PONE-D-22-16415Anti-leishmanial Physalins - Phytochemcial Investigation, In vitro Evaluation against clinical and MIL Resistant L. tropica strains and In silico Studies PLOS ONE

Dear Dr. Yousuf,

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 12 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Shahid, Ph.D.

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 suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service. 

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services.  If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file)”

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"“The research leading to these results has, in part, received funding from UK Research and Innovation via the Global Challenges Research Fund under grant agreement ‘A Global Network for Neglected Tropical Diseases’, grant number MR/P027989/1.” 

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"“Sammer Yousuf and M. Iqbal Choudhary, researcher leading to these results has, in part, received funding from UK Research and Innovation via the Global Challenges Research Fund under grant agreement ‘A Global Network for Neglected Tropical Diseases’, grant number MR/P027989/1.” 

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

5. Please ensure that you refer to Figures 5, 7, 8, and 9 in your text as, if accepted, production will need this reference to link the reader to the figure.

6. We note you have included a table to which you do not refer in the text of your manuscript. Please ensure that you refer to Tables 2, 4, and 6in your text; if accepted, production will need this reference to link the reader to the Table.

[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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: General comments

The authors, in continuation of their work, have used different components extracted from physalis minima (isolated and structurally characterized). Anti-leishmanial effects of physalins were explored against L.tropica and L.major, the causative agents of ACL and ZCL, respectively. Although the work is extensive major revisions are needed before being considered for publication.

Specific comments

Title

- Mixed capital and small wording should be corrected according to the journal's style.

Abstract

- 97 countries should be 98 countries (L28-494)

- Exclude 3T3 and BJ in the keywords (L62).

Introduction

- L75, the expected figure for 7 countries is wrong as written (>6,000) are 80% of cases globally?

- Solanaceae (L83) should be written in italic font.

M+M

- Document a standard reference for performing TLC (L102-127).

- Exclude “of the Department of Botany University of Karachi (L131-132). It is a repetition of the above line (L130).

- Present a standard ref. for extraction and isolation processes (134-156).

- The authors did not use an MTT assay to check the viability of promastigotes. Did you check for viability before performing the treatments to make sure they are all viable (motile) because you counted them by eyes?

- RPMT should always be written complete (RPMI-1640, L250)

- The authors visually counted the promastigote stage, why did they use DAPI stain? How did it support the study (L259-260)?

- Correct 37оC (L282).

- Correct 5×104 cells/ml (L283).

- Write the results in the past tense (L423 and other places).

- For cytotoxicity assays on fibroblasts, the authors should calculate the CC50 values, and then to determine the safety index; one should calculate the selectivity index (SI). SI=CC50/IC50≥1, non-toxic.

- If the authors do not use this equation how can one say the compound is safe?

- Be consistent in writing words, the authors have repeatedly used anti-leishmanial in L439 used anti leishmanial (correct).

- Mention why you docked with phosphoglucose isomerase?

- Why the authors did not use a uniform and standard positive control like AmpB throughout the experiments so that one could be able to compare the outcome? All of the positive controls even AmpB, pentamidine, and MIL are the second-line drugs, except AmBisome (a liposomal form of AmpB is used to treat Indian and Mediterranean type kala-azar (visceral leishmaniasis due to L.donovani and L.infantum, respectively). The authors could use meglumine antimoniate (Glucantime®) and if resistance is a common phenomenon then AmpB would be OK, otherwise using different positive controls is not realistic. Miltefosine does not affect L. tropica it is only active at around 83%-85% against L.major and L.infantum.

- Another important thing the authors should know is that: promastigotes are the extracellular stage in the gut of sand flies (biological vectors) and culture media. The authors could use intra-macrophage amastigotes (the clinical stage inside the phagocytic cells in a vertebrate host such as humans and wild mammals). Promastigotes are biochemically and molecularly different from amastigotes (Leishman bodies) in several aspects: they are more resistant and aerobic stage, although extracellular.

- L434, Cytotoxicity of physalin against human fibroblast as given in Table 5 are CC50. These values should be used in the equation above to calculate the SI index as the measure of safety (SI=CC50/IC50≥1, safe), otherwise how can you tell the component is safe?

- This section is not well organized, and it should be titled more specifically according to the journal’s style.

Discussion

- AmpB is not the drug of choice for L.tropica and L. major. It is rather a liposomal form OF AmpB(AmBisome used against VL). It is all right you used other second-choice drugs because you had no access to meglumine antimonite (Glucantime®) or sodium stibogluconate(Pentostam®), but you should have used similar positive controls to be able to compare the activity of each component.

- The authors should not point out the Figs in the discussion (L490).

- Close the sentence in L490.

- I am not export on docking reactions and you pay attention to other referees for this purpose.

- How could you tell that the difference between the test results and the untreated control group is significant, or how could you predict the significant difference among groups. The author should mention the statistical test and define the P-value.

- the discussion is too weak and should be enriched and compared with similar or closely-related issues.

