The right amygdala and migraine: Analyzing volume reduction and its relationship with symptom severity

Shota Kosuge; Yuri Masaoka; Hideyo Kasai; Motoyasu Honma; Kouzou Murakami; Nobuyuki Yoshii; Keiko Watanabe; Takaaki Naito; Miku Kosuge; Misa Matsui; Daiki Shoji; Syunsuke Sakakura; Hidetomo Murakami; Masahiko Izumizaki

doi:10.1371/journal.pone.0301543

Peer Review History

Original SubmissionDecember 21, 2023
7 Feb 2024 Decision Letter - Keisuke Suzuki, Editor PONE-D-23-42993The Right Amygdala and Migraine: Analyzing Volume Reduction and Its Relationship with Symptom SeverityPLOS ONE Dear Dr. MASAOKA, 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. Please submit your revised manuscript by Mar 23 2024 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, Keisuke Suzuki, MD, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This study was supported by a JSPS KAKENHI Grant (Number 20K03482)" We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 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: "JSPS KAKENHI Grant (Number 20K03482)" Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following financial disclosure: "JSPS KAKENHI Grant (Number 20K03482)" Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: The authors are encouraged to revise the manuscript in accordance with the reviewers' comments. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know Reviewer #2: No ****** 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: No Reviewer #2: No ****** 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: I could not quite follow the meaning of "MIDAS scores influenced by MSQ outcomes which were further affected by depression, anxiety etc." I thought it may be better for the authors to focus on the relationship between MRI finding and each questionnaire/assessments rather than the relationship between the questionnaire/assessments. Please review the manuscript carefully as there seemed to be typos or incomplete sentence. For example, I assume you do not need 5) in page 4, lint 74. Reviewer #2: Overview and general recommendation: Numerous neuroimaging studies have indicated gray matter volume reductions in migraine patients. And chronic migraine patients have different biomarkers compared to episodic migraine. The present study aimed to elucidate the association between gray matter volume changes and factors. The study hypothesized that there was a relationship between (1) gray matter volume (GMV) and migraine symptom severity and (2) GMV and psychological status. To test hypotheses, authors performed functional magnetic resonance imaging (fMRI) on patients and healthy controls. Group comparisons for intracranial volume were employed to validate structural differences between migraine sufferers and healthy controls. Then, path analyses were performed to study the interactions between variables. The findings suggest that the chronicity and severity of migraine headaches might specifically affect the right amygdala. The study highlights a complex interrelation that migraine disability is influenced by the quality of life, which is, in turn, affected by psychological states. On the one hand, the present study contributes to neuroimaging and clinical research on migraine. The authors exhibit proficiency in data processing and analysis, and their findings extend the relationship between clinical symptoms and psychological status. However, upon closer examination, some areas for improvement in the paper were identified. Certain study aspects were described in insufficient detail, and some crucial points must be addressed. Major concern: 1. Language. The author is requested to revise the entire text to make it consistent. (1) In the abstract, the author describes the use of 140 brain regions as the region of interest for analysis, but in the text, the author describes the use of 137 regions (page 6, line 111); (2) Add original word of the abbreviation "MG" (page 3, line 45); (3) Authors need to pay attention to whether the fifth point in the inclusion and exclusion criteria is written (page 4, line 74). 2. Quality control. It is recommended that the authors use tools to detect MRI image quality. The authors must explain the necessity of manually adjusting the segmentation [1]. If there is no statistical difference, please use the automated tool to ensure reproducibility; if there is a difference, it is recommended to perform a quality control evaluation in the supplementary material [2]. 3. Statistical Analysis. (1) The author used 137 regions of interest. Did he use multiple comparison corrections in the analysis? Please add relevant content, and if not used, please explain. (2) Did the author test that the data is normal distribution? For example, using the Shapiro–Wilk test, (3) The migraine subjects recruited by the author were divided into migraine with aura (n = 11) and migraine without aura (n = 22). In the covariance analysis, is there " migraine with aura” a covariate? (4) Did the author conduct a collinearity test to ensure all variables are independent? 4. Interpretability. In the path analysis, the correlation between MIDAS and right amygdala was r=-0.38, a low correlation; the correlation between CESD and right pallidum volume was r=-0.35, which was also a low correlation. Please explain in the discussion: what is the difference between low and high correlation here? Does the low correlation indicate that the right amygdala volume is not closely related to MIDAS? References: [1] Vahermaa V, Aydogan DB, Raij T, et al. FreeSurfer 7 quality control: Key problem areas and importance of manual corrections. Neuroimage. 2023;279:120306. doi:10.1016/j.neuroimage.2023.120306 [2] Klapwijk ET, van de Kamp F, van der Meulen M, Peters S, Wierenga LM. Qoala-T: A supervised-learning tool for quality control of FreeSurfer segmented MRI data. Neuroimage. 2019;189:116-129. doi:10.1016/j.neuroimage.2019.01.014 ****** 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. 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.0301543.r001
Revision 1
28 Feb 2024 Author Response Thank you for your useful comments and suggestion. We corrected and added the manuscript according to your comments. Reviewer #1 Question 1: I could not quite follow the meaning of "MIDAS scores influenced by MSQ outcomes which were further affected by depression, anxiety etc." I thought it may be better for the authors to focus on the relationship between MRI finding and each questionnaire/assessments rather than the relationship between the questionnaire/assessments. Answer: Thank you for your suggestions. We modified the relationship between MIDAS, MSQ and other exogenous variables in the abstract section and a section created “Assumption from the path model” in the discussion. Since each path between MIDAS and MSQ, and MSQ and CED-D were significantly impacted, we would like to comment in the form of a proposal. We made modifications as follow: P2, L38: Abstract Our path model suggests a complex relationship whereby migraine disability is strongly influenced by quality of life, which is, in turn, affected by psychological states, such as anxiety and depression. P16, L272~ Assumptions from the path model The path analysis also revealed several mediating factors that connected MG severity to lower amygdala volume. Our findings suggested that the MIDAS score, which represents MG severity, is shaped by patients’ quality of life, as measured by the MSQ. The MSQ is, in turn, influenced by exogenous factors, such as depression, anxiety, and the right pallidum volume. The association between the right pallidum volume and depression in MG patients was an intriguing result. This suggested that those with elevated depression levels tended to have lower pallidum volumes. The pallidum is a structure within the basal ganglia that plays a pivotal role in motor control and cognitive and emotional functions [32]. Depressed patients have been shown to exhibit structural and functional anomalies in the pallidum, caudate, putamen, amygdala, and anterior cingulate [33]. The cortico-striatal-pallidum-thalamic and limbic circuits are considered vital in the pathophysiology of depression [34]. Although the right pallidum did not directly influence the right amygdala in our model, its effect was mediated by the influence of depression, the MSQ score, and the MIDAS score on amygdala volume. The importance of the pallidum in these relationships highlights its potential as a therapeutic target for improving anxiety, depression, and quality of life in patients with MG. Moreover, this may facilitate a new approach to managing patients with MG, whereby physical symptoms as well as emotional and psychological symptoms are considered linked to MG severity. Self-management strategies, such as relaxation techniques, may also be key to regulating anxiety and depression [35]. Question 2: Please review the manuscript carefully as there seemed to be typos or incomplete sentence. For example, I assume you do not need 5) in page 4, lint 74. Answer: We corrected typos and incomplete sentences throughout the manuscript. Thank you for your useful comments and suggestion. We corrected and added the manuscript according to your comments. Reviewer #2 Major concern: Question 1: Language. The author is requested to revise the entire text to make it consistent. (1) In the abstract, the author describes the use of 140 brain regions as the region of interest for analysis, but in the text, the author describes the use of 137 regions (page 6, line 111). Answer: The manuscript revised the entire text, and we all corrected