Dermal formulation based on carbopol and Gum Arabic improves skin retention of indomethacin

Hiroko Otake; Ogata Fumihiko; Yosuke Nakazawa; Manju Misra; Masanobu Tsubaki; Naohito Kawasaki; Noriaki Nagai

doi:10.1371/journal.pone.0326051

Peer Review History

Original SubmissionFebruary 9, 2025
9 Feb 2025 Author Response https://doi.org/10.1371/journal.pone.0326051.r001
Decision Letter - Giovanni Signore, Editor PONE-D-25-07074Dermal formulation based on carbopol and gum arabic improves skin retention of indomethacinPLOS ONE Dear Dr. Nagai, 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. My personal evaluation of the manuscript, in line with those of the reviewers, is that with a careful and well performed revision of the issues raised the manuscript could be a valuable addition to PLOS One Please submit your revised manuscript by May 09 2025 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 also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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: No 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: 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: The manuscript attempts to evaluate an indomethacin gel loaded with microparticles and nanoparticles; however, the study lacks scientific rigor and is not well-supported by valid reasoning from previous literature. Several major issues need to be addressed before this work can be considered for publication. (1) Lack of Justification for Polymer Selection and Comparison The study primarily focuses on the effect of a polymer, but the justification for its selection is weak. Instead of comparing your results to well-established studies in the literature, you have only compared them with your previous work. This approach does not provide a strong scientific basis for the study. (2) Poorly Written Results Section The results are not well-detailed, particularly for thermogravimetric analysis (TGA). There is no in-depth discussion of thermal degradation, weight loss patterns, or any comparative analysis. Such details are essential to support the stability of the formulation. (3) Lack of Polymer-Drug Compatibility Data Fourier Transform Infrared Spectroscopy (FTIR) data is missing or inadequately discussed. FTIR is a crucial tool for assessing polymer-drug interactions, and without this analysis, the compatibility of the polymer with indomethacin remains uncertain. (4) Incomplete Evaluation of Formulation Safety and Stability Skin irritation studies are absent, which is a significant limitation since any topical formulation must be evaluated for its safety on the skin. Stability data is also missing. Long-term and short-term stability studies are essential to establish the robustness of the formulation over time. (5) Image quality The figure quality is poor and the formulation pictures are blurred. Also, the graphical abstract is not self-explanatory. The different size of arrows will be difficult to elucidate by a young researcher or new reader. Overall, the manuscript is weak and does not provide a comprehensive or scientifically sound evaluation of the formulation. The lack of literature-based justification, inadequate results, missing polymer-drug compatibility studies, and absence of crucial safety and stability data significantly weaken the study. The manuscript requires substantial revisions before it can be considered for further review. Reviewer #2: The study could contribute to pharmaceutical sciences and dermatological formulations by providing an improved method for delivering indomethacin through the skin. In this paper, authors evaluated penetration, retention, drug delivery and systemic absorption of indomethacin, using two different gel formulations, based on microparticles or nanoparticles. The claim is well supported by the current literature. The details of the methodology are sufficient to allow the experiments to be reproduced. Experiments are appropriate and well-designed, although data analysis is not always rigorous, leading to conclusions that are not fully substantiated. Although in its current form the paper is unsuitable for publication, it shows good potential and authors are encouraged to resubmit a revised version. The manuscript is overall well organized and written in a clear scientific English. A certificate of editing is available. Please, see the attached document for further comments and requests of review. ****** 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: Yes: Gemma Sardelli, PhD ******** [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. Attachments Attachment Submitted filename: Review PONE-D-25-07074.docx https://doi.org/10.1371/journal.pone.0326051.r002
Revision 1
4 Apr 2025 Author Response We carefully revised our manuscript according to the suggestions of the reviewers, and details are as follows. <Q and A for Reviewer 1> Q1. Lack of Justification for Polymer Selection and Comparison: The study primarily focuses on the effect of a polymer, but the justification for its selection is weak. Instead of comparing your results to well-established studies in the literature, you have only compared them with your previous work. This approach does not provide a strong scientific basis for the study. A1. These are excellent points. This study employs the bead milling method to prepare drug nanocrystals using gum arabic as an additive, and evaluates the dermal drug behavior when these nanocrystals are incorporated into a Carbopol gel. To clarify the research purpose, we have added a statement and reference on the usefulness of Carbopol gel as a base in the development of dermal formulations. These revisions have addressed the previously noted lack of justification for polymer selection and comparison. We appreciate your valuable feedback (line 85-92, Reference 19-22). Reference Hatem S, Kamel AO, Elkheshen SA, Nasr M, Moftah NH, Ragai MH, et al. Nano-vesicular systems for melanocytes targeting and melasma treatment: In-vitro characterization, ex-vivo skin retention, and preliminary clinical appraisal. Int J Pharm. 