PONE-D-24-02571Effect of matcha green tea on cognitive functions and sleep quality in older adults with cognitive decline: A randomized controlled study over 12 monthsPLOS ONE

Dear Dr. Uchida,

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.

The most pressing points to address are from Reviewer 2 who focused on the statistical aspects of the study and who raises several discrepancies from the CONSORT guidelines. Please ensure that you familiarise yourself with these criteria and address Reviewer 2's points in turn. Reviewer 1 and 3 raise some pertinent issues relating to definitions, language-use and areas that require expansion in the Introduction and Discussion sections.  Please submit your revised manuscript by May 25 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,

Tamlyn Julie Watermeyer

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

Authors with competing interests

Enter competing interest details beginning with this statement:

I have read the journal's policy and the authors of this manuscript have the following competing interests: [Kazuhiko Uchida serves as a board member of MCBI Inc. Kohji Meno, Tatsumi Korenaga, Liu Shan, and Hideaki Suzuki are employees of MCBI Inc. Yoshitake Baba, Chika Tagata, Yoshiharu Araki, Shuto Tsunemi, Kenta Aso, Shun Inagaki, Sae Nakagawa, Makoto Kobayashi, and Takanobu Takihara are employees of ITO EN , LTD. This research received no external funding. ]

   

We note that one or more of the authors are employed by a commercial company. 

a. 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, 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. 

b. 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.

3. In the online submission form, you indicated that The data cannot be shared publicly without permission from all authors. Access to the data is available through the Institutional Data Access/Ethics Committee (contactable via the corresponding author) for researchers who meet the criteria for accessing confidential data.

All PLOS journals now require all data underlying the findings described in their manuscript to be freely available to other researchers, either a. In a public repository, b. Within the manuscript itself, or c. Uploaded as supplementary information.

This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If your data cannot be made publicly available for ethical or legal reasons (e.g., public availability would compromise patient privacy), please explain your reasons on resubmission and your exemption request will be escalated for approval. 

[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

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

Reviewer #3: 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: No

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: This was a very interesting article. I have included some minor amendment below:

-Data statement – no explanation as to why data can’t be shared. States without permission from all authors but no explanation as to why all authors wouldn’t give permission. Is data owned by a third party?

- Typo on page 6, line 3, double comma

- Line 8 – could some context be given as to what is being classified as ‘middle age’ here.

- Within intervention section it would be useful to provide some justification as to why this dosage was decided upon.

- For statistical analysis is there justification as to why per protocol participants were analysed rather than Intent To Treat. CONSORT recommends reporting both in parallel, so some acknowledgement of ITT would be beneficial here, were the results the same?

- The results are interesting and placed well within previous literature. However there were no effects on primary outcomes and this should be acknowledged somewhere in the discussion.

Reviewer #2: Important note: This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ [like medical importance, relevance of the study, ‘clinical significance and implication(s)’ of the whole study, etc.] are to be evaluated [should be assessed] separately/independently. Further please note that any ‘statistical review’ is generally done under the assumption that (such) study specific methodological [as well as execution] issues are perfectly taken care of by the investigator(s). This review is not an exception to that and so does not cover clinical aspects {however, seldom comments are made only if those issues are intimately / scientifically related & intermingle with ‘statistical aspects’ of the study}. Agreed that ‘statistical methods’ are used as just tools here, however, they are vital part of methodology [and so should be given due importance]. I look at the manuscript in/with statistical view point, other reviewer(s) look(s) at it with different angle so that in totality the review is very comprehensive. However, there should be efforts from authors side to improve (may be by taking clues from reviewer’s comments). Therefore, please do not limit the revision only (with respect) to comments made here.

COMMENTS: Kindly note that your ABSTRACT is well drafted (in my opinion), but is ‘assay type’. It is preferable [refer to item 1b of CONSORT checklist 2010: Structured summary of trial design, methods, results, and conclusions] to divide the ABSTRACT with small sections like ‘Objective(s)’, ‘Methods’, ‘Results’, ‘Conclusions’, etc. which is an accepted practice of most of the good/standard journals [including this one, though ‘The PLoS One Guidelines to Authors’ did not specify an Abstract format, it is desirable]. It will definitely be more informative then, I guess, whatever the article type may be. Since your ‘Article Type: Clinical Trial’, it very much desirable.

