PONE-D-22-24149Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic RatsPLOS ONE

Dear Dr. Bushra Ahmed Hamdi,

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PLOS ONE

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Additional Editor Comments:

PONE-D-22-24149

"Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats"

Original Submission

Reviewer Recommendation Term: Major Revision

Rate Review: 0

Custom Review Question(s): Response

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. Partly

2. Has the statistical analysis been performed appropriately and rigorously? 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. 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. No

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) Thank you for inviting me to review this manuscript. I have the following comments:

1. Please, concise the abstract. It is too long.

2. The sentence lines 36-38 is an incomplete. Please, revise it.

3. Line 86: please, write β-cells in all manuscript sections. Not B-cells.

4. Line 193: Please, add ‘’Ethical approval’’ subtitle.

5. Line 215: replace the word ‘’divided’’ with the word ‘’allocated’’.

6. Line 232: in ‘’Experimental design’’, explain that 32 diabetic rats allocated into four groups other than the control non-diabetic (the fifth group).

7. Line 249: Please, replace the word ‘’measurement’’ with ‘’determination’’.

8. In Table 1, the authors used MRC1 Antibody as a human peptide, while your experiment was done on rats. Please, explain?????

9. In molecular docking, please, make it with protein of enzymes only, where you will determine if the ligands act as inhibitors or not.

10. Another comment regarding the molecular docking, the author made docking with protein of other species other than rat. Please, repeat it with rat proteins because your experiment was done on rats. But if you did not find the protein of rats, you can use the protein of human and make sequence alignment in https://www.uniprot.org/ to determine the degree of similarity between rat and human sequences of the same protein.

11. Please, write the pKi in Table 6 and 7.

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.

No

Confidential to Editor

1. Do you have any potential or perceived competing interests that may influence your review? Please review our Competing Interests policy and declare any potential interests that you feel the Editor should be aware of when considering your review. If you have no competing interests, please write "I have no competing interests." Thank you for inviting me to review this manuscript. I have the following comments:

1. Please, concise the abstract. It is too long.

2. The sentence lines 36-38 is an incomplete. Please, revise it.

3. Line 86: please, write β-cells in all manuscript sections. Not B-cells.

4. Line 193: Please, add ‘’Ethical approval’’ subtitle.

5. Line 215: replace the word ‘’divided’’ with the word ‘’allocated’’.

6. Line 232: in ‘’Experimental design’’, explain that 32 diabetic rats allocated into four groups other than the control un-diabetic (the fifth group).

7. Line 249: Please, replace the word ‘’measurement’’ with ‘’determination’’.

8. In Table 1, the authors used MRC1 Antibody as a human peptide, while your experiment was done on rats. Please, explain?????

9. In molecular docking, please, make it with protein of enzymes only, where you will determine if the ligands act as inhibitors or not.

10. Another comment regarding the molecular docking, the author made docking with protein of other species other than rat. Please, repeat it with rat proteins because your experiment was done on rats. But if you did not find the protein of rats, you can use the protein of human and make sequence alignment in https://www.uniprot.org/ to determine the degree of similarity between rat and human sequences of the same protein.

11. Please, write the pKi in Table 6 and 7.

2. Did you receive any assistance in preparing this review (e.g. from a post-doc or graduate student)? If yes, please include their name below.

3. If accepted, do you think this submission should be highlighted on the PLOS ONE website? PLOS ONE does not evaluate manuscripts based on perceived significance or readership. We aim to provide tools for readers to filter and evaluate our publications. (optional)

Do you want to get recognition for this review on Publons?

If you opt in, your Publons profile will automatically be updated to show a verified record of this review in full compliance with the journal’s review policy. If you don’t have a Publons profile, you will be prompted to create a free account. Yes

 

PONE-D-22-24149

"Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats"

Original Submission

Reviewer Recommendation Term: Major Revision

Rate Review: 0

Custom Review Question(s): Response

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. No

2. Has the statistical analysis been performed appropriately and rigorously? 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. 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. No

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’s comments

I have read the manuscript entitled “Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats” several times and I have the following issues raised before the manuscript can be considered for possible publication

1. In the title the plant name is not the corrected way of writing. Authors should confirm and check the accepted using http://mpns.kew.org/mpns-portal/?_ga=1.111763972.1427522246.1459077346 or http://www.plantsoftheworldonline.org/ or http://www.worldfloraonline.org/.