to 140 brain regions throughout the manuscript. Questions 2: Add original word of the abbreviation "MG" (page 3, line 45) Answer: We added the abbreviation “ MG” as follow. P3, L2~ Introduction Migraine (MG), a prevalent neurological disorder marked by severe headaches and an array of neurological symptoms, poses a significant public health challenge worldwide [1,2]. Question 3: Authors need to pay attention to whether the fifth point in the inclusion and exclusion criteria is written (page 4, line 74). Answer: We corrected as follow: P4, L68-73. Thirty-three patients with MG participated in the study. Eligibility criteria were as follows: 1) aged 21 to 60 years, 2) experienced MG attacks for a minimum of 2 years, 3) no clear structural pathology or hyperintense white-matter lesions on previous magnetic resonance imaging (MRI) scans, 4) no comorbid basal ganglia disorder or history of other neurological disorders. Question 4: Quality control. It is recommended that the authors use tools to detect MRI image quality. The authors must explain the necessity of manually adjusting the segmentation [1]. If there is no statistical difference, please use the automated tool to ensure reproducibility; if there is a difference, it is recommended to perform a quality control evaluation in the supplementary material [2]. Answer: Thank you for your suggestion. We also had the automated freesurfer data so we compared pre- and post-manual quality control. We added following statistic results, explanations and a reference you suggested in the method. Also mean and standard deviation from each brain regions extracted from original Freesufer data were indicated in the Supplemental information. P6, L104~ Manual corrections were confined to eliminating non-brain tissue that appeared within the cortical boundary, according to our written guidelines established to ensure consistency. Most errors were located in the paracentral lobule and near the cerebellar tentorium. After any necessary adjustments were made, the images were reprocessed using the recon-all command. According to quality control studies of structural images [26], differences between pre- and post-quality control measurements are small. Nevertheless, we ensured this by comparing all 140 gray matter volumes from the stats files using a paired t-test and a reliablility analysis between pre- and post-manual quality control. There were no differences in gray matter volumes between pre- and post-manual quality control (all P > 0.05) across all participants. We confimed that Cronbach’s alpha was all >0.9 (all mean and standard deviation were indicated in Supplementary Table 1-5). We used the automatically extracted volume to ensure reproducibility. [26] Vahermaa V, Aydogan DB, Raij T, et al. FreeSurfer 7 quality control: Key problem areas and importance of manual corrections. Neuroimage. 2023;279:120306. doi:10.1016/j.neuroimage.2023.120306 Question 5: Statistical Analysis. (1) The author used 137 regions of interest. Did he use multiple comparison corrections in the analysis? Please add relevant content, and if not used, please explain. Answer: We added sentences as follow: P8.L131~ The main objective here was to examine the relationship between symptom severity and the neural substrates underlying symptom severity. Thus, we constrained our hypotheses according to the anatomical connectivity relevant to the MIDAS. As such, gray matter volume differences between MG patients and controls were analyzed using ANCOVA without multiple comparison correction (e.g., family-wise error correction) to identify brain regions relevant to MG. Question 6: Did the author test that the data is normal distribution? For example, using the Shapiro–Wilk test Answer: We performed Shapiro–Wilk test for MIDAS, MSQ, MoCA-J scores, CESD and STAI scores to confirm normality of distribution. For ANCOVA, equality of hometogeneity of variance was confirmed by Levene’s test. We added sentences as follow: P7, L123~ All data were first analyzed using the Shapiro–Wilk test to test the normality of the distribution. Differences in age, years of education, and MIDAS, MSQ, and MoCA-J scores (Shapiro–Wilk, P < 0.05) were analyzed using the non-parametric Mann–Whitney test, and CESD and STAI scores (Shapiro–Wilk, P > 0.05) were analyzed using parametric t-tests. P11, L180~ The homogeneity of variance was equal across groups as confirmed by Levene’s test (all P > 0.05) in the ANCOVA, which indicated that parametric analysis was suitable. Question 7: The migraine subjects recruited by the author were divided into migraine with aura (n = 11) and migraine without aura (n = 22). In the covariance analysis, is there " migraine with aura” a covariate? Answer: We added following sentences in the statistical analysis section. P7, L128~ Because there was no difference in volume between patients with MG with or without aura (as noted in the results section), the presence of aura was not included as a covariate for the ANCOVA. Question 8: Did the author conduct a collinearity test to ensure all variables are independent? Answer: We added following senteces indicated collinearity test results. P12, L195~ For the multiple regression analysis, we examined multicollinearity for each independent variable to ensure the variables were not correlated. The variance inflation factor was < 5 for all independent variables (left pallidum: 3.2, right pallidum: 2.9, and bilateral amygdala: 1.6). Question 9: Interpretability. In the path analysis, the correlation between MIDAS and right amygdala was r=-0.38, a low correlation; the correlation between CESD and right pallidum volume was r=-0.35, which was also a low correlation. Please explain in the discussion: what is the difference between low and high correlation here? Does the low correlation indicate that the right amygdala volume is not closely related to MIDAS? Answer: Thank you for your questions. It is very important to note about each coefficient but we did not indicate details. Straight arrows indicate the direction of causal relationships between variables, and indicate standardized beta coefficients. On the other hand, double-headed curved arrows indicate correlations between exogenous variables, indicated r. We added following descriptions in the statistical analysis, results sections and figure legends. Statistical Analysis, P8, L148~ The structural equation modeling software IBM SPSS Statistics Amos (version 23.0, IBM Corp.) determined the statistical significance of the path coefficients. Straight arrows linking the variables indicate the direction of causal relationships between variables. Double-headed curved arrows indicate correlations between exogenous variables. Standardized beta coefficients are indicated by straight arrows. Results, P13, L223~ In the path model (Fig 2), the three exogenous variables (i.e., state anxiety score, CESD score, and right pallidum volume) were modeled as being correlated, as depicted by the double-headed arrows (indicated r as a correlation coefficient). The endogenous dependent or independent variables were connected by one single-headed arrow (indicated as betas [standardized regression weights]). There was a covariate relationship between the CESD score and state anxiety score (r = 0.81, P < 0.001) and the right pallidum volume (r = −0.35, P = 0.01). The CESD score had a direct effect on the MSQ score (β = 0.58, P < 0.001). The MSQ score had an impact on the MIDAS score (β = 0.71, P < 0.001), and the MIDAS score had an impact on the right amygdala (β = −0.38, P = 0.002). This suggested that a higher MIDAS score corresponded with a smaller right amygdala volume. Figure legends, P14, L230~ Fig 2. Path analysis with standardized regression weights (β; single-headed arrows) and correlation coefficients (double-headed arrows) for the migraine (MG) group. The Migraine Disability Assessment (MIDAS) score followed a standardized direct path to the right amygdala, which suggested that a higher MIDAS score had an impact on the right amygdala volume. The MIDAS score was directly influenced by the Migraine Specific Quality of Life Questionnaire (MSQ) score, and the MSQ score, in turn, had a direct path originating from the Epidemiologic Studies Depression Scale (CESD) score. The CESD score was correlated with the state anxiety score and the right pallidum volume. Attachments Attachment Submitted filename: Response_to_reviewers .doc https://doi.org/10.1371/journal.pone.0301543.r002
18 Mar 2024 Decision Letter - Keisuke Suzuki, Editor The Right Amygdala and Migraine: Analyzing Volume Reduction and Its Relationship with Symptom Severity PONE-D-23-42993R1 Dear Dr. MASAOKA, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at http://www.editorialmanager.com/pone/ and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Keisuke Suzuki, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 4. 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 ****** 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 ****** 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: Authors addressed to the commnets accordingly. Thank you very much for your hard work upon revising the manuscript. ****** 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 ******** https://doi.org/10.1371/journal.pone.0301543.r003
Formally Accepted
24 Mar 2024 Acceptance Letter - Keisuke Suzuki, Editor PONE-D-23-42993R1 PLOS ONE Dear Dr. Masaoka, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Keisuke Suzuki Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0301543.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .