In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis

Nataliya Smith; Debra Saunders; Megan Lerner; Michelle Zalles; Nadezda Mamedova; Daniel Cheong; Ehsan Mohammadi; Tian Yuan; Yi Luo; Robert E. Hurst; Beverley Greenwood-Van Meerveld; Rheal A. Towner

doi:10.1371/journal.pone.0239282

Peer Review History

Original SubmissionFebruary 28, 2020
12 Apr 2020 Decision Letter - Christopher R. Weber, Editor PONE-D-20-05854 In vivo Assessment of Bladder Hyper-Permeability and Using Molecular Targeted Magnetic Resonance Imaging to Detect Claudin-2 in a Mouse Model for Interstitial Cystitis PLOS ONE Dear Dr. Towner, 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. ============================== This manuscript was carefully reviewed by one reviewer and as a whole the manuscript very well received. However, the reviewer pointed out some important control experiments which will require significant additional experimentation. Given the care that the reviewer took in the review, we have decided that a second reviewer will not be needed at this time. For the manuscript to be acceptable, please address the reviewer critiques as well as the following 6 points. IHC staining may be acceptable if it can be demonstrated in images that claudin-2 is indeed at the tight junctions. If not, then IF should be considered because it may give a better signal, as suggested by reviewer. Quantification of all IHC and/or IF is required. For the figure 2 images, please consider whether there is an orientation difference or whether there is a difference in the nature of the umbrella cell layer. Consider choosing a better representative image if there are not overt histological differences. Scale bars should be present in all microscopic images. Check the labeling of Y axes. If it is percent, label as percent Avoid the word leaky when discussing claudin 2 and use the word hyperpermeability instead since it is a selectively permeable pathway. ============================== We would appreciate receiving your revised manuscript within 6 months. 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, Christopher R. Weber, MD, PhD 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 noticed you have some minor occurrence of overlapping text with previous publications, which needs to be addressed: http://www.painful-bladder.org/pdf/2017-01_Newsletter.pdf https://link.springer.com/article/10.1007%2Fs00404-017-4364-2 https://www.medicinenet.com/interstitial_cystitis/article.htm https://doi.org/10.1523/ENEURO.0381-16.2017 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 manuscript of Smith and colleagues describes a novel technique to evaluate urothelial permeability and detect claudin-2 using magnetic resonance imaging. This approach was tested in the URO-MCP-1 mouse model of interstitial cystitis (IC), which exhibits increased urothelial permeability. IC diagnosis is based upon symptoms and exclusion of mimicking diseases. Therefore, this method, if validated, could have a significant impact in the diagnosis of this condition. Below are some comments that might help to improve the manuscript. Major A major concern is the specificity of the antibody-based probe towards claudin-2. A control with a probe with inactivated antibody, a probe with a control antibody or the biotin-albumin-Gd-DTPA should be performed. Fig. 1 shows that using the Gd-DTPA probe the authors observe change in permeability between controls and LPS treated mice. Because the backbone of the antibody-based probe is Gd-DTPA, the question is whether this probe is binding to claudin-2 or just simply detecting changes in permeability. Please provide a detailed description of the animal model in the introduction, this will help the reader. Additional information should be provided about the method for coupling the claudin-2 antibody to the probe and the purification of the probe. Authors are encouraged to provide enough information so that the experiment can be duplicated. How was the mass of the probe determined? Page 9, line 156. List the reagents produced by Vector Labs. State the concentration of claudin-2 Atb used. Number of animals and statistical test used should be indicated in the figure legends. Page 11, line 198. The images showed in Fig. 2 should be improved. It looks that the umbrella cell layer is gone in the LPS treated bladder. Is this correct? If possible, authors should quantify urothelial damage. Claudin-2 only appears to be expressed in umbrella cells in control and across the whole urothelium in mice treated with LPS. Unfortunately, the immunohistochemical staining does not allow to define the cellular localization of claudin-2, which is relevant for its detection with the antibody-based probe. Authors should consider using immunofluorescence to assess whether Cldn2 is expressed in the plasma membrane and /or tight junctions. The number in the panels of Fig.2 do not correspond with the figure legend. Page 13, last paragraph. The facts that the authors observe a signal by immunohistochemistry does not mean that the probe is detecting claudin-2 in the tissue. If the urothelium is leaky, the probe will diffuse and can be covalently attached to the tissue during fixation. This issue should be addressed. Page 14, line 248. The statement is incorrect. There is no evidence that the overexpression of claudin-2 inhibits the synthesis of tight-junction proteins. The increase in claudin-2 expression in mice treated with LPS should be quantified by western-blot. Minor Page 7, line 114. remove and retained for 10 min. Please rephrase last sentence of the conclusions. ****** 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 [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.0239282.r001
Revision 1
