NMR spectroscopy-based analysis of gallstones of cancerous and benign gallbladders from different geographical regions of the Indian subcontinent

Mohd Adnan Siddiqui; Navneet Dwivedi; Mohammed Haris Siddiqui; S. V. Rana; Anil Sharma; N. R. Dash; Rebala Pradeep; Ranjit Vijayahari; Anu Behari; V. K. Kapoor; Neeraj Sinha

doi:10.1371/journal.pone.0286979

Peer Review History

Original SubmissionJanuary 1, 2023
23 Feb 2023 Decision Letter - Thanh-Danh Nguyen, Editor PONE-D-23-00049NMR SPECTROSCOPY-BASED ANALYSIS OF GALLSTONES OF CANCEROUS AND BENIGN GALLBLADDERS FROM DIFFERENT GEOGRAPHICAL REGIONS OF THE INDIAN SUBCONTINENTPLOS ONE Dear Dr. Sinha, 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 Apr 09 2023 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. 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M.A.S. acknowledges IUL MCN ………… N.D. acknowledges financial assistance from the Department of Science and Technology (DST), Government of India (IF170726). N.S. acknowledges funding from CBMR, Lucknow." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments 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 and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "NO" Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We note that Figure 1 in your submission contain copyrighted images. 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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: Overall, the authors have got the NRM spectrum from patient gallstones from various regions in India. The results have shown the different in Gallstones components in NMR. There are some recommends and questions for the authors. Recommendation: It is not necessary to have figure 1 and 2 in the manuscript. Authors could add Indian map. On the map, authors provide the location of collected samples, number, samples, sexuality, type/color/image of gallstones. Author should have the period of collecting samples in Materials and methods Question: 1. In line 223-224, Authors have pointed out the mis-classification by morphology of gallstone. This may happen. So do the author compare between the NMR spectrum and morphology/ color/type of gallstone in the study? How many precents of correction when diagnosis by looking at the morphology/color of gallstones by Doctor? 2. Authors have found many chemical in gallstones. Do the authors take samples, analyze and confirm the chemical in the gallstone by another methods? 3. Identifying type of gallstone is very important, sometimes, patients don’t need to remove the gallbladder or gall track. So is it possible to perform the Magnetic resonance spectrum (MRS) or MRI in patients to find out the component of gallstones? Authors may consider the time, magnetic force for scanning. Reviewer #2: The manuscript by Siddiqui et al. describes the characterization of gallstones from India using NMR spectroscopy. Major changes: pg. 138-139Authors should provide SEM images of cross-sections of all the studied gallstones. Optical images could be provided to show the overall morphological differences. pg.313-315 It's not clear if Figure 6's spectra correspond to the gallstones from CC and GBC patients. Please rephrase the corresponding text and Figure caption. Add labels in Figure 6. pg. 343-350 Please discuss the effect of trace elements during the crystallization process. Minor changes: pg. 24 Explain GS abbreviation in the abstract and in its first appearance in the main text. pg. 85 Leave space between scanning electron microscopy and (SEM) pg. 91-99 Discuss the work in the previous regions of India about gallstones pg. 105 Lowercase Solid-State. Apply to all text. pg. 108 Lowercase Solution-State. Apply to all text. pg.163 Superscript 1 in 1H NMR. Apply to all text. pg.203-205 Could you provide percentages of the cholesterol pg. 223-231 This information should be mentioned earlier in the introduction pg. 240-242 Please refer to the specific differences between the regions and add references pg. 247 What do you mean by molecules? Calcium carbonate is a inorganic structure. You could add mineral. pg. 373 Complete the missing information ****** 6. 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Revision 1
18 May 2023 Author Response Editor Comments 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Response: We have followed PLOS ONE's style guidelines for the preparation of the revised manuscript. 2. Thank you for stating the following financial disclosure: "NO" At this time, please address the following queries: a) Please clarify the sources of funding (financial or material support) for your study. List the grants or organizations that supported your study, including funding received from your institution. b) State what role the funders took in the study. If the funders had no role in your study, please state: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” c) If any authors received a salary from any of your funders, please state which authors and which funders. d) If you did not receive any funding for this study, please state: “The authors received no specific funding for this work.