PONE-D-19-34544

Enhanced oxidative stress and damage in glycated erythrocytes

PLOS ONE

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CT is a recipient of a fellowship grant from the Ministère de l'Education Nationale, de

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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

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

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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

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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

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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 ID PONE-19-34544 presents interesting data on the effects of glycation on measures of erythrocyte structure, oxidative state and uptake/breakdown by endothelial cells. In general the manuscript is well-written.

Major Comments:

-Have the antibodies been validated for the dot blots and western blots? Were positive or negative controls performed?

-It is interesting that the western blots showed an initial increase in AGEs following incubation with G5 and G25, but not with G137 whereas the dot blots showed significantly higher AGEs at G25 and G137.

-Line 247: These HT50 data are shown in figure 1C, not figure 3. It is interesting that the HT50 trend is no longer seen with the G137 dose. Might that be related to the loss of AGE increase at the highest dose as well?

-Discussion material (references) should not appear in the results section of a manuscript. As currently written, the results section reads more like a combined results and discussion section. Justification for the various assays could also be moved to the methods describing them.

-Line 277 mentions a progressive increase in DCF fluorescence. However, only the highest dose of glucose produced significantly greater fluorescence than G0. Therefore, I do not think one can conclude that the fluorescence was progressively increased.

-Line 288: mentions that peroxidase activity was significantly reduced in G137 cells compared to G25. However, the table indicates a significant difference between G137 and G0 cells.

-Figure S6 seems to show variability in the amount of glycation within the diabetic samples with only 2 showing increased glycation. Were these samples from 4 different subjects?

-Line 330 mentions a significant reduction in SOD in the diabetic samples. However, this significance is not shown in table 4.

-Lines 336-337: mentions that the phosphatidylserine exposure was significantly higher in diabetic samples. This conclusion cannot be made as the p-value was not significant (0.051). Moreover, the conclusion regarding this variable in the discussion is likewise an overstatement (lines 349-351).

-It is interesting that the in vitro exposed cells showed reduced LLVY with glycation whereas this variable was increased in the diabetic samples. What could explain this difference? It appears the in vitro experiments do not accurately model this variable.

Minor Comments:

-Lines 100, 224: Did you mean “fructosamine” as opposed to “frutosamine”?

-Inter- and intra-assay coefficients were provided for the free radical-induced hemolysis test, but not for other assays.

-Have the SOD and catalase activity assays been previously validated? (ie. Provide citations)

-What is meant by “fixed erythrocytes”, line 168? Do you mean affixed to the plate?

-Table 1 and figures should be able to stand alone from a paper. Please define G0, G5, G25, and G137 in the table legend.

-Figure 2: The labelling is confusing as the group of 4 figures do not need separate A-D labels. Consider labeling A-D, simply “A”.

-Line 281-282: It is mentioned that 4-HNE was higher in G25 and G137 erythrocytes compared to G5. Were levels not also higher than G0?

-lines 362-363: “glycated erythrocyte lysis when glycated”??

-Line 373: delete “Pr”

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Reviewer #1: No

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PONE-D-19-34544R1

Enhanced oxidative stress and damage in glycated erythrocytes

PLOS ONE

Dear Dr. Bourdon,

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. These are minor revisions. 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 Jun 19 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,

Ping Song, Ph.D

Academic Editor

PLOS ONE

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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

Reviewer #2: (No Response)

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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

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The highlight of this manuscript is relatively comprehensive comparison on the structure change, redox status and the EC phagocytosis of erythrocytes under the condition of glycation between in vivo and invitro. But there are still some parts needed to be improved and further explained. Some of the results showed opposite phonotype in in vitro and in vivo, which should be well discussed.

Majors:

In the hemolysis study, the results indicate that low concentrated glucose incubation had a protective membrane-stabilizing effect, which means an increased “flexibility” of the cell membrane or less fragile membrane. However, in the test of deformability, elongation index was reduced after glucose incubation, which suggests a reduced “flexibility” of the cell membrane and/or cytoskeleton. How does the authors explain these possible “conflict” phonotypes?

In some papers (for example PMID: 31704555), erythrocytes from diabetes showed impaired hemolysis resistance, which is opposite the phenomenon observed in this manuscript. Do the in vitro and in vivo glycations lead to different hemolysis results?

The high glucose concentration may change the osmolarity of the solution and making the solution hypertonic, therefore the cells may already in a shrank state in the initial stage, which may cause influence on the results. Did the authors detect the cell morphology with microscopy?

The proteasome activity detected in in vitro and in vivo showed opposite results in the manuscript. When treated with high glucose level, impaired proteasome activity was detected. But the proteasome activity from the diabetic erythrocytes showed an increased activity. How does the author explain these results?

Minors:

The data analysis used in Table2 & 3 should be One-way ANOVA.

If possible, can the author add the AOPP data back?

In the method section, water was used to induce lysis of the erythrocytes sticking on the surface of ECs. EC adhesion is not very strong. Does this method also wash away some EC and affect the quantification of internalized erythrocytes?

The figures need to be reorganized. The supplementary figures can be integrated in regular figure as well.

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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.

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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.

Enhanced oxidative stress and damage in glycated erythrocytes

PONE-D-19-34544R2

Dear Dr. Bourdon,

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.

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Kind regards,

Ping Song, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have addressed the concerns raised by two reviewers. The manuscript is acceptable in PLOS ONE.

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: 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 #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: They authors have addressed all the comments. But the figures still need reorganization to be appropriated displayed in a printed page. A single figure should is composed of multiple well-arranged graphs. The authors' figures still display as individual graph which does not meet the requirement of publication. Please look at the figure pattern in other published PLOS papers.

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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