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: ANALYSIS OF MICROBIOTA IN ELDERLY PATIENTS WITH ACUTE CEREBRAL INFARCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study by Huang et al aims to identify microbiota changes in patients with acute cerebral infarction so as to identify a causal relationship. Despite a relevant attempt, there are several discrepancies associated with the manuscript:

Authors argue that there are no published reports identifying a causal link between Cerebral Infarction and microbiota (Line 171). As far as this reviewer can tell, a recent report has identified several microbial changes in the same line of work (Ref: MOLECULAR MEDICINE REPORTS 16: 5413-5417, 2017) which is in sharp contrast to work presented in this paper. In this reviewer’s opinion, authors need to discuss the significance of their results in wake of any relevant published work, especially as stated above.

Lines 188 and 200 directly contradict each other. Please proof read.
Experimental design: There is no mention of how the blood biochemistry assays were performed. Authors have neither described the results of variations in the blood tests (provide a separate bar diagram and not in Table 1) and nor discussed any statistical changes within the normal and disease groups. As per Table 1, it seems a significance test is applied but it is not clear. For instance, in Table 1, the parameter Cr has p value of 0.13 which means that the difference is non-significant. This implies that the parameter is not a marker of the disease and thus finding a correlation would be meaningless. Please address this issue.

The statistics applied is not clear. For instance, Table 1 suggests three different significance tests were performed. Why so? Also, while reporting values please mention the relevant SD or SEM and not the range. Preferably, report with a bar diagram or a box-whisker plot.

I also have a concern with the huge variation in sampling numbers. The control group has only 9 subjects while as many as 31 subjects of cerebral infarction were considered. This could skew the data interpretation. For instance, abundance of Blautia in both normal and patients seems to have a similar range, despite the observed significant change. Please elaborate this in relation to the chosen statistical method.

The data on correlation between bacteria taxa and presumed risk factors is very crude. The correlation observed is very poor as R2 value is closer to 0/0.1 and hence doesn’t appear to be very significant. In general, the blood tests and correlation performed render very little conclusive data. Please discuss.

State the median age of sample subjects in the methods section.

Split Table 1. Only include subject characteristics, while blood biochemistry results should be reported separately.
Validity of the findings: Please discuss a causative link between gut dysbiosis and how it could actually promote risk of infarction to emphasize your results.

While discussing results, please state any changes observed in terms of percent increase or decrease preceding with statistical significance and p value.

Please state a hypothesis of your work in the introduction section and how you plan to check it. Then based on your observations, discuss whether you accept or reject your hypothesis in the discussion section.

State limitations of the study in a separate paragraph.

Please state Ethics committee approval number with date.
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: ANALYSIS OF MICROBIOTA IN ELDERLY PATIENTS WITH ACUTE CEREBRAL INFARCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper on analysis of microbiota in patients with Acute Cerebral Infarction is interesting. The authors have analyzed the contribution of gut microbiota (GM) in the development of acute cerebral infarction (ACI). Authors compared the GM and blood samples of 9 healthy individuals and 31 individuals with ACI and demonstrated that (1) abundances of Blautia obeum, Streptococcus infantis and Prevotella copri play role in the development of ACI, (2) a negative correlation between Blautia and WBC, and a positive correlation between Streptococcus and creatinine and lipoprotein and, (3) desired biological pathways in GM of patients with ACI are enriched.
Though, this study claims that B. obeum, S. infantis and P. copri play role in development of ACI, I find results unconvincing. Sample size is small. In figure 2B, it seems that if number of healthy persons will be increased, there will not be any significant difference in the abundance of B. obeum between healthy individuals (N) and patients (P). Similarly, for the abundance of S. infantis and P. copri, several patients have relative abundances similar to the healthy individuals. Also figure 3B does not show obvious differences in the average pathway abundance between N and P. These scenarios make me doubtful to say that these bacteria can influence the development of AIS. Along this line, authors also claim that overall composition of GM is similar in healthy individuals and patients, and P. copri does not have any relation to any disease risk factors. What we see here in results could result from the differences in dietary habits and age. It is advised that manuscript should report and interpret above facts, and authors should avoid from making any solid claims.

Minor comments:
1.	Introduction needs to be more descriptive.
2.	Line 126: define OTU.
3.	Line 130: define PLS-DA.
4.	Line 145: correct (Figure 2.)
5.	Line 155: correct (Figure. 3)
6.	Line 158: change “inhabited” to “present” or “prevalent” or as needed.
7.	Line 179: correct reference style “colon[16].”
8.	Line 200: it seems that “relatively low abundance” should be “relatively high abundance”.
9.	Mention following in full when they appear first time in the manuscript: Blautia obeum, Streptococcus infantis, Prevotella copri, acute ischemic stroke.
10.	Change Acute Ischemic Stroke to Acute Cerebral Infarction in the text, which would be consistent with the manuscript title.


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: ANALYSIS OF MICROBIOTA IN ELDERLY PATIENTS WITH ACUTE CEREBRAL INFARCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have responded to earlier issues. However, there are several grammatical errors that need to be corrected before publication.
Experimental design: The median age of subjects is reported as 61 years. I advise authors to incorporate the word 'elderly/aged' in the manuscript title so as to accurately reflect the study context.
Validity of the findings: No comment
Additional comments: Authors have improved the manuscript based on suggested changes. Further changes recommended are general in nature.


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: ANALYSIS OF MICROBIOTA IN ELDERLY PATIENTS WITH ACUTE CEREBRAL INFARCTION
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have justified my previously raised issues. The revised manuscript has numerous typos and I suggest that authors should thoroughly re-review the manuscript for typos.

Minor corrections
1. At multiple places genus and species are not italic.
2. Genus and species should be written in full when they appear first time, and afterword genus should be abbreviated.
3. Change “Lipoprotein” to “lipoprotein”, when it appears in the middle of a sentence.
4. Line 102: Full stop is not properly spaced at the end of sentence “…coronary heart disease and renal dysfunction”.
5. Lines 107-108: air tight should be airtight.
6. Line 120. Properly place the comma at “The venous blood samples”.
7. Lines 130-131: TaKaRa should be Takara.
8. Line 142: Full stop is not properly spaced at the end of sentence “…categorical variables using SPSS”.
9. Line 152: “dysfunctionin” should be “disfunction”.
10. Line 239: “leading to production of” should be “leading to the production of”.
11: Line 243: Space after full stop “2013).There is growing evidence”.
12: Line 199: trimethylamine oxide should be TMAO.
13: Line 273: MTAO should be TMAO.


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: COASTAL FISH ASSEMBLAGES AND PREDATION PRESSURE IN NORTHERN-CENTRAL CHILEAN LESSONIA TRABECULATA KELP FORESTS AND BARREN GROUNDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: While the English is generally very good there are areas that could be improved for clarity (see below). It is also worth noting that you can generally get away with not putting ‘the’ in front of a large number of words. I generally read aloud and if it doesn’t affect meaning get rid of ‘the’. The authors also use ‘herein’ throughout the manuscript and this at times isn’t helpful or breaks up flow. I would suggest not using this term.

The introduction provides a good background to the study. Specific comments below:
Line 50 heatwaves is one word
Line 53 Add the before absence
Line 57 Suggest changing to Grazing activity is…..
Line 58 Delete These
Line 66 Suggest changing sentence structure to “ In these cases, high predator abundances control grazers leading to increased algal biomass.
Line 70 Delete ‘however’ and replace with “Outside of protected areas….
Line 76 Delete “mentioned as” and replaced with “considered”
Line 78-80 It is not clear to me why there is discussion of sea stars when they are not the focus of the study. Moreover, given their role of predators of urchins it is interesting that they are not mentioned being potentially responsible for predation observed in this study. Were these species observed by the videos or are they present at the study locations?
Line 83-86 Suggest restructuring this sentence to “All these species have been subject to intense fishing activity with population declines…..
Line 88 Are the authors suggesting that the role of fish predation in controlling urchin numbers is less well known? I suggest that the authors clarify this point.
Line 89-91 This sentence needs a reference

The figures and tables are relevant, but I feel that more information is required in many of the legends and the figures 4-7 are messy in places.
Table S1 KF and BG need to be defined in the table legend. Also I am guessing that O should be W for a paper written in English
Figure 1 The authors might consider including different coloured symbols to denote different management regimes
Figure 4-7 The authors need to explain what the point symbols represent (I am guessing raw data). I also found their placement a little random. Have they been jittered? If so I wonder if it would be better to jitter in the vertical rather than the horizontal? Also the lines, boxes and error bars need to be defined to enable the reader to interpret the data.
Fig 5 & 6 I suggest the authors explain that the numbers on the outliers represent FO and Nmax respectfully.
Fig 7 Suggest rephrasing to Percentage of a) T. niger (urchin) and b) P. laevigatus (crab) consumed at …….

