Theoretical step approach with ‘Three-pillar’ device assistance for successful endoscopic transpapillary gallbladder drainage

Michihiro Yoshida; Itaru Naitoh; Kazuki Hayashi; Yasuki Hori; Akihisa Kato; Kenta Kachi; Go Asano; Hidenori Sahashi; Tadashi Toyohara; Kayoko Kuno; Yusuke Kito; Hiromi Kataoka

doi:10.1371/journal.pone.0281605

Peer Review History

Original SubmissionAugust 3, 2022
18 Oct 2022 Decision Letter - Alejandro Piscoya, Editor PONE-D-22-21361Theoretical ‘Step’ Approach with ‘Three-pillar’ Device Assistance for Successful Endoscopic Transpapillary Gallbladder DrainagePLOS ONE Dear Dr. Naitoh, 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 Dec 02 2022 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. 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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 financial disclosure: " NO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” 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: Yes 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: No ****** 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: Many thanks for giving me the opportunity to review this manuscript. In the submitted study the authors evaluated the clinical role of endoscopic transpapillary gallbladder drainage in patients with acute cholecystitis. In this regard four clinical scenarios were defined that might impair technical access to the gallbladder and therefore proper drainage. A total of 115 patients was retrospectively included evaluating conventional ETGBD technique (Classical ETGBD, N=50) and strategic ETGBD with optional Three-pillar assistance (Strategic ETGBD, N=65). The overall technical success rate was significantly higher for the strategic approach 96.9% compared to the classical approach. In addition, technical methods were evaluated for each clinical scenario in the strategic approach. Overall assessment: The study is of great interest for the endoscopic treatment of patients with acute cholecystitis. The manuscript is technical sound, the data properly support the stated conclusion. Nevertheless, some major points should be addressed: Methods: - The authors already published some studies referring to the same classification system and technical options. I assume that there is a significant overlap of the patients recruited in this retrospective study compared to the enumerated publications (reference numbers 16 - 19). The potential overlap of patients needs to be clearly listed/defined. - The word "step" for the different clinical scenarios/situations is confusing to my opinion. I would prefer to scenario or category 0-4 for the different "steps". Results: - An additional correlation of the "steps" to the severity grade of acute cholecystitis and to the respective technical success rates would be helpful to elucidate potential bias that is caused by the inflammation itself. - The authors only refer to the technical success rates. What about the clinical success rates? Did the patients benefit from the endoscopic interventions? That should be addressed additionally. - Figure 3b: In the legend the pictograms of "conventional", "SG" and "Flex GW" cannot be distinguished (at least in the pdf that was send) Discussion: Technically, three access routes are available to drain the gallbladder in acute cholecystitis as alternative to surgery: (i) Transcutaneous GB-drainage (ii) transluminal GB-drainage (cholecysto-duodenostomy or cholecysto-gastrostomy with LAMS) (iii) transpapillary drainage The authors should discuss the other options in more detail. Especially, what are the pros and cons when comparing the transluminal to the transpapillary approach. Please suggest a respective patient selection as a clinical guidance. Reviewer #2: This study is evaluating a theoretical step strategy for the ETGBD. The authors concluded that it would improve the technical success rate of ETGBD. It was well written, but there were several points to be clarified. 1. In mild acute cholecystitis, it is not usually necessary to do GB drainage. All the patients with mild cholecystitis underwent GB drainage? What is the strategy for acute cholecystitis in your institution? During the study period, PTGBD or EUS-GBD was not performed at all? 2. In the comparison of procedure time, you compare the procedure time defined as time from starting seeking the CD to completion of stent deployment. As the novel approach requires SG insertion before starting the seeking the CD, it was not fair to compare the procedure time under defined conditions. Please provide the overall procedure time for both groups. 3. In table 2, it looks procedure time was shorter in success strategic ETGBD without any pillar than in success conventional ETGBD. What is the main reason for this difference? 4. When did you decide to use each pillar in each step. For example. how long did you continue to seek CD without SG? 5. Please provide the cost of each procedure. It is very important point to select new strategic ETGBD. ****** 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: Yes: Prof. Dr. Mark Ellrichmann 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.0281605.r001
Revision 1
