PONE-D-20-06143

Effect of sugar metabolite methylglyoxal on horse keratinocytes: an ex vivo model for laminitis

PLOS ONE

Dear Dr. Vercelli,

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.

Reviewer 1 has identified a number of questions that need to be addressed regarding aspects of the methods and analyses included in your study. Please ensure that you provide detailed responses to each of these questions and concerns, as well as addressing the presentational issues raised.

Please submit your revised manuscript by May 21 2021 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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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.

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We look forward to receiving your revised manuscript.

Kind regards,

Jamie Males

Senior Editor

PLOS ONE

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1. Thank you for stating in your Funding Statement:

The present study was funded with ex 60% fund of the University of Turin.

Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.

Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

3. We noticed you have some minor occurrence of overlapping text with the following previous publications, which needs to be addressed:

- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605361/

- https://onlinelibrary.wiley.com/doi/full/10.1002/dmrr.2811

- https://beva.onlinelibrary.wiley.com/doi/abs/10.2746/042516406778400565

- https://www.sciencedirect.com/science/article/abs/pii/S0165242711000201?via%3Dihub

The text that needs to be addressed involves:

- Lines 297-302

- Lines 303-310

- Lines 315-326

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

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: 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

Reviewer #2: 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: The authors have submitted a manuscript describing an ex vivo model of laminitis that they have created using a glucose metabolite (methylglyoxal) applied to lamellar explants. Such a model, if it could be validated, would be extremely useful for research in this area and has, in fact, been sought for some years now by researchers in the field. This could be a valuable contribution to those efforts - I have some questions and comments for the authors about their work, listed below (by line number in the manuscript):

Line 1 - 'Effect of the sugar metabolite...'; ex vivo should be italicized

Abstract:

Line 15 - '...synthesized during the digestion process in horses...'

Line 16 - '...excessive levels could lead to alterations in the hoof lamellar structure.'

Line 18 - '...were applied to hoof explants (HE), which...'

Line 19 - 'Microscopic'

Line 20 - '..post-MG application.'

Line 21 - '...(MMP)-2 and -14, and tissue inhibitor of metalloproteinases (TIMP)-2'

Line 22 - '...at each time point for all...'

Line 23 - '...mimicking laminitis. The separation force test revealed...'

Line 26 - '...significant weakness, and samples...'; 'In the same samples, high levels of MMP-2 and -14 and low levels of TIMP-2 were present.

Line 27 - 'All results support that high levels of MG could induce irreversible...'

Introduction:

Line 33 - 'It is characterized by damage/deterioration of the lamellar tissue...'

Line 37 - '...acute or chronic, and it is not usually readily reversible.'

Lines 38 - 39 - 'The most common etiology of laminitis cases involves gastrointestinal or metabolic disease.'

Line 41 - '...hormonal influences and weight/pressure are both involved..'

Line 46 - would add comma after 'isoforms'

Line 48 - '...disturbances resulting from ingestion...'

Line 49 - '...role in sepsis-related laminitis...'

Line 50 - '...used as highly digestible...'

Line 52 - '...glucose liberation...'

Line 54 - '...followed by colic and laminitis.' (founder is redundant here, I believe)

Line 56 - 'In this scenario, release of toxins from the hindgut is suspected to occur, which in theory induces degradation of the lamellar basement membrane...'

Line 58 - '...infiltration into the lamellae.'

Line 59 - '...can lead to rapid, devastating changes that result in the elaboration of toxins and other substances:...'

Line 60 - 'Gram-negative and -positive...'

Line 61 - '...stress, leading to ...'; has this substance been shown to appear in equine serum/plasma following induction of a carbohydrate overload model of laminitis? That would seem to be central to the premise here, given the proposed route of exposure of the lamellae to this substance in vivo.

Lines 64-65 - Would include some context-specific criteria where lactate may serve as a biomarker of laminitis risk (not laminitis itself) - certain situations are associated with hyperlactatemia but not laminitis risk (such as intense aerobic exercise), so would restrict this in some way to situations in which this is more likely to be the case (e.g., sepsis).

