Epithelial changes of congenital intestinal obstruction in a rat model

Quentin Ballouhey; Laurent Fourcade; Laurence Richard; Camille Bellet; Chaharazed El Hamel; Jean Michel Vallat; Franck Sturtz; Sylvie Bourthoumieu

doi:10.1371/journal.pone.0232023

Peer Review History

Original SubmissionSeptember 19, 2019
2 Jan 2020 Decision Letter - Shree Ram Singh, Editor PONE-D-19-26433 Epithelial changes of congenital intestinal obstruction in a rat model PLOS ONE Dear Dr Ballouhey, 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. We would appreciate receiving your revised manuscript by Feb 16 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Shree Ram Singh, 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 3. Thank you for including the following funding information within the acknowledgements section of your manuscript; "This work was supported by a grant from the UHC of Limoges, APREL (Appel à projet Recherche Equipes Emergentes et Labellisées, 2013), funded by the ARS Limousin." 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: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." [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: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: Manuscript appears to be well explained and contains all the statistically significant supporting data. It would be good to mention the number of animals used for each group, while explaining materials method (though it has been mentioned later in results). Please, carefully read the manuscript and prefer to avoid using repetitive words (like "impaired" used twice in a single sentence). Also avoid repetitive use of "the" at many places(like "The ratio of the target genes to the reference genes"). Rest seems to be fine. Reviewer #2: The manuscript entitled “Epithelial changes of congenital intestinal obstruction in a rat model” by Ballouhey et.al has described the effect of surgically induced atresia in rat embryos. Different models of atresia have been reported, however, most of them focused on morphological changes. The authors tried to advance previous reports on morphological changes by taking a gene expression profile-based approach together with morphological examinations. However, in my opinion, the manuscript fails to address the specific scientific hypothesis. The conclusions, and the title of the manuscript are not supported by data. Major concerns: 1. Overall representation of the manuscripts looks more incoherent. The result sections are very confusing and redundant. 2. Sample size for most of the data are very small (n=3). Previous studies by Fourcade et. al. had used n>10 in the rat model. 3. Table 2; The statistical power of this data is not so convincing considering the small sample size (n=3). It is difficult to assume the gaussian distribution of the data with the small sample size and with high variation. Therefore, I suggest non parametric test for this set of data. 4. Table 3: number of deferentially expressed gene does not make any scene unless the they suggest any biological function or pathway. I suggest a gene ontology analysis of the deferentially expressed gene to better represents the biological changes. 5. Is the gene expression data deposited in any data bank? 6. Figures should be more organized and properly labelled for the clarity of the readers. Minor concerns Please provide HPRT and TBP primer sequence in table 1. ****** 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0232023.r001
Revision 1
4 Mar 2020 Author Response Response to Reviewer 1 Reviewer 1: Manuscript appears to be well explained and contains all the statistically significant supporting data. It would be good to mention the number of animals used for each group, while explaining materials method (though it has been mentioned later in results). Please, carefully read the manuscript and prefer to avoid using repetitive words (like "impaired" used twice in a single sentence). Also avoid repetitive use of "the" at many places (like "The ratio of the target genes to the reference genes"). Rest seems to be fine. We thank Reviewer 1 for their very useful comments. As suggested in the comments, we have changed the following: -“Impaired” has been replaced with “Altered” in the introduction section -The sentence “The ratio of the target genes to the reference genes was calculated and expressed as 2-ΔCt.” Was modified to “Target gene/reference gene ratios were calculated and expressed as 2-ΔCt.” -The style of the entire manuscript style has also been checked. Response to Reviewer 2 Reviewer 2: The manuscript entitled “Epithelial changes of congenital intestinal obstruction in a rat model” by Ballouhey et al. has described the effect of surgically induced atresia in rat embryos. Different models of atresia have been reported, however, most of them focused on morphological changes. The authors tried to advance previous reports on morphological changes by taking a gene expression profile-based approach together with morphological examinations. However, in my opinion, the manuscript fails to address the specific scientific hypothesis. The conclusions, and the title of the manuscript are not supported by data. Response to Reviewer 2 1. Overall representation of the manuscripts looks more incoherent. The result sections are very confusing and redundant. Author’s response: We apologize for the fact that Reviewer 2 found the Results section to be confusing. In light of this remark, we have changed the followings points in the Results paragraph: -“Analysis of global gene expression” section has been moved in the first part of the paragraph. -The presentation of the table illustrating the morphometric results has been modified. -Throughout the entire paragraph, “Microvilli” has been substituted with “Villi” as Microvilli can only been detected by electron microscopy. -The “Enterocyte cell brush border” section has been modified and the last sentence has been deleted “Sucrase isomaltase…(S1 Table)”. 