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: The authors received no specific funding for this work. The clarification statement has been made in the cover letter also. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "Acknowledgments: The authors acknowledge the 800MHz & 600 MHz NMR facility at the Centre of Biomedical Research (CBMR), SGPGIMS Campus, Lucknow. The authors acknowledge Dr S.K. Mandal for help in the statistical analysis of the study. M.A.S. acknowledges IUL MCN ………… N.D. acknowledges financial assistance from the Department of Science and Technology (DST), Government of India (IF170726). N.S. acknowledges funding from CBMR, Lucknow." We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments 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 and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "NO" Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: The authors received no specific funding for this work and required changes has been made in the “Acknowledgements section”. 4. We note that Figure 1 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright. We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission: a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license. We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text: “I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.” Please upload the completed Content Permission Form or other proof of granted permissions as an ""Other"" file with your submission. In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].” b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only. Response: The Figure 1 from has been replaced with the new Figure (Indian Map) in the revised manuscript, as suggested by Reviewer #1. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Response: We have included the text at the end of manuscript as per PLOS ONE's supporting information guidelines. Reviewer #1: Recommendation: 1. It is not necessary to have figure 1 and 2 in the manuscript. Response: The suggestion is appreciated and we have removed the Figure 1 and Figure 2. 2. Authors could add Indian map. On the map, authors provide the location of collected samples, number, samples, sexuality, type/color/image of gallstones. Response: Thanks for providing valuable suggestion. We have incorporated the Indian map in the revised the manuscript on page no. 5. 3. Author should have the period of collecting samples in Materials and methods. Response: The suggestion is acknowledged and the period of collecting the samples has been mentioned in the revised manuscript line no. 163-164. Questions: 1. In line 223-224, Authors have pointed out the mis-classification by morphology of gallstone. This may happen. So do the author compare between the NMR spectrum and morphology/ color/type of gallstone in the study? How many precents of correction when diagnosis by looking at the morphology/color of gallstones by Doctor? Response: We agree with the reviewer comments. NMR spectrum and its comparison with type of stone is presented in SI Figure No 1. The diagnosis of gallstones diseases based solely on the morphology and color of the stones can be unreliable. The appearance of gallstones can provide some clues as to their composition. The accuracy of diagnosis depends greatly on the experience level and expertise of the doctor performing the diagnosis. 2. Authors have found many chemical in gallstones. Do the authors take samples, analyze and confirm the chemical in the gallstone by another methods? Response: NMR spectroscopy is the chosen method, and it is a powerful technique for the characterization of compounds, including those present in gallstones. We have utilized both, the solution-state NMR and solid-state NMR technique to provide comprehensive insight of the major component of the gallstones. Along with this, we have used scanning electron microscopy (SEM) to reveal the morphological and structural differences between the different types of stone included in the study i.e. cholesterol stone, mixed stone and pigment stone. The SEM images showing the plate like cholesterol crystals with laminar and radial arrangement and confirmed the presence of cholesterol in the gallstones. Our results are in the same line with previous studies (reference no 18 in the revised manuscript). 3. Identifying type of gallstone is very important, sometimes, patients don’t need to remove the gallbladder or gall track. So is it possible to perform the Magnetic resonance spectrum (MRS) or MRI in patients to find out the component of gallstones? Authors may consider the time, magnetic force for scanning. Response: Yes, it is possible to use Magnetic Resonance Imaging (MRI) or Magnetic Resonance Spectroscopy (MRS) to identify the type of gallstone. MRI can provide high-resolution images of the gallbladder and gallstones, allowing clinicians to determine the composition of the stone. One of the study that uses the MRI for the analysis of different compositions of gallstones shows that on T1-weighted 3D fast spoiled gradient-echo images, of the pigment gallstones were hyper-intense and the cholesterol gallstones were hypo-intense. So, we can say that based on the differences of