Acute Kidney Injury Induces Lung Damage via Mitochondrial DAMPs by Activating TREM-1 and cGAS-STING Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors  analyze  how ischemia–reperfusion–induced acute kidney injury (IR-AKI) cause acute lung injury (ALI), highlighting the role of mitochondrial damage–associated molecular patterns (mtDAMPs). Through murine models, proteomic profiling, and cell culture experiments, the study pinpoints TREM-1 and the cGAS–STING pathway as major mediators of the inter-organ inflammatory response. The research question is clinically appropriate, and the data are intriguing.

The following comments aim to further strengthen the manuscript:

Major Comments

1. The animal model data are compelling, but the discussion should better address the potential translation to human settings. Are there existing or ongoing clinical studies exploring TREM-1 or cGAS–STING inhibition in AKI/ALI? Highlighting the therapeutic importance of dual targeting, these pathways could strengthen the translational impact.
2. The evaluation of lung function relies on BALF analysis, W/D ratio, and flow cytometry. Adding histological lung injury scoring  would make the assessment more comprehensive and align it with standard ALI methodologies.
3. The use of AMJ2-C8 and MM14.LU cell lines raise concerns about physiological relevance. The inclusion of human-derived or primary cell models would improve the biological validity of the findings.
4. Regarding sample size and statistical power- some animal experiments appear to include n = 3 per group, which may be underpowered. Increasing to n ≥ 5 would improve the reliability and statistical robustness of the conclusions.
5. While mtDNA is highlighted, other mtDAMP components—such as formyl peptides, TFAM, and cardiolipin—should also be discussed. If possible, other fractionation or quantitative assays (e.g., mass spectrometry) could help define the mtDAMP profile more clearly.
6. Inclusion of a schematic diagram that summarizes the mtDAMP–TREM-1–cGAS–STING pathway and its relationship to ALI would enhance the clarity and visual impact of the manuscript.

Minor Comments

1. Clarify the rationale for using 200 µg/mL mtDAMPs in vitro and in vivo—was this based on prior dose–response data or physiological relevance?
2. The Introduction and figure legends contain grammatical and stylistic errors.
3. Spell out “mitochondrial damage–associated molecular patterns (mtDAMPs)” at first mention in the Introduction section.
4. Figures appear crowded and inconsistently formatted; standardize layout, labeling, and scaling for better readability.

 

Comments on the Quality of English Language

 

 

Author Response

Authors  analyze  how ischemia–reperfusion–induced acute kidney injury (IR-AKI) cause acute lung injury (ALI), highlighting the role of mitochondrial damage–associated molecular patterns (mtDAMPs). Through murine models, proteomic profiling, and cell culture experiments, the study pinpoints TREM-1 and the cGAS–STING pathway as major mediators of the inter-organ inflammatory response. The research question is clinically appropriate, and the data are intriguing.

The following comments aim to further strengthen the manuscript:

 

Response: We greatly appreciate the time and expertise dedicated to improving the quality of our manuscript. Below, we provide a point-by-point response to each comment and outline the revisions we have made accordingly.

 

 

Major Comments

1. The animal model data are compelling, but the discussion should better address the potential translation to human settings. Are there existing or ongoing clinical studies exploring TREM-1 or cGAS–STING inhibition in AKI/ALI? Highlighting the therapeutic importance of dual targeting, these pathways could strengthen the translational impact.

 

Response: Both TREM-1 and cGAS-STING pathway are just gain attention recently, there are no existing or ongoing clinical studies focus on TREM-1 or cGAS-STING pathway in AKI/ALI. We highlighted the therapeutic importance of dual targeting the pathway (Line 450-451), and added more relevant content in the discussion section.

 

 

1. The evaluation of lung function relies on BALF analysis, W/D ratio, and flow cytometry. Adding histological lung injury scoring  would make the assessment more comprehensive and align it with standard ALI methodologies.

 

Response: The acute lung damage characterized by 1. W/D ratio change (edema); 2. Inflammation, 3 neutrophil infiltrations, 4. Lung tissue morphology change. Any three of them can determine the injury happened in the lung. We have presented the 1, 2, and 3 in the manuscript. The reason we did not show the histological lung injury score is that we only reperfusion the mice for 24 hours after IR-AKI surgery, we believe the time is not enough to develop the morphology changes. To strength our analysis, we added the neutrophil to macrophage ratios, as shifts in this balance are a well-established hallmark of acute lung injury. (Fig. 1F & Fig. 4D)

 

1. The use of AMJ2-C8 and MM14.LU cell lines raise concerns about physiological relevance. The inclusion of human-derived or primary cell models would improve the biological validity of the findings.

 

 

Response: The human derived or primary cell models sure could improve the biological validity of our findings. However, the TREM-1 only express on the myeloid cells-1, and to validity the lung damage, we will still need another human-derived primary cell line, it will rise the difficulty for cell co-culture, most of the primary cells using different culture media, and it happens that the two cell lines we picked use the similar media.

 

1. Regarding sample size and statistical power- some animal experiments appear to include n = 3 per group, which may be underpowered. Increasing to n ≥ 5 would improve the reliability and statistical robustness of the conclusions.

 

Response: The only reason of some assays we only used n=3 is because the sample limit, and most of the assays, such as Proteome frofiles and WB, we all double confirmed with real time - qPCR.

 

 

1. While mtDNA is highlighted, other mtDAMP components—such as formyl peptides, TFAM, and cardiolipin—should also be discussed. If possible, other fractionation or quantitative assays (e.g., mass spectrometry) could help define the mtDAMP profile more clearly.

 

Response: There are a lot of undefined content in the mtDAMPs, we won’t be able to identify all of them. For our project, we only focus on the TREM-1 and cGAS-STING pathway. So we added the relevant content such as HMGB1, HSP70 and eCIRP et al. to the discussion section. The mechanisms will be more clear to our project: TREM-1 been activated by HMGB1, HSP70, and cGAS-STING pathway been activated by mtDNA.  Line:423-432.

 

1. Inclusion of a schematic diagram that summarizes the mtDAMP–TREM-1–cGAS–STING pathway and its relationship to ALI would enhance the clarity and visual impact of the manuscript.

Response: Yes, that is true. And we just put the schematic diagram in the first beginning of our manuscript as a Graphical Abstract. We can change the graphical abstract to a figure in the manuscript if the journal does not need one.

Minor Comments

1. Clarify the rationale for using 200 µg/mL mtDAMPs in vitro and in vivo—was this based on prior dose–response data or physiological relevance?

 

Response: We added the rational for using 200 µg/mL in the discussion section. (Line: 456-458)

 

 

1. The Introduction and figure legends contain grammatical and stylistic errors.

 

Response: We corrected the grammatical and stylistic errors and label red.

 

1. Spell out “mitochondrial damage–associated molecular patterns (mtDAMPs)” at first mention in the Introduction section.

 

Response: We corrected that (Line: 67).

 

 

1. Figures appear crowded and inconsistently formatted; standardize layout, labeling, and scaling for better readability.

 

 

Response: We rearranged the figures to enlarge every single picture, make sure every image is clear to readers.

Reviewer 2 Report

Comments and Suggestions for Authors

The study attempts to explain the association of acute kidney injury with acute lung injury. mtDAMPs were shown as the main mediator in this study. The study is large, with a large number of results, but the text itself has flaws that must be corrected in order for the article to be published.

 

The introduction should introduce the reader to the topic. The paper itself in the title highlights the importance of the TERM 1 protein, as well as the activation of the cGAS-STING signaling pathway. However, both of these terms were not mentioned in the introductory part, and I consider them very important for understanding this topic and this study. The data and information provided in the introduction are of a general nature and should be more related to the paper itself and its specific topic.

In addition, in the "Introduction" section, it is necessary to add references in two places:

• At the end of the sentence in line 46, add references for the statement made.
• At the end of the sentence "Data from patients and animal models suggest that the pathophysiological mechanisms are complex and involve multiple pathways, but these mechanisms have not been fully clarified." Add reference.

 

The method materials are excellent and clearly written. The reader can follow the course of the experiment. The study is large and there is a lot of material. The statistics were done correctly and correctly.

 

The results are the best part of the article. I am positively surprised by the amount of data the authors presented, but the presentation of the results itself must be better. Due to the amount of data, the images and graphs are very small, and at high magnifications the quality of the image deteriorates.

Figure 1, split at least into two pictures. Black dots and triangles on parts A, B, C and E should be extinguished. I assume that these are the results of individual samples, but in this case they are unnecessary and create confusion in the picture.

Figure 2- Now we have white dots and triangles follow the instructions from the comment above.

