Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A WITHIN-SUBJECT COMPARISON OF DIFFERENT RELAXATION THERAPIES IN ELICITING PHYSIOLOGICAL AND PSYCHOLOGICAL CHANGES IN YOUNG WOMEN
Review round: 1
Reviewer: 3

Output:
Basic reporting: see below
Experimental design: see below
Validity of the findings: see below
Additional comments: This is an interesting trial that was carefully planned and well-conducted. I agree with the authors that the comparison of different relaxation interventions is indeed of great interest. A number of limitations apply, however, as outlined here:

1.	The first two sections of the introduction seem unnecesassary. The authors probably want to justify their use of a female-only sample, but conceptually, some of the arguments that are made are not well-founded (e.g., the two sexes do not necessarily face different sources of stress; they face the same sources, but they might respond differently, which is something completely different). Even if it were the case, then one would still expect the authors to include a male sample just to test those hypothesized sex differences. The second section deals with the benefits of relaxation in the context of pregancy and breast-feeding, but since this was not a topic of the current study, I really don´t see how this might justify the use of a female-only sample. I agree that relaxation techniques are relevant in these conditions, but that´s something that should be part of the discussion, not the introduction.

2.	In relation to issue number 1, the introduction should be used for providing a compelling rationale of the selection of the relaxation techniques (why these and why not others?) as well as the dependent variables. The latter is really important, because the reader is kind of suprised to learn about the main outcome variables only in the methods section; however, the authors need to introduce and justify their outcomes already in the introduction, based on sound theoretical reasoning.

3.	The main limitation is the lack of control of reproductive variables. The authors should have, at least, assessed cycle phases and controlled for a potential effect on the main outcome variables. Also, it is unclear whether the use of hormonal contraceptives was exclusionary as there is evidence that HC might influence some of the outcome variables.

4.	There is evidence that stress reactivity is lower in the morning compared to afternoon hours (e.g. cortisol). The time window from 9am to 1pm seems quite broad, particularly given the fact that there is a spike in cortisol at noon, and that having eaten lunch might affect relaxation parameters. It is doubtful, therefore, that participants examined in the morning can be compared to those examined at lunch hours. Also, I was wondering whether the same time was held constant within-person (e.g. the same person always coming to the lab at 9am).

5.	Half of the participants „reported regularly using a form of relaxation“ (line 230). This is usually an exclusionary criterion, so I was wondering why the authors allowed for this variable. Did those individuals differ from the ones not practicing relaxation techniques?

6.	The authors should adhere to standard CONSORT reporting for trials, as it is important to know the flow of recruiting, inclusion/exclusion of participants, drop-outs, etc.

7.	A couple of smaller issues: a) the abstract states that all interventions increased PR, whereas in the discussion it is stated that all interventions, except RL and control, increased PR; the authors should check for inconsistencies between abstract and manuscript; b) the authors state (line 90f.) that the use of psychotherapy in this population is challenging, but do not provide a reason for this statement; c) throughout the manuscript, „interventions“ should be used instead of „therapies“; „relaxation techniques“ would be even better, as this adequately describes the interventions used in this study; d) the sample size is relatively small; an a priori power analysis is usually done for all main outcome variables, not just one (HR in this case); also, I was wondering about the (clinical) relevance of 0.75 mean difference in HR; the authors should better justify their power analysis – or call their study a „pilot study“; e) importantly, all analyses need to be controlled for multiple comparisons, resulting in p level adjustment; this might lead to some findings not being significant anymore; f) three out of four relaxation techniques were auditory-based; the authors should discuss whether this might affect the physiological pathways they are assuming, and whether and how it is ok to compare these techniques to light exposure which is something completely different.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A WITHIN-SUBJECT COMPARISON OF DIFFERENT RELAXATION THERAPIES IN ELICITING PHYSIOLOGICAL AND PSYCHOLOGICAL CHANGES IN YOUNG WOMEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I appreciate your making the changes I suggested. It is a better article, more able to be understood and implemented by other clinicians.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A WITHIN-SUBJECT COMPARISON OF DIFFERENT RELAXATION THERAPIES IN ELICITING PHYSIOLOGICAL AND PSYCHOLOGICAL CHANGES IN YOUNG WOMEN
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments
I do not have any further particular concerns to express about the manuscript. Authors addressed sufficiently all points raised by the three reviewers.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SMRT SEQUENCING OF THE FULL-LENGTH TRANSCRIPTOME OF THE RHYNCHOPHORUS FERRUGINEUS (COLEOPTERA: CURCULIONIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article could use some editing, some sentences need to be improved for clarity, such as lines 157-158, and also some vague, yet over-promissing sentences (e.g. 77-79) need rephrasing. The English reads clearly as non-native, but is serviceable with minor editing.
Experimental design: The Methods section need major changes, and some parts of the methodology is not clearly written or just absent. This is a major issue for any attempt of reproducing the results, and also does not clearly describes what was done.

Beginning at line 123, the sequencing section of the methodology describes how the authors extracted the DNA and the PacBio sequencing, but it should also include which sequencing kit was utilized, and which generation of PacBio machine was used. It matters as it informs what fragment size could have been obtained, and the amount of data generated. The authors mention more than once that they used Illumina sequencing technology to check for sequencing errors, and it is a good and safe thing to do. But the authors never mention how much data they generated for Illumina reads, what sequencing machine and kit were used or any details whatsoever. This is a major flaw that needs correction. I could find some of this information by accessing the raw data NCBI link, but it needs to be on the manuscript, specially since not all details are sent to NCBI.

Some other methods were very vague and unclear. The “Functional annotation of transcripts” (line 149), and “Identification of TFs and lncRNAs” (line 162) only list the databases used in these steps, but fail to mention what exactly was done. The functional annotation, for example, only describes all major databases and vaguely mention they were used (line 150) to “annotate”  the transcripts function, but never tells the method, programs, and parameters used. This is not only bad for the reader if they want to reproduce such steps, but also completely non-descriptive of whatever was done. I believe this is unacceptable in a manuscript.

The CDS prediction (lines 157-159) are confusing and make no sense. They should be rewritten.

On line 171 (AS analysis) the authors mention “De Bruijn diagram” instead of  “De Bruijn graph”, which is mathematically not the same thing and should be corrected.

In the Results section (line 187) the authors mention again the Illumina data, and point to Table 1, which is very short and not really informative. Table 1 should either be expanded or just written down in text form since it’s two lines only. It also contain a typo.
Validity of the findings: This is a very straightforward manuscript that offers to the community new transcriptome data for a species that doesn’t have an assembled genome yet. While it is a description paper, I would like to see certain results further discussed in the Discussion section. For example, the LncRNA prediction has its amount described, but not really explored or explained in the Discussion.