Line 67 - '...which precedes and enables remodeling...'

Line 70 - '...have shown that under specific...'

Line 72 - '...making them an attractive model for the...'

Line 77 - '...different exposure time points.'

Materials and Methods:

Line 81 - the authors might comment in the discussion about how widely these data might be extrapolated to other breeds/types of horses

Line 82 - '...were enrolled in the study.'

Line 84 - would add a comma after 'Italy)'; would remove the commas after 'collected' and 'consent'

Line 88 - would remove 'symptoms and' from this sentence; what signs of laminitis, specifically, were evaluated (would list them)? Also, would specify whether the limb was a front or hind limb.

Line 89 - would add a comma after 'joint'; '...within 45 minutes of slaughter and..'

Line 101 - would use 'lamellae' instead of 'laminae' consistently throughout

Line 114 - '48 hour incubation'

Lines 116-117 - would include how survival was evaluated here (not just in the Results section)

Line 117 - '...used for the rest of the study.'

Line 125 - '...integrity of the samples.'

Line 130 - '...lamellar site were considered valid measurements.'

Lines 130-131 - I'm not sure what the authors are trying to say here, it's a bit unclear; consider rewording this sentence.

Line 136 - '...then embedded in paraffin, and 3-um slices were mounted...'

Line 142 - 'epidermal'

Line 144 - '...or severe and extensive (Grade...'

Lines 146 - 147 - This sentence is unclear - what particular characteristic of the SEL was evaluated?

Line 149 - how long after the force testing did RNA extraction occur? Was RNA extracted from any samples that were not subjected to force testing? Can the authors comment on the likely influence of this testing on the mRNA concentrations of some of their target genes in the sample tissue (i.e., are they known to be influenced by stretch, if sufficient time had elapsed between force testing and RNA extraction)?

Line 157 - can remove the comma after 'USA)'

Line 162 - '...MMP-14, and TIMP-2 transcripts...'

Line 164 - 'Equus caballus'

Line 169 - '...gene accession numbers, and...'

Line 174 - '...(ACTB), and...'

Line 175 - how was this determined?

Line 177 - '...triplicate, and template...'

Line 179 - can remove comma after 'averaged'

Line 187 - 'All the data were analyzed with a commercially available software program...'

Lines 190-193 - 'The separation force test and q-PCR data were analyzed using one-way ANOVA and Tukey's...'

Line 192 - How were the distributions of the histology score data analyzed?

Line 196 - '...was accepted at values...'

Results:

Lines 200-201 - Were any more sensitive/quantifiable measures of autolysis used to evaluate this tissue? This seems like a very subjective assessment.

Line 202 - Can the authors include a figure displaying representative histologic sections?

Line 203 - '...in a few samples...'

Line 204 - 'lamellae'

Line 205 - '...changes, and histological...'

Line 206 - 'structures; all samples...'

Lines 208-209 - '...in the preliminary assay, samples were kept in DMEM+ for all subsequent experiments.'

Line 212 - '...on all samples...'

Line 213 - '...at predetermined time points.'

Line 215 - '...separation of the HE lamellar structures could be...

Line 221 - '...can be observed.'

Line 222 - 'However, these differences did not reach the level of statistical significance.'

Line 225 - 'methylglyoxal'; '(N=45 per time point)'

Line 235 - '...due to alteration of...'

Lines 237, 238, 241 - do the authors mean 'epidermal' instead of 'epithelial' in these instances?

Lines 251-252 - 'Nevertheless, these differences did not reach the level of statistical significance.'

Line 256 - would avoid commenting on trends, if possible

Line 266, 270 - 'MG' is missing in the last line of the legend for Figure 6 and Figure 7

Line 267 - 'Tissue inhibitor of metalloproteinases-2 (TIMP-2) gene...'

Discussion:

Line 274 - would add a comma after 'solution'; can the authors discuss any other papers that have attempted to characterize/use a lamellar explant model of laminitis, for the purposes of comparing their model to others? (I believe there are at least a few out there currently.)

