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I have examined the revised version of the manuscript and find that concerns raised in the previous review have been reasonably addressed.
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
The revised manuscript has been examined by one of the two reviewers who had looked at the original submission. Please address the comments raised in the new review. Regarding comment #1, the suggestion of the reviewer is not mandatory but I think it will be good if you can provide such that. RTPCR of M1/M2 marker genes should not be too difficult.
Additionally:
1. Please provide more details of siRNA experiments – transfection reagent, duration if transfection treatment, etc.
2. Can you deposit the raw sequencing data in a public database such as one run by NCBI or EBI? Accession number of such deposition should be noted in the manuscript.
In this article, the author systematically elaborated the mechanism of Low-intensity pulsed ultrasound in the treatment of periodontitis, and found that Low-intensity pulsed ultrasound can regulate the macrophage polarization via Wnt2b/AXIN/β- catenin pathway. This manuscript contains some interesting discussions, providing a deep insight into the link between Low-intensity pulsed ultrasound and macrophages. The study is within the journal's scope, and I found it to be well-organized and clear stated. However, there are some suggestions necessary to make the article complete and fully readable. For these reasons, the manuscript requires minor changes.
MINOR COMMENTS：
1.In this manuscript, the author used flow cytometry to verify the polarization of macrophages, and the results were in line with the prediction, but the test method was too simple. Therefore, it is suggested that the author increase experiments to verify the polarization of macrophages, such as PCR and WB technology.
2.In this manuscript, the author found that the Wnt signaling pathway and HIPPO signaling pathway participated in the inhibition of M1 polarization. Please explain why only the Wnt signaling pathway was selected for subsequent experiments
3.In the figure 6, there are two confusing groups in the experimental group, the “RAW+LPS+LIPUS+Wnt2b siNC”group , and the results of the two groups are also different. Please check the results carefully.
4.There are several syntax errors in the manuscript, which requires a standardized syntax.
Finally, this manuscript shows a basic structure, properly divided and looks like very informative on this topic. Furthermore, discussions are complete and easy to read. In conclusion, this manuscript is densely presented and well organized, based on rigorous argument. The authors were lucid in their style of writing, making it easy to read and understand the message, portrayed in the manuscript. However, there are some minor concerns in this manuscript, and I have comments for these concerns, for improvement before acceptance for publication. The article is accurate and provides relevant information on the topic, and I have some major points to make, that may help to improve the quality of the current manuscript and maximize its scientific impact. I would accept this manuscript if the comments are addressed properly.
no comment
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The manuscript has been reviewed by two referees. Based on their comments and my examination of the manuscript, I am asking for a significant revision of the manuscript. All comments of both referees should be addressed appropriately with changes to the manuscript or rebutted in a response-to-review letter. A very important concern is the way the manuscript has been prepared – organization of the information in the manuscript for continuity (flow), with rationales described and findings discussed in context of existing literature is critical. The entirety of Results require re-writing. Just summarizing statistics is inadequate. Why something was done, what the results mean, etc. have to be noted. Finally, many details are missing from Methods. They include, but are not limited to, sources of LPS and IL4, vendor product identifiers for antibodies used in flow cytometry and Western, sequences and source of the siRNAs. Additionally, the RNA sequencing data with sample annotation data should be submitted to a public repository.
In the manuscript, the authors demonstrated that low intensity pulsed ultrasound can effectively regulate macrophage polarization, and successfully predicted and verified the mechanism of action of low intensity pulsed ultrasound. This manuscript contains some interesting discussions, providing a deep insight into the link between Wnt2b/AXIN/β-catenin pathway and periodontitis. The study is within the journal's scope, and I found it to be well-organized and clear stated. However, there are some suggestions necessary to make the article complete and fully readable. For these reasons, the manuscript requires minor changes.
MINOR COMMENTS：
1.In the List of abstract, the authors used “restore the inhibitory eûect of LIPUS”, which is an inaccurate academic term, please revise it.
2.In the figure 1 legend, the author used 10ng/ml LPS to stimulate RAW 264.7, which is inconsistent with the description in the method section. Please give a reasonable explanation or modification.
3.In the figure 7B, the content of TNF- α in the control group can reach 60000pg/ml, and compared with the RAW+LPS+Wnt2b siNC group , the content of TNF-α in the RAW+LPS+Wnt2b siNC group is only slightly increased, which is not in line with the records in the previous literatures. Please give an explanation or modification.
4.There are several syntax errors in the manuscript, which requires a standardized syntax.
Finally, this manuscript shows a basic structure, properly divided and looks like very informative on this topic. Furthermore, the discussions are complete and easy to read. In conclusion, this manuscript is densely presented and well organized, based on rigorous argument. The authors were lucid in their style of writing, making it easy to read and understand the message, portrayed in the manuscript. However, there are some minor concerns in this manuscript, and I have comments for these concerns, for improvement before acceptance for publication. The article is accurate and provides relevant information on the topic, and I have some major points to make, that may help to improve the quality of the current manuscript and maximize its scientific impact. I would accept this manuscript if the comments are addressed properly.
no comment
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many latest references are available. it can be included in introduction and discussion.
Satisfactory
How does this study add to the existing findings?
Results should be supported with adequate references in the discussion
It is a good study. Requires changes.
GENERAL
1. There are many studies available on LIPUS in periodontics. How does this add to the existing literature. Is there anything novel in this study?
2. Authors should pay attention to language and grammar. Abstract, introduction and discussion have to be carefully rewritten. Some points to be considered
ABSTRACT
Background has to be changed. LIPUS mechanisms are many, and clear. Structure the abstract accordingly.
INTRODUCTION
There is no continuity.
Some are in present tense, some in past tense,- please check grammar
LIPUS is always used in a non invasive manner only, so that can be removed
Introduction can follow this order
• Firstly, a paragraph on periodontal disease,
• LIPUS with its beneficial properties
• LIPUS in medical fields followed by dental, especially periodontal
• Wnt /β Catenin pathway in general and in periodontitis
• How Wnt signaling modulates macrophage polarisation
• Aim of the study
MATERIALS & METHODS
1. Were the standard guidelines for the invitro studies followed. Please add which guideline was followed?
2. Why the term macrophage like polarisation is used? RAW cells are either polarised in to M1 or M2… “macrophage like polarisation” can be removed
3. Include the reference for the LIPUS protocol used
RESULTS
Can be presented better.
Mean±SEM. SEM is not a descriptive statistic and generally Mean ± SD is preferred. Please check that
DISCUSSION
Has to be structured properly. It is more like a literature review. The first paragraph can summarise the results of the study and the following paragraphs can substantiate all the results with appropriate references. Write about the limitations also.
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