2. Sample size for most of the data are very small (n=3). Previous studies by Fourcade et al. had used n > 10 in the rat model. Author’s response: Indeed, the sample size was n = 3 or = 4, as the guidelines for animal experimentation stipulate that as few animals as possible are used. The aim of the experiment was to use a variety of techniques rather than a large number of animals. The transcriptomic experiment required n = 3 for each group, which meant using more than 18 operated animals, not including the high mortality rate during the surgical procedures. In previous published studies, the number of animals was higher but only a single technique was used, for example Fourcade et al. only performed an optical microscopy analysis. 3. Table 2: The statistical power of this data is not so convincing considering the small sample size (n=3). It is difficult to assume the gaussian distribution of the data with the small sample size and with high variation. Therefore, I suggest non parametric test for this set of data. Author’s response: We fully agree with this remark. Consequently, we have opted to use a non-parametric test and we have made the following changes: -The sentence corresponding to the statistical analysis has been changed in the Methods section: Differences in the area between the proximal and the distal segment have been analyzed quantitatively using the Wilcoxon test. -The data in the Results section corresponding to Table 3 have been changed. A qualified statistician has now reviewed the statistics of the manuscript and this individual has hence been added to the list of authors. 4. Table 3: number of deferentially expressed gene does not make any scene unless they suggest any biological function or pathway. I suggest a gene ontology analysis of the deferentially expressed gene to better represents the biological changes. Author’s response: the objective was not to produce biologically significant results but to determine which segment was the most impaired. In accordance with the reviewer’s comment, we have made the following modification: -The beginning of the Introduction section has changed to “The aim of our study was to determine whether the proximal or the distal segment was the most impaired and …to analyze…” -The Results section has also been modified and the “Analysis of global gene expression” section along with the related Table (Table 3 becoming Table 2) has been moved in the first part of this paragraph. 5. Is the gene expression data deposited in any data bank? Author’s response: Yes, the data are available online. The microarray analysis of this study was performed at the genomic platform (GENOMIC’S) of the Institut Cochin in Paris (22 Rue Mechain, 75014 Paris) and it has been assigned accession number E-MTAB-5981 in ArrayExpress. 6. Figures should be more organized and properly labelled for the clarity of the readers. Author’s response: We apologize for the fact that Reviewer 2 found our pictures to be in want of improvement. In accordance to this, all of the patterning schematics of the figures have been changed to improve their appearance: the labelling of the Figures 1 and 2 has been modified. A legend has been added for each picture and the scale bars have been modified. 7. Minor concerns. Please provide HPRT and TBP primer sequence in table 1. Author’s response: the HPRT and TBP primer sequences are now provided in Table 1. Attachments Attachment Submitted filename: Response to reviewers.doc https://doi.org/10.1371/journal.pone.0232023.r002
2 Apr 2020 Decision Letter - Shree Ram Singh, Editor PONE-D-19-26433R1 Epithelial changes of congenital intestinal obstruction in a rat model PLOS ONE Dear Dr Ballouhey, Thank you for submitting your revised 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 minor points raised by a reviewer on your revised manuscript. We would appreciate receiving your revised manuscript by May 17 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Shree Ram Singh, Ph. D. Academic Editor PLOS ONE [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 #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: The authors have significantly improved the revised manuscript. I would suggest that the following analysis to be included in the manuscript for better elucidation of the intestinal differentiation in control embryos. As the authors have performed microarray with different stages of control samples, namely ED15, ED17, ED19 and E21, it is important to show differential gene expression of all three stages (i.e. ED17,19, 21) compare to ED15 and associated pathways instead of just number of genes in ED21 vs ED15 (table 3). ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0232023.r003
Revision 2
6 Apr 2020 Author Response Response to Reviewer 2 Reviewer 2: Manuscript appears to be well explained and contains all the The authors have significantly improved the revised manuscript. I would suggest that the following analysis to be included in the manuscript for better elucidation of the intestinal differentiation in control embryos. As the authors have performed microarray with different stages of control samples, namely ED15, ED17, ED19 and E21, it is important to show differential gene expression of all three stages (i.e. ED17,19, 21) compare to ED15 and associated pathways instead of just number of genes in ED21 vs ED15 (table 2). Author’s response: We thank Reviewer 2 for their very useful comments. We fully agree with this remark. Consequently, we have implemented the intermediate results of microarray E15-17-E19-E21 for kinetic controls in Table 2. We have implemented the intermediate results of microarray E15-17-E19-E21 kinetic controls for specific pathways in Table S1. Attachments Attachment Submitted filename: Response to reviewers.doc https://doi.org/10.1371/journal.pone.0232023.r004
7 Apr 2020 Decision Letter - Shree Ram Singh, Editor Epithelial changes of congenital intestinal obstruction in a rat model PONE-D-19-26433R2 Dear Dr. Ballouhey, We are pleased to inform you that your revised manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Shree Ram Singh, Ph. D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0232023.r005
Formally Accepted
8 Apr 2020 Acceptance Letter - Shree Ram Singh, Editor PONE-D-19-26433R2 Epithelial changes of congenital intestinal obstruction in a rat model Dear Dr. Ballouhey: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Shree Ram Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0232023.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .