in the footpad could have let to the substantial growth required for lung metastasis without impairing the animal mobility. Could it be that cells where actually injected in the fat pad of the mice and this is just a misspelling in the text?*\n\n*In this case, the different in the tissue residence NK cells could also potentially explain why 4T1 are not cleared in the fat pad like the B6 cells are in the footpad.*\n\n*The authors should comment on the difference in the in clearance of the cells at the injection site in Figure 1C VS Figure 1E.*\n\nResponse: We apology the erratum in the legend.\n\nFigure 1C was performed to examine whether NK cells in the lung could be exhausted or inert 14 days after the inoculation of B16F10 cells. In this experiment, Akaluc-expressing B16F10 cells were inoculated to monitor the bioluminescence for 24 hrs.\n\nIn figure 1E, we used Akaluc-expressing 4T1 breast cancer cells because 4T1 cells inoculated into footpad can be spontaneously metastasized to the lung (Kamioka et al., 2017). We observed the bioluminescence of 4T1 cells in the lung for up to 20 days.\n\nRef: Kamioka, Y., Takakura, K., Sumiyama, K., and Matsuda, M. (2017). Intravital FRET imaging reveals osteopontin-mediated polymorphonuclear leukocyte activation by tumor cell emboli. Cancer Sci 108, 226-235.\n\n*Reviewer #3 (Significance (Required)):*\n\n*The present work is highly relevant to the field of cancer metastasis. While it is known that NK are responsible for the first line of defence against metastatic seeding, most of the studies focuses on how they are suppressed or influenced by other immune cells. The present study provides a very accurate description of their mechanism of action, how they depend in the interaction with the endothelial cells and highlight the novel aspect of thrombin in inducing cancer cells NK resistance. What cause thrombin activation is the next relevant question, by in my opinion this study is complete and important.*\n\n*My field of expertise is cancer metastasis and their interaction with the immune system and I personally enjoy very much reading this work.*\n\nResponse: We thank the reviewer for favorable comments and appreciate the effort to evaluate our work."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784v1","doi":"10.1101\/2021.01.15.426784"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#3\n\n#### Evidence, reproducibility and clarity\n\n **Summary**\r\n\r\nIchise et al., present a solid work describing the modality and time frame of action of NK control over seeding metastatic cells within the lung vasculature. Th authors use a variety of technique able to dissect how NK patrol lung vasculature, that they interact with cancer cells as they interact with the endothelial cells and they activate a ERK dependent activation leading to calcium influx in cancer cells leading to their death. The data support the notion that this NK control occur over an early time frame, 4h after cancer cells arrival and is mediated by Necl expression on cancer cells. After this time point cancer cells show a thrombin dependent loss of Necl expression on their surface and therefore become resistant to NK control.\r\n \r\n**Comments:**\r\n\r\nThe data presented are supporting the conclusions. This work utilizes a variety of elegant strategy combining reporter strategy with in vivo imaging to assess the phenomenon of interaction, ERK activation, Calcium Inflax and Apoptosis activation directly in the lung.\r\nIn term of experiments, I found the work thorough and complete. \r\nThe data a presented well overall and the statistics seems adequate.\r\nI only have few suggestions:\r\n\r\nSupplementary Figure S3, show the use of antiLy6G to deplete neutrophils in the lungs of C57BL\/6 mice injected with melanoma B16F10 cells. It was recently shown that this antibody is not efficient in depleting neutrophils in this background, but only lead neutrophils to internalise the Ly6G so they cannot be detected by FACS. As shown in Boivin et al 2020 http:\/\/doi.org\/10.1038\/s41467-020-16596-9) neutrophils depletion in C57BL\/6 mice can be achieved by using antiGr1 antibody. Therefore, if the authors aim to show this additional control, which I also agree is really good to have, I suggest performing the experiment accordingly to the best-known practice.\r\n\r\nFigure 1E: in the text the experiment is described as 4T1 Akaluc cells were inoculated into the foot pad of BALB\/c mice with either control antibody or &#x03B1;AGM1, but the legend states that mice subcutaneously injected with B16 Akaluc cells into footpad. \r\nAs B16 melanoma cells are not in BALB\/c background, I assume the legend needs to be corrected as the cells should be 4T1, however I wonder if injecting 4T1 breast cancer cells in the footpad could have let to the substantial growth required for lung metastasis without impairing the animal mobility. Could it be that cells where actually injected in the fat pad of the mice and this is just a misspelling in the text? \r\nIn this case, the different in the tissue residence NK cells could also potentially explain why 4T1 are not cleared in the fat pad like the B6 cells are in the footpad. \r\n\r\nThe authors should comment on the difference in the in clearance of the cells at the injection site in Figure 1C VS Figure 1E.\r\n\n\n#### Significance\n\n The present work is highly relevant to the field of cancer metastasis. While it is known that NK are responsible for the first line of defence against metastatic seeding, most of the studies focuses on how they are suppressed or influenced by other immune cells. The present study provides a very accurate description of their mechanism of action, how they depend in the interaction with the endothelial cells and highlight the novel aspect of thrombin in inducing cancer cells NK resistance. What cause thrombin activation is the next relevant question, by in my opinion this study is complete and important. \r\n\r\nMy field of expertise is cancer metastasis and their interaction with the immune system  and I personally enjoy very much reading this work.\r\n\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784v1","doi":"10.1101\/2021.01.15.426784"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#2\n\n#### Evidence, reproducibility and clarity\n\n The authors use in vivo imaging techniques to investigate the killing of lung metastasis by NK cells. They demonstrate that the cleavage of CD155 may result in resistance of killing by NK cells and suggest that this could be an immune evasion mechanism of metastatic tumor cells. \r\nOverall, the subject is highly relevant, and the in vivo imaging is an interesting and highly relevant technique. However, the message, that tumor cells escape the killing by NK cells by cleavage of CD155 is interesting, but not yet fully supported by the data.\r\n\r\n**Major comments:**\r\n\r\n1.\tFigure 6: To support their main claim the authors would need to transfect the tumor cells with a CD155 mutant, which cannot be cleaved by Thrombin and show that these tumor cells can no longer escape NK cell-mediated killing. This experiment is straight forward and feasible. Another important experiment along this line would be the use the CD155\/CD112 deficient tumor cells (Which the authors use in figure 4) in the experiments shown in figure 1. One would expect that tumor control by NK cells within the first 24h is absent when using these tumor cells.\r\n2.\tFigure 5: The demonstration that ERK is activated in this in vivo setting is novel. However, ERK activation is not DNAM-1 specific and the ERK inhibitor is significantly less effective that the depletion of NK cells. Therefore, the relevance of these data to the main message of the manuscript is unclear and the figure could be omitted.\r\n3.\tIn general, the issue of NK cell exhaustion should be addressed in more detail. The experiments do not address serial killing activity of NK cells and more data is needed to show that it is not an exhaustion of NK cells but the cleavage of CD155 from the tumor cells that prevents further killing.\r\n\r\n**Minor comments:**\r\n\r\n4.\tFigure 1C: The relevance of this experiment needs to be better explained.\r\n5.\tFigure 3A: What does SHG stand for?\r\n6.\tFigure 3: Please add a statistical analysis for these experiments.\r\n7.\tFigure 4: The use of the caspase-3 and the calcium sensors may detect different cytotoxic mechanisms used by the NK cells. While caspase-3 can be activated by death receptor and perforin\/granzyme B mediated killing, the calcium sensor may report mostly on perforin mediated membrane damage. These killing mechanisms have different kinetics and are differentially used during serial killing by NK cells. This should be addressed (at least in the discussion).\r\n\r\n\n\n#### Significance\n\n Investigating the in vivo cytotoxicity of NK cells against tumor cells by using live imaging technologies is highly relevant for the understanding of the dynamic relationship between tumor and killer cells. Therefore, the subject of this manuscript and the technologies used are very relevant, as in vivo killing activities do not always translate to the in vivo setting.\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-24","createdOn":"2021-03-24","completedOn":"2021-03-24","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.15.426784v1","doi":"10.1101\/2021.01.15.426784"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#1\n\n#### Evidence, reproducibility and clarity\n\n The authors aimed to understand the control and the elimination of disseminated tumor cells by NK cells within the lung, their main question being how pulmonary NK cells are able to prevent tumor cells from colonization in the lung.\r\n\r\nTo dissect this question, Hiroshi Ichise and colleagues took advantage of the ultra-sensitive bioluminescence whole body imaging system combined with intravital two-photon microscopy technology involving genetically-encoded biosensors tumor or NK cells to explore the behavior and functional competences of NK cells in an experimental lung metastasis model.\r\nFirst, the authors have monitored the fate of intravenously injected B16-Akaluc cells from 5 min to 10 days and observe that tumor cells decrease rapidly within the first 12-24 hours. In parallel, they performed asialoGM1+ and NK1.1+ cells depletion by injection of depleting anti-aGM1 and anti-NK1.1 antibodies in order to see the involvement of these populations on the elimination of the disseminated tumor cells. They conclude that a rapid decrease of the tumor cells is mediated by NK cells. Consisting with this first data, the authors observe also the same early NK cells mediated impact on two other syngenic mouse tumor cell lines : the BRAFV600E melanoma and the colon adenocarcinoma MC-38. \r\n\r\nIn a second part, the authors dissected NK cell dynamic behaviors in the pulmonary capillaries by taking advantage of the NKp46iCRExrosa26dtTomato mice where NKp46+ cells are fluorescents and performed 2P intravital imaging to follow the in situ the NKp46+ cells behavior. They could nicely observe that NK cells arrive from the capillaries and patrol on the lung epithelial cells in a stall-crawl-jump manner. Moreover, they also show that the attachment to the pulmonary capillaries is mediated by LFA-1. In the presence of B16F10 tumor model, they observe that NK cells stay longer in the capillaries and increase their duration time of crawling indicating that NK cells stay in contact longer with tumor cells.\r\n\r\nThe authors then explored the NK-mediated tumor killing in the lung by measuring tumor cell apoptosis using B16F10-SCAT3 cells (which leads to visualize caspase 3 activation) and  Ca2+ influx in tumor cells expressing two Ca2+ sensors, GCaMP6s and R-GECO. They could observe casp3 activation but also Ca+ influx on tumor cells within few minutes after encountering NK cells. They also observe that evasion of NK cell surveillance is mediated by Nectin-5 and Nectin-2 expressed on tumor cells.\r\n\r\nThen, they focus on NK cell activation by looking at ERK activation. To do so, they have isolated NK cells from Tg mice expressing a FRET-based ERK biosensor and performed in vitro killing assay against B16-R-GECO tumor cells but also in vivo experiments. For the in vivo experiments, they have developed reporter mice whose NK cells express the FRET biosensor for ERK. They observe that ERK-dependent NK cell activation contributes to the elimination of disseminated tumor cells within the first few hours but not after 24hours. Indeed, theu observe that B16F10-Akaluc tumor cells are equally eliminated when injected 24h after a first injection of B16F10 or PBS in mice. The authors concluded that tumor cell acquire the capacity to evade NK cell surveillance after 24h rather than a hypothesis toward NK cells loose tumoricidal activity over time.\r\nFinally, the authors have explored their last result on the potential tumor cell evasion of the NK cell surveillance. They show that this NK cell evasion is mediated by the shedding of cell surface Necl-5. They next show that clivage of extracellular domain of Necl-5 was mediated by thrombin in vitro and that anti-coagulation factors such as Warfarin, Edoxaban or Dabigatran Etexilate promote tumor elimination as observed by the bioluminescence experiments. This loss prevents the NK cell signaling needed for effective killing of tumor targets.\r\nHowever, most of the results remain correlations and have not been formally demonstrated or miss controls.\r\nB16F10 is a well known and characterized NK cell target in a in vivo model so the first part is not really knew except the in situ behavior of NK cells within the lung capillaries. The new mecanism of thrombin-mediated shedding of Necl-5 causing evasion from NK Cell surveillance is really concentrated on the last figure (Fig N{degree sign}6) and some supplemental experiments are mandatory and needed to really confirm this affirmation.\r\n\n\n#### Significance\n\n There are several points to address to improve the significance of these data.\r\n\r\n**Major points**\r\n\r\n1) A global point : 3 mice\/group is to small to analyse and interprete data because of the heterogeneity of the mice. Mean +\/- SEM have to represented instead of SD.\r\n\r\n2) The authors used the well known polyclonal anti-asialoGM1 Ab to deplete NK cells. AsialoGM1 is also expressed by ILC1, T, NKT and gd+T cells but also basophils (Trambley J et al., Asialo GM1(+) CD8(+) T cells play a critical role in costimulation blockade-resistant allograft rejection. JCI, 1999). The authors checked the involvement only for the basophils. They have to check the depletion of each of these populations specifically in the lung to assume that the depletion impact only the NK cells or they must change their conclusion on the entire manuscrit and say that not only NK cells is responsible and involved in the control of the disseminated tumor cells but maybe also ILC1, NKT and or gd+T cells.\r\n\r\n3) Lines 133 to 136 : The authors say that they &#x00AB; did not observe any significant difference in the relative increase of the bioluminescence signal between the control and &#x03B1;AGM1-treated mice, implying that NK cells eliminate disseminated melanoma cells primarily in the acute phase (&lt; 24hrs) of lung metastasis &#x00BB; Please comment because the depletion of asGM1+ cells impact also the growth of the tumor until 8 days (fig 1B-E-G)\r\n\r\n4) Fig S3A-B : The authors say that basophils express aGM1 so they performed basophils involvement on the elimination of B16F10 tumor cells with depleting aCD200R3 mab. They also checked the involvement of neutrophils and monocytes. They observed that basophils, neutrophils and monocytes are not involved on the B16F10 elimination. But what is the hypohesis to assess the role of neutrophils and monocytes ? Moreover, they did not explore Basophil roles in the other models including  MC-38, BRAFV600E and 4T1 tumor cells.\r\n \r\n5a) Fig 1D : Missing control : the author must add the WT Balbc + a-AGM1 as control.\r\n\r\n5b) Lines 154 to 156 : the authors say that &#x00AB; T cell immunity does not contribute to tumor cell reduction &#x00BB; because tumor cells are eliminated in the nu\/nu mice as efficiently as in the WT Balbc mice. This is not correct because they are looking in a window that correspond to innate immunity activation (up to 24h) so they cannot talk about T cell immunity, the adpative response will come more later around 8 days after.\r\n\r\n6) Line 159 : (refer to point #2) To affirm that NK cells is critical and involved in the elimination of the disseminated tumor, authors have to perform experiment in a model of NK cell deficiency. The most relevant nowaday is the NKp46ICRExrosa26DTA mice that are deficients in NK cells but also ILC1 cells. Indeed, the authors have used the NKp46iCre mice model for other questions.\r\n\r\n7a) Fig 2F : IC missing\r\n\r\n7b) Lines 181-182 : Authors conclude that the effect of anti-LFA-1 on NK cells adhesion to the pulmonary endothelial cells is mediated primarily by LFA-1. It is not totally true because it is partially mediated as observed in the fig 2F. So authors should change their conclusion and precise that the involvement is partially mediated by LFA-1.\r\n\r\n8) Fig S5B-C-D and S7: The authors talk about tumor cell death. But they are analyzing Ca2+ influx in vitro so it is a little bit different from the cell death. I'm wondering how the cell death is mesured espacially in the fig S5D and S7?\r\n\r\n9) Fig 4H and lines 232-233 : the authors conclude that &#x00AB; damage to tumor cells is dependent on the engagement of DNAM-1 on NK cells &#x00BB;. There is any experiment performed to affirm this point so the authors cannot maintain this conclusion. First, the authors only analyzed Ca2+ influx at a specific time point. So this result only show that Nectin-5 and\/or Nectin-2 expressed by B16F10 is involved in the Ca2+ influx following NK cell contact but there is any data on DNAM-1 contribution. So, the role on the NK cells and specifically DNAM-1+ NK cells have not been adressed here. To answer to that question, the author have to perform in vivo model of engrafted WT vs Necl-5\/2 ko B16F10 in a WT vs DNAM1 deficient NK cells mouse model to ascertain the contribution of Necl-5\/2-DNAM-1 on NK cells. Moreover, survival curve and bioluminescent experiments would be very appreciated.\r\n\r\n10) Lines 253-254 : the authors talk about tumor apoptosis but they are looking at Ca2+ influx. So, they should change their conclusion or show killing experiment.\r\n\r\n11) Fig 6 : the authors conclude that the trombin dependent shedding of Necl-5 causes evasion of NK cells surveillance. Moreover, all experiments are correlations and do not implicate in the same experiment Necl-5, DNAM-1+ NK cells and trombin or anti-coagulation factors. So, as in the comment #9, to adress this point, the authors should inject WT vs Necl-5 deficient B16F10-Akaluc into WT vs NK cell depleted mice and monitor the bioluminescence of the tumor cells within 24h following injection of anti coagulation factors as in the fig 6H. Moreover,  the monitoring of the survival curve and the number of the lung metastasis would be also very important and informative to really answer to this point.\r\n\r\n**Minor points**\r\n\r\n1) Fig 2E: The authors assess the involvement of LFA-1 and MAC-1 on the NK cells attachement to the the pulmonary endothelial cells. But there is other adhesion molecules that are known to be expressed by NK cells as  for example CR4 (CD11c\/CD18). So, the attachement of NK cells could be also due to this molecule. \r\n\r\n2) Lines 190 to 197 : Authors should put this methodology part in the &#x00AB; material and method &#x00BB; in order to be more clear on the message they want to deliver.\r\n\r\n3) line 228 : There is any hypothesis or explanation regarding  the use of Necl5\/Necl2 deficient B16F10. Why authors decided to go and explore this pathway ? Authors could add some transition sentence and explanation to help readers.\r\n \r\n4) The author could performed the same experiment as in Fig S7D and assessed ERK activation of DNAM+ vs - NK cells against WT vs Necl-5\/Necl-2KO R-GEKO B16F10 cells.\r\n\r\n5) Line 283 : Thanks to reformulate the sentence. Check the firgures associated with the text.\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637v1","doi":"10.1101\/2020.10.08.331637"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3:**\n\nThe authors hypothesized lower GABA levels in older adults would influence cortico-cortical phase relationships more than cortico-muscular phase relationships during performance of a bimanual motor task. To this end, they evaluated the mediating role of endogenous bilateral sensorimotor cortex GABA content in relation to behavioral performance and patterns of interhemispheric and cortico-muscular electrophysiological phase coherence during a bimanual motor control task. The central finding was that the mediating influence of right M1 GABA on the relationship between cortico-cortical electrophysiology and behavior diverged between the younger and older groups, with lower endogenous GABA concentrations potentially benefitting bimanual motor performance in young adults and hindering performance in older adults. The result was specific to right M1 GABA, raising questions about hemispheric asymmetry, and behavioral performance differed substantially between groups, possibly influencing the sensitivity of the analyses of the electrophysiological phase relationships. Moreover, several earlier studies suggest endogenous M1 GABA content relates to cortico-muscular excitability measurements, other than phase synchrony, and it is unclear what distinguishes phase synchrony from these other measurements. The behavioral, MRS, and electrophysiological methods employed are fairly well-established and are combined in a novel manner. The Bayesian moderated mediation analysis represents a new approach to evaluating relationships between these measures under the moderating influence of age. The central questions concerning the roles of cortical endogenous GABA in bimanual control, and in age-related changes in motor control more generally, are important for determining the neural computations underlying flexible and precise behavior.\n\n1) The total number of finger taps within the 2000 ms transition epoch likely differed between groups and could influence the ISPC measures. It would be helpful to rule out this possibility by examining relationships between ISPC measures and the total number of taps.\n\n2) The differences between right and left M1 are somewhat surprising and merit further attention, particularly given the cortico-cortical ISPC results. The interpretation provided in the discussion (lines 607-618) is not particularly satisfying since this asymmetry is a critical feature of a key result. Can the authors leverage their own data to provide further insight into why RM1 GABA+ may be more likely to exhibit a relationship than LM1 GABA+? Would analyzing the behavioral data separately for the left and right hands provide further insight? Does the non-dominant hand lag behind the dominant hand, and\/or is it more susceptible to errors?\n\n3) There were some general issues concerning the GABA+ data:\n\n   a. Figure 2a suggests an interaction in the pattern of variance in the GABA+ data between the Young and Older groups for the LM1 and RM1 voxels. Is this interaction in variance significant, and if so, what might this mean for the M1 GABA+ results? Specifically, Young show greater variance for LM1, and Older show greater variance for RM1. Also, Young appear to show considerably lower variance for RM1 than LM1. However, the data in Figure 2 supplement 2 suggest that variance in the Young is similar between LM1 and RM1. Do these numbers accurately reflect the data depicted in Figure 2a?\n\n   b. It would be helpful to show the difference spectra in Figure 2 supplement 1b with separate plots for Young and Older.\n\n   c. Figure 2, supplement 1a: Was the LM1 voxel more dorsal and medial than the RM1 voxel?\n\n4) The authors interpret the decrease in failure and increase in error rate across the task in the Older group as an indication of a loss of precision over time. Alternatively, might this pattern also arise because these participants are becoming faster at correcting their errors (i.e. within 2000 ms), avoiding trials from being categorized as a failure? More generally speaking, it would be helpful if the authors provided additional information about the cumulative error rate trials and what behavior looked like on these trials.\n\n5) The authors should provide further justification for the assignment of age as the moderator and GABA+ as the mediator in their statistical model. Conceptually, it seems these factors could be reversed.\n\n6) Several studies have established relationships between transcranial magnetic stimulation measures of cortico-muscular excitability and endogenous GABA+ content in the dominant M1. The manuscript would benefit from further discussion of the relationship of the phase connectivity measurements used here in comparison to these other previous studies.\n\n7) It is not clear that data or analysis code are available.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637v1","doi":"10.1101\/2020.10.08.331637"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2:**\n\nI like this type of multimodal study, and I think that the rationale for the study is good. I am not, however, convinced about the results\/conclusions provided. Here are my main points:\n\nI don't agree with your conclusion that the mediating role of GABA changes in aging. This requires longitudinal data, the cross-sectional approach in this study can only conclude differences between groups since only 1 time point is available.\n\nNo age interaction, this is surprising to me since there are age differences?\n\nCompensatory explanation: Is there a correlation with performance? If there isn't, the proposal of compensatory mechanisms is unclear since it is then not obvious what the compensation is for?\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.08.331637v1","doi":"10.1101\/2020.10.08.331637"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1:**\n\nThe authors have acquired a substantial multimodal dataset and have used careful statistical approaches throughout. The data are acquired and analysed using appropriate methods.\n\nOverall, this is an impressive body of work that aims to answer an interesting question. However, a number of questions over the methods and interpretation make the authors' conclusions difficult to justify.\n\nWhen comparing between older and younger adults it would also be helpful to know the amount of grey matter in the voxels of interest. It might be expected that older adults might have more atrophy and therefore lower GABA+, than younger adults and this should be controlled for in the statistical models. The authors have put assumptions into their quantification, which are reasonable but are still assumptions. It would be helpful to directly test for a difference in grey matter fraction in the voxel between the two groups, and include this in the model if necessary.\n\nThe authors then look at behaviour, where they use a previously described task which consists of bimanual tapping, with switching between two patterns. The results are complex as there are a number of behavioural metrics, and no clear pattern emerges. While older adults produced more errors in continuation, they also produced more fully correct switching transitions. Older subjects were slower than younger adults in all trials. While this task produces a very rich dataset, which is helpful for analysing complex behaviour, it is not clear how each metric should be interpreted in terms of the underlying neural mechanisms, and how they can be usefully combined, could be given.\n\nIn terms of connectivity, the authors found no significant group X task difference between in-phase and anti-phase conditions. They therefore look at the groups and tasks separately. They show different changes in connectivity between age groups in different frequency bands, for example between left and right M1 in the alpha\/mu and beta, between EMG and left M1 in the theta band. I am not sure that describing EEG-EMG connectivity as cortico-spinal is strictly accurate - there may be a number of other factors in this -corticomuscular would seem to be more precise. The frequency bands used are not typical, and it would be helpful to have an a priori explanation of which are being tested and why - as well as details about correction for multiple comparisons across these bands.\n\nFinally, the authors bring their GABA, behaviour and connectivity metrics together in a number of mediation analyses. They demonstrate a relationship between cortico-cortical connectivity and behaviour, which is mediated by age.\n\nThe authors describe their finding of higher GABA+ in the occipital cortex as a posterior-anterior gradient, which I think is not justified by the results - there could be a number of other reasons for this, for example that different functional networks have different GABA+ levels, which is not related to their anatomical position. With only three voxels it is difficult to make a general claim such as this, and this should probably be reworded.\n\nThe authors state that higher GABA+ indicated neural system integrity and better functioning in the older group. This seems to be rather over-interpreting their results - there are many other metrics of integrity and functioning that have not been assessed here. I would suggest rewording.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.09.03.20184051#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.09.03.20184051v2","doi":"10.1101\/2020.09.03.20184051"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Author Response to Public Reviews**\n\n> **Reviewer #1 (Public Review):**\n\n> [...] What is left unclear is what is unique about the fibrotic substrate in ESUS patients in comparison to AFib patients in the future.\n\nWe thank the reviewer for these reasonable and accurate critiques. In the revised version of our manuscript, we offer a more in-depth analysis of potential cohort-scale differences in the spatial distribution of fibrosis between ESUS and AFib patients and how that might affect the\noverall arrhythmogenicity of fibrotic remodeling between the two populations. We further acknowledge comprehensive understanding of pathophysiological consequences of fibrosis in ESUS will require much more research in the future. Our plans include analysis of how fibrosis might affect LA hemodynamic properties and the likelihood of clot formation. Future work (both clinical and computational) will also be needed to test the hypothesis generated by the present study that ESUS patients lack the triggers needed to initiate AFib. We have added clarifying text to the Discussion section of our manuscript to acknowledge these two points (see lines 286-289, 367-368).\n\n> **Reviewer #2 (Public Review):**\n\n> [...] 1) As the authors point out, clinical studies have revealed that the fibrotic burden in ESUS patients is similar to those with aFib. The question is why then, do so few ESUS patients exhibit clinically detectable arrhythmias with long-term monitoring. The authors hypothesize and their data support the notion that while the substrate is prime for pro-arrhythmia in ESUS patients, a lack of triggering events may explain the differences between the two groups.\n\nWe thank the reviewer for their kind comment about the level of anatomical and structural variability in our study. We concur that additional analysis of fibrosis spatial pattern properties (local fibrosis density and entropy, as calculated in our previous work) on a region-wise basis\nbetween AFib\/ESUS and inducible\/non-inducible models would add significant value to our work. Accordingly, we have made significant additions to the text including a completely new figure.\n\n> 2) I think the authors could go further in describing why this is surprising. Generally, severe fibrosis is thought to potentially serve as a means or mechanism for pro-arrhythmic triggers. This is because damage to cardiac tissue typically results in calcium dysregulation. When calcium overload occurs in isolated fibrotic tissue areas, or depolarization of the resting membrane potential due to localized ischemia allows for ectopic peacemaking, we might expect that the diseased\/fibrotic tissue is itself the source of arrhythmia generation. I think the novel finding here is that this notion may be a simplification, and the sources of arrhythmia generation may be more complex and may need to come from outside the areas of fibrosis. I think this is a big deal.