Effect of a Silk Sericin and Methylsulfonylmethane (MSM) Blends on Inflammatory Response and Wound Healing
, Jeong-Ok Lim 2,3,* and Jang-Soo Suh 1,4,*Round 1
Reviewer 1 Report
This manuscript is very interesting because the authors studied the synergistic effect of silk sericin/methylsulfonylmethane blends at optimum concentrations of both compounds on the suppression of the inflammatory response and promotion of healing through regulating NFkB/COX-2/iNOS pathway, suggesting that sericin/MSM blends as natural products are promising wound healing agents. However, points should be cleared before acceptance to this journal. The comments are as followed.
Major comments
1) The title should be changed to "Synergistic effect of silk sericin/methylsulfonylmethane blends on inflammatory response and wound healing" because both compounds are just mixed and the authors did not check the complexation between them.
2) Complexes in all sentences including Figure captions should be changed to blends.
3) Complexed in all sentences should be changed to blended.
Minor comments
1) Acrylamide on page 2-92 should be acrylamide.
2) Na2CO3 on page 3-102 should be Na2CO3.
3) Glutamine on page 3-120, 3-127 and 4-150 should be glutamine.
4) Na3VO3 on page 4-135 should be changed to Na3VO3.
5) Result on page 4-164 should be Results.
6) COX2 on page 7-224 should be COX-2.
7) NOS on page 8-252 should be iNOS.
8) if on page 9-286 should be of.
9) The sentences on pages 12-335 to 12-336 should be changed to "the sericin/MSM blends represent good biomaterials that exert ~~~ and promote ~~~".
10) Please check the author-name of Sp N on page 13-385.
11) Please check the author-name of Young-Joon Surh YJ on page 13-408.
Author Response
Point 1: The title should be changed to "Synergistic effect of silk sericin/methylsulfonylmethane blends on inflammatory response and wound healing" because both compounds are just mixed and the authors did not check the complexation between them.
Response 1: Thank you for your comment. We changed the title to “Synergistic effect of silk Sericin/methlysulfonylmethane blends on inflammatory response and wound healing”.
Point 2: Complexes in all sentences including Figure captions should be changed to blends.
Response 2: We changed the entire words “complex” to “blends”.
Point 3: Complexed in all sentences should be changed to blended.
Response 3: According to your comment, we changed the entire words “complexed” in all sentences to “blended”.
<Minor comment>
Point 1: Acrylamide on page 2-92 should be acrylamide.
Response 1: We changed “Acrylamide” to acrylamide according to your comment.
Point 2: Na2CO3 on page 3-102 should be Na2CO3.
Response 2: We have corrected the numbers to subscripts.
Point 3: Glutamine on page 3-120, 3-127 and 4-150 should be glutamine.
Response 3: We edited according to your comment.
Point 4: Na3VO3 on page 4-135 should be changed to Na3VO3.
Response 4: We have corrected the numbers to subscripts.
Point 5: Result on page 4-164 should be Results.
Response 5: We edited according to your comment.
Point 6: COX2 on page 7-224 should be COX-2.
Response 6: We changed “COX2” to “COX-2” according to your comment.
Point 7: NOS on page 8-252 should be iNOS.
Response 7: We changed “NOS” to “iNOS” according to your comment.
Point 8: if on page 9-286 should be of.
Response 8: It looks like this comment is not complete. We ask you to check this.
Point 9: The sentences on pages 12-335 to 12-336 should be changed to "the sericin/MSM blends represent good biomaterials that exert ~~~ and promote ~~~".
Response 9: We have corrected the sentence according to your comment.
Point 10: Please check the author-name of Sp N on page 13-385.
Response 10: We check the author’s name again and there was nothing wrong. Thanks for your check.
Point 11: Please check the author-name of Young-Joon Surh YJ on page 13-408.
Response 11: We edited “Young-Joon Surh” into “Surh YJ”.
Author Response File: 
Author Response.docx
Reviewer 2 Report
1. In the Material and methods, under the description of the western blot assay, please state clearly to amount of protein loaded per sample for SDS separation. Also, it is strange that all primary antibodies were diluted at 1:1000. Please specify the dilution factor for each of the used antibodies.
2. Under the wound healing assay lines 149, please clarify the seeding density of the L929 cells. The seeding cell density should be checked for errors. A cell viability stain such as AM calcine should be used to show cell viability, and to make it easy to visualize the cells to better interpret the assay.