The structure conforms to peerj standards and I can confirm that the raw material has been supplied
Experimental design: The research question is well defined and the methods are generally described in enough detail (but see specific comments on how to improve clarity). The design is adequate, although not ideal and the authors need to be careful how they interpret the results. For example, there are sites where both kelp and barrens areas are located within 1 km and then an additional two sites: one a barrens site and one a kelp site. Obviously a more balanced design with site pairs would have been better.

The study seeks to compare fish richness and the FO and NMax of the three most abundant species. They do this by using baited video (analysing 80 still frames over a 40 minute period) that are positioned horizontally and downward. In addition they tether two prey items (urchins and crabs) to plates within the field of view of the videos. Video footage is used to identify species removing the prey items (only 40 mins of film) and then predation is measured after 24 hours.

The data for the study is collected over two days at each sites, but the full dataset is collected over one year with data collected across all seasons. The authors mention seasonality in the discussion and given that fish vary so much in space and time I suggest that the authors provide some evidence base in the methods/discussion to suggest that while the data were collected over very different seasons that the data is still comparable because the fish they were studying are non-migratory or don’t vary in terms of abundance through time. It might be worth the authors stating that any such migratory fish were excluded.

My other major concern regards the placement of the cameras. The authors state that this is within 10m of each other, which is very close and certainly means that fish would have been able to swim between cameras therefore invalidating assumptions of independence. I believe this deployment needs some justification.

Specific comments
Line 99 Suggest starting sentence “The study…” as this isn’t describing the predation assays
Line 116 What does deep-type refer to in the context of barrens?
Line 118-120 Two study sites were separated by 500m - to 2km. It would be useful for the authors to explain and provide an evidence base for whether fish identified in the study could move between these sites.
Line 120 Remove miles around and change to kilometres
Line 145 Delete to take and replace with allowed images to be taken….
Line 147 Delete allowed taking and replace with took
Lines 149-153 It would be useful to include references to support the statement made regarding the camera set up and pros and cons. I also think the authors need to make it clear that the horizontal camera only provides information within the field of view rather than providing information for the whole surrounding area.
Line 154 Should be mesh. And what is an eternity plate?
Line 158 Add us after allowed
Lines 160-162 Suggest the authors restructure. At each study site, five vertical and five horizontal structures were randomly distributed.
Line 167 Suggest here and throughout that the authors refer to the crabs and urchins as prey items rather than bait.
Lines 167-170 The authors need to make it clear that this design was not used at BG.
Line 180 How did the authors determine where 2m was in the field or view?
Line 183 Often people exclude a period of time at the beginning of the video because of potential disturbance in placing the cameras. Are the authors sure that disturbance may not have affected the early frames used in their study.
Predation assays  - I think it is important that the authors make it clear that there is only video evidence of predation events for the first 40 mins. After this period the authors do not have a definitive knowledge of which species was responsible for predation events after this time.
Line 204 Change to monofilament fishing line
Data analysis – The authors need to make it clear to the reader that the two days data were combined for all analyses. Also I suggest that the authors refer to habitat (2 levels: kelp forest and barrens) instead of community as it is a little confusing.
Line 228 I am a little  confused with the design used for the FO and Nmax analysis. Where does species list and replicates come from?
Validity of the findings: I believe the authors need to be very careful with the interpretation of their findings. There are a number of statements made in the discussion, which are just not backed up by the data. I therefore think that the discussion needs a significant restructure to reflect what can be said based on the outcomes of this research. The results, as presented, suggest only limited significant effect i.e. one site being higher lower than another rather than a consistent response across habitats. This therefore means there is little support for some of the general statements which suggest consistent patterns. I believe the authors need to honest regarding their data and admit the patterns are not hugely clear.

Lines 343-362 Discuss how no take zones can lead to increased richness. However, the data presented in this paper doesn’t support this. The one protected zone did not have richness, FO or Nmax compared to the fished sites so the statements made here are not justified by the data.
Lines 363-369 There was no difference in predation on barren versus kelp areas therefore it is impossible to say that prey were more accessible on barren grounds
Lines 371-375 I really don’t think the data presented in Fig 6 supports the statement for a slight tendency for higher FO in kelp forests. There was no significant difference and FO in kelp forests were just highly variable.
Lines 390-391 Again this pattern is not general there was only one significant pair wise comparison between habitats for the two most common species with no differences across other sites and habitats. For the third most abundant there was no effect of either location or habitat.
Lines 407-410 This needs to toned down as it wasn’t tested for in this study. Is information known on what size fish take the urchins put out in this experiment?
Lines 411-418 The authors raise issues regarding seasonality and metabolic rates, but more importantly highlight that because data was collected at different times of the year it could have impacted on the observed responses. The issue of sampling during different time periods needs addressing and especially whether it could impact expectation regarding measures of richness, FO and Nmax.
For all stats in the results only the p-values are provided. This isn’t enough information and I suggest that the authors either include in-text stats information or provide output tables in the SOM that are referred to in the main text. Also it appears that there were only limited significant post-hoc pair-wise comparisons. In places it is cleary stated that there were no other significant differences. I just encourage the authors to ensure this is the case everywhere.
Abstract Line 35-38 I suggest the authors need to rephrase this last sentence as it is just not supported by the data presented.

Minor comments
Lines 253-261 I suggest that the authors make it clear that some of the fish mentioned were only found at one site. I think the last sentence is a little strong when the authors are presenting qualitative data and the difference is only once species. When this data is formally analysed there is no significant difference.
Line 262-267 I was surprised at the low richness in all sites and habitats. It came as a surprise to me and I wonder if it is worth just making this general statement.
Line 271 Refer to the figure
Line 275-278 Surely the main test is the most powerful and if your post-hoc tests of a significant main test are coming back non-significant than the post-hoc tests you are using is too conservative.
Line 298 Suggest restructuring sentence to “Predation pressure for both types of prey items did not vary across locations or habitats
Lines 304-306 Suggest restructuring this sentence for greater clarity
Additional comments: The research presented is interesting, but I just think that the results have been over interpreted.


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: COASTAL FISH ASSEMBLAGES AND PREDATION PRESSURE IN NORTHERN-CENTRAL CHILEAN LESSONIA TRABECULATA KELP FORESTS AND BARREN GROUNDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present paper aims to understand he differences in predation pressure in two contrasting habitat types commonly occurred in temperate reefs (i.e., kelp forests and urchin-dominated barren grounds). Authors compare species richness, abundance and predation impacts of predatory reef fishes in the two contrasting habitat types using underwater video analysis. While the subject of interests matters in terms of potential mechanism that may underlie community shifts in kelp forest ecosystems, I consider that the manuscript have potential to be published after improving some areas and aspect of the study which I will summarize below: a) the problem (motivation) raised in the introduction, need better arguments centered in the hypothesis tested; b) this is linked to the materials and methods (hence experimental design and overly analyzed data) where I found that the manuscript has some editorial problems in terms of simplifying what wants to explain particularly. there are many paragraphs explaining something that could easily be explained in one short paragraphs; c) I found where most work has to be done in order to provide a compelling evidence of a lack of difference in these contrasting habitat types is in explaining and justifying the methodology and data analyzes: 1) vertical views versus horizontal views of data frame, insufficient number of cameras, 2) MxN of 30 second per frame do not avoid double counting of reef fishes (please see Malcolm, H., & Speare, P. (2003). Potential of video techniques to monitor diversity, abundance and size of fish in studies of marine protected areas. Aquatic Protected Areas-what works best and how do we know, 455-464), and 3) selected tethered prey, one prey requires large carnivores to enter your small experimental arena and there are no potential cage (or structures built in controls) and as much as I acknowledge (to be honest) to have plenty of my own articles cited in this manuscript  (except one, Perez-Matus et al. 2016 MEPS where we compare, among other stuff, tethering of cryptic reef fish for a short period of time in order to avoid escapes and finding differences in predation pressure in kelp versus non kelp habitat) I found invaluable a critique of tethering experiments to estimate predation pressure in the field without sensitivity (behavioral analysis) (please refer to Aronson RB, Heck KL Jr (1995) Tethering experiments and hypothesis testing in ecology. Mar Ecol Prog Ser 121: 307−309). These points raised above definitely leads to d) interpretation of data, although I found the discussion a bit lengthy authors tried to explain their results.

