A Potential Role for the Receptor for Advanced Glycation End-Products (RAGE) in the Development of Secondhand Smoke-Induced Chronic Sinusitis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study utilized human and animal specimens to demonstrate the relationship between secondhand smoke exposure and RAGE expression levels in sinus tissue. Specific recommendations are as follows:

1. It is a good idea to start basic research with clinical samples. However, cases with sinusitis (smoking or non-smoking) are missing in this study. Moreover, the sample of smoking patients was not described clearly: what was the total number of patients? Information on the age, gender and underlying disease of the patients? How many years of smoking? Are there any patients included in secondhand smoke exposure if the topic of this article is to investigate secondhand smoke Induced chronic sinusitis?

2. Current results can only suggest that smoke exposure is associated with RAGE expression, but there are no relevant results to prove that it can lead to sinusitis. Conversely, does inhibiting RAGE expression have an effect on alleviating smoking-induced sinusitis?

3. The results section shows only representative images, no statistical plots, and no description of sample size or statistical parameters. It is recommended that the authors provide raw data and images.

4. Human and animal related ethical approvals need to be supplemented.

Comments on the Quality of English Language

N/A

Author Response

Reviewer 1

This study utilized human and animal specimens to demonstrate the relationship between secondhand smoke exposure and RAGE expression levels in sinus tissue. Specific recommendations are as follows:

 

Suggestions for improvement

1. It is a good idea to start basic research with clinical samples. However, cases with sinusitis (smoking or non-smoking) are missing in this study. Moreover, the sample of smoking patients was not described clearly: what was the total number of patients? Information on the age, gender and underlying disease of the patients? How many years of smoking? Are there any patients included in secondhand smoke exposure if the topic of this article is to investigate secondhand smoke Induced chronic sinusitis?
   3. We agree that starting such an article with clinical samples adds application and relevance. The figures were out of order and the resubmission now shoes human samples from chronic sinusitis (CS) patients in Figures 1, 2, and 3. We procured samples from a tissue bank that had ethical approvals to obtain the tissues initially. Furthermore, we did not show any samples of human tissues from smokers. We did not have samples from smoking patients, therefore the number of patients, age, gender, years of smoking, primary vs. secondhand human smokers, etc. does not apply to the current research.  We have added some additional details about the human samples we obtained from a commercial tissue bank in the revised manuscript.
2. Current results can only suggest that smoke exposure is associated with RAGE expression, but there are no relevant results to prove that it can lead to sinusitis. Conversely, does inhibiting RAGE expression have an effect on alleviating smoking-induced sinusitis?
   1. We agree that proof that RAGE causes CS is missing, but there is a strong correlation between SHS exposure, RAGE, and CS… therefore, we have added important clarifications in the discussion.
3. The results section shows only representative images, no statistical plots, and no description of sample size or statistical parameters. It is recommended that the authors provide raw data and images.
   1. We agree that the images shown are representative of a larger immunohistochemical battery of experiments. We decided to show representative images to convey the general histology (H&E) and qualitative differences in RAGE or S100s. Accordingly, we have no statistical quantified measures of these qualitative assessments. Importantly, we have made edits to the manuscript’s methods section.
4. Human and animal related ethical approvals need to be supplemented.
   1. Thanks for this recommendation. We have added more details to the manuscript regarding the approvals for animal use at our institution.  We procured microscope slide samples of sinusitis and control patients from a commercia tissue bank, so we were not required to obtain human use approvals. Regardless, we have added details in the revision that we hope are sufficient.

Reviewer 2 Report

Comments and Suggestions for Authors

This paper describes about an assessment of the potential role for receptors for advanced glycation end products (RAGE), an inflammatory receptor expressed in tissues exposed to secondhand smoke (SHS). It is an interesting paper, but there are some unclear points.

1. Figure 5 is shown first and the figure numbers should be rearranged in the order listed.

2. In Figure 1, arrows should indicate where changes were observed.

3. The correlation between CS onset and tobacco smoke exposure levels should be stated from a quantitative perspective.

4. RAGE expression and histological changes in CS should be explained in comparison between smokers and passive smokers.

5. What AGEs are contained in tobacco smoke?

6. What chemicals in tobacco smoke cause CS?

7. What substances in SHS induce RAGE?

8. The above questions should be addressed in the text.

Author Response

Reviewer 2

This paper describes about an assessment of the potential role for receptors for advanced glycation end products (RAGE), an inflammatory receptor expressed in tissues exposed to secondhand smoke (SHS). It is an interesting paper, but there are some unclear points.

 

Suggestions for improvement

1. Figure 5 is shown first and the figure numbers should be rearranged in the order listed.
   1. We believe the figures were erroneously positioned during formatting. The figure order is correct in the current resubmission.
2. In Figure 1, arrows should indicate where changes were observed.
   1. We have edited the figure to include designations of tissue alterations.
3. The correlation between CS onset and tobacco smoke exposure levels should be stated from a quantitative perspective.
   1. The smoking research presented in this manuscript is in a mouse model only. We have added important edits to the discussion that describes quantifying characteristics in a human smoking cohort in the discussion that should be helpful to readers.
4. RAGE expression and histological changes in CS should be explained in comparison between smokers and passive smokers.
   1. We stained for RAGE and presented qualitative changes in mouse lung tissues. All mice were exposed to passive, or SHS. We will perform a follow up study that includes a human time course where CS changes over time will be evaluated in primary and passive smokers.
5. What AGEs are contained in tobacco smoke?
   1. AGEs are reactive, cross-linking moieties that form following reactions between reducing sugars and amino groups within proteins, lipids, and nucleic acids. In fact, while no particular AGE dominates, myriad are formed in tobacco smoke. We have edited the discussion.
6. What chemicals in tobacco smoke cause CS?
   1. Specific chemicals in tobacco smoke that cause CS are not known
7. What substances in SHS induce RAGE?
   1. The specific chemicals that induce rage expression are not identified, nor was the characterization of such the primary focus of the current research.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

The article refers the important and current problem, and his medical implications cause  that it contains in the thematic profile of the periodical Current Issues in Molecular Biology. The work has an experimental character, carrying in new cognitive elements from the sphere of basic sciences.

The summary answers contents of the work and contains appropriate key words.

Introduction – the manner of representing is clear and argumentative.

Material and methods - the methods used in the research are correctly described, but the information about statistical analysis raises doubts.

"Results" does not contain any quantitative (numerical) data, and their absence does not allow full appreciation of the article.

Graphical results presentation  – good and rich graphical illustration of results

Discussion - the authors conclusion is not well known. Quality of discussion is worse because lack of reliability (statistical data).

References - More than 70% of the references are older than 10 years.

Became visible small shortcomings in the text and there is no reason, with them to dispute whether outright to reject. I have indicated the comments in the attached manuscript.

Based on my review, the above mentioned manuscript (cimb-2756742) can be accepted for publication in the Current Issues in Molecular Biology after inconsiderable adjustments. Logical association of information are clear. Subject is interesting to the journal, the paper do read well.

Comments for author File: Comments.pdf

Author Response

Reviewer 3

The article refers the important and current problem, and his medical implications cause  that it contains in the thematic profile of the periodical Current Issues in Molecular Biology. The work has an experimental character, carrying in new cognitive elements from the sphere of basic sciences. Based on my review, the above-mentioned manuscript (cimb-2756742) can be accepted for publication in the Current Issues in Molecular Biology after inconsiderable adjustments. Logical association of information are clear. Subject is interesting to the journal, the paper do read well

 

