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: MODERATELY LOW NITROGEN APPLICATION MITIGATE THE NEGATIVE EFFECTS OF SALT STRESS ON ANNUAL RYEGRASS SEEDLINGS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The language is readable, but there are still many errors. I suggest that the manuscript should be extensively modified by a native English speaker.
The article includes sufficient introduction and background with relative references.
I did not see Figure legends of Fig. S1 and S2.
I cannot find Table S1, which should show the primers.
Raw data are provided.
The sections are right.
Experimental design: N application can alleviate the salt stress on plants. The authors aim to reveal how N concentrations affect the growth and photochemical parameters of feeding annual ryegrass under low salt stress. That is an important question in the environment science field.
The authors systematically analyzed the growth and photochemical parameters of feeding annual ryegrass at different treatments. The methodology is reliable and clearly described.
However, one problem is that the authors did not properly organize the data. In some figures, the x axis is N concentrations, while in others the x axis is salt concentrations.
Validity of the findings: Repeats were properly performed.
The data were statistically analyzed.
The conclusion is related to the main question asked by the research and supported by the data.
Additional comments: The following are specific comments and suggestions. The authors should be aware that the errors are much more than listed below. Therefore, the manuscript has to be carefully and extensively modified.
1.	Line 18: damage on plants
2.	Line 23: full name of “OJIP”
3.	Line 42: remove “studies have shown that”
4.	Line 47: reduce its damage; change evolvement to evolution
5.	Line 53: change regulate to improve
6.	Line 54: rephrase the sentence “The inorganic N …”
7.	The authors analyzed the effects of N concentrations on plant growth. They show the data of 2, 5, 10 mM in Fig.1 but the data of 0.5, 2, 5 mM in Fig. S1. Why not put these data in one figure?
8.	Please mark the x axis in Fig. S1D.
9.	Line 264: remove “the”
10.	Line 277-279: The authors compared their results to the findings of Sagi et al., 1997. However, they should convert salinity to NaCl concentration for comparison.
11.	Line 300: The results are not from Fig. 1
12.	Figure 3 legend: Rephrase the sentence “*indicate parameters statistically significant between different N levels under the same NaCl level.”
13.	Line 326-327: what it represents?
14.	Line 332: is consistent with
15.	Acknowledgement: The authors should mention funding sources and people who helped them prepare the manuscript, instead of 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: MODERATELY LOW NITROGEN APPLICATION MITIGATE THE NEGATIVE EFFECTS OF SALT STRESS ON ANNUAL RYEGRASS SEEDLINGS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a very meaningful manuscript with professional language, structure, and figures.
Experimental design: Rigorous experimental design.
Validity of the findings: The results are solid and valid.
Additional comments: I suggest adding “seedings” to the topic since the research object is the seedlings rather than the whole growth period.

To update some new literatures beyond the classic literatures to support the research results.


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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors use clear and professional English throughout the article. However, there are areas throughout the manuscript that can be cleaned up a bit. For example, in the Abstract on line 28 and 29, “The true myrtle, Myrtus communisis, is an evergreen perennial small tree that occurs in Europe, Africa, and Asia with a typical circum-Mediterranean geographic distribution.” This sentence could be cleaned up by describing the tree as a “small perennial evergreen tree” and removing the word “typical”.

I do not think this article provides sufficient background information to support a reason for conducting this research. The author's premise is that no one has ever used a “metabarcoding” approach to characterize the foliar fungal endophyte communities associated with this particular host nor has any one used network analysis to characterize the fungal community for this host. The majority of the introduction discusses the plant host. However, there needs to be more justification as to why this research is relevant and how it fits into the broader field of knowledge.

Their paragraph on endophytes (lines 72-78) needs to be expanded. There is a large body of literature on foliar fungal endophytes, but the authors only cited one article (Arnold 2008) in this paragraph besides their own articles (Vaz et al.). Their following paragraph (lines 79-87) which provides their reasoning for using high throughput amplicon sequencing over culture dependent techniques needs more background reference. I believe Zimmerman and Vitousek 2012 (PNAS, Fungal endophyte communities reflect environmental structuring across a Hawaiian landscape) was one of the first foliar fungal endophyte studies to use high throughput amplicon sequencing over culture dependent techniques.

Additionally, their paragraph on network analysis needs a lot more information and a more clear explanation of network theory and bipartite networks. In addition to Toju et al. (2014) article on fungal-plant interaction using bipartite networks, Cobian et al. (2019; ISMEJ, Plant-microbe specificity varies as a function of elevation) uses bipartite networks to study the effects of elevation on foliar fungal endophyte host specificity.

Figure 1: It would be more professional if the authors produced their own map using R or some GIS application instead of a Google Earth screenshot, additionally, it would be helpful for the readers if the transect line and sampling locations were shown on the map as well.

Figure 2: I would not recommend using this type of figure because they are very messy and difficult to interpret. A heat map as in Figure 3 may be a better way to show the fungal-plant interactions.

Figure 3: The quality of this figure needs to be improved. Also, it appears that there may be the same color green that represents 0 reads and somewhere around 2500 reads, this is the color of most of the squares. I would also suggest using a different color scheme since people with red-green color blindness would likely see no differences in these colors, and in fact, there really does not seem to be any differences in most.

The authors made the raw data available and accessible.

I do not think that the authors presented a clear hypothesis or question in their article. Therefore, the results of this article do not support any clearly stated hypothesis.
Experimental design: The research is within the Aims and Scope of PeerJ. However, there was no clear or well-defined research question or hypothesis in the introduction. Additionally, the methods were not clearly explained.

The authors do not indicate how many host plants they sampled or how far apart individual trees were from one another. They indicated that they collected five leaves per individual tree and then surface sterilized the leaves. However, they only used six leaf fragments after sterilization to extract DNA, and they do not explain the size of the leaf fragments. Also, they explain that the six leaf fragments were placed in silica-gel, but they do not provide a reason why. Finally, they do not indicate if or how leaf tissue was homogenized before DNA extraction.

The bioinformatics subsection in the Methods section needs a lot more detail. For example, lines 145-148 needs to explain how they did their quality filtering, how they performed chimera sequence removal, what percentage of sequence similarity did they use for clustering, etc. Also, they never reported the results of their bioinformatics which usually indicates the number of sequences they started with, how many sequences remained after quality control, how many OTUs were obtained after clustering.

For the bipartite networks (lines 150-158), the authors need to explain how their networks were constructed. Additionally, they need to explain the different indices they are testing and what the different indices tell us about the interactions between the fungi and host plants. This paragraph needs to be explained in much more detail.

As written, I do not thing the Methods section provided sufficient detail for someone to replicate this experiment.
Validity of the findings: Given the issues with this article, I am not providing any comments regarding the findings as I am uncertain of their validity.
Additional comments: When talking about foliar fungal endophytes, the authors use about three different terms. I would be a lot more helpful for the reader if the authors only chose one term.

Although the term “basidiomycotan” is not incorrect, I recommend using the term basidiomycete when referring to fungi within the basidiomycota.


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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written and well referenced for the most part although problems arose in guild assignment of sequences, as discussed more fully under ‘Validity of Findings’.
Experimental design: The authors state that the foliar, endophytic fungi of Myrtus communis have not been studied with metabarcoding and bipartite network analysis. So, they undertook to fill this gap.  They were not testing a particular hypothesis that could have been falsified by their findings. Methods are described clearly enough, although local plant communities should have been noted, for reasons discussed below.
Validity of the findings: General Comments: I first compared their list of fungal taxa, mostly genera, with the list of 33 non-synonymous genera obtained for Myrtus communis from a search of the USDA SMML fungal databases. Myrtus communis is grown widely in many parts of the world so the 33 genera represent global records.  None of the 33 were found as foliar endophytes in this study! This complete lack of overlap was surprising although it passed without comment from the authors. I then started to examine the authors’ guild assignments of Table 5 more closely.  The first thing to strike me was their finding of Phragmidium which the authors assigned, via FUNGuild apparently, to the ‘saprotroph’ category. That is just wrong; Phragmidium is a genus of rust fungi, obligate, biotrophic parasites, that attack plants in the Rosaceae.  Phragmidium is not known to successfully parasitize Myrtus communis. An explanation of its amplification in this study must thus involve a source of its spores (infected Rosa species?) that might have been near the sampled Myrtus communis. As we know from the work by Michelle C. Heath and others, spores of rust fungi can germinate on a non-host and even substomatal space can be colonized. That level of colonization would not be washed off in reported protocols.  If that is the explanation for the Phragmidium finding here, then one would have to question additional findings since other plants could also have been sources of the other reported fungi. I also wondered why the finding of Nicoletti et al was not duplicated here, and passed without comment from the authors: Neofusicoccum australe as endophyte in M. communis.
Regarding the authors’ conclusions, I was unconvinced not only due to my doubts about their findings.  A second concern was that their ‘hypothesis’ could not have been rejected  if their conclusion were “in accordance with the hypothesis stating that the endophytism is a life-history strategy” of whichever fungi were found.
Additional comments: "Culture-dependent" work is mentioned in the Abstract but no Results are reported.