As a final comment I feel that they have a good working material (e.g handling time, effective consumption, first arrival time to the experimental arena) but authors are not taking all the relevant information that can be obtained from them and they should take advantage of footage and comments such as in lines 256-258 and other should be incorporate in the fancy statistical procedure (will get back to that shortly).
Experimental design: The experimental design is straight forward, I know how hard it is to work in the system and having few o more replicated means a lot of work in the field, but how many cameras (replicates did you used? at what depth?) But given the great difference between the sites in terms of density and morphologies of the kelp forest, I think that the work should be re-analyzed not in two categories only (KF and BG) but in factors of density of the kelp (probably three categories of this). There are some flaws in explaining how data was analyzed and the rationale of using such as framework (using replicates as random parameter to avoid over dispersion is somewhat misleading) and lastly I  do not understand the justification of using two techniques to estimate abundance. I think that each one allows to obtain different useful information for the work. In terms of The generalized linear mixed effects model (GLMM) approach need to be better described as it seems that you conducted two different analyses (see lines 217-241). I still think that I feel that it is not appropriate for the data. I would like to see a very concise description of the model validation (see Alan Zurr and colleagues outline these nicely). I would expect to see the following checks: Check for outliers and influential points, linearity between the predictor variables and the quasipoisson- or quasibinomial-transformed response variable, Check for multicollinearity among predictor variables by, for example, variance inflation factor (VIF) analysis, Check for spatial autocorrelation of model residuals using, for example, spline correlograms. In reference to the GLMMs, what is meant by “sample replicates treated as random effects to avoid  autocorrelation", if so these should be nested in locations? I think that site need to be treated as a random effect due to site-level clustering of the data ( I will suggest doing that) and the degree of clustering should be reported by, for example, the intraclass correlation coefficient (ICC) or the Variance Partition Coefficient (VPC) variance of higher level divided by the sum of variances of site and the higher-level variance of our predictor variable.
If using GLLMs, need to report the residual df on the model df, also the effect sizes and p - values. I think you don't have much zeros so poisson error is ok, but pressure over dispersion. I recommend you use all the length of the videos, specify the rationale of using vertical and horizontal views (there are in different scales), speared frames for 5 min (or more than a min) to avoid double counts. Also if you know the scale of you trapezoid frames, in your plates you can calculate the size of the fish (see lines 292-293) and then convert to biomass (see Perez-Matus et al. 2014 RBMO).
Validity of the findings: Much of the introduction in centered on the community effect of predation of urchins but few on the use of the porcelain crab which is not a grazer. I think this need to be justified as per lines 206-208 This would serve for a prey preference analysis, however both have very different exposures to their predators in the environment. For the rest, since it is not part of the question, the most convenient and informative would have been using grazer species that depredate the kelp, and therefore a niche overlap with the other tethered prey. Let, in terms of tether how can you estimate the escapes?

Need to be explained why horizontal versus vertical views
Avoid some redundancy in methods (see lines 163)
Also, richness was evaluated but no rationale explained for that, in lines 176 was conveyed that not all species were counted but some reported.
Additional comments: I have tried to be constructive in my assessment of the ms, I would like to see this study out but a re-analysis of the videos and the data can be helpful to provide information about te predation pressure and differences between BG vs KF. I consider your paper valuable as most of the fishing on KF may potentially be transformed to BG s and data is valuable in terms of the interactions and potential prediction of maintenance of contrasting communities.

Good luck in revising this manuscripts


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: COASTAL FISH ASSEMBLAGES AND PREDATION PRESSURE IN NORTHERN-CENTRAL CHILEAN LESSONIA TRABECULATA KELP FORESTS AND BARREN GROUNDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors present a nice experimental approach to study the effect of fish on grazers to explain contrasting communities (barren grounds vs kelp forests). In general, the authors made a good job managing the existing literature, but some local literature are needed at least from my perspective in different subjects:
i)	The authors must cite a highly relevant kelp-grazing paper for Chile Dayton 1985 Ecol Monogr Vol 55
ii)	The discussion should mention that Tegula atra can reduce kelp recruits but also other competing algae (Henríquez et al. JPhycol 2011)
iii)	There are previous studies comparing recruitment success between non-kelp kelp habitats (Almanza et al.  Mar Biol Res 2012 and Almanza et al. 2013 Int J Env and Sust Dev)
In addition, the authors need to be more precise in some statements as the authors indicate that:
i)	The authors indicate “Kelp forest are declining globally” citing Krumhansl et al 2016 (Line 47-48) which is not completely the case as only a proportion of the places in Chile is showing this trend and actually in Chile only in the central part of the country.
ii)	The authors indicate that “In the absence of forests there are extensive patches of ….. called barren grounds”.  The paper cited ibn the same study (Filbee-Dexter & Scheibling 2014) show that and alternative state is produced monopolized by turf algae which are not a barren ground.
In addition, please add in the figure legends what are the box plots and dots indicating (SD, range, other?).
Statistical analysis. In the text the authors provide only then the p values (e.g. lines 282 and 283). There is a need to provide more detailed statistical information. Add a table or include it in the parenthesis.
Finally, the Abstract must be modified. The first sentence is not completely valid (see Filbee-Dexter & Scheibling 2014) and the second sentence must also take into account that human action can be part of the explanation and not only the direct impact of grazers (this is acknowledged in the introduction (line 52) but the authors should not bias the abstract).
Some additional minor elements:
i)	Line 73; provide the Lessonia species name,
ii)	Line 176; The authors use abbreviation FO and MaxN but not for species richness. If the authors use abbreviations for these variables used for the 3 ones or for none of them.
Experimental design: The experimental design and the number of study sites is acceptable considering the logistic difficulties to carry out this type of studies. I only demand some more detailed statistical information in the text.
Validity of the findings: The finding's that this manuscript provide about the relevance of barren grounds are valid and offer an explanations in relation to the  importance of barter grounds for foraging. The Conclusion section is clear for me.
Additional comments: By answering the points raised in the "Basic reporting" section the paper should be ready for a final review.


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: FINE-ROOT TRAITS OF ALLELOPATHIC RICE AT THE SEEDLING STAGE AND THEIR RELATIONSHIP WITH ALLELOPATHIC POTENTIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: The current article has a sound experimental design and clear results, while a more sufficient research background, a thorough discussion, and a less speculate conclusion are needed.
Experimental design: The experiments design was simple to follow. However, working hypotheses were not dressed clearly.

The authors should give sufficient background why the definition of fine roots in
the current article is 0.2mm, which is not common in this field.
Experiment methods should have citations to show its validity. If the authors inserted any modification, you should also describe where and why the original protocol had been modified.

The statistical analysis focused only on species-variation but not development stages, e.g. data in table one are suitable for two-way ANOVA.
Validity of the findings: 1. The correlation between root traits and allelopathy potential is novel and interesting.
2. The author should pay attention that neither the previous studies nor the current study probably indicated any causality between these factors.

3. Most of the results were clear and rigorous except that the comparison of total phenolic acids among allelopathic rice species and non-allelopathic one which I would like to suggest a comparison not on whole plant base but root mass (or length) base.

4. The discussion on the correlation between root traits and allelopathy potential is not sufficient. Possible mechanisms and significance of this correlation should be included in the discussion.

5. The function of each phenolic acids was not touched in the discussion while the authors reported the species variation of phenolic acids in detail.

6. The authors apparently ignored the variations of root traits among allelopathic rice and non-allelopathic one, which also have interesting implications. For example, the specific root-biomass of Le was highest while its surface area was low, in another word, the specific root area of Le was low. Thus, on the root-area-base, non-allelopathic rice had even less impact on its surrounding.
Additional comments: The finding of this article is novel and will contribute new information to the root traits area.
However, I would like to suggest the authors change the display of their current results to the take-home-message easy to follow.
Make a less speculate conclusion about the correlations between fine root traits and allelopathy potential.
The introduction and discussion of the current article were not well-structured to provide background for the common audience to follow the scientific significance.
Please see annotated PDF for more detailed suggestions.


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: FINE-ROOT TRAITS OF ALLELOPATHIC RICE AT THE SEEDLING STAGE AND THEIR RELATIONSHIP WITH ALLELOPATHIC POTENTIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is not written very well. English language used is not professional. For example, Line 54, previous studies have shown that..... Line 65, Rice yield can be increased by controlling the direction of root extension. Authors should pay more attention to English language. One of the effective ways is reading more papers.
The authors tried to tell a story about the relationship between root traits (such as morphological and root growth) and the potential weed-suppressive ability. In general, authors have cited few important references about the potential weed-suppressive ability. However, references about root traits are not sufficient.
The structure is not good.The introduction is very hard to follow so that I can not get a clearer picture in my mind of the story that authors want to tell. There are many issue in figures and tables. For instance, I can not separate thinner (or fine) roots with the range of 0~0.2mm from  thicker (or coarse) roots with the range of 1.0~2.0mm.
Authors did not show the ANOVA results about the relationship between root traits and the total contents of eight phenolic acids or I missed. More importantly, authors expressed so-called weed-suppressive ability as the total contents of phenolic acids rather than the inhibition effect. authors should analyse the relationship between root traits and the inhibition effect.
Experimental design: This original work is within the scope of the journal. However, authors do not explain the significance of their work. People know that the allelopathic compounds are very hard to measure. If there are good relationships between root traits and allelopathic compounds, people can predict the content with root traits. On the other hand, people can select varieties for the green agriculture. Authors should mention this.
Authors did not describe the experimental design very well, especially the inhibition effect using root exudates. Authors should describe methods with many important references and sufficient detail. For instance, authors should show clearly how many plant roots were scanned, one or all per pot.
Validity of the findings: According to the introduction authors wrote, few studies investigated the relationship between root traits (root distribution and biomass) and the potential weed-suppressive ability. This work could improve our understanding of the association of the potential weed-suppressive ability with root morphological traits. However, there are serval issues about statistics. Authors should show the ANOVA results (p values and method such as Pearson correlation) as a table or figure in the text to detect the linkage between root traits and phenolic acids. Authors also should detect the linkage between root traits and the inhibition effect which is an important data to support the conclusion.
Additional comments: In the introduction, authors should give us some information about fine root traits of allelopathic rice because they analysed their effect on the inhibitory effect on weeds in the following text.
Authors should correct the technical issues in the text and references carefully, such as Line 120, Line 312, 314, etc.


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: FINE-ROOT TRAITS OF ALLELOPATHIC RICE AT THE SEEDLING STAGE AND THEIR RELATIONSHIP WITH ALLELOPATHIC POTENTIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript needs thorough grammar editing. Even the abstract had several mistakes.
The authors cited some of the most recent researches but failed to construct own hypotheses based on them.
The description of the results was very hard to follow, especially those in Table 1 and 2.
The discussion was extremely simple and did not match the introduction. Many topics and significance raised in the introduction were not mentioned again in the discussion.
Experimental design: The experimental methodology was sound.
Yet, there's no clear testable hypothesis for the experimental design.
Validity of the findings: The main findings are interesting and the authors made no speculate conclusions.

However, some of the results lacked proper statistical analysis.
The description of the output of a two-way ANOVA is very hard to follow.
Additional comments: Introduction
The current introduction did not explain the research purpose well.
The introduction ought to address why there are possible correlations between root traits and allelochemical potential.
The aims of this research laced a clear hypothesis. In this research, why root traits are important to the release pattern of root exudates to the surrounding soils should be the take-home message.

Material and methods
Why is the average value of root diameter used as a category standard of the fine roots?
Based on Figure S1, the distribution of the raw diameter data of all scanned fine roots should not be a normal distribution but a skewed one, which means the proportions of adventitious roots were different among varieties so that average value may not be an informative parameter for a group of roots not along the definition of thinner roots in this research.

Results
The result part should be improved.
The description of Table 1 was too hard to follow. I think the author probably interpreted the two-way ANOVA in a wrong way.
There's no statistic result supporting these statements in Fig. 4 or any other part of this manuscript!

Discussion
The authors should describe why the current research is needed and how does it relate with the previous researches listed here.

Please see the details in the annotated PDF.


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

Input:
Title: SEX- AND TISSUE-SPECIFIC EXPRESSION OF ODORANT-BINDING PROTEINS AND CHEMOSENSORY PROTEINS IN ADULTS OF THE SCARAB BEETLE HYLAMORPHA ELEGANS (BURMEISTER) (COLEOPTERA: SCARABAEIDAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is written well, however one more round of proofreading is required (typos, missing articles, etc.). The Introduction provide sufficient background.  The Discussion section is too short and should be expanded. Overall, discussion section is too conclusive and does not provide significant insight (the same information was already covered in Results). Authors might consider merging this section with Results. Next, it is not enough to just state that results were similar to other studies. Authors should elaborate, providing quantitative comparison.
Experimental design: Experimental design seems to be appropriate and fit to the objective of the study. Sufficient details are provided.
Validity of the findings: Conclusions are too general (more details are needed). For example, authors might consider adding the information about the main patterns they have observed and discuss the difference observed for different tissues in different sexes.
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: SEX- AND TISSUE-SPECIFIC EXPRESSION OF ODORANT-BINDING PROTEINS AND CHEMOSENSORY PROTEINS IN ADULTS OF THE SCARAB BEETLE HYLAMORPHA ELEGANS (BURMEISTER) (COLEOPTERA: SCARABAEIDAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Basic reporting is sound, and writing is clear and professional.

In the abstract, and throughout the manuscript, statements based on correlation should use appropriate language. For example, "...suggesting that they are involved in the sexual recognition process..." is not based on any direct evidence, so it sounds more assertive than the evidence provided. Similarly for, "that this CSP participates in the oviposition
process".

Lines 19-21: How does transcriptome help to study cryptic habits? The rational is not very clear, and should be expanded or edited.
Experimental design: How many replicates were done for RNA-seq? The information about biological replicates should be added to materials and methods. Without enough biological replicates, the de-novo assembly will be less robust.

Line 162-164: "NCBI (ncbi.nlm.nih.gov) protein 163 sequences of OBPs, CSPs, GRs, ORs, IRs, ORCo, SNMPs, ODEs and PDEs from 954 insect 164 species were downloaded to build databases." How exactly was that done? More details should be added to the methods.
Validity of the findings: No comments.
Additional comments: Figure 3, 4: A Scale should be added to the tree to make it a phylogeny. Otherwise it would just represent the relationship.

Authors should discuss why a large % of genes have no GO terms association assignment?


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: SEX- AND TISSUE-SPECIFIC EXPRESSION OF ODORANT-BINDING PROTEINS AND CHEMOSENSORY PROTEINS IN ADULTS OF THE SCARAB BEETLE HYLAMORPHA ELEGANS (BURMEISTER) (COLEOPTERA: SCARABAEIDAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have addressed raised issues.
Experimental design: Authors have addressed raised issues.
Validity of the findings: Authors have addressed raised issues.
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: SEX- AND TISSUE-SPECIFIC EXPRESSION OF ODORANT-BINDING PROTEINS AND CHEMOSENSORY PROTEINS IN ADULTS OF THE SCARAB BEETLE HYLAMORPHA ELEGANS (BURMEISTER) (COLEOPTERA: SCARABAEIDAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Both English and scientific clarity has improved in the revised version. The phylogeny figures have also been improved.
Experimental design: Authors provided more details on how sequences were collected and clarified the library construction method, improving the reproducibility of the results.
Validity of the findings: No comments
Additional comments: It will be better to include a brief explanation on the missing GO terms in the methods, and mention that the replicates are not 'true' biological replicates.


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: GENETIC DIVERSITY OF GLIADIN-CODING ALLELES IN BREAD WHEAT (TRITICUM AESTIVUM L.) FROM NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The article is written in grammatically correct, professional English. The text is clear, unequivocal and technically regular form, corresponding to professional standards of politeness and manner of expression.
2. The introduction presents very accurate and detailed results from previous investigations, which are in the context of the current study and underline its up-to-date nature.
3. The structure of the manuscript corresponds to the form in PeerJ's "Standard sections".
4. The figures are four. They are correctly numbered and well described, but Figures 2, 3 and 4 need further corrections:
a / Figure 2:
- to improve the quality of polyacrylamide gel electrophoresis / there is a part of the gel – an artifact in Lanes 4-5 - that disturbs the image quality.
- to indicate with arrows the components /bands/ with which the three electrophoretic gliadin spektra of the polymorphic variety Lutescence 65 (Lanes 1-3) differ from one another.
b / Figure 3:
- above each bar on the graph, the frequency of the corresponding allele should be indicated.
Recommendation: It is better the values of the frequencies of gliadin alleles to be also presented in a separate table. In this way, the exact values of the frequencies of the rare alleles will be seen.
c / Figure 4:
- In the "Results" section, a table of statistical parameters should be included on the basis of which the gliadin dendrogram has been obtained.