signal intensity of gallstones, the 3D fast spoiled gradient-echo T1-weighted imaging is able to diagnose the composition of gallstones (reference no.19 in revised manuscript). However, it is important to note that not all hospitals and medical facilities may have the equipment and expertise to perform MRS. Additionally, MRI and MRS scans can be time-consuming and may not be suitable for all patients, especially those with certain medical conditions (with pacemaker) or who cannot tolerate the magnetic force used in the scans. Reviewer #2: 1. Pg. 138-139 Authors should provide SEM images of cross-sections of all the studied gallstones. Optical images could be provided to show the overall morphological differences. Response: We appreciate the suggestion to add SEM images and optical images to provide further insight into the morphological differences between the gallstones studied. Mainly three types of gallstones from different geographical regions and etiology have been analyzed which are cholesterol stone, mixed stone, and pigment stone. To show the morphological and structural differences between them the SEM analysis of these gallstones have been included in the revised manuscript on page 9-10. 2. pg.313-315 It's not clear if Figure 6's spectra correspond to the gallstones from CC and GBC patients. Please rephrase the corresponding text and Figure caption. Add labels in Figure 6. Response: Thank you for your valuable suggestion. Figure 6 shows the type of polymorphs of cholesterol (monohydrate crystalline with amorphous form and anhydrous form) which can be present in either CC or GBC gallstones, that can also be understood by Table 1. No specific form of cholesterol is associated with either CC or GBC gallstones. Figure caption of Figure 6 has been reframed for clear understanding on line no. 353-356. 3. pg. 343-350 please discuss the effect of trace elements during the crystallization process. Response: We appreciate the suggestion and the role of trace elements in the formation of gallstones is incorporated in the manuscript in line no. 383-395. 4. pg. 24 Explain GS abbreviation in the abstract and in its first appearance in the main text. Response: Thanks for highlighting this. The GS abbreviation is now mentioned in the abstract section page no. 2 line no. 44. 5. pg. 85 Leave space between scanning electron microscopy and (SEM). Response: The suggested correction has been made in the revised manuscript line no. 110. 6. pg. 91-99 Discuss the work in the previous regions of India about gallstones. Response: Thanks for the suggestion. The work from previous studies have been incorporated on page no. 4 line no. 104-106 and page no. 12 line no. 297-302. 7. pg. 105 Lowercase Solid-State. Apply to all text. Response: The suggestion is appreciated and changes has been incorporated in the revised manuscript. 8. pg. 108 Lowercase Solution-State. Apply to all text. Response: The suggestion is appreciated and changes has been incorporated in the revised manuscript. 9. pg.163 Superscript 1 in 1H NMR. Apply to all text. Response: The suggestion is appreciated and changes has been incorporated in the revised manuscript. 10. pg.203-205 Could you provide percentages of the cholesterol. Response: The percentage of cholesterol in each type of gallstone varies: pure cholesterol stones: (≥70% cholesterol), mixed stones: (30-70% cholesterol), and pigment stones: (≤30% cholesterol). This has been incorporated in line no. 231-233. 11. pg. 223-231 This information should be mentioned earlier in the introduction. Response: The suggestion is appreciated and the information has been mentioned in the introduction section on page no. 4 line no. 100-104 of the revised manuscript. 12. pg. 240-242 Please refer to the specific differences between the regions and add references. Response: Thanks for the suggestion. The suggestion has been incorporated on page no. 11 line no. 278-280. 13. pg. 247 What do you mean by molecules? Calcium carbonate is a inorganic structure. You could add mineral. Response: Authors would like to apologize for the mistake. The appropriate changes has been made in the revised manuscript. 14. pg. 373 Complete the missing information. Response: The suggestions have been incorporated in the acknowledgement section. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0286979.r002
29 May 2023 Decision Letter - Thanh-Danh Nguyen, Editor NMR spectroscopy-based analysis of gallstones of cancerous and benign gallbladders from different geographical regions of the Indian subcontinent PONE-D-23-00049R1 Dear Dr. Sinha, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Thanh-Danh Nguyen, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0286979.r003
Formally Accepted
14 Jun 2023 Acceptance Letter - Thanh-Danh Nguyen, Editor PONE-D-23-00049R1 NMR spectroscopy-based analysis of gallstones of cancerous and benign gallbladders from different geographical regions of the Indian subcontinent Dear Dr. Sinha: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Thanh-Danh Nguyen Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0286979.r004

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