In addition, there are few issues/observations about which I have different opinion. Such observations/concerns are given below [if acceptable/agreed, try to incorporate them (consider positively wherever possible)]:

First is regarding choice of ‘Sample size’. One of the very important items in CONSORT guidelines is ’How sample size was determined (Item 7a)’ and expected/desired to described in ample details including all assumptions and software used. In fact, it is very surprising to note the ‘absence’ of the important term “CONSORT’’ despite that the manuscript is on ‘A randomized controlled clinical study/trial’. It is well-known that while reporting [findings from and even planning that is in ‘protocol’ of] ‘Clinical Trial’ one should follow/cover CONSORT guidelines.

In ‘Discussion’ it is just said/mentioned that “In this study, unbiased randomization was conducted, and the trial was implemented as a triple-blind study”. Regarding these important issues more discussion/detailed account is expected. Few important items {and all ‘Clinical Trials’ must follow} in CONSORT guidelines are ‘How sample size was determined (Item 7a)’,’ Random Sequence generation (Item 8a)’, ‘Allocation concealment (Item 9)’, Blinding (Item 11a). Since your article type is ‘Clinical Trial’, you are supposed to cover these items in the report or even in ‘Protocol’.

You may know that ‘Permuted Block Randomization’ ensures same group sizes (not simple randomization) and that randomization is a process [not only sequence generation] which includes ‘Allocation Concealment’}. Which ‘Randomization’ procedure is used in this study is not specified but almost equal group sizes are obtained. It must be ‘Permuted Block Randomization’ but such clear mention is desired.

In ‘Results-Participants’ section (as well as in Figure-1), you may use the word ‘screened’ in place of ‘recruited’ [As shown in Fig 1, we recruited 939 community-dwelling older adults aged 60–85 years and 124 participants were enrolled], in my opinion. May please confirm as word ‘recruited’ implies that 939 participated in trial. It may be known to you that when random allocation/assignment is used/done, any statistical comparison of baseline characteristics is not required. In this context [refer to Table-1], I request authors to read following note pasted from one famous standard textbook on ‘Medical Research Methodology’:

To provide a description of baseline characteristics is entirely reasonable (since it is clearly important in assessing to whom the results of the trial can be applied), however, statistical comparison of baseline characteristics when random allocation/assignment is used/done [often for good/standard/leading journals these days] is not required, because even if P-value(s) turn(s) out to be significant (while comparing baseline characteristics despite random allocation), it is, by definition, a false positive as you then are supposed to be testing ‘randomization’ then, which in any single trial may not balance all baseline characteristics (particularly when sample sizes are small). Remember that ‘randomization’ is a sort of ‘insurance’ and not a guarantee scheme. Authors may please refer to following articles:

References:

1. Stuart J. Pocock, et al., ‘Subgroup analysis, covariate adjustment and baseline comparisons in clinical trial reporting: current practice and problems’, Statistics in medicine, 2002; 21:2917–2930 [Particularly page 2927]

2. Harrington D, et al., ‘New guidelines for statistical reporting in the journal’, N Engl J Med 2019;381:285-6

[Important message (indirectly/ultimately indicated) from these articles: Never do any comparison with respect to ‘baseline’ characteristics {by applying statistical significance test(s)}, when allocation is done randomly].

However, Statistical comparison [only with respect to important/indicated variables] of baseline characteristics may be performed, to find out if analysis adjustment (say stratified analyses or else) is required with respect to these variables.

What is the purpose of Table-3? Why Between group comparison is performed separately at ‘Baseline’ and ’12-months’? What is/are interpretation of insignificant (both) P-vale(s)? Why not the comparison using ‘change scores’? In all the tables, test name is indicated only for few ‘variables/parameters’ {by giving symbol as superscript on P-value (last column)}. Is not it required to indicate test name in case of (i.e. with respect to) other/remaining variables/parameters? Or is it left for readers to guess? Remember/mind you that this is a scientific/academic document and so all details should be clearly/correctly communicated (do not take readers’ for granted). Most of the findings are reported without properly interpreting them or given relevance to study (example: page 26).

Only limitation of the study [A limitation of the current study is the small number of participants] mentioned on page 32 is agreed. However, does that mean {according to authors} there are none others? As pointed out in ‘important note’ above “This review pertains only to ‘statistical aspects’ of the study and so ‘clinical aspects’ should be assessed separately/independently. In my opinion, to make this article acceptable some amount of re-vision (re-drafting) may be needed. However, please do not limit the revision only (with respect) to comments made here. ‘Major revision’ is recommended.

