PONE-D-23-15586Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massaPLOS ONE

Dear Dr. Sunithi Gunasekera,

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"This work was supported by Swedish Research Council's Linkage grant (2017-05416) awarded to S.G.  Marine sponge samples collected through the scientific surveys with the R/V Dr. Fridtjof Nansen as a part of the collaboration between the EAF-Nansen Programme and the Government of Sri Lanka were included in the study. The EAF Nansen Programme is a partnership between the Food and Agriculture Organization of the United Nations (FAO), the Norwegian Agency for Development Cooperation (Norad), the Institute of Marine Research (IMR), Norway, and partner countries and institutes e.g. National Aquatic Resources Research and Development Agency (NARA), Sri Lanka. We thank the EAF Nansen Programme and the Government of Sri Lanka for their immense support. We would like to thank the crew and scientists who participated in the Dr. Fridtjof Nansen Survey, and the National Aquatic Resources research and development Agency (NARA) for supporting the study in many ways especially, K. G.S. Nirbadha, Chaminda Karunarathne and Chaminda Prasad for assisting sponge sample collections. Additionally, this study made use of the NMR Uppsala infrastructure, which is funded by the Department of Chemistry–BMC and the Disciplinary Domain of Medicine and Pharmacy, Uppsala University."

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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: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

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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

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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

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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: In this paper the authors have identified 35 sponge specimens from Sri Lanka and have identified 19 species to have cytotoxic and antibacterial activities. They have identified potent antibacterial and cytotoxic compounds from the aqueous extracts of Aciculites and Acanthella species. Extraction from Stylissa massa led to identification of bromopyrrole alkaloids with anti-bacterial activities. They have identified these bioactive compounds from less studied biodiversity hotspot in Sri lanka which could potentially have clinical use if characterized thoroughly.

Although they have identified several compounds with antibacterial and cytotoxic activity, the authors haven’t shown the mode of action of these drugs and their broad range activities against different bacteria or cell lines. Also the writing of the paper and the representative figures needs to be revisited and improved as it is hard for the readers to get the grasp of important results that are represented in table.

Major comments:

1. Results are not clearly represented for both antibacterial assay and cytotoxic assay. For table 2, Results of disk diffusion and microdilution assay would be best represented suing bar graphs as the numbers are difficult to interpret between species when represented in table.

2. Although the compounds are tested against 4 species only the results are shown for E. coli and S. aureus but not for Pseudomonas and Bacillus that were tested.

3. The final concentration of the compounds tested for diffusion assay is 20 ug/ml while the positive control ampicillin and gentamycin are used at the concentration of 10 ug/ml. Similar concentrations should be used for control and test compounds for comparison and dilutions of different concentrations must be tested.

4. For the compounds 1 and 2 tested why was the MIC was too high at 62.5 uM although it was previously shown to have antibacterial activity. Is it due to the mode of isolation from the extracts. Pure compounds (commercially synthesized) of 1 and 2 should be tested for anti-bacterial and cytotoxic effects to nullify any effect that has occurred during isolation.

5. As a follow up of previous point, though the mass and the structure has been determined by NMR, are the compounds tested with pure isolates of scepterin and hymenin to ascertain that both the isolated one from Stylissa massa and the pure compounds are same and runs similarly in HPLC?.

6. The authors have described the cytotoxic effects only in lymphoma cell lines. The effect of compounds on other cell lines including the non- carcinogenic ones like Hela must be tested as well to show that effect of tested compounds are specific to oncogenic cell line.

7. The mode of action of these antibacterial and cytotoxic compounds are not determined or discussed at all in the Discussion section. The effect of similar compounds on anti-bacterial and cytotoxic effects, the pathway it affects must be discussed.

8. Can the extracts from Aciculites and Acanthella species be repeated and scaled up as they had most antibacterial and cytotoxic activity?

Minor Comments

Line 27 – Change evaluated in to evaluated for

Line 32-33- Remove the sentence in parantheses as the details aren’t necessary for the abstract

What does (1) 2, 3 mean why it is parenthesized although the name has been described.

Line 39- Porifera –porifera

Line 564-565 – Why is Petrosia mentioned in paranthesis

Line 565- Remove paranthesis.

Table 1- What does voucher number, museum number and the Division Reg number mean in the table?

Table 1- what does the last column refer to what is COI? Is the numbers in the column refer to genbank accession number? How are the other species identified that doesn’t have any number eg: Row 1, Row 3 etc.

Table 2: It contains a lot of information. It would be good to represent them in graph for different assays or for different species.

.Line 302 and 304- Mention the actual final concentration instead of fold dilutions.

Reviewer #2: The systematic approach you employed in screening antibacterial and cytotoxic activities of marine sponges showcases the depth of your scientific rigor. The clarity and precision with which you detailed your methods and results enhance the reproducibility of your study.

Furthermore, your adeptness in isolating bromopyrrole alkaloids from Stylissa massa adds significant value to the field. Your findings contribute not only to the understanding of marine natural products but also to the potential development of novel therapeutic agents.

Congratulations on your exceptional work, and I eagerly anticipate seeing your research in print.

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Reviewer #1: No

Reviewer #2: Yes: Lama Al_Darwish

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PONE-D-23-15586R1Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massaPLOS ONE

Dear Dr. Gunasekera,

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 Jan 18 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,

Awatif Abid Al-Judaibi, PhD

Academic Editor

PLOS ONE

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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.

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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 #3: 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 #3: Yes

**********

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

Reviewer #1: N/A

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 #1: Yes

Reviewer #3: 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 #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 #1: The manuscript has been revised and all of the comments have been addressed.

The following points needs to be revised.

Line 408 and 423- Change “literature values” to “Previously published values”.

Line 456- Change A, orientalis to A. orientalis

Combine lines 513 to 516 to a single sentence.

Reviewer #3: Dear Authors

It is recommended to carry out future in vivo animal studies to confirm the in vitro enzyme assay

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Reviewer #1: No

Reviewer #3: Yes: Mahmoud A. Al-Sha'er

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Screening for antibacterial and cytotoxic activities of Sri Lankan marine sponges through microfractionation: Isolation of bromopyrrole alkaloids from Stylissa massa

PONE-D-23-15586R2

Dear Dr. Sunithi Gunasekera,

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.

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Kind regards,

Awatif Abid Al-Judaibi, PhD

Academic Editor

PLOS ONE