I understand this is mostly a preliminary report, or a descriptive paper, but I would like a further exploration of the data acquired by the authors, perhaps a comparison with other Coleoptera transcriptome data currently available could enrich the discussion section.
Additional comments: This is a short, descriptive, paper that provides the community with a new, useful, dataset. I appreciate the effort into publishing a manuscript with mostly new data that will serve the scientific community. I just wish the authors would put a little extra effort into making the methods more clear and detailed for the reader and perhaps discuss the findings in light of other available datasets.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SMRT SEQUENCING OF THE FULL-LENGTH TRANSCRIPTOME OF THE RHYNCHOPHORUS FERRUGINEUS (COLEOPTERA: CURCULIONIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article should be rewriten. Several language mistakes, lack of proper punctuation, even some sentences starting with a nonsense word in lowercase.
Weak reference library.
The author was more committed to reinforce the advantages of third generation sequencing  than exploring his hypothesis. In fact, the hypothesis only appears in the introduction.
The figures and tables legends were poorly written.
They should compare and discuss about another Colleoptera transcriptomes, argue and convince the reader that his hypothesis was corroborated by the results.
There is no Discussion. The author repeated some results and joint some occasional citations.
How could this transcriptome help the pest control, after all?
Experimental design: The experimental design was clear, but the Methods were too briefly described.
Too much abbreviations. You should always write the entire name and then the abbreviation, between parentheses, in the very first time it appears in the text. When you have so many names do abbreviate, you may even write a brief glossary.
Several tools were used, but they were weakly described. It is important always describe "why" you chose that tool, how this tool benefits your strategy. If you just cite one tool after another, the reader will not trust your methods.
Validity of the findings: There is a lot of results, and they could generate a good article, but they are immature. It is very important to have the extra information that the author describes, as transcription factors, lncRNA, but it is necessary to connect each other, show the consistency of the results as a whole.
A lot was said about the advantages of third generation sequencing strategy.
Most transcriptomes published up to now were assembled using second generation sequencing and complementary sequencing strategies to achieve the best results. Each sequencing technology has its benefits and disadvantages. The strategy is not better because a specific sequencing technology was adopted, it is better when the strategy that you adopted generated the results you were expecting. It would be advisable to call attention to the complemented long reads with the short reads, the use of short reads to adjust the high error rate of the long reads.
The full-length transcriptome described should be compared to some Arthropoda short reads assembled transcriptomes and another hybrid strategies, to show to the readers the evidence that the long reads full-length transcriptome could be advantageous, indeed.
Additional comments: Invest more in consistency. If you have some nice interesting new methodoly, describe it and show how it was important to achieve the results. In the results, show how they connect to each other. The results should tell the reader a story. The discussion must go back to the initial hypothesis, it is the moment when you stop telling the story and start talking about the repercussions, negative and positive insights, convince the reader that the content of you story was really interesting and reliable.
After all, how this results elucidated the metabolic resilience of this weevil in order to help future studies concerning red palm management and pest control?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SMRT SEQUENCING OF THE FULL-LENGTH TRANSCRIPTOME OF THE RHYNCHOPHORUS FERRUGINEUS (COLEOPTERA: CURCULIONIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I reviewed an updated version of the manuscript after the first reviewing round. The manuscript was much improved by the authors, who addressed all the points raised in the first review. The authors explored more the dataset and provided all the information that was asked.
Experimental design: The authors expanded the methodology text, adding much needed details for all steps. It is now much clearer to understand and follow.
Validity of the findings: As mentioned in the previous revision, this is a descriptive paper, which aims mostly to provide the community an interesting and valid dataset, and the authors describe this novel data in a simple, straightforward way.
Additional comments: This short, straightforward, paper got much improved after the first revision round. The methods and results are much better explained, and the authors address all questions raised by the previous review. I think this manuscript provides novel data that will be much appreciate and useful to the specialists in the field.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SMRT SEQUENCING OF THE FULL-LENGTH TRANSCRIPTOME OF THE RHYNCHOPHORUS FERRUGINEUS (COLEOPTERA: CURCULIONIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: The paper was properly re-written. Good language and clear sentences. Improved and consistent bibliographic references.
Experimental design: Improved and sufficiently rewritten. The experimental design was clearly described as in the fisrt version, but now it is sufficiently described.
Validity of the findings: Important results and much better exploration of them. The authors efficiently synthesized the results obtained.
Additional comments: I congratulate the authors for the excelent revision. It is clear that the authors were dedicated to improve this importante paper.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Global protein expression changes induced by pamidronate in RAW 264.7 cells as determined by IP-HPLC” by Lee et al analyzed protein expression changes with IP-HPLC technology in RAW 264.7 cells treated with pamidronate. The authors showed that pamidronate treatment induced RARP-1 and FAS-mediated apoptosis signaling, while suppressed inflammation, cellular differentiation, survival, angiogenesis, and osteoclast genesis signaling. The manuscript was properly written. Figure quality is ok.  But the manuscript still needs a minor revision before it can be accepted for publication.

Though the authors have utilized the technology for several times, how can the authors make sure that the 218 antibodies are all specific? The authors should at least explain in the manuscript.

Second, the authors only used 218 antigens to do IP, so this is not a true global protein expression change, the authors should change the manuscript title to a much proper way.
Experimental design: Experimental design is ok.
Validity of the findings: I noticed that the authors had published another paper on Scientific Reports in 2018, investigating protein expression changes of RAW264.7 treated with dialyzed coffee extract. So, what is the specificity of RAW264.7 cells treated with pamidronate compared with dialyzed coffee extract? It is better the authors compare these two treatments in this manuscript.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Introduction section does not provide accurate background information. Moreover, the authors' statements are not supported by adequate citations / references. The reviewer provides examples of statements made by the authors which need to be revised:

1) The following statement is inaccurate: “Bisphosphonates inhibit the digestion of bone by causing osteoclasts to undergo apoptosis and thereby inhibit bone loss”
-BPs antiresorptive actions are not limited to induction of osteoclast apoptosis. BPs impair osteoclasts’ ability to form a ruffled border, to adhere to the bone surface, and to synthesize protons necessary for bone resorption. Furthermore, BPs suppress osteoclast activity by decreasing osteoclast progenitor development and recruitment.

2) The following statement is inaccurate: “In osteoporosis and Paget's disease, the most popular first-line bisphosphonates are alendronate and risedronate, but when they are ineffective or digestive tract problems develop, intravenous pamidronate may be used”
-IV zoledronic acid is the first-choice treatment for Paget disease because of its efficacy and ease of administration. The choice of zoledronic acid as the initial agent for most patients with active Paget disease is consistent with both the 2014 clinical practice guidelines of the Endocrine Society and the 2019 Paget's Association guidelines.

3) The following statement is inaccurate: “They also change bone ultrastructures, e.g., they obliterate haversian canaliculi and deposit irregular and thick reversal lines”
-BPs have been shown to obliterate haversian canals, not haversian canaliculi.

4) The authors make the statement, “In addition, bisphosphonate-related osteonecrosis of the jaw (BRONJ) may develop in patients who have used bisphosphonates long term (Marx et al. 2005; Ruggiero et al. 2004)”
 -The authors need to address the incidence of BRONJ in patients being administered oral bisphosphonates versus IV bisphosphonates. BP route of administration critically influences the incidence of BRONJ.
-The authors should cite the specific research studies which have shown that long-term use of bisphosphonates is a risk factor for BRONJ.

5) The following statement is inaccurate: “BRONJ is a microbial infection that occurs after dental extraction or surgery and constitutes a severe complication of suppurative and necrotizing osteomyelitis in jaws (Chirappapha et al. 2017; Choi et al. 2017; Park et al. 2009)”
-The pathophysiology of BRONJ is currently unclear. BRONJ has been attributed to infection, suppressed bone turnover, vascularity, genetic predisposition, etc.
-BRONJ is not always caused by dentoalveolar surgery. BRONJ can occur spontaneously.
-The current evidence does not support the view that BRONJ is a complication of suppurative and necrotizing osteomyelitis in jaws.

6) The authors make the statement, “Pamidronate (pamidronate disodium or pamidronate disodium pentahydrate) is a nitrogen-containing bisphosphonate and used to prevent bone loss due to steroid use or to treat certain cancers with propensities for bone, such as multiple myeloma”
-The reviewer is unaware that pamidronate is used to treat steroid-induced bone loss. Please cite / reference the society guidelines demonstrating this clinical use.

7) The following statement is inaccurate: “Due to its ability to sequester calcium in bone, pamidronate is frequently used to treat high calcium levels”
-BPs do not sequester calcium in bone. BPs inhibit calcium release from bone by impairing osteoclast-mediated bone resorption.

8) The authors make the statement, “Furthermore, multiple trials have shown that IP-HPLC can be used to rapidly determine multiple protein levels accurately (< ±5% standard deviation) and reproducibly.”
-The authors need to provide citations / references supporting this statement.

9) The authors make the statement, “When pamidronate is injected into blood vessels, it is immediately neutralized by cationic molecules like albumin and calcium and engulfed by macrophages, which suggests its various pharmacologic effects may be associated with the cellular functions of pamidronate-laden macrophages.”
-The reviewer is not aware that when pamidronate is injected into blood vessels, it is neutralized by albumin and calcium and engulfed by macrophages. Please provide citations / references supporting this statement.
Experimental design: The reviewer does not feel that the statistical analyses are acceptable, which is explained below.

1) Based on the authors' explanation that the data is expressed as proportions / percentages, the control cultures will always = 100% and therefore will not accurately reflect standard deviation.
2) The authors explain that analyses were repeated two to six times until standard deviations were ≤±5%. Experiments should have been repeated at least three times in order to have n-values = 3 per group.
3) The chi-square test is not an appropriate statistical test. Chi-square test is used when evaluating the relationship between two categorical variables (i.e., race, sex, age group, educational level). The study does not evaluate categorical variables.

The data should be re-analyzed applying appropriate statistical analyses methods.
Validity of the findings: The reviewer cannot evaluate the validity of the findings since the data were not analyzed appropriately.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 1
Reviewer: 3

Output:
Basic reporting: The balance between the process of osteoblasts and osteoclasts is critical for maintaining the integrity of bone mass. Earlier studies demonstrate bisphosphonates potential to inhibit osteoclasts and suppressing proinflammatory cytokines. In this manuscript, author have investigated the effect of Pamidronate on the expression of multiple genes involved in range of cellular pathways. This manuscript is well conceptualized and written, data extracted through IP-HPLC method further signifies the quantitative assessment of biological fluids to obtain multiple readouts.
Experimental design: This is very compressive study, still authors need to conduct some additional experiments to improve the outcomes of this study. Some examples where the authors could provide more insight includes-
1.	Figure 1- It is better to provide DIC images without colour background to observe the cell morphology, also include scale bar.
2.	Figure 1- Authors should provide the complete dose-time response analysis to validate 6.5 μM concentration was best dose for this study. Given that most of the protein induction was observed at 48 h of treatment, a dose-response analysis needs to be included.
3.	For cell proliferation assay authors should provide some additional biochemical based studies.
Validity of the findings: Figure 2 & 5- results shown here an increase in expression of tumor suppressor p53 and pro-apoptotic protein suggesting more cell death, however till 24 h more proliferation observed, please explain these opposite observation.
Additional comments: This study will highlight the potential of Pamidronate in regulating the expression of multiple pathways through tuning the expression of respective proteins. Still authors need to add some more data that will further enhance the outcomes of this manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: 1) In response to the reviewer's question "Based on the authors' explanation that the data is expressed as proportions / percentages, the control cultures will always = 100% and therefore will not accurately reflect standard deviation", the authors responded "The proportional data of IP-HPLC need to do multiple repeats to compare different assay series. In data sheet, the standard deviations of control assays were not listed to get proper graph plot, but calculated simultaneously".
-The reviewer recommends that the authors include the standard deviations of control assays. All underlying data should be provided.

2) In response to the reviewer's question "The chi-square test is not an appropriate statistical test. Chi-square test is used when evaluating the relationship between two categorical variables (i.e., race, sex, age group, educational level). The study does not evaluate categorical variables", the authors responded "In this study, every protein assay is independent with each other, and more, the repeated rate of each protein assay is relatively low, only 39.9% of protein assays is adequate for n-value. However, the protein assays showed standard error, √(σ^2/n). Therefore, it is agreed that the great variability of protein expression data is inadequate for statistical analysis of Chi-square test and probability (p-value) t-test, but still can be simply evaluated by standard error of the mean in this study. The data of protein expression levels were reanalyzed by standard error concept."
-The authors revised the manuscript text from "Results were analyzed using the Chi-squared test" to "Results were analyzed by measuring standard error (s=±√(σ^2/n))", but it does not appear that the data were reanalyzed since none of the study outcomes changed. Furthermore, it is unclear to the reviewer how results can be analyzed by standard error.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: EXTENSIVE PROTEIN EXPRESSION CHANGES INDUCED BY PAMIDRONATE IN RAW 264.7 CELLS AS DETERMINED BY IP-HPLC
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors have improved the text and the overall quality of the article.
Experimental design: Authors have included in situ proliferation data, explain rational to select 48 h of treatment for most of the studies.
Validity of the findings: Authors have explained in the text regarding RAW264.7 response to pamidronate for proliferation and apoptotic instigation is based on expression of p53 and Fas associated genes.
Additional comments: This revised manuscript is very much improved, and authors have added some contents according to reviewers' comments. I have no further comment.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON BETWEEN RADIOFREQUENCY ABLATION AND SUBLOBAR RESECTIONS FOR THE THERAPY OF STAGE I NON-SMALL CELL LUNG CANCER: A META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Major language editing is necessary.

ABSTRACT
line 12 This research aims to compare SLR and RFA for treatment of stage I NSCLC, applying the...
line 32 And There were significant statistical differences... (one-, three- and five-year)... (one- and three-year). [three-year or three years survival]
line 34 the postoperative complications (hemoptysis, ...) are in foavour of SLR...

INTRODUCTION
line 55 scientists and physician (or medical doctor)

line 57 Though, surgical treatment provides patients with the best chance of total cure and long-term survival. This sentence is not clear. I suggest to cancel it, adding "offering the best chance of cure" at the end of the second sentence.

The cornerstone of therapy in NSCLC patients is surgical resection and the “gold standard” surgical approach for stage I NSCLC is lobectomy with systematic mediastinal lymphadenectomy,  offering the best chance of cure

line 63 are often old (not:predisposed to advanced age), and affected by atherosclerotic cardiovascular disease, pulmonary dysfunction and other  comorbidities related with cigarette smoking.

Line 86 Sub-lobar resection (SLR), also known to as limited resection, is preferred for
69 patients that cannot tolerate lobectomy with systematic mediastinal
70 lymphadenectomy specifically to preserve pulmonary function. SLR can be
71 conducted by anatomical segmentectomy or non-anatomical wedge resection, through an open thoracotomy or with a VATS approach

Line 73 placement of an electrode into the lesion and generation of high-dose energy to cause
74(not: tumor to undergo) coagulation necrosis (not:and cell death)[
Fernando’s[9] study
75 showed that RFA is a choice for the patients with 76 stage I-II non-small cell lung cancer, who are not suitable for operation. RFA can also be used for local control of
77 peripheral lesion in patients with more advanced stage  in combination with
78 other treatment.

NOT: With the aim of exploring whether RFA can get the survival comparable to 84 that by SLR to stage I non-small cell lung cancer patients,
YES: we performed this research,
85 based on patients with stage I non-small lung cancer who underwent SLR or RFA to
86 evaluate both the postoperative complication and survival rates.

MATHERIALS AND METHOD
line 103 (1) Comparison of the survival situation and post-operative complication rates of SLR and RFA in patients with stage I NSCLC
104

105 (2) Articles that contain the survival data of the reports that presentations presented (?) at major
106 radiology and thoracic surgery academic conference (RSNA, AATS and EACTS) or
107 study published in peer-reviewed publications;

126 standard. the (you have already said that they are two) investigators also

RESULTS
I suggest to describe firstly Study characteristics, secondly Assessment of complication, and then assessment of survival.
I suggest to use this plot to develope the DISCUSSION

 DISCUSSION

The application of less invasive treatment for
217 NSCLC is especially important given that only 20% of NSCLC are suitable for
218 lobectomy and some less invasive approaches are currently being developed for
219 patients with early-stage NSCLC whose basic conditions preclude them from being
220 cured in accordance with the standard of care.

I suggest to semplify the above mentioned sentence.: considering that only 20% of NSCLC patients are suitable for lobectomy due to poor clinical conditions, some less invasive approaches are currently being developed: RFA and SLR
NOT: The choice of a modality relays more on the 222 theoretical knowledge and personal custom of the doctor than on the proof.  In the next page you say that many authors reserved RFA for patients that are deemed medically inoperable. I don't think that the treatment choise depends on personal custom of physiscians. I shouldn''t be.
.
I suggest to briefly describe the advantages and disadvantages of each procedure

...RFA is a percutaneous treatment performed with local anaestesia and conscious sedation

NOT (you have already said that): RFA induces heat denaturation of cellular proteins causeing tumor cell death, which 224 is used to provide palliation and could achieve excellent local control for patients
225 with inoperable NSCLC[24, 25].Those who advocate RFA stress it present some 226 definitive advantages over surgery.

RFA can be conducted percutaneously, avoiding 227 a thoracotomy for those who refuse surgical resection or with severe co-morbidities.
The RFA has less influence on the cardio-pulmonary function and  lower cardiac abnormality rate. it’s also
229 associated with a significant decrease in length of stay
NOT:  our results also reveal the 230 RFA group have a lower cardiac abnormality rate

Another   feature of RFA
231 is that it allows ablation of lesions without major damage to peripheral normal 232 tissues[26].
The complications of RFA were relatively minor.
 I suggest to briefly descibe the most frequent complications and the mean post-operative stay reported in the 5 reports you analysed, adding FIGURE 4-citation here
(I suggest to compare survival rates of the 5 reports, later)

This topic is unnecessary, remove it: Hu and associates initial explored the mechanism of RFA for 242 the treatment of primary pulmonary malignancy and lung metastasis from liver 243 cancer: upregulating tumor suppressor miRNAs (let-7a and miR-34a) and
244 downregulating onco-miRNAs (miR-21)[31].

