Comprehensive transcriptomic analysis of prostate cancer lung metastases

Alireza Saraji; Katharina Wulf; Janine Stegmann-Frehse; Duan Kang; Anne Offermann; Danny Jonigk; Mark Philipp Kuehnel; Jutta Kirfel; Sven Perner; Verena Wilbeth Sailer

doi:10.1371/journal.pone.0306525

Peer Review History

Original SubmissionDecember 28, 2023
6 Mar 2024 Decision Letter - Sumit Kumar Hira, Editor PONE-D-23-41725Comprehensive transcriptomic analysis of prostate cancer lung metastasesPLOS ONE Dear Dr. Saraji, 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. The study on prostate metastases gene expression profiling using FFPE samples is critiqued for not incorporating control specimens, a step crucial for identifying uniquely expressed genes between bone and lung metastases. Adjusted p-values are recommended for precise identification of differentially expressed genes, minimizing false positives. Additionally, standardizing mRNA quantification using RPKM/FPKM is suggested to ensure data reliability. The method for assessing RNA integrity, based on fragment length, is deemed inadequate; calculating the RNA Integrity Number (RIN) is proposed for more accurate quality assessment. The article's low-resolution figures also require improvement for better interpretation. Further suggestions include adding a patient demography table for contextual clarity, providing explicit criteria for mRNA regulation cut-offs, validating top dysregulated genes through secondary techniques like qRT-PCR, and offering a clearer rationale for selecting chemotactic genes in the analysis of altered pathways. These recommendations aim to bolster the study's methodological rigor, clarity, and the comprehensiveness of its findings on the gene expression profiles of prostate metastases. Please submit your revised manuscript by Apr 20 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, Sumit Kumar Hira, 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: "This work was funded by Deutsche Forschungsgemeinschaft (DFG) Schwerpunktprogramm µBone SPP2084 (to VS and SP). Duan Kang was supported by China Scholarship Council at the University of Luebeck (No. 202008440263)." Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: No ****** 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 study by Saraji et al. demonstrated the comprehensive transcriptomic analysis of prostate cancer lung metastases. The authors should modify the manuscript by considering following points. 1.The authors have used FFPE samples of prostate metastases samples and compared transcriptome profile of bone metases and lung metases samples.But authors have not used any control/adjacent control specimens to derive the specific differentially expressed genes between the subsets. Justify. 2.Regarding the statistical analysis of gene expression data, adjusted p value should be calculated to identify the differentially expressed genes in each category of samples. 3. mRNA count should be expressed as RPKM/FPKM which is the standard protocol for RNA sequencing data. 4.Here, RNA integrity with at least 90% of the fragments longer than 100 nucleotides were considered as suitable for gene expression analysis. Instead of the parameter, RIN should be calculated for the samples which is the standard protocol for gene expression analysis. 5.The resolution of the figures are very poor. That should be modified. Reviewer #2: In this manuscript, the authors have comprehensively explored transcriptomic alterations in PCa lung metastases in comparison to primary PCa and PCa bone metastases. They have reported that upregulation of immunogenic response genes and downregulation of epithelial-mesenchymal transition genes were associated with PCa lung metastases, thereby, suggesting that lung metastases from PCa have a higher immunogenic character, which may increase patients' susceptibility to immunotherapeutic treatments. The structuring of the manuscript is well-conceived, easy to follow, and the literature survey in the related field is comprehensive. However, the reviewer found some ways to do the manuscript better for the publication purpose. The comments from the reviewer are listed below: Comment 1: The article should include a patient demography table. Comment 2: It is not clear from the manuscript which log2 Fold cut-off value has been considered for determining mRNA up- and down-regulation. Additionally, the author should include a table or a supplementary Excel file listing all upregulated and downregulated genes. Comment 3: The dysregulated candidate gene expression (Top 10 up and down) should have been validated by a secondary technique (say qRT-PCR) in the same sample-set. Comment 4: The authors can be clearer in explaining the choice of chemotactic genes in the result section under the subheading "Altered pathways and biological processes in the metastatic microenvironment," since Figure 2a shows additional altered pathways and biological processes in the metastatic microenvironment. ****** 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. Attachments Attachment Submitted filename: review 29224.docx https://doi.org/10.1371/journal.pone.0306525.r001
Revision 1
8 Apr 2024 Author Response Responses to the Academic Editor from the authors Dear Academic Editor, Thank you very much for taking the time to review our manuscript “Comprehensive transcriptomic analysis of prostate cancer lung metastases”, (Manuscript ID: PONE-D-23-41725). We appreciate the academic editor and reviewer's warm thoughts and comments and we already provided and uploaded rebuttal letters for each reviewer separately. Please find the detailed point by point responses to the academic editor below. The corresponding corrections highlighted with the tracked changes in the revised manuscript and re-submitted files accordingly. Your sincerely, The Authors Point by Point Response to the Academic Editor: 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 Response 1. We appreciate this precious this comment. In order to follow the instruction, we have already re-checked our manuscript according to your journal style requirement, we have now made few changes inside the manuscript with tracked changes in the “revised manuscript” version accordingly. Furthermore, all figures have been already qualified and enhanced using “PACE” software accordingly. 2. Thank you for stating the following financial disclosure: "This work was funded by Deutsche Forschungsgemeinschaft (DFG) Schwerpunktprogramm µBone SPP2084 (to VS and SP). Duan Kang was supported by China Scholarship Council at the University of Luebeck (No. 202008440263)." Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. Response 2. We again appreciate this comment. In order to follow the instruction, we have already inserted the amended statement in our revised manuscript with tracked changes according to your instruction as following: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.. Response 3. We again thank for this comment. In order to follow the instruction according the guidelines, we have already inserted captions for the supporting information (Supplementary file 1 and 2) in our revised manuscript highlighted with tracked changes. Your sincerely, The Authors Attachments Attachment Submitted filename: Response to Reviewer #2.docx https://doi.org/10.1371/journal.pone.0306525.r002
7 May 2024 Decision Letter - Sumit Kumar Hira, Editor PONE-D-23-41725R1Comprehensive transcriptomic analysis of prostate cancer lung metastasesPLOS ONE Dear Dr. Saraji, 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 Jun 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, Sumit Kumar Hira, Ph.D. Academic Editor PLOS ONE Additional Editor Comments: Despite its potential significance, the manuscript fails to meet the standards required for publication in its present form. Unfortunately, the manuscript lacks the appropriate statistical methodologies and analyses required to support its claims effectively. [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) 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 #1: No Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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: The authors had not addressed the points regarding statistical analysis which is the most important criteria for such transcriptome analysis. Reviewer #2: (No Response) ****** 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 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.0306525.r003
Revision 2
5 Jun 2024 Author Response Responses to the respected reviewer One (Manuscript ID: PONE-D-23-41725, “Comprehensive transcriptomic analysis of prostate cancer lung metastases” Dear Reviewer One, Thank you very much for taking the time to review this manuscript. We sincerely appreciate your warm thoughts and comments. Here are the comprehensive point-by-point answers to your comments, accompanied with the highlighted tracked changes in the revised manuscript and re-submitted files where appropriate. Your sincerely, The Authors Point by Point Response to the Reviewer #1: The study by Saraji et al. demonstrated the comprehensive transcriptomic analysis of prostate cancer lung metastases. The authors should modify the manuscript by considering following points. Comment 1. The authors have used FFPE samples of prostate metastases samples and compared transcriptome profile of bone metastases and lung metastases samples. But authors have not used any control/adjacent control specimens to derive the specific differentially expressed genes between the subsets. Justify. Response 1. We appreciate this precious reviewer's comment. We sincerely regret the unwelcome disparity! As a reference and control, primary PCa was accidentally reported as "PCa bone metastasis" in the result section, despite the fact that we had already noted this in the abstract and conclusion sections! Actually, for this study we have taken primary PCa as reference “setting primary prostate cancer” as a reference versus PCa lung metastases samples but meanwhile we also compared our results from PCa lung metastases to PCa bone metastasis and there we found that 209 genes were significantly upregulated, and 100 genes were significantly downregulated in prostate cancer lung metastases as we described in the result part. We apologize for the mistake in mentioning mentioned bone metastases instead of primary PCa! In order to correct the above-mentioned discrepancy and mistakes, we have now corrected and inserted the number of our primary PCa FFPE as control in the part “Method-material/Cohort” line 89.90 and also correction in the part “Result-1” in the lines 152 accordingly in the revised manuscript. Comment 2. Regarding the statistical analysis of gene expression data, adjusted p value should be calculated to identify the differentially expressed genes in each category of samples. Response 2. We thank the reviewer's comments. Given that the FDR (False discovery rate) technique improved and adjusted the features of the p-value distribution. Furthermore, FDR helped us filter out a subset of DEGs while preserving the genes of interest (supplementary table 1). However, one point we want to emphasise specifically is that if the FDR value > 0.05 and P value < 0.05, we will still use the p-value. Additionally, according to the Nanostring TM facilities while using nsolver to calculate all samples one can chose the FDR option output as the adjusted p-value. In order to answer this comment in another way, we also used the Benjamini-Hochberg’s method. Furthermore, we then used following formula to calculate the initial FDR, which is expressed as FDR and also correlated FDR in the supporting information table 2 (supplementary table 2). FDR(x)=P(x)m/x According to the value of X (rank value of p-value) from large to small, FDR(x)=min {FDR(x), FDR(x+1)} We now created and uploaded a supplementary excel file named 2 represented our data based on the adjusted p value or FDR. Please refer to the supplementary 2 for the details. Comment 3. mRNA count should be expressed as RPKM/FPKM which is the standard protocol for RNA sequencing data. Response 3. We again appreciate this reviewer's comment. Nanostring TM is an alternative to traditional microarray technology and RNA-seq technology. The principle of Nanostring TM technology is based on the direct measurement of the barcode of the fluorescent molecule on the probe after the nucleic acid molecule hybridizes to the probe. According to the official manual of nCounter; nCounter Pro Analysis System User Manual (nanostring.com), we can use the companion software in order to obtain differentially expressed genes without converting the raw count expression matrix to RPKM/FPKM (e.g., E.Shenderov et al., 2023, PMID: 37012549). In addition, statistically speaking, regardless of whether FPKM is a necessary presentation method, we only used gene expression data in our experiments for differential analysis, and we did not use these data for survival analysis. However according to Nanostring again, variance analysis does not require FPKM normalization of the original data. In this study we only use gene expression matrix to analyse the expression differences of each gene (PMID: 37012549). Comment 4. Here, RNA integrity with at least 90% of the fragments longer than 100 nucleotides were considered as suitable for gene expression analysis. Instead of the parameter, RIN should be calculated for the samples which is the standard protocol for gene expression analysis. Response 4. We again appreciate this reviewer's comment. As we have also mentioned in the Response 4, according to the standard protocol provided by the manual nCounter Pro Analysis System User Manual (nanostring.com), we only need to measure the purity of mRNA and do not need to test the RNA integrity number to obtain samples that meet the standard criteria for the quality requirements. However, in our previous publication (Saraji et al., 202, PMID: 34529723) we have already reported a comparative and comprehensive analyses for RNA purity and integrity between classical bioanalyzers (Qubit TM, nanodrop®) and Nanostring TM and there we showed that in Nanostring TM the expressed genes which is produced by nCounter output already passed the RNA quality criteria including RIN, RNA binding density and RNA fragmentation threshold (Saraji et al., 202, PMID: 34529723). Comment 5. The resolution of the figures are very poor. That should be modified. Response 5. We thank the reviewer for this attentive comment. In order to ensure the quality and clarity of the figures, we have now enhanced the resolution of our figures in a maximum possible quality (600dpi) and re-corrected the figures quality with using “PACE” software and re-uploaded them again on the online system accordingly. Your sincerely, The Authors Attachments Attachment Submitted filename: Response to Reviewer One.docx* https://doi.org/10.1371/journal.pone.0306525.r004
20 Jun 2024 Decision Letter - Sumit Kumar Hira, Editor Comprehensive transcriptomic analysis of prostate cancer lung metastases PONE-D-23-41725R2 Dear Dr. Saraji, 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, Sumit Kumar Hira, 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 #1: 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 #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: 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 authors have addressed the specific comment raised by the reviewers and the manuscript may be accepted in the revised version. ****** 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 ******** https://doi.org/10.1371/journal.pone.0306525.r005
Formally Accepted
27 Jun 2024 Acceptance Letter - Sumit Kumar Hira, Editor PONE-D-23-41725R2 PLOS ONE Dear Dr. Saraji, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Sumit Kumar Hira Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0306525.r006

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