\n\nPatients with stroke were excluded from the AFib cohort because the etiology of stroke in our AFib cohort was not explicitly adjudicated to be cardioembolic, other ischemic such as atherosclerotic, or haemorrhagic and therefore would not allow us to draw reliable conclusions\nregarding the role of the atrial substrate in stroke in this population. A separate issue is the fact that the cell- and tissue-scale electrophysiology in models reconstructed from ESUS patients was based on the same representation as those used in AFib models. In fact, this\nwas a deliberate design choice to ensure that our modeling results represented a \u201cworst case scenario\u201d for the potential impact of fibrosis in patients with ESUS. Given the fact that our aim is to determine whether there are any differences in the pro-arrhythmic capacity of fibrotic substrate in ESUS and AFib groups, we believe that this is a suitable and justifiable modeling choice \u2013 modeling fibrosis differently in the two populations would be difficult to justify due to a lack of good experimental data and would introduce more confounding factors.\n\nNevertheless, we agree this is a relevant limitation of our study and we have added an acknowledgement of that fact to our revised manuscript (see lines 361-365).\n\n> 3) An acknowledged limitation of the study is the assumption of fixed conduction velocity and action potential duration\/effective refractory period. Bifulco et al. base this assumption on previous studies by the group (e.g. L312), which, however, concluded that reentrant driver locations and inducibility are sensitive to changes of action potential and conduction velocity (Deng et al.). For conduction velocity, wider ranges have been reported since the publication of the supporting reference (35) in 1994, e.g. Verma et al.; Roney et al.\n\nThe reviewer\u2019s point is well taken. Accordingly, we have added qualifying language pertaining to RD localization analysis in our Discussion (see lines 323-326). Having said that, we do not think this issue stands to fundamentally change our top-line interpretation of the findings from simulations, as it pertains to the idea that fibrosis in ESUS might plausibly be latent proarrhythmic substrate. The point of the paper by Deng et al. was to analyze sensitivity of reentrant driver localization to altered cell- and tissue-scale electrophysiological properties, not the concept of inducibility per se. It is thus likely that if our entire study were repeated with\n\u00b110% CV or APD (both within normal physiological range for average fibrotic atrial tissue) the take-home message would be the same.\n\n> 4) The number of pacing sites is rather low for a comprehensive in silico arrhythmia inducibility test but likely a good balance of coverage and computational feasibility considering that the primary goal of this research was to check whether the two groups of models show differences when undergoing the same (but not necessarily exhaustive) protocol.\n\nWe would argue that 15 sites in the LA alone is comparable in coverage to prior studies in biatrial models (e.g., 30 LA\/RA sites in Zahid et al. [2016] Cardiovasc Res; 40 LA\/RA sites in Boyle et al. [2019] Nat Biomed Eng). We would further stress that our decision to use these specific sites was based on our motivation to simulate triggered activity (i.e., rapid pacing)\nexclusively from sites identified as common clinically relevant trigger locations documented in AFib patients (see ref. [14] by Santangeli et al. [2016] Heart Rhythm). If we were to instead pace from randomly distributed atrial sites as in prior work, we would jeopardize our ability to\ndraw conclusions on the potential relevance of our simulations to the real-world susceptibility of atrial fibrotic substrate in ESUS patients to ectopic beats originating from realistic locations.\n\n> 5) The discussion does a good job in putting the results into context. Two interesting observations that deserve more attention are that i) the Inducibility Score was always higher for AFib vs. ESUS (Figure 6A, no statistical test performed). However, this did not translate to a difference in silico arrhythmia burden (inducibility). ii) Reentrant drivers were about twice as likely to localize to the left pulmonary veins than the right pulmonary veins in the AFib models (Figure 6D).\n\nRegarding the first point (i), with corrections made to the fiber mapping process, the statement regarding uniformly higher IdS values in AFib models is no longer true. Moreover, with our revised analysis there is no significant difference in the region-wise inducibility rates (P=0.45). The reviewer\u2019s second point (ii) still stands and is even more pronounced with a ~3x higher rate of localization to the LPV vs. RPV areas in AFib models. Notably, our new region-wise analysis of fibrosis spatial pattern (see new Fig. 6 and our response to major points 4 and 5 above) shows that LPV regions in AFib models in this cohort were much more likely to have the combination of high fibrosis density and entropy previously shown to be highly favorable to reentrant driver localization. However, we recognize that a more fulsome analysis will be required to draw truly meaningful conclusions on this subject in the context of either AFib or ESUS patients; this has been briefly noted in our Limitations section (see lines 332-335).\n\n> 6) The study succeeded in answering the question it posed in the sense that no marked difference was found between the ESUS and AFib models. This leads to the question what the stroke-inducing mechanism is in the ESUS patients. A hypothesis for future work could be that the fibrotic infiltrations in the ESUS patients reduce the hemodynamic efficacy of the left atrium and render clot formation (e.g. in the atrial appendage) more likely in this way.\n\nThe reviewer\u2019s comment is duly noted and entirely consistent with our plans for future work. In fact, we recently published a white paper (Boyle et al. [2021] Heart) outlining a vision to combine electrophysiological models of the left atrium with biomechanics and hemodynamics simulation to comprehensively understand how fibrosis might influence clot formation. Our revised Discussion emphasizes this exciting trajectory for future work (see lines 370-372).\n\n> 7) The negative finding in this study (no difference between groups) does not naturally allow us to draw clinical implications for diagnosis or stratification. Additional ways to put the hypothesis proposed by the authors (fewer arrhythmogenic triggers in the ESUS patients) to test could be to consider readouts\/surrogate measures of the autonomic nervous system.\n\nWe have noted in our Discussion (see lines 286-289) that future work could test the hypothesis arising from this project via electrocardiographic monitoring in ESUS patients with different levels of fibrosis. Concerning the idea of using direct readouts of autonomic tone, we chose to leave this out since we are unaware of any clinically available systems. The usefulness of surrogate measurements (e.g., heart rate variability) in this context also remains unclear.\n\n> **Reviewer #3 (Public Review):**\n\n> [...] 1) As the authors point out, clinical studies have revealed that the fibrotic burden in ESUS patients is similar to those with aFib. The question is why then, do so few ESUS patients exhibit clinically detectable arrhythmias with long-term monitoring. The authors hypothesize and their data support the notion that while the substrate is prime for pro-arrhythmia in ESUS patients, a lack of triggering events may explain the differences between the two groups.\n\nWe thank the reviewer for these kind remarks. It is encouraging to have our results interpreted so elegantly and accurately. We are excited to test this new hypothesis (and others prompted by the peer review process for this manuscript) in future studies.\n\n> 2) I think the authors could go further in describing why this is surprising. Generally, severe fibrosis is thought to potentially serve as a means or mechanism for pro-arrhythmic triggers. This is because damage to cardiac tissue typically results in calcium dysregulation. When calcium overload occurs in isolated fibrotic tissue areas, or depolarization of the resting membrane potential due to localized ischemia allows for ectopic peacemaking, we might expect that the diseased\/fibrotic tissue is itself the source of arrhythmia generation. I think the novel finding here is that this notion may be a simplification, and the sources of arrhythmia generation may be more complex and may need to come from outside the areas of fibrosis. I think this is a big deal.\n\nThis is an excellent point and we strongly concur that the \u201ctrigger-centric\u201d interpretation of the pathophysiological consequences of fibrotic remodeling should be reconsidered. To further reinforce this fact, we ran additional simulations to rule out the possibility that there might be exaggerated resting membrane potential depolarization in AFib but not in ESUS, which might\nprovide an alternative explanation for the clinical manifestation of arrhythmia in the former but not the latter. Our new results support the point raised by the reviewer and, in our opinion, increase the overall impact of the work. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048v1","doi":"10.1101\/2020.10.25.20219048"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Author Response:**\n\n>**Reviewer #1 (Public Review):**\n\n>This paper presents the exciting statement that increasing viral loads within a community can be used as an epidemiological early-warning indicator preceeding increased positivity. It would be interesting to support this claim to present both Ct and positivity on the same graph to demonstrate that indeed, declining Ct can be used as an early marker of a COVID-19 epidemic wave. Percentage of positive test data should not only include the ones obtained in the present study but should be compared with \"national data\" as the present study design includes a bias in patients selection that might not reflect the \"true\" situation at the time. Only with this comparison, we could claim that the present study design could predict COVID-19 epidemic waves.\nA correlation of Ct with clinical evidence to rank the confidence of positive results is also included and further support the high specificity of the RT-PCR for detecting SARS-CoV-2 (99.995%).\n\n>In a serological investigation, it was observed that some of these RT-PCR-positive cases do not appear to seroconvert and that possible re-infections might occures despite the presence of anti-spike antibodies. Although, reported on few individuals and therefore to be taken with extreme caution, this add some piece of information to the current unknown of the serological response of COVID-19 patient and would be of uttermost importance in the context of the current vaccination campaign.\n\nWe do not agree that this study is biased in terms of patient selection \u2013 it invites randomly selected\nhouseholds to join the survey and is in fact the major source of unbiased surveillance data in the 4\nnations of the United Kingdom."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048v1","doi":"10.1101\/2020.10.25.20219048"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThis paper presents the exciting statement that increasing viral loads within a community can be used as an epidemiological early-warning indicator preceding increased positivity. It would be interesting to support this claim to present both Ct and positivity on the same graph to demonstrate that indeed, declining Ct can be used as an early marker of a COVID-19 epidemic wave. Percentage of positive test data should not only include the ones obtained in the present study but should be compared with \"national data\" as the present study design includes a bias in patients selection that might not reflect the \"true\" situation at the time. Only with this comparison, we could claim that the present study design could predict COVID-19 epidemic waves.\nA correlation of Ct with clinical evidence to rank the confidence of positive results is also included and further support the high specificity of the RT-PCR for detecting SARS-CoV-2 (99.995%).\n\nIn a serological investigation, it was observed that some of these RT-PCR-positive cases do not appear to seroconvert and that possible re-infections might occur despite the presence of anti-spike antibodies. Although, reported on few individuals and therefore to be taken with extreme caution, this add some piece of information to the current unknown of the serological response of COVID-19 patient and would be of uttermost importance in the context of the current vaccination campaign."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.25.20219048v1","doi":"10.1101\/2020.10.25.20219048"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThe authors present a systematic and complete study of Ct (cycle threshold) values in RT-PCR tests and gene-specific positivity for the UK ONS infection survey. There are very few datasets like this for any viral pathogen, regardless of pandemics. The patterns are fascinating and thought-provoking.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843v1","doi":"10.1101\/2021.01.22.427843"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nThis study provides a concept of circuit organization of a pathway from the brainstem to the primary somatosensory (S1) and motor (M1) cortices through the thalamus to control the hand\/forelimb movements. Previous studies reveal detailed circuit organization of ascending somatosensory pathways in the whisker system. In contrast, much less is known about circuit organization of another ascending pathway controlling the hand\/forelimb movements, although it is known that there are some similarities and differences between two different somatosensory systems.\n\nThis paper revealed detailed circuit organization of the ascending pathways including the lemnisco-cortical and corticocortical pathways to control the hand\/forelimb movements. The strength of this study is to use a variety of sophisticated techniques, such as optogenetics, trans-synaptic viruses, both anterograde and retrograde viruses, mouse genetics, and electrophysiology, to map the neural circuits in details. The circuit was revealed by electrophysiology together with optogenetics, which is very convincing. In addition, the detailed circuit organization revealed by this study will provide an important information for future behavioral studies. The weakness is the limitation of trans-synaptic viruses. For example, pseudorabies viruses move between multiple neurons, so to interpret the results may be complicated. Although behavioral analyses have not been performed in this study, it is beyond the scope of this study and future study will follow up the behavioral analyses."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843v1","doi":"10.1101\/2021.01.22.427843"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThis study traces the detailed excitatory connections of mouse forepaw sensorimotor circuits from the spinal cord, through brainstem, thalamus, sensory and motor cortical areas, and their motor outputs. This is a welcome and important contribution, considering the technical advantages of mice for circuit cracking and the increasing number of labs studying the functions of their limbs. Although the structure and function of forelimb sensorimotor circuits have been extensively studied in primates, they have been relatively neglected in the rodent, especially compared to the enormous scope of research that has been done on the rodent vibrissae system over the past 50 years. This study uses a variety of contemporary methods to reveal important similarities and differences between the forelimb and vibrissae sensorimotor circuits.\n\nOverall, the results do not hold major surprises, although this is itself a noteworthy result. The authors did identify a few qualitative and quantitative differences between the forelimb circuit and the parallel vibrissae-related circuit; the functional significance of these differences is as yet unclear.\n\nThe weaknesses of the manuscript are few and minor. The study would have been stronger if it had performed comparable, parallel experiments on the hand and vibrissae circuits, however the scope of the study is already ambitious and strong enough as it stands. I do have a question about the identity of the cortical L4 neurons that were recorded, and this issue should be discussed."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843v1","doi":"10.1101\/2021.01.22.427843"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nSensorimotor integration is required for the accurate execution of volitional movements, but the neural circuits underlying sensorimotor integration are still not fully understood. The whisker system of the rodent has emerged as one model of sensorimotor integration with many recent studies focused on the synaptic organization of the underlying circuitry. Here, Yamawaki et al report results regarding the synaptic organization of the ascending sensory pathways related to mouse forelimb somatosensory and motor cortex. Using anatomical and functional approaches, they elucidate the circuitry from the cuneate nucleus through thalamus to forelimb S1 and M1. This work complements recent studies in the mouse of other aspects of the forelimb sensorimotor pathways and leads to informative comparisons to the circuit organization of the whisker system. The studies are well executed and well explained. The use of multiple approaches compensates for the limitations of each individual technique, although some limitations such as any effects of viral tropism are difficult to overcome. Overall, this work contributes to a better understanding of the wiring diagram of sensorimotor circuits in the mouse."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427843v1","doi":"10.1101\/2021.01.22.427843"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis paper will be of interest to those studying the sensorimotor functions of the hand and forelimb. It traces the anatomy and strength of excitatory pathways from the spinal cord, through the brainstem, thalamus, somatosensory and motor cortex, and descending outputs. The methods are contemporary, and include multiple viral tracing, genetic targeting, and transsynaptic strategies, optogenetic and electrophysiological methods; the data are compelling; and the paper is clear and concise.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426394#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426394v1","doi":"10.1101\/2021.01.12.426394"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Joint Public Review:**\n\nWorker bees perform specialised tasks: young workers nurse larvae, older ones forage for either nectar or pollen. Behaviours - including these specialist ones - arise when a stimulus (nectar, pollen or larvae) exceeds a certain 'response threshold' of the organism. This threshold can be modulated by neuropeptides to alter behaviour.\n\nThe study first shows that response thresholds to task-related stimuli differ among nurse bees, nectar and pollen foragers. Pollen foragers are most responsive to sucrose and pollen, and nurse bees most responsive to chemical stimuli of larvae. Then, taking a proteomic approach, they identify a neuropeptide, Tachykinin related protein (TRP), to be expressed in a task-specific pattern: low in nurse bees and highest in the nectar foragers.\n\nThis work provides valuable resource information on the abundance of brain neuropeptides in two species of bees. The study is exceptional in its breath of techniques used and the addition of manipulative experiments which are difficult to do in honey bees. Through their studies the authors identify a neuropeptide that modulates response thresholds of bees.\n\nThe study would have been exceptional if the authors had included studies on the expression of the tachykinin receptor. The level of tachykinin expression increases between nurse bees and foragers, but does not involve changes in spatial expression (Takeuchi et al., 2004 ref. 56). So, it is likely that the specificity of the effects of tachykinin are due to differences in the spatial expression of the receptor."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426394#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426394v1","doi":"10.1101\/2021.01.12.426394"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThe authors provide convincing evidence that tachykinin signaling is involved in regulating response thresholds of task-specific stimuli only in the respective behavioral specialist. For example tachykinin signaling affects responses to pollen in pollen foragers and responses to larval chemical cues only in nurse bees. The study highlights the importance of peptide signaling in social behaviors in insects for the first time.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924v1","doi":"10.1101\/2020.10.23.351924"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This rebuttal was posted by the corresponding author to *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Reply to the reviewers\n\n> \n> __Response to reviewers: Woodcock et al. 2021__\n\n> __*Reviewer 1 (Evidence, reproducibility, and clarity):*__\n\n> __*Summary*__* \n>  *The authors resolved the biosynthesis of trehalose and alpha-glucan in *Pseudomonas aeruginosa* and the role of these two compounds in osmotic and desiccation stress.\n\n> >We thank the reviewer for their positive review of our manuscript. Our responses to their specific queries are interspersed below.\n\n> __*Major comments:*__\n\n> + *Are the key conclusions convincing?*\n> Yes\n\n> + *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? *\n> No\n\n> + *Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. *\n> Not necessary, comprehensive coverage of research topic.\n\n> + * Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. *\n> Not applicable\n\n> + *Are the data and the methods presented in such a way that they can be reproduced?*\n> Yes\n\n> + * Are the experiments adequately replicated and statistical analysis adequate? *\n> Yes, everything is adequate but just one subtle concern: check the significance of the number of digits in the entries listed in Table S3. Revise Table S3.\n\n> >Table S3 has been revised as requested. The data in this table is now presented correct to one decimal place.\n\n> __*Minor comments:*__\n\n> + *Specific experimental issues that are easily addressable. *\n> Not applicable (Table S3: see above)\n\n> + * Are prior studies referenced appropriately?*\n> No. Refs. 18- 32: The subjects of 'trehalose' and 'osmotic stress' have already been addressed in the *Pseudomonas* field and should be referenced. The authors cite work carried out on trehalose and osmotic stress on phylogenetically distant microorganisms, but do not cite related work from the *Pseudomonas* field which I consider to be inappropriate. Similarly, trehalose biosynthesis in *Pseudomonas* has not only been covered by refs. 47 and 48.\n\n> >This is a fair comment. The focus of our introduction came from a desire to concentrate specifically on the metabolism and intracellular function of trehalose\/\u03b1-glucan in *Pseudomonas*. In hindsight, we acknowledge that our introduction is a little too narrowly focussed. We have expanded the introduction and discussion sections to include additional discussion of trehalose in *Pseudomonas* and its regulation in the CF lung.\n\n> + *Are the text and figures clear and accurate?*\n> Extremely well written manuscript and prepared figures\n\n> + * Do you have suggestions that would help the authors improve the presentation of their data and conclusions? \n>  *Revise the list of references and discuss more thoroughly your novel findings in the light of existing knowledge in the *Pseudomonas* field.\n> >Please see previous comment relating to the literature.\n\n> __*Reviewer 1 (Significance):*__\n\n> __*Significance*__\n\n> + * Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.* \n>  Conceptual advance: The authors identified and characterized the enzymatic pathway of trehalose and alpha-glucan biosynthesis in *Pseudomonas aeruginosa* and its role to cope with osmotic and desiccation stress. The authors' conclusions do not correspond with recently published peers' work; hence they should discuss in more detail why they consider their data to be more accurate to discern the role of trehalose to contain desiccation and osmotic stress in *P. aeruginosa*.\n> >Please see previous comment relating to the literature. In general, the published work to date on trehalose in *Pseudomonas* spp. does not consider GlgE pathway-mediated link to \u03b1-glucan that we characterise in this paper. Our work demonstrates that synthesis and metabolism of the two molecules are implicitly linked in species where the GlgE pathway is present, and they cannot be considered in isolation. For this reason we are very confident that our study represents the most accurate model to date for trehalose and \u03b1-glucan metabolism and their associated phenotypes in *P. aeruginosa*. We have therefore emphasised that the role of trehalose in *Pseudomonas* spp. should be re-evaluated in light of our findings.\n\n> + * Place the work in the context of the existing literature (provide references, where appropriate).* \n>  Existing literature focusing on trehalose, osmotic stress, desiccation stress in the *Pseudomonas *field not cited by the authors:\n> >>>\n\n> + These papers are of variable scientific quality, but the conceptual work by Hallsworth and the work by Behrens on the PA metabolome in CF lungs are worth discussing. All other work provides pieces of information on function and biosynthesis of trehalose up to now known by the *Pseudomonas* community. The authors resolved the function of the GlgA operon which will be definitely appreciated.\n> >We thank the reviewer for these helpful suggestions. We have reviewed these papers carefully and have incorporated several, including the papers from Hallsworth and Behrens into the revised manuscript.\n\n> __*Strengths of the manuscript:*__\n\n> + Meticulously planned and carefully executed experiments, not a single experimental flaw\n> + Very high technical quality of experiments and primary data\n> + Comprehensive coverage of the research topic\n> + Excellent presentation in text and illustrations\n> __*Only weakness:*__\n\n> + Insufficient consideration of peers' published work on trehalose and its role in stress response in *P. aeruginosa*\n> >Please see previous comment relating to the literature.\n\n> + * State what audience might be interested in and influenced by the reported findings.* \n>  Scientists working in the fields of glycoconjugate and carbohydrate research, biochemists, microbiologists with interest in metabolic pathways, stress response and\/or *Pseudomonas*.\n> __*Reviewer #2 (Evidence, reproducibility, and clarity):*__\n\n> It will be difficult for me to write a review of this paper and for the authors to make sense of my review because the manuscript's pages \/ lines are not numbered\u2026\n\n> >We apologise to the reviewer for this oversight.\n\n> __*Summary \n>  *__The authors carried out a comprehensive characterization of the metabolism of trehalose in Pseudomonas aeruginosa PA01, using techniques of biochemistry, reverse genetics, and bioinformatics. The main findings include that the disaccharide trehalose is synthesized in this organism from branched chain \u03b1-glucans and that the catabolism of trehalose proceeds via another disaccharide, maltose and is fed back into the synthesis of \u03b1-glucans. Trehalose and \u03b1-glucans have been implicated in conferring resistance to abiotic stresses in other organisms. The authors show that mutants that are blocked in the synthesis of trehalose are sensitive to high salinity but are normal with respect to their sensitivity to desiccation, whereas mutants impaired in the accumulation of \u03b1-glucans are sensitive to desiccation without being unduly sensitive to osmotic stress. These results indicate that trehalose and \u03b1-glucans have different roles in abiotic stress-tolerance.\n\n> __*Major points*__\n\n> This manuscript describes an impressive amount of careful work and presents new insights into the metabolism of trehalose, maltose, and \u03b1-glucans. However, the authors should address the following major comments before the paper is accepted.\n\n> >We thank the reviewer for their thorough and positive assessment of the manuscript. We address their specific points below.\n\n> + Discussion: the authors state that \"trehalose protects *Pseudomonas* ssp. against osmotic stress, most likely due to its role as a compatible solute.\" According to Table 2, *P. aeruginosa* grown in the medium of low osmolarity accumulated 0.13% trehalose per gram dry weight, i.e. ~4 \u03bcmol \/ g dry weight. Assuming that the dry weight \/ wet weight ratio of *P. aeruginosa* is the same as that of *P. putida*, which is ~1\/3 (PMID: 6508285), the concentration of trehalose in the cells calculates to be ~2 mM. It is not plausible that trehalose could be significant as compatible solute at this low concentration.\n> +  \n> + One way out could be if the accumulation of this disaccharide were increased by osmotic stress. The authors should also measure the trehalose content of cells grown in medium containing 0.85 M NaCl. In case of positive results in this experiment, it would be interesting to determine the effects of osmotic stress on the levels of trehalose biosynthetic and catabolic enzymes, but this would not be necessary for the acceptance of the paper.\n> >This is a fair point. To address this, we measured the trehalose and maltose-1-phosphate levels for PA01 grown in the presence of 0.85 M NaCl. We saw a highly significant increase in the abundance of trehalose, compared to growth on standard M9 media. This strongly suggests that trehalose accumulates under conditions of osmotic stress as suggested by the reviewer. These new results have been added to the relevant sections of the manuscript (M&M, results, table 2 and discussion). The student (Danny Ward) who conducted these new experiments has been added to the author list.\n\n> + However, there is also an extensive literature suggesting that trehalose has antioxidant functions e.g. PMID: 29241092 (the first paper that came up in Google search for \"trehalose as antioxidant\"). The authors should discuss this possible alternate role of trehalose.\n> >The reviewer is correct that trehalose has well-documented antioxidant functions in various species. We have modified the introduction to address this. To maintain the focus of our manuscript on bacteria we have used a different example to that suggested by the reviewer.\n\n> + It is not described adequately in the Materials and Methods how the cellular contents of trehalose and maltose-1-phosphate (M1P) were determined.\n> >The Materials and Methods section has been revised to include more details of this method.\n\n> + I found the growth curves in Figure 8, especially in panel B, to be uninterpretable. The authors should spread these data into more panels or use some other method to make them clearer.\n> >We have expanded the legend for Figure 8 to describe more fully what is going on in this figure. The results in Figure 8 are grouped according to the operons in which each set of genes is located. As such, the graphs contain unequal numbers of curves, with 8B containing the most and 8C only showing data for WT and \u0394*glgP*.\n\n> + The statement \"The GlgA and GlgE proteins . . . enable two alternate mechanisms for linear \u03b1-glucan biosynthesis\", which is echoed a number of times in the manuscript, seems to create the impression that there are two de novo pathways of synthesis of these polysaccharides. However, as shown in Figure 1, the GlgA pathway is the only route to the net synthesis of \u03b1-glucans, and GlgE is only part of a recycling pathway. Therefore, it cannot be true that \"the vast majority of \u03b1-glucan accumulated by *P. aeruginosa* will be produced by GlgE\".\n> >We have revised this section to further clarify what we mean when we state that the majority of \u03b1-glucan accumulated by *P. aeruginosa* will be produced by GlgE. Our data suggest that there is a big difference between the *generation* of \u03b1-glucan (conducted by both GlgA and GlgE) and its *accumulation* (flux through GlgA generated \u03b1-glucan is high, so only GlgE generated \u03b1-glucan can accumulate to generate large polymers).\n\n> + The authors state that \"MalQ disproportionates (sic) \u03b1-glucan with glucose to produce maltose.\" Figure 1 shows that GlgE uses an \"acceptor\", which I assume could be glucose. How is free glucose synthesized? Could cells grown on a non-carbohydrate as sole carbon source make free glucose?\n> >P. a*eruginosa* is able to carry out gluconeogenesis, so it can produce glucose from non-carbohydrate carbon sources if necessary.\n\n> >Our data show that GlgE acceptor preference gets lower as the acceptor molecule gets shorter. It is possible to detect GlgE activity without an acceptor. In this case we see a lag, implying M1P hydrolyses slowly at first and priming with glucose is also slow. Eventually however, the products get long enough for the reaction to take off. MalQ will work with DP2 or longer as the donor and DP1 or longer as the acceptor, moving one glucose unit at a time.\n\n> + Pedantic point, but \"disproportionation\" means an oxidation-reduction reaction in which two identical molecules are used to produce two different molecules (https:\/\/en.wikipedia.org\/wiki\/Disproportionation). The reaction catalysed by MalQ does not involve electron transfer. Don't the authors mean that this enzyme is a glycosyl transferase?\n> >We have checked this, and our use of disproportionation in the manuscript is correct. The definition of disproportionation is any desymmetrizing reaction of the following type: 2 A \u2192 A' + A\", and is not limited to redox reactions. MalQ carries out a reaction of this type when presented with a maltooligosaccharide.\n\n> + The authors state that TreS had \"a very high Km for trehalose (>100 mM)\". In view of the low concentration of trehalose (Point 1, above), the physiological relevance of this suggested activity is questionable.\n> >See response to question 1 above. As trehalose levels are elevated under osmostress conditions this concern becomes less critical. It is of course true that conditions* in vitro* may not fully reflect cellular conditions and that this activity may be higher *in vivo*, but this is a general limitation of all protein biochemistry studies. The important point here is that trehalose synthase activity is detected for PA01 TreS.\n\n> + Explain better what \"predicted mean log10(CFU) means.\n> >The predicted mean refers to the value of log10(CFU) predicted by the statistical model we use. We have clarified this in the relevant sections of the manuscript.\n\n> + Can the authors suggest how \"\u03b1-glucan protects PA01 against desiccation\"?\n> >Without further investigation we can only speculate as to how \u03b1-glucan confers desiccation tolerance in PA01. One possibility is that \u03b1-glucan functions as a hydrogel, like the exopolysaccharide alginate, trapping water molecules and slowing their evaporation. Alternatively, it may confer a structural role akin to that of trehalose, preventing the loss of cell integrity as water levels decrease. We now address these possibilities in the discussion.\n\n> + Can *P. aeruginosa* metabolize exogenous trehalose or maltose? If the authors know either way, they should mention it. If they don't know, I am not suggesting that they should test this for this paper, but it would be interesting to know whether these compounds would induce the expression of the trehalose or maltose catabolic enzymes or repress the relevant biosynthetic enzymes.\n> *>P. aeruginosa* is able to metabolise exogenous maltose and trehalose. While the experiments that the reviewer suggests are certainly interesting, in our view *tre\/glg* gene regulation is beyond the scope of the current manuscript. This field is certainly worth investigating in the future, however.\n\n> __*Minor points*__\n\n> + First page under \"Results\": \"phosphomutase\" should be \"phosphoglucomutase\"?\n> >Changed as requested.\n\n> + Discussion: insert \"*P. syringae*\" before \"*Pto*\".\n> >Changed as requested.\n\n> + Materials and Methods: describe how ADP was quantified in the maltokinase assay.\n> >The materials and methods section has been updated as requested.\n\n> __*Reviewer 2 (Significance):*__\n\n> __*Significance*__\n\n> Until this work, the biosynthesis of trehalose has been most extensively characterized in *Escherichia coli*, in which it has been shown that this disaccharide is made by the reaction of glucose-6-phosphate and UDP-glucose to give trehalose-6-phosphate and dephosphorylation to trehalose, catalysed by OtsA and OtsB. The authors discovered a very different pathway in *P. aeruginosa* in which the synthesis of trehalose goes through \u03b1-glucans as intermediates.\n\n> Because trehalose and \u03b1-glucans are needed for osmotic stress- and desiccation-tolerance, respectively, this work is of significance to researchers studying abiotic stress resistance.\n\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924v1","doi":"10.1101\/2020.10.23.351924"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#2\n\n#### Evidence, reproducibility and clarity\n\n Review of manuscript \"Trehalose and &#x03B1;-glucan mediate distinct abiotic responses in Pseudomonas aeruginosa\" by S. D. Woodcock et al. \r\n\r\nIt will be difficult for me to write a review of this paper and for the authors to make sense of my review because the manuscript's pages \/ lines are not numbered.  I will do my best write a review, but for the future, I urge this Journal to print the text on pages in which the lines are numbered or require this of the authors.  \r\n\r\n**Summary.**\r\n\r\nThe authors carried out a comprehensive characterization of the metabolism of trehalose in Pseudomonas aeruginosa PA01, using techniques of biochemistry, reverse genetics, and bioinformatics.  The main findings include that the disaccharide trehalose is synthesized in this organism from branched chain &#x03B1;-glucans and that the catabolism of trehalose proceeds via another disaccharide, maltose and is fed back into the synthesis of &#x03B1;-glucans.  Trehalose and &#x03B1;-glucans have been implicated in conferring resistance to abiotic stresses in other organisms.  The authors show that mutants that are blocked in the synthesis of trehalose are sensitive to high salinity but are normal with respect to their sensitivity to desiccation, whereas mutants impaired in the accumulation of &#x03B1;-glucans are sensitive to desiccation without being unduly sensitive to  osmotic stress.  These results indicate that trehalose and &#x03B1;-glucans have different roles in abiotic stress-tolerance. \r\n\r\n**Major points.**\r\n\r\nThis manuscript describes an impressive amount of careful work and presents new insights into the metabolism of trehalose, maltose, and &#x03B1;-glucans.  However, the authors should address the following major comments before the paper is accepted.  \r\n\r\n1.\tDiscussion: the authors state that \"trehalose protects Pseudomonas ssp. against osmotic stress, most likely due to its role as a compatible solute.\" \r\n\tAccording to Table 2, P. aeruginosa grown in the medium of low osmolarity accumulated 0.13% trehalose per gram dry weight, i.e. ~4 &#x03BC;mol \/ g dry weight.  Assuming that the dry weight \/ wet weight ratio of P. aeruginosa is the same as that of P. putida, which is ~1\/3 (PMID: 6508285), the concentration of trehalose in the cells calculates to be ~2 mM.  It is not plausible that trehalose could be significant as compatible solute at this low concentration.  \r\n\tOne way out could be if the accumulation of this disaccharide were increased by osmotic stress.  The authors should also measure the trehalose content of cells grown in medium containing 0.85 M NaCl.  In case of positive results in this experiment, it would be interesting to determine the effects of osmotic stress on the levels of trehalose biosynthetic and catabolic enzymes, but this would not be necessary for the acceptance of the paper.   \r\n\tHowever, there is also an extensive literature suggesting that trehalose has antioxidant functions e.g. PMID: 29241092 (the first paper that came up in Google search for \"trehalose as antioxidant\").  The authors should discuss this possible alternate role of trehalose.  \r\n\tIt is not described adequately in the Materials and Methods how the cellular contents of trehalose and maltose-1-phosphate (M1P) were determined.  \r\n2.\tI found the growth curves in Figure 8, especially in panel B, to be uniterpretable.  The authors should spread these data into more panels or use some other method to make them clearer.  \r\n3.\tThe statement \"The GlgA and GlgE proteins  . . . enable two alternate mechanisms for linear &#x03B1;-glucan biosynthesis\", which is echoed a number of times in the manuscript, seems to create the impression that there are two de novo pathways of synthesis of these polysaccharides.  However, as shown in Figure 1, the GlgA pathway is the only route to the net synthesis of &#x03B1;-glucans, and GlgE is only part of a recycling pathway.  Therefore, it cannot be true that \"the vast majority of &#x03B1;-glucan accumulated by P. aeruginosa will be produced by GlgE\".  \r\n4.\tThe authors state that \"MalQ disproportionates (sic) &#x03B1;-glucan with glucose to produce maltose.\"  Figure 1 shows that GlgE uses an \"acceptor\", which I assume could be glucose.  \r\n\tHow is free glucose synthesized?  Could cells grown on a non-carbohydrate as sole carbon source make free glucose?\r\n\tPedantic point, but \"disproportionation\" means an oxidation-reduction reaction in which two identical molecules are used to produce two different molecules (https:\/\/en.wikipedia.org\/wiki\/Disproportionation).  The reaction catalyzed by MalQ does not involve electron transfer.  Don't the authors mean that this enzyme is a glycosyl transferase? \r\n5.\tThe authors state that TreS had \"a very high Km for trehalose (&gt;100 mM)\".  In view of the low concentration of trehalose (Point 1, above), the physiological relevance of this suggested activity is questionable. \r\n6.\tExplain better what \"predicted mean log10(CFU) means. \r\n7.\tCan the authors suggest how \"&#x03B1;-glucan protects PA01 against desiccation\"?\r\n8.\tCan P. aeruginosa metabolize exogenous trehalose or maltose?  If the authors know either way, they should mention it.  If they don't know, I am not suggesting that they should test this for this paper, but it would be interesting to know whether these compounds would induce the expression of the trehalose or maltose catabolic enzymes or repress the relevant biosynthetic enzymes. \r\n\r\n**Minor points.**  \r\n\r\n9.\tFirst page under \"Results\": \"phosphomutase\" should be \"phosphoglucomutase\"?  \r\n10.\tDiscussion: insert \"P. syringae\" before \"Pto\".  \r\n11.\tMaterials and Methods: describe how ADP was quantified in the maltokinase assay.\n\n#### Significance\n\n **Significance.**\r\n\r\nUntil this work, the biosynthesis of trehalose has been most extensively characterized in Escherichia coli, in which it has been shown that this disaccharide is made by the reaction of glucose-6-phosphate and UDP-glucose to give trehalose-6-phosphate and dephosphorylation to trehalose, catalyzed by OtsA and OtsB.  The authors discovered a very different pathway in P. aeruginosa in which the synthesis of trehalose goes through &#x03B1;-glucans as intermediates.  \r\n\tBecause trehalose and &#x03B1;-glucans are needed for osmotic stress- and desiccation-tolerance, respectively, this work is of significance to researchers studying abiotic stress resistance.  \r\n\r\nThe Reviewers' guidelines stipulate that Reviewers should define their fields of expertise.  \r\n\r\nMy credentials are: a) I have been solicited to review this paper, and b) I have publications in osmotic stress adaptation and trehalose biosynthesis in Enterobacteriaceae. \r\n\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-25","createdOn":"2021-03-25","completedOn":"2021-03-25","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.23.351924v1","doi":"10.1101\/2020.10.23.351924"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#1\n\n#### Evidence, reproducibility and clarity\n\n **Summary:**\r\n\r\nProvide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.\r\n\r\nThe authors resolved the biosynthesis of trehalose and alpha-glucan in Pseudomonas aeruginosa and the role of these two compounds in osmotic and desiccation stress.\r\n\r\n**Major comments:**\r\n\r\n&#x2022;\tAre the key conclusions convincing?\r\n\r\nyes\r\n\r\n&#x2022;\tShould the authors qualify some of their claims as preliminary or speculative, or remove them altogether?\r\n\r\nno\r\n\r\n&#x2022;\tWould additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.\r\n\r\nNot necessary, comprehensive coverage of research Topic\r\n\r\n&#x2022;\tAre the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.\r\n\r\nNot applicable\r\n\r\n&#x2022;\tAre the data and the methods presented in such a way that they can be reproduced?\r\n\r\nyes\r\n\r\n&#x2022;\tAre the experiments adequately replicated and statistical analysis adequate?\r\n\r\nYes, everything is adequate but just one subtle concern: check the significance of the number of digits in the entries listed in Table S3. Revise Table S3.\r\n\r\n**Minor comments:**\r\n\r\n&#x2022;\tSpecific experimental issues that are easily addressable.  \r\n\r\nnot applicable (Table S3: see above)\r\n\r\n&#x2022;\tAre prior studies referenced appropriately? \r\n\r\nNo.  Refs. 18- 32: The subjects of 'trehalose' and 'osmotic stress' have already been addressed in the Pseudomonas field and should be referenced. The authors cite work carried out on trehalose and osmotic stress on phylogenetically distant microorganisms, but do not cite related work from the Pseudomonas field which I consider to be inappropriate. Similarly, trehalose biosynthesis in Pseudomonas has not only been covered by refs. 47 and 48. \r\n\r\n&#x2022;\tAre the text and figures clear and accurate?\r\n\r\nExtremely well written manuscript and prepared figures\r\n\r\n&#x2022;\tDo you have suggestions that would help the authors improve the presentation of their data and conclusions? \r\n\r\nRevise the list of references and discuss more thoroughly your novel findings in the light of existing knowledge in the Pseudomonas field \r\n\r\n\n\n#### Significance\n\n **2. Significance**\r\n\r\n&#x2022;\tDescribe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.\r\n\r\nConceptual advance: The authors identified and characterized the enzymatic pathway of trehalose and alpha-glucan biosynthesis in Pseudomonas aeruginosa and its role to cope with osmotic and desiccation stress. The authors' conclusions do not correspond with recently published peers' work, hence they should discuss in more detail why they consider their data to be more accurate to discern the role of trehalose to contain desiccation and osmotic strass in P. aeruginosa. \r\n\r\n&#x2022;\tPlace the work in the context of the existing literature (provide references, where appropriate).\r\n \r\nExisting literature focusing on trehalose, osmotic stress, desiccation stress in the Pseudomonas field not cited by the authors \r\n\r\nPazos-Rojas LA, Mu&#x00F1;oz-Arenas LC, Rodr&#x00ED;guez-Andrade O, L&#x00F3;pez-Cruz LE, L&#x00F3;pez-\r\nOrtega O, Lopes-Olivares F, Luna-Suarez S, Baez A, Morales-Garc&#x00ED;a YE, Quintero-\r\nHern&#x00E1;ndez V, Villalobos-L&#x00F3;pez MA, De la Torre J, Mu&#x00F1;oz-Rojas J. Desiccation-\r\ninduced viable but nonculturable state in Pseudomonas putida KT2440, a survival\r\nstrategy. PLoS One. 2019 Jul 19;14(7):e0219554. doi:10.1371\/journal.pone.0219554.\r\n\r\nWang T, Jia S, Dai K, Liu H, Wang R. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose. Can J Microbiol. 2014 Sep;60(9):599-604. doi: 10.1139\/cjm-2014-0330. \r\n\r\nHarty CE, Martins D, Doing G, Mould DL, Clay ME, Occhipinti P, Nguyen D, Hogan DA. Ethanol Stimulates Trehalose Production through a SpoT-DksA-AlgU-Dependent Pathway in Pseudomonas aeruginosa. J Bacteriol. 2019 May 22;201(12):e00794-18. doi: 10.1128\/JB.00794-18. \r\n\r\nCross M, Biberacher S, Park SY, Rajan S, Korhonen P, Gasser RB, Kim JS, Coster MJ, Hofmann A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa. FASEB J. 2018 Oct;32(10):5470-5482. doi: 10.1096\/fj.201800500R. \r\n\r\nWang T, Jia S, Dai K, Liu H, Wang R. Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose. Can J Microbiol. 2014 Sep;60(9):599-604. doi: 10.1139\/cjm-2014-0330. \r\n\r\nBehrends V, Ryall B, Zlosnik JE, Speert DP, Bundy JG, Williams HD. Metabolic adaptations of Pseudomonas aeruginosa during cystic fibrosis chronic lung infections. Environ Microbiol. 2013 Feb;15(2):398-408. doi: 10.1111\/j.1462-2920.2012.02840.x\r\n\r\nBehrends V, Ryall B, Wang X, Bundy JG, Williams HD. Metabolic profiling of Pseudomonas aeruginosa demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance. Mol Biosyst. 2010 Mar;6(3):562-9. doi: 10.1039\/b918710c. \r\n\r\nMatthijs S, Koedam N, Cornelis P, De Greve H. The trehalose operon of Pseudomonas fluorescens ATCC 17400. Res Microbiol. 2000 Dec;151(10):845-51. doi: 10.1016\/s0923-2508(00)01151-7. \r\n\r\nvan der Werf MJ, Overkamp KM, Muilwijk B, Koek MM, van der Werff-van der Vat BJ, Jellema RH, Coulier L, Hankemeier T. Comprehensive analysis of the metabolome of Pseudomonas putida S12 grown on different carbon sources. Mol Biosyst. 2008 Apr;4(4):315-27. doi: 10.1039\/b717340g. \r\n\r\nHallsworth JE, Heim S, Timmis KN. Chaotropic solutes cause water stress in Pseudomonas putida. Environ Microbiol. 2003 Dec;5(12):1270-80. doi:\r\n10.1111\/j.1462-2920.2003.00478.x.\r\n\r\nBall P, Hallsworth JE. Water structure and chaotropicity: their uses, abuses and biological implications. Phys Chem Chem Phys. 2015 Apr 7;17(13):8297-305.\r\ndoi: 10.1039\/c4cp04564e\r\n\r\nCray JA, Russell JT, Timson DJ, Singhal RS, Hallsworth JE. A universal measure of chaotropicity and kosmotropicity. Environ Microbiol. 2013 Jan;15(1):287-96. doi: 10.1111\/1462-2920.12018. \r\n\r\nChin JP, Megaw J, Magill CL, Nowotarski K, Williams JP, Bhaganna P, Linton M, Patterson MF, Underwood GJ, Mswaka AY, Hallsworth JE. Solutes determine the temperature windows for microbial survival and growth. Proc Natl Acad Sci U S A. 2010 Apr 27;107(17):7835-40. doi: 10.1073\/pnas.1000557107.\r\n\r\nThese papers are of variable scientific quality, but the conceptual work by Hallsworth and the work by Behrens on the PA metabolome in CF lungs are worth discussing. All other work provides pieces of information on function and biosynthesis of trehalose up to now known by the Pseudomonas community. The authors resolved the function of the GlgA operon which will be definitely appreciated.\r\n\r\n**Strengths of the manuscript:** \r\n\r\n&#x2022;\tMeticulously planned and carefully executed experiments, not a single experimental flaw\r\n&#x2022;\tvery high technical quality of experiments and primary data\r\n&#x2022;\tcomprehensive coverage of the research topic\r\n&#x2022;\texcellent presentation in text and illustrations\r\n\r\nonly weakness: \r\n&#x2022;\tinsufficient consideration of peers' published work on trehalose and its role in stress response in P. aeruginosa\r\n\r\n&#x2022;\tState what audience might be interested in and influenced by the reported findings.\r\n\r\nScientists working in the fields of glycoconjugate and carbohydrate research, biochemists, microbiologists with interest in metabolic pathways, stress response and\/or Pseudomonas\r\n\r\n&#x2022;\tDefine your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.\r\n\r\nReviewer's expertise: Pseudomonas genomics and physiology, respiratory tract infections, solid background in biochemistry and molecular biology\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665v1","doi":"10.1101\/2021.01.21.427665"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nIn some species, supporting cells (SCs) of the cochlea can replace hair cells and thus restore hearing. In the mouse, neonatal SCs can also produce hair cells; however, this property is lost during early postnatal life. This study sought to test whether forced expression of two transcription factors normally associated with OHC development, Atoh1 and Ifzh2, can induce adult mammalian supporting cells to take OHC-like properties. Using Cre-dependent expression in mice, the authors showed that co-expression of Atoh1 and Izfh2 could induce a small number of adult SCs to express the OHC-specific gene, Prestin. This conversion was significantly enhanced when existing OHCs were ablated, in this case using a Prestin-DTR mouse model generated by the authors. A detailed phenotypic analysis combined with single cell RNA-sequencing (scRNA-seq) supports the idea that Atoh1\/Izfh2 can partially convert adult SCs into OHC-like cells. However, the conversion is not complete, with immature bundles and a gene signature that resembles P1 OHCs (and sometimes E16 OHCs) more than P7\/P30 OHCs or P60 SCs. Accordingly, the new OHCs are not sufficient to restore hearing in the Prestin-DTR mouse model. Together, these data encourage optimism that adult SCs can be steered along the OHC path, though clearly more manipulations will be needed to produce mature, functional OHCs.\n\nThe main weakness of the study is the scRNA-seq analysis, which depends on very small sample sizes. Suggestions to improve upon the analysis are listed under Specific Recommendations."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665v1","doi":"10.1101\/2021.01.21.427665"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThe goal of this study is to devise a means of promoting adult mouse auditory sensory cell development from supporting cells (SCs), as occurs naturally in birds and fish following sensory cell death. Previous studies indicated that activating Atoh1, an early acting transcription factor that specifies sensory cell fate during embryogenesis, was not sufficient for such regeneration. The authors hypothesized that adding a second transcription factor, Ikzf2, which maintains outer hair cell (OHC) fate, would synergize with Atoh1 and push adult SCs to differentiate as OHCs. They tested this hypothesis by over-expressing both Atoh1 and Ikzf2 in supporting cells after killing the endogenous OHCs in adult cochleae. The authors showed that the induced cells first express the general HC marker, Myo6, and only later become Prestin-positive, much as occurs during normal development. Unfortunately, these induced OHC-like cells had abnormal stereocilia and did not restore auditory (ABR) thresholds. Moreover, there was a loss of IHCs (the primary auditory receptors) suggesting that much more is needed to induce a real OHC and to protect IHCs than simply inducing the two selected transcription factors. Single-cell RNAseq (scRNA-seq) results showed that the induced OHC-like cells are enriched for HC genes and depleted for SC genes, but overall are most similar to neonatal HCs as defined in published scRNA-seq data from other groups. Overall, the scRNA-seq data did not offer a clear path forward, other than to identify and test additional transcription factors that might push the induced cells to the next stage. Nevertheless, the extent of SC transformation is impressive and has not been seen in previous approaches. This is an important contribution to our understanding of the control of OHC gene expression and differentiation contributed by two important transcription factors.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665v1","doi":"10.1101\/2021.01.21.427665"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nMature mammalian hair cells in the cochlea do not regenerate after damage. The outer hair cells of the cochlea, which function to amplify sound, are particularly susceptible to damage. Ectopic activation of two key transcription factors for outer hair cell formation, Atoh1 and Ikzf2, in damaged adult cochlea is sufficient to convert supporting cells into hair cells expressing Prestin, which is an essential protein mediating outer hair cell functions. Although there is no functional recovery in these transgenic mice based on auditory brainstem response, this study paves the way for future design of models for hearing recovery. The main concern is the identity of the OHC-like cells drawn from the small sample size in the scRNA-seq experiments.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.21.427665v1","doi":"10.1101\/2021.01.21.427665"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis manuscript demonstrated the effectiveness of combined activation of Atoh1 and Ikzf2 in converting adult supporting cells to outer hair cell (OHC)-like cells in a mouse model, in which the OHCs were selectively ablated with diphtheria toxin. The authors showed that while the number of regenerated hair cells was low and there was no functional recovery based on ABR, these OHC-like cells do express Prestin and exhibit a genetic profile that resembles nascent hair cells. This paper will be of great interest to researchers interested in hearing restoration, as well as regenerative biology.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495v2","doi":"10.1101\/2020.11.16.384495"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Author Response:**\n\n>**Evaluation Summary:**\n\n>This paper will be of considerable interest to anybody focusing on highly sensitive T cell antigen recognition. It uses an extended experimental protocol and analytical methods to assess very low T cell receptor binding affinities, and to determine how T cells discriminate between self- and non-self antigens. The main conclusions are well supported by the presented analysis and provide a novel view on a previously considered concept.\n\n>**Reviewer #1 (Public Review):**\n\n>The presented manuscript takes a comprehensive and elaborated look at how T cell receptors (TCR) discriminate between self and non-self antigens. By extending a previous experimental protocol for measuring T cell receptor binding affinities against peptide MHC complexes (pMHC), they are able to determine very low TCR-pMHC binding affinities and, thereby, show that the discriminatory power of the TCR seems to be imperfect. Instead of a previously considered sharp threshold in discriminating between self and non-self antigen, the TCR can respond to very low binding affinities leading to a more transient affinity threshold. However, the analysis still indicates an improved discrimination ability for TCR compared to other cell surface receptors. These findings could impact the way how T cell mediated autoimmunity is studied.\n\n>The authors follow a comprehensive and elaborated approach, combining in vitro experiments with analytical methods to estimate binding affinities. They also show that the general concept of kinetic proofreading fits their data with providing estimates on the number of proofreading steps and the corresponding rates. The statistical and analytical methods are well explained and outlined in detail within the Supplemental Material. The source of all data, and especially how the data to analyze other cell surface receptor binding affinities was extracted, are given in detail as well. Besides being able to quantify TCR-pMHC interactions for very low binding affinities, their findings will improve the ability to assess how autoimmune reactions are potentially triggered, and how potent anti-tumour T cell therapies can be generated.\n\n>In summary, the study represents an elaborated and concise analysis of TCR-pMHC affinities and the ability of TCR to discriminate between self and non-self antigens. All conclusions are well supported by the presented data and analyses without major caveats.\n\n\n>**Reviewer #2 (Public Review):**\n\n>The paper revisits the question of ligand discrimination ability of TCRs of T cells. The authors find that the commonly held notion of very sharp discrimination between strongly and weakly binding peptides does not hold when the affinities of the weak peptides are re-measured more accurately, using their own new method of calibration of SPR measurements. They are able to phenomenologically fit their results with a ~2 step Kinetic Proofreading model.\n\n>It is a very carefully researched and thorough paper. The conclusions seem to be supported by the data and fundamental for our understanding of the T cell immune response with potentially very high impact in many scientific and applied fields. The calibration method could be of potential use in other cases where low affinities are an issue.\n\n>As a non-expert in the details of experimental technique, it is somewhat difficult to understand in detail the Ab calibration of the SPR curve - which is a central piece of the paper. The main question is - what are the grounds (theoretical and\/or empirical) to expect that the B_max of the TCR dose response curve will continue to be proportional to the plateau level of the Ab. Figure 1D does suggest that, but it would be hard to predict what proportionality shape the curve will take for lower affinity peptides. Given that essentially all the paper claims rest on this assumption, this should explained\/reasoned\/supported more clearly.\n\nWe have revised the relevant Results and Methods sections to provide additional information. This information should clarify the expected relationship between Bmax and W6\/32 binding. We emphasise that we have only interpolated within the curve and therefore, have not relied on any assumptions about the relationship between these two values outside of the empirical curve that we have generated.\n\n>On the theoretical side - I think the scaling alpha\\simeq 2 in Figure 2 is indeed consistent with a two-step KPR amplification. However, there are some questions regarding the fitting of the full model to the P_15 of the CD69 response. As explained in the Supplementary Material the authors use 3 global and 2 local parameters resulting in 37 (or 27) parameters for 32 data points. To a naive reader this might look excessive and prone to overfitting. On the other hand, looking at Figure S8 shows the value ranges of lambda and k_p are quite tight. This is in contrast to gamma and dellta that look completely unconstrained.\n\nWe have revised the relevant Results section to explicitly indicate that the number of data points ex- ceeds the number of free parameters, which together with the ABC-SMC results, should provide additional confidence that we are not over-fitting.\n\n>Finally, one of the stated advantages of the adaptive proof-reading model is that it is capable of explaining antagonism. It is hard to see how a 'vanilla\" KPR model is capable of explaining antagonism.\n\nWe have added a discussion paragraph to discuss antagonism, which cannot be explained by the basic KP model that we found is sufficient to explain our data on antigen discrimination in the presence of self pMHCs on autologous APCs. We describe how the methods we have employed can be used to study antagonism.\n\n>**Reviewer #3 (Public Review):**\n\n>Pettmann et al. aimed at significantly improving the accuracy of SPR-based measurements of low affinity TCR-pMHC interactions by including a 100% binding control (injecting of a conformation-specific HLA-antibody) in the surface plasmon resonance protocol. Interpolating with the information of saturated pMHC binding on the chip The authors arrive at KDs for low affinity binders that are significantly higher than the previously reported constants. If correct, this has considerable ramifications for the interpretations of the results obtained from functional assays measuring the T cell response towards pMHCs featured in a titrated fashion. Unlike what was put forward by earlier reports, the authors conclude that the discriminatory power of TCRs is far from perfect, as T cells still respond to low affinity pMHC-ligands without a sharp affinity threshold. This is also because they managed to detect T cells responding to even ultra-low affinity ligands if provided in sufficient numbers.\n\n>The body of work convinces in several regards:\n\n>(i) It is exceedingly well thought out and introduces a quality of analytical strength that is absent in most of the literature published thus far on this topic.\n\n>(ii) At the same time theoretical arguments are bolstered by a large body of experimental \"wet\" work, which combines a synthetic approach with cellular immunology and which appears overall well executed.\n\n>(iii) The data lead to hypotheses in the field of T cell antigen recognition in general and in the theatre of autoimmunity, cancer and infectious diseases.\n\n>There are a few aspects that may limit the impact of the study. I have listed them below:\n\n>(i) The study does not provide kinetic data for the low affinity ligand-TCR binding but rather argues from the position of affinities as determined via Bmax. This limits somewhat the robustness of the statements made with regard to kinetic proofreading.\n\nWe agree with this statement and are hoping to directly measure off-rates in the future. We note that in the published literature, including our own work, point mutations to the peptide generally modify the off-rate with only minor impact on the on-rate. An example of this can be found in Lever et al (2016) PNAS where point mutations led to 100,000-fold change in the off-rate but only a 10-fold change in the on-rate. This likely explains why antigen potency is often well-correlated with affinity when using point mutations to the peptide.\n\n>(ii) Thresholds for readouts were arbitrarily chosen (e.g. 15% activation). It appears such choices were based on system behavior (with the largest differences observed among the groups) but may have implications for the drawn conclusions.\n\nWe have chosen 15% in order to capture the ultra-low affinity pMHCs in our potency plots and have now added a sentence for why we have chosen this particular threshold. We did explore different thresholds but found that they produced similar values of \u03b1. The precise threshold could change the estimate of \u03b1 if the shape of dose-response curves was dependent on antigen affinity but we did not find any evidence for this within our data.\n\n>In summary, the work presented contributes to demystifying the link between TCR-engagement and (membrane proximal) signaling. It also provides a fresh perspective on the potential of TCR-cossreactivity.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495v2","doi":"10.1101\/2020.11.16.384495"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nPettmann et al. aimed at significantly improving the accuracy of SPR-based measurements of low affinity TCR-pMHC interactions by including a 100% binding control (injecting of a conformation-specific HLA-antibody) in the surface plasmon resonance protocol. Interpolating with the information of saturated pMHC binding on the chip The authors arrive at KDs for low affinity binders that are significantly higher than the previously reported constants. If correct, this has considerable ramifications for the interpretations of the results obtained from functional assays measuring the T cell response towards pMHCs featured in a titrated fashion. Unlike what was put forward by earlier reports, the authors conclude that the discriminatory power of TCRs is far from perfect, as T cells still respond to low affinity pMHC-ligands without a sharp affinity threshold. This is also because they managed to detect T cells responding to even ultra-low affinity ligands if provided in sufficient numbers.\n\nThe body of work convinces in several regards:\n\n(i) It is exceedingly well thought out and introduces a quality of analytical strength that is absent in most of the literature published thus far on this topic.\n\n(ii) At the same time theoretical arguments are bolstered by a large body of experimental \"wet\" work, which combines a synthetic approach with cellular immunology and which appears overall well executed.\n\n(iii) The data lead to hypotheses in the field of T cell antigen recognition in general and in the theatre of autoimmunity, cancer and infectious diseases.\n\nThere are a few aspects that may limit the impact of the study. I have listed them below:\n\n(i) The study does not provide kinetic data for the low affinity ligand-TCR binding but rather argues from the position of affinities as determined via Bmax. This limits somewhat the robustness of the statements made with regard to kinetic proofreading.\n\n(ii) Thresholds for readouts were arbitrarily chosen (e.g. 15% activation). It appears such choices were based on system behavior (with the largest differences observed among the groups) but may have implications for the drawn conclusions.\n\nIn summary, the work presented contributes to demystifying the link between TCR-engagement and (membrane proximal) signaling. It also provides a fresh perspective on the potential of TCR-cossreactivity."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495v2","doi":"10.1101\/2020.11.16.384495"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThe paper revisits the question of ligand discrimination ability of TCRs of T cells. The authors find that the commonly held notion of very sharp discrimination between strongly and weakly binding peptides does not hold when the affinities of the weak peptides are re-measured more accurately, using their own new method of calibration of SPR measurements. They are able to phenomenologically fit their results with a ~2 step Kinetic Proofreading model.\n\nIt is a very carefully researched and thorough paper. The conclusions seem to be supported by the data and fundamental for our understanding of the T cell immune response with potentially very high impact in many scientific and applied fields. The calibration method could be of potential use in other cases where low affinities are an issue.\n\nAs a non-expert in the details of experimental technique, it is somewhat difficult to understand in detail the Ab calibration of the SPR curve - which is a central piece of the paper. The main question is - what are the grounds (theoretical and\/or empirical) to expect that the B_max of the TCR dose response curve will continue to be proportional to the plateau level of the Ab. Figure 1D does suggest that, but it would be hard to predict what proportionality shape the curve will take for lower affinity peptides. Given that essentially all the paper claims rest on this assumption, this should explained\/reasoned\/supported more clearly.\n\nOn the theoretical side - I think the scaling alpha\\simeq 2 in Figure 2 is indeed consistent with a two-step KPR amplification. However, there are some questions regarding the fitting of the full model to the P_15 of the CD69 response. As explained in the Supplementary Material the authors use 3 global and 2 local parameters resulting in 37 (or 27) parameters for 32 data points. To a naive reader this might look excessive and prone to overfitting. On the other hand, looking at Figure S8 shows the value ranges of lambda and k_p are quite tight. This is in contrast to gamma and dellta that look completely unconstrained.\n\nFinally, one of the stated advantages of the adaptive proof-reading model is that it is capable of explaining antagonism. It is hard to see how a 'vanilla\" KPR model is capable of explaining antagonism."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495v2","doi":"10.1101\/2020.11.16.384495"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThe presented manuscript takes a comprehensive and elaborated look at how T cell receptors (TCR) discriminate between self and non-self antigens. By extending a previous experimental protocol for measuring T cell receptor binding affinities against peptide MHC complexes (pMHC), they are able to determine very low TCR-pMHC binding affinities and, thereby, show that the discriminatory power of the TCR seems to be imperfect. Instead of a previously considered sharp threshold in discriminating between self and non-self antigen, the TCR can respond to very low binding affinities leading to a more transient affinity threshold. However, the analysis still indicates an improved discrimination ability for TCR compared to other cell surface receptors. These findings could impact the way how T cell mediated autoimmunity is studied.\n\nThe authors follow a comprehensive and elaborated approach, combining in vitro experiments with analytical methods to estimate binding affinities. They also show that the general concept of kinetic proofreading fits their data with providing estimates on the number of proofreading steps and the corresponding rates. The statistical and analytical methods are well explained and outlined in detail within the Supplemental Material. The source of all data, and especially how the data to analyze other cell surface receptor binding affinities was extracted, are given in detail as well. Besides being able to quantify TCR-pMHC interactions for very low binding affinities, their findings will improve the ability to assess how autoimmune reactions are potentially triggered, and how potent anti-tumour T cell therapies can be generated.\n\nIn summary, the study represents an elaborated and concise analysis of TCR-pMHC affinities and the ability of TCR to discriminate between self and non-self antigens. All conclusions are well supported by the presented data and analyses without major caveats."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.11.16.384495v2","doi":"10.1101\/2020.11.16.384495"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis paper will be of considerable interest to anybody focusing on highly sensitive T cell antigen recognition. It uses an extended experimental protocol and analytical methods to assess very low T cell receptor binding affinities, and to determine how T cells discriminate between self- and non-self antigens. The main conclusions are well supported by the presented analysis and provide a novel view on a previously considered concept.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067v2","doi":"10.1101\/2020.05.04.076067"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nSun et al have assembled, modified, and applied a series of existing gene editing tools to tissue-derived human fetal lung organoids in a workflow they have termed \"Organoid Easytag\". Using approaches that have previously been applied in iPSCs and other cell models in some cases including organoids, the authors demonstrate: 1) endogenous loci can be targeted with fluorochromes to generate reporter lines; 2) the same approach can be applied to genes not expressed at baseline in combination with an excisable, constitutively active promoter to simplify identification of targeted clones; 3) that a gene of interest could be knocked-out by replacing the coding sequence with a fluorescent reporter; 4) that knockdown or overexpression can be achieved via inducible CRISPR interference (CRISPRi) or activation (CRISPRa). In the case of CRISPRi, the authors alter existing technology to lessen unwanted leaky expression of dCas9-KRAB. While these tools have previously been applied in other models, their assembly and demonstrated application to tissue-derived organoids here could facilitate their use in tissue-derived organoids by other groups.\n\nLimitations of the study include: \n\n1) is demonstrated application of these technologies to a limited set of gene targets; \n\n2) a lack of detail demonstrating the efficiency and\/or kinetics of the approaches demonstrated.\n\nWhile access to human fetal lung organoids is likely not available to many or most researchers, it is probable that the principles applied here could carry over to other organoid models."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067v2","doi":"10.1101\/2020.05.04.076067"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThere is now a considerable body of knowledge about the genetic and cellular mechanisms driving the growth, morphogenesis and differentiation of organs in experimental organisms such as mouse and zebrafish. However, much less is known about the corresponding processes in developing human organ systems. One powerful strategy to achieve this important goal is to use organoids derived from self-renewing, bona fide progenitor cells present in the fetal organ. The Rawlins' lab has pioneered the long-term culture of organoids derived from multipotent epithelial progenitors located in the distal tips of the early human lung. They have shown that clonal cell \"lines\" can be derived from the organoids and that they capable of not only long-term self-renewal but also limited differentiation in vitro or after grafting under the kidney capsule of mice. Here, they now report a strategy to efficiently test the function of genes in the embryonic human lung, regardless of whether the genes are actively transcribed in the progenitor cells. The strengths of the paper are that the authors describe a number of different protocols (work-flows), based on Crisper\/Cas9 and homology directed repair, for making fluorescent reporter alleles (suitable for cell selection) and for inducible over-expression or knockout of specific genes. The so-called \"Easytag\" protocols and results are carefully described, with controls. The work will be of significant interest to scientists using organoids as models of many human organ systems, not just the lung. The weaknesses are that they authors do not show that their lines can undergo differentiation after genetic manipulation, and therefore do not provide proof of principle that they can determine the function in human lung development of genes known to control mouse lung epithelial differentiation. It would also be of general interest to know whether their methods based on homologous recombination are more accurate (fewer incorrect targeting events or off target effects) than methods recently described for organoid gene targeting using non homologous repair."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067v2","doi":"10.1101\/2020.05.04.076067"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThe authors demonstrate applications including fluorescent marking of membranes with GFP or monomeric RFP, reporter alleles for convenient assessment of differentiation status based on fluorescence, and targeted gene knockout. They also demonstrate conditional gene knockdown and induction with tight control achieved by engineering a protein destabilizing domain. The design of the constructs is clever and imparts the ability to leverage iterative FACS to enrich successfully targeted cells, particularly useful when targeting alleles that are not actively expressed by the progenitors. The work is well done and clearly presented."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.05.04.076067v2","doi":"10.1101\/2020.05.04.076067"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nIn this paper Sun and colleagues aimed to demonstrate the feasibility of using CRISPR-based gene editing techniques applied to tissue-derived human fetal lung organoids. While previous studies have used CRISPR-Cas9 to perform knock-in or knock-out studies in organoids (such as intestinal, hepatic or tumor organoids), this is the first report to apply these tools to a tissue-derived lung organoid model. A major strength of this report is the additional use of CRISPRi and CRISPRa technologies. The work is well done, clearly presented and makes an important contribution to the literature.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)*\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442v2","doi":"10.1101\/2020.10.19.20215442"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nMoncla et al. investigated the transmission of mumps virus in Washington, USA during an outbreak in 2016-2017. They sequenced viral genomes from infected individuals in Washington and elsewhere within the United States and used phylogenetic approaches to understand the origins and patterns of spread exhibited by the virus during the outbreak. They observe a large fraction of cases in individuals who are part of the Marshallese community, and identify a link to a similar outbreak in the Marshallese community in Arkansas. They develop a method for determining the role of the Marshallese community in the Washington outbreak that is robust to sampling bias and size. This method is well thought-out and presented and demonstrates that the outbreak in Washington state was sustained by transmission within this particular community. This paper provides a thoughtful approach to dealing with sampling issues that are often overlooked in phylogenetic studies. By consulting with a public health professional from within the affected (Marshallese) community, the authors are able to contextualize their results and demonstrate the underlying issues that may have contributed to mumps spread within the state. \n\nWorking with public health advocates from affected communities is exceptionally important for long term public health impact, and this paper sets an example that should be followed by others in the pathogen genomics field. The methodology used to determine mumps transmission patterns in Washington is sound and the conclusions are well explained. However, some additional context on the issues and potential pitfalls of source-sink analyses based on phylogenetic inference would help improve this already solid paper. Specifically: \n\n1) The authors seem to assume a somewhat random sample throughout Washington state. They state that given a low sampling proportion they do not expect to have captured infection pairs, which seems reasonable. However, they then go onto assume that their sample is primarily comprised of samples from long, successful transmission chains. This is a reasonable assumption if there is no major difference in accessibility of samples from long transmission chains and shorter ones (for example, decreased access to healthcare). Could this impact the assumption of sampling primarily from long transmission chains? It seems from the data collected in this outbreak that this was not the case for mumps in Washington but addressing this assumption clearly (and potential ways to interrogate it) could make their methodology more applicable to other pathogen studies. \n\n2) There are many examples of phylogenetic analyses that have led to conclusions about pathogen sources and sinks that were later shown to be wrong because of oversampling or other sampling biases. The authors address unequal sampling between clades, but additional contextualization of the problem and how this approach is different may help strengthen the methodology presented in the paper. \n\n3) The authors present compelling evidence that the mumps outbreak in Washington state was sustained by the Marshallese community, and state that mumps did not transmit efficiently among the general Washington populace. That said, there were several other mumps outbreaks in the United States in the same 2016-2017 time period. Was there something different about Washington state that prevented mumps transmission outside of the Marshallese community? Were there no other close-knit communities (universities, prisons, other cultural communities, etc.) affected? It just seems surprising that the Marshallese community was the only community sustaining transmission at a time where many different types of communities were affected across the United States. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442v2","doi":"10.1101\/2020.10.19.20215442"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nIn this manuscript, Moncla et al. undertake a large sequencing and phylogenetic study to investigate the underlying epidemiology of the 2016-2017 Washington State Mumps epidemic. The authors generate 110 sequences and include 166 novel sequences in their analysis. This data set represents over a quarter of the publicly available Mumps genomes from North America. \n\nThey then apply a mixture of phylogenetic methods and intuitive data analyses to uncover, that \ni) Mumps was imported into Washington at least 13 times. \nii) A disproportionate amount of transmission occurred in the Marshallese community in WA with limited transmission in the non-Marshallese community. \niii) These heterologous transmission dynamics might be explained by historical and current health disparities within the community, but are not due to low vaccination coverage. \n\nThese conclusions are supported by a wide array of carefully controlled phylogenetic methods. The authors explore the sensitivity of their findings to sampling bias. Additionally, the conclusion that transmission occurred disproportionally within the Marshallese community is supported by multiple implementations of the structured coalescent as well as, more coarse but intuitive methods such as the rarefaction analysis and the \"descendent\" analysis in Figure 4. The \"descendent\" analysis complements the structured coalescent models and highlights how tips that are close to internal nodes inform the \"state\" of those unsampled ancestors. Each internal node represents an unsampled ancestor, and if transmission rates are higher in one population, then samples from that population are more likely to be close to those ancestors. The approach captures these processes; however, calling downstream tips \"descendants\" is unfortunate, as it is unknown if the tips that have \"descendants\" are direct ancestors of their \"descendants\" in the transmission chain. Inferring transmission dynamics from divergence trees is difficult, and variants of this approach are likely to be useful in other systems. \n\nThe finding that transmission disproportionally occurred in the Marshallese community leads the authors to propose several possibilities for why this may be. The authors should be commended for reaching out to Marshallese health advocates in this process and including the community in their study. This context is a major strength of the study. \n\nBoth the data generation and data analysis are achievements that advance our understanding of the epidemiology of Mumps. As can be seen in the tree in Figure 1 the 2016-2017 epidemic in North America was seeded by at least two divergent lineages that appear to have all contributed to the same outbreak. The large number of sequences contributed by this study will help future work uncover the dynamics that drive Mumps epidemics at larger scales. The findings also highlight how large outbreaks can persist in highly vaccinated populations and how an array of phylogenetic approaches can be employed to uncover the underlying population heterogeneity behind an outbreak. To have both of these achievements in the same manuscript sets this work apart. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442v2","doi":"10.1101\/2020.10.19.20215442"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nIn this study, Moncla et al. used genomic data to analyse a mumps outbreak in Washington, in order to draw inferences about the epidemiological factors driving the outbreak. Some important strengths of the analysis include sophisticated sequencing and modeling techniques to reconstruct chains of transmission during the outbreak, which support the conclusions that the mumps virus was introduced several times in Washington from other North American regions during the outbreak, and that the Washington Marshallese community was particularly at risk of mumps infection and transmission during this time. Limitations of the analysis include potential for sampling bias, where the sample may not be entirely representative of mumps outbreak cases, and a sample size that is too low to allow sufficient statistical power to assess the impacts of age and vaccination status on transmission. The work has potential public health impacts in terms of identification of a vulnerable community and points to social networks as the primary risk factor for potential future respiratory virus outbreaks. The analysis methods could be potentially applied for the phylodynamic analysis of other infectious disease outbreaks. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.10.19.20215442v2","doi":"10.1101\/2020.10.19.20215442"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nThis interesting phylogenetic analysis of a mumps outbreak in Washington will be of interest to a wide audience, especially those working at the intersection of pathogen genomics and public health. An array of classic and novel phylogenetic approaches supports the conclusions that mumps was introduced several times in Washington during the outbreak, and that the Washington Marshallese community was particularly at risk of mumps infection and transmission despite high vaccination coverage. Inferences regarding the role of age and vaccination status are however less conclusive given the small sample size. Consultation with a community health advocate from the affected communities helps contextualize the results. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.21249713#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.21249713v2","doi":"10.1101\/2021.01.12.21249713"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Author Response to Public Reviews**\n\n> **Reviewer #1 (Public Review):**\n\n> [...] The deficiencies of this study are:\n> \n> 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\n\nWe agree with the reviewer that this is a very specific cohort, largely urban, and with higher levels of education than average. We further agree that the utility of this cohort is not in making general statements about the population, but rather in deriving specific insights for\nwhich the cohort is best suited. We enumerate some of them that are present in this manuscript:\n\na) It is as important to understand the relative degree of spread between Indian cities, where a combination of denser population and indoor lives has led to the greatest spread of disease. Since pandemics are typically self-limiting, regions with greater spread are further along the course and can expect declines faster. This provides useful insight for public health strategy. While our cohort does not necessarily represent the average population, it is similar between cities, something that is not true for any other survey. The ICMR national serosurvey is a random selection of\ndistricts and is heavily rural biased.1 While that is important, that is not where fast growing outbreaks are likely based on a very outdoor life and lower density. Other city-wise serosurveys are variable in target population as well as methodology and cannot be easily compared.2-5 Thus our data is the first that permits comparison between many important urban regions of India, showing which regions were more advanced along the course and where future outbreaks were still likely. We note here that some of the regions identified by this survey as high risk such as Kerala, interior Maharashtra, amongst others, are where the outbreaks continued until much later.\n\nb) The CSIR cohort has the added advantage of greater baseline data and repeated access, we are able to determine antibody stability, as shown, and possible\ncorrelates.\n\nc) The cohort is well suited to understanding clinical associations of SARS CoV2 infections such as symptom rate and severity amongst its participants as well as\nassociations of infection risks (using seropositivity as an imperfect surrogate). \n\n> 2) The presentation of Figure 1 was quite confusing, especially the colour coding\n\nFigure 1 was made to represent cities with CSIR labs where the sero-survey was carried out in different colour coding formats to have a quick understanding of prevalence. The cities with sero-prevalence greater than 10 percent were coded as green, while cities with seroprevalence between 5-10 percent values were coded as yellow. Cities with less than 5 percent sero-positivity were depicted as red for these may turn up into hotspots or rise of cases may be higher in these cities later when sero-positivity is used as an indirect surrogate of infection. Though, these cities while truly may not represent the state population, the state\ncolour were coded as a gradient blue in respective format to reflect increased sero-positivity in a darker shade according to city sero-positivity. We realize that colour coding of states may have created a confusion and have removed this in the revised manuscript.\n\n> 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.\n\nThe Pune city sero-surveillance which has been pointed out by the reviewer was a survey of Pune\u2019s five most affected sub-wards and not the Pune population in general. 6 Despite all the limitations, which we accept in the prior comment, our overall crude positivity rate of 10% is very similar to that of the ICMR national serosurvey, and in general the patterns we see are along the lines of what is known about severity of outbreaks. Thus, there is no real evidence to the contrary that would establish inaccuracy of the trends seen by us, and we\nrespectfully note that surprising findings may be the most valuable ones. In fact, seeing current trends of rising cases in Maharashtra, including in Pune, when compared to other cities, our survey values may have been more correct. \n\n> 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.\n\nWe agree that for R of about 2, one would not expect a decline at sero-positivity of about 10%. However, it is worth noting that general seropositivity during the declining phase of the outbreak has been in this range for not just India, but also in major western European cities, New York, amongst other. This has three explanations. First, the highly exposed community\ncontaining the high-contact spreaders gets infected first, with higher seropositivity, thus effectively shortening or blocking transmission chains. We too note a much higher seropositivity amongst public transport users who may better represent this sub-population. Second, R0 of 2-3 is the potential of this virus. R-effective after measures are put in place may be much lower. Better compliance with masking in India may have been important. Last, the fraction of population immune at baseline is unknown but has been variably estimated at 20-30% from T cell reactivity studies as well as closed area breakouts such as ships. This is a speculative area but may help understand the results. \n\nWe agree that we do not directly account for testing rate, which is difficult to adjust for and can affect TPR despite that fact that TPR already is one way of adjusting for different levels of testing. Since our data is a trend across different geographies, but for 30 days bracketing the sample collection, different testing rate would not in itself explain the very strong inverse association of seropositivity with TPR. Given that high seropositivity areas are likely more advanced in the course of the pandemic, we favour that as the explanation. This is after noting the issues with overall seropositivity as a surrogate of population immunity as above.\n\n> 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\n\nWe very well agree with the statement that much of South-Asia is particularly non-vegetarian and when we started analyzing our data, it was observed that our cohort had a 70:30 ratio for non-vegetarian population to vegetarian population which was in agreement with what\nnationwide surveys have concluded in the past and hence our cohort was not biased in terms of sampling for this variable. We hereby in this work have tried to demonstrate seropositivity as an indirect surrogate of infection and the data was not analyzed in respected of\nzonal distribution and was analyzed for the entire cohort where we obtained the said observation. At this stage, we cannot speculate on the role a vegetarian diet may play in decrease sero-positivity amongst vegetarian individuals but could possibly relate it to antiinflammatory effects and effect of high fibre diet in protecting gut mucosa against viral invasion. Existing studies have only speculated on the role diet could play and there are no affirmative or largely biochemical studies to provide further evidence on this cause effect\nrelationship. We also did a multi-collinearity analysis to study if diet was related to any other variable being studied but we didn\u2019t find any such association.\n\n> 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.\n\nWhen we analyzed the data, we also hypothesized for the above stated bias; a person\u2019s occupation or job reflecting indirectly the socio-economic status can have an influence on diet preferences, but we didn\u2019t obtain such a finding. In our cohort also, outsourced staff had\nhigher non-vegetarianism than staff. Against 70:30 ratio of non-vegetarianism to vegetarianism, for the entre cohort, outsourced staff had 83 percent non-vegetarianism while staff had 66 percent, but sero-positivity amongst non-vegetarians in both the groups had higher sero-positivity of 17.25 and 8.77 percent respectively against sero-positivity of 11.89 and 6.05 percent amongst vegetarian people. We also did a logistic regression and collinearity assessment through VIF score but did not observe any such association and\nhence this was not acting as a confounder. For females, we rather didn\u2019t found this association and only found transport and occupation to be significant, hence to a certain extent it is the crowding environment and occupational exposure which stand as major exposure variables when both the genders are taken into consideration.\n\n> 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.\n\nWe agree with this statement and have stated that outsourced personnel and staff who have to travel and specifically utilize public means of transport are exposed to a higher risk. We regret if this was not clear. The subgroup of people who use public transport reflect a more generalizable sub-population from within the cohort, with all associated risks and confounders. While we attempt a regression to separate a few of them, that is not the primary focus of this work.\n\n> 8) The correlations with blood group don't seem to match what is known from elsewhere\n\nWe are unsure of what the reviewer is matching our data with, but have tried to explain why we consider our results to be broadly concordant. As advised elsewhere, this has not been detailed in the revised manuscript. Data for 7496 individuals was available for their Blood\nGroup type and serological status. Blood Group (BG) distribution amongst total samples collected was similar to national reference based on a recent systematic review. Hence the sample characteristics of our cohort were similar to the national population reference.\nThrough the literature available, it has been observed that \u2018O\u2019 BG type has less risk of getting infected which was observed in our study also. In our study, BG type O was associated with a lower sero-positivity rate, with an OR of 0\u221976 (95 % CI 0\u221964 -0\u221991, p=0\u2219018) vs Non O blood group types with a overall sero-positivity of 7.09 percent which was less than the cohort wide sero-seropositivity. BG type AB and B had higher chances of\ntesting sero-positive is what has been observed by available literature which was corroborated in our findings too. In regard to available literature; BG A has a higher risk of getting infection and this was contrary to our finding where we obtained a favourable OR in\nfavour BG type A albeit it was not significant on statistical testing.\n\n> 9) The statement that \"declining cases may reflect persisting humeral immunity among sub-communities with higher exposure\" is unsupported. What sub-communities?\n\nWe regret the lack of clarity. The wording has now been corrected, it just refers to subgroups of population with high levels of exposure.\n\n> **Reviewer #2 (Public Review):**\n\n> 1) The extrapolation of the study results to the country may not be completely acceptable with the basic difference from the country's urban rural divide and a largely agricultural economy. The female gender is underrepresented in the study cohort, and no children have been included.