- Also, in the title, streptozotocin-diabetic rats should be streptozotocin-induced diabetic rats

2. Abstract

-Did the author induced hypoglycemic associated inflammatory disorders

-the abbreviation of [OAE] does not represent methanolic extract of Onopordum acanthium (OAE).

- In the abstract line 28-30 should be rephrased and express correctly

-methods section not written properly. How many rats were used? How many groups did the authors used? Was there no standard drug group. If there was, how was the drug administered.

-line 36-38 not clear. Revise appropriately.

- Not enough biochemical assays was done to justify the study

-Under results insulin content, FBS and body weight alone is not enough to justify the study. More parameters need to be provided.

-line 42-43; STZ induced hyperglycemia associated inflammatory by the first sentence under background and objective shows it induces hypoglycemia associated inflammatory damage. Please clarify.

3. Introduction

-the accepted name of the plant should be written correctly.

- On the plant the anti-inflammatory activity has been reported before. What makes your work different from others.

-line 118 please revise

-line 122-123 should be moved above ……’To the best of our knowledge…..

Line 125-132 sentence does not make any sense. Authors should strive for clarity at all times. Revise

4. Methods

- The dosages of OAE 200 and 400 was not justified as well as the glibenclamide dose.

-DPPH assay to determine the antioxidant activity is not sufficient. Assays such as FRAP, Fe chelation, TAC, NO and OH radical scavenging should be carried out.

-Fasting blood glucose and body weight not enough.

-More parameters like serum insulin, glycated hemoglobin, HOMA-IR and HOMA-β should be carried out.

-in silico studies, the grid box for the docking should be provided.

-how were the ligands and proteins prepared. This should be separated and stated out clearly.

-Instead of the molecular docking, why didn’t the authors analyze for the induced-fit docking. Was the docking to the active site of the proteins or the authors did blind docking.

5. Discussion

- More recent studies can be used to improve the discussion especially the in silico section

6. Conclusion

-The analysis done is not sufficient enough to justify the conclusion made as well as the objectives of the study.

6. Conflict of interest

- The statement ‘The authors received no funding for this work’ is not appropriate here.

General comments

Authors should subject the manuscript to a native English speaker as most of the sentences are difficult to comprehend and also disjointed.

Thank you.

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. No

Confidential to Editor

1. Do you have any potential or perceived competing interests that may influence your review? Please review our Competing Interests policy and declare any potential interests that you feel the Editor should be aware of when considering your review. If you have no competing interests, please write "I have no competing interests." No potential or perceived competing interests that may have influence my review.

2. Did you receive any assistance in preparing this review (e.g. from a post-doc or graduate student)? If yes, please include their name below. NO

3. If accepted, do you think this submission should be highlighted on the PLOS ONE website? PLOS ONE does not evaluate manuscripts based on perceived significance or readership. We aim to provide tools for readers to filter and evaluate our publications. (optional) Yes, on a more specific subject area page (e.g. Biochemistry, Atmospheric Science)

Do you want to get recognition for this review on Publons?

If you opt in, your Publons profile will automatically be updated to show a verified record of this review in full compliance with the journal’s review policy. If you don’t have a Publons profile, you will be prompted to create a free account.

Yes

 

PONE-D-22-24149

"Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats"

Original Submission

Reviewer Recommendation Term: Major Revision

Rate Review: 0

Custom Review Question(s): Response

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. Partly

2. Has the statistical analysis been performed appropriately and rigorously? 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. 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. No

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) Thank you for inviting me to review this manuscript. I have the following comments:

1. Please, concise the abstract. It is too long.

2. The sentence lines 36-38 is an incomplete. Please, revise it.

3. Line 86: please, write β-cells in all manuscript sections. Not B-cells.

4. Line 193: Please, add ‘’Ethical approval’’ subtitle.

5. Line 215: replace the word ‘’divided’’ with the word ‘’allocated’’.

6. Line 232: in ‘’Experimental design’’, explain that 32 diabetic rats allocated into four groups other than the control non-diabetic (the fifth group).

7. Line 249: Please, replace the word ‘’measurement’’ with ‘’determination’’.

8. In Table 1, the authors used MRC1 Antibody as a human peptide, while your experiment was done on rats. Please, explain?????

