PONE-D-22-30479Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola giganticaPLOS ONE

Dear Dr. Beg,

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 Apr 22 2023 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,

Paulo Lee Ho, 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. Thank you for stating the following financial disclosure:

“No funding was available for this project”

At this time, please address the following queries:

a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution.

b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

c) If any authors received a salary from any of your funders, please state which authors and which funders.

d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.”

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

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4.  PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

[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: No

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: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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

Reviewer #1: The manuscript is interesting, and important to the field since Fasciola sp infection is difficult to do a diagnosis because of the low presence of eggs of the parasite in feces and treatment is also problematic. So, it is important to discover new tools for diagnosis and treatment. Despite that, the manuscript has some problems that should be improved. The questions are:

Major:

1. In figure 1a, Where are the positive and negative controls of PCR? What is the size of the amplicon? Is it specific? Was it sequenced?

2. In figure 2b, did the researchers apply any statistical approach in this experiment?

3. In figures 3a and 3b how can I prove that is a mitochondrial protein extract? I suggest a control using an antibody against mitochondrial marker protein.

4. In this phrase "For the immunological characterization of MAO-A in F. gigantica worms a number of studies were performed". How many? Please specify, the number of studies made.

5. In figure 4b, where is lane 2?

6. The results which are shown in figure 7 sound interesting, but the authors should provide loading controls ( ponceau stained membrane and/or a mitochondrion antibody marker with present no alteration with inhibitor treatment. Without this, is not possible to do the affirmation made by the authors in the text.

7. It seems that the anti-MAO-A primary antibody was purchased from Sigma Aldrich. This antibody was raised in which organism? Fasciola? If not raised in Fasciola, what is the degree of identity of the epitope?

Minor:

1. In the phrase in the Introduction section: "Till now, MAO has been reported in parasitic helminths belonging to different classes, namely", please change "till" to "Until".

2. In Materials and methods in the PCR section: Please provide PCR reaction components concentrations

3. Please provide a link to the PCR amplification with other results.

Reviewer #2: PONE-D-22-30479

Title: “Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola gigantica.”

Minor Revision

The publication does not overstate anything and addresses the titular claims well. It is a well referenced manuscript and authors have nicely mentioned drug resistance story and explained the present gaps in the characterization of MAO in helminths. Furthermore, characterization through multi-omics approach validates most of the findings. I believe molecular characterisation of parasite is a strong pivot of this manuscript because identification of field-procured parasite just on the basis of morphological identification could give false positive results. Although, I have no doubt on experimentation but the quality of manuscript can be improvised by small changes which I have enumerated below:

1. It is mentioned that MAO activity in mitochondria is significantly higher compared to whole homogenate but there is no significant asterisks on activity bars. Kindly put significant marks in Fig 2a.

2. Authors have forgotten to put lane 2 western blot in Fig 4b. Kindly fix that.

3. In Abstract, abbreviate the full form of ‘WHO’ as ‘World Health Organization’ because it is not a good idea to introduce abbreviated words in abstract because it is the first text the audience reads.

4. Almost at all the places spelling of ‘Dependent’ is wrong. Kindly replace “Dependent” with “Dependant”. For spellings, stay with either UK English or US English in a manuscript and not a mixture of both in a single publication.

5. Avoid using same adjective in a single sentence such as in this statement “The strong intensity of bands in western blots and the spots in Dot-blots indicate strong immunogenic nature of the MAO protein” strong is used two times as an adjective. Then again after two three lines “clearly indicating that the tropical liver fluke possesses strong MAO-A activity” the use of word “strong” comes into play. Kindly replace it with different synonyms.

6. The statement in 3rd paragraph of introduction “thus making MAO an indispensable biomolecule for developing the host-parasite interaction for the survival and perpetuation” does not make any sense or it is too complex. Kindly use simpler text for a better understanding for a wider range of audience.

7. Kindly rephrase the last paragraph statement in introduction “Previously researchers have reported and localized MAO in helminth parasites but data is still in dearth about the complete picture of this enzyme in a specific parasite”.

8. In the conclusive remarks “Considering the present results, it can be elucidated that MAO-A is an important neurophysiological enzyme having a high molecular mass and is abundantly present in the mitochondria of tropical liver fluke, F. gigantica” refrain from using unspecific words such as “high molecular mass” instead of that mention the size of band observed.

**********

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: Yes: Dr. Shahanwaz Rehman

**********

[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: Minor Revision PONE-D-22-30479.pdf

Characterization of Monoamine Oxidase-A in tropical liver fluke, Fasciola gigantica

PONE-D-22-30479R1

Dear Dr. Beg,

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,

Paulo Lee Ho, 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

**********

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: Yes

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy 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: 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: 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: The manuscript is very well written, depicting the complete story in a lucid manner. The queries and comments are very well answered and introduced in the revised manuscript.

Thank You

**********

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: Yes: Dr. Shahnawaz Rehman

**********