 

Line 267 "TREM-1 is a pattern recognition receptor whose activation by DAMPs leads to the

release of pro-inflammatory cytokines and chemokines. Damaged mitochondria release mtDAMPs, including mtDNA, and the cGAS-STING signaling pathway is known to play a crucial role in mtDNA induced inflammation in sepsis-induced AKI [20,21]." This kind of narrative is more appropriate for the "Introduction" section.

 

Line 274 "Altogether, these results suggest that IR-AKI damages kidney mitochondria, resulting in the release of mtDAMPs to the lung, which subsequently activates the TREM-1 and cGAS-STING pathways, ultimately leading to acute lung injury." This kind of narrative is better suited to a "discussion" or "conclusion" section.

Figures 3 and 4 have a similar problem as the previous figures, the WB images are blurry.

 

The discussion is too short and does not explain the results obtained. It is imperative to expand the discussion and analyze the results obtained in this study with similar studies. The discussion serves to explain the results. Also, it should be emphasized what this study shows for the first time, why it is important for publication. My suggestion is to write the discussion from the beginning.

 

The paper should be considered after the revision. The results are excellent and provide a good basis for further research into the problems caused by AKI. The aspect is important not only for basic science but also has great potential for further clinical research.

Author Response

The study attempts to explain the association of acute kidney injury with acute lung injury. mtDAMPs were shown as the main mediator in this study. The study is large, with a large number of results, but the text itself has flaws that must be corrected in order for the article to be published.

Response: We greatly appreciate the time and expertise dedicated to improving the quality of our manuscript. Below, we provide a point-by-point response to each comment and outline the revisions we have made accordingly.

The introduction should introduce the reader to the topic. The paper itself in the title highlights the importance of the TERM 1 protein, as well as the activation of the cGAS-STING signaling pathway. However, both of these terms were not mentioned in the introductory part, and I consider them very important for understanding this topic and this study. The data and information provided in the introduction are of a general nature and should be more related to the paper itself and its specific topic.

Response: We appreciate the reviewer’s suggestion. We have added some background information regarding the TREM-1 and cGAS-STING signaling pathway in the introduction section. Line: 66-79.

 

In addition, in the "Introduction" section, it is necessary to add references in two places:

• At the end of the sentence in line 46, add references for the statement made.

 

Response: We have added the references at the end of the sentence in line 46.

 

• At the end of the sentence "Data from patients and animal models suggest that the pathophysiological mechanisms are complex and involve multiple pathways, but these mechanisms have not been fully clarified." Add reference.

 

Response: We have added the references at the end of the sentence in line 54.

 

The method materials are excellent and clearly written. The reader can follow the course of the experiment. The study is large and there is a lot of material. The statistics were done correctly and correctly.

 

The results are the best part of the article. I am positively surprised by the amount of data the authors presented, but the presentation of the results itself must be better. Due to the amount of data, the images and graphs are very small, and at high magnifications the quality of the image deteriorates.

Figure 1, split at least into two pictures. Black dots and triangles on parts A, B, C and E should be extinguished. I assume that these are the results of individual samples, but in this case they are unnecessary and create confusion in the picture.

Response: We rearranged the figures to enlarge every single picture, make sure every image is clear to readers. For the Bar-dot plot, every dot represented an individual data point, and we believe that it provides more information than a standard bar chart by showing the mean and variability together. We also formatted every figure to white dots for control and triangle for AKI.

Figure 2- Now we have white dots and triangles follow the instructions from the comment above.

Response: We formatted every figure to white dots for control and triangle for AKI.

 

Line 267 "TREM-1 is a pattern recognition receptor whose activation by DAMPs leads to the

release of pro-inflammatory cytokines and chemokines. Damaged mitochondria release mtDAMPs, including mtDNA, and the cGAS-STING signaling pathway is known to play a crucial role in mtDNA induced inflammation in sepsis-induced AKI [20,21]." This kind of narrative is more appropriate for the "Introduction" section.

Response: We have added the information to introduction section (Line 72-74).

 

Line 274 "Altogether, these results suggest that IR-AKI damages kidney mitochondria, resulting in the release of mtDAMPs to the lung, which subsequently activates the TREM-1 and cGAS-STING pathways, ultimately leading to acute lung injury." This kind of narrative is better suited to a "discussion" or "conclusion" section.

Response: We corrected that (Line: 467-469).