Line 284 - '...but according to our results, FBS and L-glutamine are necessary for tissue preservation.'

Line 290 - '...hoof lamellae, rendering them less robust and unable...'

Line 291 - '...untreated samples are instead able to bear and remain intact.'

Line 301 - would add a comma after 'features'

Line 311 - would add a comma after 'concentration'

Line 315 - would remove comma after 'detected'

Line 320 - '...has been shown to be regulated in breast cancer, where...'

Line 321 - '...with increased risk of malignancy...'

Line 326 - '...in the past, yielding important...'

Line 333 - would remove comma after 'intermediates'

Line 341 - '...subjects show higher levels...'

Line 342 - '...compared with those of healthy subjects.'

Line 347 - 'Escherichia coli' - should be italicized

Line 348 - 'induce the laminitis process...'

Line 350 - '...suggests that endotoxins are unlikely to induce laminitis...'

Line 351 - 'more likely merely contribute to the...'

Lines 358-359 - can the authors clarify here how their approach and evaluations mitigated the effects of variable diffusion of nutrients and oxygen in explants?

Line 362 - '...following their harvest, and that sections could be used for ex vivo studies.'

Line 367 - would list specific MMP's here

Lines 367-368 - I don't think that MMP mRNA expression necessarily correlates well with enzymatic activity, so would probably refine this statement a bit.

Line 367-368 - 'Further studies are needed to advance our understanding...'

Figure 1 - 'Sample preparation'; 'Histology/histology'

Figure 2 - 'Outer'; 'Lamellae'

Figure 4 - 'Histological score'

Figures 5, 6, and 7 - 'Concentration of Methylglyoxal (mM)'

Reviewer #2: Congratulations for this important research work. It is not easy to culture hoof explants of horses mainly by secondary bacteria contamination. I only suggest you to replace the figure 1 by another one more explicative and less tedious. I also suggest to add a set of photographs showing the method for explant procurement in a sequential fashion (step by step). This technique is very important and useful for equine laminitis researchers around the world.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-06143R1

Effect of sugar metabolite methylglyoxal on horse keratinocytes: an ex vivo model for laminitis

PLOS ONE

Dear Dr. Vercelli,

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 Jul 10 2021 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. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Kanhaiya Singh, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Please address the suggestions made by Reviewers 2 and 3.

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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: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: (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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #2: Yes

Reviewer #3: 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 author have addressed the majority of the questions and comments that I had included in a review of the previous version of this manuscript, and I believe that it is improved. I have a few additional questions, by line, below:

Title - 'Effect of the sugar metabolite...'; would use 'equine lamellar explants' instead of 'horse keratinocytes' in the title, to more accurately describe the tissue that was used; '... model of laminitis'

Abstract:

Line 18 - '...and excessive levels absorbed into peripheral blood could be delivered to the foot and lead to...'

Line 23 - can remove the comma after '14'

Line 25 - can remove 'a' after 'mimicking'

Line 27-28 - this sentence (the one that begins with 'In the same...') is incomplete and should be reworded

Introduction:

Line 41 - '...resembling the human diabetic...'

Line 49 - please add a space between 'from' and 'ingestion'

Line 55 - '...caecal pH, often followed by colic and laminitis...'

Line 59 - '...AGEs accumulate in significant amounts in the hoof lamellar tissue in the acute...'

Line 61 - 'using the hyperinsulinemic model...'

Line 63 - '...MG) causes the formation of AGEs, leading...'

Line 66 - '...intermediate, it is converted...'

Line 72 - 'In equines, feeding large amounts of fermentable carbohydrates with subsequent acidosis increases the level of plasma D-lactate...'

Line 75 - '...D-lactate can be considered a...'

Line 84 - '...making them an attractive model...'

Line 87 - '...of MG on equine lamellar explants in an ex vivo...'

Materials and Methods:

Line 99 - '...typical stance, increased digital pulse, and increased hoof capsular warmth).'

Line 100 - '...the right fore distal limb...'