...SLRs require a general anaeshesia. (this fact can be a disadvantage because many patients can tolerate a general anaesthesia) They can be performed through a thoracotomy or with a Video assisted thoracoscopic surgery (recently several authors reported different VATS techiniques to localise lung toumors avoiding the necessity of open thoracotomy [Surg Endosc. 2012 Apr;26(4):914-9. doi: 10.1007/s00464-011-1967-8 ] [J Thorac Cardiovasc Surg. 2016 Aug;152(2):535-544.e2. doi: 10.1016/j.jtcvs.2016.04.052 ] [Clin Radiol. 2017 Oct;72(10):898.e7-898.e11. doi: 10.1016/j.crad.2017.05.015 ][Radiol Med, 125 (1), 24-30  Jan 2020 DOI: 10.1007/s11547-019-01077-x]
SLR is associated with higher complication rate in respect to RFA..

after discussing about complications, I suggest to analyse survival and disease-free interval rates of RFA and SLR, trying to explain the reasons (example: 1 different stage clinical for RFA and pathologiacl for SLR; 2 incomplete toumor ablation for RFA; 3 patients medical conditions ..)

This part is excessive and redundant. I suggest to simplify it underlining that RFA can not assure the complete toumor ablation, while surgival resection can provide limph node staging.

REMOVE THIS: Those who are against the RFA stress that SLR could provide better oncologic 246 outcomes in respect to RFA. One of the key deficiencies for RFA is the difficulty in 247 achieving thoroughly lesion destruction[32]. As the expected temperature for 248 treatment cannot be completely achieved throughout the lesion due to periphery of 249 the lesion is far away from the core of ablation. On the contrary, insufficient RFA 250 can induce the growth of non-small cell lung
 ancer via up-regulating HIF-1α 251 through the PI3K/AKT signals[33].
Experimental design: the authors clearly descibe the aim of the study . the issue is relevant and interesting

the literature review apperared performed in a rigorous way, with high ethical standard
Validity of the findings: interesting finding, supported by the results of the study
Additional comments: major language editing is necessary


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON BETWEEN RADIOFREQUENCY ABLATION AND SUBLOBAR RESECTIONS FOR THE THERAPY OF STAGE I NON-SMALL CELL LUNG CANCER: A META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.    The English language should be improved throughout the manuscript to ensure that an international audience can clearly understand your text. Grammatical mistakes such as preposition usage (Line20: “is aimed at assessing” not “is aimed to assessing”), misuse of pronoun reference (Line62, 297: “patients who are” not “patients which are”), confusing uses of articles (Line71-72: “RFA is a minimally” not “The RFA is a minimally”), as well as singular and plural errors (Line192: “Assessment of complications” not “Assessment of complication”) should all be rectified.
2.    References should be added to Table 1, Line76-78, Line195-204, and Line 269.
3.    Line267-269: The language should be improved as the current phrasing makes comprehension difficult.
4.    Line 269-274: I suggest that you move these analyses of bias to the “limitations” part also in the “Discussion” section.
5.    Line 291: “randomized clinical trials (RCTs)” not “RTC”
6.    Figure 1 showing the process of study selection needs more detail. I suggest you provide reasons together with the number of articles in the exclusion process.
7.    Table 1 presented with basic characteristics of patients’ needs improvement. I suggest that you list the characteristics of each group of patients in single rows and remember to provide a consistent display of numbers among different studies (For example: "median ± SD" for Age). Descriptive data such as tumor sizes, indicators of patients' clinical condition (ECOG, FEV1 or FEV1%), and numbers of patients who have received adjuvant treatments could also be added to the table.
Experimental design: 1.    Your most important issue is that the extent of operation for patients in the surgery group was “lobectomy or pneumonectomy” in one of your included studies (by Kim et al) which did not meet the including criteria. You have to exclude this study from your work.
2.    Line 104: I suggest that you should mention the system you chose to identify the stage of included patients.
3.    Line110-111: For the selection of clinical outcomes included in your study, I have the following suggestions. First, I suggest that both disease-free survival (DFS) and progression-free survival should be included since RFA probably cannot eliminate tumors. Second, "distant metastasis" may be considered together with the "local recurrence" when evaluating the efficacy of treatments, both of which were involved in the study by Ambrogi et al. The number of patients you extracted for meta-analysis was also the sum of the two cases. Please reconfirm the data included in the meta-analysis.
4.    Line134-136: Disease-free survival seems to be a concept used to describe the period after a successful treatment during which there are no signs and symptoms of the disease that was treated.
5.    Line166-191: As far as I know, most of the included studies in your work provided with survival rates or survival curves. Some studies even did not present with specific survival data of the year that you have chosen in your meta-analysis. Please explain in the “Methods” section how you extracted the number of patients of the year of one, three, and five for analysis. Besides, please reconfirm the accuracy of the data in your meta-analyses.
6.    Line 174-175: Heterogeneity was classified into grades with low, medium and high in your study. You should better use these terms instead of "some".
Validity of the findings: The studies included by the authors on this issue were comprehensive but less representative. One of the studies included by the authors is not suitable for the study. Some of the data extracted by the authors for analysis seemed to be less accurate. Both of the two reasons result in the final results of this meta-analyses to be less robust or reliable.
Additional comments: The authors focused on comparisons on efficacy between RFA and sub-lobar resection in stage I NSCLC patients. The main objective of this manuscript has been straightforwardly achieved. This Reviewer believes that this manuscript could be of relevance to clinicians in this field. Nevertheless, there are still several major points to be justified and some minor points to be corrected.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON BETWEEN RADIOFREQUENCY ABLATION AND SUBLOBAR RESECTIONS FOR THE THERAPY OF STAGE I NON-SMALL CELL LUNG CANCER: A META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This meta-analysis has performed systematic review of literature which compared the prognosis of RFA and that of SLR in NCLC patients in stage I. This study has clinical value and the results are promising. However, there are still some issues should be addressed.
1.	Besides the treatment methods, what about the other risk factors influencing the outcome of patients with NCLC? What about the proportions of these risk factors in patients of your included researches?
2.	It’s all known that the therapeutic effect of RFA is closely related to tumor size, so what is the tumor size of the tumors included in the study? Was there any difference between the two groups? These should be analyzed and discussed.
3.	Minor suggestion for the “Abstract” section, “In cases of stage I NSCLC, surgical resection is preferred due to its higher survival rates and disease control as opposed to radiofrequency ablation. The RFA option could be considered a valid alternative for inoperable and high-risk patients, due to its short hospital stay and low invasiveness” Should be the content of “conclusions”, but not “results”.
4.	When discussing the mechanism of RFA, the authors described that it’s “the upregulating rumor suppressor miRNAs and downregulating onco-miRNAs (Lines 241-244). However, we all known that the main mechanism of RFA for treating tumors should be thermally induced tumor cell death, and the molecular mechanism mentioned above should occur in areas where the temperature is not high enough to kill cells. Therefore, this argument tends to confuse readers.
5.	Some grammar mistakes need serious correction, for example, “There are two different minimally invasive approaches, sub-lobar resection (SLR) and radiofrequency ablation (RFA), which are performed for stage I non-small cell lung cancer treatment” (Lines 18-20).
6.	Full text should be checked and corrected for spelling errors, for example, “RAF” (Line 226).
7.	In Lines 417-418, the figure legend of Figure 3 should be “…disease free survival rate…”, but not “…disease survival rate…”.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON BETWEEN RADIOFREQUENCY ABLATION AND SUBLOBAR RESECTIONS FOR THE THERAPY OF STAGE I NON-SMALL CELL LUNG CANCER: A META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article was well-written, but there are still some minor points to be improved:
1. In lines 97 and 189, it was written "adapted". I think they were typos and the intention was to write "adopted".
2. There're some grammar errors in the manuscript, so the English writing should be polished further.
Experimental design: The research question was novel and well defined. Analyses were performed with a high technical and ethical standard.
Validity of the findings: The results were presented in a convincing and rigorous manner. Limitations of this work were also fully discussed by the authors.
Additional comments: This work investigated the optimal choice from RFA and sub-lobar resection in stage I NSCLC patients, which would be an interesting topic for thoracic surgeons. The article was well written, and the results of the meta-analyses were presented in a convincing and rigorous manner. I have only minor comments to make.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INSIGHTS ON EARLY MUTATIONAL EVENTS IN SARS-COV-2 VIRUS REVEAL FOUNDER EFFECTS ACROSS GEOGRAPHICAL REGIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The ‘Basic Reporting’ features from PeerJ are generally well met. The writing does includes more than the usual number of typos or poorly constructed sentences. For example, the sentence starting on line 262 needs revision (“One interesting feature of the mutations in this limited number of samples is the evolutionary pressure undergone helicase gene.”). Also see line 266 (“…making it unfeasible to drug this protein…”)
Experimental design: The experimental design features from PeerJ appear to be met.
Validity of the findings: The validity of findings features from PeerJ also appear to be met.
Additional comments: The submission by Farkas et al. (Insights on early mutational events…) is a straightforward report on the point substitutions found in an early set of SARS-CoV-2 virus genomes downloaded from public databases. The main findings are that, within their sampling scheme, (1) a set of point mutations, with high allelic frequencies, distinguish viruses from Washington state in the US relative to the first genomes from China, (2) a set of Australian virus genomes also showed clonal mutations but these were more heterogeneous, and (3) some of the point substitutions occurred in genome locations targeted by PCR primers. They warn that PCR primers will need continual evaluation to provide reliable tests for infection.
	Line 63 states, “…it is likely that pangolin species are a natural reservoir…” That is not my reading of the recent studies. I’d refer the authors to Zhang and Holmes 2020.  Cell; https://doi.org/10.1016/j.cell.2020.03.035 and references therein, which suggest that pangolins are better characterized as a possible intermediate host in transmission of coronaviruses between bats and humans. Bats are better characterized as natural hosts for coronaviruses. Pangolins populations (endangered) have not been extensively evaluated for coronaviruses.
	The interpretation of the findings are limited and reasonable, though I don’t think the existence of point mutations alone constitutes “evolutionary pressure” (line 263), which usually connotes directional selection. If using the term the authors would need to show that the changes are not random in their distribution and overall population effects.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INSIGHTS ON EARLY MUTATIONAL EVENTS IN SARS-COV-2 VIRUS REVEAL FOUNDER EFFECTS ACROSS GEOGRAPHICAL REGIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The Basic reporting of the study is correct but please see "General comments for the author".
Experimental design: The Experimental design of the study is correct but please see "General comments for the author".
Validity of the findings: The Validity of the findings of the study is correct but please see "General comments for the author".
Additional comments: This study investigates the presence of founder effects in the current expansion of the SARS-CoV-2 virus using on early mutational events. The study is interesting but I find several aspects that should be clarified. My recommendation is accept after revisions.

Major comments

I am wondering about the nucleotide diversity of the multiple sequence alignment of the 436 genome sequences downloaded from the NCBI. My impression (but just an impression) is that the nucleotide diversity is low, and in this case it could affect the detection of founder effects because lack of genetic information.

I am wondering if the results could change with the consideration of more genome sequences in the studied dataset, particularly sequences from other locations (i.e., Europe).

Minor comments

In some places along the manuscript it is mentioned that this virus is rapidly evolving worldwide. However it is unclear if this affirmation is based on the number of infections, number of mutations or number of fixed mutations (substitutions), etc. Is there any estimation about the genetic evolutionary rate of this virus to affirm that this is rapidly evolving virus variant?. If not I suggest modify the text.

In some places along the manuscript a sentence like the following is “Mutations in the helicase and orf1a coding regions from SARS-CoV-2 were predominant, among others, suggesting that these proteins are prone to evolve by natural selection”. I do not see the relationship between “the number of mutations” and “natural selection”. Natural selection can occur at any genetic region based on molecular adaptation (i.e., evolutionary constraints from the function), not on the number of mutations. Slowly evolving genetic regions are also subjected to the action of natural selection. I suggest modify the text.

The “Results” section repeats some information already presented in the “Methods” section. It needs to be rewritten avoiding repetitions.

Since the text mentions specific protein-coding regions (i.e., helicase, pol, etc), it would be interesting for readers to add a figure (either for the main text or for the supplementary material) illustrating the different regions of this virus, just something like, https://en.wikipedia.org/wiki/Structure_and_genome_of_HIV#/media/File:HIV-genome.png and highlighting the regions mentioned in this study. This could help readers to find the location of the studied regions in the whole genome. This is a suggestion.

It could be nice for readers if they can download the analyzed multiple sequence alignment (436 genome sequences downloaded from the NCBI) directly, from a link given by the authors (i.e., to GitHub or any other datasets repository). Indeed, note that the journal asks us (reviewers) about the raw data used in the study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: INSIGHTS ON EARLY MUTATIONAL EVENTS IN SARS-COV-2 VIRUS REVEAL FOUNDER EFFECTS ACROSS GEOGRAPHICAL REGIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: There is not enough justification for using the selected samples for analyses in order to confirm/refute that the identified mutations represent "founder" effects in the corresponding countries.
Experimental design: The experimenal design is flawed and cannot be used to test the hypotheses posed by the authors.
Validity of the findings: The conclusions are not sustained by the data presented because these have not been analyzed adequately.
Additional comments: This manuscript presents an analysis of a very limited number of SARS-CoV-2 isolates from China, USA and Australia selected from the availability of sequencing projects deposited at the NCBI SRA repository. Raw reads from 30 samples were used to identify variants against the reference used for mapping (one of the reference genomes initially obtained in Wuhan). Then, the frequency of these variants was estimated by downloading sequences from the corresponding countries deposited in GenBank. The analysis proceeds with a detailed characterization of each mutation as inferred from the annotation of the corresponding loci and its effects in the deduced protein sequence when in a CDS.
In my opinion, this study has several major flaws that lead me to recommend its rejection. Firstly, there is no clear definition of what the authors understand by “founder mutation” and how this is established in the corresponding populations. From their logic, it seems that founder mutations are those found in the 30 samples finally used for analysis, with no justification for why these and not other samples are the ones that seed the corresponding clades in each country. In addition, there is a no provision for population genetic nor phylogenetic analyses of the fate of each of these mutations when compared with the extant variability inferred for samples for each of the clades where the samples are included. Using a few samples from a bioproject (all of which have only a few samples) to establish any “founder effect” lacks logic and justification.
There are several additional issues on how to analyze population data but lack of information on the phylogenetic and epidemiological relationships among the viral sequences used in the analyses makes the conclusions of this manuscript completely unjustified.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH EXPRESSION OF STROMAL SIGNATURES CORRELATED WITH MACROPHAGE INFILTRATION, ANGIOGENESIS AND POOR PROGNOSIS IN GLIOMA MICROENVIRONMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: High expression of stromal signatures correlated macrophage infiltration, angiogenesis and poor prognosis in glioma microenvironment is well structured and well written.
I have made some considerations and would like the authors to review, read and restructure the following suggested points:
1.How about the protein level and the RNA transcript level of HSPG2, FOXF1, KDR, COL3A1, SRPX2  in the clinic samples?
2. question 1 should also be discussed in the discussion section.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH EXPRESSION OF STROMAL SIGNATURES CORRELATED WITH MACROPHAGE INFILTRATION, ANGIOGENESIS AND POOR PROGNOSIS IN GLIOMA MICROENVIRONMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: The topic is of clinical importance. However,  the methods are not solid enough to draw the conclusion. The major weakness is lacking of tissue examination for glioma infiltrating macrophages. In general, the scientific merit is not enough to be published in the current content.
Experimental design: 1. The clinical data from CGGA and TCGA may need to be stratified according to current standard prognostic factors for further analysis of stromal signature.