 

Figures 3 and 4 have a similar problem as the previous figures, the WB images are blurry.

 Response: We rearranged the figures to enlarge every single picture, make sure every image is clear to readers. The WB images are clear now.

The discussion is too short and does not explain the results obtained. It is imperative to expand the discussion and analyze the results obtained in this study with similar studies. The discussion serves to explain the results. Also, it should be emphasized what this study shows for the first time, why it is important for publication. My suggestion is to write the discussion from the beginning.

Response: We discussed more results in the discussion section as you suggested (Line: 426-436, line 454-469).

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors examined the acute lung injury associated with ischemia/reperfusion induced acute kidney injury and identified the release of mtDAMPs from kidney after I/R injury played a major role to induce lung inflammation and the subsequent injury. The association of lung injury with AKI is an important clinical problem while the mechanism is not well-understood. This study reported a new mechanism about this organ crosstalk with TREM-1 and cGAS-STING co-activation in lung mediated by macrophages, though there are still some concerns about the STING activation evidence, method description, and data analysis. 

(1) In Fig. 2B, the authors quantified mitochondrial damage from EM images. A detailed method description for this semi-quantification is needed. 

(2) In Fig. 3D, there are two bands in the STING blot. Which band is the specific band of STING? In addition, more evidence may be needed to support the cGAS-STING activation such as the examination of STING or TBK1 phosphorylation. 

(3) In Fig. 6J, they authors provided representative images showing the damage caused by mtDAMPs in a wound healing model. Some semi-quantification of the bronchus cell number or cell layer confluence may help to confirm the damage and healing speed difference. 

(4) In this study, the authors identified the activation of both TREM-1 and cGAS-STING pathways. While single pathway suppression only showed marginal protection of lung injury, the inhibition of both pathways showed much more significant effect. The potential mechanism of the collaboration or crosstalk between these two pathways is interesting and may be discussed.  

Author Response

In this study, the authors examined the acute lung injury associated with ischemia/reperfusion induced acute kidney injury and identified the release of mtDAMPs from kidney after I/R injury played a major role to induce lung inflammation and the subsequent injury. The association of lung injury with AKI is an important clinical problem while the mechanism is not well-understood. This study reported a new mechanism about this organ crosstalk with TREM-1 and cGAS-STING co-activation in lung mediated by macrophages, though there are still some concerns about the STING activation evidence, method description, and data analysis. 

 Response: Thanks for your valuable comments and suggestions to improve our manuscript.

 

(1) In Fig. 2B, the authors quantified mitochondrial damage from EM images. A detailed method description for this semi-quantification is needed. 

 Response: We have added the description in the method section. Line 145-150.

 

(2) In Fig. 3D, there are two bands in the STING blot. Which band is the specific band of STING? In addition, more evidence may be needed to support the cGAS-STING activation such as the examination of STING or TBK1 phosphorylation. 

 Response: The WB bands of STING antibody have different form in different cell line or tissue, both one band or two bands are normal. I have attached the picture of the venders website and the link to the antibody: (https://www.cellsignal.com/products/primary-antibodies/mouse-reactive-sting-pathway-antibody-sampler-kit/16029)

We appreciate the reviewer’s suggestion to examine the p-STING and TBK1, this project only focused on if the cGAS-STING pathway been activated or not. Even though we found out the cGAS-STING pathway contributed to the AKI-induced lung injury, there are still a lot of questions need to be answered, such as how cGAS-STING modulate the macrophage with TREM-1 together, what is the mechanisms of cGAS-STING pathway and TREM-1 co-inhibition improves the cell behaviors. We will measure all the cGAS-STING pathway downstream proteins and their phosphorylations by that time.

(3) In Fig. 6J, they authors provided representative images showing the damage caused by mtDAMPs in a wound healing model. Some semi-quantification of the bronchus cell number or cell layer confluence may help to confirm the damage and healing speed difference. 

 Response: Thank you for the thoughtful and constructive suggestion. Our cytotoxicity assay already demonstrated that, mtDAMPs were able to reduce 40% of the cell numble in the first 5 hrs, and continuing increase to 50%. That will also happen when we run migration assay.

(4) In this study, the authors identified the activation of both TREM-1 and cGAS-STING pathways. While single pathway suppression only showed marginal protection of lung injury, the inhibition of both pathways showed much more significant effect. The potential mechanism of the collaboration or crosstalk between these two pathways is interesting and may be discussed.  