Line 112 - '...dermal lamellae, and the bone.'

Line 120 - can remove 'of' in front of 'Fetal'

Line 126 - 'Sample'

Line 142 - would use 'lamellae' instead of 'laminae' throughout the manuscript

Line 143 - 'broken at'

Line 149 - 'One sample from each time point and from each MG concentration...'

Line 150 - would add a comma after 'paraffin'; '...slices were mounted...'

Line 153 - '...microscope, and images were captured...'

Line 160 - '...evaluated for lesions of the secondary...'

Line 164 - 'RNA-Later solution (Ambion), and the stratum lamellatum was disrupted using...'

Line 177 - '...MMP-14, and TIMP-2 transcripts, qPCR...'

Line 179 - 'designed from Equus caballus...'

Line 200 - '...were designed from Equus caballus...'

Line 205 - 'one-way ANOVA and Tukey's multiple...

Line 207 - '...their distributions were analyzed using two-way ANOVA...'

Results and discussion:

Line 221 - '...all structures; all samples were...'

Line 225 - 'characteristics'

Line 236 - 'predetermined'

Line 245 - '...for 48 h can be observed.'

Line 258 - '...of all layers and were statistically...'

Line 267 - 'concentrations'

Line 280 - can remove the comma after 'concentration'

Line 296 - '...as a suitable viable model system for investigating...'

Line 307 - 'with our results, FBS and...'

Line 309 - '...period, supporting the viability of the samples...'

Line 310 - 'Other papers have evaluated the possibility of using hoof explant samples but focus their attention on different metabolic goals (e.g.,...'

Line 312 - 'To the authors' knowledge, this is the first paper focusing specifically on the role of MG (...'

Line 315 - 'to modification of the hoof lamellae...'

Line 316 - '...that untreated samples are able to bear...'

Line 317 - '...confirmed that higher concentrations of MG and a longer...'

Line 319 - '...were confirmed by histological...'

Line 322 - 'The results of gene expression of MMPs...'

Line 325 - '...matrix remodeling, more severe histological features, and...'

Line 337 - '...starch overload or other causes, increases...'

Line 346 - can remove the comma after 'MMP2' here

Line 349 - 'yielding'

Line 363 - '...various pathologies; for example,...'

Line 370 - '...not sufficient to induce laminitis in vitro.'

Line 373-374 - '...more likely contributing to the disease's...'

Line 383-385 - '...(Breton horses) that were relatively young; it would be interesting to apply the same ex vivo model of laminitis to even older horses of different breeds, such as pony breeds, to assess any differences...'

Line 388 - '...could be used for ex vivo studies.'

Line 393 - would use 'supported' instead of 'confirmed' here

Supplemental data:

Line 519 - would use 'lamellar' instead of 'laminar' here

Reviewer #2: Congratulations. This is an important research work. In general, you addressed the concerns raised during the review process.

Reviewer #3: In this paper, the authors are examining the effects of excessive methylglyoxal on the hoof lamellar structure with an aim to identify the mechanism behind the Laminitis disease. They use an ex vivo experimental design and perform various types of analysis - macroscopic analysis, histological analysis, separation force test and gene expression. They identify that high levels of methylglyoxal could induce irreversible damage in the hooves which mimicks laminitis in an ex vivo model.

Overall, the findings of the manuscript are well-supported by the data and the methods used are appropriate. The authors have also addressed the reviewers comments adequately. However, there are some points to consider that I have outlined below:

1. In the revised manuscript, the authors have renamed the ‘Results’ section as ‘Results and Discussion’, however, the discussion has not been incorporated with the results. It is still presented as a separate section (minus the heading). The layout of this section will have to be changed significantly if the authors want to keep the ‘Results and Discussion’ heading. If not, I would recommend adding the ‘Discussion’ heading and keeping ‘Results’ separate.

Minor concerns:

1. In the abstract, the authors mention ‘Microscopical and histological analysis’, however, in the rest of the manuscript, there is ‘Macroscopical analysis’. Please correct accordingly.

