Dear Dr. Sekiya,

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 Aug 17 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.

• 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,

Xindie Zhou

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. 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.

3. Thank you for stating the following in the Competing Interests section:

“Tsukasa Kitahashi, Kentaro Nakamura, and Ryo Kogawa are employees of FUJIFILM Corporation. The remaining authors declare no competing interests.”

We note that one or more of the authors are employed by a commercial company: FUJIFILM Corporation

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. 

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

4. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and in Supporting Information files.

Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition).

For example, authors should submit the following data:

- The values behind the means, standard deviations and other measures reported;

- The values used to build graphs;

- The points extracted from images for analysis.

Authors do not need to submit their entire data set if only a portion of the data was used in the reported study.

If your submission does not contain these data, please either upload them as Supporting Information files or deposit them 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.

If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access.

5. 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?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The study asks whether major histocompatibility mismatch in synovial-derived MSCs will block the chondroprotective effect previously shown by the same group in a different rat OA induction model.

The study also asks whether the synovial-derived MSC’s ability to express HGF is necessary for their therapeutic effect.

The major findings of the study are that synovial derived MSCs appear to still have a chondroprotective effect despite MHC mismatch; and that this effect was not observed if synovial derived MSCs with siRNA induced HGF knockdown were used.

Overall comments:

The strengths of the study are the well-controlled designs, reproducibility obtained, and careful analyses. The weaknesses of the study are the lack of translational relevance of the ACLT model as a clinical model of human OA induction in which damage is much more common in the femoral condyle; the lack of translational relevance of the MSC injection regimen as a clinical injection regimen where repeated injections would not be feasible; and lack of consideration of other signals affected by HGF knockdown in the MSCs that may be impairing their activity versus the author’s inference that the chondroprotective activity of the cells is due exclusively to HGF knockdown.

Specific comments:

Describe size of needle used in MSC injections.

Describe if the MHC mismatched cell injections led to any immune response in the animal – eg, presence of lymphocytes in the synovium should be examined

The authors have previously identified Prg4 and BMP2,6 as additional potentially pro-chondrogenic factors expressed by synovial MSCs. Were the levels of Prg4 and BMP2,6 mRNA and protein also decreased in the cells by HGF knockdown? Without this information, it is an overstatement to attribute the chondroprotective

Reviewer #2: This manuscript presents a study aimed at determining the implication of hepatocyte growth factor (HGF) in the therapeutic effects of synovial derived mesenchymal stem cells (MSCs) in a rat osteoarthritis (OA) model. To do this, both in vitro and in vivo studies have been conducted. First, HGF expression is silenced, and it is demonstrated that this silencing has a negative effect for the chondrogenic differentiation of synovial MSCs in vitro. Second, both HGF-silenced and control MSCs are administered into rat OA knees (as well as no-cells control), and the effects of these administrations is measured by macroscopic and histological findings in cartilage. Of note, the in vivo study is designed for the MSC administration to be MHC-mismatched, using two different rat strains as donors (ACI) and recipients (Lewis).

Overall, the manuscript is well written, the goals are clear (but could be more precisely defined), the study design and methods are appropriate (they could be complemented, though), and the results are generally clearly presented. Limitations are discussed and conclusions are based on the results. In general, this is an interesting and formally adequate manuscript, however some points need attention prior to further considering it for publication.

Please see below main general comments, and later more specific comments pointed out along the manuscript. I hope these points can help authors making the most of their manuscript, but if they disagree with any comment/suggestion, please kindly explain why.

General comments:

- I strongly recommend putting more emphasis into the novelty of the study. The introduction is very brief and, in my opinion, it does not provide enough context to understand the relevance of the present work. Previous studies have already assessed the role of HGF in MSC mechanisms in vitro and in vivo, and some of them have also assessed knockdown cells in different disease models, so the authors should answer very clearly in the introduction: what is the novelty of our study? What does our study adds to the current knowledge?

- To assess the effect of MHC-mismatched administration is not an actual goal of the study, but part of its design. While I fully agree that allogeneic MHC-mismatched administration is the most realistic scenario for clinical use, and thus it is important to assess MSCs in this scenario, this study does not really provide information about the effect of MHC-mismatching because no MHC-matched (or syngeneic) control is used. Thus, I recommend placing the focus towards accounting for this factor by selecting a mismatched scenario, but not towards unveiling the effect of such scenario. This should be reflected along the whole manuscript but especially in the goals (end of introduction), study design (beginning of the methodology) and conclusions.

- Evaluation of the in vivo study outcome mostly relies on histopathological assessment, and only cartilage is assessed. It could have helped elucidating HGF-mediated mechanisms to include more biochemical parameters about cartilage composition, and also assessing synovium as a key target of MSC therapy to modulate inflammation in OA. I consider that this should at least be discussed as a limitation.

- Linked to the previous point, it should be emphasized that this is an observational study but not a mechanistic one. This study reveals that HGF plays some role on the outcome of MSC therapy in OA, but does not provide information about which is this role. Thus, any claim about mechanisms should be done very carefully. In addition, regarding the in vitro part of the study, only chondrogenesis is assessed, however (as authors state in the discussion), MSC therapeutic effects in OA are mainly mediated via paracrine signalling and not by direct differentiation, so in vitro assays could have also been designed more in that direction. I consider that this should at least be discussed as a limitation.

Specific comments:

Introduction:

- In addition to what has been commented under general comments, I would recommend including in the introduction a brief explanation about the rational for choosing these two strains of rats (ACI and Lewis) to establish a MHC-mismatched transplantation scenario.

- Goals at the end of the introduction section should be adjusted as suggested under general comments, especially regarding the MHC-mismatching not being a goal itself but a study condition. Focus should be placed on the role of HGF in MSC therapeutics from an observational perspective.

Material and methods:

Animals:

- Any particular reason to use all male donors and all female recipients? Using half males and half females as recipients could have allowed to also accounting for the sex effect.

Isolation of rat synovial MSCs

- Under the previous section “Animals”, in lines 86-87, it is stated that "To establish MHC-mismatched synovial MSCs, 30 male ACI rats (...) were purchased", however here it is mentioned that 15 rats were used to create a pool of synovial MSCs. What the other 15 ACI male rats were used for?

- If I properly understood, all in vitro and in vivo assays were conducted with passage 0 and passage 1 cells. Were these passages selected for any particular reason? Early passages such as 0 and 1 may present higher cell heterogeneity compared to later passages, which are more frequently used for therapeutic purposes to provide a more homogeneous cell population.

- Were synovial MSCs characterised by any means? i.e immunophenotype, tri-lineage differentiation?

Analysis of Hgf expression

- At what time-points was HGF gene expression and protein secretion assessed? For gene expression it is stated "in the cryopreserved stock". Does it mean in the cells frozen after 24h of transfection? For protein secretion (ELISA), time points are not stated but in Results (figure 1) one can see day 1, 3 and 7. Please ensure that experiment design is explained with enough detail under methodology to match with what is presented under results.

- Since the synovial MSCs used consisted of a pool from 15 donors, I guess only this one biological (pooled) replicate was used, and that what is represented in figures are technical replicates. For example, for ELISA results (fig 1.C) three replicates are represented, but for gene expression it is not clear. Please explain these methodological details about biological and technical replicates under methodology section.

- Line 137: analysis method for gene expression should be 2^-ΔΔCt instead of 2^-ΔCt.

- Please briefly explain how the HGF ELISA was performed. Even if it was done according to kit's manufacturer's instructions, a brief explanation should be provided including any procedure on the supernatants (centrifugation, filtration, freezing...), any dilution of the samples, points in the standard curve, absorbance length and equipment used for absorbance reading, as well as negative controls used (i.e. what is represented as "w/o MSCs" in figure 1.C). This information cannot be exclusively placed in the figure captions but needs to be in the main text so the reader can get the necessary details on it.

In vitro chondrogenesis assay

- It is my understanding, according to the results (figure 2) that both HGF-silenced MSCs and control MSCs were used in chondrogeneic assays, however this is not detailed in the methodology. Please state it clearly in the methodology, the reader needs to get clear and direct information from the text and not to guess based on figures. Please also state the number of biological and technical replicates, as mentioned for gene expression/protein secretion of HGF.

- Please briefly describe the immunostaining procedure, even if previously described in bibliography (primary antibody clone, secondary antibody if applies, antibody dilution, etc.).

Anterior cruciate ligament transection (ACLT) surgery and MSC injection

- In line 89 (Animals section), it is stated that 34 female Lewis rats were purchased. Based on the abstract, 20 knees were included per group. According to this section, both knees of each rat were used. One can infer that 30 rats were used, divided into three groups of 10 rats per groups, and using both knees of each rat, thus making 20 knees per group. Two comments about this:

o This information about group distribution should be clearly explained in this section with exact number of animals and knees, and not left to be inferred by the reader from different parts of the manuscript.

o If 34 rats were purchased but 30 were used for the assay, what the other 4 rats were used for? One may guess that extra animals were purchased in case animals in the study needed to be replaced, but this should not be inferred but crystal clear explained. The destination of the unused/discarded animals (guess euthanasia?) should also be disclosed.

- Please briefly explain anaesthetic and pain management protocols for this particular ACLT surgery.

- Please explain procedure for intraarticular injections in rats. Were these done under general anaesthesia? With which needle size? Volume injected per joint? Vehicle substance? Were cells administered directly after thawing or were cultured for a few days after thawing to readjust conditions? If the former, viability of cells post thawing?

Macroscopic evaluation

- Line 171: even if it might be obvious, please state that at this time point (8 weeks) animals were euthanized as explained before for tissue harvesting.

Statistical analysis

- How was normality assessed, e.g. Shapiro Wilk, Kolmogorov Smirnov...? Since non-parametric tests were used (Kruskall Wallis, Mann-Whitney) I guess that normality was checked and not met. Please clarify.

- In addition, when three groups are compared (e.g. control, HGF-silenced MSCs, control MSCs), the customary strategy is to run a test like Kruskall-Wallis with a posthoc such as Dunns or similar, and not to subsequently conduct pairwise analysis like Mann-Whitney. Please revise and consider amendment.

- Why customary p<0.05, p<0.01 and p<0.001 were not used but instead arbitrary p value levels are considered? It is not technically incorrect, but a bit odd to make this choice.

Results

Hgf knockdown

- Figure 1a does not provide much information. It could be removed or, if kept, it should be enriched with further details like a whole study design representation of the in vitro assays (gene expression, protein secretion, chondrogenesis), the n of animals, etc.

- Figure 1b: the amplification plot is not needed and just the table should be kept. As mentioned in methodology, number of biological and technical replicates should be included. I guess it was only 1 replicate and thus there is no mean or deviation to show in terms of gene expression. In the table, it would be useful to include value 1 for the reference sample, even to represent these numbers (1 and 0.3) in a bar graph for visually representing the downregulation of HGF.

- Figure 1c: Were statistically significant differences found among negative control (whatever it is, w/o cells), control MSCs and HGF-silenced MSCs in terms of HGF secretion? Or along the time i.e. among 1, 3 and 7 days? This should also be explained in the main text.

In vitro analysis

- Were statistically significant differences found between control and silenced-HGF MSCs in terms of weight or diameter of the chondrogeneic pellets produced?

- Number of biological and technical replicates in the chondrogeneic assay?

- Figure 2: Even if quantification of histological parameters in chondrogeneic pellets has not been performed, there are some differences between control and silenced HGF MSCs that can be qualitatively described in the main text, such as different staining intensity of Safranin O, for example.

- Figure 2 caption needs to provide more detail into all the data presented (types of staining and immunostaining).

Discussion

- Lines 414 – 416: “The significant reduction in MSC therapeutic efficacy following Hgf knockdown suggests that this growth factor plays a crucial role in orchestrating the complex interplay of these protective mechanisms”. This is an overstatement. First, even though the score reduction was statistically significant over control, such reduction was not so dramatic. Second, and more importantly, from these results it cannot be concluded which exact mechanisms plays HGF in relation to the multimodal action of MSCs, since other mediators and their interactions have not been assessed (please also see general comments).

- Lines 420 – 424: The choice of the mismatched scenario using two rat strains should be explained in the introduction or in the study design section so the reader can understand the suitability of the mismatched design from the beginning of the manuscript.

- Lines 424 – 425: This sentence should be revised. As already mentioned, the lack of MHC-matched control (i.e. syngenic) does not allow inferring if the cells would have promoted more or less effect depending on the matching degree. Maybe matched cells would have not elicited more therapeutic effects in the conditions of this study, so it cannot be inferred that it is a positive results that mismatched cells still elicited therapeutic effect.

**********

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.

Dear Dr. Sekiya,

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 Oct 18 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.

• 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 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,

Xindie Zhou

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: Authors have done a remarkable job revising the manuscript, which has significantly improved. It is particularly worth of mentioning the great work with the Introduction, which is convincing yet concise and clearly highlighting the main points. I consider that the manuscript is in good shape for acceptance, yet a few minor points need attention:

- Regarding the procedure for MSC injection, the vehicle substance is not yet disclosed. May it be CP-1 High Grade cryoprotectant used for freezing the cells? If so, please clearly state.

- Regarding the sex of the animals of the study: I understand that it is a common approach to use males as donors and females as recipients in order to track the cells using Y chromosome markers, and I think this can be a valid point when designing the study, even though tracking of male donor cells was not pursued. I also understand that including a gender dimension in the in vivo studies can be challenging and require extra resources. However, I feel that the statement “we believe the sex difference did not influence thetherapeutic outcomes observed” (lines 559-560 of the Discussion) is not supported by evidence (e.g. doi: 10.21037/atm-23-1546) and thus should be removed.

- In line 480 of the Discussion, the statement “even in the presence of strong allogeneic immune responses” is an assumption, since the immune response against the MSCs was not evaluated. Please consider removing this sentence to avoid misleading information.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: 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

MHC-mismatched synovial mesenchymal stem cell injections delay knee osteoarthritis progression through hepatocyte growth factor secretion in rats

PONE-D-25-18549R2

Dear Dr. Sekiya,

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. For questions related to billing, please contact billing support .

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,

Xindie Zhou

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewer #2:

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

**********

Reviewer #2: Authors have appropriately addressed the last few pending points. I have no further comments and consider the manuscript suitable for publication.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: No

**********