5 Nov 2022 Author Response We are pleased that you are interested in our paper, and reviewer’s comments are extremely helpful for our manuscript to become a more substantial paper. We have answered all reviewer’s comments adequately. We highlighted the changes to the revised manuscript by colored text ‘Revised Manuscript with Track Changes’. A point-by-point reply to the Journal Requirements and the reviewer’s comments is as follows. Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We ensured PLOS ONE’S style requirements and modified file naming. 2. Thank you for stating the following financial disclosure:　"NO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."　At this time, please address the following queries:　d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” We did not receive any funding for this study. We would like to change this statement in the online submission form as follows: The authors received no specific funding for this study. Review Comments to the Author Reviewer #1 Methods: - The authors already published some studies referring to the same classification system and technical options. I assume that there is a significant overlap of the patients recruited in this retrospective study compared to the enumerated publications (reference numbers 16 - 19). The potential overlap of patients needs to be clearly listed/defined. Thank you for the suggestion. We added a list of patients recruited in this study, as S1 Appendix, to clarify the overlap of patients compared to the enumerated publications (Ref.16-19). - The word "step" for the different clinical scenarios/situations is confusing to my opinion. I would prefer to scenario or category 0-4 for the different "steps". As suggested, we reworded ‘step’ for the different clinical situations to ‘Category 0-4’. Thank you for the suggestion. Results: - An additional correlation of the "steps" to the severity grade of acute cholecystitis and to the respective technical success rates would be helpful to elucidate potential bias that is caused by the inflammation itself. We added the correlation of the ‘steps (Category)’ to the severity grade of acute cholecystitis and to the respective technical success rates in S1 and S2 Tables. I appreciate your pointing. - The authors only refer to the technical success rates. What about the clinical success rates? Did the patients benefit from the endoscopic interventions? That should be addressed additionally. The clinical success rate among patients in whom technical success was achieved was 91.7% (33/36) and 93.7% (59/63) in the Classical and Strategic ETGBD groups, respectively, and the difference lacked statistical significance (p=0.711). We added the clinical success rates in the ‘Procedural outcomes’ part (Results part). - Figure 3b: In the legend the pictograms of "conventional", "SG" and "Flex GW" cannot be distinguished (at least in the pdf that was send) Thank you for pointing. We revised them for the clear-cut pictograms. Discussion: Technically, three access routes are available to drain the gallbladder in acute cholecystitis as alternative to surgery: (i) Transcutaneous GB-drainage (ii) transluminal GB-drainage (cholecysto-duodenostomy or cholecysto-gastrostomy with LAMS) (iii) transpapillary drainage. The authors should discuss the other options in more detail. Especially, what are the pros and cons when comparing the transluminal to the transpapillary approach. Please suggest a respective patient selection as a clinical guidance. We agree with this point. As suggested, we added the discussion about the other options in detail, including the pros and cons in the top of the Discussion part. Thank you for the suggestion. Reviewer #2: 1. In mild acute cholecystitis, it is not usually necessary to do GB drainage. All the patients with mild cholecystitis underwent GB drainage? What is the strategy for acute cholecystitis in your institution? During the study period, PTGBD or EUS-GBD was not performed at all? Thank you for pointing. In our practical applications, fundamental strategies for the patients of AC in whom are unfit for early surgery are as follows:1) PTGBD is the first-line drainage procedure. 2) When PTGBD cannot be performed in patients with an anatomically inaccessible GB, those at risk of self-removal of the drainage tube, EUS-GBD or ETGBD is performed as the second-line drainage procedure. 3) In patients with possible CBD stones, suspected GB cancer, serious coagulopathy and thrombocytopenia, or ascites, ETGBD is preferentially performed. We added these explanations of the strategy in the Discussion part. 2. In the comparison of procedure time, you compare the procedure time defined as time from starting seeking the CD to completion of stent deployment. As the novel approach requires SG insertion before starting the seeking the CD, it was not fair to compare the procedure time under defined conditions. Please provide the overall procedure time for both groups. You raised an important point. We agree the importance of the overall procedure time. But, because of the nature of the retrospective study, the overall procedure time involved many participation factors such as the difficulty of biliary cannulation, the presence and extent of CBD stones requiring the extraction, and with or without the necessity of pathological examination, which must have a huge effect on the overall procedure time. So, we defined the procedure time as time from starting seeking the CD to completion of stent deployment to exclude these possible influencing factors. We will leave the analysis of the overall procedure time to future prospective studies. Thank you for pointing. 