2. No tissue examination for validation of macrophage infiltration predicted by CIBERSORT.
Validity of the findings: The macrophage infiltration. especially for the M2 infiltration  needs to be validated.
Additional comments: None.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH EXPRESSION OF STROMAL SIGNATURES CORRELATED WITH MACROPHAGE INFILTRATION, ANGIOGENESIS AND POOR PROGNOSIS IN GLIOMA MICROENVIRONMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comments
Additional comments: I congratulate the authors for using public available data sets to conduct highly innovative and impactful research on gliomas. In the paper, the authors provide the websites where the CGGA and TCGA data sets are downloaded. However, it would be more more useful to the general readers if the authors can provide more descriptive detailed information about the data used in the paper, particularly for those downloaded from the Chinese CGGA website. More detailed descriptive metadata identfiers are useful to future general readers. The study design is well thought through and comments and discussion are useful and insightful. My suggestion is to provide more details on the statistical analyses. Examples include (1) provide more details on the model the at generated the classifier on CGGA and based on the classifier the ROC and AUC is calculated. Also, the classifier used in the TCGA datset, is there any model fitting used again, or just using the same classifier obtained from the CGGA dataset? If the same approach is used with TCGA as training and CGGA as validation, are the AUCs similar? (2) Are Kaplan-Meier curves to compare high versus low groups all based on median cut off? If not, specify the method used; (3) Are the p-values comparing the Kaplan-Meimer curves log-rank tests? Improve the description of the statistical analyses used can help the future readers; a last minor point, (4) the authors made novel contribution on impact of cells at tumor microenvironment on giloma prognosis, there is a recent publication by Sun X. et al (2019, Journal of Translational Medicine, vol 17, article 159) which discussed a macrophages-related gene signature predictive of survival of glioma patients also using the CGGA and TCGA datasets. It might be of some interest for the authors to comment on whether there is any connections between the signatures.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH EXPRESSION OF STROMAL SIGNATURES CORRELATED WITH MACROPHAGE INFILTRATION, ANGIOGENESIS AND POOR PROGNOSIS IN GLIOMA MICROENVIRONMENT
Review round: 1
Reviewer: 4

Output:
Basic reporting: 1. Citation for CGGA and TCGA are missing.
2. The tool for applying the GSEA is not mentioned
3. R, R packages (survival and pROC) GO, KEGG, STRING and Cytoscape are not cited properly.
4. Method and cutoff for dividing samples into high/low infiltration (of immune cells) and expression (of the five genes) are not mentioned.
5. Figure 4A-D. The figures show a significance value (-log10 FDR) but no estimate (like, a fraction of differentially expressed features in each term) which might be useful to show. Arguably, the raw p-values should be used since these are the per-gene quantity unlike the FDR values.
6. The generation of PPI network and the stromal gene signature are not described in enough details in the methods section. What exactly was the use for MSigDB? Please, clarify.
7. Tables describing the clinical characteristics of the CGGA and TCGA sets might be useful to add to the manuscript.
8. Figure 6G is not reference in the text.
Experimental design: The selection of 102 core genes and five stromal genes from the functional 3 gene sets wasn’t fully described or justified. This was done apparently based on their correlation with angiogenesis (Line 139). Arguably, considering a larger number of genes could improve the predictive value of the signature. Further validation of the stromal signature in the TCGA dataset may be required. Other than the ROC curve, the expression of the individual genes and their association might be useful to study.
Validity of the findings: The five stromal gene signature correlates with the tumor grade, but not the histology! Similarly, the signature correlates with the grade but not the response to treatment (Radio or chemo). Please, discuss.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HIGH EXPRESSION OF STROMAL SIGNATURES CORRELATED WITH MACROPHAGE INFILTRATION, ANGIOGENESIS AND POOR PROGNOSIS IN GLIOMA MICROENVIRONMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript addressed most of the issues raised in the review. In particular
1. Missing references and methodological details were added
2. The method for selecting core genes and the signature was clarified. Moreover, the expression of the five genes was also added to the manuscript.
3. The correlation between the proposed signature and the histology/treatment was further discussed. These correlations suggests that the proposed signature has a potential for clinical applications.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE EFFECT OF LANDSCAPE ON FUNCTIONAL CONNECTIVITY AND SHELL SHAPE IN THE LAND SNAIL HUMBOLDTIANA DURANGOENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: all comments on the pdf file
Experimental design: all comments on the pdf file
Validity of the findings: all comments on the pdf file
Additional comments: Great study

I have a morphometric code in R that I can share if you desire. I mention in my review the use of R for this analysis. Please contact the journal if you are interested in accessing the R code.

I think the manuscript is in a very good shape except for some further analysis in the morphometric side: adding a procrustes analysis and adding deformation grids with the change in shape to figure 1.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE EFFECT OF LANDSCAPE ON FUNCTIONAL CONNECTIVITY AND SHELL SHAPE IN THE LAND SNAIL HUMBOLDTIANA DURANGOENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very interesting study where authors evaluate the effect of climate and vegetation cover on the functional connectivity of populations of Humboldtiana durangoensis, a land snail from Mexico, based on information of a previous study in which evidence of population structure was found for the species. Also, the effect of climate in shell size and shape was assessed aimed to distinguish conservation units (ESUs). Although there is no general consensus on how to define ESUs, the authors use herein an interesting approach. The manuscript is clearly written and is in good shape, figures are relevant, and raw data are available as supplemental information.
Experimental design: The research objective is clearly defined, and methods are technically sound and appropriately detailed.
Validity of the findings: Authors evaluated the functional connectivity for the species and provide evidence suggesting that vegetation rather than climate has influenced the availability of suitable habitats, which have decreased since the Pleistocene to present, hypothesizing that functional connectivity has been promoted by the presence of temperate and cold forests. They also found evidence of a relationship among shell size and shape with climatic variables, explained by the latitude and altitude where the populations examined occur. Authors also suggest the seven genetic groups identified in a previous paper should be considered ESUs based not only on the genetic component, but also on the phenotypical differences. Overall, this kind of information is very welcome for the molluscan fauna from Mexico for which conservation efforts are scarce.
Additional comments: - Some references listed in the main text are not in the reference list and vice versa.
- One of the main advantages of geometric morphometrics (GM) is to describe the variation in shape, excluding the size component. In this paper when using GM, shape variation is only described in relation to “size” (e.g. larger shells, higher spirals). In my opinion it would better to describe shape variation in terms of shape (e.g. conical, rounded, squared…).
- I have introduced minor comments and suggestions directly into the attached MS.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE EFFECT OF LANDSCAPE ON FUNCTIONAL CONNECTIVITY AND SHELL SHAPE IN THE LAND SNAIL HUMBOLDTIANA DURANGOENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the authors made great changes to the article. I loved how they added the deformation grids to their figures. I still think figure S1 should be a figure in the main manuscript and not in the supplementary materials: it's the study system, it deserves a place as a figure in the main manuscript, it makes it easier to understand the deformation grids and the morphometric analysis, it's a beautiful picture. I think figure S1 should be figure 1.
Experimental design: The comments on my first review were followed by the authors and they did a great job
Validity of the findings: As I mentioned the first time, I think the findings are clear and important.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE EFFECT OF LANDSCAPE ON FUNCTIONAL CONNECTIVITY AND SHELL SHAPE IN THE LAND SNAIL HUMBOLDTIANA DURANGOENSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have revised the manuscript and addressed all reviewers’ suggestions.  I have no additional comments, and I think the manuscript should be accepted. Congratulations to the authors for their excellent work.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors has reported A new species of freshwater crab of the genus
Qianguimon, as the fifth species of this genus. The results have  been well present. Moreover, molecular analysis added to morphological results has been validated finding. However, in the discussion section, i observed no discussion about findings. I have submitted a series of corrections  in the attached version of paper. I emphasis that the discussion section should revise according the comments of attached version. Finally I recommend to publish the article after a major revisions.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors used compound sentences in the parts of living color and ecology. These parts should be rewritten and checked by a native speaker of English.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The main comment is the author should show the DNA results, such as the minimum interspecific divergence between the new species and other species of this genus. And provide a table of genetic distances within the new species and between five species of Qianguimon.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Comments in attached pdf.
Experimental design: Comments in attached pdf.
Validity of the findings: Comments in attached pdf.
Additional comments: Comments in attached pdf.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A NEW SPECIES OF FRESHWATER CRAB OF THE GENUS QIANGUIMON HUANG, 2018 (DECAPODA: BRACHYURA: POTAMIDAE) FROM GUANGXI, SOUTHERN CHINA
Review round: 2
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I have just the recommendations (personal point of view):
1) It is not necessary to use so thick scale bars and the letters used in figures seem to large in the ratio of the photo/drawing area.
2) Dataset is reduced, but I mean, for the confirmation of the new species is just enough to use sequences of the congeners and one or two close genera as the outgroup.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ENHANCEMENT OF SPECIFIC T-LYMPHOCYTE RESPONSES BY MONOCYTE-DERIVED DENDRITIC CELLS PULSED WITH E2 PROTEIN OF HUMAN PAPILLOMAVIRUS 16 AND HUMAN P16INK4A
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: All experiments should have correct control groups.
Validity of the findings: No comment.
Additional comments: High-risk human papillomavirus (HPV) infection is known to be a necessary factor for cervical and anogenital malignancies. Cervical cancers account for over a quarter of a million deaths annually. Despite the availability of prophylactic vaccines, HPV infections remain extremely common worldwide. Furthermore, these vaccines are ineffective at clearing pre-existing infections and associated preinvasive lesions. As cervical dysplasia can regress spontaneously, a therapeutic HPV vaccine that boosts host immunity could have a significant impact on the morbidity and mortality associated with HPV. In this manuscript, Dr. Sunthamala and colleagues looked at the role of the HPV16 E2 and p16INK4a protein in stimulation of T lymphocytes, and wanted to provide a strategy for further in vivo study of stimulation of T lymphocytes for therapy of HPV-associated cancer. The study provides the conclusion, which showed that monocyte-derived dendritic cells pulsed with both proteins enhances specific response of both CD4+ and CD8+ T lymphocytes. Although, the current study is interesting but there are some major concerns that need to be addressed.

Major concerns:
1. “Materials and methods – Isolation of CD14+ peripheral blood mononuclear cell (PBMC) and cultivation of monocyte-derived dendritic cells (MDCs)” Line 131. Were each volunteers’ PBMC separated or mixed for subsequent experiments? Please state clearly.
2. “Results – HPV16 E2 and p16INK4a proteins induced phenotypic maturation of MDCs” Line 230-231, Figure 2A. “We observed that CD14+ monocytes cultured with rhGM-CSF and rhIL-4 (Fig 2A) can be induced to develop from immature to mature dendritic morphology.” However, there were characteristics of immature MDCs before stimulation in figure 2A!
3. “Results – HPV16 E2 and p16INK4a proteins induced phenotypic maturation of MDCs” Line 226-233, Figure 2A and B. It will be better to present the photos of before and after treatment in four groups, which were negative control (PBS or something), positive control (rhGM-CSF and rhIL-4 treatment), HVP16 E2 treatment (best condition, might 250 ng/ml) and p16INK4A treatment (best condition, might 150 ng/ml).
4. “Results – HPV16 E2 and p16INK4a proteins induced phenotypic maturation of MDCs” Line 234-239, Figure 2C to F. It is necessary to present the positive control (rhGM-CSF and rhIL-4 treatment) in all charts, and it will be better using “Negative control” than “Untreated”.
5. “Results – HPV16 E2 and p16INK4a proteins induced phenotypic maturation of MDCs” Line 226-239, Figure 2. To make the evidence stronger, it will be better to present the expression level of CD83 before and after treatment with HPV16 E2 (best condition, might 250 ng/ml) and p16INK4A (best condition, might 150 ng/ml) respectively in more cases of mature DCs, which separate from 5-8 other volunteers respectively.
6. “Materials and methods – Evaluation the role of MAPK pathways on maturation and activation of MDCs” Line 155-158. “The specific kinase inhibitors were p38 MAPK inhibitor (SB203580, 50 μM), ERK inhibitor (PD98059, 50 μM), and JNK inhibitor (SP600125, 20 μM).” Why use the concentration of these inhibitors? Please state clearly and provide references.
7. “Results – HPV16 E2 and p16INK4a proteins stimulate the maturation and activation of MDCs via MAPK pathway” Line 248-251, Figure 3C and D. It is necessary to present the levels of IL-1β and IL-12 before and after treatment with HVP16 E2 (best condition, might 250 ng/ml) and p16INK4A (best condition, might 150 ng/ml) respectively.
8. “Discussion” Line 317-319. “We found that HPV16 E2 and p16INK4a proteins stimulate the secretion of IL-1β and IL-12 which was partially suppressed by MAPK inhibitors (Figs 3C and 3D).” I did not see any data shown that PV16 E2 and p16INK4a proteins stimulate the secretion of IL-1β and IL-12.
9. “Results – MDCs pulsed with HPV16 E2 and p16INK4a stimulate CD4+ and CD8+ T lymphocytes to produce IFN-γ” Line 256-260, Figure 4A and B. It is necessary to present the populations of CD4+ and CD8+ T lymphocytes with negative control (non-stimulating the maturation of MDCs). It also need negative control in Figure 4C and D.
10. “Results – MDCs pulsed with HPV16 E2 and p16INK4a stimulate CD4+ and CD8+ T lymphocytes to produce IFN-γ” Line 265-266. “This indicates that HPV16 E2 might exhibit higher immunogenicity than the combination of HPV16 E2 and p16INK4a.” To make the evidence stronger, it will be better to present the experiment in more cases of mature DCs, which separate from 5-8 other volunteers respectively.
11. “Results – CD4+ and CD8+ T lymphocytes co-response to combination of HPV16 E2 and p16INK4a pulsed MDCs” Line 265-266. “Suggesting that the stimulation with combination of HPV16 E2 and p16INK4a clearly enhances response of both CD4+ and CD8+ T lymphocytes.” To make the evidence stronger, it will be better to present the experiment in more cases of mature DCs, which separate from 5-8 other volunteers respectively.

Minor comments:
1. The ABSTRACT need to be rewrite. It does not need to contain much background information in the ABSTRACT section.
2. Figure 3C and D. What is the concentration of HPV16 E2 and p16INK4a in “E2+p16” groups?
2. Figure 4. What is the concentration of HPV16 E2 and p16INK4a in “E2+p16” groups?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ENHANCEMENT OF SPECIFIC T-LYMPHOCYTE RESPONSES BY MONOCYTE-DERIVED DENDRITIC CELLS PULSED WITH E2 PROTEIN OF HUMAN PAPILLOMAVIRUS 16 AND HUMAN P16INK4A
Review round: 1
Reviewer: 2

Output:
Basic reporting: The abstract should be structured.

The introduction is too long, especially the last paragraph, where the authors should shortly describe the general aim. Some technical aspects mentioned here would be fit better the Materials and Methods section, maybe before the subsections describing the specificities.
Experimental design: Methods are overall clear and the experimental procedures are reasonable. The authors declared they used 5 healthy volunteers to get the blood, but they repeated the experiment three times: each time did they make the experiments with the purified cells from all of them?
By the way, did the authors assess the HLA genotype of the healthy donors?
Validity of the findings: Again, from the figures and legends, it is not clear how the cells of the 5 healthy volunteers have been used.
Additional comments: See above comments. In general, the manuscript is clearly written. A few experimental details should be clarified.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ENHANCEMENT OF SPECIFIC T-LYMPHOCYTE RESPONSES BY MONOCYTE-DERIVED DENDRITIC CELLS PULSED WITH E2 PROTEIN OF HUMAN PAPILLOMAVIRUS 16 AND HUMAN P16INK4A
Review round: 1
Reviewer: 3

Output:
Basic reporting: Dendritic cells (DCs) are potent antigen-presenting cells of the immune system and their maturation status is critical in terms of both cytokine secretion and stimulating resting T cells. Several in vitro approaches to stimulate DCs maturation pulsed though foreign antigenic proteins has been tested and employed in T cells activation. In this manuscript, author investigated that human PBMCs stimulated with tumor associated protein HPV E2 and p16INK4a differentiated into mature DCs leading to T cell activation.  This manuscript is well conceptualized and written, however limited experimentation to confirm integrity of purified proteins and lack of any data that could indicate the maturating DCs are not contaminated with other cells types weaken the effectiveness of this study.
Experimental design: Authors need to provide some strong biochemical and biophysical characterization of the purified protein, given that both the proteins are not of bacterial origin and chances of them to form inclusion bodies are very high that leads to synthesis of immature proteins. Immune signaling from NF-kB pathways drives secretion of major inflammatory mediators, nothing described from NF-kB pathway further diluting the outcome of this study. Additionally, it is required to monitor the surface markers for CD3, CD19 and CD14 to rule out the possibility of other cell type contamination post DCs maturation. Some other points are-
1.	Figure 1C & D- better to provide SDS staining with un-induced control, also in supplementary figure for uncropped WB of protein it seems that the gel background of marker lane and protein lane has different contracts settings.
2.	Figure 2A & B- Post 48h of protein incubation, the status of protein integrity is a major concern in culture medium. It is not tested whether proteins have no disintegration in culture media post incubations.
3.	Figure 2- what is the status of B cell and macrophages population post protein treatment.
4.	Figure 3- measuring expression of total MAPK are not a good readout to confirm the induction of signaling pathways, it is better to analyze the phosphorylation status of these proteins.
5.	Figure 3D in PBS lane cell exposed to combined protein treatment has less IL12, compared to individual treatments, any explanation?
Validity of the findings: Authors have analyzed that around 60% immature DCs are converted to mature DCs at the peak concentration of proteins, still 40% cells were unable to respond which can be seen in Figure 4A & B with no significant induction of CD4+ and CD8+ cells population. Moreover, authors have not provided any positive control with protein treatment to compare the outcomes from matured DCs.
Additional comments: Authors have shown the in vitro maturation of DCs through protein pulsing followed by activation of T cells population, still authors need to add some more data that will further enhance the outcomes of this manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ENHANCEMENT OF SPECIFIC T-LYMPHOCYTE RESPONSES BY MONOCYTE-DERIVED DENDRITIC CELLS PULSED WITH E2 PROTEIN OF HUMAN PAPILLOMAVIRUS 16 AND HUMAN P16INK4A
Review round: 1
Reviewer: 4

