Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARγ ortholog Eip75B

Joseph Blommer; Megan C. Fischer; Athena R. Olszewski; Rebeccah J. Katzenberger; Barry Ganetzky; David A. Wassarman

doi:10.1371/journal.pone.0258873

Peer Review History

Original SubmissionJune 30, 2021
2 Aug 2021 Decision Letter - Efthimios M. C. Skoulakis, Editor PONE-D-21-21417 Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARg ortholog Eip75B PLOS ONE Dear Dr. Wassarman, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers and I concur that this is an interesting and timely manuscript. However there are a few questions by both reviewers that need to be addressed as outlined below and aim to clarify and improve the manuscript Please submit your revised manuscript by Sep 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: [The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.] Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript the authors test the impact of feeding flies a ketogenic (KD) vs high carbohydrate diet (CMYD) in the 24 hrs after TBI. The system they use is the so-called HIT device to administer TBI. They find that animals on a the CMYD diet or a yeast diet have standard high mortality after 24 hrs, whereas animals on a KD have low mortality similar to feeding animals water. They provide evidence that the animals are eating because starved animals have a high mortality. They show that the protective effect of a KD is conferred upon a range of genetic backgrounds, and then provide molecular insight by showing that the effect is dependent on Eip75B gene function, which is the homolog of PPAR-gamma. These findings suggest that KD, by activating Eip75B activity, mitigates the deleterious outcomes of TBI. These data are consistent with some mammalian findings. Overall this is an interesting and appropriate manuscript. There are just a few points that need to be addressed.. In Figure 2, are the lifespans of the different strains on KD significantly different? The RAL441 and RAL391 look like they might be. Thus, whereas on a CMYD diet, the mortality is marked different, on a KD diet it may still be different but overall dramatically better. In figure 3, at first it is rather confusing why these lifespans are so dramatically different from those in figure 1. They could clarify this by making the point more clearly that the flies are cultured differently. That is… ”Lifespan was determined…for 24 h following TBI but then surviving flies were cultured on solid CMYD.” Also, some of their arguments about lifespan (median lifespan between injured and uninjured animals; Fig 3) seem sketchy because the uninjured CMYD animals have a better median lifespan than KD animals. So the animals survive better after TBI if on a KD for 24 hrs, but after that, the KD lifespan is compromised. So it seems like there are pros and cons. How do they explain or interpret this. Since only 1 of two Eip75B alleles showed loss of the KD benefit, the significance of Eip75B function seems questionable also. Maybe those results are due to genetic background. Can they show somehow that the alleles are of a strength consistent with their interpretation that the MI04895 allele is less severe? This needs another allele, and/or controls for background, or some other way of validating the findings to be properly interpreted. Perhaps they could measure activation of PPAR-gamma to at least show correlation. In any case, this point needs to be strengthened. Reviewer #2: This is an interesting paper by Blommer et al that provides novel experimental data indicating that a ketogenic diet can mediate beneficial effects in a fly model of traumatic brain injury. My comments are as followed: 1. Please make sure that gene names are consistently italicized. 2. According to the Alliance of Genome Resources, Eip75B is an ortholog of the following human genes: NR1D2, PPARA, PPARG, NR1D1, and PPARD. It may be worthwhile to mention that PPARG is not the sole ortholog of this fly gene. 3. In the Introduction, I would mention that traumatic brain injury is a known risk factor dementia. This will help highlight the broader importance of your research. There are multiple papers one could cite for this, such as this recent systematic review and meta-analysis (PMID: 33044182). 4. In the Introduction, it may be useful to mention that there is a growing interest in the interplay between lipid metabolism and aging. Dr. Anne Brunet has published excellent reviews on this topic. 5. It would be helpful if the authors can explicitly list the increase in median and mean lifespan in response to a ketogenic diet. For those looking to do systematic reviews and/or meta-analyses in the future, this information could be useful. It may be simplest to do this in the form of a table that summarizes all of the lifespan results (i.e., p-value, median increase, mean increase). 