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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: In their rebuttal, the authors "agreed with the Reviewer 2 and discussed this complete lack of overlap based on the very strong selection caused by traditional isolation methods based on standardized culture media for fungi. Please, see Lines: 314-328 in the Discussion."  I don't find this argument convincing because many of the fungi that they are reporting here (e.g., Aurantiporus) are as culturable as the fungi and oomycetes reported in the USDA SMML databases for Myrtus communis. It is also misleading to call the SMML fungi and oomycetes 'culture-dependent'. They are culturable but they do not depend on culture to affect Myrtus communis in ecologically significant ways.  The authors report Phragmidium, a rust fungus that has no ecologically significant role in M. communis (and the DNA of which may have come from neighbouring Rubus), but do not report Austropuccinia psidii, a rust fungus that does attack M. communis, at least in some parts of the world. Was there heart rot caused by Aurantiporus in the trees sampled for leaf endophytes?  Or, could spores from basidiomata of Aurantiporus on nearby Acer or Populus or Salix (etc.) have blown over and stuck to the Myrtus leaves as the Phragmidium spores evidently did? If Phragmidium was just trace DNA and not ecologically meaningful in leaves of M. communis, then could not this also be true of other reported taxa in Table 6?
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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Pag. 2, Line 67: The authors should consider describing better the what is a myrtucommulones, because it is not clear in the sentence.
Pag. 3 Line 109: Remove one of the words: with or since from the sentence.
Pag 5. Line 156: I believe that the correct term to be used would be disinfested.
Pag. 7 Line 230: Replace 212,167 for 212.167.
Pag. 7 Lines 241-244: The english language here should be improved. Using the words “not only” and “but also” made the sentence very confusing. Please re-write.
Pag. 9 Line 335: The citation Thomas et al., 2012 is not in the reference section. Also, you said our study and cites another author, please review that.
Pag. 9 Lines 331-335: Considering that your work highlight the benefits of using culture-independent high-throughput sequencing methods instead of culture-dependent methods, but in Pag. 10 Lines 343-353 is suggested that the genus Aurantiporus sp.  found in this work could belong to two different species or an entirely new species. So, don’t you think that this represents a lack of a precise identification that could be benefited from a study using a culture-dependent method? Maybe we should not prefer one technique over another but use both techniques to obtain a more precise and detailed work.
Pag. 10 Lines 356-358: The english language here should be improved. Using the words “there has long been” made the sentence very confusing. Please re-write.
Experimental design: I believe the authors should make their justification clearer in the text, since I was little confused about the purpose of the work. The objective of the work was to describe a fungal community diversity in a plant of medicinal interest in an area that is endangered and little studied? Because fungal endophytes are important for plants? Or was it to demonstrate the efficacy of a metagenomic technique? How all of this is connected?
Validity of the findings: The authors propose two questions in the end of the introduction section, which are:
(i) Are the bipartite network-level patterns of Myrtus communis-foliar fungal endophytes similar to the networks of other foliar fungal endophytes and their distinct hosts in different biomes? and (ii) Are the fungal trophic modes and guilds related to the particular bioclimatic features of the Mediterranean sclerophyllous forests ecoregion?
However, the first question was answered very indirectly, and the second question was not answered in the discussion or in the conclusions section. Please review that.
Additional comments: This is an interesting work with important metagenomic data. The paper is well written, and without major errors in relation to the English grammar. The results were clearly described, the discussion is well based, and the literature is updated. However, the authors proposed to answer two questions, but they answered both questions in an indirect and inconclusive way. Also, the paper seems very descriptive, since the authors does not provide a clear justification for the importance of this type of work. For this reason, it would be interesting for the authors to explain the purpose of the research more clearly in the text and even provide suggestions on how they could expand this research by using other types of techniques and performing other analysis.


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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The topic addressed in this study is interesting, but as it is presented, it is not new. In the introduction and discussion, a lot of information is missing regarding the novelty of the work. The introduction is not well structured; as suggestion, first briefly describe the broad research and then narrow down to your particular topic. Sometimes the text is also not well linked; for example, the paragraph (lines 79-87) is absolutely not related with the previous sentence about fungal endophytes. My second major concern is regarding the main goal and the ecological novelty of the study. Clear objectives of the study are missing here. Furthermore, I don´t see any ecological novelty in this work. It´s not clear what ecological question the authors seek to answers by using this bipartite networks, and what´s novel of that. Bipartite networks are usually used to represent ecological communities. But the authors here just worked with one plant species. It would have been nice to see the structure of fungal endophyte communities along an array of plant species in this Mediterranean biome. The article is in general well written and clear to follow.
Experimental design: Methods are described with enough details. Authors are using novel bioinformatic data here; nevertheless I miss a nice ecological hypothesis in the introduction, which properly justify the use of bipartite networks in this study.
Validity of the findings: - Results are clear and well explained. But to the lack of an ecological hypothesis in the introduction, it´s difficult to see the novelty of the results.
- I miss a deep discussion of the results as well as the biological relevance of them. For example, what´s the ecological relevance in this kind of interactions that individuals of M. communis and foliar endophytic fungi are very low connected, with low nestedness, and moderately high specialization? Findings should be set the into the context of the literature, and then into broader theory.
- It would be nice that authors, together with weaknesses of culture-dependent methods, add some information about weaknesses of a metagenomic approach.
- The importance and novelty of this research with respect to the existing ones should be highlighted. Similarly, the potential importance/benefits of dominant fungal endophytes for the studied plant species should be included. This aspect, although speculative, should be included in the discussion.
- Figure 1 should be moved to supplementary material.
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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 4
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: FOLIAR MYCOENDOPHYTOME OF AN ENDEMIC PLANT OF THE MEDITERRANEAN BIOME (MYRTUS COMMUNIS) REVEALS THE DOMINANCE OF BASIDIOMYCETE WOODY SAPROTROPHS
Review round: 4
Reviewer: 2

Output:
Basic reporting: I thank the authors for the corrections made; the manuscript improved markedly.
I'm still not completely convinced with the structure of the introduction; nevertheless it  improved regarding the last version. The discussion has also considerably improved, showing the ecological relevance of the data. The new version of the manuscript highlights much better the novelty of the work, and it is in general very well written.
Experimental design: Methods are described with enough details.
Validity of the findings: Results are clear and well explained.
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: IDENTIFICATION OF A SIX-GENE METABOLIC SIGNATURE PREDICTING OVERALL SURVIVAL FOR PATIENTS WITH LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: The figures are a bit crowded and would benefits of a bit more explanation and additional legend descriptions.

Some additional files could be merge as some just contains 3 or 4 entries on a excel sheet.
Additional comments: The paper is well written and provide analysing supporting the conclusions.
This work tries fo fill a gap in the study of LUAD prognosis and thus propose to face a valable research question.
The authors made a good use of available databases and performed relevant analysis in order to identify these genes.
More details about the metabolic pathways involved and how these 6 genes could interfere would bring more strength to 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: IDENTIFICATION OF A SIX-GENE METABOLIC SIGNATURE PREDICTING OVERALL SURVIVAL FOR PATIENTS WITH LUNG ADENOCARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: In the manuscript ‘Identification of a six-gene metabolic signature predicting overall survival for patients with lung adenocarcinoma’, using a bioinformatics approach the authors provide some interesting insights concerning the role of a six-gene metabolic signature in the prediction of survival in LUAD patients. Overall, it is a robustly conducted study and a well-written manuscript. However, some minor corrections need to be made before it is accepted to be published.

1.	The world ‘prognoses’ should be substituted by ‘prognosis’ throughout the text.
2.	In the ‘Gene Set Enrichment Analysis’ section of the results, the metabolism-related enriched pathways should be described further, including some of the key genes involved.
3.	Tables S6 and S7 should be moved in the main text, potentially as one merged table.
4.	Figure 1 should have at least a brief legend.
5.	The legend in figure 3 should provide some more details, i.e. what each graph corresponds to in each panel (A and B). A clearer alignment of the graphs would be useful, as well.
6.	The quality of figure 8 should be improved. The right panel is almost impossible to read without zooming in. Alternatively, some of the data could be presented in the form of a table.
7.	Figure 9 is redundantly complicated. It would be clearer if the figure was focused only on LUAD, instead of incorporating so many different cancer types. It may be worth keeping only some relevant cancer types, e.g. other lung cancers or cancers where LUAD could metastasise to.
8.	The legend of figure 10 needs elaboration/details. Show which protein shows elevated/ decreased expression for each corresponding transcript of the six-gene signature.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article suggested the Risk factors for new-onset atrial fibrillation in patients with chronic obstructive pulmonary disease. In general, this article bears significance and innovation. I recommend revision for this article. The following comments are provided for the authors' consideration before accept.
Experimental design: The Experimental design is good, as least in part.
Validity of the findings: The findings are helpful for the cardiologists and respiratory specialists.
Additional comments: This article suggested the Risk factors for new-onset atrial fibrillation in patients with chronic obstructive pulmonary disease. In general, this article bears significance and innovation. I recommend revision for this article. The following comments are provided for the authors' consideration before accept.

Major consideration
1. The search strategy is terrible and the authors shall used a combination of MeSH, Emtree and text word, and give a detailed search strategy including exp time, literature number of each database.
e.g. PubMed: please use methods of MeSH and text word.
Embase: Emtree
as well as Cochrane and ClinicalTrials.gov
2. The type setting is terrible and must be in accordance with the style of the journal.
3. The assessment of risk of bias is missing.
4. The subgroup analysis is missing. E.g. Retrospective cohort AND case-control; Asian OR Euro-America.
5. The funnel plot is missing.
6. The keywords is missing.

Minor consideration
1. The language needs revision by a native speaker, e.g. American Journal Experts.
2. Please give reference for Quantitative Analysis.
3. Please provide a PRISMA Checklist.
4. Inclusion/exclusion criteria shall be in detail.
5. Please cite the following literature in appropriate sites throughout the manuscript for a better understanding of background and discussion, and marked them in red clearly in the reference. PMID:
COPD: 30659954
atrial fibrillation: 28400980


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous English, good structure and sufficient literarure
Experimental design: Research question well defined. The data are well-presented, the study is methodologically right and the results are very interesting and fill an identified knowledge gap.
Validity of the findings: Important results, conclusions well-stated.
Additional comments: This study was designed to identify risk factors for new-onset AF in patients with COPD, and to propose recommendations for preventing AF in COPD patients and to assist clinical decision making.

The data are well-presented, the study is methodologically right and the results are very interesting. The study reports that cardiovascular and demographic factors are related to new-onset AF in patients with COPD. Despite the fact that cardiovascular related factors of non-COPD-associated AF are also considered as risk factors for  new-onset AF during COPD, influences are stronger in COPD patients. Interestingly, co-morbid  factors (diabetes mellitus, prior beta blocker use, hypertension, peripheral vascular disease and hepatic failure) identified in non COPD-associated AF are not associated with an increased risk of new-onset AF during COPD. This highlights the importance of distinguishing risk factors for AF in patients with COPD and the potential need for different preventive interventions.

The study deserves to be published.

Minor points

1) In my opinion I think that the title of the study should not include the phrase “based on 8,072,043 participants”. The title should be “Risk factors for new-onset atrial ﬁbrillation in patients with chronic obstructive pulmonary disease: a systematic review and meta-analysis”.
2) In page 14, line 276, “expiratory”, instead of “expiatory”.
3) In page 14, line 281, “streptococcus-induced pneumonia”, instead of “streptococcus pneumonia-induced pneumonia”.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Some of the wording is awkward and needs attention. For example, “In qualitative analysis, comparison of the risk factors for COPD-associated and identified non-COPD-associated AF.” (Lines 56–58). Careful proofreading by a native English speaker should identify and correct these issues. There are also various typos (e.g. “andthe” in Table 1) that could also be identified and corrected in this way.

Note a typo in each of Figures S1, S2, S3 (“ommited”).

There are sometimes missing spaces, e.g. the typo above, Line 138’s “StataSE14.0”, Table 2 and Figure 2’s “Black race(vs White)”, etc.

Sometimes the manuscript talks about “gender” (the social construct) and sometimes “sex” (the biological status), e.g. Table 1 includes both.

Rather than stating databases “including” those listed on Lines 106–107, an exhaustive list of databases should be provided at this point in the text.

There seems to be some potential confusion when describing the database searches (Lines 105–109) and review article checks (Line 107) which do not involve keyword searches (at least not in the same way).
Experimental design: When multiple adjusted models were identified, assuming this occurred during data extraction, how was the “adjusted” OR (Line 114) determined? Was this the most adjusted model and how were models that adjusted for variables potentially on the causal pathway as sensitivity analyses, for example, handled as part of this process for identifying the data to be extracted? Note that the most appropriate causal model could differ between risk factors also (a variable that is a confounder or competing exposure for one association could be a mediator or collider for another association).

While I commend the authors for contacting authors of studies where they were not able to extract necessary information (Lines 129–130), how often did this situation arise and how often did authors provide the requested data?

It should be made clearer what variables were examined for inter-rater reliability (Line 166, is this simply for studies being selected yes/no?) and exactly when this was done (pre-discussion?)

I would have preferred a more theory-driven/principled approach for deciding between using a fixed or a random (not “randomized”, Line 140) effects model as the interpretation of these is different. It would not, in particular, make sense to me to use both approaches for different exposures from the same study unless there was a clear and well-explained mechanism that would justify treating the factors differently. While statistical tests for and measures of heterogeneity are appealing in many ways, I would suggest a more principled approach be taken here (Lines 138–143) and suggest looking at sections 9.5 and 9.6 from the Cochrane Handbook. Can the authors justify their empirical strategy here?
Validity of the findings: The phrase “strongest risk factors” (e.g. Line 54) is problematic for me as the meaning of “strongest” here depends on the reader’s interpretation (for example, this could refer to the magnitude of the effect for some particular comparison involving that factor or the statistical significance of an association involving that factor). Neither of these can fully inform the clinical interpretation of the potential risk factors (for some variables at least) which will also potentially include modifiability and ease of assessment for screening variables. The use of a 50% increase in odds for identifying important factors is also potentially problematic for at least some variables as this depends crucially on the categories used for categorical variables (including the level used as the reference) and the units used for continuous variables (and note that this is not a 50% increase in “likelihood” as stated on Line 147, c.f. Line 186). For the former, an OR of 1.8 for one non-reference level and an OR of 1.1 for another non-reference level would, for equal numbers in each non-reference level, average out as less than 1.50 for a pooled non-reference level despite one category meeting this criterion (compared to the reference and also compared to a pooled category involving the lower risk non-reference level) showing also the importance of how dichotomous variables are determined. Did any of the studies report on results using three (or more) categories and if so how were these addressed (e.g. ethnicity as Black, White, and Hispanic or Other)? Or were all binary variables included using the same two labels and definitions throughout (this might also inform the choice of fixed versus random effects models)? Similarly, for the latter, an OR of 1.042 for one unit of a continuous variable would represent an OR of 1.51 for a 10 unit increase or a comparison between categories with means 10 units apart, but less than 50% higher odds for any smaller increment. Categorising continuous variables, such as age, cannot entirely avoid this problem as the number and widths of categories can potentially make the reported OR either greater or less than 1.5. I appreciate the desire to identify substantial risk factors, but the approach used here seems fraught to me outside of clear dichotomous variables (e.g. presence/absence of a well-defined co-morbidity). Was this always the case?

At the same time, this approach to identifying important factors ignores the precision/lack of precision communicated by the widths of the CIs. An OR of 2 with 95% CI limits of 0.5 and 8 would meet the criterion of OR > 1.5 despite the large uncertainty. Restricting these factors to only those statistically significant would then ignore an OR of 4 with 95% CI limits of 0.99 and 16.16 despite most of the 95% CI being above the criterion value (the interpretation here would be facilitated by credible intervals instead).

At the same time, the interpretation of any risk factor depends crucially on other variables included in the model. The clearest interpretation of the observed ORs depends on all necessary confounders being included but no colliders or intermediate variables, with competing exposures included to enhance precision. I appreciate that you cannot control the models used in each of the included studies, but I do feel that more consideration of these points (and perhaps some sensitivity analyses informed by them) is needed along with some incorporation of this as a limitation in the discussion. I wonder if an “ideal” DAG (directed acyclic graph) might not be useful for readers to appreciate how you see the causal associations that inform the interpretation of the published results, and such a model would seem likely to be of great use to researchers in this area.
Additional comments: My apologies for my slow review in these trying times.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed questions successfully.
Experimental design: The authors have addressed questions successfully.
Validity of the findings: The authors have addressed questions successfully.
Additional comments: The authors have addressed questions successfully.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous English, good structure and sufficient literarure.
Experimental design: Research question well defined. The data are well-presented, the study is methodologically right and the results are very interesting and fill an identified knowledge gap.
Validity of the findings: Important results, conclusions well-stated.This study investigated the differences between the risk factors of COPD-associated AF and the risk factors of non-COPD-associated AF. The authors reported that demographic characteristics have a significant impact on new-onset AF during COPD. Older age (over 65 years of age), men and whites are at higher risk of developing AF. Cardiovascular related factors (CAD, HF and CHF) are dominant factors as well, in this setting. Of note, co-morbid factors (DM, prior beta blocker use, hypertension, PVD and hepatic failure) identified in non-COPD-associated AF are not associated with increased risk of new-onset AF during COPD. Therefore, it seems to be of outmost importance to distinguish risk factors for AF in patients with COPD and to take appropriate preventive measures, especially in patients with a history of cardiovascular disease.
Additional comments: The authors have carefully revised the manuscript and performed the necessary changes according to the reviewers’ comments. The data are presented in detail, the study is methodologically right and the results are very interesting.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 2
Reviewer: 3

Output:
Basic reporting: Some specific/minor comments are below:

Line 49: I think you mean “included” rather than “including” here and don’t need the immediately preceding comma.

Line 146: Thank you for explaining that this was not necessary. I think some readers would also appreciate this information as a note around here.

Line 167: “wide” would be a clearer description than “large” when it comes to CIs (although, all other things being equal, larger ORs will also have wider CIs).

Lines 168–169: While the 95% CI includes/does not include the null value == non-/statistical significance rule works for linear models, it doesn’t quite work for binary outcomes as the standard error differs for the hypothesis test and the CI, so it is possible to generate data where the 95% (as you note on Line 167) CI for the OR is strictly above or below 1 and it is not statistically significant, and where the CI includes 1 but is statistically significant at the 0.05 level (as you note on Line 162).

Line 168: Similarly, “narrow” rather than “small”.

Lines 349–351: I think you mean that a limitation was that there was substantial heterogeneity. In response to this limitation, you then performed these additional analyses.

Line 365: Rather than “required” (which is past tense), do you mean “require”, or perhaps “recommend” or “suggest”.

Line 366: Rather than “the directed acyclic graph”, perhaps “causal modelling approaches such as directed acyclic graphs” as it is the process that matters rather than the specific technique used to achieve this.

Lines 366–367: Rather than “to distinguish the confider, collider, and mediator in risk factors”, perhaps “to distinguish between confounders, colliders, and mediators when considering causal risk factors”?

Table 1: Your column “Study Inception” shows the year the study started (its inception) and the year it finished (its conclusion), perhaps “Study period” would be more correct? Rather than “Adjustment” do you mean this to be a full set of “Independent variables”? For Miriam Warnier’s study, you list “sex, age,…” in the variables when you usually do this in the opposite order.

Figure 2: There seems to be a space in “bromide” (i.e. “brom ide”).

PRISMA checklist: You are missing a space after “page” for the first two items. You also vary capitalisation (e.g. “page” sometimes and others “Page”) in this column.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: Thank you for your constructive and informative responses and the edits to your manuscript. I feel that your work is much improved, although I do still have some questions remaining.

Sorry, but it’s still not absolutely clear to me how you determined the “adjusted model”. Did none of the articles from which data was extracted include more than one model where multiple variables were included? If so, this fact could be added around Line 145. The same point would apply to any article using forward selection, stepwise, backward elimination, LR test-based, or IC-based approaches where variables would not appear in the “adjusted” model if they were excluded because of these empirical model building strategies. The same reassurance, or warning, could be added around Line 125, 130, or 145. I note that in Table 1, for the “Adjustment” variables for Harsha Ganga’s study, you say “clinical variables that were significantly associated with AF” which would be a problem if they have used empirical variable selection, as it sounds like they have, but also this doesn’t tell the reader what was included in their model. The same point applies to “all other independent variables associated with AF” for Rashid Nadeem’s study. How did you decide between “age (<64, 65–74, and >75)” for Wei-Syun Hu’s study in Table 1 as this would provide two ORs but there was no overlap between the study IDs for >65 and >75 in Figure 2?

While I appreciate the removal of “strongest” for associations involving the independent variables, I’m still not completely convinced by the OR of 1.5 and above threshold (e.g. Line 48) for continuous variables (age here) that have been categorised. Perhaps as a better illustration, there is presumably some reference and non-reference age groups with an OR < 1.5 and others with an OR > 1.5 (including the two presented in Figure 2). So “age” (Line 49) by itself doesn’t have an OR of > 1.5, but “age over 65” would qualify (as you use on Lines 56–57 and 226–227, as examples). Related to this, it’s not clear to me what “education” in Figure 2 means. Is this completing high school versus not, per year of formal education, university/colleague education versus not, or something else? (I noted that “education” was mentioned in two of the studies in Table 1.) What does “Low HRQoL” mean in operational terms (I appreciate its semantic meaning, but what is the line between low and not-low HRQoL here)?

Also, if an OR > 1.5 is of interest, presumably ORs < 0.66… would also be of interest (as this depends only on the reference/non-reference group choice). I think it would be useful to add this around Lines 48, 165, 226, and 253.

You have very helpfully provided the reference categories for some variables (race, sex, urbanity, etc.) in Figure 2 (and for all relevant variables in Table 2). Can you add these for age (even if it is trivial for the age groups provided), education and Low HRQoL in Figure 2, along with some explanation of what they mean in the methods (HRQoL is mentioned by name on Line 254, but without explanation, and not listed in Table 1) and education is only listed in Table 1 without any further explanation.

I wasn’t able to see how the study IDs in Figure 2 linked to the actual studies. My apologies if I’m overlooking the obvious here.

Education is apparently from [16] only but there are two studies in Table 1 with education included (Kuang Ming Liao and Tomasz Rusinowicz). There are many studies in Table 1 with age as a covariate (18 I think), but only four in Figure 1, why is that? Similarly there are 16 with sex, but only 3 in Figure 1, which seems a very low rate to have included in the meta-analysis. Three studies mention smoking (one under “tobacco consumption”) and three mention BMI in the “Adjustment” column, what were the reasons for why these factors could not be looked at more closely? If these are due to the data extraction requirements, this seems important as a limitation to be added to the discussion.


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 3
Reviewer: 1

Output:
Basic reporting: Thank you for your thoughtful responses to my questions and the constructive revisions to your manuscript. I have a number of mostly minor queries and suggestions based on the revised manuscript.

Line 51: Remember that additivity (where +50% would be the inverse of -50%) is on the log-scale  and not the exponentiated/ratio scale. If the reference group had odds of 0.10 and the comparison group had odds of 0.15, this would be an OR of 0.15/0.10 = 1.50 (and so meet this criterion if the inequality was non-strict). However, if you reverse the reference/comparison groups, leaving the magnitude of the effect unchanged, the OR would then be 0.10/0.15 = 0.667 (3dp), a 33.3% (3dp) decrease in odds rather than a 50% decrease. The inverse of a 50% increase (Line 48) or an increase of one-half would be a one-third decrease and not a 50% decrease (c.f. Line 51). See also Lines 169–170 and 266 for the same point in the main text.

Lines 83–84: I looked at Mapel, et al. 2005 for these figures and the closest I could find for the 14.3% stated here was “Among COPD patients hospitalized in 1998, the prevalence of coronary artery disease, congestive heart failure, and atrial fibrillation were very high (33.6%, 24.4%, and 14.3%, respectively)” which given their focus on veterans didn’t seem to align with “For patients with COPD, the worldwide prevalence…” (Lines 82–83), is for those hospitalised only, and didn’t specify “new-onset” (Line 83, for which shouldn’t “prevalence” on Line 83 be “incidence”, c.f. Lines 85–86?). I couldn’t find a mention of the 40% mentioned on Line 84, but perhaps this is in a table or figure that I’m overlooking or you derived it from values in the article. See also Line 301 for the same 40% and Line 300 for a 17% overall prevalence (which I also couldn’t find in Mapel, et al. 2005 which is included here as a reference alongside Patel et al.). Also the journal title on Line 490 should be “COPD” and not “Copd”.

Line 88: Perhaps related to the above, I couldn’t find the 5.7% mortality in Mapel, et al. (or the 2.2%, but I’m assuming this is from the other reference?) despite the reference on Lines 89–90. If I’m not overlooking anything here, it might be worth checking that each reported number in the introduction and discussion can be located in the reference(s) provided. My sincere apologies if I’m overlooking these values in the references.

Line 136: Rather than “inconsistency”, perhaps “disagreement” here?

Lines 136–137: While consulting the third reviewer is a good step, it doesn’t in itself guarantee that the disagreement would be resolved. Perhaps “…would be consulted to resolve the disagreement.” You state this more explicitly on Line 142 for data extraction, so this edit would make the text for study selection more similar to that.

Line 137: I think you mean “inter-rater” here (i.e., between raters, as stated on Line 138) rather than “intra-rater” (i.e., within a single rater). You use the correct “inter-rate” on Line 195.

Line 137: Perhaps add “analysis” here to read: “An inter-rater reliability ANALYSIS was conducted to”, or “Inter-rater reliability was CALCULATED to…”

Line 153: Perhaps “used” rather than “conducted” here.

Line 155: Is “all authors” here referring to “J Liu and J Lu” (Line 151) plus all seven other authors? That seems a very large number of authors to resolve inconsistencies.

Lines 158–159: I’m not entirely sure what this sentence is intended to communicate in terms of the options chosen in Stata.

Line 160: Spurious “in” in “in the in”.

Line 165: Perhaps “used” rather than “prepared” here.

Line 171: Perhaps “95% CIs were used…of the ORs.”.

Lines 171 and 172: Perhaps add “A”/”a” before “[w]ide CI”, “narrow CI”, and “95% CI spans” here.

Lines 172–173: As explained previously, this “rule” doesn’t apply when the standard error differs between CIs (based on observed values) and hypothesis tests (based on null values). On Line 257, you say that “All of the un-pooled ORs showed the 95% CI and P value with consistency.” which I interpreted as meaning that the sentence here (Lines 172–173) was satisfied in Figure 2. However, note the presence of some “very near misses” in the pooled analyses, in particular short acting b-agonists (where the limit is 1.00–1.71 but the p-value is not sufficiently close to 0.050, being 0.040, to be explained by rounding) and long-acting b-agonists (where the CI is 0.96–2.34 and so the p-value should be just above 0.05 rather than the reported p=0.646) and violations in the unpooled analyses for venous thromboembolism (95% CI 1.17–1.44, so not including 1.0, but p=0.092) and OSA (95% CI 0.85–5.69, so including 1.0, but p<0.001).

Lines 196–200: Since you list the references for these 20 studies immediately below (Lines 200–207) broken down by study type, this initial listing of the references seems unnecessary.

Line 216: “and ONE scoring six of nine”.

Line 222: In all but here and one other instance (Figure 2 abbreviations), you have a space between “95%” and “CI”.

Lines 229, 237, and 337: Here you use beta for beta-agonists, but note you use “b” in Figure 2.

Lines 251–252: Here you give only the sensitivity analysis I-squared statistic, but on Lines 253 and 254 you give both the overall and sensitivity analysis values.

Lines 253 and 254: Here you only give the sensitivity analysis ORs, but on Line 252 you give both the overall and the sensitivity analysis ORs.

Line 268: Should the “+” symbol be “and” or “alongside”? See also Figure 2.

Line 286: Missing comma between “75” and “acute” in “age over 75 acute care encounter”.

Lines 332 and 333: This is perhaps stylistic, but the “1” in FEV1 is usually a subscript.

Lines 361–362: I think readers will appreciate a short statement here about the effects of removing Desai et al. as a sensitivity analysis (summarising Lines 248–255). Note that “Appendix 3. Sensitivity analysis” doesn’t seem to show these sensitivity analyses, but rather appears to show the individual studies. I’m not confident that I’m interpreting these supplementary figures correctly so perhaps more explanation can also be added to the supplement as well.

Line 371: You say “intermediate variables” here and on Line 377 you say “mediators”. Either is fine, but perhaps the reader will find this easier to follow if you use the same term in both places.

Table 1: Note (b) about education could delete “The”.
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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 4
Reviewer: 1

Output:
Basic reporting: See below.
Experimental design: See below.
Validity of the findings: See below.
Additional comments: Thank you for your very constructive responses to my queries and your thoughtful revisions.

I really have only one remaining query, albeit in two parts. First, you have added “Studies that reported ORs with 95% CIs and P values showed inconsistency were excluded.” (Lines 132–133) as part of your exclusion criteria, but this isn’t entirely clear in its intention. Do you mean that these were excluded on the basis that they were likely to have problems with their analyses and/or reporting (leading to the inconsistency) or something else?

Related to this, there are several reasons for why CIs and p-values can give (slightly) different interpretations of results in some cases when we move away from general linear models. As a trivial example, “csi 30 20 20 30, or” in Stata will show a RR (which for a rare outcome approximates the OR, although we are usually interested in the opposite direction for this approximation) where the 95% CI includes 1.0 (being 0.997, 2.256) but the p-value is below 0.05 (being 0.046). The OR has a 95% CI that is compatible with the reported p-value (being 1.016, 4.982 for Cornfield’s interval; or 1.011, 5.008 for Woolf’s interval), but the RR and its CI limits can of course be converted to ORs using the observed or an assumed prevalence where the incompatibility will again exist. Adding the exact option will report a two-sided Fisher’s exact test p-value (0.0713), which is compatible with the RR CI but not the OR CIs. In short, there are multiple ways of obtaining 95% CI limits for ORs (including Wald-based and from profiling the likelihood) and multiple ways of obtaining p-values (including those from Wald, LR, and score tests). Other issues can arise for more complex models, particularly, I believe, those involving weights and/or non-linear associations. Unless you wanted to go into detail and provide references for the potential sources of these inconsistencies, and I’m not aware of specific research on this issue for meta-analyses, I suggest deleting the text “In binary outcomes, however, when the standard error differs between CIs (based on observed values) and hypothesis tests (based on null values), it is possible to generate data with the 95% CI and P value showed inconsistency. This kind of situation basically does not happen in meta-analysis, as it can be avoided in original studies.” With the resolution of this issue in Table 2, this seems much less important to the reader now in any case. Of course, you could also expand on this section instead, but I suspect that this would become more of a distraction to readers.

I will also note a few small language issues.

Line 46: Here you say “during COPD”, where I think you mean “in COPD patients” (as you use on Line 44 and elsewhere), or perhaps “for those with COPD”. See also Lines 54, 55, 56, 61, 66, 97, 101, 229, 285, 286, 292, 298, 303, 313, 346, 402, and 408, and the caption for Figure 2 (and possibly elsewhere).

On Line 84 you give a category of “60–80%” % predicted FEV1, and on Line 85 you give FEV1 ≥ 80% % predicted as a separate category. Based only on this text, it’s not clear which category exactly 80% would belong to. The same point arises again on Lines 86 and 87 (although the “<60%” on Line 86 is entirely clear about the natural of the interval) and Lines 342 and 343.

Line 110: “referred” should be “Preferred” (being the “P” in “PRISMA”)

For Line 140, I suggest changing this to refer to the “the third reviewer (J Liu)” (as you did before) rather than saying that “it would be consulted” (it’s unclear what “it” is here).

In the revision to Lines 172–173, the added sentence: “As the inverse of a 50% increase in odds would be a 33.3% decrease in odds.” is a fragment (not a complete sentence). This could be corrected by removing “As”, by simply connecting it with the previous sentence without the current period (and either with or without a comma) and capitalisation, or the same but as a parenthetical remark.

Line 365’s “indicates” is a very strong word to use for results from observational studies. Perhaps “suggests” or similar would be more appropriate. It might be worth checking for any other overly strong causal language throughout the manuscript.

Line 369: “response” should be “respond”.

Line 370: “source” would read better as “sources” (there could be more than one).

Line 403: I suggest deleting “related to” so this reads “The dominant factors are cardiovascular (CAD, HF and CHF)…” (or you could reword this in another way.)


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: RISK FACTORS FOR NEW-ONSET ATRIAL FIBRILLATION IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY DISEASE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 5
Reviewer: 1

Output:
Basic reporting: No further comments.
Experimental design: No further comments.
Validity of the findings: No further comments.
Additional comments: Thank you for your revisions and responses. I have no additional comments to make about your 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: IDENTIFICATION OF SIGNIFICANT GENES SIGNATURES AND PROGNOSTIC BIOMARKERS IN CERVICAL SQUAMOUS CARCINOMA VIA BIOINFORMATIC DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: (1) Figures and tables need more improvement. Figure 1 shows wrong aspect ratio. The texts of last few lines in Figure 2 are hard to tell between the background color. Almost all figures have low resolution. Especially, the label of every node in Figure 3A isn’t clearly. Column Table 1 needs better description. E.g. “Expression” and “Genes Name”.

(2) Section “Materials & Methods” shows lots of bioinformatics analysis methods but lacks the citation of them.
Experimental design: Major comments:
(1) There is a lack of innovation in integrated bioinformatical analysis part. The main analysis pipeline is highly similar with Identification of key candidate genes and small molecule drugs in cervical cancer by bioinformatics strategy (https://doi.org/10.2147/CMAR.S171661).

(2) In section “Identification of differentially expressed genes”, you choose DEGseq as the DEG analysis tool. First, the citation of DEGseq is incorrect. You must check all citations carefully! Then, As “How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?” (https://doi.org/10.1261/rna.053959.115) said, DEGseq has unsatisfied effect between these similar tools such as edgeR and DESeq2. So could you explain why you choose DEGseq in this section?

Minor issues

(1) It needs more solid results to support the discuss. For example, the conjecture “highly expressed RFC4 may suggest early stage CESC, which indicates better overall survival” needs further comparison about the expression of RFC4 in samples at different stages.
Validity of the findings: no comment
Additional comments: This paper identified three differentially expressed genes on the prognosis of CESC by integrated bioinformatical analysis such as Gene Ontology, KEGG pathway analysis, PPI network and Kaplan-Meier survival analysis. Then, RFC4 was validated to be significantly associated with the occurrence and prognosis of CESC using clinical samples by quantitative transcription PCR, Western Blot and immunohistochemistry. Although the clinical validation of RFC4 has scientific merit, this paper needs further improvement to be ready for publication.


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: IDENTIFICATION OF SIGNIFICANT GENES SIGNATURES AND PROGNOSTIC BIOMARKERS IN CERVICAL SQUAMOUS CARCINOMA VIA BIOINFORMATIC DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors focused their attention in the identification of "significant gene signatures and prognostic biomarkers in CESC via integrated bioinformatical analysis". Although the authors used a reliable database (GEO) and molecular assays were conducted to validate their findings,  I do have some questions and comments for the authors. In particular, extensions, modifications and better explanations of their methods and results are suggested. In addition, a linguistic revision is needed.
Experimental design: 1. The manuscript is entitled "Identification of significant genes signatures and prognostic biomarkers in cervical squamous carcinoma via integrated bioinformatical analysis". What does "signficant genes signatures" means? Is it means "differentially expressed genes"? Furthermore, the microarray data obtained from GEO seems not original data, and the authors do not need to process the data by any bioinfomatics methods. Therefore, it should be a data mining analysis. For the above reasons, the title should be modified.
2. For K-M survival analysis, the authors should illustrate how to discretize the continuous gene expression. Furthermore, to estimate the hazard ratio of gene expression on patient survival, the authors should check the proportional hazards assumption.
3. The follow-up data should be provided, so that the readers can replicate the findings of the manuscript.
4. For the differential expression analysis, the standard should be consistent and do not need to described twice (in the methods and results).
5. The results of the enrichment analysis should be well explained, not merely list them out.
Validity of the findings: This work find that 3 genes were differentially expressed and associated with patient survival. Furthermore, the authors validated 1 gene in clinical specimens form RNA and protein level. Overall, this study is lack of innovation and workload.
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: IDENTIFICATION OF SIGNIFICANT GENES SIGNATURES AND PROGNOSTIC BIOMARKERS IN CERVICAL SQUAMOUS CARCINOMA VIA BIOINFORMATIC DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: It can be accepted.


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: IXODES SCAPULARIS MICROBIOME CORRELATES WITH LIFE STAGE, NOT THE PRESENCE OF HUMAN PATHOGENS, IN TICKS SUBMITTED FOR DIAGNOSTIC TESTING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors present an interesting study on the structure of microbial communities in the black legged tick Ixodes scapularis, a species of major medical interest in North America. While the diversity of bacteria endosymbionts and tick-borne pathogens (TBP) have been extensively studied in this tick species, the authors produced novel results using field collected specimens. They notably concluded that life stages, but not TBP presence/absence, is the strongest driver of microbiome composition. I particularly appreciated the use of statistics – an approach too rarely used in this kind of metabarcoding study. The text is globally well written, and the rationale is clear. However, I have some concerns, detailed below.
Experimental design: -	Materials and Methods: What about the negative controls? Controls are needed at each step of molecular protocols (extraction control, PCR control, kit control, etc). They are pivotal in metabarcoding studies because they allow the identification of bacterial contaminants that are widely present in the lab. Without these controls, it remains extremely difficult to identify contaminant bacteria, and thus to remove them from dataset before further analyses.
-	By the way, rarefaction curves comparing the number of 16S reads and the number of detected bacterial taxa are not presented. Did they showed plateaued asymptotes for each library? If not, it will indicate that sequencing depth was not enough to detect all bacterial taxa present. So, it is important to show that all DNA samples have produced good quality of data.
-	Materials and Methods: Some important details on tick specimens must be indicated there. The sex of adult and feeding status should be indicated in this section - not only at the end of the discussion (l.372). Most importantly, there is no detail on origins of samples: Were they from the same location? Were they collected during the same season? Over several years? Since spatiotemporal features covariate with structure of microbial communities in ticks, these details are of importance. To resume: Are these samples homogeneous from their origins? If not, it introduced some noise that deserve discussion.
Validity of the findings: -	As indicated in the discussion (l.372, and not in the Materials and Methods…), specimens are engorged. So, it means some detected TBP in tick specimens may be not ‘true’ members of tick microbiota but rather transitional passengers (only present in the blood meal and unable to further infect tick tissues). Alternatively, these TBP could have been recently acquired with this last blood meal, and have simply not enough time to interact with microbes present in the gut of ticks. This may explain why there is no apparent interaction between TBP and structure of microbial communities.
-	Engorged ticks may contain a lot of blood. Since the specimens used in this study were removed from humans, I expect that they were more or less engorged: substantial variation can exist between specimens. Such interindividual heterogeneity should be discussed because there are two consequences of this. First, blood is a powerful inhibitor of PCR and microbes are typically more difficult to amplify using fully engorged specimens. In this context, less 16S bacterial reads could be obtained from fully engorged specimens, introducing a bias when compared with less engorged specimens. Second, large amount of blood will massively dilute resident microbes and potentially introduce more TBP if patient was infected. Again, it may introduce another bias when compared with less engorged specimens. These variations could be included in analyses.
-	Line 404-410: the authors indicate that their ticks are potentially contaminated by bacteria from human skin because they have not cleaned the cuticles before extraction. They however stated that it is not important since internal environment (gut and ovaries) must harbor far more microbes than external surfaces (cuticles). Unfortunately, this is not entirely true. Most studies aiming to characterize tick microbiome wash specimens with ethanol, buffer or bleach. A recent analysis in another American tick species showed that cuticles consistently harbor a greatest diversity of microbes than internal organs, and that these microbes are a substantial source of contamination in metabarcoding studies (Binetruy et al 2020, Parasites & Vectors). So, the authors should softened their conclusion on this aspect.
Other comments:
-	Line 50: ‘obtained’ or ‘produced’ rather than ‘collected’.
-	Lines 107-107: This is entirely true for hard ticks (and thus including I. scapularis) but not for soft ticks that have several nymphal stages (and thus take more blood meals during their development).
Additional comments: This work is worthy of interest. However, they are some important concerns that must be considered before acceptance.


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: IXODES SCAPULARIS MICROBIOME CORRELATES WITH LIFE STAGE, NOT THE PRESENCE OF HUMAN PATHOGENS, IN TICKS SUBMITTED FOR DIAGNOSTIC TESTING
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figure legends should be added. Numerous relevant references are missing.
Experimental design: More methods needed for tick collection and processing.
Validity of the findings: Results should be placed into context of well-documented 16S low-biomass sequencing artifacts. Statistical tests, sample sizes, sample details, etc should be included in figure legends.
Additional comments: The current manuscript by Gil et al examines microbial diversity associated with wild-caught Ixodes scapularis ticks, using up-to-date 16S rRNA amplicon sequencing methods and careful analysis. The authors break down their ticks into groups based on detection of tick-borne pathogens by qPCR, and find that pathogen infection is not associated with an altered microbiome. In contrast, they find that life stage is associated with differences in alpha and beta diversity. The paper is well written, and the figures are well put together and clear. Overall, while there have been a number of tick microbiome papers in recent years, the question of differences in the microbiome as it relates to specific pathogen infection is unresolved and quite interesting.

Major comments:
-	More detail is required on methods regarding handling/storage of ticks prior to DNA extraction. Were the ticks alive or dead, and if dead for how long and stored under what conditions? These points could potentially influence the viability and abundance of tick-associated bacteria. Were ticks washed or externally sterilized prior to DNA extraction? This has commonly been performed in other papers to remove external contaminants. If washing was not performed, this needs to be acknowledged and the interpretation of detected microbes being internal needs to be revised.

-	I am concerned about the finding of higher diversity in nymphs. Nymphs are much smaller than adult ticks, and therefore the bacterial biomass associated with nymphs is correspondingly less than that of adults. There is a well-documented effect of biomass/input DNA on diversity metrics in the broader microbiome literature (see de Goffau et al 2018 Nature Microbiology; Erb-Downward et al 2020 mBio; Salter et al 2014 BMC Biology; and other relevant papers cited by these works. Also see Hammer et al 2019 FEMS Microbiology Letters for a discussion of best practices for microbiome analysis of low-biomass arthropod samples.). I am therefore concerned that the major finding of this paper (that diversity decreases with life stage) is an artifact of differences in input bacterial biomass between lifestages. Proposed methods for assessing/dealing with low biomass may not be feasible with the small nymphs used examined in this study (e.g. visual detection of bacteria via fixation, FISH, and imaging). The authors could use qPCR and known standard to roughly quantify bacterial copy number in nymphs (see Fig 1 Hammer et al PNAS 2017 for an example of this, also Couper 2019). Minimally, I would like to see the authors acknowledge and discuss this well-documented biomass-deep sequencing phenomenon in the Discussion.

-	The authors should provide detailed figure legends for each figure that explain (among other things) the sample sizes, statistical tests performed, etc. E.g. for Figure 1, are the data combined nymphs and adults?

-	Check spelling throughout, e.g.:
o	Table S4, “Analasma”
o	Figure 2, “diveristy”

-	The authors should cite a number of significant and relevant published references which are missing from this manuscript and are important for placing this work within the current state of knowledge:

o	Landesman et al 2019 FEMS Microbiology and Ecology; Landesman et al 2019 Ticks and Tick Borne Diseases. Both of these papers examine Eastern US-collected I. scapularis nymphal ticks, with the FEMS paper looking at over 400 samples.
o	Couper et al 2019 Ecology and Evolution. Found low diversity in Ixodes microbiomes (7 OTUs). Methods for reducing false signal from contamination.
o	Ross et al 2017 ISME found that alpha diversity differences between adult ticks (in particular between female and male ticks) were correlated with total bacterial 16S abundance by qPCR. This finding is in line with many studies demonstrating that low input biomass leads to artificially high alpha diversity driven by contaminants.


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: IXODES SCAPULARIS MICROBIOME CORRELATES WITH LIFE STAGE, NOT THE PRESENCE OF HUMAN PATHOGENS, IN TICKS SUBMITTED FOR DIAGNOSTIC TESTING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors responded to all reviewers' comments and modified the manuscript accordingly. Thank you for that. There are several typos to correct, eg at lines 81 (‘Babesia’), 113 (dot and space). Please also make clear that the microorganisms B. microti and B. miyamotoi do not belong to the same lineage, eg using term such as Ba. microti and Bo. miyamotoi.
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: IXODES SCAPULARIS MICROBIOME CORRELATES WITH LIFE STAGE, NOT THE PRESENCE OF HUMAN PATHOGENS, IN TICKS SUBMITTED FOR DIAGNOSTIC TESTING
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have expanded their citation of the literature in this resubmission, as recommended in the first review.
Experimental design: The authors have nicely revised their original manuscript with attention to reviewer concerns pertaining to low biomass samples.
Validity of the findings: No comment
Additional comments: The authors have done a nice job revising their manuscript in response to reviews. I think it is very much improved and should be accepted. I have a minor comment. Supplemental figure 3 (which is nice) has overlapping colors for some of the taxa (Klebsiella-Rickettsia, Family_comamonadaceae-Sphingomonas-Stenotrophomonas, etc). If possible I recommend expanding the color palette to allow for more differentiation, perhaps by using less "hot" colors. Finally, I was unable to see find the supplemental figure legends.


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: PHOSPHO-ISLANDS AND THE EVOLUTION OF PHOSPHORYLATED AMINO ACIDS IN MAMMALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript is well written and referenced.  It is also logically structured and well focused.

A few small issues I noticed:

line 109 and others – HMR used several times before definition

line 120 – ratention → retention
line 147 – mouse. → mouse

line 232 - “We consider the HMR set to be enriched in accurately predicted phosphosites”, wording conflicts with my understanding; the previous paragraph describes this set as the union of intersections between human-mouse and human-rat experimental annotations.  These are then used to MAKE predictions by transfer (as stated in the last sentence of the paragraph), but are not themselves predictions.

Fig 1C,D – Data exceed axis

Fig 2A,B,C,D – no axis, no ticks, some small illegible text on B and D.  A bit over stylized in my opinion, conventional axes for C and D would be more clear

Paragraph beginning 291 – incorrect references to supplementary figure 8

line 319 – No supplementary figure S8E

Supplementary Figures – two figures named S9
Experimental design: Generally good; selected datasets, computational methods, and analysis are appropriate.

One issue with design:  It is not clear why DR phosphosites are used for island modelling but OR phosphosites are excluded.  This is not justified in the text.  This division seems to complicate interpretation somewhat, since disorder and island are entangled in the model.  Why not model all phosphosites, then compare with disorder in the analysis.

Also, please clarify how DRs are defined, particularly when comparing homologs (do both need to be disordered, just one?)

Finally, line 423, Are non-phosphorylated ST amino acids required to be in DRs as well?  Not clear, but it seems that they should be to control for DR associated amino acid bias.
Validity of the findings: In general, conclusions are clear and supported by data.

This reviewer is a bit confused by finding related to predicted phosphosite islands throughout the manuscript.  Are these only phosphosite islands predicted in DRs?  Please clarify.

Related, the statement on line 315 is troubling: “We do not observe phospho-islands in ordered regions.”  This seems inaccurate since the data seems to suggest the opposite, since the distance distribution matches islands in DRs.  (1) This would not be an issue if all phosphosites were modeled, then compared with DRs. (2) An unexplored implication of this is that single sites are much more common in DRs than ORs, this seems unexpected and worth exploring, or at least discussing.

A bit of speculation, VSL2B is fairly sensitive to aromatics, including tyrosine, are OR islands enriched in Y relative to DR islands?
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: PHOSPHO-ISLANDS AND THE EVOLUTION OF PHOSPHORYLATED AMINO ACIDS IN MAMMALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written and thoroughly referenced throughout.

Acronyms should be defined explicitly at the first occurrence (HMR, HMM, etc)

Some spelling errors throughout (residues, amino acids etc) – please check this

In Fig 2,3,5 axes labels should be clarified. Figure 5 caption can be made more detailed.
Experimental design: The manuscript discusses the phenomenon of non-uniform distribution of phosphosites along protein sequences. The authors present an interesting evolutionary analysis to identify and characterize the properties of phosphorylation clusters (termed phospho-islands). While there is thorough comparison and discussion of the results in the context of existing literature, new results could be emphasized more clearly. In particular, can the authors extend their discussion of the implications of their findings related to the evolutionary patterns in phosphosites more generally?
Validity of the findings: The analysis is comprehensive and statistically sound. I believe two aspects of the data analysis should be addressed further. Specifically:

1) Prediction of disordered regions should be discussed in more detail. The authors should note the diversity of predictive methods for IDPs/IDRs. Would the variability in IDP definitions and prediction methods alter the results observed? How do the authors define DR and OR here? For varied definitions, would results still be statistically significant? In particular, as related to Figure 4 and associated discussion.

2) Notably, the authors should motivate why they use the Viterbi method (from 1967) for calculating phosphorylation clusters. Again, how sensitive are the results to the model?
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: DISTRIBUTION OF PHYLOGENETIC GROUPS, ADHESIN GENES, BIOFILM FORMATION, AND ANTIMICROBIAL RESISTANCE OF UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM HOSPITALIZED PATIENTS IN THAILAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, Tewawong et al. study the phylogenic grouping, antimicrobial resistance, adhesin gene presence and biofilm-forming capability of 208 Thai UPEC isolates. The work contains a considerable amount of information that will support UTI research in this area. The authors note in the Introduction that there has been a previous study on UPEC in Thailand but that this study provides novel data on phylogenic groups and biofilms.

The paper is well-structured and very well-referenced. Sufficient context for all aspects of the work is provided in the Introduction. The paper is self-contained and the authors write in clear and appropriate English throughout. Ethical approval was granted for the study. I thank the authors for including the raw dataset and uncropped images with this manuscript.

Minor typographical issues:

Line 58: There should be a comma after FimA.

Line 126: Should “cefoperazone/sulbactam” be written as “cefoperazone-sulbactam” for consistency with the other dual antibiotic discs used?

Line 238: Missing word – “This finding demonstrates that biofilm formation may be associated with [WHICH?] phylogenetic group.”

Fig 1B: “The pie chart show” does not need to be stated in the legend.

Table 4: Percentage scores are given in the table in parenthesis, therefore column headers should preferably be written “n = X (%)” rather than “n = X, %”.
Experimental design: A reasonable method of selection for the E. coli isolates is given, though there should be more detail on the confirmation methodology, given how closely-related E. coli is to some other species and the variety of biochemical differences between E. coli strains and pathovars.

Full details of primers and PCR conditions, antibiotic testing and Congo red biofilm analyses are provided. One aspect of concern is that the paper cited for Congo red biofilm assay relates to a Gram positive organism (Staphylococcus epidermidis) and not a Gram negative organism such as E. coli. Since Congo red reacts with various Gram negative cell-surface structures (e.g. Curli fimbriae, PMID: 26485271; type III secretion systems, PMID: 1802051) and is used as a specific assay for those molecules, I am not certain whether this methodology has been appropriately applied.

Issues to be addressed:

Lines 103-104: Please give brief details of the “standard biochemical tests” used to confirm the isolates were E. coli.

Lines 134-139: As noted above, I have concerns regarding the use of a Staphylococcus assay to test for biofilm formation in E. coli. The authors should comment on how this protocol was adjusted or adapted to fit the present study (or at the very least, whether this was considered at all). It would be preferable if the authors could show images of controls, e.g. well-characterised high- and no-biofilm producing strains, or cite a previous E. coli study. See also comments on Fig S2, below.

Lines 140-143: Data for statistical tests are well-tabulated in the Results section but there is no mention of adjusting for multiple comparisons. Was this carried out?

Fig 1A: The authors should state what negative control was used, e.g. water only or DNA from an unrelated species.

Fig S2: There is a typographical error (“very blank” rather than “very black”) on the image. Moreover, the rest of the segments are labelled with their biofilm properties (e.g. weak/none) rather than their colour – please make this consistent. The figure legend and method description in lines 137-139 suggest that “very black” should be “weak” biofilm formation, which does not correlate with the labelling on the image, where the “weak” labels seem to be on “almost-black” streaks. The authors should change the order of the biofilm descriptors so that they match the order of the colours in both the legend and methods section.
Validity of the findings: In general, I find the authors’ conclusions are reasonable given the data, despite my concerns regarding the biofilm assay. The Discussion is broad and takes into account a significant amount of previous work in the field, particularly when drawing links to past PCR-based studies. There are some minor issues as outlined below.

Lines 197-198: The authors explain the sex imbalance in the study using reasonable biological explanations, but since these explanations were not actually tested during the study, such a statement should be qualified by saying “most likely” or similar.

Lines 220-222: The authors state that three strains could not be assigned to phylogenetic groups “due to large-scale recombination events from two different groups or to genome plasticity driven by loss and gain of genes “. Since the authors did not perform an analysis that would enable them to make this claim and are simply relying upon PCR of a few small number of genes, their statement should again be speculative, as above.

Lines 241-243: The authors have stated that their p value cutoff is 0.05, so it is reasonable for them to make this statement, however it would be appropriate for the authors to mention their sfa result (p = 0.06) here, given how close it is to significance.

Line 247: Adhesin genes such as fimH are under strict control by phase variation in many strains (and some are “locked on” or “locked off” through mutation). The authors should note in their Discussion that the presence of adhesin genes certainly does not imply their expression. It would have been far more informative if the authors had been able to correlate expression of these genes rather than just their presence/absence by PCR – so a comment on this in the Discussion seems appropriate.

Lines 263-264: I’m not sure what claim the authors are making here – could this be rephrased?

Lines 265-266: Did the authors perform a statistical analysis to back up this claim? If so, please cite the relevant p values in the Results section – otherwise consider rephrasing this statement.
Additional comments: n/a


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: DISTRIBUTION OF PHYLOGENETIC GROUPS, ADHESIN GENES, BIOFILM FORMATION, AND ANTIMICROBIAL RESISTANCE OF UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM HOSPITALIZED PATIENTS IN THAILAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is well written, language is professional and easy to follow, with enough background information that make the big picture of this work very clear. A sentence in the end of introduction that evidences the main question of this work and how it fills a knowledge gap in the field be appreciated.
Experimental design: The material and methods are very well described with sufficient detail or referenced when appropriate.
Validity of the findings: This work analyzed a large collection of UPEC samples for phylogenetic groups, presence of adhesins, biofilm formation and antimicrobial resistance and it provides interesting results that were analyzed in a very clear and organized way.  If there is a weakness, is that it misses statistical analysis (as noted below in general comments for the author), to show significance of the findings and give the work a higher impact.
Additional comments: Specific comments for Results section

Please mention table 2 in results section
Was the association between phylogenetic groups and adhesins statistically significant? It is not clear if statistical analysis was performed in this case? IF not, it would be interested to see if these associations were significant. Same applies to the relationship between antimicrobial resistance genes and phylogenetic groups.
The correlation found between pap adhesin gene and biofilm formation seems to be negative in this case, as the prevalence of pap was lower in strong biofilm formers than in weak and moderate. Please make it clear in the text (lines 192-194)
Table 1: OK
Table 2: not mentioned in the text (line 166); missing stats analysis
Table 3: missing stats analysis
Table 4: missing stats analysis
Table 5: OK
Table 6: OK
Figure 1: OK
Figure 2: OK. Seems like there could be a significant difference in resistance to ciprofloxacin, norfloxacin and levofloxacin between biofilm producer and non producer, but there is no statistical analysis to support it.
Supplemental material
Table S1 and S2: please fix title of table to start with “Sequence of oligonucleotides…” or “Sequence of primers…”.
Figure S2: according to the results section, “very black” correspond to weak biofilm. However, in this figure you have “very black” and “weak” as different things. Please clarify.


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: DISTRIBUTION OF PHYLOGENETIC GROUPS, ADHESIN GENES, BIOFILM FORMATION, AND ANTIMICROBIAL RESISTANCE OF UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM HOSPITALIZED PATIENTS IN THAILAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised the manuscript as recommended and I have no further scientific concerns. There are some minor issues with English that have crept in as a result of the modifications, so the authors may wish to double-check the wording of the revised text. For example, the use of plural vs singular words is sometimes incorrect (e.g. line 187: "association" should be "associations"; line 142: "organism were" should be "organisms were").
Experimental design: The authors have clarified aspects of their experimental design that I queried previously. The manuscript has been much improved by these additional details.
Validity of the findings: The authors have revised their manuscript's Discussion appropriately; in particular, they have noted where their conclusions are drawn from the wider literature as opposed to their own results. Again, this improves the manuscript considerably.
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: DISTRIBUTION OF PHYLOGENETIC GROUPS, ADHESIN GENES, BIOFILM FORMATION, AND ANTIMICROBIAL RESISTANCE OF UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM HOSPITALIZED PATIENTS IN THAILAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: No additional comments.
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: COMPARATIVE GENOMIC ANALYSIS OF THE PRINCIPAL CRYPTOSPORIDIUM SPECIES THAT INFECT HUMANS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See the general comments section
Experimental design: See the general comments section
Validity of the findings: See the general comments section
Additional comments: The report describes the results of the phylogenetic studies of Three Cryptosporidium species based on genome-wide SNV analysis. The report is straightforward and well prepared. I have mostly minor issues for the authors to consider.

Major comments:
1.	L224-227 and Fig 1. The reason for the high SNVs within C. hominis and C. meleagridis is not clear. Was the C. parvum IOWA genome used as reference? This this is the case, the data are really not on intraspecies diversity as the descriptions implied.
2.	The reason for different clustering by C. hominis isolate 30976 should be discussed in the context of genetic recombination. See: Guo, Y., Tang, K., Rowe, L.A., Li, N., Roellig, D.M., Knipe, K., Frace, M., Yang, C., Feng, Y., Xiao, L., 2015. Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum. BMC genomics 16, 320.

Other suggestions:
L22, L266. No need to capitalize the word Apicomplexan.
L24. Delete the word The.
L47. A parenthesis symbol is missing before “Furthermore”.
L137-138. The reads were extended. What does it mean?
L187, L209, L229. These subtitles are for the Results section. Please modify them.
L290. Delete the word coding.
Table 2. What is the differences between the last two columns of the table, which are both labeled as “Mean coverage of read mapping (X) “.