Protease-activated receptor 2 deficient mice develop less angiotensin II induced left ventricular hypertrophy but more cardiac fibrosis

Albrecht Meyer zu Schwabedissen; Silvia Vergarajauregui; Marko Bertog; Kerstin Amann; Felix B. Engel; Christoph Daniel

doi:10.1371/journal.pone.0310095

Peer Review History

Original SubmissionJanuary 4, 2024
25 Apr 2024 Decision Letter - Luis Eduardo M Quintas, Editor PONE-D-24-00413Protease-activated receptor 2 deficient mice develop less angiotensin II induced left ventricular hypertrophy but more cardiac fibrosisPLOS ONE Dear Dr. Daniel, 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 Jun 09 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Luis Eduardo M Quintas, Ph.D. Academic Editor PLOS ONE Journal requirements: 1. When submitting your revision, we need you to address these additional requirements. 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. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. 3. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 4. We note that you have indicated that there are restrictions to data sharing for this study. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Before we proceed with your manuscript, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., a Research Ethics Committee or Institutional Review Board, etc.). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. You also have the option of uploading the data as Supporting Information files, but we would recommend depositing data directly to a data repository if possible. We will update your Data Availability statement on your behalf to reflect the information you provide. 5. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 6. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: Partly ******** 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: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: 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: Meyer zu Schwabedissen et. al conduct a study evaluating the effects of PAR2 deficiency on ang II-induced cardiac injury and find that it promotes fibrosis but reduces left ventricular hypertrophy. The manuscript could be strengthened if the following points are addressed: - What would explain the increase in p-ERK1/2 in the PAR2 KO healthy control animals? These results are drastically different than your other groups. -Would sequencing the heart tissue provide insights into potential mechanisms regulating the enhanced fibrosis in the PAR2 KO Ang II groups? - Given the variance it is hard to determine if there are true differences in P-Smad2/3, could this be measured by western blot instead? Same with the P-ERK1/2? -Are blood pressure measurements, hemodynamics or echocardiography measurements possible to determine physiological and functional consequences of PAR2 deficiency? Reviewer #2: Schwabedissen et al. investigated roles of protease activated receptor 2 (PAR2) on the heart using PAR2-KO mice. The mice were treated with angiotensin II (Ang II) for 4 weeks using osmotic minipumps, and then various in vivo studies were performed. They found that left ventricular hypertrophy (LVH) after Ang II treatment was inhibited in PAR2-KO mice, although cardiac interstitial fibrosis was significantly higher in PAR2-KO mice compared to wild-type mice. They concluded that PAR2 has multiple effects in AngII-stimulated cardiac tissue and its role in cardiac remodelling appears to be ambivalent. Although this paper is well-written and provides some scientific implications, there are severe concerns in the present study. Major criticism: 1. Important findings in the present study is related to cardiac perivascular fibrosis. PAR2 deficiency with Ang II treatment enhanced cardiac perivascular fibrosis. However, the authors only showed historical data and their data are very descriptive without any mechanistic insights. At least some in vitro studies are required. Would it be possible to perform in vitro studies using isolated cardiac fibroblasts from PAR2-deficient mice? 2. The authors showed that cardiac hypertrophy induced by Ang II treatment was inhibited in PAR2-KO mice. How was this inhibition related with cardiac perivascular fibrosis? This is a critical question in this paper. 3. Although FGF23 mRNA expression levels in heart were shown in Figure 1, this is not enough. How were alpha-MHC and/or BNP mRNA expression levels in heart? 4. The authors showed that P-ERK1/2-positive cells in heart were significantly increased in PAR2-KO mice, but they were not increased in all other groups (Figure 2A). Why did these happen? Why did Ang II treatment decrease P-ERK1/2-positive cells in the heart frm PAR2-KO mice? More clear explanation and/or additional experiments were required to confirm this result. 5. The number of experiments or mice should be described in the main text and figure legends. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0310095.r001
Revision 1
6 Aug 2024 Author Response Reviewer #1: Meyer zu Schwabedissen et. al conduct a study evaluating the effects of PAR2 deficiency on ang II-induced cardiac injury and find that it promotes fibrosis but reduces left ventricular hypertrophy. The manuscript could be strengthened if the following points are addressed: - What would explain the increase in p-ERK1/2 in the PAR2 KO healthy control animals? These results are drastically different than your other groups. This observation also surprised us the most. Other studies have shown that MAP kinases play an important role in the regulation of hypertrophy and fibrosis. We therefore investigated this in our study. However, we did not expect that P-ERK1/2 will be increased in the healthy PAR2 deficient mice. Other studies have shown that activation of PAR2 leads to increased phosphorylation of ERK1/2, so it is surprising that we observed the opposite, increased activation in the healthy PAR2-deficient animals. Since ERK1/2 can also be activated by other pathways, the observed response could also be a kind of compensation. Such compensations can occur especially in knock-out animals. Our experiments with isolated fibroblasts, newly included in the revised manuscript, showed the strongest phosphorylation of ERK1/2 in the unstimulated fibroblasts treated with a PAR-2 inhibitor (see Figure 2D). However, these results reached not the level of significance. -Would sequencing the heart tissue provide insights into potential mechanisms regulating the enhanced fibrosis in the PAR2 KO Ang II groups? While it cannot be ruled out that sequencing of cardiac tissue may provide answers to the mechanism, we would hypothesize that it is primarily differences in signal transduction of stimuli that occur. These are very often not regulated at the level of gene regulation but at the level of post-translational modification, so that they would not be detected by gene sequencing. However, differences in signal transduction might influence expression of other genes. - Given the variance it is hard to determine if there are true differences in P-Smad2/3, could this be measured by western blot instead? Same with the P-ERK1/2? Unfortunately, we do not have any frozen tissue left from the experiment to carry out Western blot analyses for the phosphorylation of Smad 2/3 and ERK1/2. -Are blood pressure measurements, hemodynamics or echocardiography measurements possible to determine physiological and functional consequences of PAR2 deficiency? In fact, in our experimental setting, we planned to include measurements of blood pressure. We started with intra-arterial measurements. However, we could not measure blood pressure in the PAR2ko mice because we could not fix the catheter in the carotid artery, as the arteries seem to be more fragile compared to the corresponding wild-type mice. After failing to obtain a measurement in 4 PAR2ko animals, we refrained from further measurements. However, there are published data examining blood pressure in wild-type and PAR2-deficient mice after stimulation with angiotensin II and reporting no significant differences between the two genotypes, only that some individuals of the PAR2-deficient mice tended to develop lower blood pressure values [1]. The fact that the observed effects in the PAR2-deficient patients are most likely not due to an altered blood pressure was added in the revised version of the manuscript with a reference to the publication by McGuire et al. Reviewer #2: Schwabedissen et al. investigated roles of protease activated receptor 2 (PAR2) on the heart using PAR2-KO mice. The mice were treated with angiotensin II (Ang II) for 4 weeks using osmotic minipumps, and then various in vivo studies were performed. They found that left ventricular hypertrophy (LVH) after Ang II treatment was inhibited in PAR2-KO mice, although cardiac interstitial fibrosis was significantly higher in PAR2-KO mice compared to wild-type mice. They concluded that PAR2 has multiple effects in AngII-stimulated cardiac tissue and its role in cardiac remodelling appears to be ambivalent. Although this paper is well-written and provides some scientific implications, there are severe concerns in the present study. Major criticism: 1. Important findings in the present study is related to cardiac perivascular fibrosis. PAR2 deficiency with Ang II treatment enhanced cardiac perivascular fibrosis. However, the authors only showed historical data and their data are very descriptive without any mechanistic insights. At least some in vitro studies are required. Would it be possible to perform in vitro studies using isolated cardiac fibroblasts from PAR2-deficient mice? As suggested by the reviewer, we performed in vitro experiments with primary cardiac fibroblasts. We would have liked to perform these experiments with fibroblasts from PAR2-deficient mice, but at present we do not have homozygous PAR2-deficient animals available, as we regularly backcross the animals. Instead, with the help of colleagues, we have isolated cardiac fibroblasts from the hearts of neonatal rats and stimulated them with AngII. We investigated PAR2-dependent effects by adding a blocking anti-PAR2 antibody (SAM11) and a PAR2 inhibitor (AZ3451). To exclude the possibility that the addition of the antibody had non-specific effects, an isotype control was used. Gene expression analyses for TGF-ß and matrix molecules were performed as read-outs and signal transduction, in particular the phosphorylation of ERK1/2 and Smad 3, was investigated by immunofluorescence microscopy. Western blot analyses were not performed, as it was not possible to generate sufficient material for various analyses from the neonatal hearts available. To our surprise, the experiments with cardiac fibroblasts showed that the expression of TGF-ß and possibly as a consequence also of collagen 1 and fibronectin was upregulated by the addition of the two PAR2 inhibitors (Figure 6). However, this effect was not further enhanced in AngII-stimulated fibroblasts. The in vitro experiments show that in the short observation period of 24 h, which was chosen to capture the early signal transduction as far as possible, not all genes are increased in their expression by AngII, but only collagen 1 and collagen 4 and also SMA. Furthermore, the significant increase in expression that we observe in the unstimulated cells for TGF-ß, collagen 1 and fibronectin is not observed for SMA and collagen 4. This shows that not all matrix molecules are affected by PAR2 in the same way, or at least with different kinetics. Overall, the in vitro experiments confirmed that PAR2 deficiency or inhibition can also have pro-fibrotic effects. However, the mechanism is still unclear and needs to be clarified in future studies. 2. The authors showed that cardiac hypertrophy induced by Ang II treatment was inhibited in PAR2-KO mice. How was this inhibition related with cardiac perivascular fibrosis? This is a critical question in this paper. There are numerous studies showing that cardiac hypertrophy is usually associated with fibroblast activation and fibrosis [1, 2], and that both hypertrophy and fibrosis can be reduced by, for example, RAAS blockade [3]. It is therefore unusual that these two processes appear to be separate in our study. In most cases, the two processes of cardiac hypertrophy and fibrosis are coupled, i.e. a reduction in hypertrophy is usually associated with a reduction in fibrosis. However, inhibition of hypertrophy alone may also lead to adverse cardiac effects in the setting of sustained volume overload [4]. If the absence of PAR2 only affects hypertrophy, an activation of fibroblasts would be conceivable, as previous studies in this model with PAR2-deficient mice have shown that blood pressure is not significantly reduced even in PAR2-deficient mice [5]. This means that the fibroblasts may continue to be stimulated by the elevated blood pressure. However, more data are needed to prove this hypothesis. These considerations were included in the discussion in the manuscript. 3. Although FGF23 mRNA expression levels in heart were shown in Figure 1, this is not enough. How were alpha-MHC and/or BNP mRNA expression levels in heart? As suggested by the reviewer, we examined other markers of hypertrophy including BNP and cardiac troponin expression in the mouse hearts. Indeed, cardiac troponin was significantly increased in wt mice treated with AngII and expression was normalized to control levels in PAR2-deficient mice (Figure 1D). A similar trend was observed in the expression analysis of BNP (Figure 1F). However, no significant differences were observed for BNP. 4. The authors showed that P-ERK1/2-positive cells in heart were significantly increased in PAR2-KO mice, but they were not increased in all other groups (Figure 2A). Why did these happen? Why did Ang II treatment decrease P-ERK1/2-positive cells in the heart from PAR2-KO mice? More clear explanation and/or additional experiments were required to confirm this result. This observation was also completely unexpected to us, and we tried to address this phenomenon in the in vitro experiments as well. Interestingly, the experiments with the isolated fibroblasts mirrored the observations from the animal experiments. Also in cell culture, the highest phosphorylation was observed in the samples with PAR2 inhibitor without AngII stimulation, whereas after AngII administration rather fewer cells showed phosphorylation of ERK1/2 (Figure 2D). As mentioned above, more induction was also observed for other targets in the samples that were not stimulated with AngII (Figure 6). Unfortunately, we do not yet have a conclusive explanation for this observation. 5. The number of experiments or mice should be described in the main text and figure legends. As suggest, we included number of experiments and numbers of mice also in the legends. [1] Liu Q, Li HY, Wang SJ, Huang SQ, Yue Y, Maihemuti A, Zhang Y, Huang L, Luo L, Feng KN, Wu ZK. Belumosudil, ROCK2-specific inhibitor, alleviates cardiac fibrosis by inhibiting cardiac fibroblasts activation. Am J Physiol Heart Circ Physiol. 2022;323:H235-h47. [2] Chalise U, Hale TM. Fibroblasts under pressure: cardiac fibroblast responses to hypertension and antihypertensive therapies. Am J Physiol Heart Circ Physiol. 2024;326:H223-h37. [3] Garvin AM, De Both MD, Talboom JS, Lindsey ML, Huentelman MJ, Hale TM. Transient ACE (Angiotensin-Converting Enzyme) Inhibition Suppresses Future Fibrogenic Capacity and Heterogeneity of Cardiac Fibroblast Subpopulations. Hypertension. 2021;77:904-18. [4] Crozatier B, Ventura-Clapier R. Inhibition of hypertrophy, per se, may not be a good therapeutic strategy in ventricular pressure overload: other approaches could be more beneficial. Circulation. 2015;131:1448-57. [5] McGuire JJ, Van Vliet BN, Halfyard SJ. Blood pressures, heart rate and locomotor activity during salt loading and angiotensin II infusion in protease-activated receptor 2 (PAR2) knockout mice. BMC Physiol. 2008;8:20. Attachments Attachment Submitted filename: Point to point answer to the reviewers.docx https://doi.org/10.1371/journal.pone.0310095.r002
26 Aug 2024 Decision Letter - Luis Eduardo M Quintas, Editor Protease-activated receptor 2 deficient mice develop less angiotensin II induced left ventricular hypertrophy but more cardiac fibrosis PONE-D-24-00413R1 Dear Dr. Daniel, 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, Luis Eduardo M Quintas, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #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: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes 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: (No Response) Reviewer #2: The authors performed many additional experiments and responded very well. There is no further comment on this paper. ****** 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.0310095.r003
Formally Accepted
29 Sep 2024 Acceptance Letter - Luis Eduardo M Quintas, Editor PONE-D-24-00413R1 PLOS ONE Dear Dr. Daniel, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Luis Eduardo M Quintas Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310095.r004

Open letter on the publication of peer review reports

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

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

Learn more at ASAPbio .