9. In molecular docking, please, make it with protein of enzymes only, where you will determine if the ligands act as inhibitors or not.

10. Another comment regarding the molecular docking, the author made docking with protein of other species other than rat. Please, repeat it with rat proteins because your experiment was done on rats. But if you did not find the protein of rats, you can use the protein of human and make sequence alignment in https://www.uniprot.org/ to determine the degree of similarity between rat and human sequences of the same protein.

11. Please, write the pKi in Table 6 and 7.

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. No

Confidential to Editor

1. Do you have any potential or perceived competing interests that may influence your review? Please review our Competing Interests policy and declare any potential interests that you feel the Editor should be aware of when considering your review. If you have no competing interests, please write "I have no competing interests." Thank you for inviting me to review this manuscript. I have the following comments:

1. Please, concise the abstract. It is too long.

2. The sentence lines 36-38 is an incomplete. Please, revise it.

3. Line 86: please, write β-cells in all manuscript sections. Not B-cells.

4. Line 193: Please, add ‘’Ethical approval’’ subtitle.

5. Line 215: replace the word ‘’divided’’ with the word ‘’allocated’’.

6. Line 232: in ‘’Experimental design’’, explain that 32 diabetic rats allocated into four groups other than the control un-diabetic (the fifth group).

7. Line 249: Please, replace the word ‘’measurement’’ with ‘’determination’’.

8. In Table 1, the authors used MRC1 Antibody as a human peptide, while your experiment was done on rats. Please, explain?????

9. In molecular docking, please, make it with protein of enzymes only, where you will determine if the ligands act as inhibitors or not.

10. Another comment regarding the molecular docking, the author made docking with protein of other species other than rat. Please, repeat it with rat proteins because your experiment was done on rats. But if you did not find the protein of rats, you can use the protein of human and make sequence alignment in https://www.uniprot.org/ to determine the degree of similarity between rat and human sequences of the same protein.

11. Please, write the pKi in Table 6 and 7.

2. Did you receive any assistance in preparing this review (e.g. from a post-doc or graduate student)? If yes, please include their name below.

3. If accepted, do you think this submission should be highlighted on the PLOS ONE website? PLOS ONE does not evaluate manuscripts based on perceived significance or readership. We aim to provide tools for readers to filter and evaluate our publications. (optional)

Do you want to get recognition for this review on Publons?

If you opt in, your Publons profile will automatically be updated to show a verified record of this review in full compliance with the journal’s review policy. If you don’t have a Publons profile, you will be prompted to create a free account.

Yes

[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: No

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

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: Yes

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: No

Reviewer #3: No

**********

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: The work entitled “Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats” is well designed and it contains different techniques including histology and immunohistochemistry, HPLC analyses, antioxidant activity and Molecular docking for testing of the anti-diabetic and cardio protective impact of extract from leaves of the O. acanthium. According to the authors’ results, the extract ameliorates negative effects of STZ. Finally, Authors discussed and proposed that rich bioactive compounds in the extract might have protective roles against to diabetic complications sourced from STZ. In my opinion, the manuscript can be accepted for publication but some minor remarks should be considered by authors before publication.

Minor comments:

Line 18-19: Delete the sententence "Streptozotocin (STZ) induces hypoglycaemia associated with inflammatory disorders." in Background and objective

Line 72: change the word “Diabetic” as “Diabetes”

Line 110: correct “( O.acanthium)” as “(O. acanthium)”

Line 136-137: Please indicate the name of the plant taxonomist who identified O. acanthium with a sentence.

Line 183: correct “per cent” as “percent”

Line 205: correct “Rattus norvegicus Domestica” as “Rattus norvegicus domestica” and as being italic.

Line 268: correct “deparaffinisation” as “deparaffinization”

Line 649: correct “Gallic“ as “gallic”

Line 687: correct “MiR-126” as “miR-126”

Reviewer #2: Reviewer’s comments

I have read the manuscript entitled “Onopordum acanthium extract attenuates pancreatic β-Cells and cardiac inflammation in streptozotocin-diabetic Rats” several times and I have the following issues raised before the manuscript can be considered for possible publication

1. In the title the plant name is not the corrected way of writing. Authors should confirm and check the accepted using http://mpns.kew.org/mpns-portal/?_ga=1.111763972.1427522246.1459077346 or http://www.plantsoftheworldonline.org/ or http://www.worldfloraonline.org/.