5. Unmatched data was provided in Supplementary material 1/2 in accordance with the PeerJ requirement. It is necessary to change the description of Supplementary material because the table contains data of the Gliadin allele composition of varieties/lines from Kazakhstan as well as varieties originating from other countries. In the Supplementary Material1 the origin of each variety/line should be indicated. This is necessary to demonstrate the results in Table 1 as well as the results of the cluster analysis presented in Figure 4.
Experimental design: Primary studies are very original and relate to the purpose and scope of  the PeerJ . The main objectives / research issues are well defined. Detailed and modern up-to date research has been outlined and analyzed the results of previous studies. The following additions are required:
- in  the  section Materials  and  Methods / Wheat germplasm and geographic locations
 it is advisable to present in a table the varieties, lines and standards included in the study along with their origin.
- in the section Materials and Methods / Electrophoresis and identification of Gli alleles,
 it has been shown that polyacrylamide gel electrophoresis was performed by the method of Metakovsky & Novoselskaya, 1991. In order to reproduce the assay according to the PeerJ requirements, it is necessary to indicate known details of how the electrophoretic method is performed in one separate laboratory, for example: the concentration of the separating polyacrylamide gel, the duration of electrophoresis, the temperature and power of the assay, the methods of fixing, the coloring/discoloration of the electrophoretic plates, and used equipment.
Validity of the findings: The results obtained are very well described and discussed. They are substantiated and supported by the results of previous research by the same scientific team as well as by other authors. Conclusions are well-formulated, supporting the results in conformity with the main purpose of the study.
Additional comments: This study is very original and useful for the wheat breeding in terms of the fact that certain gliadin alleles, which are found in high frequency in varieties and lines, show very good cold-resistance, tolerance to dryness conditions and high grain quality. The results are also proven with data from the previous studies. This manuscript supports the biochemical markers based on storage proteins as an alternative method involving cheaper and simpler methods that can be successfully used by breeders to purposefully create genotypes with concentrated gliadin alleles and desirable traits.


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: GENETIC DIVERSITY OF GLIADIN-CODING ALLELES IN BREAD WHEAT (TRITICUM AESTIVUM L.) FROM NORTHERN KAZAKHSTAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Literature references, sufficient field background/context provided.
Professional article structure, figures, tables.
Self-contained with relevant results to hypotheses.
Experimental design: Original primary research within Aims and Scope of the journal.
Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Rigorous investigation performed to a high technical & ethical standard.
Methods described with sufficient detail & information to replicate.
Validity of the findings: Data is robust, statistically sound, & controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Protein markers, such as the electrophoretic spectra of wheat gliadins, have not lost their relevance for assessing the economically valuable traits of varieties and their ecological and geographical correspondence to certain regions. Electrophoresis spectra of gliadins are not altered by environmental conditions, are easily reproducible and very useful for wheat germplasm identification in addition to DNA markers. Extensive material on the allelic composition of wheat gliadins in northern Kazakhstan and other territories has been seriously and interestingly analyzed.


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: T CELL RECEPTOR SIGNALING PATHWAY AND CYTOKINE-CYTOKINE RECEPTOR INTERACTION AFFECT THE REHABILITATION PROCESS AFTER RESPIRATORY SYNCYTIAL VIRUS INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: Pass. The manuscript is in clear and unambiguous English. Need improved proofreading due to some grammarical errors.
Experimental design: The acute and relapsing phase data seem from different patients. Is there a way to include same patient acute vs. relapsing dataset?

Pass
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: T CELL RECEPTOR SIGNALING PATHWAY AND CYTOKINE-CYTOKINE RECEPTOR INTERACTION AFFECT THE REHABILITATION PROCESS AFTER RESPIRATORY SYNCYTIAL VIRUS INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: •	Qian et al. have pulled an RSV patient sample dataset from Jong et al. 2016 (E-MTAB-5195) to compare gene changes between 39 acute phase samples vs. 21 recovery timepoint samples. In this reinterpretation of the Jong et al. dataset, the authors have identified genes that associate with the sample time-point (RNA set 1) and staging (RNA set 2). In RSV infection, a subset of individuals will be hospitalized due to severe infection complications and the Jong et al. 2016 dataset is taken specifically from individuals who are hospitalized. The authors should clarify these points and add additional information to the introduction to discuss hospitalization and severe RSV infection.
•	The authors provide an immunological background of long-noncoding RNAs (lncRNAs) and immune mediators in the context of RSV. The authors should more clearly define what is known between acute and recovery stages of the in the introduction. This information will help shape their overall goal of assessing acute vs recovered patient samples.
Experimental design: •	The authors have clearly stated the forms of analysis and corresponding programs that were utilized for the analysis of the Jong et al. 2016 dataset. However, the authors do not discuss how their analysis differs from the Jong et al. 2016 report. For instance, why have the authors chosen a cutoff of “|log fold change (FC)| < 0.263” compared to the Jong et al. 2016 cutoff of “absolute fold change (FC) threshold of 2”? The authors should clearly define the reasoning for their choices and compare these to the primary Jong et al. 2016 analysis to differentiate their analyses.
•	“RNA set 1” and “RNA set 2”, and the relationship to the color modules, are not clearly defined early in the paper. The color modules are not clearly defined in the paper. Please provide additional information on how these groups are generated, different, and defined earlier in the paper.
Validity of the findings: •	Qian et al. have performed differential gene analysis on the Jong et al. 2016 dataset to define differences between acute infected samples and recovery samples. As part of this analysis, Qian at al. have identified a unique set of genes that are different between acute vs recovery that may help inform rehabilitation mechanisms during RSV infection. However, there are key deficiencies in the analysis and discussion of the reinterpretation of the Jong el al. 2016 dataset. The Jong et al. 2016 dataset split hospitalized RSV patients into several categories that affected clustering including: age, sex, mild RSV, moderate RSV, and severe RSV. The authors have not commented how their data analysis took these factors into account. Jong et al. demonstrated that healthy controls and recovered samples are similar (1 differential expressed gene), and it is unclear how the authors analysis of acute vs recovered samples differs from the Jong et al. analysis to healthy controls. Please provide additional information how the analysis and findings of the study herein compare to the Jong et al. 2016 study.
Additional comments: •	Qian et al. have provided an extended analysis of acute RSV infected samples vs recovery samples from the Jong et al. 2016 study (E-MTAB-5195). The differentially regulated genes identified in this study are discussed in the context of immune responses that might foster the rehabilitation process. However, it is not clear how the results herein are different from the Jong et al. analysis of the same data set. Further, the authors do not discuss why their analysis yielded a different set of differentially expressed genes than the Jong et al. study.
•	Taken together, at this time this manuscript is not suitable 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: T CELL RECEPTOR SIGNALING PATHWAY AND CYTOKINE-CYTOKINE RECEPTOR INTERACTION AFFECT THE REHABILITATION PROCESS AFTER RESPIRATORY SYNCYTIAL VIRUS INFECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript the authors utilized a pre-existing dataset obtained from a previously published study to examine the blood samples from infants undergoing an acute RSV infection as well as samples obtained following resolution of the infection. A total of 2489  differentially expressed RNAs were identified between the acute and recovery phase of the infection and were further interrogated using pathway analyses.  Several RNAs associated were T cell receptor signaling were identified that may be involved in the recovery phase as well as RNAs involved in the IL-1 and IL-18 cytokine and cytokine signaling pathways.
Experimental design: The experimental design is straightforward analysis of a pre-existing dataset generated from a previous study.
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: GROWTH-PROMOTING CHARACTERISTICS OF POTENTIAL NITROGEN-FIXING BACTERIA IN THE ROOT OF AN INVASIVE PLANT AGERATINA ADENOPHORA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: l 151. Root sterilization is normally performed with intact roots. After sterilization, the roots are subjected to fragmentation. Root fragments may spill off internal contents and the sterilization process may reach the root interior.

l 154. Why did the authors used NA culture medium to check for surface sterilization, while using YMA to isolate reNFB? Please, clarify.

l 156-163. A common practice when isolating root bacteria is to proceed serial dilution from root extract. Normally, endophytic bacteria are isolated in 10^-5 to 10^-8 dilution. Why did not the authors proceed with serial dilution?

l 167-171. Was the cultivation of isolates made in nitrogen-free solid medium? Most of endophytic-nitrogen fixing bacteria are not able to growth (fixing nitrogen) in these conditions due to contact with high concentration of oxygen in the air. It includes taxonomic groups identified in this work. The normal procedure is to use semi-solid media. Please, clarify the procedure.

l 179. What were the reagents and concentrations in PCR mix reaction tube?

l 187-189. How exactly were the sequences filtered for chimeric bases? Why was variation in sequence length not allowed? "...cut out chimaeric bases to make each sequence 1351 pb long...". Does the 1351 represent the alignment length (in alignment positions), instead of sequence length (in bp)?
How many isolate sequences were generated? From OTUs in supplementary material (peerj-33237-Data.xlsx) it seems that 131 sequences were produced. However, in GenBank database only 23 sequences are available. Why? Although sequences may be very similar, they represent different isolated colonies. Also, clustering sequences at 97% identity means that differences exist within clusters that should be showed. Please, make all sequences available in GenBank database and provide all accession numbers in the manuscript.
How was the clustering analysis performed (e.g., software used)?
Versions and references for software and databases must be provided.