- The docking technique is employed to predict the tentative binding affinity of the ligand-receptor complex ahead of time. So this should preferably be presented at the beginning of the M+M.

Miscellaneous

- The abbreviation should be used at a first appearance for the most commonly used terms such as cutaneous leishmaniasis (CL) in the abstract and other places.

- After using Leishmania tropica or L.major the genus should be used in form of abbreviation throughout the manuscript (L.tropica or L.major). Mixed using of the species wording is used and this should be corrected consistently.

- The Old and New world should be written with capital first initials (i.e., introduction).

- The scientific name of organisms either parasite ( l97-98) or sand flies should be printed in the capital font ( e.i., Lutzomyia ( i.e., L70). In some places, Leishmania is not italic or with a small l (L477, L505).

- In-vitro or in vitro (L478).

- Phlebotomine should be written with small letters in the middle of a sentence (L70).

- The authors initially defined Miltefosine as MIL, then again used Miltefosine in L57. They must be consistent in using abbreviation forms? In some places, miltefosine is written with capital in other places with small (L418).

- Amphotericin B is written with small and capital ”A”(413 and many places). Be consistent in writing words.

Reviewer #2: The manuscript titled: "Anti-leishmanial Physalins - Phytochemcial Investigation, In vitro Evaluation against clinical and  MIL  Resistant L. tropica strains and In silico Studies" was aimed to describe the in vitro cytotoxicity and antileishmanial activity of the physalins from Physalis minima using normal fibroblast (3T3) and BJ (human fibroblast) cells lines and promastigotes L. major and L. tropica. For this, the authors used methodologies widely described and accepted by different authors in the world. The experiments are properly written, which makes them easy to reproduce. The results obtained allow conclusions to be made around the proposed objective and are rightly discussed. In my opinion, this manuscript is sustainable for publication in Plos One.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

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

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-16415R1Anti-leishmanial Physalins - Phytochemcial Investigation, In vitro Evaluation against clinical and MIL Resistant L. tropica strains and In silico StudiesPLOS ONE

Dear Dr. Yousuf,

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 Sep 29 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Shahid, Ph.D.

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 #1: (No Response)

**********

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

**********

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

Reviewer #1: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

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: No

**********

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: Specific comments

-The title still needs corrections as I displayed by track changes.

-In the abstract, the authors abbreviated cutaneous leishmaniasis in two places (L 27-30).

- The authors did not use an MTT assay to check the viability of promastigotes. Did they check for viability before performing the treatments to make sure they are all viable (motile) because they counted them by eyes?

- The authors visually counted the promastigote stage, why did they use DAPI stain? How did it support the study?

- Mention why you docked with phosphoglucose isomerase.

- Why the authors did not use a uniform and standard positive control like AmpB throughout the experiments so that one could be able to compare the outcome? All of the positive controls even AmpB, pentamidine, and MIL are the second-line drugs, except AmBisome (a liposomal form of AmpB is used to treat Indian and Mediterranean type kala-azar (visceral leishmaniasis due to L.donovani and L.infantum, respectively). The authors could use sodium stibogluconate (Pentostam®) or meglumine antimoniate (Glucantime®) and if resistance is a common phenomenon then AmpB would be OK, otherwise using different positive controls is not realistic. Miltefosine does not affect L. tropica it is only active at around 83%-85% against L.major and L.infantum.

- AmpB is not the drug of choice for L.tropica and L. major. It is rather a liposomal form OF AmpB (AmBisome used against VL). It is all right you used other second-choice drugs because you had no access to meglumine antimonite (Glucantime®) or sodium stibogluconate(Pentostam®), but you should have used similar positive controls to be able to compare the activity of each component.

- I am not export on docking reactions and you pay attention to other referees for this purpose.

- How could you tell that the difference between the test results and the untreated control group is significant, or how could you predict the significant difference among groups? The author should mention the statistical test and define the P-value. In the results whenever a significant level is seen the p-value should be pointed out.

- The docking technique is employed to predict the tentative binding affinity of the ligand-receptor complex ahead of time. So this should preferably be presented at the beginning of the M+M.

- After using Leishmania tropica or L.major the genus should be used in form of abbreviation throughout the manuscript (L.tropica or L.major). Mixed using of the species wording is used and this should be corrected consistently.

- The Old and New World should be written with capital first initials (like World).

- The scientific name of organisms either parasites or sand flies should be printed in the capital font ( e.i., Lutzomyia). In some places, Leishmania is not italic or with a small l.

- Subtitles are not uniformly written and they are written with mixed capital and small ones, whereas the journal style is different. I advise the authors once again to review the journal’s instructions or one of the newly published Plos One articles and follow through and make the corrections.

**********

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

**********

[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: Physalins-Plos One Word file August 14-2022.docx

Anti-leishmanial physalins - phytochemcial investigation, in vitro evaluation against clinical and MIL- Resistant L. tropica strains and in silico studies

PONE-D-22-16415R2

Dear Dr. Yousuf,

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,

Mohammad Shahid, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: