Association of NUDT17 rs9286836 and rs2004659 variants with breast cancer risk in Bangladeshi Women

Md. Shajid Hossain Rafi; Md. Shalahuddin Millat; Md Abdul Barek; Syed Masudur Rahman Dewan; Mohammad Shahriar; Zabun Nahar; Mohammad Safiqul Islam

doi:10.1371/journal.pone.0344584

Peer Review History

Original SubmissionNovember 6, 2025
9 Dec 2025 Decision Letter - Milad Khorasani, Editor Dear Dr. Islam, 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 23 2026 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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If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. [Note: HTML markup is below. Please do not edit.] 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 Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: No Reviewer #2: No Reviewer #3: 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 Reviewer #3: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** Reviewer #1: 1. General Comments The study is well-designed with comprehensive data collection and appropriate analysis. The study provides valuable preliminary data on breast cancer genetic susceptibility in Bangladeshi women. However, several sections need some clarification and improvements. 2. Specific Comments Abstract • This section is clearly described but needs to avoid excessive inclusion of numerical values from results and rather focus on significant findings. • Try and narrative results for logistic regression then long OR/CIs numbers. Introduction • This section is detailed and backed with relevant literature. As for the section of NUDIX gene, the authors must focus on its role specifically with regards to breast cancer. • Need to strengthen the justification for selecting rs9286836 and rs2004659 SNPs and emphasize the need for this analysis in Bangladeshi women. Materials and Methods Methods are well-detailed however, need to clarify; • Selection criteria for study subjects and whether cases/controls were matched. • Include genotype call validation (e.g., random re-genotyping for QC). • In the sample size estimation paragraph do include actual sample sizes. • In statistical analysis do indicate how Hardy–Weinberg equilibrium was tested. Also clarify Bonferroni correction (pre-specified or post hoc). If possible do consider adjusting logistic regression models adjusted for potential confounders (e.g., age, BMI, reproductive factors) for stronger outcomes. Results • Overall results are well organized but could be meaningful by summarizing them with relevant details. • Data analysis presented in tables is comprehensive and well structured but needs to highlight statistically significant results e.g. of rs2004659 SNP and avoid overinterpretation for rs9286836 SNP. • Gel electrophoresis figures are more suited to the supplementary section. Discussion • The results interpretation is well aligned and descriptive but avoid emphasis on some of the non-significant results and speculative statements. • The functional implications of rs2004659 results need to be carefully discussed as the analyses are in silico and not experimentally validated. • Inclusion of a study limitations paragraph will strengthen the manuscript. • It’s worth comparing the Bangladeshi population with the Chinese cohort but do focus on the potential genetic variability of both ethnic groups. • In the conclusion section, avoid repeating results and focus on the main findings. Language and Style • The English is fluent and formal, but a few sentences are long and could be simplified. • Maintain consistent use of “breast cancer” instead of abbreviations (BC) unless used frequently in tables. • Check punctuation, spacing, and consistent decimal formatting (e.g., “p = 0.0098” rather than “p=0.0098”). • Ensure consistent use of scientific italics for gene symbols (NUDT17). Figures and Tables • Figures quality could be improved by enhancing resolution and clearer labelling. • The rarely used sociodemographic variables presented in table 2 could be moved to Supplementary materials. References • All references are properly selected and up-to-date. • Authors must ensure PLOS ONE style formatting. • Citing papers on role of NUDIX enzymes to oxidative stress response in cancer will be a plus. Reviewer #2: PONE-D-24-22063R1 Association of Vitamin D levels and Vitamin D receptor gene polymorphism with obesity in Bangladeshi school-going children: A cross-sectional study PLOS ONE My comments: This is a very interesting study which will attract the attention of scientists and readers globally. However, manuscript requires a major revision and can be considered for publication after addressing the following comments. 1. In abstract authors require to add this sentence “An association between the NUDT17 polymorphisms rs9286836 and rs2004659 and the risk of breast cancer in Bangladeshi Women has not been studied previously and thus, this study aimed to determine this correlation. It will clarify the novelty of the study. 2. How was the number of BC participants determined? Sample size is small and should be at least 400 or more to be statistically valid according to the existing number of BC patients in Bangladesh. Please justify. 3. There are four SNPs; rs9286836, rs2004659, rs10910830, and rs10910829 probable binding determinants associated with the regulation of NUDT17 gene expression. Why only two SNPs rs9286836, rs2004659 were studied? I would recommend investigating the association of rs10910830, and rs10910829 with BC risk as well. 4. Genotyping was performed using tetra-primer ARMS-PCR for rs9286836 and PCR–RFLP for rs2004659, why two different techniques were used? Please explain. 5. Hardy-Weinberg equilibrium should be determined for allelic and genotypic model of each SNP. Include this in the statistical evaluation section and its findings in the result section. 6. In the introduction, please include the study conducted in Chinese women by Han et all., 2024 to reveal this association as a first line of the last paragraph. 7. In the discussion, include detail on allelic association with BC risk from the study conducted by Han et all, 2024 (Title of the study: Association between NUDT17 polymorphisms and breast cancer risk) and compare your study findings with their findings. 8. In the discussion section, please include references for this statement “Previous studies on the NUDT17 rs9286836 and rs2004659 polymorphisms suggested a possible correlation between these genetic variants and cancer susceptibility”. 9. In the in the first line of conclusion, please replace “individuals” by “women”. Reviewer #3: In introduction section: 1. Please add the background information regarding the association of SNPs of interest with breast cancer seen in other ethnicity (if available). 2. Avoid using the information regarding the current study method in the last part of the introduction, shift them to methodology. State the objective only. Methodology: 1. Why the own study data was not used during in silico gene expression analysis? Result: 1. It is better to describe as 'no association' for rs9286836 in stead of mentioning 'associated but not statistically significant'. Overall: 1. Please write the full form when first using the acronyms/abbreviations. 2. I wonder why you have no co-authors from NICRH. How did you manage sample and data collection without collaborating with them? ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Dr. Raushanara Akter Reviewer #3: Yes: Mashfiqul Hasan ******** [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.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. Attachments Attachment Submitted filename: Review Report_PONE-D-25-57284.pdf Attachment Submitted filename: 30.11.2025-Review Report_Plos One.docx https://doi.org/10.1371/journal.pone.0344584.r001
Revision 1
9 Feb 2026 Author Response PONE-D-25-57284 Association of NUDT17 rs9286836 and rs2004659 Variants with Breast Cancer Risk in Bangladeshi Women PLOS One Editor: 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'. 2. Thank you for stating the following financial disclosure: “The study was partially financially supported by the Department of Pharmacy at the University of Asia Pacific.” 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 note that funding information should not appear in any section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript. 4. We note that there is identifying data in the Supporting Information file < Supplementary materials.rar>. Due to the inclusion of these potentially identifying data, we have removed this file from your file inventory. Prior to sharing human research participant data, authors should consult with an ethics committee to ensure data are shared in accordance with participant consent and all applicable local laws. Data sharing should never compromise participant privacy. It is therefore not appropriate to publicly share personally identifiable data on human research participants. The following are examples of data that should not be shared: -Name, initials, physical address -Ages more specific than whole numbers -Internet protocol (IP) address -Specific dates (birth dates, death dates, examination dates, etc.) -Contact information such as phone number or email address -Location data -ID numbers that seem specific (long numbers, include initials, titled “Hospital ID”) rather than random (small numbers in numerical order) Data that are not directly identifying may also be inappropriate to share, as in combination they can become identifying. For example, data collected from a small group of participants, vulnerable populations, or private groups should not be shared if they involve indirect identifiers (such as sex, ethnicity, location, etc.) that may risk the identification of study participants. Additional guidance on preparing raw data for publication can be found in our Data Policy (https://journals.plos.org/plosone/s/data-availability#loc-human-research-participant-data-and-other-sensitive-data) and in the following article: http://www.bmj.com/content/340/bmj.c181.long. Please remove or anonymize all personal information (<specific identifying information in file to be removed>), ensure that the data shared are in accordance with participant consent, and re-upload a fully anonymized data set. Please note that spreadsheet columns with personal information must be removed and not hidden as all hidden columns will appear in the published file. 5. 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. 6. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. Response: The revised manuscript has been prepared in accordance with the Editor's and reviewers' suggestions. The patients’ and controls' IDs have been removed from the ‘Research data’ file and provided with just numbering as patient and control IDs. Funder information has been moved to the cover letter. 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: Yes Reviewer #2: Yes Reviewer #3: Yes ________________________________________ 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No Reviewer #3: Yes ________________________________________ 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ________________________________________ 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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: 1. General Comments The study is well-designed with comprehensive data collection and appropriate analysis. The study provides valuable preliminary data on breast cancer genetic susceptibility in Bangladeshi women. However, several sections need some clarification and improvements. Response: We appreciate the positive evaluation. Following the reviewer’s suggestions, we have revised the Abstract, Introduction, Methods, Results, Discussion, and presentation of figures and tables to improve clarity, focus, and compliance with PLOS ONE standards. 2. Specific Comments Abstract • This section is clearly described but needs to avoid excessive inclusion of numerical values from results and rather focus on significant findings. • Try and narrative results for logistic regression then long OR/CIs numbers. Response: Thank you for this comment. We have revised the abstract to reduce the emphasis on detailed numerical values and instead focus on the main findings that are most relevant biologically and clinically. The results are now described in a narrative manner, particularly for the logistic regression analyses, with emphasis placed on the statistically significant protective effect of the rs2004659 variant, rather than listing multiple odds ratios and confidence intervals. 3. Introduction • This section is detailed and backed with relevant literature. As for the section of NUDIX gene, the authors must focus on its role specifically with regards to breast cancer. • Need to strengthen the justification for selecting rs9286836 and rs2004659 SNPs and emphasize the need for this analysis in Bangladeshi women. Response: In addition, the introduction has been refined to better contextualize the role of the NUDT17 gene specifically in breast cancer. We have strengthened the rationale for selecting rs9286836 and rs2004659 by highlighting prior evidence, their potential functional relevance, and the lack of population-specific data. The need to evaluate these variants in Bangladeshi women is now more clearly stated, given the distinct genetic background and the limited existing data from this population. 4. Materials and Methods Methods are well-detailed however, need to clarify; • Selection criteria for study subjects and whether cases/controls were matched. • Include genotype call validation (e.g., random re-genotyping for QC). • In the sample size estimation paragraph do include actual sample sizes. • In statistical analysis do indicate how Hardy–Weinberg equilibrium was tested. Also clarify Bonferroni correction (pre-specified or post hoc). If possible do consider adjusting logistic regression models adjusted for potential confounders (e.g., age, BMI, reproductive factors) for stronger outcomes. Response: We thank the reviewer for these valuable suggestions. We clarified the inclusion/exclusion criteria and explicitly stated age, sex, and BMI matching between cases and controls. Specifically, we have clarified the selection criteria and matching strategy for cases and controls, added a description of genotype call validation for quality control, explicitly stated the actual sample size used in the a priori power calculation, and expanded the statistical analysis section to describe Hardy-Weinberg equilibrium testing and the pre-specified use of Bonferroni correction. We also clarified the rationale for reporting crude logistic regression models and addressed potential confounding. 5. Results • Overall results are well organized but could be meaningful by summarizing them with relevant details. • Data analysis presented in tables is comprehensive and well structured but needs to highlight statistically significant results e.g. of rs2004659 SNP and avoid overinterpretation for rs9286836 SNP. • Gel electrophoresis figures are more suited to the supplementary section. Response: We appreciate the reviewer's constructive suggestions. The results section has been revised to provide a clearer summary of the major findings, with emphasis on the statistically significant association observed for rs2004659 and careful wording for the non-significant result for rs9286836. Interpretations for rs9286836 have been limited to descriptive statements without speculative inference. The gel electrophoresis figures (Figures 1 and 2) have been moved to the Supplementary Information (S1 Fig and S2 Fig) as recommended. 6. Discussion • The results interpretation is well aligned and descriptive but avoid emphasis on some of the non-significant results and speculative statements. • The functional implications of rs2004659 results need to be carefully discussed as the analyses are in silico and not experimentally validated. • Inclusion of a study limitations paragraph will strengthen the manuscript. • It’s worth comparing the Bangladeshi population with the Chinese cohort but do focus on the potential genetic variability of both ethnic groups. Response: Thank you for these comments. We have gone through the discussion and made several small but important changes. We reduced the attention given to rs9286836, where the results were not significant, and removed wording that could be taken as speculative. We also made it clearer that the functional interpretation of rs2004659 comes from in silico expression data and has not yet been tested experimentally. A separate paragraph on study limitations was added to explain the constraints of the sample size and the use of public databases. The comparison with Chinese studies was revised as well, so that it now reflects possible differences between the two populations rather than assuming the same genetic effects. These changes were made to keep the discussion accurate and appropriately cautious. 7. In the conclusion section, avoid repeating results and focus on the main findings. Response: The conclusion section has been modified according to the reviewer’s suggestion. 8. Language and Style • The English is fluent and formal, but a few sentences are long and could be simplified. •Maintain consistent use of “breast cancer” instead of abbreviations (BC) unless used frequently in tables. • Check punctuation, spacing, and consistent decimal formatting (e.g., “p = 0.0098” rather than “p=0.0098”). • Ensure consistent use of scientific italics for gene symbols (NUDT17). Response We carefully reviewed the entire manuscript to improve readability and consistency. Several long or complex sentences were simplified without altering their scientific meaning. The term “breast cancer” is now used consistently throughout the text, with abbreviations avoided except where necessary in tables. Punctuation, spacing, and decimal formatting were standardized across the manuscript (e.g., p = 0.0098), and gene symbols, including NUDT17, were checked to ensure consistent use of scientific italics. 9. Figures and Tables • Figures quality could be improved by enhancing resolution and clearer labelling. • The rarely used sociodemographic variables presented in table 2 could be moved to Supplementary materials. Response: All figures were revised to improve resolution and visual clarity, with clearer labels and legends to enhance interpretability. Additionally, these figures have now been moved to the supplementary materials, as per your previous suggestion. In response to the suggestion regarding Table 2, sociodemographic variables that were not directly relevant to the genetic association analyses were moved to the Supplementary Materials (S1Table), allowing the main tables to focus on variables central to the study objectives. 10. References • All references are properly selected and up-to-date. • Authors must ensure PLOS ONE style formatting. • Citing papers on role of NUDIX enzymes to oxidative stress response in cancer will be a plus. Response: In response to the reviewer’s suggestion, the manuscript already cites key studies describing the role of NUDIX family enzymes in oxidative stress and nucleotide sanitization, including foundational biochemical work on MTH1/MTH2 and oxidized nucleotide metabolism [24,30,31], as well as recent evidence linking NUDT17 polymorphisms to breast cancer susceptibility [25]. Reviewer #2: PONE-D-24-22063R1 Association of Vitamin D levels and Vitamin D receptor gene polymorphism with obesity in Bangladeshi school-going children: A cross-sectional study PLOS ONE My comments: This is a very interesting study which will attract the attention of scientists and readers globally. However, manuscript requires a major revision and can be considered for publication after addressing the following comments. Response: Thank you very much for your comments. Although the title is not the title of our manuscript, all the comments are relevant to our manuscript. We sincerely thank the reviewer for the constructive and detailed comments. Several of the points raised by you (Reviewer #2) overlap with those raised by Reviewer #1 and have already been addressed in the revised manuscript with tracked changes. For clarity, we respond to each comment individually below and indicate where revisions were made. 1. In abstract authors require to add this sentence “An association between the NUDT17 polymorphisms rs9286836 and rs2004659 and the risk of breast cancer in Bangladeshi Women has not been studied previously and thus, this study aimed to determine this correlation. It will clarify the novelty of the study. Response: We agree wi Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0344584.r002
23 Feb 2026 Decision Letter - Milad Khorasani, Editor Association of NUDT17 rs9286836 and rs2004659 Variants with Breast Cancer Risk in Bangladeshi Women PONE-D-25-57284R1 Dear Dr. Safiqul Islam, 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. For questions related to billing, please contact billing support . 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, Milad Khorasani, PhD Academic Editor PLOS One Additional Editor Comments (optional): 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: The authors have tried to address all the comments/suggestions raised by the reviewers. The current version of the revised manuscript looks much improved. Reviewer #2: (No Response) ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No Reviewer #2: Yes: Dr. Raushanara Akter ******** Attachments Attachment Submitted filename: COMMENTS on Article (PONE-D-25-57284R1) _PlosOne_Dr. Akter.docx https://doi.org/10.1371/journal.pone.0344584.r003
Formally Accepted
Acceptance Letter - Milad Khorasani, Editor PONE-D-25-57284R1 PLOS One Dear Dr. Islam, 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. Milad Khorasani Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0344584.r004

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