Response: We added more discussion content in the discussion section and mentioned the potential mechanism of the collaboration between these two pathways(Line 426-436, 454-455, ).

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript by Tian et al. presents a well-designed experimental study investigating how AKI can cause lung damage through mitochondrial DAMPs that activate the TREM-1 and cGAS-STING pathways. I found the paper well written, methodologically detailed, and scientifically sound. I have only a few suggestions for improvement.

• Please add a clear research gap in the Introduction; explain briefly why TREM-1 and cGAS-STING were chosen.
• The mtDAMPs mixture should be described in more detail, including what components were present and how much mtDNA or protein was included.
• It’s not explained whether mtDAMPs directly activate TREM-1 and cGAS-STING or if other immune signals are involved. Some extra experiments or discussion would help support this claim.
• Several abbreviations in the abstract and the main text (TREM-1, DAMPs, HEPES, PVDF, dsDNA) are used before being defined.

Author Response

The manuscript by Tian et al. presents a well-designed experimental study investigating how AKI can cause lung damage through mitochondrial DAMPs that activate the TREM-1 and cGAS-STING pathways. I found the paper well written, methodologically detailed, and scientifically sound. I have only a few suggestions for improvement.

• Please add a clear research gap in the Introduction; explain briefly why TREM-1 and cGAS-STING were chosen.

 

Response: We would like to sincerely thank the reviewer for the thoughtful and constructive feedback. We added the background information regarding TREM1 and cGAS-STING in the Introduction section. Line: 67-79.

 

 

• The mtDAMPs mixture should be described in more detail, including what components were present and how much mtDNA or protein was included.

Response: We described the mtDAMPs mixture’s components in discussion section (Line: ). The concentration we used were determined using BCA protein Assay kit as we described in method section (Line: 187). The mtDAMPs we used for animal experiment were 200ug IP as descripted in line 313. For the cell experiment, the concentration of mtDAMPs were 200 ug/ml as labeled in the figures (Fig 5 & 6).

 

• It’s not explained whether mtDAMPs directly activate TREM-1 and cGAS-STING or if other immune signals are involved. Some extra experiments or discussion would help support this claim.

 

Response: We added the content in the discussion section as you suggested.Line:429-436, 446-447.

 

• Several abbreviations in the abstract and the main text (TREM-1, DAMPs, HEPES, PVDF, dsDNA) are used before being defined.

Response: We corrected this. TREM-1 (Line: 67); DAMPs(Line:65); HEPEs(Line:177); PVDF(Line:204); dsDNA(We deleted dsDNA, used mtDNA instead, Line 75)

Reviewer 5 Report

Comments and Suggestions for Authors

See PDF below.

Comments for author File: Comments.pdf

Author Response

The study is well designed, the experiments are comprehensive, and the findings provide novel mechanistic insights into kidney–lung crosstalk following IR-AKI. The data are generally clear and support the conclusions.

However, a few minor revisions are needed before acceptance.

 

Response: Thank you for the constructive and insightful feedback. We have carefully addressed each of your suggestions. 

 

 Experimental Details & Figures:

• Figure 1E: The authors present neutrophil infiltration as the percentage of neutrophils among total immune cells, which is appropriate for showing the relative abundance of neutrophils within the inflammatory compartment. However, this alone can be misleading in tissues such as the lung, where alveolar macrophages typically dominate under basal conditions. To strengthen the analysis, the authors should also report absolute neutrophil numbers and/or neutrophil-tomacrophage ratios, as shifts in this balance are a well-established hallmark of acute lung inflammation.

Response: We analyzed the neutrophil to macrophage ratio and added into figure 1 (Fig 1 f). 

 

• Figure 3A: The manuscript reports the use of a Proteome Profiler array to survey 40 cytokines, chemokines, and acute-phase proteins in lung tissue; however, the rationale for selecting these specific analytes is not provided. Please clarify whether this panel was chosen based on biological relevance to IR-AKI/ALI, prior literature, or simply reflects the contents of a commercial kit. For transparency and reproducibility, include a complete list of analytes (with spot positions), the exact panel and catalog number, protein input per membrane, number of biological replicates, and the quantification/normalization method (e.g., normalization to positive controls, background subtraction).