Output:
Basic reporting: Manuscript is well written and figures, legends are neat. Discussion can be improved.
Experimental design: Expt design was ok but could be more rigorous with controls
Validity of the findings: please see pdf comments and word doc concerns that need to be addressed
Additional comments: Sunthamala et al use recombinant HPV proteins to induce DC maturation that they show can enhance CD4/CD8 T cell responses in the manuscript herein.
The manuscript is written well. The scope of the study is limited but still important enough to encourage science and research.
Certain aspects of the manuscript need reinforcement with controls and some clarifications to warrant publication.
Points to address:
1. The authors should show that LPS contamination has no role in the phenotype. Use of TLR4 inhibitor (TAK242) or IRAK4 inhibitor can address this
2. Maturation of DC with CD83 should be supported with additional facs for CD80, CD86 and MHC-I/II. Costimulation and antigen presentation both are required for Tc response so these might be changed by the HPV protein treatment of DCs
3. Authors should include important control of untreated DC when comparing each HPV protein or combination. This is included in CD83 figure panel but missing in fig 3c,d and 4.
4. Cytokine IL12 and IL1b data are not convincingly explained. Why is there no difference in IL1b. Additionally, IL12 is only reduced by MAPK inh in p16, is there explanation for this? Why does E2+p16 decrease IL12 compared to p16 alone (compare PBS group black bars in fig 3d
5. if fig 4e, comparison should be done to untreated DC and not  just between E2 v p16.
6. LPS treated DCs can be used as positive control in expt
7. fig 4e and 4f there is discrepancy: E2 increases IFNg in 4e (1:10) but in 4f E2 alone has no difference with p16 alone (compare no block groups). Also why only E2+p16 shows phenotype with HLA block needs clarifications.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ENHANCEMENT OF SPECIFIC T-LYMPHOCYTE RESPONSES BY MONOCYTE-DERIVED DENDRITIC CELLS PULSED WITH E2 PROTEIN OF HUMAN PAPILLOMAVIRUS 16 AND HUMAN P16INK4A
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: High-risk human papillomavirus (HPV) infection is known to be a necessary factor for cervical and anogenital malignancies. Cervical cancers account for over a quarter of a million deaths annually. Despite the availability of prophylactic vaccines, HPV infections remain extremely common worldwide. Furthermore, these vaccines are ineffective at clearing pre-existing infections and associated preinvasive lesions. As cervical dysplasia can regress spontaneously, a therapeutic HPV vaccine that boosts host immunity could have a significant impact on the morbidity and mortality associated with HPV. In this manuscript, Dr. Sunthamala and colleagues looked at the role of the HPV16 E2 and p16INK4a protein in stimulation of T lymphocytes, and wanted to provide a strategy for further in vivo study of stimulation of T lymphocytes for therapy of HPV-associated cancer. The study provides the conclusion, which showed that monocyte-derived dendritic cells pulsed with both proteins enhances specific response of both CD4+ and CD8+ T lymphocytes. The paper is significantly improved and all concerned raised by the reviewer have been addressed. I think it is suitable for publication at this point for this version of revised manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RECENT ADVANCES IN BIOCHAR APPLICATION FOR WATER AND WASTEWATER TREATMENT: A REVIEW
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: The Conclusions section should include the main aspects of biochar modification.
Additional comments: This manuscript reviews the recent advances on biochar applications in water and wastewater treatment. A brief discussion is also conducted on the sorption mechanisms of mainly certain organic pollutants. Finally, a brief discussion on various biochar modification and their effect on the structure, surface properties, and sorption efficiency is presented. The manuscript is well written and scientifically well organized. It is potentially interesting for the readers of the Journal and could be considered for publication after changes.

Specific Comments

1.	Since a number of referenced papers concern the use of biochar for the removal of contaminants form water or aqueous solutions the title of the article should be changed to: “Recent advances in the application of biochar for water and wastewater treatment: A review”
2.	The effect of pyrolysis temperature on biochar surface and sorption capacity on metals and PAHs as well basic modification of biochar should be benefited by referring to relevant literature on of this topic such as:
a.	Preparation and Characterization of Biochar Sorbents Produced from Malt Spent Rootlets." Industrial & Engineering Chemistry Research, 54(39), 9577-9584.2015.
b.	Effect of Chloride and Nitrate Salts Effect on Hg(II) Sorption by Raw and Pyrolyzed Malt Spent Rootlets. Journal of Chemical Technology and Biotechnology, 92(8), 1912-1918. 2017.
c.	Phenanthrene Removal from Aqueous Solutions Using Well-Characterized, Raw, Chemically Treated, and Charred Malt Spent Rootlets. Journal of Environmental Management, 128, 252-258. 2013.
3.	In the section of inorganic ions a brief discussion on the mechanisms for nitrogen and phosphorus removal should be useful for the reader.
4.	Line 463. Please clarify what watershed treatment means ?
5.	Lines 469-472. Please explain how the addition of biochar into soil reduces runnof and increases runnof time.
6.	Modification of biochar. The authors discuss in detail the various methods for biochar modification. In my opinion a comparative discussion is needed at the end of the section on how to select the appropriate modification method according to the target contaminant(s). Which are the future directions for biochar modification?
7.	The Conclusions section should include the main aspects of biochar modification.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RECENT ADVANCES IN BIOCHAR APPLICATION FOR WATER AND WASTEWATER TREATMENT: A REVIEW
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well-written with minor editing mistakes but the use of the terminology related to sorption can be further improved.  Please see suggestions in the annottated pdf file.
Sufficient references are used but some key references are missing and suggestions are provided in the annottated pdf file.
The article is well-structured, the figures are useful and another figure is proposed to organize the last section that is on concerns and future suggestions.
Experimental design: Manuscript content is within the Aims and Scope of the journal.
Rigorous investigation was performed but still some key references are missing.  See annottated pdf file.
The structure of the review is well-prepared however some points need to be better highlighed and should be on new separate subsections.   See annottated pdf file.
Validity of the findings: The information is well-presented and well-discussed with future concerns and suggestions provided at the end of the manuscript.
The current use of biochar or similar material in wastewater facilities is not sufficinetly -described.
Additional comments: The use of the sorption terminology requires attention and should be carefully used throughout the manuscript.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: RECENT ADVANCES IN BIOCHAR APPLICATION FOR WATER AND WASTEWATER TREATMENT: A REVIEW
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript is well-written.
Experimental design: No comment.
Validity of the findings: No comment
Additional comments: The authors responded very well to reviewer comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: THE ASSOCIATION OF EGF RS2237051 VARIANT, SERUM EGF LEVELS AND GENERALIZED AGGRESSIVE PERIODONTITIS: A PRELIMINARY STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The background section of the abstract needs to be improved.
The connection between the presence of studies of EGF in saliva and gingival crevicular fluid, and the need to study serum EGF levels and polymorphism in patients with GAgP is not clear.

The authors should describe in more detail their findings in the context of what is already known in the literature concerning EGF, EGF polymorphisms, and phenotypes correlated to what reported by them - e.g. BMI alterations, inflammatory diseases, matrix turnover/pathologies associated to matrix modulation. This would strengthen their results and the relevance of their manuscript.
Experimental design: The authors should clarify why they assess serum levels of EGF, and not EGF in saliva / gingival fluid for their study.

The gaps the study wishes to fill are not clear. Do the authors imagine EGF as a therapeutic target? as a prognostic marker for periodontitis?

The study is purely correlative, and as such the experimental design properly described.
Validity of the findings: The authors report purely correlative findings, and do not overstate the implications of their results.

Is the higher BMI in GAgP patients something reported in the literature? Is there a real correlation?

Is there a known correlation between EGF levels, or EGF polymorphisms, and BMI?

What is the effect of the EGF polymorphisms described on the function of EGF itself? The authors should discuss what is the possible way via which the polymorphisms could actually influence the parameters described in the work.
Additional comments: No additional comments