\n\nWe agree with the reviewer that this is a specific cohort, largely urban. We also agree that a cohort of people utilizing public transport would be better representative and we are following the individuals as the cohort enables to follow them and get further insights. We further agree that the utility of this cohort is not in making general statements about the population,\nbut rather in deriving specific insights for which the cohort is best suited. We enumerate some of them that are present in this manuscript. (See also response to Reviewer #1.)\n\n> 2) The observations regarding corelates of sero-positivity such as diet smoking etc would need specifically designed adequately powered studies to confirm the same. The sample size for the three and six months follow up to conclude stability of the humoral immunity, is small and requires further follow-up of the cohort. The role of migration of labour helping the spread of the pandemic simultaneously to all parts of the country though attractive may not explain lower rates in states like UP and Bihar where maximum migrants moved to.\n\nWe agree that the observations in regard to diet and smoking are only hypothesis generating and need specifically designed studies to confirm the findings. We have also mentioned in the manuscript that associations found between seropositivity and some of the parameters should be confirmed with studies specifically designed for this purpose. We are following up more individuals at three and six months to ascertain the stability of the antibodies. Maximum migrants in the early phase moved to UP and Bihar and it would indeed be expected that seeding would be higher there. While known cases were low for these states, the seropositivity data supports that seeding did occur but may have gone undetected. The ICMR Aug-Sept serosurvey data, for example, shows seropositivity in districts of these states to be higher than those Gujarat or Rajasthan.\n\n> 3) A large chunk of seropositive data set has been removed representing the big cities of Delhi and Bengaluru while correlating Test Positivity Rate citing duration as the reason. However, these cities also had different testing strategies and health infrastructure and hence are important.\n\nWe agree that some data was removed. This was because the sample collection was extended over a longer interval, making point estimates meaningless for some labs, especially CSIR-IGIB which conducted many mini-surveys. For Delhi, only IGIB has been removed and other labs are still kept in the analysis. The graph directionality and trends remain same when analyzed with the excluded data. On keeping Bengaluru data, R square doesn\u2019t change to second decimal place and remains same. When adding back the data from CSIR-IGIB, the R square is 0.32, maintaining the directionality and trend.\n\n> 4) Test positivity rate depends on testing strategy and type of test used; whether RTPCR or the Rapid Antigen Test and the ratio of the two tests was different in different parts of the country.\n\nThis is a very well taken point, but the data was taken as a surrogate from a third party website and the further breakup of positivity rate was not available. It should of course be done ideally with one type of test only but this was not possible. Our larger point is that for a given part of the country TPR went down when seropositivity went up. This is relevant even if different parts of the country used different ratios of the test.\n\n> **Reviewer #3 (Public Review):**\n\n> [...] Weaknesses: While it is a pan-India survey, the population is not quite representative of general population of the country. CSIR labs are mostly in cities, and most of the employees use private transport. So the results cannot be generalized to the country as a whole. Restricting to people using public transport would be a better representation, although it still would not be fully representative.\n\nWe agree with the reviewer that this is a specific cohort, largely urban. We also agree that a cohort of people utilizing public transport would be better representative and we are following the individuals as the cohort enables to follow them and get further insights. We further agree that the utility of this cohort is not in making general statements about the population, but rather in deriving specific insights for which the cohort is best suited. We enumerate some of them that are present in this manuscript. (See also response to Reviewer #1.)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955v2","doi":"10.1101\/2020.12.08.413955"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nIn this incredibly detailed effort, Hulse, Haberkern, Franconville, Turner-Evans, and coauthors painstakingly and patiently reveal the connectivity of central complex neurons within one \"hemibrain\" EM-imaged connectome of a fruit fly. This is best read as one of a series of such detailed papers including Scheffer et al., 2020 (which introduces the dataset) and Li et al., 2020 (which focuses on the mushroom body). \n\nThe authors achieve two major goals. First, they present a full account of all neurons (by type) present in the central complex and the connections between them (including to and from regions outside the central complex). By necessity, this work only examines such connections within a single animal from whose brain the hemibrain volume was imaged. Nonetheless, the relatively conserved morphology of fly neurons (at the scale of which regions they form arbors within) allows the authors to confidently relate their neurons to known examples from genetically labeled lines imaged at the light level. (And in some cases, they are able to show that some neurons with similar morphology can then be further subdivided into different types on the basis of their connectivity). Importantly, the hemibrain dataset contains both sides of the central complex, allowing for a complete analysis. \n\nSecondly, the authors contextualize the observed connectivity patterns within the known functions of the central complex (particularly navigation and sleep\/arousal). Appropriately and importantly, they offer detailed explanations for how the circuitry observed can support these functions. In some cases, particularly in their discussion of the fan body, they show how the connectivity patterns can support multiple variations of models of path integration (and more broadly how its architecture supports vector computation in general). These analyses make their central complex connectome a useful map - there is little doubt that it will inspire many future experiments in the fly community. \n\nThe only limitations of this work are rooted in the nature of the source material: it's only one animal's brain and because it's EM-based there's often no way to know whether a given cell type (if new) is even excitatory or inhibitory (though, notably, the authors take care to note where this is the case and to offer alternate interpretations of the circuit function). Synaptic strength is another relative unknown (not to mention plasticity rules or modulatory influences). For EM-based connectomes, the number of synapses made between two neurons is considered the basis for determining whether or not they are meaningfully connected. However, this precise number can vary as a function of how complete the reconstructions are (generally, as proofreading progresses, more synapses are found). This work improves on prior hemibrain studies by carefully demonstrating that it is possible to set a threshold on the relative fraction of synaptic contributions within a region in order to identify meaningful connections. (That is, they find that as the number of synapses discovered increases, the relative contribution remains relatively constant). \n\nThis is a massive work. There are 75 figures, not including supplements, and numerous region and neuron names to keep track of (not to mention visualize). It is impossible to read in a single sitting. So for the purposes of this public review, I highly recommend to any reader that they first find the region of the paper they're interested in and skip to that to view in side-by-side mode. The \"generally interested\" reader is best served by reading through the Discussion, which has more of the structure-function analyses in it and then referring to the Results as their curiosity warrants. \n\nScheffer et al., 2020 is available here: <https:\/\/elifesciences.org\/articles\/57443#content>\nLi et al., 2020 is available here: <https:\/\/elifesciences.org\/articles\/62576#content>\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955v2","doi":"10.1101\/2020.12.08.413955"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nIt is difficult to overestimate the importance of this paper. The full connectome of the Drosophila central complex is both the beginning and the end of an era. It provides the first comprehensive dataset of arguably the most enigmatic brain region in the insect brain. This endeavor has generated ground truth data for years of functional work on the neural circuits the connectome outlines, and constitutes an unparalleled foundation for exploring the structure function relations in nervous systems in general. This will be of great importance far beyond work on the Drosophila brain, and will have far reaching implications for comparative research on insect brains and likely also smoothen the path toward understanding navigation circuits in vertebrate nervous systems. Based on presented data, the paper develops overarching ideas (at exquisite detail) of how sensory information is transformed into head direction signals, how these signals are used to enable goal direction behavior, how goals are represented, and how internal state can modulate these processes. The connectome enables the authors to base these ideas and their detailed models on actual biological data, where earlier work was forced to indirectly infer or speculate. While significantly going beyond models of central-complex function that existed previously, the authors have to be much credited for incorporating huge amounts of existing knowledge and data into their interpretations, not only work from Drosophila, but also from many other insects. This makes this paper not only an invaluable resource on the connectome of the Drosophila central complex, but also a most comprehensive review on the current state of the art in central-complex research. This unifying approach of the paper clearly marks a reset of central-complex research, essentially providing a starting point of hundreds of new lines of enquiry, probably for decades to come. \n\nGiven the type and amount of data presented, the paper is clearly overwhelming. That said, it also clearly needs to be presented in the way it was done, mostly because no single aspect of the function of this neuropil makes as much sense in isolation as it makes sense when viewed in conjunction of all its other functions. The complexity of the neural circuits discussed is clearly reflected in the enormous scope of the paper. Nevertheless, the authors have done a fantastic job in breaking the circuits and their function down into digestible bits. The manuscript is very systematic in its approach and starts with sensory pathways leading to the CX, covering the clearly delineated head direction circuits and then moving on to the more complex and less understood parts, always maintaining a clear link between structure and function. As function is necessarily based on previous work, including that from other species, the results part is interwoven with interpretation, but this is clearly necessary to keep the text readable. The authors have made considerable efforts to provide additional introductions and summaries whenever needed, almost creating nested papers embedded within the overall paper. \n\nThe figures are equally overwhelming as the text at first sight, but when taking the time to digest each one in detail, they present the data in a rich and clear manner. The figures are often encyclopedic and will serve as reference about the central complex for years. The summary graphs that are presented in regular intervals are welcome resting places for the reader, helping to digest all the detailed information that has preceded or that will follow. \n\nThe analysis performed in the paper is excellent, comprehensive and should set the standard for any future work on this topic. Also, the text is very honest about the limits of the conclusions that can be reached based on this kind of data, which is important in generating realistic and feasible hypotheses for future experiments. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.08.413955v2","doi":"10.1101\/2020.12.08.413955"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nIt is difficult to overestimate the importance of this paper. The full connectome of the Drosophila central complex is both the beginning and the end of an era. It provides the first comprehensive dataset of arguably the most enigmatic brain region in the insect brain. This endeavor has generated ground truth data for years of functional work on the neural circuits the connectome outlines and constitutes an unparalleled foundation for exploring the structure function relations in nervous systems in general. While significantly going beyond models of central-complex function that existed previously, the authors have to be much credited for incorporating huge amounts of existing knowledge and data into their interpretations, not only work from Drosophila, but also from many other insects. This effort makes this paper not only an invaluable resource on the connectome of the Drosophila central complex, but also a most comprehensive review on the current state of the art in central-complex research. This unifying approach of the paper clearly marks a reset of central-complex research, essentially providing a starting point of hundreds of new lines of enquiry, probably for decades to come. \n\n(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.09.11.289058#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.09.11.289058v2","doi":"10.1101\/2020.09.11.289058"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Author Response:**\n\n>Reviewer #1:\n\n>This manuscript reports the results of two timing experiments. The experimental paradigm asks participants to judge the time of target items in an unfilled interval between two landmark stimuli. In experiment 1, there is one item that must be judged. In experiment 2, there are two items to be judged. The basic empirical result is that relative order judgments in experiment 2 are more accurate than one might expect from the absolute timing judgments of experiment 1. A model is presented.\n\n>My overall reaction is that this paper does not present a sufficiently noteworthy empirical result. I can't imagine that there is a cognitive psychologist studying memory who would be surprised by the finding that relative order judgments in the second experiment are more accurate than one might expect from the absolute judgments in experiment 1. On the encoding side, in these really short lists (with no secondary task), there is nothing preventing the participant from noting and encoding the order as the items are presented (not unlike the recursive reminding). On the retrieval side, we've known for a very long time that judgments of serial position use temporal landmarks (see for instance a series of remarkable studies by Hintzman and colleagues circa 1970).\n\n>Methodologically, this paper falls short of the standards one would expect for a cognitive psychology paper. There are basically no statistics or description of the distribution of the effect across participants. Although I'm pretty well-convinced that the basic finding (distributions in experiment 2 are different from experiment 1), I could not begin to guess at an effect size. The model is not seriously evaluated. The bimodal distributions are a large qualitative discrepancy that is not really discussed.\n\n>Although the title of the paper invites us to understand these results as telling us something about episodic memory, the empirical burden of this claim is not carried. Amnesia patients (and animals with hippocampal lesions) show relatively subtle differences in timing tasks. There is no evidence presented here, nor literature review, to convince the reader of this point.\n\nReviewer #1 \n\nWe regret that the reviewer did not focus on the main results of the paper, and limited their remarks to just one analysis comparing the relative order precision to the one predicted from the naive assumption on independent absolute time judgements for each item. This analysis was done to confirm that relative order is quite precisely remembered for short lists that is indeed not surprising, but we still did it in order to get a quantitative estimate of ordering mistakes that we needed for our Bayesian experiments. Another purpose was to filter out the participants that don\u2019t pay attention to the task (a common problem when performing experiments over the internet). \n\nRegarding the title of the paper, we are not aware of similar experiments as ours done with amnesic patients. However we take the reviewer's point that the relation of our experiments to episodic memory as usually understood is not direct, so we took the word 'episodic' off the title in the revised version. We also added statistical analysis of the results.\n\n>Reviewer #2:\n\n>In this manuscript, the authors set out to measure participant's decisions about when an item occurred in a short list of 3 or 4 items, where the first and last items were always at the beginning and end, respectively. They report two behavioral studies that examine time judgments to items in the intermediate positions. They show that time judgments (when did you see X item using a continuous line scale) are always a little off but, more importantly, they tend to be anchored to other items presented. The results are interesting and add to our knowledge of the representation of time in the brain mainly by introducing a new paradigm with which to study time. Within the broader context of research on timing capacities, it should not be surprising that participants do not have a continuous representation of time that lasts beyond traditional time interval training of a few hundred milliseconds to a few seconds. Furthermore, research has also shown that 'events' that require attentional resources do morph our perception and memory for time. So while the paradigm is worth expanding on, the behavioral results are not surprising given this past literature. I do feel however that this work is an important first step in developing a more firm model of memory for time.\n\nReviewer #2 \n\nIndeed, as mentioned above in response to Reviewer #1, we are not surprised that subjects don't remember well the absolute presentation time, especially when several items were involved. Exactly what they remember is the main point of this study, and the model is quite crucial in understanding what we believe is our novel result about how ordinal and absolute time representations interact in memory. The reviewer did not seem to appreciate this; rather they re-formulated our results as time judgments (when did you see X item using a continuous line scale) being 'anchored' to other items presented. We are not sure what this exactly means, probably that on average the difference between reported times of different items stayed almost constant for each presentation conditions. However our study not only presented this result but showed how it follows from the Bayesian theory. \n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816v1","doi":"10.1101\/2021.01.22.427816"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nThe authors investigated pupillary response looking at the changes corresponding to perceptual events (spontaneous or physical changes) and contrasting them with requirements of over reporting (changes were reported or ignored). They demonstrate that the former is associated with a rapid constriction and re-dilation, whereas the latter shows an opposite effect with dilation being followed by re-constriction. The particular strength of the work is in no-report conditions using on OKN-based inference about timing of perceptual events that allowed for this dissociation to be observed, whereas manual report conditions allowed for a direct comparison with prior work. The analysis and control experiments are very thorough showing that reported results are unlikely to be explained other factors such as saccades or blinks. \n\nThe study makes a significant contribution but proposing a no-report paradigm for identifying perceptual events that should work for any multistable display. The fairly rapid pupil constriction event could provide an easy to detect and temporally reliable marker of perceptual switches, expanding ways the multistability data is collected. The same approach could also be useful for no-report studies of visual awareness in general. \n\nThe ability to decompose pupillary response into two components - perception and over manual response - will also be useful for studying neural correlates of spontaneous perceptual switches, as it could help to better understand switch-time activity in various frontal and parietal regions. Here, also some regions are associated with active response, whereas other with perception, distinction that could be potentially better understood based on the idea that only the former involves noradrenaline-affected processing. \nMy main worry methodologically is the under and overestimation of mean switch rate via OKN (figure 1C). OKN estimates are all within .4-.8 range, whereas for self-report rates differ from 0.2 to over 1. Further analysis would be helpful. I think it would be helpful if the authors elaborated on what kind of switches went unreported (or, conversely, what kind of events led to false alarms): switches before very short dominance phases (could be to fast to report via key presses), to return transitions, etc. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816v1","doi":"10.1101\/2021.01.22.427816"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nPupillometry is an increasingly accessible tool for the non-invasive readout of brain activity. However, our understanding of pupil-control circuits and of the relationship between changes in pupil size and perception, cognition or action, is far from complete. Therefore, any measurements that further this understanding are of great interest to a wide audience in the field of psychology and neurobiology. \n\nThis study used pupillometry to explore the neural processing that underlie perception and dissociate those from action-related neural processing. The authors use a novel and comprehensive task design, centered on binocular rivlary, that is likely to find wider use among researchers studying the neural processes that underlie perception and action. They used a non-invasive method (pupillometry) to disscociate putative processes and circuits that might drive perceptual switching. They found changes in pupil size that are reliably different depending on the task: for example - between the conditions that require reporting a perceptual switch versus not reporting it and between rivalrous and explicit changes in the visual stimulus. \n\nSuch approaches can be very useful in deciphering which of the myriad factors that can affect pupil size are in fact active under specific, controlled conditions and thus provide a basis for guided, direct measurements of these specific brain regions. \n\nOverall, this study is well-conceived and executed. However, I have some questions and concerns about the analyses and conclusions made from the results shown. In general, I would encourage the authors to try and include more of what we do know about neuromodulation and the cortical control of pupil pathways to frame the hypothesis and interpret the results. Further, it is unclear to me whether the constriction\/dilation dissociation is tenable with the presented data and analyses. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816v1","doi":"10.1101\/2021.01.22.427816"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nBrascamp and colleagues address pupil-size changes around perceptual switches in perceptual multistability. Several previous studies have found pupil dilation around or after the switch and some have found pupil constriction, though the latter was typically less robust. Moreover, while most previous studies included some controls for the effect of reporting and for the physical stimulus change, to my knowledge, so far, no study has fully crossed the factors report\/no-report and endogenous\/exogeneous switch. In the present study, this gap is filled using a binocular-rivalry stimulus and an OKN-based no-report paradigm. This allows the authors to isolate the constriction component from the dilation component and interestingly they find the constriction more robustly tied to the perceptual switch, while the dilation component is mostly related to the response. Experiments are soundly conducted and analysed and results are interpreted with appropriate care. Since the results challenge frequent interpretations as to why perceptual switches in multistability may cause pupil-size changes, the paper is of high relevance to the fields of pupillometry and multistability, but also to other areas where pupillometry is used as index of perceptual and cognitive processes. I only have some minor questions and requests for clarification with regard to result presentation and interpretation. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.22.427816v1","doi":"10.1101\/2021.01.22.427816"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nPupillometry is an increasingly accessible tool for the non-invasive readout of brain activity. However, our understanding of pupil-control circuits and of the relationship between changes in pupil size and perception, cognition or action is incomplete. Therefore, any measurements that further this understanding are of great interest to a wide audience in psychology and neurobiology. This study used pupillometry to explore the neural processing underlying perception and dissociate them from action-related neural processing. Results reveal changes in pupil size that are reliably different depending on the task. Such approaches can be very useful in deciphering which of the myriad factors that can affect pupil size are active under specific, controlled conditions. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer 3 agreed to share their names with the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124v1","doi":"10.1101\/2021.01.10.426124"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #4 (Public Review):** \n\nUsing a transgenic line of Platynereis, in which GFP is expressed under the control of cis-regulatory elements for r-opsin, the study isolates r-opsin expressing cells from the head (eye photoreceptors) and trunk region (a population of segmentally repeated r-opsin expressing cells associated with the parapodia) by FACS. Subsequent RNA-Seq establishes that both populations of cells express genes for all components of the rhabdomeric phototransduction cascade, while the population of trunk sensory cells additionally expresses genes encoding proteins involved in mechanosensation. Using heterologous expression in a mammalian cell line, it is shown that the Platynereis r-opsin responds to blue light via coupling to G\u03b1q suggesting that it mediates photoresponses via a canonical rhabdomeric phototransduction cascade. Transcriptomic analysis of an r-opsin mutant created by TALEN mediated gene editing then reveals that expression levels of the mechanosensory Atp2b channel are modulated by protracted exposure to blue light, a response abolished in the mutant. Behavioral analysis further suggests that undulatory movements of the worms are equally altered under these illumination conditions. Taken together this suggests that the r-opsin expressing trunk sensory cells act as both photo- and mechanoreceptors and that their mechanosensory properties are modulated in response to light. \nIn combining the transcriptomic analysis of cell types with experimental studies of gene function and behavioral analyses, this study provides exciting new insights into the evolution of sensory cells. Several prior studies have found co-expression of photosensory and mechanosensory proteins in sensory cells of various bilaterians, and comparative studies suggested that photo- and mechanosensory cells may share a common evolutionary origin. However, the current study goes far beyond these findings in establishing a direct functional link between photo-and mechanosensation in a population of sensory cells suggesting that these sensory cells function as multimodal cells and that their mechanosensory properties are altered in response to light. Furthermore, the behavioral data (based on a novel machine-learning based tool of analysing the animals' movement) suggest that these cells have a behaviorally relevant function. Because r-opsin was found to be expressed in mechanoreceptors not only in lophotrochozoans (including Platynereis) but also in ecdysozoans and vertebrates (although functional studies are lacking here) and r-opsins belong to a large family of opsins, almost all of which are responsive to light, the present study suggests that r-opsins may have an ancestral bilaterian role in modulating mechanosensory function in response to light (in addition to their purely photosensory role in the photoreceptors of the eyes). Light-independent functions of r-opsin as recently revealed in Drosophila may, thus, be secondarily derived. \n\nThe study is very carefully conducted and well presented. The only minor flaw is that in its present form, the discussion of the evolutionary implications of the finding lacks in clarity and specificity. The authors here often refer ambiguously to an \"ancient\" or \"ancestral\" role of r-opsins without specifying the lineage referred to (ancestral for lophotrochozoans? bilaterians? eumetazoans? metazoans?). The discussion should, therefore be revised with an explicit phylogenetic framework in mind. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124v1","doi":"10.1101\/2021.01.10.426124"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nOpsin proteins are ancient light-sensitive molecules found in photoreceptor cells throughout the animal kingdom. Recent discoveries including those made in the current paper have revealed that besides r-opsins, some classes of photoreceptor cell also express genes that are found in mechanosensory cells, and that r-opsins have both light-dependent and light-independent effects on mechanical force transduction or motion. A question remains as to whether or not: 1) a protosensory cell of animals existed which contained both photoreceptor and mechanoreceptor-like features and, 2) whether the original function of opsin included light-dependent mechanosensory features? The authors consider three competing hypotheses for the cellular evolution of photoreceptor and mechanosensory function. Two of the hypotheses envision either photo- or mechanosensory function for opsins evolving first, the third imagines them evolving simultaneously. The authors note that the majority of what we know about rhabdomeric opsins comes from studying the eye photoreceptors of the fruit fly, Drosophila melanogaster. But might this kind of photoreceptor have functions that are derived compared to the ancestral photoreceptor cell? To investigate this question, the authors turn to the non-model system, Platynereis dumerilii, which has both head and non-head photoreceptors. Here the authors use 1) a fluorescent cell sorting method to perform RNA profiling of eye and trunk photoreceptor cells of a mutant marine worm and find evidence of co-expression of photo- and mechanosensory genes in photoreceptor cells. They also compare the genes that are expressed in Platyneris photoreceptors with genes expressed in Drosophila JO (hearing organ in flies), Zebrafish lateral lines and mouse IEH (inner ear hair) cells, and again they find some commonly-expressed genes. 2) The authors use cell culture to express the opsin, demonstrate that it interacts with G-alphaq, and that it's peak sensitivity is in the blue range. 3) They use in situ hybridization to validate the RNA-seq and detect select enriched transcripts in the photoreceptor tissues. 4) They use a new method, which should be widely useful to other researchers, to detect undulation behavior of the opsin mutant vs. wildtype worms and show that the mutant worm behavior is perturbed in altered light cycles. Taken together, the authors suggest that an ancient light-dependent function of opsin was linked to mechanosensation and that light-independent mechanosensory functions of opsins evolved secondarily. The interpretation is somewhat reasonable given the available data but does not yet entirely rule out other possibilities (see below). \n\nThis paper is a tour-de-force and a really impressive collection of experiments which examines the function of r-opsin in Platyneris. There's lots of innovation here from the use of fluorescent cell sorting and cell-specific RNA-Seq on a non-model system to the deep-learning based approach to examining behavior. Overall, the authors' interpretation of their data seems reasonable however I do believe a even stronger case could be made that what we are talking about is shared ancestry vs. recent recruitment if the authors made phylogenetic trees of the numerous TRE genes that are enriched between Drosophila JO and mouse IEH cells. If a significant number of these genes were true orthologs vs. paralogs across all three species then this would provide stronger evidence of an ancient light-dependent mechanosensory function for r-opsin. GO enrichment terms, while intriguing and suggestive, don't go far enough into the weeds. Also, I think the estimate of there being only 12 genes involved in making a photoreceptor cell able to detect light is probably an underestimate, as this ignores, for example, the understudied molecular machinery required for chromophore metabolism and transport. At the very least, the work should help inspire vigorous debate between vision and auditory neuroscience communities (which do not usually converse with one another) to more carefully consider the ways in which their systems overlap and why. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124v1","doi":"10.1101\/2021.01.10.426124"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nRhabdomeric Opsins (r-Opsins) are well known for their role in photon detection by photosensory cells which are commonly found in eyes. However, r-Opsin expression has also been detected in non-photosensory cells (e.g., mechanosensors), but their function(s) in these other sensory cells is less well understood. To explore the function of r-Opsins outside the context of an eye\/head (non-cephalic function) as well as to investigate the potential evolutionary path by which sensory systems that rely on r-Opsins have evolved, Revilla-i-Domingo et al. have investigated gene expression in two distinct subsets of r-Opsin expressing cells in the marine bristle worm Platynereis dumerilii : EP (eye photoreceptor) and TRE (trunk r-opsin1 expressing) cells. The authors also generate two Pdu-r-Opsin1 mutant strains in order to investigate how the loss of r-Opsin function affects gene expression and behavior. \n\nThe question of what role r-Opsins play outside of photoreceptors is an interesting one that remains poorly understood. In this manuscript, the authors demonstrate a powerful protocol for FACS sorting and sequencing different cell populations from an important evolutionary model organism. \n\nThe transcriptomic analysis presented here demonstrates that both the cephalic EP cells and the non-cephalic TRE cells express components of the photosensory transduction pathway. This observation, together with heterologous cell expression data presented demonstrating sensitivity of Pdu-r-Opsin1 to blue light, suggests that both EP and TRE cells are likely to be light sensitive. The authors also suggest that they observe \"mechanosensory signatures\" in the transcriptomes, which, together with the analysis of undulatory movements in headless animals, lead them to suggst that r-Opsin in TRE cells functions as an evolutionarily conserved light-dependent modulator of mechanosensation, a conclusion that is not well-supported by the data presented. \n\nOverall, many of the conclusions drawn from the transcriptome data are inferential and based on weak evidence. Key limitations are listed below: \n\n1) The apparent overlap between the phototransduction and mechanosensory systems has already been shown (in Drosophila for instance) and the current work adds limited information to this story, and what is added is weakened by the absence of functional and physiological analyses. This is particularly true for supporting the claims of mechanosensory signatures in these cells. For example, genes whose expression is suggested in the text as being indicative of a mechanosensory function (glass and waterwitch) are, in fact, expressed in multiple sensory cell types. Glass (gl) is a transcription factor best known for regulating the expression of phototransduction proteins in photoreceptors. The function of waterwitch (wtrw) is not fully understood, but it is broadly expressed in sensory cells in Drosophila. It would be more compelling if mechanotransduction channels like Piezo and NompC were expressed in the TREs, but there is no mention of this. \n\n2) The suggestion that the TRE cells share similarity with the mechanosensitive mammalian inner ear is provocative, but lacks strong support. For instance, physiological characterization of the response properties of these sensory cells or identification of anatomical similarities analogous to the stereocilia upon which hair cell mechanosensitivity is based would greatly increase plausibility of this claim. Particularly for a species that diverged from mice and flies many hundreds of millions of years ago, speculation based largely on transcriptome analysis is risky. Careful validation is required as identified genes might not share a conserved function with their assigned orthologs in mice and Drosophila. \n\n3) The current analysis lacks sufficient power to make compelling claims with regard to potential ancestral protosensory cells. The investigators are examining a single species of marine worm and doing so without detailed anatomical and functional studies of the r-Opsin-expressing cells in the worm. \n\n4) The behavioral experiments require more functional data to interpret unambiguously. The data indicate that r-opsin1 is required for light to surpress the undulation of decapitated worms. Does this mean that the TREs are photosensors whose activity inhibits locomotion or that the TREs are light-sensitive mechanosensors ? \n\n5) It is assumed that the TREs constitute a homogenous cell population, but this is not demonstrated. This means that the TREs could be a mixed population (for example, distinct sets of photosensors and mechanosensors) and some of the TRE-expressed genes identified could be expressed in different specific subset of TREs. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124v1","doi":"10.1101\/2021.01.10.426124"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nStrengths and Weaknesses. The authors did quite a lot to establish gene expression and function of the annelid's trunk cells and compare them to photoreceptors of the annelid's eye. They isolated the cells with FACS and characterized gene expression in detail, they knocked down r-opsin with TALEN in the trunk and found a significant difference in a crawling response, and they express the opsin in cell culture to confirm wavelength and G-protein sensitivity. As a potential link between light sensitivity and mechano-sensitivity, they report r-opsin function and light intensity influence expression of atp2b2, a gene that modulates neuronal sensitivity in other organisms. Wavelength and G-protein activation data are valuable because I can think of few or no other organisms in the entire group of lophotrochozoan animals, where this level of experimental manipulation could be done. In short, a strength of this manuscript is the detailed characterization of the trunk receptor cells, which express r-opsins. The authors have brought much evidence to the claim that these TRE cells have both light and mechano-sensitive gene expression and function. Based on these findings in an annelid worm, I believe the paper is a significant advance, and of interest to a broad audience by adding to a growing set of discoveries of similar hybrid sensory cells. \n\nIf a hybrid mechano\/photo-receptor is indeed an ancient cell type in bilaterians, this would bring many evolutionary implications for sensory biology. However, in these evolutionary interpretations is where I find a weakness of the manuscript. Namely, with only a handful of species shown thus far to have the hybrid cell type - and many differences in detail about these cell types in different organisms - we can not yet make firm conclusions about whether the multi-functional cells were ancestral. I believe other interpretations are equally valid (and still interesting) and should be given more consideration. Namely, it seems possible that photo- and mechan- sensory processes \"joined forces\" (e.g. through separate co-option events) in new cell-types, multiple times during evolution. The current manuscript loosely indicates ancestral multi-functionality is more parsimonious. However, no detail is given about that. I suppose the authors mean a single origin of hybrid cell types requires fewer evolutionary transitions than multiple origins. However, such a parsimony count does not count the transitions requiring loss of phototransduction in mouse hearing and do not count transitions to loss of mechanosensitivity in eye photoreceptor cells. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.10.426124v1","doi":"10.1101\/2021.01.10.426124"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nThis manuscript presents an investigation of receptors in the trunk of Platynereis annelids that express genes involved in both photoreception (e.g. r-Opsin) and mechanosensation. This is particularly interesting in light of other work in model organisms like flies that uncovered broadly similar results. The authors compare gene expression of fly Johnston Organ cells and mouse hearing cells to the worm receptors. Because Platynereis is distantly related to flies and mice, the authors suggest this \"hybrid\" sensory receptor could be very old and homologous across many animals. The question of what role r-Opsins play outside of photoreceptors is an interesting one that remains poorly understood. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #4 agreed to share their names with the authors.*)"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138v4","doi":"10.1101\/2020.12.02.408138"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This rebuttal was posted by the corresponding author to *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Reply to the reviewers\n\nWe are grateful to the editors at Review Commons and to the reviewers for their thoughtful attention to our manuscript. Our work presents data showing that deletion of the apoptosis regulator *Mcl-1* in CNS stem cells that give rise to neurons and glia resulted in specific degeneration of the white matter, beginning after postnatal day 7 (P7). Cellular analysis shows that oligodendrocytes were depleted while astrocytes persisted. Co-deletion of apoptosis effectors *Bax* or *Bak* rescued different aspects of the *Mcl-1* deletion phenotype, confirming the role of apoptosis. Based on these observations, we conclude that oligodendrocytes require MCL-1 to prevent spontaneous apoptosis, and that MCL-1 depletion results in leukodystrophy, which resembles severe cases of the human disorder Vanishing White Matter Disease (VWMD). We further suggest that MCL-1 deficiency, caused by the eIF2B mutations of VWMD, may play a critical role in VWMD pathogenesis.\n\nThe reviewers questioned the similarity of the *Mcl-1* deletion phenotype to VWMD and were not convinced that MCL-1 deficiency is integral to VWMD. Based on reviewer feedback, we concede that a firm link to VWMD is not supported by the available data. We consider, however, that our findings that MCL-1 is required for oligodendrocyte survival and white matter stability remain highly significant. Accordingly, we propose to revise the work as suggested by Reviewer 1 to highlight the insight our data provide as to apoptosis regulation in glia and its importance for brain development, and to revise the title, as suggested by Reviewer 3, to remove the specific reference to VWMD.\n\nIn the revision, we will make clear that the comparison to specific leukodystrophies is hypothetical and will require extensive follow-up experiments that are suggested by the findings of this work, as described in the reviews. Revising our work by removing the assertion that our data strongly implicate MCL-1 in VWMD pathogenesis will address the main reviewer concern, strengthen the logical flow, and highlight the potential for MCL-1 to be broadly relevant to white matter pathology. The significance of our findings that oligodendrocytes depend on MCL-1 protein to prevent their spontaneous apoptosis, and that MCL-1 deficiency produces white matter degeneration, will not be altered by these changes. Our data will continue to show that MCL-1 dependence is a physiologic vulnerability of oligodendrocytes that sets them apart from astrocytes and neurons and that this vulnerability is sufficient to cause white matter-specific brain degeneration when MCL-1 expression is blocked.\n\nThe other issues raised by the reviewers are all tractable and can be addressed with new experiments that we can complete in a short time-frame, such as studies of retinal pathology and addition immunohistochemistry studies, or with changes to the text. We consider that with these revisions, the manuscript will be an important contribution to understanding glial biology and the pathogenesis of white matter-specific disorders. We describe in detail below our responses to reviewer feedback and planned changes to the manuscript.\n\n*Reviewer comments are in italics *and our responses are in plain text.\n\n*Reviewer #1 (Evidence, reproducibility and clarity (Required)):**\n \n \\*\\*Comments\\*\\*\n *\n *1. MCL-1 promotes the survival of different cell lineages through its ability to inhibit the pro-apoptotic proteins BAK and BAX, the main effectors of cell death in mammalian cells. By depleting brain cells of MCL-1, apoptosis is promoted in these cells, as confirmed by histopathology of the mouse brain. This is, however, a nonspecific process. Depletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue and general knock-out is known to cause embryonic lethality. So, it is no surprise that knock out of MCL-1 in brain cells leads to a brain disease.*\n\nWhile we acknowledge many important points in this review, this first point is based on a premise that is inaccurate. Based on published data, we respectfully disagree with the statement that *\u201cDepletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue*\u201d. Most cells do not require an anti-apoptotic protein to prevent spontaneous apoptosis; cells that depend on anti-apoptotic proteins are specifically referred to as \u201cprimed for apoptosis\u201d (*1-5*). Our conditional deletion genotypes ablated *Mcl-1* in neurons of the forebrain and cerebellum and in all subtypes of glial cells. The loss of oligodendrocytes in our *Mcl-1*-deleted mice shows that a specific subset of white matter cells in the postnatal brain require MCL-1. Together with the increase in apoptosis and the rescues by co-deletion of Bax or Bak, these data demonstrate that cells within the oligodendrocyte lineage are primed for apoptosis in a manner that is restricted by MCL-1. In contrast, we have shown in published data that we cite in this manuscript that conditional deletion of *Mcl-1* cerebellar granule neurons, the largest neuronal population in the brain, does not cause apoptosis (*6*); these data provide direct evidence that large populations of cells in the brain do not depend on MCL-1. We therefore disagree with the characterization of the brain-specific *Mcl-1* deletion phenotype as \u201cnon-specific\u201d.\n\n\n\n * The white matter disease is interpreted as similar to VWM; VWM is specifically investigated and MCL-1 is found to be decreased in VWM brain tissue. The decrease is most likely nonspecific. Decrease in MCL-1 is most likely part of a general mechanism of degeneration of brain tissue or white matter. That is a different but also important conclusion. It is essential that other progressive leukodystrophies and acquired brain diseases with tissue degeneration, such as encephalitis, are investigated as well to see whether MCL-1 is also decreased in these disorders. If so, the MCL-1 decrease in white matter disease and other brain degenerative disease should be described as a final common pathway rather than specifically applicable to VWM.*\n\nWe agree that MCL-1 is likely to be a final common point in multiple disease processes that affect white matter. As described in our response to point 3 below, we are persuaded by the reviewers that the proposed similarity of the *Mcl-1* deletion phenotype and VWMD is not sufficiently supported by the available evidence. We will revise the text to make clear that we consider that impaired MCL-1 is \u201clikely part of a general mechanism of degeneration of\u2026 white matter\u201d.\n\n\n\n * Adding to point 2 is the fact that the pathology of the brain-specific MCL-1 knock-out mouse does not resemble the pathology of VWM at all. The central features of VWM are abnormal astrocyte morphology with astrocytes having a few stunted processes, lack of reactive astrogliosis, lack of microgliosis, increase in number of oligodendrocytes and presence of foamy oligodendrocytes. The increase in oligodendrocytes in VWM may be such that the high cellularity leads to diffusion restriction on MRI. Bergmann glia are typically ectopic, but not reduced in number. By contrast, the brain-specific MCL-1 knock-out mouse is characterized by decreased numbers of oligodendrocytes, increased numbers of microglia, reactive astrogliosis, decreased numbers of Bergmann glia and ectopic granule cells. No morphological abnormalities of oligodendrocytes and astrocytes are observed. So, histopathologically the only shared feature is preferential involvement of the brain white matter.*\n\nWe are persuaded by the reviewers that our assertion of a high degree of similarity between the *Mcl-1* deletion phenotype and VWMD was not adequately supported by our available data. In the revision, we will state that a role for MCL-1 deficiency in VWMD pathogenesis is hypothetical, and that additional studies beyond the scope of this project will be needed to test this hypothesis. However, we reassert that the white matter specificity of the *Mcl-1*-deletion phenotype is important.\n\nThe reviewer accurately characterizes the pathology of the *Mcl-1* deletion phenotype and notes \u201cthe preferential involvement of the white matter\u201d. We consider that the preferential involvement of white matter, and of oligodendrocytes within the white matter are highly significant. We will revise the work to focus on the *Mcl-1* deletion phenotype, the white matter specificity, and the potential relevance to diverse white matter-specific disease.\n\nWhile we concede that more data would be needed to firmly connect MCL-1 to VWMD, we do not agree that the *Mcl-1* phenotype \u201c*does not resemble the pathology of VWM at all\u201d. *There is a diversity of published observations of pathology in VWMD and not all published reports support the descriptions in the reviewer comment. This diversity of findings is highly relevant to our work. For example, while autopsy studies of humans with end stage VWMD show lack of microgliosis (*7*), studies of mice with a mutation known to cause VWMD in humans, that clearly recapitulate VWMD, show robust microgliosis earlier in the disease process (*8*). These different observations raise the possibility that microgliosis occurs during the period of active neurodegeneration or at least that in murine brain, the VWMD process activates a microglial reaction. Either interpretation would support a likeness between *Mcl-1*-deleted mice and VWMD mouse models. Another study of cerebellar pathology in twin human fetuses with characteristic VWMD mutations showed complete absence of Bergmann glia (*9*). We propose in the revision to address the reviewer\u2019s concerns by presenting the diversity of perspectives on microglial reaction and Bergmann glial changes in VWMD, including all of the citations above.\n\n\n\n * The clarity of the work would benefit from a different approach to introduce the study. It would help the reader to know that (1) gray matter cell specific Mcl-1 deletion in mice did not cause apoptosis and (2) apoptosis may have different effector proteins. This important information is now in the discussion. The switch to another cell type in the brain (hGFAP+ cells) would be logical and the significance of the work may improve. When approaching the topic from the field of leukodystrophies one would not necessarily think of deleting the Mcl-1 gene, especially as this gene is not associated with any known leukodystrophy and tends to associate with preneoplastic and neoplastic disease.*\n\nWe appreciate these suggestions, which we agree will enhance the logical flow and the significance, in line with our response to point 3. We will revise the Introduction as suggested.\n\n\n\n * The authors claim that the ISR is activated in VWM, which means that eIF2\u03b1 phosphorylation levels are increased, general protein synthesis is decreased and a transcription pathway is regulated by ATF4 and other factors. However, this is not what is seen in VWM. Increased eIF2\u03b1 phosphorylation and reduced general protein synthesis are not observed in VWM; strikingly, the level of eIF2\u03b1 phosphorylation is reduced, general protein synthesis appears at a normal rate, and only the ATF4-regulated transcriptome is continuously expressed in VWM astrocytes. *\n\nThis point is not well-settled, as published studies show that the ISR is activated in VWMD despite decreased eIF2\u03b1 phosphorylation (*10, 11*). Published scRNA-seq studies of mice with VWMD mutations moreover, show that the ISR transcriptome is activated in oligodendrocytes, as well as neurons, endothelial cells and microglia (*8*). We will address this concern in the revision by citing these published reports that show both decreased eIF2\u03b1 phosphorylation and lines of evidence that support ISR activation.\n\n*Fritsh et al. show that MCL-1 protein synthesis is reduced by increased eIF2\u03b1 phosphorylation due to reduced translation rates at the Mcl-1 mRNA and not due to differences in Mcl-1 mRNA levels. *\n\nWe agree with this interpretation of Fritsh et al, which is fully compatible with our proposed mechanism. We suggest that ISR activation in VWMD decreases translation of Mcl-1 mRNA, leading to reduced MCL-1 protein expression. MCL-1 protein is rapidly degraded and may therefore be a more sensitive detector of impaired translation than other readouts. We currently cite published work documenting altered translation in VWMD in the manuscript and in the revision will add the reference Moon et al, which is directly on point (*11*).\n\n*One would a priori not expect to find altered MCL-1 synthesis rates in the mildly affected VWM mouse model Eif2B5R132H\/R132H. *\n\nThe model does not show reduced global translation under normal conditions, but rather hypo-activity of eIF2B affects the translation of specific mRNAs (*12*). We will make this point clear in the revision.\n\n*Actually, ISR deregulation has not been reported in the Eif2B5R132H\/R132H VWM mouse model. The authors need to rephrase this part of their study taking this information into account, when explaining their experiments and interpreting their results. *\n\nConsistent with the data that the ISR is activated in VWMD, mice show ATF4 up-regulation and other evidence of ISR activation (*13*) and impaired responses to physiologic stress (*14, 15*). In the revision, we will add these citations. To address the reviewer concerns, we will state in the revision that ISR activation is one of many potential mechanisms of reduced MCL-1 expression.\n\n*The authors now imply that their study adds mechanistic insight into the VWM field and that is not the case.*\n\nAs we describe in response to point 3, we will acknowledge in the revision that the assertion that MCL-1 deficiency causes VWMD is hypothetical.\n\n*In addition, Figure 7C shows differences in actin signal rather than MCL-1 signal, suggesting that transfer of the actin protein from the gel to the blot was not optimal for the middle lanes. MCL-1 protein may thus not be reduced in these samples from Eif2B5R132H\/R132H VWM mice.*\n\nWe stand by our Western blot data that show that MCL-1 levels are lower in the Eif2B5R132H\/R132H VWM mouse model, coincident with the onset of symptoms. The Western blot shown is a representative image that includes 3 biological replicates for each condition and of a total of 12 mice. The quantification demonstrates the reproducibility of the finding.\n\n\n\n * Can the authors show in which cell type was apoptosis found (lines 315-316)? Their study uses the hGFAP - Cre mouse model to generate conditional Mcl-1 knock-out mice. The original paper by Zhuo et al. describing the hGFAP - promoter mouse model suggests that Mcl-1 expression is also affected in neurons and ependymal cells. The authors can investigate this further to assess which cell types (1) are sensitive to apoptosis by Mcl-1 deletion and (2) depend on Bax and Bak.*\n\nApoptosis may occur at different times in different cell populations, and asynchronous apoptosis can be difficult to detect at any point in time, which can complicate the suggested studies. Despite significant effort, we have not been able to co-localize any markers with dying cells in our model.\n\nTo address the question of neuronal involvement, the revised manuscript will refer to prior published studies (*16-18*) which show that *Mcl-1* deletion affects forebrain neural progenitors. In this context, we will discuss that our *Mcl-1* deletion studies show that significant neural progenitor populations survive prenatal *Mcl-1 *deletion and generate appropriate cortical and hippocampal architecture in *Mcl-1-*deleted mice at P7, prior to the onset of white matter degeneration.\n\nTo identify involved glial cells, we quantified the cells that were depleted or persisted in the Mcl-1 deleted brain. These studies identified oligodendrocytes and Bergmann glial as cell types depleted during P7-P15, when postnatal degeneration occurs in *Mcl-1* deleted mice. In contrast, astrocytes persisted, indicating that astrocytes are not MCL-1-dependent. In the review, we will add new data quantifiying the immature, PDGFRA-expression subset of oligodendrocytes, which will increase the specification of which cells are depleted by *Mcl-1* deletion.\n\nWe share the reviewer\u2019s interest in the question of which subsets of *Mcl-1* dependent cells are rescued by co-deletion of *Bax* or *Bak*. As known markers may not be sufficient to distinguish these subsets, we consider that scRNA-seq studies are an ideal approach to identify these subsets and their specific gene expression patterns. However, these studies are outside the scope of the present work, which establishes that specific white matter cells depend on *Mcl-1*.\n\n\n\n * Heterozygous deletion of Bak greatly reduces the number of Bak-expressing cells (Fig. 3C, line, 331-333). Authors need to explain this remarkable finding. *\n\nAs we state in the text, the reduced Bak expression in the heterozygous Bak +\/- mice is consistent with a gene dosage effect, which has been observed for other genes.\n\n*Please provide raw IHC data. *\n\nOur IHC data is \u201craw\u201d in the sense of unaltered. We are happy to include a supplementary figure with additional low power and high-power images of BAK staining.\n\n*Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful. *\n\nTo address this point, we have successfully performed double labeling with antibodies to BAK and with antibodies to the oligodendrocyte marker SOX10 and the astrocyte marker GFAP. We will add these images to the revision. These images show that BAK+ cells include oligodendrocytes and astrocytes. The position and morphology of the BAK+ cells show that they are not neurons.\n\n*In addition, what does the Western blot signal for the BAX protein represent in Bax homozygous knock out mice (Fig. 3C)? *\n\nWe will add text stating that the small residual BAX protein detected in the conditional Bax-deleted mice can be attributed to BAX expression in cells outside the *Gfap *lineage, including endothelial cells, vascular fibroblasts, and microglia.\n\n*Can the percentage of BAX+ cells in Mcl-1\/BaxdKO corpus callosum be determined, similarly as was done for BAK? Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful here as well. The legend of Fig. 3D does not state what staining is shown (H&E?).*\n\nWe were not able to label BAX protein in individual cells using immunohistochemistry. In contrast, BAK immunohistochemistry worked well, allowing us to analyze the cellular distribution of BAK protein. We will revise the legend in 3D to state the staining is H&E.\n\n\n\n * What explains the strong GFAP expression in processes of Mcl-1 KO astrocytes? Are these cells refractory to apoptosis or to hGFAP-driven Cre expression and recombination? Do they lack BAK or BAX or other apopotic-regulating protein? Or do specific factors compensate for the loss of MCL-1?*\n\nAs we discuss in our response to point 1 above, not all cells require MCL-1 to prevent spontaneous apoptosis. The persistence of GFAP+ astrocytes in *Mcl-1*-deleted mice shows that astrocytes do not require MCL-1 to maintain their survival. These data do not mean that these astrocytes are refractory to apoptosis, but rather they are not primed for apoptosis in a way that is critically restricted by MCL-1. We will add a discussion of these implications to the revision.\n\n\n\n * Which developing symptoms do the authors refer to in line 468? Please specify and introduce appropriate references.*\n\nWe will add a description of symptoms to the revision.\n\n\n\n * The definition of leukodystrophies given in the paper is outdated. Leukodystrophies are not invariably progressive and fatal disorders. For more recent definition of leukodystrophies see Vanderver et al., Case definition and classification of leukodystrophies and leukoencephalopathies, Mol Genet Metab 2015, and van der Knaap et al., Leukodystrophies a proposed classification system based on pathology, Acta Neuropathol 2017.*\n\nWe appreciate this advice. We will revise the Introduction accordingly and cite the recommended work.\n\n\n\n * It is not correct that there is no specific targeted therapy clinically implemented to arrest progression of the disease in any leukodystrophy. Perhaps hematopoietic stem cell transplantation is not specific targeted, although curative if applied in time in adrenoleukodystrophy and metachromatic leukodystrophy, but certainly genetically engineered autologous hematopoietic stem cells would qualify the definition. In any case, the suggestion that no leukodystrophy is treatable is not correct.*\n\nWe appreciate this correction. We will revise the text to provide a more detailed description of treatment options while underscoring the need for mechanistic insight. \n \n Reviewer #2 (Evidence, reproducibility and clarity (Required)):\n \n *In this manuscript, the authors characterize the phenotype associated with brain-specific deletion of the mcl-1 gene in mice as a model for vanishing white matter-like disease in humans. Unfortunately, the gfap gene is expressed in many cell types during development which are outside of the intended cell type for this study, so functional data presented from the mutant mice is open to interpretation. The authors have not ruled out other interpretations of their results. The authors need to address major shortcomings in their data interpretation by addressing the following issues.*\n\nWe appreciate that concerns related to vision and hearing in the *Mcl-1* deleted mice, and address these concerns as described below.\n \n *On line 57, the authors indicate that seizures are common in leukodystrophy. This is controversial. Patients may have attacks that look like seizures, but without EEG recordings there is no way to distinguish these events from myoclonus. The authors should note this ambiguity.*\n\nWe will note this ambiguity in the revision.\n *\n On line 58, the authors indicate the absence of treatments for leukodystrophies. The authors should review the following articles: PMID: 7582569, 15452666 and 27882623, and moderate the text.*\n\nWe will cite these papers and moderate the text as recommended\n \n *The methods section is lacking in details in several areas. For example beginning line 136, there is virtually no indication of the MRI details without going to secondary literature. The authors should provide a brief description including magnet strength, type of imaging and the general sequence, software used to collect and analyze the images. *\n\nWe will include these details in the revision.\n\n*Were the brains actually harvested fresh, where mechanical stresses easily deform brain structure, prior to immersion fixation for 48h? This could be troubling despite the method being previously published.*\n\nBrains were harvested fresh and drop fixed. We have extensive experience over more than ten years in handling brain tissue from neonatal mice and subsequently analyzing MRI images and sections. These methods have allowed us to make quantitative volumetric comparisons of the 3-dimensional architecture of the developing brain using MRI in prior studies, that detected genotypic differences in brain growth without confounding fixation artefact (*19*). We can confirm that no mechanical stress of handling can reproduce the white matter specific changes that we see in the Mcl-1-deleted brain. We did not detect any abnormalities in control brains subjected to the same handling techniques.\n *\n Beginning on line126, the authors could at least indicate the fixative details and whether the mice were perfused or tissue was immersion fixed. Compare this lack of detail with the description of lysis buffer beginning on line 158.*\n\nWe will add fixation details to the revision.\n \n *Behavioral testing at young ages is rather problematic regarding data interpretation. For example, open field testing (Fig. 2B) at postnatal day 7, which relies on visual cues, is rather dubious when mice do not open their eyes until 12-13 days after birth. How would the pups know if they were in the middle of an open field and exhibit thigmotaxis, even if they were capable of the behavior at such a young age? Thus, the P7 data likely cannot be interpreted in terms of the knockouts being normal.*\n\nWe fully agree with the reviewer on the challenges with behavioral analysis of such young mice. The rationale for the open field test was that, at P7, mouse pups are gaining greater control of hind limb function, which can be observed as a transition from pivoting in one place to forward locomotion. Thus, we measured the number of pivots and distance traveled in the open field as indicators for maturation of motor function. Center time was presented to show that, at P7, both WT and knockout mice stayed in the middle (i.e., the groups were at the same stage of limited mobility). We consider that these measures, together with geotaxis and latency to righting (Table 1), provide a developmentally-appropriate neurologic assessment for an age when behaviors are very limited. We will make clear in the revision that these specific tests must be considered together in order to be informative.\n\n*By P14, when the mutants exhibit a phenotype, they are already significantly underweight, which can lead to non-specific phenotypes such as retinal dysfunction or degeneration. Did the authors look for pathological changes in the retina?*\n\n\n *Further, GFAP is expressed in retina of many vertebrate species (PMID 1283834) which would inactivate mcl1 in that tissue and possibly lead to blindness. Indeed, the table at the following link provides a list of tissues in which the gfap-cre transgene is expressed during development. The authors need to address this major issue.\n **http:\/\/www.informatics.jax.org\/allele\/MGI**:2179048?recomRibbon=open*\n\nWe appreciate this suggestion and we will look for pathology in the retina and optic nerve. Such pathology, if we find it, is likely to be specific, as the optic nerve is myelinated and we have already noted extensive myelination abnormalities in the Mcl-1-deleted mice. If we find retinal or optic nerve abnormalities, we will note the potential for these abnormalities to impact on open field testing.\n \n *For the startle response, which relies on normal hearing, did the authors check to determine if the mutants are deaf? This is very difficult at such a young age, especially prior to tight junction assembly in the lateral wall at around P14. Again, GFAP is expressed in the cochlea at an early age (see PMID 20817025) and may have caused degenerative pathology in this tissue. The authors need to address this major issue.*\n\nThe reviewer brings up the potential issue of deafness as a confounding factor for acoustic startle testing. Our results showed that startle responses in the mutant mice were increased at P14, which clearly indicates the mice were able to hear the acoustic stimuli. Further, at P14 and P21, both WT and knockout mice had orderly patterns of prepulse inhibition, providing confirmation of good hearing ability at each timepoint. We will make these points clear in the revision.\n \n \n *Reviewer #2 (Significance (Required)):\n \n Unknown.*\n\nThe reviewer has not raised specific issues with the significance. We consider the significance of our work to be the finding that oligodendrocyte-lineage glial cells depend on MCL-1 and thus are primed for apoptosis, such that disrupting MCL-1 expression results in catastrophic degeneration of the cerebral white matter. Addressing the reviewer\u2019s concerns described in the section on Evidence, reproducibility and clarity will support this significance.*\n \n \n Reviewer #3 (Evidence, reproducibility and clarity (Required)):\n \n Cleveland et al. tried to argue that brain-specific depletion of apoptosis regulator MCL-1 reproduces Vanishing White Matter Disease (VWMD) in mice. The authors show that brain-specific MCL-1 deficiency leads to brain atrophy, increased brain cell apoptosis, decreased oligodendrocytes, decreased MBP immunoreactivity, and activation of astrocytes and microglia. It is known that VWMD is a hypomyelinating disorder caused by mutation of eIF2B subunits, which displays severe myelin loss but minimal oligodendrocyte apoptosis or loss in the CNS white matter. In fact, a number of studies show increased oligodendrocyte numbers in the CNS white matter.*\n\nPublished reports show decreased normal oligodendrocytes and increased immature oligodendrocyte populations *(20)**.*\n\n*The characteristic oligodendrocyte pathology is foamy oligodendrocytes (Wong et al., 2000), rather than apoptosis. *\n\nFoamy oligodendrocyte pathology and increased oligodendrocyte apoptosis are not mutually exclusive. The above referenced paper, Wong et al, in addition to foamy oligodendrocytes, also describes a \u201cdecrease in numbers of cells with oligodendroglial phenotype, both normal and abnormal\u201d (*21*); this decrease is compatible with increased apoptosis. Moreover, published reports specifically describe apoptotic oligodendrocytes in human brains with VWMD (*22*). To address this point, we propose to include both of these citations in the revision to reference foamy oligodendrocyte pathology in VWMD and to state that this pathologic finding does exclude a role for apoptosis in VWMD pathogenesis.\n\n*Since the CNS pathology of brain-specific MCL-1 deficient mice is drive by brain cell apoptosis, the relevance of this mouse model to **VWMD** is very limited. *\n\nWhether apoptosis plays a mechanistic role in VWMD is less clear than this comment suggests, as described in multiple publications (*22, 23*).\n\n*The title of this manuscript is misleading, and should be changed. *\n\nWe accept that our statement that *Mcl-1*-deletion recapitulates VWMD is premature and not adequately supported by the available data. We will revise the title, introduction and discussion accordingly, to focus on the white matter specificity of the *Mcl-1*-deletion phenotype.\n\n*Moreover, there are a number of major concerns.**\n 1. Figure 1 clearly shows severe atrophy of neocortex in Mcl-1 cKO mice; however, the white matter appears largely normal in the cerebellum and brain stem. Mcl-1 cKO mice also display ventricular dilation and possible atrophy of corpus callosum. The authors should discuss severe atrophy of neocortex in Mcl-1 cKO mice and the possibility that ventricular dilation and corpus callosum atrophy result from severe atrophy of neocortex?*\n\nThe cortical atrophy that the reviewer notes begins after P7 and is minimal at P14 when white matter loss is already pronounced. At P21, when there is clear cortical thinning, the white matter loss is extreme. Based on the time course, we consider that the white matter loss is the primary pathology, and the cortical thinning is secondary. Importantly, glial cells populate the cortex as well as the white matter and our cellular data show that oligodendrocytes are reduced in the cortex as well as in the white matter structures. Based on these lines of evidence, we consider that the primary cell type affected is the oligodendroglial population of the glia. We will add a discussion along these lines to the revision.\n\nWe agree that the brain stem is preserved. Our data show that the hGFAP-Cre promoter is least efficient in the brain stem and midbrain regions (Sup Fig.1). We will note this differential efficiency in the revision.\n\n\n\n * The motor and sensory tests in Figure 2 are potential interesting, but their relevance to myelin abnormalities is limited. The authors should perform the behaviors tests that are highly relevant to myelin abnormalities.*\n\nThe tests presented show progressive neurologic impairment, correlating with the onset of neuropathology. In the revision we will note that ataxia and tremor are common features of leukodystrophies and the *Mcl-1*-deleted mice show both ataxia and tremor.\n\n\n\n * It is well expected that there are increased apoptotic cells in the brain of Mcl-1 cKO mice. The authors should perform double labeling to demonstrate which cell types undergo apoptosis: neurons, oligodendrocytes, or other cell types? On the other hand, Figure 3A shows that there are substantial apoptotic cells in the cerebral cortex, which is consistent with severe cerebral cortex atrophy in Mcl-1 cKO mice, suggesting neuron apoptosis in the cerebral cortex. Neuron apoptosis would further rule out the relevance of Mcl-1 cKO mice to VWMD.*\n\nThese studies would be of interest, but we have not been able to co-label apoptotic cells in the *Mcl-1*-deleted mice with any marker. In the advanced state of apoptosis when dying cells are detectable by TUNEL staining, the relevant marker proteins have been degraded beyond recognition by IHC. In contrast, the apoptotic marker cleaved caspase-3, which is positive earlier in the apoptotic process and might allow marker co-labeling, was not detectably elevated in the *Mcl-1*-deleted mice. We attribute the lack of cleaved caspase-3+ cells to the asynchronous nature of the increased cell death, and to the short duration in which dying cells are cleaved caspase-3+. While double label studies of dying cells have been problematic, our studies quantifying each cell type provide information to address the reviewer\u2019s question. Our cell counts show clearly that oligodendrocytes are the primary cell type reduced in number in the Mcl-1 deleted mice.\n\n\n* Figure1, 4 the authors use H&E staining to demonstrate white matter loss. H&E staining is good to show general CNS morphology; however, it is impossible to use H&E staining to quantify the integrity of the white matter. The authors should perform specific staining to quantify white matter loss in the mouse models.*\n\nOur MBP stains later in the paper are used to quantify white matter loss.\n\n\n\n * Figure 5, MBP IHC is good to show general myelin staining, but is not a reliable assay to quantify myelin integrity in the CNS. The authors should perform electron microscopy analysis to quantify myelin integrity in the CNS in the mouse models.*\n\nOur studies of MBP staining show that the myelinated area in cross sections is significantly reduced in the *Mcl-1*-deleted mice. Electron microscopy studies cannot show whether the myelinated area is reduced and studies of myelin integrity are not needed to prove that reduced oligodendrocytes correlate with reduced myelination.\n\n\n* Figure 6, SOX10 is a marker of oligodendrocytes and OPCs. The authors should quantify the number of oligodendrocytes (using oligodendrocyte markers, such as CC1) and the number of OPCs (using OPC markers, such as NG2). Does deletion of BAK or BAX reduce oligodendrocyte apoptosis in the CNS of Mcl-1 cKO mice?*\n\nWe agree that this is an important question, and we are working to quantify OPCs in the *Mcl-1*-deleted mice by counting cells labelled with the OPC marker PDGFRA. We will add these data to the revision and discuss their significance when we know what they show.\n\n\n\n * The authors show that the level of MCL-1 is comparable in brain lysates of wildtype and eIF2B5 R132H\/R132H mice at the age of 7 months, and moderately decreased in eIF2B5 R132H\/R132H mice at the age of 10 months. VWMDis a developmental disorder. Similarly, brain-specific MCL-1 deficiency causes developmental abnormalities in the CNS. The normal level of MCL-1 in 7-month-old eIF2B5 R132H\/R132H mice strongly suggests that MCL-1 is not a major player involved in the pathogenesis of VWMD. Does brain-specific MCL-1 deficiency starting at the age of 10 months (using CreERT mice) cause CNS abnormalities in adult mice?*\n\n*\n *We agree that Mcl-1 deletion in our model disrupts postnatal brain development. Our studies show that in early life, oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis. It is an interesting, but separate question whether *Mcl-1* deletion induced in the adult would also cause a similar phenotype. The suggested studies would take over a year to conduct, and while they are of interest, they are not required to prove our main point, which is that developmental leukodystrophies may result from the dependence of oligodendrocytes on MCL-1. In the revision, we will state that our comparison on the *Mcl-1*-deletion phenotype to VWMD is hypothetical, and that additional studies are needed to test this hypothesis.\n\n\n* Does MCL-1 deletion exacerbate the pathology in eIF2B5 R132H\/R132H mice? Moreover, does MCL-1 overexpression rescue the pathology in eIF2B5 R132H\/R132H mice? These two experiments are necessary to demonstrate the involvement of MCL-1 in **VWMD**pathogenesis.*\n\n*\n *We agree that these are interesting and important studies; however, these studies will require years to complete and extensive resources. These studies are not needed to show that Mcl-1 deletion produces early onset white matter degeneration, which is our main point. As in our response to point 7 above, we will state in the revision that our comparison on the Mcl-1-deletion phenotype to VWMD is hypothetical, and list these experiments as follow up studies that are needed to test this hypothesis.\n\n\n *Reviewer #3 (Significance (Required)):\n \n The study will not significantly advance the understanding of VWMD pathogenesis.*\n\nWe recognize that our assertion of a direct relevance to VWMD was premature, and that additional studies, beyond the scope to this project, are needed to determine if MCL-1 deficiency contributes to VWMD pathology. We agree that the available data do not yet inform VWMD pathogenesis, but these data may become relevant to VWMD as follow-up studies are conducted. The data remain highly relevant to the broad group of leukodystrophies as they demonstrate a physiologic vulnerability of oligodendrocytes that sets them apart from astrocytes and neurons, and thus may play a role in disorders in which oligodendrocyte pathology is central.\n\n\n *Neuroscientists may be interested in the reported findings.*\n\nWe appreciate the reviewer noting the significance for neuroscience.\n \n *My field of expertise: oligodendrocyte, myelin, neurodegeneration, ER stress*\n\nReferences cited:\n\n1. K. A. Sarosiek, C. Fraser, N. Muthalagu, P. D. Bhola, W. Chang, S. K. McBrayer, A. Cantlon, S. Fisch, G. Golomb-Mello, J. A. Ryan, J. Deng, B. Jian, C. Corbett, M. Goldenberg, J. R. Madsen, R. Liao, D. Walsh, J. Sedivy, D. J. Murphy, D. R. Carrasco, S. Robinson, J. Moslehi, A. Letai, Developmental Regulation of Mitochondrial Apoptosis by c-Myc Governs Age- and Tissue-Specific Sensitivity to Cancer Therapeutics. *Cancer Cell* __31__, 142-156 (2017).\n2. R. Dumitru, V. 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S. van der Knaap, Leukoencephalopathy with vanishing white matter: a review. *J Neuropathol Exp Neurol* __69__, 987-996 (2010)."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138v4","doi":"10.1101\/2020.12.02.408138"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#3\n\n#### Evidence, reproducibility and clarity\n\n Cleveland et al. tried to argue that brain-specific depletion of apoptosis regulator MCL-1 reproduces Vanishing White Matter Disease (VWMD) in mice. The authors show that brain-specific MCL-1 deficiency leads to brain atrophy, increased brain cell apoptosis, decreased oligodendrocytes, decreased MBP immunoreactivity, and activation of astrocytes and microglia. It is known that VWMD is a hypomyelinating disorder caused by mutation of eIF2B subunits, which displays severe myelin loss but minimal oligodendrocyte apoptosis or loss in the CNS white matter. In fact, a number of studies show increased oligodendrocyte numbers in the CNS white matter. The characteristic oligodendrocyte pathology is foamy oligodendrocytes (Wong et al., 2000), rather than apoptosis. Since the CNS pathology of brain-specific MCL-1 deficient mice is drive by brain cell apoptosis, the relevance of this mouse model to VWMD is very limited. The title of this manuscript is misleading, and should be changed. Moreover, there are a number of major concerns.\r\n1.\tFigure 1 clearly shows severe atrophy of neocortex in Mcl-1 cKO mice; however, the white matter appears largely normal in the cerebellum and brain stem. Mcl-1 cKO mice also display ventricular dilation and possible atrophy of corpus callosum. The authors should discuss severe atrophy of neocortex in Mcl-1 cKO mice and the possibility that ventricular dilation and corpus callosum atrophy result from severe atrophy of neocortex? \r\n2.\tThe motor and sensory tests in Figure 2 are potential interesting, but their relevance to myelin abnormalities is limited. The authors should perform the behaviors tests that are highly relevant to myelin abnormalities.\r\n3.\tIt is well expected that there are increased apoptotic cells in the brain of Mcl-1 cKO mice. The authors should perform double labeling to demonstrate which cell types undergo apoptosis: neurons, oligodendrocytes, or other cell types? On the other hand, Figure 3A shows that there are substantial apoptotic cells in the cerebral cortex, which is consistent with severe cerebral cortex atrophy in Mcl-1 cKO mice, suggesting neuron apoptosis in the cerebral cortex. Neuron apoptosis would further rule out the relevance of Mcl-1 cKO mice to VWMD.\r\n4.\tFigure1, 4 the authors use H&E staining to demonstrate white matter loss. H&E staining is good to show general CNS morphology; however, it is impossible to use H&E staining to quantify the integrity of the white matter. The authors should perform specific staining to quantify white matter loss in the mouse models.\r\n5.\tFigure 5, MBP IHC is good to show general myelin staining, but is not a reliable assay to quantify myelin integrity in the CNS. The authors should perform electron microscopy analysis to quantify myelin integrity in the CNS in the mouse models.\r\n6.\tFigure 6, SOX10 is a marker of oligodendrocytes and OPCs. The authors should quantify the number of oligodendrocytes (using oligodendrocyte markers, such as CC1) and the number of OPCs (using OPC markers, such as NG2). Does deletion of BAK or BAX reduce oligodendrocyte apoptosis in the CNS of Mcl-1 cKO mice?\r\n7.\tThe authors show that the level of MCL-1 is comparable in brain lysates of wildtype and eIF2B5 R132H\/R132H mice at the age of 7 months, and moderately decreased in eIF2B5 R132H\/R132H mice at the age of 10 months. VWMD is a developmental disorder. Similarly, brain-specific MCL-1 deficiency causes developmental abnormalities in the CNS. The normal level of MCL-1 in 7-month-old eIF2B5 R132H\/R132H mice strongly suggests that MCL-1 is not a major player involved in the pathogenesis of VWMD. Does brain-specific MCL-1 deficiency starting at the age of 10 months (using CreERT mice) cause CNS abnormalities in adult mice?\r\n8.\tDoes MCL-1 deletion exacerbate the pathology in eIF2B5 R132H\/R132H mice? Moreover, does MCL-1 overexpression rescue the pathology in eIF2B5 R132H\/R132H mice? These two experiments are necessary to demonstrate the involvement of MCL-1 in VWMD pathogenesis.\r\n\n\n#### Significance\n\n The study will not significantly advance the understanding of VWMD pathogenesis. \r\n\r\nNeuroscientists may be interested in the reported findings.\r\n\r\nMy field of expertise: oligodendrocyte, myelin, neurodegeneration, ER stress\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138v4","doi":"10.1101\/2020.12.02.408138"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#2\n\n#### Evidence, reproducibility and clarity\n\n In this manuscript, the authors characterize the phenotype associated with brain-specific deletion of the mcl-1 gene in mice as a model for vanishing white matter-like disease in humans. Unfortunately, the gfap gene is expressed in many cell types during development which are outside of the intended cell type for this study, so functional data presented from the mutant mice is open to interpretation. The authors have not ruled out other interpretations of their results. The authors need to address major shortcomings in their data interpretation by addressing the following issues.\r\n\r\nOn line 57, the authors indicate that seizures are common in leukodystrophy. This is controversial. Patients may have attacks that look like seizures, but without EEG recordings there is no way to distinguish these events from myoclonus. The authors should note this ambiguity.\r\n\r\nOn line 58, the authors indicate the absence of treatments for leukodystrophies. The authors should review the following articles: PMID: 7582569, 15452666 and 27882623, and moderate the text.\r\n\r\nThe methods section is lacking in details in several areas. For example beginning line 136, there is virtually no indication of the MRI details without going to secondary literature. The authors should provide a brief description including magnet strength, type of imaging and the general sequence, software used to collect and analyze the images. Were the brains actually harvested fresh, where mechanical stresses easily deform brain structure, prior to immersion fixation for 48h? This could be troubling despite the method being previously published. \r\n\r\nBeginning on line126, the authors could at least indicate the fixative details and whether the mice were perfused or tissue was immersion fixed. Compare this lack of detail with the description of lysis buffer beginning on line 158.\r\n\r\nBehavioral testing at young ages is rather problematic regarding data interpretation. For example, open field testing (Fig. 2B) at postnatal day 7, which relies on visual cues, is rather dubious when mice do not open their eyes until 12-13 days after birth. How would the pups know if they were in the middle of an open field and exhibit thigmotaxis, even if they were capable of the behavior at such a young age? Thus, the P7 data likely cannot be interpreted in terms of the knockouts being normal. By P14, when the mutants exhibit a phenotype, they are already significantly underweight, which can lead to non specific phenotypes such as retinal dysfunction or degeneration. Did the authors look for pathological changes in the retina? \r\n\r\nFurther, GFAP is expressed in retina of many vertebrate species (PMID 1283834) which would inactivate mcl1 in that tissue and possibly lead to blindness. Indeed, the table at the following link provides a list of tissues in which the gfap-cre transgene is expressed during development. The authors need to address this major issue.\r\nhttp:\/\/www.informatics.jax.org\/allele\/MGI:2179048?recomRibbon=open\r\n\r\nFor the startle response, which relies on normal hearing, did the authors check to determine if the mutants are deaf? This is very difficult at such a young age, especially prior to tight junction assembly in the lateral wall at around P14. Again, GFAP is expressed in the cochlea at an early age (see PMID 20817025) and may have caused degenerative pathology in this tissue. The authors need to address this major issue.\r\n\r\n\r\n\n\n#### Significance\n\n Unknown.\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138#review","permalink":"","policy":"","provider":"Review Commons","asserter":"Review Commons","assertedOn":"2021-03-26","createdOn":"2021-03-26","completedOn":"2021-03-26","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.02.408138v4","doi":"10.1101\/2020.12.02.408138"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Note:** This preprint has been reviewed by subject experts for *Review Commons*. Content has not been altered except for formatting.\n\nLearn more at [Review Commons](https:\/\/reviewcommons.org)\n\n\n-----\n\n\n### Referee \\#1\n\n#### Evidence, reproducibility and clarity\n\n **Comments**\r\n\r\n1.\tMCL-1 promotes the survival of different cell lineages through its ability to inhibit the pro-apoptotic proteins BAK and BAX, the main effectors of cell death in mammalian cells. By depleting brain cells of MCL-1, apoptosis is promoted in these cells, as confirmed by histopathology of the mouse brain. This is, however, a nonspecific process. Depletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue and general knock-out is known to cause embryonic lethality. So, it is no surprise that knock out of MCL-1 in brain cells leads to a brain disease. \r\n2.\tThe white matter disease is interpreted as similar to VWM; VWM is specifically investigated and MCL-1 is found to be decreased in VWM brain tissue. The decrease is most likely nonspecific. Decrease in MCL-1 is most likely part of a general mechanism of degeneration of brain tissue or white matter. That is a different but also important conclusion. It is essential that other progressive leukodystrophies and acquired brain diseases with tissue degeneration, such as encephalitis, are investigated as well to see whether MCL-1 is also decreased in these disorders. If so, the MCL-1 decrease in white matter disease and other brain degenerative disease should be described as a final common pathway rather than specifically applicable to VWM. \r\n3.\tAdding to point 2 is the fact that the pathology of the brain-specific MCL-1 knock-out mouse does not resemble the pathology of VWM at all. The central features of VWM are abnormal astrocyte morphology with astrocytes having a few stunted processes, lack of reactive astrogliosis, lack of microgliosis, increase in number of oligodendrocytes and presence of foamy oligodendrocytes. The increase in oligodendrocytes in VWM may be such that the high cellularity leads to diffusion restriction on MRI. Bergmann glia are typically ectopic, but not reduced in number. By contrast, the brain-specific MCL-1 knock-out mouse is characterized by decreased numbers of oligodendrocytes, increased numbers of microglia, reactive astrogliosis, decreased numbers of Bergmann glia and ectopic granule cells. No morphological abnormalities of oligodendrocytes and astrocytes are observed. So, histopathologically the only shared feature is preferential involvement of the brain white matter.\r\n4.\tThe clarity of the work would benefit from a different approach to introduce the study. It would help the reader to know that (1) gray matter cell specific Mcl-1 deletion in mice did not cause apoptosis and (2) apoptosis may have different effector proteins. This important information is now in the discussion. The switch to another cell type in the brain (hGFAP+ cells) would be logical and the significance of the work may improve. When approaching the topic from the field of leukodystrophies one would not necessarily think of deleting the Mcl-1 gene, especially as this gene is not associated with any known leukodystrophy and tends to associate with preneoplastic and neoplastic disease. \r\n5.\tThe authors claim that the ISR is activated in VWM, which means that eIF2&#x03B1; phosphorylation levels are increased, general protein synthesis is decreased and a transcription pathway is regulated by ATF4 and other factors. However, this is not what is seen in VWM. Increased eIF2&#x03B1; phosphorylation and reduced general protein synthesis are not observed in VWM; strikingly, the level of eIF2&#x03B1; phosphorylation is reduced, general protein synthesis appears at a normal rate, and only the ATF4-regulated transcriptome is continuously expressed in VWM astrocytes. Fritsh et al. show that MCL-1 protein synthesis is reduced by increased eIF2&#x03B1; phosphorylation due to reduced translation rates at the Mcl-1 mRNA and not due to differences in Mcl-1 mRNA levels. One would a priori not expect to find altered MCL-1 synthesis rates in the mildly affected VWM mouse model Eif2B5R132H\/R132H. Actually, ISR deregulation has not been reported in the Eif2B5R132H\/R132H VWM mouse model. The authors need to rephrase this part of their study taking this information into account, when explaining their experiments and interpreting their results. The authors now imply that their study adds mechanistic insight into the VWM field and that is not the case. In addition, Figure 7C shows differences in actin signal rather than MCL-1 signal, suggesting that transfer of the actin protein from the gel to the blot was not optimal for the middle lanes. MCL-1 protein may thus not be reduced in these samples from Eif2B5R132H\/R132H VWM mice. \r\n6.\tCan the authors show in which cell type was apoptosis found (lines 315-316)? Their study uses the hGFAP - Cre mouse model to generate conditional Mcl-1 knock-out mice. The original paper by Zhuo et al. describing the hGFAP - promoter mouse model suggests that Mcl-1 expression is also affected in neurons and ependymal cells. The authors can investigate this further to assess which cell types (1) are sensitive to apoptosis by Mcl-1 deletion and (2) depend on Bax and Bak. \r\n7.\tHeterozygous deletion of Bak greatly reduces the number of Bak-expressing cells (Fig. 3C, line, 331-333). Authors need to explain this remarkable finding. Please provide raw IHC data. Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful. In addition, what does the Western blot signal for the BAX protein represent in Bax homozygous knock out mice (Fig. 3C)? Can the percentage of BAX+ cells in Mcl-1\/BaxdKO corpus callosum be determined, similarly as was done for BAK? Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful here as well. The legend of Fig. 3D does not state what staining is shown (H&E?).\r\n8.\tWhat explains the strong GFAP expression in processes of Mcl-1 KO astrocytes? Are these cells refractory to apoptosis or to hGFAP-driven Cre expression and recombination? Do they lack BAK or BAX or other apopotic-regulating protein? Or do specific factors compensate for the loss of MCL-1?\r\n9.\tWhich developing symptoms do the authors refer to in line 468? Please specify and introduce appropriate references. \r\n10.\tThe definition of leukodystrophies given in the paper is outdated. Leukodystrophies are not invariably progressive and fatal disorders. For more recent definition of leukodystrophies see Vanderver et al., Case definition and classification of leukodystrophies and leukoencephalopathies, Mol Genet Metab 2015, and van der Knaap et al., Leukodystrophies a proposed classification system based on pathology, Acta Neuropathol 2017.\r\n11.\tIt is not correct that there is no specific targeted therapy clinically implemented to arrest progression of the disease in any leukodystrophy. Perhaps hematopoietic stem cell transplantation is not specific targeted, although curative if applied in time in adrenoleukodystrophy and metachromatic leukodystrophy, but certainly genetically engineered autologous hematopoietic stem cells would qualify the definition. In any  case, the suggestion that no leukodystrophy is treatable is not correct.\n\n#### Significance\n\n see above\r\n\n\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/836338#review","permalink":"","policy":"","provider":"","asserter":"","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/836338v4","doi":"10.1101\/836338"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"Version 4 of this preprint has been **peer-reviewed and recommended by [Peer Community in Ecology](https:\/\/ecology.peercommunityin.org)**.\n\nSee [the peer reviews and the recommendation](https:\/\/doi.org\/10.24072\/pci.ecology.100075).\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899v3","doi":"10.1101\/2021.01.29.428899"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):** \n\nThis manuscript sheds new light on the regulation and function of a signaling network comprised of the adaptor molecules Cas and BCAR3. The data presented in the manuscript are generated through rigorous experimentation, frequently with the use of multiple approaches to arrive at the stated conclusions. \n\nMinor concerns: \n\n1) Figure 3e. The authors state that \"SOCS6 binds BCAR3 and Cas independently\" (bottom of page 7). However, while they show that the EE BCAR3 mutant binds to SOCS6 under conditions when it does not bind to Cas, they do not show the reciprocal interaction in this paper. Their previous paper (J Cell Sci 2014) suggests that SOCS6 binding to Cas may be independent of BCAR3 but neither that paper nor the current manuscript explicitly examine that. Unless there is direct evidence that SOCS6 can bind to Cas in the absence of BCAR3, perhaps it would be more accurate for the authors to limit their conclusion by saying that \"SOCS6 binds to BCAR3 independently of Cas.\" \n\n2) Figure 8a and c. Without showing a Western blot to address total pools of phosphorylated Cas, it is not clear whether the depletion in pY165 is targeted to the pool of Cas present in adhesions or to a diminution in phosphorylation of the total pool of Cas in the cell. At a minimum, the authors would need to clarify that phosphorylation at Y165 of Cas in the pool of Cas that is localized to adhesions is reduced in the presence of Y117F, R177K, or the EE mutant of BCAR3. \n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899v3","doi":"10.1101\/2021.01.29.428899"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):** \n\nThis is a well-written paper describing the co-recruitment of p117-BCAR3 and Cas to adhesion sites for activation of lamellipodial ruffling and the subsequent ubiquitin-dependent degradation. The completeness of the description of the cycle is a major success of this article and warrants publication. I didn't find major holes in their arguments and they did document that this pathway was not universal but there were possibly analogous signaling processes with other players. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899v3","doi":"10.1101\/2021.01.29.428899"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):** \n\nSummary: The study by Steenkiste focuses on the formation of adaptor protein complexes at sites of integrin receptor adhesion in the modulation of in vitro membrane ruffling and cell movement. The authors are studying the role of BCAR3 (also termed AND34 or NSP1) protein regulation by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). This is one of many adaptor proteins localized to adhesion sites. Studies are being performed on MCF10A or Hela cells to knockdown (siRNA) or over-express tagged protein constructs. By proteomics, a new phosphorylation site was identified (BCAR3 Y117). Mutagenesis showed that BCAR3 Y117 is important for enhancement of in vitro cell movement under conditions where the cullin-5 E3 ligase has also been reduced by siRNA expression. \n\nOpinion: The authors provide support for a \"co-regulatory\" model whereby the recruitment of BCAR3 to adhesions acts in part to modulate another adaptor protein tyrosine phosphorylation, p130Cas. This is associated with enhanced cell migration. The data presented are generally supportive of the conclusions and consistent with previous studies of BCAR3 and p130Cas. However, an unresolved issue is why cell phenotypes are dependent on cullin-5 knockdown or otherwise investigated by BCAR3 mutant over-expression. Cul5 loss can alter multiple aspects of cell signaling and the transient knockdown or inducible over-pression assays are a limited primary means of investigation. As multiple protein domains and post-translational modifications modulate the BCAR3-p130Cas complex, the authors did not establish a strong mechanistic linkage between newly-identified BCAR3 Y117 phosphorylation, SOCS6 binding, and a CUL5-dependent cell phenotype. Additionally, some of the experimental conditions (+\/- EGF in growth media) are difficult to connect to EGF receptor activation and or signaling. "},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.29.428899v3","doi":"10.1101\/2021.01.29.428899"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:** \n\nThis study focuses on the formation of adaptor protein complexes at adhesion sites and their links to in vitro membrane ruffling and cell movement. Specifically, the authors study the role of the adaptor BCAR3 protein which is regulated by post-translational mechanisms (ubiquitin degradation and tyrosine phosphorylation). The authors propose a \"co-regulatory\" model whereby the recruitment of BCAR3 to adhesions acts to modulate p130Cas tyrosine phosphorylation and cell migration. This manuscript would be of particular interest to cell and cancer biologists interested in the molecular regulation of cell migration. \n\n(*This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.*)\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848v2","doi":"10.1101\/871848"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nSummary:\n\nFrey et al develop an automated decoding method, based on convolutional neural networks, for wideband neural activity recordings. This allows the entire neural signal (across all frequency bands) to be used as decoding inputs, as opposed to spike sorting or using specific LFP frequency bands. They show improved decoding accuracy relative to standard Bayesian decoder, and then demonstrate how their method can find the frequency bands that are important for decoding a given variable. This can help researchers to determine what aspects of the neural signal relate to given variables.\n\nImpact:\n\nI think this is a tool that has the potential to be widely useful for neuroscientists as part of their data analysis pipelines. The authors have publicly available code on github and Colab notebooks that make it easy to get started using their method.\n\nRelation to other methods:\n\nThis paper takes the following 3 methods used in machine learning and signal processing, and combines them in a very useful way. 1) Frequency-based representations based on spectrograms or wavelet decompositions (e.g. Golshan et al, Journal of Neuroscience Methods, 2020; Vilamala et al, 2017 IEEE international workshop on on machine learning for signal processing). This is used for preprocessing the neural data; 2) Convolutional neural networks (many examples in Livezey and Glaser, Briefings in Bioinformatics, 2020). This is used to predict the decoding output; 3) Permutation feature importance, aka a shuffle analysis (<https:\/\/scikit-learn.org\/stable\/modules\/permutation_importance.html><https:\/\/compstat-lmu.github.io\/iml_methods_limitations\/pfi.html>). This is used to determine which input features are important. I think the authors could slightly improve their discussion\/referencing of the connection to the related literature.\n\nOverall, I think this paper is a very useful contribution, but I do have a few concerns, as described below.\n\n\nConcerns:\n\n1) The interpretability of the method is not validated in simulations. To trust that this method uncovers the true frequency bands that matter for decoding a variable, I feel it's important to show the method discovers the truth when it is actually known (unlike in neural data). As a simple suggestion, you could take an actual wavelet decomposition, and create a simple linear mapping from a couple of the frequency bands to an imaginary variable; then, see whether your method determines these frequencies are the important ones. Even if the model does not recover the ground truth frequency bands perfectly (e.g. if it says correlated frequency bands matter, which is often a limitation of permutation feature importance), this would be very valuable for readers to be aware of.\n\n2) It's unclear how much data is needed to accurately recover the frequency bands that matter for decoding, which may be an important consideration for someone wanting to use your method. This could be tested in simulations as described above, and by subsampling from your CA1 recordings to see how the relative influence plots change.\n\n3)\n\n   a) It is not clear why your method leads to an increase in decoding accuracy (Fig. 1)? Is this simply because of the preprocessing you are using (using the Wavelet coefficients as inputs), or because of your convolutional neural network. Having a control where you provide the wavelet coefficients as inputs into a feedforward neural network would be useful, and a more meaningful comparison than the SVM. Side note - please provide more information on the SVM you are using for comparison (what is the kernel function, are you using regularization?).\n\n   b) Relatedly, because the reason for the increase in decoding accuracy is not clear, I don't think you can make the claim that \"The high accuracy and efficiency of the model suggest that our model utilizes additional information contained in the LFP as well as from sub-threshold spikes and those that were not successfully clustered.\" (line 122). Based on the shown evidence, it seems to me that all of the benefits vs. the Bayesian decoder could just be due to the nonlinearities of the convolutional neural network."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848v2","doi":"10.1101\/871848"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nIn the current manuscript, Frey et al. describe a convolutional neural network capable of extracting behavioral correlates from wide-band LFP recordings or even lower-frequency imaging data. Other publications (referenced by the authors) have employed similar ideas previously, but to my knowledge, the current implementation is novel. In my opinion, the real value of this method, as the authors state in their final paragraph, is that it represents a rapid, \"first-pass\" analysis of large-scale electrophysiological recordings to quickly identify relevant neural features which can then become the focus of more in-depth analyses. As such, I think the analysis program described by the authors is of real value to the community, particularly as it becomes more commonplace for labs to acquire multi-site in vivo recordings. However, to maximize its utility to the community, several aspects of the analysis need clarification."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/871848v2","doi":"10.1101\/871848"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nFrey et al. describe a convolutional neural network capable of extracting behavioral correlates from wide-band LFP recordings or even lower-frequency imaging data. The analysis program described by the authors provides a rapid \"first pass\" analysis using raw, unprocessed data to generate hypotheses that can be tested later with conventional in-depth analyses. This approach is of real value to the community, particularly as it becomes more commonplace for labs to acquire multi-site in vivo recordings.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660v1","doi":"10.1101\/2021.02.24.432660"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nIn this manuscript, the authors use high-resolution live imaging to investigate how progenitor cells travel through an embryo to a distant site for differentiation and organ formation. The test case is the movement of dorsal forerunner cells (DFCs) in the zebrafish embryo, which give rise to a transient organ called Kupffer's vesicle that functions to establish the left-right body axis. DFCs are derived from enveloping layer (EVL) cells ~5 hours post-fertilization (hpf) and then move towards the vegetal pole of the embryo. They ultimately end up in the tailbud where they differentiate into epithelial cells to form Kupffer's vesicle between 10-11 hpf. Live imaging convincingly shows that EVL cells undergo apical constriction and delaminate from the EVL layer to form DFCs. Some DFCs remain connected to the EVL via ZO-1 enriched tight junction-like apical attachments. The authors propose that spreading of the EVL layer 'drags' the underlying DFCs towards the vegetal pole via these apical attachments. Supporting this model, EVL and DFCs co-migrate with the same speed and directionality, and perturbation of an actomyosin ring network in the yolk syncytial layer (YSL) disrupts movement of both EVL and DFCs. Between 8-9 hpf DFCs detach and are uncoupled from the EVL. The authors show that E-cadherin is necessary for DFC-DFC adhesion, and additional imaging experiments show that DFCs can extend long protrusions that 'capture' detached DFCs to facilitate clustering. Taken together, these data suggest an interesting drag mechanism for guiding progenitor cell movements, however the results presented do not fully demonstrate this mechanism, and alternative mechanisms were not thoroughly tested."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660v1","doi":"10.1101\/2021.02.24.432660"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThis work analyses the movement of the dorsal forerunner cells (DFCs) and its interaction with the extra-embryonic enveloping layer (EVL). By doing high-resolution time lapse microscopy the authors characterize the movement of the DFCc showing that they delaminate from the epithelium by apical constriction but they remain attached to the superficial EVL. By doing laser ablations they show that the movement of the DFCc depends on the attachment and vegetal displacement of the EVL. However, they show that with some frequencies some DFCc are detached from the rest of the cluster, leading to some random movement or even being left behind and differentiating into other cell types. Importantly, they investigate an additional mechanism to explain the movement of the DFCc detached cells. They show that single cells generate protrusions that connect them with the DFCc cluster forming an E-cadherin junction. This paper makes an important contribution by adding some new mode of migrations during development. Most of the conclusion are supported by the experiments."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660v1","doi":"10.1101\/2021.02.24.432660"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nPulgar et al. describe an interesting mechanism explaining how directed motion of group of cells maintain their migratory path as a group of cells. Incomplete delamination allows here to maintain coordinated cell movements amongst the DFC. The story is self-contained, logical, well-written and just in general very nice. The mechanism described belongs to the so-called mechanical drag which is a new type of multicellular locomotion and may be a general feature involved in many morphogenetic systems.\n\nThe major strength of the study is the extensive use of live imaging and analysis of dynamic events. The study provides a nice cellular mechanism in the process they described. The molecular mechanism would be the only weakness of the study.\n\nAn overall very exciting study."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.02.24.432660v1","doi":"10.1101\/2021.02.24.432660"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nIn this study, Pulgar et al. describe an interesting phenomenon addressing organ integrity in a unique example of collective cell migration. The group focused on the migration of the dorsal forunner cells (DFC), which will constitute the left-right organizer of the zebrafish. The authors show that DFCs retain apical contacts stemming from incomplete delamination and drag detached DFCs to their final destination. The study opens a number of exciting new questions related to the mechanism underlying the 'safeguards' process and the mechanism of coordination between migration and regulation of attachment.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328v1","doi":"10.1101\/2021.01.12.426328"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nThe authors study the leaf transcriptomes of males and females in 10 species of Leucadendron and infer genes expressed significantly differently between males and females (sex-biased genes, hereafter SBGs). Most SBGs in Leucadendron leaves evolved recently, suggesting that SBGs turnover (evolution and reversion) is very high because the genus is ancestrally dioecious since >10My. Using species in which the genes orthologous to SBGs are not sex-biased, the authors show that SBGs have high rates of expression evolution already before becoming SBGs. This suggests that most SBGs evolved under drift and the majority of SBGE (sex-biased gene expression) is evolving neutrally. This is confirmed by the estimated small proportion of SBGs evolving under adaptation (about 20% of SBGs have 5 fold higher expression divergence compared to polymorphism divergence, a mark of relatively recent adaptation). Also, SBGs are more tissue specific (less pleiotropic). Finally, the percentage of SBG is not correlated to the intensity of morphological dimorphism. All these findings go against the classical view that SBGE is driven by sex-specific selection for sexual dimorphism.\n\nThe analyses are very cautious with well designed controls and randomizations.\n\nThe results support well the conclusions.\n\nThis study puts forward the role of drift in sex-biased gene expression, offering a new interpretation of this common evolutionary phenomenon."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328v1","doi":"10.1101\/2021.01.12.426328"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nScharmann et al. present a study of sex-biased gene expression as a function of sexual dimorphism in leaf tissue in the genus Leucadendron. Comparative studies of sex-biased expression across clades are still relatively rare, and this analysis tests some core findings of a recent paper (Harrison et al. 2015). Overall, I like the analysis and think it could be a valuable addition to the literature on sex-biased genes. This is particularly true given the difficulty of cross-species expression comparisons and the paucity of them in plants.\n\nHowever, there are some critical differences between the Harrison paper and the one here, and I think it would be helpful if the authors present them early in the text. Specifically, Harrison et al. (2015) was primarily focused on gonad tissue, which in animals is the site of the vast majority of sex-biased genes. In contrast, the authors here focus on vegetative (leaf) tissue, which is analogous to animal somatic tissue. None of the patterns that Harrison et al. (2015) observed and report from the gonad were evidence in the somatic tissue they assessed. Also, by looking at gonadal tissue, Harrison et al. (2015) focused on the tissue that produces gametes, which are thought to be subject to some of the strongest sexual selection pressures. The fairest comparison would be flower tissue in plants, so I am unsure how much of the Harrison results would be expected to hold up in leaf samples. This doesn't mean the authors should do the analyses they present, just that they should be a little more upfront about what they might reasonably expect to find.\n\nThere is also a conflation at times in the paper between sexual dimorphism, which the authors can quantify in their leaf samples, and sexual selection. I explain this in more detail below, but to summarize here, I think the expectations for the relationship between sex-biased gene expression and sexual selection versus sexual dimorphism are somewhat distinct.\n\nFinally, I am a little concerned that the low numbers of sex-biased genes, expected from leaf tissue, offer limited power for some of the tests the authors want to do. Harrison et al. (2015) had hundreds of sex-biased genes from the gonad, and this power made it possible to detect subtle patterns. The authors have a few dozen sex-biased genes, and this makes it difficult to know whether their negative results are the result of low statistical power. That they find clear associations between pre-sex-biased genes and rates of evolution is quite impressive given this low power.\n"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328v1","doi":"10.1101\/2021.01.12.426328"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\n*A summary of what the authors were trying to achieve.\n\nThe study takes advantage of the interesting plant genus Leucadendron to compare gene expression between male vs. female in species with more or less sexual dimorphism. This question was addressed in a somewhat comparable manner in only one previous paper by Harrison et al. 2015 across six bird species. The overarching question is the role of natural selection in sexual dimorphism.\n\n*An account of the major strengths and weaknesses of the methods and results.\n\n-Beside the genus-wide comparison of whole transcriptomes across related species, which makes in itself a strong dataset, the major strength of the analysis is the phylogenetic framework that allows the authors to track the evolution of sex bias through several tens of million years of evolutionary history. Despite ancestral dioecy in the genus, very few genes show consistent sex bias across several species, with sex-bias being mostly species-specific. Two striking negative results will be of special interest to the community : 1) species with more pronounced sexual dimorphism at the morphological level do not tend to exhibit more pronounced sex-biased gene expression 2) the few genes that do show sex-biased expression were apparently recruited among those with the highest expression variance to begin with, strongly suggesting that sexual selection has not been the main force driving their expression divergence.\n\n-In my view, the main limitation of the work is the use of leaf rather than reproductive tissues, making the comparison to other studies less straightforward to interpret. It is especially important that the expectations for somatic vs gonadic tissues be made a lot clearer in the text. Also, the fact that a single leaf phenotype is measured (specific leaf area) seems arbitrary : one could imagine sexual dimorphism on many other characteristics, yet they are not considered here. The text on p.324 mentions \"striking convergence in aspects of morphological dimorphism across the genus\", but there is no way for the reader to appreciate the extent of this convergence. Finally, it would be useful to at least make some mention of the sex-determination system in these species, since the expectations would differ if some of the sex-biased genes were linked to sex chromosomes.\n\n*An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.\n\nThe analysis is mostly sound, but I am a bit concerned by the arbitrary threshold used to define SBGE. The text on p.305 says that \"This result is extremely robust to the choice of threshold\", but 1) the results are not reported so it is impossible for the readers to evaluate the basis of this assertion and 2) it is not clear whether robustness of the other results has been evaluated at all. This aspect clearly deserves more attention.\n\n*A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community.\n\nThis work will be of interest to the community, as rapid rates of expression evolution would generally be interpreted as the consequence of sex bias, whereas the phylogenetic analysis presented here instead supports the idea that the expression of genes that end up being sex biased were instead intrinsically less constrained to begin with."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2021.01.12.426328v1","doi":"10.1101\/2021.01.12.426328"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Evaluation Summary:**\n\nThis is one of the first studies to investigate sex-biased gene expresion in a broad phylogenetic context, and the first in a plant genus. The findings go against the classical view that sex-biased gene expression is driven by sex-specific selection for sexual dimorphism, and instead suggests that sex-bias preverentially evolved in genes that already had the highest expression variance to begin with. It will broadly appeal to researchers interested in the evolution of sexual dimorphism.\n\n*(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)*"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490v2","doi":"10.1101\/2020.12.27.424490"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #4 (Public Review):**\n\nThe goal of the manuscript was to add to the research on the rates of success of African American\/Black PI in their pursuit of NIH funding. The authors specifically addressed variability in funding levels of NIH Institutes and Centers(ICs). The authors were successful in identifying that there are differentials rates of award rates by IC. The authors describe that topic choice was not associated with funding after accounting for IC assignment which vary in their funding rates."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490v2","doi":"10.1101\/2020.12.27.424490"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #3 (Public Review):**\n\nThis analysis focuses on funding success for a set of NIH R01-mechanism grant applications submitted between 2011 and 2015, with a focus only on those which had white and Black Principal Investigators (PIs). It is presented as a follow-up to the previously published paper from Hoppe and colleagues in 2019, uses the same population of applications and relies on the same analysis of application text to cluster these applications by topic. The authors set out to determine how success rates associated with the application's proposed topic may be determined by the success rates associated with the Institute or Center within the NIH to which the application had been assigned for potential funding. This is a critical and important investigation that is of high potential impact. The scholarship of the Introduction and Discussion, however, fails to convey this to the reader. There are many recent publications in the academic literature that address why a disparity of funding to AA\/B investigators, and a disparity of funding of topics that are of interest to AA\/B investigators, are such critical matters for the NIH to identify and redress. Similarly, the Discussion and Conclusions sections do not suggest any specific actions that may be recommended by these findings, which is an unfortunate oversight that limits the likely impact of this work.\n\nThe significance of this work is limited by a number of methodological choices that are unexplained or have not been justified and therefore appear to be somewhat arbitrary. While it can be necessary to draw category lines in an investigation of this type, it is necessary to provide some indication of what would happen to the support for the central conclusions if other choices had been made. This includes the exclusion of multi-PI applications if the Black PI was not the contact PI, the definition of AA\/B-preferred ICs as the top quartile (particularly given the distribution of success rates within this quartile), the definition of AA\/B-preferred topics as the 15 word clusters that accounted for only half of the AA\/B applications, and the ensuing inclusion of only 27% of the AA\/B applications. Arbitrary choices to use only a subset of the data raise questions about what the conclusions would be if the entire dataset of grants assigned across all of the ICs, and on all of the topics, was used.\n\nA fundamental limitation to this manuscript is that the authors are relying on an indirect logic of analysis instead of simply reporting the success rates for applications with AA\/B and white PIs within each IC. The primary outcome deployed in support of the central conclusion is a reduction of the regression coefficient for the contribution of PI race to award success and an elimination of statistically significant contribution of research topic preferred by AA\/B applicants to the award success once IC success was partialed out. The former analysis is interpreted in imprecise terms instead of simply reporting what magnitude of effect on the white\/Black success rate gap is being described. And the latter analysis appears to show a continued significant effect of PI race on award success even when the IC success rate is included. The much more intuitive question of whether award rates for white and AA\/B applicants differ within each IC has not been addressed with direct data but the probit model outcome suggests it is still significantly different. This gives the impression that the authors have conducted an unnecessarily complex analysis and thereby missed the forest for the trees- i.e. even when accounting for IC award rates there is still a significant influence of PI race.\n\nThe manuscript is further limited by atheism omission of any discussion of how and why a given grant is assigned to a particular IC (this is exacerbated by incorrect phrasing suggesting the applicant \"submits an application to\" a specific IC) and any discussion of the amount of the NIH budget that is assigned to a given IC and how that impacts the success rate. This is, at the least, necessary explanatory context for the investigation."},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490v2","doi":"10.1101\/2020.12.27.424490"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #2 (Public Review):**\n\nThe paper by Lauer et al provides further insight into the factors that might determine why RO1 applications from AAB (African American Black) principal investigators appear to fare worse than their white counterparts. Their work is derived from an earlier analysis published by Hoppe et al that found 3 factors determined funding success among AAB PIs. These included decision to discuss at study section, impact score, and topic choice. The latter, topic choice (community and population studies) appeared to represent more than 20% of the variability in funding gaps. This raised the question of whether there was reviewer bias at study sections. In the Lauer paper, after controlling for several of these variables, the authors found that the topic choice of AABs (ie. preferred topics) were indeed important in respect to funding, but they uncovered the fact that the topic choices occurred more frequent in ICs that had lower funding rates. Thus the authors conclude that the disparity between AAB and white investigator RO1s is very dependent on topic choice which ultimately ends up in larger ICs with lower funding percentiles.\n\nOverall the paper is relatively straightforward and could be important as It provides some additional data to consider. It is in fact basically a re-analysis of the Hoppe paper, but that is reasonable since that paper left many unanswered questions. Its implications however are less clear, and these raise additional questions of importance to the extramural scientific community as well as IC leadership.\n\nOverall the reader is left with the unsettling question: Can we just wish away these disparities based on IC funding rates? (Figure 1).\n\n1) Why would topic choice of community engagement or population studies fare worse at an Institute such as AI rather than at GM if both have the relatively same proportion of preferred topics, and both have relatively high budgets compared to other institutes. Is there one or more ICs that drive the correlations between IC funding and preferred topics or PIs?\n\n2) Since only 2% of all PIs are AAB does that represents another issue of low frequency relative to the larger cohort?\n\n3) It would be valuable to know if community engagement or population studies in total do worse than mechanistic studies. The authors do admit that preferred topics of AABs in general fare worse(Figure 2, Panel B).\n\n4) Another concern is that the data are up to 2015; it has now been five years and things have changed dramatically at NIH and in society. There are now many more multiple PI applications including AABs that may not be the contact PI yet are likely to be in a preferred topic area.\n\n5) There is nothing in the discussion about potential resolutions to this very timely issue; In other words we now know that the disparity in funding is such that AAB RO1s do worse than white PIs because they are selecting topics that end up at institutes with lower funding rates. Should the institutes devote a set aside for these topic choices to balance the portfolio of the IC and equal the playing field for AABs? Are there other alternative approaches?"},{"contentType":"review","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490#review","permalink":"","policy":"","provider":"eLife","asserter":"eLife","assertedOn":"2021-03-29","createdOn":"2021-03-29","completedOn":"2021-03-29","isReviewOf":[{"contentType":"preprint","content":"https:\/\/www.biorxiv.org\/content\/10.1101\/2020.12.27.424490v2","doi":"10.1101\/2020.12.27.424490"}],"contributors":[{"name":"","affiliations":"","id":"","orcid":"","role":""}],"contentText":"**Reviewer #1 (Public Review):**\n\nThis manuscript by Lauer et al follows up on previous articles that ask the question whether there are funding disparities at the National Institutes of Health for African American or Black (AAB) investigators. The investigators breakdown the analysis by race, topic of proposal, and NIH institute-Center (IC) to which an application was assigned. They conclude that the most important factor in determining funding is the Institute assignment with lower funding rates related to the funding capacity of a particular Institute (e.g National Eye Institute vs Minority Health and Health Disparities). The present study is a welcome addition to this debate since if biases do exist, NIH needs to address these. The strengths of this manuscript are the detailed breakdown of the available data in order to evaluate for biases, the availability of data for multiple years (2011-2015) and the consideration of alternate explanations (e.g new applications vs resubmissions; single vs multi PI, etc). A weakness of the data is that if their conclusion is that Institute assignment was the main determinant of funding rates, why wasn't the approach for Institute assignment discussed? Are there possible biases in this assignment besides keyword searches? There is also the question of whether there is circular logic operating here. The Minority Health and Health Disparities received the most AAB applications but had one of the lowest funding rates. Wouldn't this Institute be expected to be one in which AAB applicants would try to direct their application to? This manuscript is sure to generate additional discussion on this topic which is an important step in trying to address the issue of potential funding disparities. However as the authors point out the fact that only 2% of the applications submitted to the NIH were from AAB investigators is of concern."}]}