6. While there is evidence that a ketogenic diet may be beneficial in specific circumstances, it may not be as impactful as other dietary interventions (e.g., caloric restriction, intermittent fasting, Mediterranean diet, plant-based diet). In the Discussion, can the authors comment on where a ketogenic diet will be specifically beneficial vs. other dietary alterations and why they think this is the case? 7. Major sections vs. sub-sections need to be demarcated more clearly. I recommend making major sections entirely capitalized (e.g., DISCUSSION) while keeping sub-sections as sentence case (e.g., KD and water appear to reduce early mortality following TBI by different mechanisms). 8. Unless the journal handles this separately, please add a Conflict of Interest section to your manuscript. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
9 Sep 2021 Author Response Responses to reviewer’s criticisms (in bold font) Reviewer #1: In this manuscript the authors test the impact of feeding flies a ketogenic (KD) vs high carbohydrate diet (CMYD) in the 24 hrs after TBI. The system they use is the so-called HIT device to administer TBI. They find that animals on a the CMYD diet or a yeast diet have standard high mortality after 24 hrs, whereas animals on a KD have low mortality similar to feeding animals water. They provide evidence that the animals are eating because starved animals have a high mortality. They show that the protective effect of a KD is conferred upon a range of genetic backgrounds, and then provide molecular insight by showing that the effect is dependent on Eip75B gene function, which is the homolog of PPAR-gamma. These findings suggest that KD, by activating Eip75B activity, mitigates the deleterious outcomes of TBI. These data are consistent with some mammalian findings. Overall this is an interesting and appropriate manuscript. There are just a few points that need to be addressed. In Figure 2, are the lifespans of the different strains on KD significantly different? The RAL441 and RAL391 look like they might be. Thus, whereas on a CMYD diet, the mortality is marked different, on a KD diet it may still be different but overall dramatically better. Previously, we found that fly lifespan affects the MI24, that is, at a given age, flies with a short lifespan have a higher MI24 than flies with a long lifespan (Katzenberger et al. (2013) PNAS). Thus, it is likely that RAL441 flies have a longer lifespan than RAL391 flies when fed solid CMYD. We agree that it would be interesting to determine if the lifespan of these lines differs when fed KD, but we believe that this is beyond the scope of the manuscript. In figure 3, at first it is rather confusing why these lifespans are so dramatically different from those in figure 1. They could clarify this by making the point more clearly that the flies are cultured differently. That is… ”Lifespan was determined…for 24 h following TBI but then surviving flies were cultured on solid CMYD.” We clarified this point in several ways. In the legend for Figure 1, we added the phrase “over the course of the experiment” to the sentence “Percent survival was determined for uninjured 0-7 day old, mixed sex w1118 flies fed water (n=240) or 0.3 cal/200 �l CMYD (n=200) or KD (n=239) over the course of the experiment.” Also, in the legend for Figure 3, we added the phrase “that is, flies in the experiment that survived 24 h feeding on 0.3 cal/200 �l CMYD or KD were fed solid CMYD throughout the rest of their lifespan” to the sentence “Percent survival was determined for uninjured and injured 0-7 day old, mixed sex w1118 flies fed 0.3 cal/200 �l CMYD or KD for 24 h following TBI and solid CMYD thereafter, that is, flies in the experiment that survived 24 h feeding on 0.3 cal/200 �l CMYD or KD were fed solid CMYD throughout the rest of their lifespan.” Also, some of their arguments about lifespan (median lifespan between injured and uninjured animals; Fig 3) seem sketchy because the uninjured CMYD animals have a better median lifespan than KD animals. So the animals survive better after TBI if on a KD for 24 hrs, but after that, the KD lifespan is compromised. So it seems like there are pros and cons. How do they explain or interpret this. We see the reviewer’s point that the data are not black and white. But we think that our main conclusion is supported by the data. Figure 3 shows that the survival curve of injured KD flies more nearly approximates that of uninjured KD flies than does that of injured CYMD flies to their control. Thus, relative to the appropriate control, the ketogenic diet is beneficial both at 24 hours and throughout the course of the lifespan for flies that survived mortality at 24 hours. Accordingly, in the Abstract we conclude “flies protected from early mortality by KD continued to show survival benefits weeks later” and in the section about Figure 3, we similarly conclude “Thus, flies that avoid mortality following TBI because of the beneficial effects of KD during a 24 h window after primary injuries continue to manifest long-term benefits of this diet weeks later.” Since only 1 of two Eip75B alleles showed loss of the KD benefit, the significance of Eip75B function seems questionable also. Maybe those results are due to genetic background. Can they show somehow that the alleles are of a strength consistent with their interpretation that the MI04895 allele is less severe? This needs another allele, and/or controls for background, or some other way of validating the findings to be properly interpreted. Perhaps they could measure activation of PPAR-gamma to at least show correlation. In any case, this point needs to be strengthened. To address this point, we examined another Eip75B allele (Eip75BKG04491) and included these data in Figure 4. Thus, there are now three Eip75B alleles or allelic combinations in which the MI24 is higher for KD than water. We also added a figure (Figure 4A) that supports the conclusion that Eip75BMI04895 is a weaker allele than Eip75BKG04491 and Eip75BBG02576. Lastly, we added analyses of male and female flies to Figure 2 (new panel 2A) that reinforce the finding that water and KD have similar effects on the MI24 of flies that are wild type for Eip75B. Based on these data, we have concluded that “The beneficial effect of KD was, however, retained in Eip75BMI04895 homozygotes (ordinary one-way ANOVA with Dunnett’s Multiple Comparison test, p=0.999), which we attribute to a presumptive weaker loss of function of Eip75B caused by this mutation. Eip75BMI04895 only disrupts three of the seven Eip75B pre-mRNA isoforms, whereas Eip75BKG04491 and Eip75BBG02576 disrupt four and five isoforms, respectively (Fig. 4A). Thus, while it remains possible that differences in genetic background underlie differences in MI24 values for Eip75B mutant flies fed water versus KD, the data support the conclusion that activation of Eip75B/PPAR� by KD triggers mechanisms that reduce early mortality following TBI.” Reviewer #2: This is an interesting paper by Blommer et al that provides novel experimental data indicating that a ketogenic diet can mediate beneficial effects in a fly model of traumatic brain injury. My comments are as followed: 1. Please make sure that gene names are consistently italicized. We believe that all of the gene names are italicized. In accord with Drosophila convention, genes and RNAs are italicized, and proteins are not italicized. 2. According to the Alliance of Genome Resources, Eip75B is an ortholog of the following human genes: NR1D2, PPARA, PPARG, NR1D1, and PPARD. It may be worthwhile to mention that PPARG is not the sole ortholog of this fly gene. Our assignment of Eip75B as the ortholog of PPAR� is based on two lines of evidence. First, BLAST search analysis of the Drosophila genome with the human PPAR��protein identified Eip75B as the most significant match. Second, data in references 38 and 39 demonstrate that Eip75B functions similarly to PPAR��Thus, we have added the sentence “The orthologous relationship is inferred both from amino acid sequence identity (i.e., Eip75B is the most significant match to human PPAR� in a BLAST search of the Drosophila proteome).” This information is provided under the section heading “Beneficial effects of KD on early mortality are mediated by the PPAR� ortholog Eip75B.” PPARA, PPARD, NR1D1, and NR1D2 may be listed as orthologs simply because they are similar in sequence to Eip75B. Often single genes in flies are represented by expanded gene families in mammals. 3. In the Introduction, I would mention that traumatic brain injury is a known risk factor dementia. This will help highlight the broader importance of your research. There are multiple papers one could cite for this, such as this recent systematic review and meta-analysis (PMID: 33044182). We appreciate the suggestions to include dementia and the interplay between lipid metabolism and aging in the Introduction. Our rationale for not including these topics as well as many others related to TBI is to focus the Introduction on topics that are critical for the reader to understand and to appreciate the significance and implications of the data that are presented. 4. In the Introduction, it may be useful to mention that there is a growing interest in the interplay between lipid metabolism and aging. Dr. Anne Brunet has published excellent reviews on this topic. Please see the response to point 3. 5. It would be helpful if the authors can explicitly list the increase in median and mean lifespan in response to a ketogenic diet. For those looking to do systematic reviews and/or meta-analyses in the future, this information could be useful. It may be simplest to do this in the form of a table that summarizes all of the lifespan results (i.e., p-value, median increase, mean increase). For readability, we have provided median lifespan numbers and p-values in the text as they were discussed. 