Strategies for improved endothelial cell adhesion in microphysiological vascular model systems

Jingyi Zhu; Halie L. Hotchkiss; Kevin L. Shores; George A. Truskey; Stacey A. Maskarinec

doi:10.1371/journal.pone.0323080

Peer Review History

Original SubmissionDecember 4, 2024
4 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0323080.r001
17 Jan 2025 Decision Letter - Ahmed El-Fiqi, Editor PONE-D-24-55852Strategies for improved endothelial cell adhesion in microphysiological vascular model systemsPLOS ONE Dear Dr. Truskey, 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 03 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. 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Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes Reviewer #2: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. 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 present a lab protocol for optimized endothelial cell seeding within microphysiological vascular model systems (vessel diameters ~0.8 mm). They explore three parameters: (1) endothelial cell seeding density, (2) rotation duration, and (3) method of flow initiation. The parameters they optimize are uniformity and stability of seeded cells upon exposure to 24 hours of fluid shear stress. While previous groups have published protocols for uniformly seeding vascular grafts, the current protocol presents a systematic effort to identify optimal seeding conditions, particularly for microvessels. Furthermore, the protocol presents a flow chamber with innovative design features including a microscopy viewing window and simple vessel clamps. Overall, the manuscript is well written and the experiments are described logically. This is a valuable contribution to the field, especially for groups working with micropysiological vascular model systems. Applicability to the broader field of vascular tissue engineering, which typically utilizes larger diameter vessels, different endothelial cell types, different substrate biomaterials, different adhesion coatings, and different flow conditions, is less certain. Specific comments are below. 1. The rotation speed of the system is described as 15-22 rph and 18-22 rph in different parts of the manuscript. Which is correct and why is there a range? 2. Do the authors expect their optimal conditions to also apply to larger diameter vessels (e.g. 3-6 mm diameter), different endothelial cell types, different biomaterial substrates, different adhesion coatings, and different flow conditions? Might it be necessary to determine optimal seeding conditions for each combination of these parameters? 3. Given that this is a lab protocol manuscript, the detailed step-by-step protocol provided in the supplement is appreciated. However, this is likely too much detail for the typical reader. I suggest providing a high-level overview of the protocol in the Methods section of the manuscript so readers have sufficient information to understand the rest of the manuscript without needing to read the lengthy supplement. 4. The viewing chamber described in Expected Results section A is difficult to understand based on a text description. Please provide a schematic figure with dimensions and labels. What is the distance between the TEBV lumen and the microscope objective? What is the “frontal housing lens?” Example images using a light microscope would be informative. 5. Please provide CAD files for the chamber, chamber holder, and mold designs in the supplement. This will allow others to fabricate similar devices. 6. In Expected Results section B, it would help to clarify the clamping force is provided by snugly assembling the chamber. 7. Please also express the cell seeding densities as cells per cm2 of seeding area. Given the cells are settling onto a surface, the number per area is more useful than the number per volume. 8. Why was en face imaging not performed to calculate cell coverage within the vessels? Why was sectioning performed and circumferential coverage calculated when the chambers nicely provide the ability to directly visualize the cell coverage on the surface of the vessels (e.g. Figure 8B)? 9. Why is there no static group for the data presented in Figure 5C-D? 10. Line 346 may have a typo: starting position in Figure 7A appears to be 90 deg. 11. It is stated that rotational speeds above 29 rph prevent cells from contacting the vessel wall. Can these data be shown? E.g. 30 rph? 12. The last section in Expected Results section C describing the forces acting on the cells reads as disconnected from the rest of the manuscript. Please relate this section back to optimal seedings conditions, especially rotational speed. 13. Please define the white arrows in the caption for Figure 10A. 14. In Figure 1A, consider showing hours/minutes rather than days on the timeline. 15. In Figure 7, consider highlighting the inner wall located at 0.04 cm. Reviewer #2: Reviewing decision for Manuscript Number: PONE-D-24-55852 This study offers an efficient protocol in tissue-engineered blood vessel modeling through the ability to monitor multiple TEBVs simultaneously, developing a viewing chamber for real-time visualization during endothelialization process, simulating the physiological conditions by gradually ramping shear stress, leading to a more stable endothelial layer, demonstrating an optimized process for seeding density and rotation duration, addressing previous gaps in EC adhesion studies and incorporating functional markers (e.g., VE-cadherin, ICAM-1) to confirm endothelial stability and functionality. All these provide an accessible workflow for vascular research. So, I recommend this paper for publication in PLOS One journal. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. 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Revision 1
