PONE-D-24-12061Effects of electroporation on Acanthamoeba

PolyphagaPLOS ONE

Dear Dr. Pessoa,

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.

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We look forward to receiving your revised manuscript.

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Alireza Badirzadeh

Academic Editor

PLOS ONE

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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

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

Reviewer #1: I Don't Know

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:  1. This article has previously been presented at the following congress: CAMPOS, M. S. Q. ; FREITAS, D. ; BARCELOS, R. ; PINTO, L. F. ; PESSOA, P. S. P. . EFFECTS OF ELECTROPORATION ON Acanthamoeba Polyphaga. 2023. (Apresentação de Trabalho/Congresso). Based on the link: https://sbpz.org.br/wp-content/uploads/2023/09/livro-inteiro-04set.pdf

2. There are some similarities in this article with other published articles.

Reviewer #2:  Abstract section

1. Lack of information regarding the methods used for performing electroporation on cysts and trophozoites.

2. Insufficient details about the presented results, including data interpretation and statistical distributions.

3. Need for technical enhancement in presenting the results in a manner that allows for more precise analysis and comprehensive interpretation.

4. Absence of sufficient information regarding the significance of the obtained results and the necessity for clearer articulation of this significance.

5. Requirement for delineating limitations and suggestions for future research in the abstract.

Introduction section

1. Line 50: "genus Acanthamoeba spp." should be "genus Acanthamoeba" as "spp." indicates multiple species, which is not necessary here.

2. Line 54: Instead of "evolutionary forms," use "life cycle stages" for more accurate terminology.

3. Line 57: Change "These are latent and resistant forms, allowing them to remain and spread in the environment, as well as becoming chronic in cases of eye disease" to "These are dormant and resistant forms, enabling them to persist and spread in the environment, and to cause chronic infections in cases of eye disease."

4. Line 62: Replace "23 different genotypes (T1-T23)" with "23 distinct genotypes (T1-T23)."

5. Line 64: "in humans are relatively uncommon" should be "are relatively uncommon in humans."

6. Line 69: Change "which can easily be confused with other ocular infections" to "often confused with other ocular infections."

7. Line 70: Replace "expanding to severe cases with more specific symptoms, severe and painful ulcers in the corneal region, which can spread to the inner layers of the eyeball" with "progressing to severe cases with more specific symptoms, including severe and painful corneal ulcers that can extend to the inner layers of the eyeball."

8. Line 74: "throughout the therapeutic process" should be "during the treatment process."

9. Line 85: "making it less time-consuming for patients" should be "reducing the treatment duration for patients."

10. Line 88: "applying electric fields" should be "application of electric fields."

11. Line 91: Change "configured to form hydrophilic pores" to "adjusted to form hydrophilic pores."

12. Line 94: "and then closes, after which cellular equilibrium is re-established" should be "and then closes, allowing cellular equilibrium to be re-established."

13. Line 95: Change "With more exacerbated pulse parameters, the electroporation process becomes irreversible, as the cell is unable to restructure itself" to "With higher pulse parameters, electroporation becomes irreversible, preventing the cell from restructuring."

14. Ensure the reference to "Pussard and Pons classification, 1977" is accurate and from a reliable source.

15. Provide more details about the safety and efficacy of the electroporation method.

16. Mentioning the potential use of electroporation to facilitate treatment should be backed by supporting studies and scientific research.

• The introduction is quite lengthy. Consider summarizing some sections and focusing on the most critical points to maintain reader engagement and clarity.

Methodology Section:

- The methodology section is generally well-organized, but there are some areas where clarity and detail can be improved. Additionally, consistent formatting and correct scientific terminology are essential.

1. Line 105: "A standardized Acanthamoeba strain was used in this study: Acanthamoeba Polyphaga (American Type Culture Collection, ATCC® 30461TM) obtained from a corneal scrape of a CA case in Texas, 1973."

• Change "CA case" to "keratitis case."

• Consider rephrasing for clarity: "A standardized strain of Acanthamoeba polyphaga (ATCC® 30461TM) was used in this study, obtained from a corneal scrape of a keratitis case in Texas, 1973."

2. Line 109: "The ATCC 30461 isolate was grown on non-nutrient agar medium supplemented with heat-inactivated E. coli (DH5α) at 25ºC."

• Remove the line break before "The ATCC 30461 isolate."

• "heat-inactivated" should be hyphenated.

3. Line 110: "After these strains had been disentangled, the non-nutrient agar plate was washed with transport saline and the wash was centrifuged at 2000 RPM for 15 minutes."

• "disentangled" is not the correct term. Use "harvested."

• Specify the exact centrifuge conditions: "washed with transport saline and centrifuged at 2000 RPM for 15 minutes."

4. Line 112: "After centrifugation, the supernatant was discarded and the amoeba pellet re suspended in 1mL of culture medium, which was then inoculated into a 25 cm2 culture flask containing 4mL of culture medium and incubated in an oven at 25ºC."

• "re suspended" should be "resuspended."

• Clarify "culture medium" to specify the type.

5. Line 114: "To obtain the trophozoite forms, the isolate was grown in PYG (proteose-peptone - yeast extract - glucose) culture medium and, for the cystic forms, it was grown in Neff encystment medium."

• Clarify: "To obtain the trophozoite forms, the isolate was grown in PYG (proteose-peptone - yeast extract - glucose) culture medium. For cystic forms, it was grown in Neff's encystment medium."

6. Line 119: "Trophozoites and cysts of the ATCC 30461 isolate were counted in a Neubauer chamber and their concentration was adjusted to obtain an initial inoculum of 5x106 trophozoites/cysts per mL of electroporation buffer (285mM sacarose, 0,7 MgCl2, 1mM KlC, 10mM HEPES, 3Mm NaOH)."

• Correct "sacarose" to "sucrose."

• Correct "0,7 MgCl2" to "0.7 mM MgCl2."

• Correct "KlC" to "KCl."

• Correct "3Mm NaOH" to "3 mM NaOH."

7. Line 122: "Thus, 100μL of this inoculum (5x105 amoebas) were added to the electroporation cuvettes exposed to an electric field of 2000 and 2500V."

• Clarify: "Thus, 100μL of this inoculum (5x105 amoebas) was added to the electroporation cuvettes, which were then exposed to an electric field of 2000 or 2500V."

8. Line 123: "Subsequently, methods for detecting electroporation involving fluorescence, described in detail below in item “Electroporation detection”, were carried out to standardize and evaluate the best voltage to be applied to Acanthamoeba cysts and trophozoites."

• "Subsequently, fluorescence-based methods for detecting electroporation were carried out as described below in the “Electroporation detection” section, to standardize and evaluate the optimal voltage for Acanthamoeba cysts and trophozoites."

9. Line 129: "The in vitro experimental model was divided into the following groups: cysts and trophozoites controls (without electroporation); group 1: cysts and trophozoites electroporated at 2000 volts; group 2: cysts and trophozoites electroporated at 2500 volts."

• Use bullet points or a clearer list format for readability:

� Controls (cysts and trophozoites without electroporation)

� Group 1: cysts and trophozoites electroporated at 2000 volts

� Group 2: cysts and trophozoites electroporated at 2500 volts

10. Line 138: "To assess the loss of cysts and trophozoites from the ATCC 30461 isolate resulting only from the shock applied, a separate analysis was first carried out counting the number of amoebas in each group before and after electroporation using Trypan blue 0.4% in a Neubauer hemocytometer."

• "resulting only from the shock applied" can be simplified: "To assess the loss of cysts and trophozoites due to the applied shock, a separate analysis was performed. Amoebas in each group were counted before and after electroporation using 0.4% Trypan blue in a Neubauer hemocytometer."

11. Line 146: "Subsequently, another analysis was carried out using the fluorescence intensity method. The electroporated cysts and trophozoites were plated in 96-well plates and stained with the fluorescent dye propidium iodide (PI; Sigma-Aldrich, USA)."

• Simplify: "Another analysis was performed using fluorescence intensity. Electroporated cysts and trophozoites were plated in 96-well plates and stained with propidium iodide (PI; Sigma-Aldrich, USA)."

12. Line 152: "The fluorescent dye 4',6-diamidino-2-phenylindol (DAPI; Sigma-Aldrich, USA), acts as a marker, coloring any cell with a blue nucleus."

• Correct spelling: "4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, USA), acts as a marker, staining the nucleus of any cell blue."

13. Line 167: "Statistical analyses were carried out using the GraphPad Prism 8.0 program. The two-way ANOVA test was used for multiple comparisons between groups. Values of p < 0.05 were considered statistically significant."

• Minor correction: "Statistical analyses were carried out using GraphPad Prism 8.0. A two-way ANOVA test was used for multiple comparisons between groups. Values of p < 0.05 were considered statistically significant."

In discussion section

1. Some concepts need to be explained in simpler terms to enhance reader comprehension.

2. Provide more specific numbers and detailed information about the experimental setup, methodology, and results. Additionally, ensure consistency in referencing previous studies.

3. Elaborate on certain aspects of the results and methods to provide a clearer understanding, especially regarding any discrepancies observed in the experiments.

4. Offer a more detailed interpretation of the results, including comparisons with similar studies and a discussion of potential implications and limitations.

5. Discuss the generalizability of the findings beyond the laboratory setting and their applicability in real-world clinical scenarios.

Overall, improvements in expressing concepts, more precise information about results, additional explanations, and a more logical interpretation of the results can enhance the paper.

As a reviewer of the manuscript, I recommend addressing the low quality of the images and graphs. It is essential that these visual elements be of higher quality to effectively convey the research findings. Therefore, I strongly advise the authors to improve the resolution and clarity of the images and graphs before finalizing the manuscript for publication. This enhancement will significantly enhance the overall presentation and comprehension of the study.

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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:  Ahmad Hosseini-Safa

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Attachments

Attachment

Submitted filename: PPC-Report-176780.pdf

<div>PONE-D-24-12061R1Effects of electroporation on Acanthamoeba

polyphagaPLOS ONE

Dear Dr. Pessoa,

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 Nov 21 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,

Alireza Badirzadeh

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.

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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: (No Response)

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: Yes

**********

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

Reviewer #1: I Don't Know

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 #1: Yes

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 #1: Yes

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 #1: 1. Since a part of this article has already been presented in the Congress, if it does not conflict with the policies of this journal, it can be printed.

2. It is better to write the sample storage and transfer method.

Reviewer #2: 1. Original Research:

The study presents novel research focused on using electroporation to treat Acanthamoeba polyphaga cysts and trophozoites, which addresses an important area in ophthalmology.

The originality of the topic, especially the use of electroporation as a potential adjunct therapy, is evident and relevant. It appears to have been presented in conferences before, but the authors clarify that this is the first full disclosure of the study.

2. Unpublished Results:

The results do not seem to have been previously published elsewhere, except for a presentation at a conference, which is common practice.

3. Technical Standards and Clarity of Methods:

The methods are described with sufficient technical detail, allowing replication of the experiment.

However, clarity in some areas of the methodology could be improved. For example, the explanation of the electroporation setup, such as voltage pulse durations and the number of pulses, could be expanded for clarity.

4. Appropriate Conclusions:

The conclusions are consistent with the data, particularly regarding the effectiveness of electroporation for Acanthamoeba polyphaga.

However, the conclusions might benefit from further elaboration on the clinical implications of the findings. While the results are promising, discussing potential hurdles in clinical application (such as safety or cost-effectiveness) would add depth.

5. Presentation and Language:

The article is mostly well-written and intelligible, but there are some minor language issues that can be improved for better readability. For instance, sentence structure could be simplified in sections like the discussion. Some scientific terminology is overly technical for a general reader, so clarification may be needed in specific areas.

Spelling and grammatical issues: There are a few minor errors, such as "re suspended" (should be "resuspended"), "sacarose" (should be "sucrose"), and "permaebilization" (should be "permeabilization").

6. Ethical Standards and Research Integrity:

The study adheres to ethical standards, although there is no explicit mention of ethics approval, which should be included if necessary for any animal or human-related procedures (although this may not apply here).

There are no competing interests or financial disclosures that affect the research.

7. Data Availability and Reporting Standards:

The authors have confirmed that all data necessary to replicate the study are included, and appropriate figures and tables support the data.

Recommendations for Improvement:

Clarification in Methods: Improve the explanation of the electroporation parameters, including the exact number of pulses and pulse duration.

Language Refinement: Fix minor grammatical and structural issues to enhance clarity.

Further Interpretation: Expand on the implications of the findings in a clinical context, and discuss potential limitations or challenges in applying electroporation for AK treatment.

Ethics Approval: Ensure that any necessary ethical statements are included, if applicable.

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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: Yes:  Fares Bahrami

Reviewer #2: No

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[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.

Effects of electroporation on Acanthamoeba

polyphaga

PONE-D-24-12061R2

Dear Dr. Palloma Santiago Prates Pessoa,

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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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,

Alireza Badirzadeh

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: "I have no concerns regarding dual publication or research ethics. The manuscript appears to comply with all necessary ethical standards. I have reviewed the submission and confirm that there is no indication of dual publication or unethical practices. The study follows appropriate guidelines for ethical research, and no issues related to research ethics were noted."

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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 #2: Yes:  Ahmad Hosseini-Safa

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