6. While there is evidence that a ketogenic diet may be beneficial in specific circumstances, it may not be as impactful as other dietary interventions (e.g., caloric restriction, intermittent fasting, Mediterranean diet, plant-based diet). In the Discussion, can the authors comment on where a ketogenic diet will be specifically beneficial vs. other dietary alterations and why they think this is the case? Unfortunately, there are very little data in the mammalian literature and no data in the Drosophila literature on the effect of other dietary interventions on TBI. Thus, we do not feel able to add any meaningful comparisons among the various diets at this time. 7. Major sections vs. sub-sections need to be demarcated more clearly. I recommend making major sections entirely capitalized (e.g., DISCUSSION) while keeping sub-sections as sentence case (e.g., KD and water appear to reduce early mortality following TBI by different mechanisms). The guidelines for style are set by PLoS One, which we are required to follow. 8. Unless the journal handles this separately, please add a Conflict of Interest section to your manuscript. Please see the response to point 7. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258873.r002
20 Sep 2021 Decision Letter - Efthimios M. C. Skoulakis, Editor PONE-D-21-21417R1Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARg ortholog Eip75BPLOS ONE Dear Dr. Wassarman, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ==============================the reviewers feel and I concur that the manuscript is ready for publication save for the clarifications presented below. It is essential in my opinion to clarify the MI24 measurements are indeed %.Please make these minor changes and submit ASAP. ============================== Please submit your revised manuscript by Nov 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Efthimios M. C. Skoulakis, PhD Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript has been improved by the revisions suggested by the Reviewers. One minor point is to make clear along the y-axes that MI24 is a percentage. It is unlabelled and not clarified in the legends. Also in the text on page on page 4, "varies from 7 to 58" should be "7% to 58% among 179...." Reviewer #2: It is surprising that the authors were unwilling to accommodate minor changes to the text that would have helped emphasize the importance of this work to a broader audience. While my recommendation is to accept the paper based on its technical soundness and its improvement from the review process, I would encourage the authors to be more flexible in the future. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0258873.r003
Revision 2
21 Sep 2021 Author Response Responses to reviewer’s criticisms Reviewer #1: This manuscript has been improved by the revisions suggested by the Reviewers. One minor point is to make clear along the y-axes that MI24 is a percentage. It is unlabelled and not clarified in the legends. Also in the text on page on page 4, "varies from 7 to 58" should be "7% to 58% among 179...." We appreciate the reviewer’s suggestion. To increase clarity, we added the following sentence to the legends for Figures 1, 2, and 4. “The MI24 represents the percent mortality of injured flies minus the percent mortality of uninjured flies 24 h following TBI.” Adding percent or % to the y-axis label is problematic because the MI24 is defined as a percent. So, MI24 percent and MI24% are redundant and percent mortality and % mortality are incorrect. Furthermore, I would like the graphs to be consistent with our prior publications. We have published graphs in nine papers with y-axes labeled MI24, and other labs have also used this nomenclature. Reviewer #2: It is surprising that the authors were unwilling to accommodate minor changes to the text that would have helped emphasize the importance of this work to a broader audience. While my recommendation is to accept the paper based on its technical soundness and its improvement from the review process, I would encourage the authors to be more flexible in the future. We thank the reviewer for the suggestion. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0258873.r004
7 Oct 2021 Decision Letter - Efthimios M. C. Skoulakis, Editor Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARg ortholog Eip75B PONE-D-21-21417R2 Dear Dr. Wassarman, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Efthimios M. C. Skoulakis, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0258873.r005
Formally Accepted
15 Oct 2021 Acceptance Letter - Efthimios M. C. Skoulakis, Editor PONE-D-21-21417R2 Ketogenic diet reduces early mortality following traumatic brain injury in Drosophila via the PPARg ortholog Eip75B Dear Dr. Wassarman: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Efthimios M. C. Skoulakis Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0258873.r006

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