Transcription Repressor Hes1 Contributes to Neuropathic Pain Development by Modifying CDK9/RNAPII-Dependent Spinal mGluR5 Transcription

Dear sir:

We really appreciate that you, sir, has given our manuscript entitled “Transcription repressor Hes1 contributes to neuropathic pain development by modifying CDK9/RNAPII-dependent spinal mGluR5 transcription (ijms-568838)” a chance to be reviewed once again for publication in the International Journal of Molecular Sciences.

Along with the revised manuscript, a reply to each point raised by the reviewers is as the below; and all the modifications we made have been indicated by red underline text for easy recognition. We hope all the modifications and answers will comply with the standard of the reviewers’ and the editors’. In case of any further comments about our manuscript, we hope the editor could kindly contact us so that we can make the necessary changes. We will appreciate any additional comment that you may have to help us to improve our manuscript.

Thank you for your consideration in this matter

Sincerely yours

Tzer-Bin Lin

Answer to reviewer 1:  

We sincerely appreciate the positive and instructive comment of the reviewer. All the comment of the reviewer and our reply are as the following:

Question: Manuscript could be improved by fixing grammar and sentence structure and increasing the clarity and description of figures.

Reply:

We sincerely appreciate the positive and instructive comments of the reviewer. And we really appreciate the reviewer kindly sympathizes that as a non-native English speaker, we have to try to communicate our idea using English. The grammar and sentence structure have checked throughout the revised manuscript; and all the descriptions indicated by the reviewer have been corrected following the comments of the reviewer.

Question: Figure 1C – Are the lower images a higher magnification of the dorsal horn images above? If so, explain that they are (or if not, explain what they are). These images look as though they have been stretched so they are wider than they should be?

Reply:

We apologize that we did not clearly explain the lower images in Figure 1C of the previous version. We have added a brief explanation for the lower images in the Figure 1C of the revised manuscript by “The lower images are higher magnification of the upper images” (Line 127, 128). We hope this modification could provide clear information about this Figure. In addition, we regret that we the format of Figure 1 make confuse. Images in Figure 1C were not stretched; nevertheless, for we showed the upper half of these image only, it looks like a stretched image.

The original images are as the following.

Question: Lines 178, 181, 216 and 219 all say “respectively. both” – is this a repeated typo? Line 319: “amerliorated by genetic trimmed-down Line 372: in air or oxygen? Were sutures applied to the control animals (and not tightened), or was it just a sham surgery without sutures around the nerve? Line 413: “tissuescut” all one word Line 427: “removed from anesthetized and cut into” – removed from anaesthetized what?

Reply:

In addition, several typos indicated by the reviewer have been corrected as the following:

“respectively. both” have all been corrected “respectively” (Line 273, 278, 348, 352) “amerliorated by genetic trimmed-down” corrected by “ameliorated by” The isoflurane was mixed by room air, and hence described as “2% in room air” (Line 599, 605, 697). We apologize that we did not clearly describe the surgical procedures of the sham-operated animals; and in the MATERIALS AND METHODS of the revised version, we have added a brief paragraph as “the surgical procedures were identical to the nerve-ligated animals, except that the nerves were not ligated” (Line 601,602). We hope this paragraph could provide useful information about our experiment protocol. “tissuescut” was corrected by “tissues were cut” (Line 645). “Fresh samples were rapidly removed from anesthetized and cut into small pieces” was corrected by “Fresh dorsal horn samples were rapidly removed from anesthetized animals and cut into small pieces” (Line 658, 659)

We are sincerely grateful in the insight comment of the reviewer; these points not only improve this manuscript but also is benefit to our future study.

Answer to reviewer 2

We sincerely appreciate the positive and instructive comment of the reviewer. All the comment of the reviewer and our reply are as the following:

Question- all abbreviation should be explained in the text (also in the abstract).

Reply:

Following the comment of the reviewer, we have explained all the abbreviations in the text when they first appear in the manuscript. We hope this modification could increase the readability of our manuscript.