Reviewer #3: The manuscript was well written and an interesting trial. I suggest some minor points to improve it:

- Introduction is well written with relevant links to supporting green tea literature. It would be useful to include rationale for the intervention timeframe (12 months). There does seem to be some literature on matcha tea interventions that haven't been included, which is more relevant than the included green tea literature - for example a paper by Sakurai (2020) used matcha for a 12 week intervention in elderly Japanese individuals.

- Page 7 line 5 - missing refernece for frequency of green tea consumption. It would also be useful here to have a brief explanation of this finding and the relation to this trial

- Page 7 line 8 - missing refernece for sleep disturbance/disorder stats, it would be more useful here to have the stats for sleep disorder prevalence in Japan rather than the US

- I am interested to know why age was stratified as >74 years old/ <74 years old, when the age range was 60-85 years. Is there a reason that 74 was chosen rather than 72? A brief comment at Page 9 line 2 would be useful

- Page 9 Line 16 - points 3 and 4 of the eligibility criteria are the same, please check

- Within eligibility criteria you mention that participants could not consume more that 7 cups of matcha per week, there is a comment within the discussion that the study did not impose restrictions on green tea intake during the trial, why was this decided? Did you collect information on habitual intake of green tea/matcha during the trial? If so, were there any differences in consumption between treatment groups? High consumption in the placebo group could explain lack of findings.

- Intervention information - I'd be interested to know what 2g of matcha equates to in comparison with consuming as a tea and/or how the catechin/theanine/caffeine content compares with green tea. I think this is particularly interesting given the citation of various green tea literature and the decision to exclude participants consuming 7+ cups matcha each week.

- Page 16 Line 2 - Please provide comment on if the repeated freezing/thawing of samples may have impacted the immunoassay results. Was this per protocol? It would also be useful here to include details of the blood sampling process. It would also be useful to include some information about the biomarkers assessed (e.g. assessing vitamin status)

- Page 23 Line 7 - compliance - earlier a pill count was mentioned, do you have the information on treatment compliance based on this? Was there a compliance cut off used for including participants in analysis?

- Within sample size calculation information I don't think it is clear why a medium-large effect size (cohen's d = 0.7) was used. Was this based off previous green tea literature? Please expand.

- Whilst this is recognised several times, consider highlighting each time that the PSQI finding is a trend only to be careful when over interpreting this finding.

I also found a few minor typos:

Page 6 Line 3 – extra comma

Page 20 line 4 – matcha misspelled

**********

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

Reviewer #3: 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.

Attachments

Attachment

Submitted filename: renamed_7c415.docx

PONE-D-24-02571R1Effect of matcha green tea on cognitive functions and sleep quality in older adults with cognitive decline: A randomized controlled study over 12 monthsPLOS ONE

Dear Dr. Uchida,

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 Jul 26 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,

Valerio Manippa

Academic Editor

PLOS ONE

Journal Requirements:

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

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: Yes

**********

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

Reviewer #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: 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 #2: COMMENTS: Since all of the comments made on earlier draft are considered positively & mostly attended satisfactorily, I recommend the acceptance after minor revision. Although many modifications could be seen clearly, few details/modifications given while addressing comments were/are not very convincing [example: account regarding choice of ‘Sample size’]. ‘Minor revision’ is recommended so that authors can re-visit earlier comments and further modify by taking clues from earlier comments. However, please do not limit the revision only (with respect) to comments made there. More improvement is expected {note that ‘clinical implications’ are valuable}.

Reviewer #3: (No Response)

**********

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 #2: No

Reviewer #3: 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.

Effect of matcha green tea on cognitive functions and sleep quality in older adults with cognitive decline: A randomized controlled study over 12 months

PONE-D-24-02571R2

Dear Dr. Uchida,

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,

Madepalli K. Lakshmana, 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 #2: All comments have been addressed

Reviewer #3: 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 #2: (No Response)

Reviewer #3: Yes

**********

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

Reviewer #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: 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 #2: (No Response)

Reviewer #3: 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 #2: COMMENTS: Further to what I stated earlier that all of the comments made on original draft were attended satisfactorily, I add that comment made on revised draft also is attended and I recommend the acceptance without any hesitation now.

Reviewer #3: (No Response)

**********

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 #2: No

Reviewer #3: No

**********