2024;665:124731. doi: 10.1016/j.ijpharm.2024.124731. Salem HF, Abd El-Maboud MM, Said ASA, Salem MN, Sabry D, Hussain N, et al. Nano Methotrexate versus Methotrexate in Targeting Rheumatoid Arthritis. Pharmaceuticals (Basel). 2022;16(1):60. doi: 10.3390/ph16010060. Patel V, Mehta TA. Betamethasone Dipropionate Nanocrystals: Investigation, Feasibility and In Vitro Evaluation. AAPS PharmSciTech. 2022;23(6) :197. doi: 10.1208/s12249-022-02346-1. Khan D, Ahmed N, Muhammad A, Shah KU, Mir M, Rehman AU. A macromolecule infliximab loaded reverse nanomicelles-based transdermal hydrogel: An innovative approach against rheumatoid arthritis. Biomater Adv. 2025;167:214093. doi: 10.1016/j.bioadv.2024.214093. Q2. Poorly Written Results Section: The results are not well-detailed, particularly for thermogravimetric analysis (TGA). There is no in-depth discussion of thermal degradation, weight loss patterns, or any comparative analysis. Such details are essential to support the stability of the formulation. A2. In order to respond to the comment, we added the TG data, and discussed for thermal degradation, weight loss patterns, and any comparative analysis in the Result. Thank you for pointing this out (line 340-347, Figure 2 and Figure 2 legend). Q3. Lack of Polymer-Drug Compatibility Data: Fourier Transform Infrared Spectroscopy (FTIR) data is missing or inadequately discussed. FTIR is a crucial tool for assessing polymer-drug interactions, and without this analysis, the compatibility of the polymer with indomethacin remains uncertain. A3. The reviewer’s comments are very important. In this study, the bead milling method was employed to prepare drug nanocrystals using gum arabic as an additive, and the transdermal drug behavior of these nanocrystals incorporated into a Carbopol gel was evaluated. To assess the crystalline structure before and after bead milling, X-ray diffraction (XRD) was utilized, while the physical properties were evaluated using differential thermal analysis (TG-DTA). Although these measurements allow for the assessment of physicochemical properties, Fourier Transform Infrared Spectroscopy (FTIR) data are usefulness to evaluate the compatibility of the polymer with indomethacin. In accordance with the given suggestions, we have added the TG data (Figure 2D). Moreover, we added the necessary of FTIR results in future studies. Thank you very much for pointing this out (line 486-491, Figure 2D). Q4. Incomplete Evaluation of Formulation Safety and Stability: Skin irritation studies are absent, which is a significant limitation since any topical formulation must be evaluated for its safety on the skin. A4. These are excellent points. In order to respond to the comment, we added the result of skin irritation (line 227-234, 357-358, 483-484, Table 1). Q5. Stability data is also missing. Long-term and short-term stability studies are essential to establish the robustness of the formulation over time. A5. In order to respond to the comment, we added the results on the degradation stability and viscosity changes after 1 months of storage in a sealed and dark environment (line 222-225, 354-357, 481-483, Table 1). Q6. Image quality: The figure quality is poor and the formulation pictures are blurred. Also, the graphical abstract is not self-explanatory. The different size of arrows will be difficult to elucidate by a young researcher or new reader A6. The reviewer’s comment is correct. We revised the figure quality and graphical abstract (Figure 8). Briefly, the size and shape of the arrows were standardized, and the differences in drug release and permeability were represented by the number of arrows (Figure 8). In addition, we also exchanged the formulation pictures in Figure 1D and Figure 8. Thank you very much for pointing this out (Figure 1D and Figure 8). Thank you for great comments. <Q and A for Reviewer 2> Q1. Line 80: incomplete sentence. Please, revise for clarity. A1. The reviewer’s comment is correct. We revised this sentence (line 80-81). Q2. Lines 318-319: The claim that particles in IMP-NC are more evenly distributed than in IMP-MC is not sufficiently supported by Image 1D alone. Further justification or quantitative analysis is needed. A2. The reviewer’s comments are very important. We have replaced the formulation images with higher-resolution versions (Figure 1D). In addition, the quantitative analysis of drug uniformity in the gel were shown in Figure 3A. We added this information in the relevant part (line 337-339, Figure 1D and 3A). Q3. Line 335: a scale bar should be added to figure 1D. A3. In order to respond to the comment, we added the scale bar (5 mm) in the Figure 1D (Figure 1D). Q4. Line 372: Instead of a multiple comparison, compare each parameter of the two formulations individually using a Student’s t-test or a non-parametric test if normality is not satisfied. A4. The reviewer’s comment is correct. We compared each parameter of the two formulations individually by using a Student’s t-test (line 413). Q5. Line 376: The text (lines 365-366) states that no significant differences were observed in the reported parameters between the two formulations, but the statistical test you used is not specified. Please, clarify. A5. In order to respond to the comment, we compared each parameter of the two formulations individually by using a Student’s t-test. We mentioned the information in the text (line 407). Q6. Line 385: ANOVA One-Way with Tukey-Kramer post hoc test is not suitable in this context, since measurements overtime