Question- in the opinion of the reviewer the list of abbreviations used in the text added at the beginning of the manuscript would increase the readability of the article

Reply:

Following the comment of the reviewer, we have added the list of abbreviations at the beginning of manuscript (Line 19-22). We hope this modification could increase the readability of our manuscript.

Question- in the whole manuscript the authors used the verbs in first person, for example: “we demonstrated”, “our finding”, ‘we reported”. In the opinion of the reviewer impersonal forms are more appropriate in scientific papers for example “in the present study it has been reported” instead of “we reported” or “the obtained results” instead of “our findings” etc.

Reply:

We really appreciate the comment of the reviewer; and following this comment, we have modified the inappropriate sentences in the revised manuscript, and we hope the description in the revised version would be more appropriate in scientific papers.

Question- in the opinion of the reviewer the short definition of allodynia and eventually clinical importance of studies conducted added in the introduction chapter would enhance the manuscript

Reply:

We sincerely agree the comment of the reviewer that to define allodynia and its clinical importance will really enhance the quality of the manuscript. Following the instructive comment of the reviewer, define the sign of allodynia and it clinical importance in the revised manuscript by “allodynia, a pain sensation caused by innocuous stimulation that is a clinical sign of neuropathic pain” (Line 72-73). We hope this modification could comply with the standard of the reviewer.

Question- figures are illegible. To small letters in legends on the graphs and microphotographs make it impossible to read the results.

Reply:

We apologize that the letters on the graphs are too small to be clearly read. Following the comment of the reviewer, we have replaced the letters in Figures of revised manuscript (from 6pt to 8pt; IHC from 6pt to 12pt); and we hope this modification could be read more easily

Question- poor quality of microphotographs concerning immunofluorescence. They are too small, and immunopositive structures are not visible at all. The publication of such microphotographs doesn't make sense. The Authors have to re-edited these photographs. Moreover, the immunopositive structures should be marked by arrows.

Reply:

We apologize that the size of immunofluorescence images is too small that structures can’t be clearly visible. Following the comment of the reviewer, we have revised these images by showing images with higher amplification. In addition, following the comment of the reviewer, all the immunopositive structures have been marked by arrows to be easily recognized.

Queston- the description of microphotographs is also not clear. The authors use the term “puncta”. In the opinion of the reviewer the Authors should determine in which structures (nerves or cells) the studied substances have been noted.

Reply:

We sincerely apologize that, as indicated by the reviewer, the term “puncta” is inappropriate in our manuscript; and we have indicated these by “neurons” in the revised manuscript. We hope this modification could comply with the standard of the reviewer.

Question- in the opinion of the reviewer the most important values of obtained results (especially in the case, when the graphs are illegible) should be added in the text of results chapter

Reply:

We sincerely agree the comment of the reviewer that the most important values of obtained results should be added in the text of results; and following the instructive comment of the reviewer, we have added several value of our results in the revised RESUTLS of the revised manuscript. We hope this modification could improve the quality of our manuscript.

Question- the reviewer noticed unnecessary lengthening of the discussion chapter by the repeat of observation obtained during the study. For example phrase in lines 315-319 from the words: “In the current study…” to words “CDK9 expression” would be replaced by “Results obtained during the present study support.”

Reply:

We sincerely agree the comment of the reviewer that the repeat of observation can be trimmed out, and following the comment of the reviewer, we have revised the paragraph by “Data in the current study provide support for the role of CDK9/pRNAPII cascade, as a novel pathogenic pathway for the progression of spinal plasticity” (Line 513-515).

Question- the age of animals used in the experiment should be added

Reply:

We regret that we did not clearly indicate the age of animals used in this study; and following the comment of the reviewer, we have added this information in the MATERIALS AND METHODS of revised manuscript (Line 586).

Question- the total number of animals included into experiments as well as the number of animals in particular experimental groups must be absolutely added

Reply:

We sincerely appreciate the reviewer has kindly comment that the total number of animals included into experiments as well as the number of animals in particular experimental groups must be indicated. Following the comment of the reviewer, we have added a short paragraph to briefly summarize the count of animals used in this study and the animals in each group in the MATERIALS AND METHODS of the revised manuscript (Line 586, 593-597). We hope this description could provide information about the animal numbers in this study.

Question- Did the bioethical commission give the agreement for the study? The authors wrote that the experiment was approved by the Institutional Review Board of Taipei Medical University. Is the agreement unambiguous with the agreement of bioethical commission? It should be clearly marked in the text. Moreover, the number and data of the bioethical agreement should be added. The experiments on animals without bioethical agreement cannot be published.

Reply:

We sincerely appreciate the reviewer has kindly comment that it is necessary to clearly indicated the animal experiments in this study was reviewed and approved by the bioethical committee. In the revised version we have described that this study was approved by the Institutional Review Board of Taipei Medical University (LAC-2017-0385) which is unambiguous with the Bioethical Committee in our campus. Moreover, our study is also reviewed and approved by the Institutional review Board of Biosafety committee of Mackay Medical College (B1060014); and number of both of these agreements has been indicated in the MATERIALS AND METHODS of the revised version (Line 588-591).

Question-Were there made the specificity tests of antibodies used in immunofluorescence method? These test should be shortly described in the manuscript.

Reply:

We regret that we did not clearly describe how we tested the specificity of antibody used in the immunofluorescence; and following the comment of the reviewer, we have added this information in the MATERIALS AND METHODS of revised manuscript by “To test the specificity of antibody used in the immunofluorescence, the molecular weight were first checked of antibody using western blot analysis. In addition, the specificity of antibody was further confirmed by the western blat analysis of samples treated with siRNA targeting Hes1 and CDK9 [16] and pRNAPII-selective inhibitor [16]. (Line 639-642)” We hope this modified could provide clear information about our study.

Question-The Authors have to add conclusion chapter at the end of the manuscript. On the one side such chapter is required in IJMS journal. On the other side the summary of the most important results obtained in the study and main conclusions shown in the short form would increase the readability of the article

Reply:

We really agree the format of the IJMS journal that it is necessary to have a paragraph of CONCLUSION in the end of the manuscript; and we have added a paragraphs in the revised manuscript (Line 577-582). We hope this paragraphs could summarize the most important results and increase readability of this study.

We sincerely appreciate the comment of the reviewer, in case of any further comment on our study, we are very glad to response as soon as possible.

Answer to academic editor

We sincerely appreciate the positive and instructive comment of the reviewer. All the comment of the reviewer and our reply are as the following:

Question: Full uncut and labeled blots are required as suppl. file for evaluation all blots need MW labeling

Reply:

We sincerely appreciate the positive and instructive comments of the editor. The full uncut and labeled blots with MW labeling have provided the revised manuscript (uncut and labeled blots, Line 833). We hope this modified could provide clear information about our study.

Question: Quantitative data are preferentially presented as scatter plots to show sample sizes and biological variability.

Reply:

We sincerely appreciate the positive and instructive comments of the editor. The Quantitative data have presented as scatter plots in the revised manuscript. We hope this modified could provide clear information about our study.

Question: IF images in Fig. are stretched, which causes morphological artefacts
Reply:

Images in Figure 1C were not stretched; nevertheless, for we showed the upper half of these image only, it looks like a stretched image.

The original images are as the following.

Question: Figure legends in part lack information about samples sizes and type of statistical tests

Reply:

We sincerely appreciate the positive and instructive comments of the editor. The figure legends about samples sizes and type of statistical tests have provided in the revised manuscript. We hope this modified could provide clear information about our study.

Question: mGluR5 agonists rather reduce nociceptive behavior by increasing release of endocannabinoids from pre-synaptic neurons

Reply:

We sincerely appreciate the positive and instructive comments of the editor. Following the comment of the reviewer, we have added DISCUSSIONT of revised manuscript (Line 567-575). We hope this modification could provide clear information about our study.