30 Jul 2020 Author Response I would like to thank the Reviewers for their thoughtful suggestions and comments. We have addressed each comment below. The changes that we have made to the manuscript were substantial, and have dramatically improved the quality of the revised manuscript. Major changes include adding immunofluorescence data, adding an IgG isotype negative control for the molecular-targeting imaging data, quantification of all data, expanding the Methods section, and addition of relevant references. Editor’s comments: For the manuscript to be acceptable, please address the reviewer critiques as well as the following 6 points. 1. IHC staining may be acceptable if it can be demonstrated in images that claudin-2 is indeed at the tight junctions. If not, then IF should be considered because it may give a better signal, as suggested by reviewer. IF was included – new Figure 3. 2. Quantification of all IHC and/or IF is required. Quantification for every figure has been included. 3. For the figure 2 images, please consider whether there is an orientation difference or whether there is a difference in the nature of the umbrella cell layer. Consider choosing a better representative image if there are not overt histological differences. Better representative images have been included. 4. Scale bars should be present in all microscopic images. Added as suggested. 5. Check the labeling of Y axes. If it is percent, label as percent Changes as suggested. 6. Avoid the word leaky when discussing claudin 2 and use the word hyperpermeability instead since it is a selectively permeable pathway. Changed. Reviewer #1: 1. A major concern is the specificity of the antibody-based probe towards claudin-2. A control with a probe with inactivated antibody, a probe with a control antibody or the biotin-albumin-Gd-DTPA should be performed. A non-specific IgG isotype contrast agent (replacing the claudin-2 antibody with a mouse IgG) has been added. 2. Fig. 1 shows that using the Gd-DTPA probe the authors observe change in permeability between controls and LPS treated mice. Because the backbone of the antibody-based probe is Gd-DTPA, the question is whether this probe is binding to claudin-2 or just simply detecting changes in permeability. The probe is intravesically administered, but then washed out with saline after distilling in the bladder for an hour. Any residual probe would be washed out and not detected. The SA-HRP data clearly shows that the probe is present in the bladder urothelium. 3. Please provide a detailed description of the animal model in the introduction, this will help the reader. Now included. 4. Additional information should be provided about the method for coupling the claudin-2 antibody to the probe and the purification of the probe. Authors are encouraged to provide enough information so that the experiment can be duplicated. How was the mass of the probe determined? Additional information on the synthesis of the claudin-2 and IgG contrast agents has been included, along with an estimation of the molecular weight. 5. Page 9, line 156. List the reagents produced by Vector Labs. State the concentration of claudin-2 Atb used. Updated and included. 6. Number of animals and statistical test used should be indicated in the figure legends. Now included. 7. Page 11, line 198. The images showed in Fig. 2 should be improved. It looks that the umbrella cell layer is gone in the LPS treated bladder. Is this correct? If possible, authors should quantify urothelial damage. We have used better representative images for Figure 2, as well as included quantification of Clnd2 expression. We have also additionally added trans-epithelial electrical resistance (TEER) data in Figure 1, that supports the increased permeability/damage to the LPS-induced mouse bladders, compared to saline controls. 8. Claudin-2 only appears to be expressed in umbrella cells in control and across the whole urothelium in mice treated with LPS. Unfortunately, the immunohistochemical staining does not allow to define the cellular localization of claudin-2, which is relevant for its detection with the antibody-based probe. Authors should consider using immunofluorescence to assess whether Cldn2 is expressed in the plasma membrane and /or tight junctions. Immunofluorescence data has been added in a new figure, including quantification. From IF images it seems that Cldn2 is expressed in both the plasma membrane and tight junctions (added in figure legend – new Figure 3). 9. The number in the panels of Fig.2 do not correspond with the figure legend. Changed. 10. Page 13, last paragraph. The facts that the authors observe a signal by immunohistochemistry does not mean that the probe is detecting claudin-2 in the tissue. If the urothelium is leaky, the probe will diffuse and can be covalently attached to the tissue during fixation. This issue should be addressed. We are stating that we detect an increase in streptavidin-horse radish peroxidase (SA-HRP) which specifically targets the biotin moiety of either the claudin-2 probe or the IgG isotype control contrast agent. This can only occur if either probe or contrast agent is present. This has been shown to be the case for several probes using the Ab-albumin-Gd-DTPA-biotin construct by the authors (of which some of these references have been cited in this manuscript). We have included a non-specific IgG isotype contrast agent as a negative control. We have also rephrased the paragraph. 11. Page 14, line 248. The statement is incorrect. There is no evidence that the overexpression of claudin-2 inhibits the synthesis of tight-junction proteins. Corrected. 12. The increase in claudin-2 expression in mice treated with LPS should be quantified by western-blot. Quantification of the IHC, immunofluorescence, and molecular-targeted probe via streptavidin horse radish peroxidase which binds to the biotin moiety of the claudin-2 probe (and the non-specific IgG isotype contrast agent) was evaluated instead. Minor 13. Page 7, line 114. remove and retained for 10 min. Corrected. 14. Please rephrase last sentence of the conclusions. Changed. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0239282.r002