3. Under the statistical description, how was multiple testing corrected for?
4. The description of cell culture should be separated from that describing the cell proliferation assay. In description, please include all important experimental details such as the number of technical and biological replicates.
5. All graphs should be revised for better visualization. The quality of the presented graphs is low. Bars should be used to denotes comparisons between the respective groups. The use of multiple symbols is confusing and hard to interpret. Please use easier to interpret shads for the graphs.
6. The Western blot results for iNOS are very unclear and difficult to properly evaluate as currently presented. I suggest qPCR be performed to complement the Western blot, to rule out any doubts.
7. The wound healing assay is not conclusive in it current state. The results show that cells repopulating the ‘wound’ is what is changing, and the wound in all treatment groups is healing. For instance, how are the wounds measured? There must be a better way to evaluate this assay. The current method raises so many other questions.
Author Response
Thank you for your thorough review and valuable comments. We made point-to-point reply to the reviewer’s comments and revisions are reflected in the revised manuscript marked in red.
Point 1: In the Material and methods, under the description of the western blot assay, please state clearly to amount of protein loaded per sample for SDS separation. Also, it is strange that all primary antibodies were diluted at 1:1000. Please specify the dilution factor for each of the used antibodies.
Response 1: We analyzed protein quantification using the Lowry method (1951). All primary antibodies were also diluted 1:1000. We have revised this content in the paper.
Point 2: Under the wound healing assay lines 149, please clarify the seeding density of the L929 cells. The seeding cell density should be checked for errors. A cell viability stain such as AM calcine should be used to show cell viability, and to make it easy to visualize the cells to better interpret the assay.
Response 2: According to your comment, we clearly state to the density of the L929 cells in lines 149. Also, Live&dead assay was additionally analyzed and revised for visualization of cell viability.
Point 3: Under the statistical description, how was multiple testing corrected for?
Response 3: We analyzed multiple testing with Dunnett's multiple comparisons tests method and revised the paper.
Point 4: The description of cell culture should be separated from that describing the cell proliferation assay. In description, please include all important experimental details such as the number of technical and biological replicates.
Response 4: According to your advice, the method was divided into cell viability and cell viability parts.
Point 5: All graphs should be revised for better visualization. The quality of the presented graphs is low. Bars should be used to denotes comparisons between the respective groups. The use of multiple symbols is confusing and hard to interpret. Please use easier to interpret shads for the graphs.
Response 5: We modified all graphs by replacing them with visually better JPG files. And the bar for comparing the respective groups was not added because it was visually too complicated. However, if you request it again, we will make corrections.
Point 6: The Western blot results for iNOS are very unclear and difficult to properly evaluate as currently presented. I suggest qPCR be performed to complement the Western blot, to rule out any doubts.
Response 6: Thanks for your comment. However, since our research team does not have qPCR equipment, it was difficult to proceed with qPCR within the revision period. iNOS western blotting has already been experimentally analyzed several times significantly, we replaced the picture with the clearest possible.
Point 7: The wound healing assay is not conclusive in it current state. The results show that cells repopulating the ‘wound’ is what is changing, and the wound in all treatment groups is healing. For instance, how are the wounds measured? There must be a better way to evaluate this assay. The current method raises so many other questions.
Response 7: The wound healing measurement method was analyzed using Image j, which is used in many studies. We changed the picture of the wound healing recovery rate in Figure 7 to visually confirm the results. Please check one more time, thank you.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
1. For better interpretation of the Western blot results, please state the amount of protein that was loaded per sample/lane
2. The graphs have not been revised as claimed.
Author Response
Response to Reviewer 2 Comments
Thank you for your thorough review and valuable comments. We made point-to-point reply to the reviewer’s comments and revisions are reflected in the revised manuscript marked in red.
Additionally, while reviewing the contents of the paper again, it was found that Sericin and MSM blends concentrations were written differently from those originally tested, so we revised them. Revised content is highlighted in yellow.
Point 1: For better interpretation of the Western blot results, please state the amount of protein that was loaded per sample/lane
Response 1: According to your comment, we states the amount of protein that was loaded per sample/lane.
Point 2: The graphs have not been revised as claimed.
Response 2: We revised all the graphs, as you clamed.
Author Response File: Author Response.docx
