A standardized method for plasma extracellular vesicle isolation and size distribution analysis

J. Nathaniel Diehl; Amelia Ray; Lauren B. Collins; Andrew Peterson; Kyle C. Alexander; Jacob G. Boutros; John S. Ikonomidis; Adam W. Akerman

doi:10.1371/journal.pone.0284875

Peer Review History

Original SubmissionSeptember 22, 2022
19 Oct 2022 Decision Letter - Vibhuti Agrahari, Editor PONE-D-22-26362A standardized method for plasma extracellular vesicle isolation and size distribution analysisPLOS ONE Dear Dr. Diehl, 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 03 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. 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, Vibhuti Agrahari Academic Editor PLOS ONE Journal requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. [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 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: No 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: N/A 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: In this manuscript, the authors describe protocols for preparing plasma samples to be subsequently processed by automated size exclusion chromatography and tunable resistive pulse sensing to determine the concentration and size distribution of EVs in plasma. Protocols of this kind can be helpful in particular for applications of EVs as biomarkers of diseases using small blood plasma volume or for analytic purposes to characterize further and study the functional activities of the isolated EV e.g., in cellular models. The authors provide substantial evidence supporting the reliability of the methods they describe. The possibility to scale up the procedures is more arguable, as other types of fractionation methods using other commercial size exclusion resins and hardware would instead be used. One primary concern I have with the manuscript is its questionable novelty as compared to other (non-cited) publications (even though I admit they were not all written as protocols) describing the way how to process blood samples and isolate (including by q-EVs) and analyze (including by TRPS) the EVs, e.g., https://pubmed.ncbi.nlm.nih.gov/20831623/; https://www.ahajournals.org/doi/full/10.1161/CIRCRESAHA.117.309417; https://www.cell.com/cell-reports-methods/pdfExtended/S2667-2375(21)00103-X). For instance, some of these papers do also provide "specific guidance on the peripheral blood draw component of human plasma isolation" and use q-EV SEC and TRPS. What is unique in the current manuscript that has not been highlighted in previous publications? Regarding the validation of the procedures, I would also be interested to know why other methods like NTA and DLS have not been used to validate the isolation and characterization methods obtained by TRPS. Other comments: Line 72: isn't ---- > is not Line 86: is it 10um that is meant? Or rather 1um? Lines 117-118: NTA is listed twice Lines 125 and below: It is debatable to claim that TRPS is the most reliable and accurate method with sub-nm precision, and is superior to the more commonly used light scattering techniques. TRPS has, like other methods, limitations for heterogeneous EV populations, and the data obtained are directly impacted by the type of membrane selected to avoid clogging. Most people would think combining techniques is suitable for characterizing heterogeneous EV populations. Therefore I do not see the need to promote TRPS (or other Izon products) in this manuscript that describes protocols. Lines 149-150: is there any risk of losing EV on the membrane of the centrifugal filter? Reviewer #2: Diehl and colleagues submitted a manuscript of protocol to explain the standardized method for the characterization of EVs in plasma. This manuscript describes detailed procedures of plasma collection, EVs isolation, and sample procedures. In this paper, the explanation of the rationale for the selection of methods (SEC and TRPS). This paper is well-organized and has a logical basis. If some requirements in the following comments are modified, this paper is going to be able to clear information for readers. Minor comments. 1. Please add the information of reference at (DOI xx) line 148. 2. Spacing between number and unit (lines 84 and 86). 3. Add unit at diameter (line 269 (Marfan 69.92)) 4. In terms of utilization of this technique, Marfan syndrome was selected to show the difference in the number and size distribution of EVs in plasma. Please add the physiological background information to explain why this syndrome could be a model as a diagnostic target. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0284875.r001
Revision 1