19 Aug 2020 Decision Letter - Christopher R. Weber, Editor PONE-D-20-05854R1 In vivo and ex vivo Assessment of Bladder Hyper-Permeability and Using Molecular Targeted Magnetic Resonance Imaging to Detect Claudin-2 in a Mouse Model for Interstitial Cystitis PLOS ONE Dear Dr. Towner, 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 address the concerns of reviewer 1 and also please show additional IF images demonstrating claudin colocalization with ZO-1 or other tight junction protein. Much of the staining appears cytoplasmic or perinuclear and it is important to show that it colocalizes with the tight junction as well. Please submit your revised manuscript by Oct 03 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Christopher R. Weber, MD, PhD 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 #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: No ****** 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 satisfactorily my previous comments. An additional comment, t-test should be used for use to compare data from two groups (Fig. 1, 2 and 3). ANOVA is generally used to compare data from more than two groups. ****** 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 [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.0239282.r003
Revision 2
19 Aug 2020 Author Response I would like to thank the Reviewers for their thoughtful suggestions and comments. We have changed the wording and included a Student’s t-test for data presented in Figs. 1, 2 and 3. Reviewer #1: The authors have addressed satisfactorily my previous comments. An additional comment, t-test should be used for use to compare data from two groups (Fig. 1, 2 and 3). ANOVA is generally used to compare data from more than two groups. We have updated the statistical analyses and wording for Figs. 1-3 in the Methods and relevant figure legends. The p-values were not substantially changed, i.e. the asterisks reported for each figure remain the same, and therefore no changes were made to the figures. Attachments Attachment Submitted filename: Response to Reviewers 081920.docx https://doi.org/10.1371/journal.pone.0239282.r004
27 Aug 2020 Decision Letter - Christopher R. Weber, Editor PONE-D-20-05854R2 In vivo and ex vivo Assessment of Bladder Hyper-Permeability and Using Molecular Targeted Magnetic Resonance Imaging to Detect Claudin-2 in a Mouse Model for Interstitial Cystitis PLOS ONE Dear Dr. Towner, 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 provide IF and/or IHC images with demonstrate claudin-2 protein expression at the tight junction. It states in the legend that it is at the tight junction, however, presently, it is difficult to discern this. There is also a lot of cytoplasmic and perinuclear staining, which should also be described in addition to the membranous staining. It may also help to include an arrow or two pointing to the tight junctions. Please submit your revised manuscript by Oct 11 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Christopher R. Weber, MD, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] [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.0239282.r005
Revision 3
31 Aug 2020 Author Response I would like to thank the Editor and Reviewers for their thoughtful suggestions and comments. We have included co-localization data for claudin-2 and ZO-1, and updated the text in the Abstract, Methods, Results and Figure 3 legend, as a result. Editor: Please provide IF and/or IHC images with demonstrate claudin-2 protein expression at the tight junction. It states in the legend that it is at the tight junction, however, presently, it is difficult to discern this. There is also a lot of cytoplasmic and perinuclear staining, which should also be described in addition to the membranous staining. It may also help to include an arrow or two pointing to the tight junctions. We have included co-localization data for claudin-2 and ZO-1 in Figure 3 and the Results section, which includes quantitative co-localization coefficient M1 and M2 data, as well as the Methods section. We have also updated the wording regarding the IF staining to say, “Claudin-2 seems to be detected in the plasma membrane, cytoplasm, per-nuclear, as well as the tight junctions, however co-localization with ZO-1 seems to be represented mainly in tight junctions (B and D).” https://doi.org/10.1371/journal.pone.0239282.r006
3 Sep 2020 Decision Letter - Christopher R. Weber, Editor In vivo and ex vivo Assessment of Bladder Hyper-Permeability and Using Molecular Targeted Magnetic Resonance Imaging to Detect Claudin-2 in a Mouse Model for Interstitial Cystitis PONE-D-20-05854R3 Dear Dr. Towner, 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, Christopher R. Weber, MD, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0239282.r007
Formally Accepted
14 Oct 2020 Acceptance Letter - Christopher R. Weber, Editor PONE-D-20-05854R3 In vivo and ex vivo Assessment of Bladder Hyper-Permeability and Using Molecular Targeted Magnetic Resonance Imaging to Detect Claudin-2 in a Mouse Model for Interstitial Cystitis Dear Dr. Towner: 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. Christopher R. Weber Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0239282.r008

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