26 Feb 2025 Author Response Dear Reviewers, We are pleased to submit our revised manuscript, “Strategies for improved endothelial cell adhesion in microphysiological vascular model systems (PONE-D-24-55852)” for consideration for publication in PLOS One Lab Protocols. We thank the reviewers for providing helpful feedback and insightful comments. Please find below our point-by-point response to the reviewers. All line numbers referenced correspond to the tracked-changes version of the manuscript. Reviewer #1 1. The rotation speed of the system is described as 15-22 rph and 18-22 rph in different parts of the manuscript. Which is correct and why is there a range? RESPONSE: We thank the reviewer for the comment. The correct rotation speed range is 18-22 rph. This range reflects the operation of the two custom-made rotators used in our study. Based on our measurements, the average time taken for one rotation is approximately 2.5 minutes, which corresponds to a rotation speed of around 24 rph under ideal conditions. We have included the simulation result for 24 rph in Fig 7C. However, slight fluctuations in speed are expected, and the reported range serves as a practical reference for others who may wish to build similar rotators. We have revised the manuscript to ensure consistency and provide this clarification (lines 115-119). 2. Do the authors expect their optimal conditions to also apply to larger diameter vessels (e.g. 3-6 mm diameter), different endothelial cell types, different biomaterial substrates, different adhesion coatings, and different flow conditions? Might it be necessary to determine optimal seeding conditions for each combination of these parameters? RESPONSE: While the presented conditions were optimized for the current system, we believe the general conclusions are applicable to other systems. However, due to differences in vessel dimensions, simulations and experiments will be necessary to determine the optimal combinations for varying parameters in other systems. We have included this information in the manuscript (lines 743-745). 3. Given that this is a lab protocol manuscript, the detailed step-by-step protocol provided in the supplement is appreciated. However, this is likely too much detail for the typical reader. I suggest providing a high-level overview of the protocol in the Methods section of the manuscript so readers have sufficient information to understand the rest of the manuscript without needing to read the lengthy supplement. RESPONSE: We have included a protocol overview in the Methods section (lines 146-161) to provide readers with sufficient information without requiring review of the supplementary protocol. 4. The viewing chamber described in Expected Results section A is difficult to understand based on a text description. Please provide a schematic figure with dimensions and labels. What is the distance between the TEBV lumen and the microscope objective? What is the “frontal housing lens?” Example images using a light microscope would be informative. RESPONSE: We have added a new schematic figure (Fig 2A) with dimensions and labels to help understand the text description (Fig 2A caption, line 207-214). 5. Please provide CAD files for the chamber, chamber holder, and mold designs in the supplement. This will allow others to fabricate similar devices. RESPONSE: All CAD files for the chamber, chamber holder, and mold designs are now included in the supplementary materials (S1 CAD files). 6. In Expected Results section B, it would help to clarify the clamping force is provided by snugly assembling the chamber. RESPONSE: The TEBV chamber should be tightly closed by hand-tightening the four screws located at the corners of the chamber. Then, use pliers to further tighten the screws evenly until the nuts can no longer be turned. We have clarified that the clamping force is achieved by snugly assembling the chamber components (lines 250-251). 7. Please also express the cell seeding densities as cells per cm2 of seeding area. Given the cells are settling onto a surface, the number per area is more useful than the number per volume. RESPONSE: In this study, the inner radius of the TEBVs was 0.04 cm, resulting in an estimated inner luminal surface area of 0.25 cm² and a lumen volume of 0.005 cm³ for a 1 cm-long TEBV (lines 526-527). Based on these calculations, we have now reported the EC seeding densities as 1.0×105 (low), 1.5×105 (medium), and 2.0×105 (high) ECs/cm² were utilized (lines 287-288). These cell seeding densities have been incorporated into the revised manuscript. 8. Why was en face imaging not performed to calculate cell coverage within the vessels? Why was sectioning performed and circumferential coverage calculated when the chambers nicely provide the ability to directly visualize the cell coverage on the surface of the vessels (e.g. Figure 8B)? RESPONSE: En face sectioning often leads to endothelial cell loss during processing due to the small size of the vessels. Additionally, the opaqueness of the collagen layer in our viewing chamber prevents accurate evaluation of endothelial coverage in situ. Therefore, sectioning and circumferential coverage calculations were deemed the most efficient and reliable methods to evaluate endothelial coverage across the vessel length. 9. Why is there no static group for the data presented in Figure 5C-D? RESPONSE: In Figure 5, we examined EC coverage after 24 hours of perfusion (Fig 5B) and 7 days of perfusion (Fig 5D). Since the static experiments did not undergo any perfusion, the static attachment conditions alone are not a suitable reference group and were therefore removed. The study in Fig 5C-D emphasizes long-term EC coverage under flow. Due to the oxygen and nutrient supply provided by flowing media, static culture of TEBVs for 7 days would result in substantial cell loss and thus was not included in this figure. Furthermore, dynamic perfusion more closely mimics the physiological conditions of blood vessels, aligning with the setup required for downstream applications. 10. Line 346 may have a typo: starting position in Figure 7A appears to be 90 deg. RESPONSE: We have corrected the typo. 11. It is stated that rotational speeds above 29 rph prevent cells from contacting the vessel wall. Can these data be shown? E.g. 30 rph? RESPONSE: We have included an additional figure (Fig 7D) to show cell trajectory under a rotational speed of 30 rph. 12. The last section in Expected Results section C describing the forces acting on the cells reads as disconnected from the rest of the manuscript. Please relate this section back to optimal seedings conditions, especially rotational speed. RESPONSE: We have revised the manuscript to better relate this section to optimal seeding conditions, particularly rotational speed (line 574-576). 13. Please define the white arrows in the caption for Figure 10A. RESPONSE: In the revised caption for Figure 10A (line 652), we have clarified that the white arrows indicate adhered monocytes on the endothelial cell surface within the TEBV lumen. This has been clarified in the figure caption. 14. In Figure 1A, consider showing hours/minutes rather than days on the timeline. RESPONSE: We have modified the timeline in the Figure 1A. 15. In Figure 7, consider highlighting the inner wall located at 0.04 cm. RESPONSE: We have modified Figure 7 to highlight the inner wall location at 0.04 cm. Reviewer #2: This study offers an efficient protocol in tissue-engineered blood vessel modeling through the ability to monitor multiple TEBVs simultaneously, developing a viewing chamber for real-time visualization during endothelialization process, simulating the physiological conditions by gradually ramping shear stress, leading to a more stable endothelial layer, demonstrating an optimized process for seeding density and rotation duration, addressing previous gaps in EC adhesion studies and incorporating functional markers (e.g., VE-cadherin, ICAM-1) to confirm endothelial stability and functionality. All these provide an accessible workflow for vascular research. So, I recommend this paper for publication in PLOS One journal. RESPONSE: We are grateful for the reviewer’s recommendation for publication and their acknowledgment of the contributions our protocol offers to vascular research. In conclusion, we hope these changes adequately respond to the reviewers’ concerns and make the manuscript significantly better. Thank you for your consideration. We look forward to hearing from you. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0323080.r003
14 Mar 2025 Decision Letter - Ahmed El-Fiqi, Editor PONE-D-24-55852R1Strategies for improved endothelial cell adhesion in microphysiological vascular model systemsPLOS ONE Dear Dr. Truskey, 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 Apr 28 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, Ahmed El-Fiqi, Ph.D. 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. Additional Editor Comments (if provided): Please, address these two remaining minor issues raised by reviewer #1 prior to decision processing [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes ******** 2. Has the protocol been described in sufficient detail? To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files. The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses. Reviewer #1: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes ****** 4. If the manuscript contains new data, have the authors made this data 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 ****** 5. Is the article 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 highlight any specific errors that need correcting in the box below. 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: Two remaining minor concerns: 1. It is still not clear why the average rotation speed is stated to be 24 rph and the range is stated to be 18-22 rph. How can it be the average speed is not within the range of speeds? 2. It is stated the raw data would be provided in a supplementary file entitled "Raw Data." However, there is no reference to this file in the text of the manuscript and it is not provided as a supplementary file. ****** 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 ******** [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.0323080.r004
Revision 2
1 Apr 2025 Author Response We thank the reviewer for providing additional feedback. Please find below our point-by-point response to the reviewers. The line numbers referenced here correspond to those in the clean manuscript. 1. It is still not clear why the average rotation speed is stated to be 24 rph and the range is stated to be 18-22 rph. How can it be the average speed is not within the range of speeds? We appreciate the opportunity to further clarify this point. Based on our prior work and the theoretical evaluation of rotational speed during endothelial cell seeding, the initial target range for the rotator system was ~18–22 revolutions per hour (rph). The original rotator, prior to modification, operated at ~ 8 revolutions per minute (rpm). Upon consultation with our machinist, we determined that the motor supplied with the original rotator was not sufficiently powerful to support the necessary gearing changes to achieve the target speed. Reaching the desired 18–22 rph would have required both replacing the motor and manufacturing a custom worm gear set. As an alternative, a standard 20:1 worm gear set was available, which reduced the speed to approximately 24 rph. After discussions with our machinist and other team members, we elected to proceed with this readily available option and retain the original motor. The system was then assembled using a custom housing integrated with the rotating assembly. We have included both values in the manuscript to provide practical references for others who may wish to build a similar system and clarified the reasoning behind the discrepancy (line 113-120). To ensure consistency, we addressed this issue in other relevant sections as needed (line 247-248, line 362, line 423-424). 2. It is stated the raw data would be provided in a supplementary file entitled "Raw Data." However, there is no reference to this file in the text of the manuscript and it is not provided as a supplementary file. Thank you for bringing this issue to our attention. We have included the raw data as the supplementary file. We have also added statement in the figure legends (line 284-285, line 336, line 456-457, line 481-482) to guide readers to the supplementary file entitled "Raw Data." In conclusion, we appreciate the reviewer’s attention to these issues and hope our explanation resolves the confusion. Attachments Attachment Submitted filename: Response_to_reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0323080.r005
3 Apr 2025 Decision Letter - Ahmed El-Fiqi, Editor Strategies for improved endothelial cell adhesion in microphysiological vascular model systems PONE-D-24-55852R2 Dear Dr. Truskey, 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, Ahmed El-Fiqi, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Minor issues have been properly addressed https://doi.org/10.1371/journal.pone.0323080.r006
Formally Accepted
Acceptance Letter - Ahmed El-Fiqi, Editor PONE-D-24-55852R2 PLOS ONE Dear Dr. Truskey, 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. Ahmed El-Fiqi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0323080.r007

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