Response: The Proteome Profiler was performed using the commercially available Mouse Cytokine Array Kit Panel A Kit(R&D systems, Minneapolis, MN: https://www.rndsystems.com/products/proteome-profiler-mouse-cytokine-array-kit-panel-a_ary006) Line: 152. It detects 40 mouse cytokines, chemokines, and acute phase proteins simultaneously, as the IR-AKI model only reperfuse the mice for 24 hours, and the damage mainly came from the cytokines and chemokines.  I have added the catalog number to the manuscript (Line: 153). It is a very popular commercial kit with 430 citations on the website, so we skipped all that information including spot positions. We descripted the protein input per membrane as 100 ug of lysate (Line 156). We added the quantification method in Line 161-163.

 

• Figure 4B: To be consistent, use the same color as 1D.

Response: We sincerely sorry for the incontinence, the color of the follow cytometry generated by the machine, we did not know how to change it.

• Figure 4C: Same as Figure 1E, report absolute neutrophil numbers and/or neutrophil-tomacrophage ratios.

Response: We analyzed the neutrophil to macrophage ratio and added into figure 4 (Fig 4 D). 

 

• Figure 5: a. To be consistent, (i) show protein levels of A and B; (ii) show RNA levels of TREM-1, cGAS, and STING of MM14.LU+AMJ2-C8 first, then show WB results and quantification of WB. The figure is missing some of that data; (iii) 5E has the wrong label.

Response:  (i) We did not measure the protein levels of A and B. The image from Fig 5C, the first three bands are actually the protein level of A. We just don’t want the figures too complicate and focus on the cell co-culture.  (ii) The reason why we did not show the RNA levels of TREM-1, cGAS, and STING of MM14.LU + AMJ2-C8 is the fact that our data suggested that TREM-1 was significantly increased; however, the change of cGAS and STING signals were not significant. We have tested different time point, but it’s difficult to find the time point both RNA level and protein level increased simultaneously. (iii) We corrected that.

 

1. Overall, 400ug/ml concentration shows no dose-dependent effect, and/or in some cases, no effect at all. Either discuss it in the manuscript, or try a different concentration, or show it in the supplementary results; otherwise, it would be distracting.

Response:  We discussed in the discussion section (Line: 459-461).

14. The rationality of choosing MM14.LU, AMJ2-C8, and the combination of MM14.LU and AMJ2-C8 cells should be discussed, and the different phenotypes between these two cell lines should also be discussed.

Response:  We discussed in the discussion section (Line: 456-465).

 

• Figure 6: a. The rationality of choosing the combination of MM14.LU and AMJ2-C8 cells should be clarified. b. Some figures are fuzzy; for example, it’s impossible to read the y-axis of 6E, the adherent cell layer is difficult to see in 6J.

Response:  a. We discussed in the discussion section (Line:462-466 ). b. We rearranged the figures to enlarge every single picture, make sure every image is clear to readers.

 

 

Discussion section:

This part reads more like a mini-review than a focused analytical discussion. Expand the interpretation of the co-targeting findings, which appear novel but are under-discussed. There is no interpretation at all. For example, are there known connections between REM-1 and the cGAS-STING pathway? Is the effect synergistic or additive?

 

Response:  We rewrite the discussion section as you suggested. Line: 426-436, 454-455

 

 Detailed comments:

Line 42-46: If the authors provide more detailed numbers related to the “mortality” (line 42, 45), their statement would be stronger.

Response:  We added the detailed numbers in the manuscript (Line 45, 50).

 

 Line 47-51: The authors state that the mechanisms are “not fully clarified,” but then immediately describe a specific causal pathway. This is conceptually inconsistent, confusing, and should be revised for clarity. If the mechanism is indeed uncertain, the authors should use more cautious language (e.g., “is thought to be,” “has been proposed”) and clearly distinguish between established features and hypothesized mechanisms. This is addressed better in the discussion section (line 383).

Response:  Thank you for your suggestion, we corrected this as you suggested (Line 56).

 

Line 326: The manuscript has one more space before “(B)”.

Response:  We deleted the extra space.

 

Line 340: The manuscript has one more space before “and”.

Response:  We deleted the extra space.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have  addressed all the issues raised in the previous review and the manuscript has been  improved .Their responses are clear, detailed and appropriate.

I have no further comments. The manuscript is now suitable for publication in its present form.

Comments on the Quality of English Language

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have accepted the suggestions. I have no further comments. The article can be published.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have answered all my questions.