Suggestions for improvement

1. The summaryanswers contents of the work and contains appropriate key words. 
   1. Thank you for this comment.
2. Introduction – the manner of representing is clear and argumentative. 
   1. Thank you for this comment.
3. Material and methods- the methods used in the research are correctly described, but the information about statistical analysis raises doubts. 
   6. Thank you for this comment—we originally assumed non-normal patterns and decided to remain with the Mann-Whitney testing. We still discovered statistical differences in the quantified data presented in the Figure 6.
4. "Results" does not contain any quantitative (numerical) data, and their absence does not allow full appreciation of the article.
   1. We began the research by showing qualitative differences in the progression of sinus related disease. We then sought to correlate disease characteristics with the presence of RAGE and its ligands. The last segment of the research is highly quantitative—we show in figure 6 statistically different expression levels for cleaved cat space three, matrix metalloprotease, and three key pro inflammatory cytokines. We are hopeful that these final quantified data support the general histology observations.
5. Graphical results presentation– good and rich graphical illustration of results 
   1. Thank you for this comment.
6. Discussion- the authors conclusion is not well known. Quality of discussion is worse because lack of reliability (statistical data).
   1. We remain hopeful, that the qualitative and quantify data together demonstrate an interesting story. We have added edits to the discussion for greater clarity.
7. References - More than 70% of the references are older than 10 years.
   1. Yes, some of the foundational aspects of CS and other related topics are older than 10 years, but we believe the manuscript is sufficiently supported by the work of others and accurate.
8. Became visible small shortcomings in the text and there is no reason, with them to dispute whether outright to reject. I have indicated the comments in the attached manuscript.
   1. Thank you very much for the attached manuscript with revisions. We have excepted all of your suggestions and have made those edits in the resubmitted draft.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors' responses to review comments are partially acceptable.

One important concern: All staining results are not convincing enough if they only show representative and regional images without providing the original images and/or statistical results covering multiple samples.

Comments on the Quality of English Language

N/A

Author Response

Reviewer 1

One important concern: All staining results are not convincing enough if they only show representative and regional images without providing the original images and/or statistical results covering multiple samples.

Response: As noted in the revised manuscript, our immunohistochemical stains included n=8 for each human group (normal control patients and CS patients) and 4 randomized images were assessed per human sample. For the mouse IHC, we evaluated 8 images per mouse and n=8 per group. As is most common, we originally planned to show representative images of the histology. Although initially performing statistical analyses of relative immunoreactivity, we felt the representative images were sufficient. However, we’ve now revised the manuscript again to include some quantification of these stains.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

As mentioned, individual, regional staining images are not convincing enough, and the current images of the control and CS groups are not exactly the same area. Actually, IHC staining can be quantified simply with softwares. The authors should provide either all original images with reduced magnification or quantified statistical graphs. Moreover, parameters such as biological replicates mentioned by the authors in their responses should also be reflected in the figure legends.

Comments on the Quality of English Language

N/A

Author Response

Reviewer 1

Initial Comment: One important concern: All staining results are not convincing enough if they only show representative and regional images without providing the original images and/or statistical results covering multiple samples.

Response: We were hopeful that showing representative images from many stained fields obtained from 8 samples per human or mouse group would have merit. We then also added details in the text showing statistical immunoreactivity using Image J software in order to quantify the stains, as requested in this comment (please see or notation in initial comment)

 

Second Comment: As mentioned, individual, regional staining images are not convincing enough, and the current images of the control and CS groups are not exactly the same area. Actually, IHC staining can be quantified simply with softwares. The authors should provide either all original images with reduced magnification or quantified statistical graphs. Moreover, parameters such as biological replicates mentioned by the authors in their responses should also be reflected in the figure legends.

Response: We quantified staining in the representative images using Image J software and these details have been added to the manuscript. The quantified differences are now also shown in the text of the Results section. Specifically, the methodology is summarized in the Materials and Methods (Histology) section and the results are noted in the Results Section on line 9 of page 5, line 15 of page 5, and line 8 of page 6. We have also noted the biological replicates and sample sizes are now also reflected in the figure legends as requested.

 

We are trying our best to respond to these follow-up requests but we have been presented with some insurmountable problems. We have lost most of our samples due to Covid issues and storage failures. Furthermore, our lab is still mostly closed due to the holiday break and we are unable to do additional experiments at this time. Because the time allowed for responses is so short, we remain optimistic that these edits are satisfactory to the Reviewer and that we are kindly able to proceed. Thanks very much for your consideration.