are not independent (aliquots at different time points come from the same sample, thus they are dependent). A repeated-measures ANOVA should be used if normality is satisfied. A6. The reviewer’s comment is correct. We reanalyzed the statistical analysis by using JMP software version 5.1 (SAS Institute). In order to respond to the comment, we compared each parameter of the two formulations individually by using a repeated-measures ANOVA. We mentioned the information in the text (line 322-324, 426). Q7. Line 391: The caption should specify that this is the in vitro approach. A7. These are excellent points. We have explicitly stated in the caption that this is an in vitro approach (line 431, 434). Q8. Line 393: As above, ANOVA One-Way with Tukey-Kramer post hoc test is inappropriate, since also these measurements are not independent each other. Use repeated-measures ANOVA if normality is met. A8. The reviewer’s comments are very important. In order to respond to the comment, we evaluated the statistical analysis using a repeated-measures ANOVA, and mentioned the information in the text (line 322-324, 434-435). Q9. Lines 395-397: You would put this sentence in the main text and discuss its implication, as it suggests that that the IMC (but not the carrier) can pass through the skin, at least in vitro. A9. In accordance with the suggestions, we mentioned that, the IMC can pass through the skin, at least in vitro. Thank you for pointing this out (line 399, 402-404, 508). Q10. Line 401: The caption is inconsistent with the graph (Fig. 6A), which shows a time scale rather than plasma IMC levels 6 hours after treatment. Please correct this inconsistency. A10. The reviewer’s comment is correct. We corrected to “24 h” from “6 h” (line 442). Thank you for great comments. Attachments Attachment Submitted filename: 250329Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0326051.r003
Decision Letter - Giovanni Signore, Editor PONE-D-25-07074R1Dermal formulation based on carbopol and gum arabic improves skin retention of indomethacinPLOS ONE Dear Dr. Nagai, 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.i agree with the reviewer that a further check should be performed on the result of the statistical analysis provided, to rule out the possibility for mistakes. Please submit your revised manuscript by Jun 14 2025 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, Giovanni Signore Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #2: (No Response) ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: 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 #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 #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 #2: The authors have addressed all the comments raised in the first round of review. Although the request to adopt a different and more appropriate method of data analysis has been accepted and the changes have been implemented in the main text and/or figure caption, the statistical significance remains exactly the same as in the previous, incorrect analysis. The authors are kindly requested to check for any typos or inconsistencies in the revised version of the manuscript, or to demonstrate the validity of the results obtained from the newly performed analysis. ****** 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 #2: Yes: Gemma Sardelli ******** [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.0326051.r004
Revision 2
1 May 2025 Author Response We carefully revised our manuscript according to the suggestions of the reviewer 2, and details are as follows. <Q and A for Reviewer 2> Q1. The authors have addressed all the comments raised in the first round of review. Although the request to adopt a different and more appropriate method of data analysis has been accepted and the changes have been implemented in the main text and/or figure caption, the statistical significance remains exactly the same as in the previous, incorrect analysis. The authors are kindly requested to check for any typos or inconsistencies in the revised version of the manuscript, or to demonstrate the validity of the results obtained from the newly performed analysis. A1. The reviewer’s comment is correct. We have appropriately corrected the symbols in Figures 4A and 5A. In addition, we have revised the corresponding descriptions in the manuscript, and carefully checked that the content is accurately reflected. Thank you very much for pointing this out (line 322, 424, 432, Figure 4A and 5A). Thank you for great comments. Attachments Attachment Submitted filename: 250502Response_to_Reviewers.docx https://doi.org/10.1371/journal.pone.0326051.r005
Decision Letter - Giovanni Signore, Editor Dermal formulation based on carbopol and gum arabic improves skin retention of indomethacin PONE-D-25-07074R2 Dear Dr. Nagai, 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 Editorial Manager® 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, Giovanni Signore 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 #2: 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 #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: (No Response) ****** 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 #2: The authors have met all the requirements, and the graphs have been appropriately adjusted in line with the revised statistical analysis. ****** 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 #2: Yes: Gemma Sardelli ******** https://doi.org/10.1371/journal.pone.0326051.r006
Formally Accepted
Acceptance Letter - Giovanni Signore, Editor PONE-D-25-07074R2 PLOS ONE Dear Dr. Nagai, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. Giovanni Signore Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0326051.r007

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