- Also, in the title, streptozotocin-diabetic rats should be streptozotocin-induced diabetic rats

2. Abstract

-Did the author induced hypoglycemic associated inflammatory disorders

-the abbreviation of [OAE] does not represent methanolic extract of Onopordum acanthium (OAE).

- In the abstract line 28-30 should be rephrased and express correctly

-methods section not written properly. How many rats were used? How many groups did the authors used? Was there no standard drug group. If there was, how was the drug administered.

-line 36-38 not clear. Revise appropriately.

- Not enough biochemical assays was done to justify the study

-Under results insulin content, FBS and body weight alone is not enough to justify the study. More parameters need to be provided.

-line 42-43; STZ induced hyperglycemia associated inflammatory by the first sentence under background and objective shows it induces hypoglycemia associated inflammatory damage. Please clarify.

3. Introduction

-the accepted name of the plant should be written correctly.

- On the plant the anti-inflammatory activity has been reported before. What makes your work different from others.

-line 118 please revise

-line 122-123 should be moved above ……’To the best of our knowledge…..

Line 125-132 sentence does not make any sense. Authors should strive for clarity at all times. Revise

4. Methods

- The dosages of OAE 200 and 400 was not justified as well as the glibenclamide dose.

-DPPH assay to determine the antioxidant activity is not sufficient. Assays such as FRAP, Fe chelation, TAC, NO and OH radical scavenging should be carried out.

-Fasting blood glucose and body weight not enough.

-More parameters like serum insulin, glycated hemoglobin, HOMA-IR and HOMA-β should be carried out.

-in silico studies, the grid box for the docking should be provided.

-how were the ligands and proteins prepared. This should be separated and stated out clearly.

-Instead of the molecular docking, why didn’t the authors analyze for the induced-fit docking. Was the docking to the active site of the proteins or the authors did blind docking.

5. Discussion

- More recent studies can be used to improve the discussion especially the in silico section

6. Conclusion

-The analysis done is not sufficient enough to justify the conclusion made as well as the objectives of the study.

6. Conflict of interest

- The statement ‘The authors received no funding for this work’ is not appropriate here.

General comments

Authors should subject the manuscript to a native English speaker as most of the sentences are difficult to comprehend and also disjointed.

Thank you.

Reviewer #3: Thank you for inviting me to review this manuscript. I have the following comments:

1. Please, concise the abstract. It is too long.

2. The sentence lines 36-38 is an incomplete. Please, revise it.

3. Line 86: please, write β-cells in all manuscript sections. Not B-cells.

4. Line 193: Please, add ‘’Ethical approval’’ subtitle.

5. Line 215: replace the word ‘’divided’’ with the word ‘’allocated’’.

6. Line 232: in ‘’Experimental design’’, explain that 32 diabetic rats allocated into four groups other than the control non-diabetic (the fifth group).

7. Line 249: Please, replace the word ‘’measurement’’ with ‘’determination’’.

8. In Table 1, the authors used MRC1 Antibody as a human peptide, while your experiment was done on rats. Please, explain?????

9. In molecular docking, please, make it with protein of enzymes only, where you will determine if the ligands act as inhibitors or not.

10. Another comment regarding the molecular docking, the author made docking with protein of other species other than rat. Please, repeat it with rat proteins because your experiment was done on rats. But if you did not find the protein of rats, you can use the protein of human and make sequence alignment in https://www.uniprot.org/ to determine the degree of similarity between rat and human sequences of the same protein.

11. Please, write the pKi in Table 6 and 7.

**********

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

**********

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Submitted filename: Reviewer Comments 17102022.docx

Onopordum acanthium L.extract attenuates pancreatic β-Cells and cardiac inflammation in streptozocin-induced diabetic rats

PONE-D-22-24149R1

Dear Dr. Bushra Ahmed Hamdi

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Giribabu Nelli

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript can be accepted for publication.

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

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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: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: Authors have completed all recommended requirements by me. Thus, the manuscript can be accepted for publication in Plos One.

Reviewer #2: No further comments. All comments raised have been addressed. Other comments raised that cannot be addressed during revision has been duly addressed in the limitations of the study.

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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

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