Investigating the role of FOX gene family in development and stress response in Labeo rohita: A multi-faceted analysis of phylogeny and genome characterization

Saima Naz; Urwah Ishaque; Ahmad Manan Mustafa Chatha; Muhammad Farooq; Qudrat Ullah; Shabana Naz; Marco Ragni; Ibrahim Alhidary

doi:10.1371/journal.pone.0323740

Peer Review History

Original SubmissionDecember 30, 2024
11 Feb 2025 Decision Letter - Bijay Behera, Editor Dear Dr. Naz, 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 Mar 28 2025 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. 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'. 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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, Bijay Kumar Behera, 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. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. Additional Editor Comments: The recent manuscript has its own merit. According to the two reviewers recommendations, my decision is to Major revision of the manuscript. Kindly see the two reviewers comment. [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? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: No ****** Reviewer #1: The article “Investigating the Role of FOX Gene Family in Development and Stress Response in Labeo rohita: A Multi-faceted Analysis of Phylogeny and Genome Characterization” is well written. The authors need to consider a few points mentioned below before publication. •The Abstract section is not justified. •L499: If possible, for secondary structure, authors are advised to use more than one program for their validation with suitable references •For using iTol, the file type used for designing the phylogeny should be mentioned. •The study only highlights the available sequences of the FOX gene in the NCBI database. The authors haven’t confirmed the 5’-UTR and 3’-Poly A tail by using RACE PCR. Using the terms in conclusion, like detailed genome-wide analysis, might not be true. If possible, avoid using terms like that. • The discussion section is well written. The authors advised to revise accordingly. Reviewer #2: Comments about the manuscript: The manuscript entitled “Investigating the role of FOX gene family in development and stress response in Labeo rohita: a multi-faceted analysis of phylogeny and genome characterization” aims to explore the evolutionary and molecular roles of FOX genes in L. rohita using computational analysis. The introduction is streamlined and clear but the other section should be improved. The manuscript may be published with a revision as detailed below. •In Abstract, authors should specifically state the need of this research. •Line No 134-135: Bootstrap consensus values were calculated for each node in the analysis……………. In Figure 1, no bootstrap values are visible. •Figure caption should be improved with details. •Discussion section should be revised by the authors. Try to correlate the results obtained with each other. •Line No 499: Authors are advised to use multiple programs for validation with appropriate references. •4.2. Phylogenetic Analysis of FOX genes superfamily: Authors should write their methodology in details. •Line No 528: Avoid the term ‘detailed genome-wide analysis of the FOX gene family’, because authors only used computational tools for this study. ****** 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. https://doi.org/10.1371/journal.pone.0323740.r001
Revision 1
27 Mar 2025 Author Response Dear Editor and Reviewers, We would like to express our sincere gratitude for the time and effort invested in reviewing our manuscript entitled “Investigating the Role of FOX Gene Family in Development and Stress Response in Labeo rohita: A Multi-faceted Analysis of Phylogeny and Genome Characterization.” Your insightful comments have been invaluable in enhancing the quality and clarity of our work. Below, we provide a detailed, point-by-point response to all comments raised. Response to Reviewer #1 Comment: “The Abstract section is not justified.” Response: We have revised the abstract to ensure full justification in accordance with the journal’s formatting guidelines. The updated abstract now meets the required presentation style. Comment: “If possible, for secondary structure, authors are advised to use more than one program for their validation with suitable references.” Response: We thank the reviewer for the suggestion. Our manuscript now specifies that FOX protein secondary structure was predicted using PSIPRED and independently validated with Pyre2, with both methods yielding consistent results. All relevant references are now provided in the manuscript. Comment: “For using iTol, the file type used for designing the phylogeny should be mentioned.” Response: We have now specified in the Methods section that the phylogenetic tree was generated using the Newick format file generated by MEGA11. Comment: “The study only highlights the available sequences of the FOX gene in the NCBI database. The authors haven’t confirmed the 5’-UTR and 3’-Poly A tail by using RACE PCR. Using the terms in conclusion, like detailed genome-wide analysis, might not be true. If possible, avoid using terms like that.” Response: We acknowledge this concern and have revised the manuscript accordingly. The text now clearly states that the study is based on computational analysis of sequences available in the NCBI database. The term “detailed genome-wide analysis” has been replaced with “comprehensive computational analysis” to more accurately reflect the scope of our work. Comment: “The discussion section is well written. The authors advised to revise accordingly.” Response: We appreciate the positive feedback. Minor revisions have been made in the discussion to enhance clarity and to improve the integration and correlation of the results. Response to Reviewer #2 Comment: “In Abstract, authors should specifically state the need of this research.” Response: The abstract has been updated to explicitly state the motivation behind our study, highlighting the significance of understanding the role of FOX genes in the developmental and stress response mechanisms of Labeo rohita. Comment: “Bootstrap consensus values were calculated for each node in the analysis……………. In Figure 1, no bootstrap values are visible.” Response: We have revised Figure 1 to include bootstrap values for each node. The figure legend has been updated to describe these values and the method of calculation in detail. Comment: “Figure caption should be improved with details.” Response: The caption for Figure 1 has been expanded to include additional details. Comment: “Discussion section should be revised by the authors. Try to correlate the results obtained with each other.” Response: We have restructured the discussion to provide a more cohesive interpretation of our findings. The revised version more clearly correlates the outcomes. Comment: “Authors are advised to use multiple programs for validation with appropriate references.” Response: In our study, FOX protein secondary structure was accurately predicted using PSIPRED and validated with Pyre2 for 2D analysis. We have now incorporated SWISS-MODEL for 3D structure validation, and relevant references have been added to the manuscript. Comment: “4.2. Phylogenetic Analysis of FOX genes superfamily: Authors should write their methodology in details.” Response: We have expanded Section 4.2 to include a detailed description of our phylogenetic analysis methodology to enhance reproducibility. Comment: “Avoid the term ‘detailed genome-wide analysis of the FOX gene family’, because authors only used computational tools for this study.” Response: In line with the feedback, we have replaced the term “detailed genome-wide analysis” with “comprehensive computational analysis” to more accurately represent our study’s scope. We believe that these revisions have significantly improved the manuscript and have addressed all the concerns raised. We appreciate the reviewers’ constructive feedback and are hopeful that the revised manuscript meets the journal’s standards for publication. Thank you for considering our revised manuscript. We look forward to your positive response. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0323740.r002
8 Apr 2025 Decision Letter - Bijay Behera, Editor Dear Dr. Naz, 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 May 23 2025 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. 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 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, Bijay Kumar Behera, 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. Additional Editor Comments: Dear Author, Now the manuscript has been revised. One minor comment is in the manuscript. Kindly see the reviewers comments and revise accordingly. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: (No Response) Reviewer #2: The authors have addressed all the comments satisfactorily. Only one issue I have found in the Revised manuscript which is mentioned below: Line no 137-138: These sequences were organized into 12 major clades ……….. But there are 11 clades. In Figure 1, they have also clustered the phylogenetic tree into 11 clades. Please rectify it. The manuscript may be accepted after fixing the issue. ****** 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 Attachments Attachment Submitted filename: Review comments 2.docx https://doi.org/10.1371/journal.pone.0323740.r003
Revision 2
11 Apr 2025 Author Response Dear Editor and Reviewers, We would like to express our sincere gratitude for the time and effort you have dedicated to reviewing our manuscript entitled “Investigating the Role of FOX Gene Family in Development and Stress Response in Labeo rohita: A Multi-faceted Analysis of Phylogeny and Genome Characterization.” Your thoughtful and constructive feedback has been instrumental in improving the overall quality and clarity of our work. Following the recent round of minor revision, we have carefully addressed the remaining comment provided by Reviewer #2, as detailed below: Response to Reviewer #2 Comment: Line no. 137–138: “These sequences were organized into 12 major clades...” But there are 11 clades. In Figure 1, they have also clustered the phylogenetic tree into 11 clades. Please rectify it. Response: The mentioned line has been corrected in the revised manuscript, changing the number of clades from 12 to 11 to accurately reflect the phylogenetic analysis and Figure 1. We have reviewed the manuscript thoroughly to ensure consistency throughout the text. We appreciate your continued support and are hopeful that the manuscript is now suitable for acceptance. Thank you once again for your guidance and constructive suggestions throughout the review process. Sincerely, Shabana Naz Department of Zoology, Government Sadiq College Women University, Bahawalpur 63100, Punjab, Pakistan. Email: drshabananaz@gcuf.edu.pk Attachments Attachment Submitted filename: Response_to_Reviewers_auresp_2.docx https://doi.org/10.1371/journal.pone.0323740.r004
15 Apr 2025 Decision Letter - Bijay Behera, Editor Investigating the Role of FOX Gene Family in Development and Stress Response in Labeo rohita: A Multi-faceted Analysis of Phylogeny and Genome Characterization PONE-D-24-60377R2 Dear Dr. Naz, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Bijay Kumar Behera, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): The manuscript entitled "Investigating the Role of FOX Gene Family in Development and Stress Response in Labeo rohita: A Multi-faceted Analysis of Phylogeny and Genome Characterization" has been accepted for publication. Reviewers' comments: https://doi.org/10.1371/journal.pone.0323740.r005
Formally Accepted
Acceptance Letter - Bijay Behera, Editor PONE-D-24-60377R2 PLOS ONE Dear Dr. Naz, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Bijay Kumar Behera Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0323740.r006

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