13 Dec 2022 Author Response We thank the reviewers for their thoughtful comments. We had addressed each one directly and have provided both a response and the amended manuscript text. This process improved the overall quality of our submission immensely. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We formatted the font size, line spacing, headings/subheadings, and placement of the figure legends to adhere to the specifications provided in the above links. 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. We have included all full blot images as specified by the editors. This has been noted in the cover letter. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. We have included the full data sets in the supplement as specified by the editors. This has been noted in the cover letter. 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 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: No Reviewer #2: Yes The protocol describes a validated method. Validation data is located in figure 2. ________________________________________ 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: N/A Reviewer #2: Yes Validation data is in figure 2. Additionally, all data is now located in the supplement. ________________________________________ 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: In this manuscript, the authors describe protocols for preparing plasma samples to be subsequently processed by automated size exclusion chromatography and tunable resistive pulse sensing to determine the concentration and size distribution of EVs in plasma. Protocols of this kind can be helpful in particular for applications of EVs as biomarkers of diseases using small blood plasma volume or for analytic purposes to characterize further and study the functional activities of the isolated EV e.g., in cellular models. The authors provide substantial evidence supporting the reliability of the methods they describe. The possibility to scale up the procedures is more arguable, as other types of fractionation methods using other commercial size exclusion resins and hardware would instead be used. Thank you for recognizing the importance of these protocols. We have clarified how we plan to leverage automation to address concerns of scalability. The following statement has been added to the introduction. “When size exclusion chromatography (SEC) is performed with automated fraction collectors, it not only isolates vesicles with high levels of purity and recovery efficiency, it also scales with each additional collector [32]. Other available methods, such as asymmetric flow field-flow fractionation (AF4), tangential flow fractionation (TFF), differential centrifugation, and precipitation are not scalable in this fashion.” One primary concern I have with the manuscript is its questionable novelty as compared to other (non-cited) publications (even though I admit they were not all written as protocols) describing the way how to process blood samples and isolate (including by q-EVs) and analyze (including by TRPS) the EVs, e.g., https://pubmed.ncbi.nlm.nih.gov/20831623/; https://www.ahajournals.org/doi/full/10.1161/CIRCRESAHA.117.309417; https://www.cell.com/cell-reports-methods/pdfExtended/S2667-2375(21)00103-X). For instance, some of these papers do also provide "specific guidance on the peripheral blood draw component of human plasma isolation" and use q-EV SEC and TRPS. What is unique in the current manuscript that has not been highlighted in previous publications? We carefully assessed each article provided and have added the following paragraph to the introduction section. We addressed uniqueness and novelty specifically. “Myriad methods can analyze EVs and each may be appropriate for specialized applications.[40-42] For our purposes, however, a combination of automated SEC and TRPS works best. Because our protocol provides a simultaneous measurements of absolute quantification and size distribution of EVs 30-300 nm, applications like flow cytometry, NTA, and DLS, are not appropriate as detection is often limited to particles greater than 100 nm (or 60 nm for NTA/DLS) in diameter or do not provide absolute quantification.[43, 44] The uniqueness of this protocol lies in the way we leverage this combination of SEC and TRPS. Specifically, this protocol provides a method for reproducible quantification and is performed in a way that allows vesicles to remain intact throughout the measurement process, thus providing novel methods for normalization in any downstream application.” Regarding the validation of the procedures, I would also be interested to know why other methods like NTA and DLS have not been used to validate the isolation and characterization methods obtained by TRPS. We appreciate that NTA and DLS are useful technologies and validation methodologies. However, our protocol is expressly built around TRPS for absolute quantification. Being that NTA and DLS do not quantify absolutely, we chose to maintain a singularly focused approach. Other comments: Line 72: isn't ---- > is not Thank you for identifying this error, it has been corrected. “Differential centrifugation protocols have been used many times, but this practice is not viable for several reasons having to do with the stress applied to vesicles by centrifugal forces (26).” Line 86: is it 10um that is meant? Or rather 1um? The original citation contained a typographical error. This has been corrected to reflect the actual size range: 50nm – 1um. “In both function and size, microvesicles are extremely heterogeneous: their size ranges between 50nm and 1µm and they shed into the extracellular milieu by the budding of the cell membrane (28).” Lines 117-118: NTA is listed twice Thank you for identifying this error, it has been corrected. “Several methods of EV quantification exist. These include resistive pulse sensing, nanoparticle tracking analysis, microscopy (scanning/transmission electron microscopy, and atomic force microscopy), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), flow cytometry, and small-angle X-ray scattering (SAXS) (27). Lines 125 and below: It is debatable to claim that TRPS is the most reliable and accurate method with sub-nm precision, and is superior to the more commonly used light scattering techniques. TRPS has, like other methods, limitations for heterogeneous EV populations, and the data obtained are directly impacted by the type of membrane selected to avoid clogging. Most people would think combining techniques is suitable for characterizing heterogeneous EV populations. Therefore I do not see the need to promote TRPS (or other Izon products) in this manuscript that describes protocols. To address concerns about perceived promotion of any one company’s product, we have amended the sentence such that it now states only the objective differences. “TRPS quantifies absolutely, whereas other commonly used light scattering techniques provide bulk estimates.” Lines 149-150: is there any risk of losing EV on the membrane of the centrifugal filter? This is an excellent point. The following statement has been added to clarify the purpose of centrifugal concentration of EVs. “While we recognize some EVs may be lost during centrifugal concentration, this step was performed solely to further concentrate the samples so that even amounts of total protein (10 �g) are analyzed by western blot.” Reviewer #2: Diehl and colleagues submitted a manuscript of protocol to explain the standardized method for the characterization of EVs in plasma. This manuscript describes detailed procedures of plasma collection, EVs isolation, and sample procedures. In this paper, the explanation of the rationale for the selection of methods (SEC and TRPS). This paper is well-organized and has a logical basis. If some requirements in the following comments are modified, this paper is going to be able to clear information for readers. Thank you for recognizing the importance of these protocols. We have addressed the following comments and enhanced the text’s overall clarity. We especially appreciate this reviewer’s careful attention to minute detail. Minor comments. 1. Please add the information of reference at (DOI xx) line 148. We appreciate reviewer 2 pointing out our mistake, and we have rectified it by listing both follow protocol related DOIs: (dx.doi.org/10.17504/protocols.io.3byl4jeb2lo5/v1, dx.doi.org/10.17504/protocols.io.ewov1ojnolr2/v1) 2. Spacing between number and unit (lines 84 and 86). Thank you for identifying the error, we have corrected the issue. “EVs are divided into two groups: exosomes and microvesicles. Exosomes are commonly reported to be 30-120 nm in size and are released from multivesicular endosomes following fusion with the plasma membrane (28). In both function and size, microvesicles are extremely heterogeneous: their size ranges between 50 nm and 1 µm and they shed into the extracellular milieu by the budding of the cell membrane (28).” 3. Add unit at diameter (line 269 (Marfan 69.92)) Thank you for identifying the error, we have corrected the issue. “…Marfan 1.47 x 1011 particles/mL), mean diameter of EV particle (control 77.75 nm, Marfan 69.92 nm)…” 4. In terms of utilization of this technique, Marfan syndrome was selected to show the difference in the number and size distribution of EVs in plasma. Please add the physiological background information to explain why this syndrome could be a model as a diagnostic target. We have explained the physiological linkage between Marfan syndrome, aortic aneurysm, and extracellular vesicle concentration. “We utilized the techniques described here to address a clinically meaningful question. In our system, we measure EV concentration and size in the plasma of healthy control patients and in Marfan patients who possessed a known thoracic aortic aneurysm. Previously, we have demonstrated that increased wall tension, as seen in aneurysm disease, leads to increased aortic fibroblast secretion of EVs. The primary cardiovascular complication associated with Marfan Syndrome is thoracic aortic aneurysm. Therefore, we are interested in determining whether EV concentration and size distribution can be leveraged as a diagnostic marker for aneurysm disease.” ________________________________________ Attachments Attachment Submitted filename: Diehl_plosone_reviewerresponse.docx https://doi.org/10.1371/journal.pone.0284875.r002
11 Apr 2023 Decision Letter - Vibhuti Agrahari, Editor A standardized method for plasma extracellular vesicle isolation and size distribution analysis PONE-D-22-26362R1 Dear Dr. Natheniel, 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, Vibhuti Agrahari Academic Editor PLOS ONE Additional Editor Comments (optional): 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 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: I thank the authors for responding to my questions and modifying the manuscript accordingly. I have no further comments Reviewer #2: Thank you for revising the manuscript. Your protocol for exosome isolation and protein sample preparation is suitable to study the differences of EVs concentration between Marfan patients and control patients. ****** 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 ******** https://doi.org/10.1371/journal.pone.0284875.r003

Open letter on the publication of peer review reports

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

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

Learn more at ASAPbio .