l 194. Taxonomic identification based on sequence similarity search is sensible to database content. What was the database selected for similarity search analysis (e.g., nr, 16S ribosomal RNA)? It seems that the taxonomic identification is based on best-blast hit. The authors must make it clear, specifying the database used and its version (or date). For example, strain CY25 was identified as Variovorax gossypii, but similarity search using megablast and "16S ribosomal RNA (Bacteria and Archaea)" show Variovorax guangxiensis as best hit. Strain WS26 was classified as Nocardioides albertanoniae, but its best hit is Nocardioides luteus. The authors should check for other differences. Taxonomic classification based solely on 16S rRNA gene sequences is not reliable below genus level. Other tools and databases may be used and the results showed in a comparative way.

l 250-251. "Tree cropland soils mixed with two humus and one vermiculite...". Do the values mean proportions of each substrate used? Please, clarify. Consider change the statement to (or equivalent)... "substrate was prepared including cropland soils, humus, and vermiculite, mixed in a proportion of 3:2:1".
Validity of the findings: In order to have the results clear and completely trustable, the authors need to clarify some methodological concerns and answer the specific questions, pointed out in this review.
Additional comments: l 134. The term "growth-promoting regulators" is better applied to refer only to phytohormones (it not include phosphate solubility and siderophore production).

l 293. Several isolates are close related to well known nitrogen-fixing bacteria within Alphaproteobacteria and Betaproteobacteria (e.g., Burkholderia and rhizobia). Although, those isolates were able to growth during 7 generations in nitrogen-free media, to confirm them as nitrogen fixer, more evidence should be provided, such as nifH amplification/sequencing and nitrogenase activity assay.

l 307-308. For each sampling region, three plants were collected and about 23 to 29 bacteria were isolated (only considering those with 16S rRNA gene sequence produced). Is this a representative sample in terms of diversity? If more isolates were taken, could new OTUs be discovered (e.g., rarefaction curve reach a plateau or not)? If not, Shannon index values may be biased.

Figure 1 (legend). "The bootstrap values were indicated at branch node, shown as MrBayes/Maximum Likelihood". Bootstrap analysis do not apply to Bayesian method.


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: GROWTH-PROMOTING CHARACTERISTICS OF POTENTIAL NITROGEN-FIXING BACTERIA IN THE ROOT OF AN INVASIVE PLANT AGERATINA ADENOPHORA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors present findings on root endophytic nitrogen-fixing bacteria in an invasive weed Ageratina adenophora through isolation, characterisation and identification, followed by the assessment of growth promotion of Ageratina adenophora seedlings through soil inoculation of selected isolates. While the authors present a fairly comprehensive study, it is the opinion of this reviewer that further major revisions are required.
As a general comment, the manuscript requires English language editing. I have found numerous grammatical errors and confusing statements throughout. Some of these statements will be listed in the specific comments below. Therefore it is highly recommended that the authors seek English language corrections with the aid of a professional editor or native English speaker.
There is an excessive use of abbreviations in the manuscript that have not been explained at first mention. Please note that the readers of this journal are from various aspects of biology, medicine and life sciences and may not be aware of certain abbreviations, which the authors may consider common knowledge.
The authors should acknowledge the role of plant secondary metabolites influencing population dynamics of rhizosphere and endophytic nitrogen-fixing bacteria and their potential roles in plant-microbial communication. This information is lacking.
Specific comments
Ln 85-86 this sentence is confusing. Please reword for clarity
Ln 89 “mutualisms” suggest changing to mutualistic relationships
Ln 173 YMA should be YM as agar is not used in liquid culture
Ln 179 “MIX” please note the name of the kit used and mention manufacturer so that the experiment can be reproduced properly by someone following this method.
Ln 226 YMA, should this be YM as well?
Ln 258 “(day per night)” should read (day/night)
Ln333 “P=0.0000”, please check your P values
Please use the specific comments as guides to correct numerous other grammatical errors. The study does isolate and characterise a number of root endophytic nitrogen-fixing bacteria that also accumulate IAA, solubilise phosphorus and produce siderophores as measures of plant growth promotion. However, a direct impact of nitrogen fixation or plant growth promotion has not been deduced from these experiments.
Experimental design: The experiments presented in the manuscript fit within the aims and scope of the journal. The gaps in current knowledge have been identified by the authors and addressed in the presented experiments. The methods have been explained in sufficient detail to replicate with the exception of the use of unexplained abbreviations and the lack of references to reagents as mentioned above.
When characterising the microbial profiles from isolates in Ageratina adenophora, it is also important to consider the endophytic microorganisms present in key native plants. As one of the claims of the study is that there is potentially recruitment of reNFB by Ageratina adenophora, a direct comparison should be made with a native plant/ plants, or as a minimum discussed in the manuscript as a limitation of the study
Specific comments
Ln 140 Authors should identify the specific locations the plant samples were collected and explain the soil properties including soil type, pH and conductivity as the soil type often has a major impact on plant growth characteristics and microbial populations
Validity of the findings: In general the findings have been statistically analysed satisfactorily to answer the research question. Plant growth promotion by the 6 isolates as tested in Ageratina adenophora seedlings was presented well and supports the hypothesis that the recruitment of reNFB may enhance plant growth promotion in this plant and potentially aiding in its invasiveness.

Specific comment: Figure 3- Comparison of study sites on plant growth promotion. Were there statistical differences between the sites? If there were no differences, please indicate as not significant.
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: GROWTH-PROMOTING CHARACTERISTICS OF POTENTIAL NITROGEN-FIXING BACTERIA IN THE ROOT OF AN INVASIVE PLANT AGERATINA ADENOPHORA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Although the methodology is better presented and clear in this version of the manuscript, the methods used for endophytic bacteria isolation was not adequate and do not guarantee the isolation of nitrogen-fixing bacteria. No further evidence for nitrogen fixation was provided. However, the authors stands the work on "root endophytic nitrogen-fixing bacteria". Further, there was not a quantification of the endophytic bacteria.
Although phylogeny is only used as a way to cluster the isolates, once the authors chose to use it, it must be performed in a correct way. But the tree showed in the manuscript suffer from some conceptual/methodological problems.
Please, see details in the attached file.
Validity of the findings: Please, see observations in "Experimental design" above, that interfere in the validity of the findings and other details discussed in the attached file.
Additional comments: Please, see the reply for all authors responses (first review) in the attached file.


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: GROWTH-PROMOTING CHARACTERISTICS OF POTENTIAL NITROGEN-FIXING BACTERIA IN THE ROOT OF AN INVASIVE PLANT AGERATINA ADENOPHORA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The clarity of the information contained within the manuscript has been enhanced through professional English editing and all queries directed to the authors have been addressed in the manuscript
Experimental design: Queries on the experimental design especially with respect to 16rRNA analysis and media selection have been addressed by the authors
Validity of the findings: Discussion of the limitations of the manuscript have been adequately addressed by the authors and errors in statistical analysis corrected.
Additional comments: Thank you for providing edits to the manuscript as requested. The manuscript has improved in quality when compared to the initial submission. I believe the comments I have provided have been addressed adequately.


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: CONSERVATION AND DIVERSITY IN EXPRESSION OF CANDIDATE GENES REGULATING SOCIALLY-INDUCED FEMALE-MALE SEX CHANGE IN WRASSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: General comments:
In this manuscript, authors addressed whether degree to which a conserved genetic machinery orchestrates sex change by comparing expression patterns of four candidate regulatory genes among protogynous three species of wrasses. Based on the qOCR results, authors suggested that conserved roles for cyp19a1a and amh as important proximal regulators of gonadal sex change in protogynous wrasses. On the other hand, in the brain, qPCR results do not support a role for brain aromatase, cyp19a1b, in initiating behavioral sex change, as expression changes for this gene trailed rapid behavioral changes. Brain isotocin expression strongly correlated with TP male-specific behaviors and the rapid behavioral changes characterizing the onset of sex change in the bluehead wrasse, but not the others. Based on these results, authors conclude that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioral sex change may be more variable. I believe that comparison among several species could shed light on the communality and diversity in sex change mechanism. So, I recommend that this manuscript would be acceptable after solving my following concerns.

In this study, I think that non-breeding ovary in temperate species is one of the important points. Authors also describe that there is ambiguity between these gonads (L. 481-482). Probably, there could be atretic oocytes in not only ET gonad but also non-breeding ovary. So, what criteria do authors apply to distinguish between non-breeding ovary and ET gonad? I recommend that histological picture showing the criteria which readers can distinguish should be added in figure 5. Ambiguous classification leads to misinterpretation of the present results. Especially, in discussion section of cyp19a1a, authors discussed that downregulation of its expression is an important event in the initiating stages of gonadal sex change in temperate species also. But, it has been reported that there is no difference of estrogen level between sexes at non-breeding season in protogynous species exhibiting seasonal breeder (Bhandari et al., 2003; Ohta et al., 2008). These facts indicate that cyp19a1a expression would be enough low level in non-breeding female. In the present study also, Fig. 7C and 7D does not show significant difference between each stage. Thus, I do not fully agree that cyp19a1a were downregulated at the initiation of gonadal sex change in temperate species.

Authors emphasized that amh act as an important initiator of male phenotype during female-to-male sex change, while it seems to me that amh expression upregulated after appearance of testicular tissue (specifically appearance of spermatid or spermatozoa). Similar results were reported in a protogynous fish (Horiguchi et al., 2018). Horiguchi et al., 2018 discussed that amh plays a role in spermatogenesis rather than initiator of male phenotype during protogynous sex change. I think that change in amh expression should occur before testicular appearance (St 3 at the latest) if amh plays an important role in initiator of testicular differentiation. What do authors think about this?

Specific coments
L. 235. There is no “Untergasser et al., 2012” in references list.

L. 277. In Table S5, there is no description about the choice of reference genes.

L. 280. I could not see anything about the explanation of normalization in Table S5.

L. 345. Authors explain that figure 5 exhibits gonad histology in Survey 1 (non-breeding season sampling). But, I wonder why Fig. 5 includes ovary of breeding season, not non-breeding. I recommend that histological picture of non-breeding ovary should be added in Fig. 5.

L. 379. Number of degrees of freedom is “4”, isn’t it?

L. 504-507. This sentence is no needed, I think.

Reference No 21 and 22 were not cited in the manuscript.

I think that most figures meet the standard quality of images. However, histological picture in Fig 6A includes something contamination and degree of staining in Fig 6B is absolutely different. I recommend that these histological pictures should be replaced.


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: CONSERVATION AND DIVERSITY IN EXPRESSION OF CANDIDATE GENES REGULATING SOCIALLY-INDUCED FEMALE-MALE SEX CHANGE IN WRASSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses by Thomas and colleagues presents a comparative study of the mechanisms underlying sex change in 3 species of protandrous fishes. The study focuses on 4 logically selected candidate genes and presents both positive and negative results. This is a useful first study of this group of fish. The paper is very clearly written, well organized, and effectively packaged with appropriate citations and discussion.  The authors MUST check the text to be sure the right supplemental table is indicated at each point (see comments 1-3 but also check the rest of the document).

1) Line 265 direct the reader to Table S4 for the annealing temperatures but that information is actually in table S3.

2) Line 277 directs the reader to Table S5 for information about chosen reference genes but that information is actually in table S6.

3) Line 280 directs the reader to Table S5 for information about the differences between normalization strategies, but table S5 gives primer sequence, amplicon size and primer efficiency.

4) Line 248-249 directs the reader “below” for details regarding the use of heterologous oligos but I find additional information only in the supplement, not below (or I’ve missed it).
Experimental design: The authors provide all raw data and R code used to analyze that data. They also provide an incredibly thorough MIQE table in the supplement.

5) I cannot find a supplemental table that demonstrates the different influence of normalization strategies. Comparing the main figures to the supplement figures, I don’t see a drastic change in the trend of the results. I am confused by lines 275-280. Perhaps by presenting the results in supplement with additional explanation this will be clear?
Validity of the findings: 6) I find it interesting that the isotocin expression of the IP males was similar to that of the females. This results supports its hypothesized role in establishing dominance. The result is stated on line 439, but I would think it could be emphasized in the discussion.

7) I would encourage the authors to make the sentence about isotocin decrease in gobies (line 538-540) explicitly state that it was a decrease in cell number. This is a minor detail but because some reports relate to cell size and some to cell number it is nice to have this information provided explicitly.
Additional comments: Thank you for an enjoyable read.


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: CONSERVATION AND DIVERSITY IN EXPRESSION OF CANDIDATE GENES REGULATING SOCIALLY-INDUCED FEMALE-MALE SEX CHANGE IN WRASSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: The majority of my initial comments were technical issues with table numbers etc. and those have all been resolved.
The authors have also appropriately added the details that I requested concerning isotocin expression in other studies.
I am excited to see this paper in press.
I made a couple notes in the resubmitted version that might be attended to prior to copy editing stages.
Line 74 mentions the results of aromatase inhibitors in "teleosts" but the papers cited are only sex changing teleosts.... If this result is generalizable to other fish, there should be reference to them, or clarify this sentence to be more specific.
Line 82 The word "become" is vague, do you mean independently co-opted in evolution or do you mean "has been identified as"
Lines 83 should "dmrt1" be "dmy" here as it refers specifically to the fish homolog? or possibly indicate "teleost homolog of dmrt1"
Line 502 the species name should be italicized.
Line 529 Cyp10a1b is included in a list that is preceded by the descriptor "neuropeptide" but it is not a neuropeptide.... just reword this sentence.
(I apologize for the delay in my review)


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

Input:
Title: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	The English used in the paper is in general clear and unambiguous, but with some less clear expressions.

•	The article has a sufficient introduction where the reason for further examination of biomarkers is explained with relevant references. The abstract does however lack a sentence or two of the background of the study. The text written under ‘Background’ is more the aim of the study. Please add ‘The aim of’ in front of This study in line 23.

•	There is however some less clear definition of diabetic nephropathy as it seems as microalbuminuria is defined as diagnostic of diabetic nephropathy. Diabetic nephropathy is defined in both type 1 and type 2 diabetes as the presence of persisting severely elevated albuminuria of more than 300 mg/24 hour (or > 200 µg/min)  or an albumin creatinine ratio >300 mg/g creatinine, confirmed in at least two out of three samples, with concurrent presence of diabetic retinopathy and absence of signs of other forms of renal disease (Parving H-H et al. Diabetic Nephropathy. In: al. MWTe, editor. Brenner and Rector: The KIDNEY. 1. Philadelphia, USA: Elsevier; 2012. p. 1411-54.)

•	The structure of the article conforms to an acceptable format which also counts the figures and tables. Raw data is available.
Experimental design: Re. Research question
•	The research question is as stated, if there is a role of multiple serum and urinary biomarkers in the early diagnosis of diabetic nephropathy. But it is not clear how “early diagnosis of diabetic nephropathy” is explained. Please add that. Many of the biomarkers have been examined in diabetic cohorts previously, why it would be relevant to include a description of the novelty of this study or knowledge gap being investigated and how this study contributes to filling that gap.

•	The study has been conducted in accordance with the prevailing ethical standards in the field.

Re. Methods
Patients:
•	The ‘Patients’ section: please add ‘cross-sectional’ after ‘In this’ line 65.
•	When was the study conducted? (year), how was type 2 diabetes defined (according to WHO criteria?), how was ‘healthy controls’ defined? From where were the patients recruited? (outpatient clinic?, general practice? Hospital?) and the healthy controls? Please add more information on details about how the participants were examined (how many days, where and by whom nurse, doctor, as part of a visit in the clinic etc) how demographic data was collected and from where.
Data collection:
•	Re. urine: was the UACR based on a first voided morning urine? Please add. Was the characterization of albuminuria group based on actual albuminuria (on the examination day) or historical albuminuria? Was the characterization based on a single or several UACR measurements? How was UACR measured?
•	For all biomarkers: please add the length of the period from sample collection to analysis and please add how the samples were stored in the meantime (at minus 80 degrees C etc)
•	Re.eGFR: based on which method was that calculated (CKD-Epi?)
Statistical analysis:
•	Quite often biomarkers are non-normally distributed, please add information on this and if skewed data, then information on log-transformation
•	Line 98 and 99: Instead of compression, I suggest the use of ‘comparisons’
•	Please explain why logistic regression analysis has been used instead of linear regression?
•	There is no information of adjustments for traditional risk factors: gender, Hba1c, eGFR, smoking, LDL, systolic BP and other conventional risk factors. Adjustment is highly recommended to give data clinical relevance.
•	ROC curves and the AUC are widely used to examine the effectiveness of diagnostic markers but can be strongly influenced by covariate variables. Please include relevant covariates in the analysis.
•	To account for the influence of urinary dilution of the biomarker concentration, please normalize urine biomarkers to the urine creatine concentration.
Validity of the findings: •	Impact and novelty not firmly assessed.
•	The recommended statistical analyses need to be performed before validity of findings can be evaluated.
•	In the introduction it is stated that there is “a dire need for more sensitive and specific biomarkers for the early diagnosis of diabetic nephropathy” (line 55-56) since “its (UACR) early diagnostic value in DN is limited as renal injury commonly precedes proteinuria”. It therefore seems contradictive to write in the conclusion (line 223-224) “The combined use of these novel biomarkers, along with the traditional marker (microalbuminuria) may improve the early detection of diabetic nephropathy”. Please clarify.
Additional comments: This manuscript by Yuan et al. examined the associations between 13 biomarkers and the early diagnosis of nephropathy in 287 patients with T2D and various degrees of albuminuria and a control group of healthy individuals. The topic of the paper is relevant, and I commend the authors for an impressing number of participants included. There are some major issues in the methodology, including the statistical analysis which should be improved as well as a more detailed description of the method section.


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

Input:
Title: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is written in clear and professional English. However, there are some parts where the language and the used terminology could be improved:

1.	The description of the calculation of AUC, sensitivity and specificity based ROC analysis on lines 101-103 should be improved.
2.	It is not clear to which analysis are lines 103-106 referring to since the same has been said already before on the “Statistical analysis” section.
3.	The meaning of the term “compression” on lines 90, 102 is not clear.
4.	Term “normoalbuminuria” is more commonly used than “normal proteinuria”.
5.	The correct spelling of “Wilcoxon rank sum” is “Wilcoxon rank-sum” and “chi-square test” is “chi-squared test”

The authors provide sufficient background on the importance for the early detection of diabetic nephropathy (DN), the short-comings of albuminuria in predicting development of DN and the need for improvement in the early detection of DN.

The article follows the acceptable format of ‘standard sections’ and figures and tables are relevant to the content of the article. The raw data has been made available as excel-files.
Experimental design: The article represent original primary research within aims and scope of the journal. The research has been conducted rigorously and to a high technical and ethical standard. The research question is well formulated but the design of the experiments does not support answering the identified knowledge gap:

1.	In the introduction it is stated that 13 biomarkers are evaluated for their potential application in the prediction of DN. It is not possible to evaluate the predictive value in a cross-sectional study setting. Some follow-up data on events related DN should be obtained for this purpose.
2.	It is unclear how the identified urinary biomarkers Tf, NGAL and TNF-alpha help in the early diagnosis of DN compared to the gold standard UACR. It is possible that they would be useful on top of UACR in predicting onset of DN, but to test this hypothesis, follow-up data would be needed.
3.	It is not clear what is the utility of determining biomarkers that are correlated with microalbuminuria which itself is a biomarker of DN.

Methods have been described in sufficient detail but there are some parts that could be clarified:

1.	It is not clear how the sensitivity and specificity in Table 3 and Table 4 have been calculated. I assume they have been evaluated at the ‘Cut-off’ but this should be clearly stated as well as how ‘Cut-off’ was defined.
2.	It should be stated how eGFR was calculated. Was it done with CKD-EPI equation or with some other equation?
3.	It should be stated for which test the p-value is given in Table 3 and Table 4.
Validity of the findings: The data is robust and statistically sound. However the are some issues:

1.	The urinary biomarkers found to be associated with microalbuminuria, namely, Tf, NGAL and TNF-alpha, have been previously shown to be associated with UACR in T2D individuals as noted by the authors. It should be clearly stated what is the added value to the literature of investigating this associations again.
Additional comments: I congratulate the authors for their well phenotyped dataset and the manuscript which is well written in professional language. The biggest weakness is that the identified knowledge gap, prediction of DN cannot be achieved with the design of the experiment. Follow-up data is needed to infer possible utility of the biomarkers in predicting onset of DN. In addition, as the authors note, urinary Tf, NGAL and TNF-alpha have been previously shown to be associated with UACR in T2D individuals. It should be clearly stated what is the added value to the literature of replicating these findings in the presented 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: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 1
Reviewer: 3

Output:
Basic reporting: see comments
Experimental design: see comments
Validity of the findings: see comments
Additional comments: In this work, Yan et al. analyze plasma and urine biomarkers in a cohort of diabetic patients carachterized by different degrees of proteinuria.
They found that a number of urine  biomarkers were increased in parallel with the increase of proteinuria. They also calculated risk with a multivariate analysis between patients with normal proteinuria and those with microalbuminuria and also estimated the diagnostic value of a few urine biomarkers  (NGAL, Tf, TNF-alpha) using a ROC curve. They concluded that these biomarkers, alone or in combination, could represent early diagnostic biomarkers od diabetic nephropathy.

The work is well written, concise and of broad potencial interest for the nephrologists community.
The background is well framed.  However there are some assumption and lacking controls that weaken the conclusions of the work.

Major.
-	As they also stated in the introduction the presence of albuminuria is not an essential condition associated with DN. Only kidney biopsy allows an etiological diagnosis of kidney damage and DN. In this work there is no histologically proven diagnosis of DN. As a consequence, the biomarkers investigated in this study can only be correlated with the degree of proteinuria and not with early or late stage of DN. In other words, microalbuminuria cannot be considered tout court a proven sign of early diabetic nephropathy.
-	The hypothesis that transferrin could be a potential diagnosis biomarker of DN is not novel (Jpn J Nephrol 37:649-654, 1995), as it is that of NGAL (Diabet. Med. 27, 1144–1150 (2010) , TNF- (NDT (2006) 21: 3428–3434).
-	In most of the studies conducted on potential novel urine biomarker for kidney diseases, their amount is normalized for the urine creatinine (Ucr), considering that the degree of urine output is extremely variable depending on water intake, room temperature, exercise… In this work , the authors did non perform a normalization of their data.  Authors should normalize their data before performing the downstream analysis.
Minor:
-	Authors should better describe the results reported in Table 2 and explicits the labels (, Wald, OR) to allow an easier understanding of the data.


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

Input:
Title: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Nothing further to add
Experimental design: Nothing further to add
Validity of the findings: Nothing further to add
Additional comments: No additional comments


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

Input:
Title: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The article is written in clear and professional English. However, there are some parts where the language could be improved as well as some small typographical errors:
1.	On lines 69-70 I suggest using normoalbuminuric, microalbuminuric and macroalbuminuric.
2.	On lines 108-109 the language is not clear. I suggest, “Differences among the groups were analyzed …”.
3.	Table 1 uses “normal proteinuria” instead of “normoalbuminuria” used otherwise in the manuscript.
4.	First paragraph of the results lines 118-129: here P<0.01 is used but according to Table 1 it should be P<0.001. Which one is correct?
5.	Line 163: Instead of “the progression of microalbuminuria”, I suggest “the progression to microalbuminuria” or “the development of microalbuminuria”
6.	Table3. The 95% CI for Tf does not include the reported AUC value?

The authors provide sufficient background on the importance for the diagnosis of early-stage diabetic nephropathy (DN), the short-comings of albuminuria in predicting development of DN and the need for improvement in the early detection of DN.

The article follows the acceptable format of ‘standard sections’ and figures and tables are relevant to the content of the article. The raw data has been made available as excel-files.
Experimental design: The article represent original primary research within aims and scope of the journal. The research has been conducted rigorously and to a high technical and ethical standard. The research question is well formulated and the knowledge gap being investigated is identified. However methods section needs more detail:
1.	Lines 130-131: How were the biomarkers shown in Table 2 chosen for analysis? Please clearly define what was considered correlation with UACR.
2.	Line 114: Please describe how sensitivity and specificity were calculated more precisely. For example “Sensitivity and specificity were evaluated at a cut-off value which maximized Youden’s index”. Assuming that Youden’s index was used?
3.	It should be stated for which statistical model and null hypothesis are the p-values in Table 3 reported to.
4.	The raw biomarker data contains values <X (where X is a number). I assume these are values that were below the detection threshold of the method used to measure biomarker concentrations. It should be stated how these values were treated in the analysis.
Validity of the findings: The data is robust and statistically sound. However the are some issues:
1.	Please discuss why the ‘Partial regression coefficient of TNF-alpha in Table 2 is negative, whereas in Table 1 the TNF-alpha seems to increase from normoalbuminuria group to microalbuminuria and macroalbuminuria group?
2.	Please discuss why the ROC analysis is not adjusted for covariates.
Additional comments: I congratulate the authors for their well phenotyped dataset and the manuscript which is well written in professional language. The presented data is interesting but the methods section still needs more detail.


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

Input:
Title: THE ROLE OF SERUM AND URINARY BIOMARKERS IN THE DIAGNOSIS OF EARLY DIABETIC NEPHROPATHY IN PATIENTS WITH TYPE 2 DIABETES
Review round: 3
Reviewer: 1

Output:
Basic reporting: For lines 69-70 I would use the form albuminuric instead of albuminuria since you are talking about  patients classified into these groups.

The footnote of the Table 1. (line 411) uses "normal proteinuria" instead of "normoalbuminuria" used elsewhere in the manuscript. I suggest using consistently one term.

On line 123 I would suggest removing "Youden’s index was calculated by “sensitivity and specificity minus 1” and adding reference for Youden's index in the previous sentence.
Experimental design: Please clarify which statistical test was used to test the hypothesis that the biomarkers were not indicative of early-stage DN (lines 118-119).
Validity of the findings: no comments
Additional comments: Thank you for answering my questions and addressing my comments. I have only few minor comments attached here.