2. In the abstract, the second last line starting with ‘In the same samples…’ (Line 29), does not make sense, it seems incomplete. Please edit.

3. Line 45, ‘…influences and weight/ pressures…’ needs to be edited for coherence.

4. Lines 48-51, need to be changed to make sense. Right now, it seems like a collection of terms.

5. Line 57, ‘this means’ should be ‘which means’.

6. Line 64, ‘hoof lamellar tissue level’, the word ‘level’ can be removed.

7. Line 70, ‘highly reactive intermediate is’, should be ‘highly reactive intermediate, it is’.

8. Line 78, ‘sequent acidosis’, do the authors mean ‘subsequent acidosis’?

9. Line 79, ‘increase the level of plasma levels’, should be ‘increase the plasma levels of’.

10. Line 81, ‘also because’ is redundant. Use either also, or because.

11. Line 89, ‘attracting model’ should be ‘attractive model’.

12. Line 100, ‘for this study purpose’ can be rephrased to, ‘for the purposes of this study’.

13. Line 105, ‘typical pain position and digital pulse and hot hoof’, one ‘and’ can be removed.

14. Line 107, ‘within 45 minutes of slaughter’ should be ‘within 45 minutes’, since the sentence is starting with ‘following slaughter’.

15. Line 114, Mungall and Pollitt is missing an in-text citation.

16. Line 131, should be ‘Fig S1’ to make it consistent with the rest of the manuscript.

17. Line 133, do the authors mean ‘Sample survival’?

18. Line 141, for ‘controls (K)’, reference the figure number.

19. Line 144, ‘the’ before structural integrity should be removed.

20. Line 150, do the authors mean ‘broken’, instead of ‘brocken’?

21. Line 172, change RNA later to RNAlater (as per manufacturers label).

22. Line 198 and Line 202, move Cq definition from line 202 to line 198.

23. Line 207, table 1 is not in line with the text.

24. Line 211-214, do not make sense. Which softwares? Which companies? Please edit. Line 211, remove ‘a’ before ‘commercially’ if you are referring to more than 1 software.

25. Line 216, ‘Turkey’s’ should be ‘Tukey’s’.

26. Line 222, define SD.

27. Line 228, should be ‘performed for all samples’.

28. Line 232-233, separate the sentences. ‘All samples’ should be a different sentence.

29. Line 248, should be ‘predetermined’.

30. Line 254-255 is very confusing. Please clarify.

31. Line 271-272, ‘and resulted statistically different’, please clarify.

32. Line 277, what do you mean by definition?

33. Line 280, should be ‘concentrations’.

34. Fig 6-8 legend, the gene descriptions should be in the text and not in the figure legends.

35. Line 328-329, please rephrase for coherence.

36. Line 330, should be ‘In our knowledge’.

37. Line 340, please edit for coherence.

38. Line 344, edit spelling to ‘correlated’.

39. Line 345, should be ‘resulting in structural weakness’.

40. Line 367, please define the ‘entire process’ so as to make the sentence complete.

41. Line 369, edit spelling to ‘yielding’.

42. Line 378, the acronym AGEs does not need to be defined again.

43. Line 391, the word ‘bacteria’ should be before E.coli and not after.

44. Line 395, rephrase for coherence.

45. Line 403-406, rephrase for coherence.

46. Line 410, remove ‘an’ before ex vivo.

47. Line 412, time-dependent would be more appropriate than ‘time-related’.

48. Figure 1, The authors can consider changing ‘force test’ to ‘separation force test’. It would then be consistent throughout the manuscript.

49. Figure 4, table headings are missing.

50. Figure 4,6,7,8, the legend for time needs to be consistent throughout. Either 24h, 48h or T24h, T48h.

51. Figure 6-8, the Y-axis should be Cq instead of Ct, for consistency between figures and text.

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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 #1: No

Reviewer #2: No

Reviewer #3: No

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Effect of sugar metabolite methylglyoxal on equine lamellar explants: an ex vivo model of laminitis

PONE-D-20-06143R2

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