3. In table 2, it looks procedure time was shorter in success strategic ETGBD without any pillar than in success conventional ETGBD. What is the main reason for this difference? Thanks for your good insight. When conventional ETGBD was performed, we did not have any effective troubleshooter such as three-pillar Device assistance. In difficult cases of conventional ETGBD, it took a lot of work and time to continue to try ETGBD without quitting. In contrast, strategic ETGBD without any pillar tended to involve easy cases. 4. When did you decide to use each pillar in each step. For example. how long did you continue to seek CD without SG? We empirically drew on the procedure time of successful Classical ETGBD (median, 17.5 min). Although it depended on the endoscopist’s preference because of the nature of the retrospective study, we basically continued to seek CD without SG for 15-20 min. 5. Please provide the cost of each procedure. It is very important point to select new strategic ETGBD. The cost of each procedure is as follows: SG, USD 2,039 (JPY 300,000); Flex-GW, USD 156 (JPY 23,000); 3-Fr Micro, USD 169 (JPY 25,000) (currency exchange rate, USD/JPY: 0.0068/1). I added this information in the Discussion part. Thank you very much for your consideration of our paper. We look forward to hearing from you again. Sincerely yours, Itaru Naitoh Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0281605.r002
22 Dec 2022 Decision Letter - Alejandro Piscoya, Editor PONE-D-22-21361R1Theoretical ‘Step’ Approach with ‘Three-pillar’ Device Assistance for Successful Endoscopic Transpapillary Gallbladder DrainagePLOS ONE Dear Dr. Naitoh, 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 Feb 05 2023 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, Alejandro Piscoya 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. 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 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 Reviewer #2: No ****** 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: The authors`sufficiently answered alls question that were raised, the manuscript was adapted accordingly. Many thanks for the opportunity to review this manuscript. Reviewer #2: It was well revised and written, but there were some point to be clarified. 1. Though this study is a retrospective nature, it would be important to show overall procedure time in both groups. 2. Please provide overall procedure time in failed cases in both groups to minimize the potential selection biases. 3. In the study periods, how many patients underwent gallbladder drainage other than ETGBD. Please provide the flow chart of the management of acute cholecystitis during this periods. ****** 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: Yes: Prof. Dr. Mark Ellrichmann 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.0281605.r003
Revision 2
23 Dec 2022 Author Response We are pleased that you are interested in our paper, and reviewer’s comments are helpful for our manuscript to become a more substantial paper. We have answered all reviewer’s comments adequately. We highlighted the changes to the revised manuscript by colored text ‘Revised Manuscript with Track Changes R2’. A point-by-point reply to the reviewer’s comments is as follows. Reviewer #2: It was well revised and written, but there were some point to be clarified. 1. Though this study is a retrospective nature, it would be important to show overall procedure time in both groups. As suggested, we added overall procedure time of both groups in the ‘Procedure time’ part (Results part) and S3 table. Thank you for the suggestion. 2. Please provide overall procedure time in failed cases in both groups to minimize the potential selection biases. As suggested, we also added overall procedure time of failed cases in the ‘Procedure time’ part (Results part) and S3 table. Thank you for the suggestion. 3. In the study periods, how many patients underwent gallbladder drainage other than ETGBD. Please provide the flow chart of the management of acute cholecystitis during this periods. In our practical applications during this period, fundamental strategies for the patients of AC in whom are unfit for early surgery (N=380) are as follows:1) PTGBD is the first-line drainage procedure (N=219). 2) When PTGBD cannot be performed in patients with an anatomically inaccessible GB, those at risk of self-removal of the drainage tube, EUS-GBD (N=46) or ETGBD (N=115) is performed as the second-line drainage procedure. 3) In patients with possible CBD stones, suspected GB cancer, serious coagulopathy and thrombocytopenia, or ascites, ETGBD is preferentially performed. We updated these explanations of the strategy in the Discussion part and added the flow chart of the management of AC during this period in S1 Figure. Thank you for the suggestion. Attachments Attachment Submitted filename: Response to Reviewers R2.docx https://doi.org/10.1371/journal.pone.0281605.r004
27 Jan 2023 Decision Letter - Alejandro Piscoya, Editor Theoretical ‘Step’ Approach with ‘Three-pillar’ Device Assistance for Successful Endoscopic Transpapillary Gallbladder Drainage PONE-D-22-21361R2 Dear Dr. Naitoh, 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, Alejandro Piscoya Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0281605.r005
Formally Accepted
1 Feb 2023 Acceptance Letter - Alejandro Piscoya, Editor PONE-D-22-21361R2 Theoretical Step Approach with ‘Three-pillar’ Device Assistance for Successful Endoscopic Transpapillary Gallbladder Drainage Dear Dr. Naitoh: 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 Professor Alejandro Piscoya Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0281605.r006

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