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: PEPTIDE-BASED NTA(NI)-NANODISCS FOR STUDYING MEMBRANE ENHANCED FGFR1 KINASE ACTIVITIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: More reference on the background of nanodiscs could be cited, e.g. Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR, which was published in Nature Methods.
Experimental design: no comment
Validity of the findings: Average diameters of the nanodiscs determined from TEM data should be based on 2D class average results. However, no 2D class average results are shown in the article.
Additional comments: 1.	The authors used DMPC as the main lipid for nanodisc formation. However, DMPC is an artificial lipid and is shorter by two carbons than the shortest native lipid. Could the authors comment on the reason of using DMPC and how physiologically relevant this study is by using DMPC?
2.	Line 170: the average diameters of the nanodiscs by TEM should be based on 2D class averages, the authors should provide the 2D class average results in the figure; in additon, it seems in Figure 2B that the nanodiscs are stacking onto each other for 1:1 ratio, while they are well separated from each other for 1:3 and 1:9 ratios. Does the authors have an explaination on this observation? How does this affect the phosphorylation? In Line 190-196, the authors stated that phosphorylation is correlated with the size of the nanodiscs based on the observation that the 1:1 ratio discs provided highest phosphorylation. Is it possible that phosphorylation is related to the stacking effect instead of the size of the nanodiscs?
3.	Line 184-186: the black should be nanodiscs only and the red should be the complex, please make sure the text matches the data in the figure; in addition, the SEC profile of FGFR1K alone should also be shown in Figure 3A for better comparison.
4.	Line 186-188: the authors performed ITC experiments to measure binding affinity between empty nanodiscs and FGFR1K. ITC experiments on nanodiscs are hard to perform due to bubble formation during stirring in the ITC cell. The authors should indicate in the experimental section on how to avoid air bubble formation during ITC experiments. In additon, proper control experiments are very important for ITC results to be reliably interpreted. The authors should include experimental design of the control experiments in the experimental section and the results of the control in the supplementary material.
5.	More evidence of complex formation between nanodiscs and FGFR1K is needed in addition to ITC results. SDS PAGE gel for the peak in Figure 3A could be presented to show the presence of both FGFR1K and 22A (if visible on gel). Or TEM images could be presented for this peak to show that FGFR1K is associated with nanodiscs.


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: MITOCHONDRIAL PHYLOGENY AND COMPARATIVE MITOGENOMICS OF CLOSELY RELATED PINE MOTH PESTS (LEPIDOPTERA: DENDROLIMUS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Literature references, sufficient field background/context provided.
Professional article structure, figures, tables. Raw data shared.
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.
Additional comments: Revision suggestions:
1.concerning the species of Dendrolimus, Fauna Sinica Vol.47(2006) should also be referenced.
2. Six individuals of three  species (D. kikuchii, D. houi and D. superans, 2 individuals for each species) were selected for sequencing in this study. Please state more clearly how did you collected 2 individuals, from one site or different sites?
3.It should be stated more clearly that from Mitochondrial phylogeny and comparative mitogenomics of closely related pine moth pests what results were different from the results of previous studies. And should also discuss the reasons.


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: MITOCHONDRIAL PHYLOGENY AND COMPARATIVE MITOGENOMICS OF CLOSELY RELATED PINE MOTH PESTS (LEPIDOPTERA: DENDROLIMUS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting ms based on a large dataset of mitochondrial genomes of an important group of pine defoliating pests in the Palaearctic region and in Asia.

I have two major issues:

1. an important species, actually the species with the largest range in Asia and occurring also in China, Dendrolimus sibiricus, is missing. I suggest in the comments how this can be addressed, basically to add it to the dataset or to use sequences published by other authors for comparison, although on a part of the genome (see Kononov et al. not cited in this ms).

2. the objectives and the discussion should be more clear about the taxonomic level to consider. I would suggest to reduce/remove the part on the family and superfamily level and to concentrate on the Dendrolimus genus. This will help to make the paper more focused, especially if D. sibiricus is added.
Experimental design: The experimental design is fine, although one important species is missing and should be added (suggestions in the pdf file)
Validity of the findings: Results should be integrated with the addition of the species mentioned above.
Additional comments: I think this ms can be considered after the recommendations are addressed.


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: MITOCHONDRIAL PHYLOGENY AND COMPARATIVE MITOGENOMICS OF CLOSELY RELATED PINE MOTH PESTS (LEPIDOPTERA: DENDROLIMUS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: It is suggested to refer to more papers for the results of phylogenetic tree construction, and discuss the differences between species and the reasons.
Experimental design: no comment
Validity of the findings: In the results and discussion, the similarities and differences between the six species of pine caterpillar based on the base composition, codon, tRNA structure and other aspects and the results of phylogenetic tree are needed more summarize. Codon usage and RSCU were similar among the different Dendrolimus species, and the Figure 3 and 4 seems uninformative. I suggest move these two figures to the supplement material or change them as comparing the Codon usage and RSCU between Lasiocampoidea and Bombycoidea.
Additional comments: Qin et. al. sequenced mitogenomes of three Dendrolimus species, and performed a very detailed analysis of the mitogenomes known in Dendrolimus spp. Based on mitochondrial genome sequences, the paper compared the base compasition, the order of the 37 genes and protein gene of initiation codon and termination codon, the characteristics of A+T rich area structure, and phylogenetic tree was constructed. The information of mitochondrial genome in lepidoptera was enriched, and the authors illustrated interspecific relationship of the six species.
1. In the Background part of Abstract, The authors should add one sentences to explain the discrimination challenging of different Dendrolimus species.
2. Line 66: The reference were cited as numbers here but name and year information in other sentences.


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: CHILDREN’S ATTITUDES TOWARDS ANIMALS ARE SIMILAR ACROSS SUBURBAN, EXURBAN, AND RURAL AREAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found no major problem. The general framework is clearly exposed. Method, data and analyses are well presented. Results are convincing. Conclusions are sound.
Experimental design: There is no real experimental design. This study is empirical. Yet large data set provides robustness.
Validity of the findings: The results were expected, the patterns are clear. I fully trust the findings. The statistics were simple, but I do not see any reason to perform sophisticated ones, this will add nothing.
Additional comments: This study reports interesting results based on questionnaire responses obtained from a large sample of children. The method used and the type of questions addressed have been somehow previously employed; but considering that few investigations of this kind have been performed, it is essential to challenge published studies. Indeed, as nicely exposed in the introduction, exploring the knowledge and possible attitudes of schoolchildren toward wildlife is a key pre-requisite to guide educational actions.  Unfortunately, the results (expectedly) show that children have a minimal knowledge regarding local fauna, and thus are disconnected from wildlife. This study therefore militates for the necessity to bring children in the field. Although this conclusion might seem obvious or over simplistic, robust study like the current one are very important because the reality is that children spend an increasing amount of time in front of their screen and thus proportionally less in the field. For convenience and for multiple other reasons, many schools encourage this trend. Overall, I enjoyed reading the manuscript. Below I propose several minor comments.

The discussion is too long, somehow repetitive and often commenting detailed results (e.g. line 440-447). Perhaps that the discussion could be more streamlined? Similarly the long conclusion is a summary of the discussion (e.g. kids love dogs). Perhaps providing few recommendations (e.g. schoolchildren should spend more time in the field) or future directions (do we really need umbrella species, like loveable mammals, during outdoor activities?) might be more appropriate.

Line 140: Although I understand why whales or wolfs were categorized as exotic animals (Humpback whales can be observed from North Carolina beaches but the schools were inland), it is less clear for bats (several widespread bat species occur inland in North Carolina). Further, bats were moved later displaced into the local group (line 149), this is confusing.

Line 275: rabbit is not an exotic species (see line 139)

Line 433 etc.: why more efforts should be devoted to the groups displaying “lower” scores? Is the objective to eliminate differences among groups? In practice, it might be complicated and resource consuming because specific actions will be needed. Improving the attitude and knowledge of all kids from a classroom seems to be more easy and likely more beneficial, despite group differences.

Meuser et al. 2009 (line 71) is not in the reference list.

Several typos and formatting problems can be found in the reference list, this should be checked.


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: CHILDREN’S ATTITUDES TOWARDS ANIMALS ARE SIMILAR ACROSS SUBURBAN, EXURBAN, AND RURAL AREAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript was clear and easy to read, with few grammatical mistakes and misspellings. I recommend more recent citations, especially in the introduction, to support the authors' claims. For instance, lines 50, 51, and 234. The hypotheses were clearly stated. One suggestion, however, is to use a better term for "development zones." In the context of studying children, this term can be easily confused with biological development. Perhaps economic development would be a less confusing term?
Experimental design: The experimental design seemed realistically simple given the type of data. The research questions were well defined and all were tested and results reported. I was confused by line 124-what was a "control" teachers and where is this discussed in the results? Unfortunately, the study had only a single rural school, which greatly limits the impact of the study and the ability to measure a gradient of the effect of economic development. I did not see where the Filgner-Killeen test was discussed in the methods.
Validity of the findings: I believe that the results and conclusions accurately reflect the findings. There were "negative findings," but that makes the study interesting. I think the authors should discuss the lack of rural schools more, and how that affects the results, conclusions, and future studies.
Additional comments: Overall, I enjoyed this manuscript, but am disappointed that the sample size of rural schools was so low, as it makes for a much weaker impact. In the introduction, the "pigeon paradox" could be explained in more detail, and in the conclusion, it would be nice to see some recommendations on how to combat disconnect with 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: THERMAL CONDITIONS AND AGE STRUCTURE DETERMINE THE SPAWNING REGULARITIES AND CONDITION OF BALTIC HERRING (CLUPEA HARENGUS MEMBRAS) IN THE NE OF THE BALTIC SEA
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The manuscript includes several violations of plagiarism mostly, e.g. Line: 53-55, Line: 55-57 or Line 89-92
- Refer to own papers, even if they are not the original source
- The reference list is not complete and inconsistent
Experimental design: It is not well described how the herring data was collected. The beginning of the spawning season is defined as the appearance of pre-spawning herring. This is contradictory, either herring are spawning or not. When herring are in pre-spawning conditions, especially on a 6 maturity stage scale, this can be a quite long time. Also using commercial catches is not the optimal solution to define spawning since fishermen only start fishing when sufficient numbers of herring occur. It does not indicate whether herring are caught throughout the whole year. The same account for the larval period, it is not mentioned how they were sampled.
Validity of the findings: Thanks a lot for sharing the raw data. However, the results presented do not correspond with the provided raw data, e.g. total number mentioned in the text 12 148 individuals is not the sum of the individuals in Table 1 (12 031), or mean WAT for week 18 is -13.3 but in Figure 3 it is ~-8.
Additional comments: Given the fact of plagiarism and the fraud of data, I recommend rejecting the manuscript. I like the idea of the manuscript and the results are worth for publishing, however, at the present state, the interpretation of the results is not very convincing.


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: THERMAL CONDITIONS AND AGE STRUCTURE DETERMINE THE SPAWNING REGULARITIES AND CONDITION OF BALTIC HERRING (CLUPEA HARENGUS MEMBRAS) IN THE NE OF THE BALTIC SEA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and structured.
Experimental design: The exp design is sound. Some additional comments on how the environmental proxy project into the ocean, and further how this proxy relates to the larger scale variability would be welcomed
Validity of the findings: Ok
Additional comments: Review of  “Thermal conditions and age structure determine the spawning regularities and condition of Baltic herring (Clupea harengus m.) in the NE of the Baltic Sea
Arula et al.

In general, I think this a well written paper. It is easy to follow and seem logical. In think the paper is worth publishing but I have some points that I would like the authors to consider.

General comments
Given my initial background in physical oceanography and ocean climate I am left with the question how much of the observed oceanic warming in the North Atlantic is reflected in the Baltic or GoR. Or is the Baltic climate basically determined by local atmospheric forcing including air-ocean heat fluxes. Also, there should be some discussion about the sensitive of proxy to the choice period (herein Jan-Mar)?

The paper show that the integrated Tair is a useful proxy for winter severity, however some idea on how this proxy projects into the ocean in terms of temperature variability between cold/warm years would be relevant.

Details

Introduction
Line 44-45 The sentence “Still, not all fish stocks are so well predictable with high accuracy”. This sentence seems a bit strange, basically most fish species are not well predictable. Also it somewhat unclear, i.e. predictable in terms of what? Please clarify.

Line 85-86: need to specify what region what is referred to here. In the air or ocean, and preferable also how much.

Line 104: define calendar week (CW) here?

Line 189-190: does the factor 100 have any effect on the statical results, is this needed?

236-237: for readability, you correlate mean average age of herring with calendar week of what? Please include.

247-248: I do not understand what is mean here, can you clarify, add some explanation?

273-274: Do this imply that also suitable prey is delayed during a cold winter, is it observed.

277-279: Does this mean that the Baltic show large inter-annual variability but no trend in WAT temperature changes? Clarify

278-279: which two periods , please specify

298-299: maybe change “ BoHS in later season” to “ BoHS later in the season”, please check

354-355: Missing word “ …it results in a more .. “


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: THERMAL CONDITIONS AND AGE STRUCTURE DETERMINE THE SPAWNING REGULARITIES AND CONDITION OF BALTIC HERRING (CLUPEA HARENGUS MEMBRAS) IN THE NE OF THE BALTIC SEA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Line 108-109: Please rephrase "  ...  twice warmer  .. " which does not make sense.

Make sure refs are correct, e.g.
line 335: (Arula 2014  should be Arula et al., ..

Conclusions points 1 and 3 should be written more clearly.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thanks for clarifying my points.
Also check the text for typos.


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: FIRST EX SITU OUTPLANTING OF THE HABITAT-FORMING SEAWEED CYSTOSEIRA AMENTACEA VAR. STRICTA FROM A RESTORATION PERSPECTIVE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper by Gina de la Fuente and co-workers is interesting since they provide a methodology based on outplanting of juveniles of algae of the species Cystoseira amentacea in places that –according to ancient data- are able to thrive but that are currently absent. The study is novel in the sense that this species has never been subject to this kind of outplanting, i grows in exposed rocky shores, it is very vulnerable to nutrient excess and other anthropogenic factors such as urbanization, and the method of attachment had never been used.

English grammar is fine but sentences are sometimes confusing and can be improved in order to enhance comprehension by the reader. The introduction is well contextualized although authors should take care in be more precise when talking about canopy forming algae in general versus Cystoseira and of the whole ocen versus the Mediterranean. I had problems in understanding what they were talking about in some sentences. Ancient literature has to be revised more thoroughly since they usually only use the most recent papers but not previous studies dating back to before 2005 or to the last century.

As it has been said already, the manuscript can be improved for clarity. Figures and tables are fine.
Experimental design: Regarding the experimental design, it is very simple as they describe a methodology and they are interested in the partial and final outputs but not in testing the different factors they have used in the methodological design. For instance they use antibiotics in culture to improve survival of recruits and early juveniles but do not have a control situation (no antibiotics). Therefore they can not test if the use of antibiotics makes any improvement in the survival/growth of the early juveniles. In my opinion the study is just a description of the method used and not a test to slightly different methods. However, I do not complain about this unless the authors do not say in the discussion that antibiotics are very useful in improving survival
Validity of the findings: The research is well defined and the aims of the paper too. Methods -in my opinion- are described well enough to be replicated.

Data is robust (it could not be other way regarding the huge amount of tiles used in the study) and therefore results are sound.
Additional comments: The writing is confusing, mainly in the results and discussion chapter and the authors should try to improve this aspect. Conclusions are not well stated and authors should also try to give a clear message of the specific results they obtained, not to limit in general aspects of the study.


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

Input:
Title: FIRST EX SITU OUTPLANTING OF THE HABITAT-FORMING SEAWEED CYSTOSEIRA AMENTACEA VAR. STRICTA FROM A RESTORATION PERSPECTIVE
Review round: 1
Reviewer: 2

Output:
Basic reporting: The basic reporting is ok, except for the use of English language which needs moderate improvement, although the meaning is general clear.

A few other points: the abstract is too short and needs more exact details of what the results were. And the figure legends need more detail (the figures + legends should be mostly understandable without looking at the rest of the text).The use of paragraph structure in parts of the introduction is unusual, maybe the small paragraphs need to be combined, as some have only one sentence.
Experimental design: 1) The research questions in the Introduction are strongly about restoration of intact functioning habitats, but these experiments are only applicable to very early stages of restoration (there is proper data only about the first 3 months). This experimental design does not thus allow much understanding of the possibility for eventual restoration success.

2) There are aims about assessment of grazers, but this part of the project is not properly developed. For example, it is not stated in the text where or when the grazers were collected from. It seems they were not collected from on top of the tiles (line 190), but I think sampling of grazers directly on the tiles would be the only way to get meaningful information. A proper hypothesis and statistical test would be ideal, e.g. comparison of grazer densities on vs off the transplanted algae.

3) Good data about size of the plants would be very useful for a project like this. The authors have some mention about measuring size of algae individuals (line 133) but again this part of the project is not well developed. In the results there is just a brief mention of sizes (line 186) with almost no details of how these measurements were made. If proper formal measurements were made, these should be displayed as a graph rather than just a series of informal photographs (Fig. 6) which are provided currently.

4) I do not think that the data about cover of algae from before to after transport to the field site will be very useful to most readers, and could be briefly mentioned in one or two sentences, or put in supplementary information.

5) The use of "cover classes" (line 152-155) is not ideal, as it seems three arbitrary categories have been chosen and the data put into them. These data are really continuous, not categorical data, so a regression analysis would be best. At the very least, the reasons for making those specific categories would need to be explained.

6) Overall, there was only limited use of statistics on the data. Analyses would be useful to see if the changes occurring over time were significant, whether the changes varied according to the different sites, correlations with grazer abundance etc. Only two tests are described (line 145-155), and the first of these (about changes before and after transportation) I do not think is very relevant (see point above).
Validity of the findings: 1) As "most of the tiles were detached" (line 131) during a storm after only 3 months, there needs to be strong emphasis on how the experimental attachment method was actually not successful. There is text which makes it seem like the attachment method worked (e.g. line 253). Most tiles detaching after 3 months I do not think could be considered successful, regardless of whether the storm that happened was "unprecedented" (line 132). Transplantation substrata must remain for many more months/years if self-sustaining populations of macroalgae will establish.

2) Not only did the method for attaching the tiles not really seem to work, but the persistence of the algae also seemed to be mostly unsuccessful. After 3 months there was a 60% drop in the number of tiles that had the presence of any macroalgae (Fig. 5). If this trend continued, all tiles would have lost their macroalgae within a few more months. The fact that the presence of the algae is declining towards zero is not discussed. Overall, the only evidence of success is some photographs (Fig. 6) but no valid data is provided to show that the scene in the photographs is representative to what occurred on average.

3) The discussion text about grazers (line 238) is not valid unless more extensive and clearly explained data on grazers are collected.
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: FIRST EX SITU OUTPLANTING OF THE HABITAT-FORMING SEAWEED CYSTOSEIRA AMENTACEA VAR. STRICTA FROM A RESTORATION PERSPECTIVE
Review round: 1
Reviewer: 3

Output:
Basic reporting: In general the MS is relatively easy to read but there are some specific sentences where the flow and/or the English language should be improved to ensure that an international audience can easily understand the text. Eg:
-	L40, Cystoseira is discussed, but the following line 41 the author refers back to all macroalgae. The author would improve the logical flow of the paper by revising the structure of this paragraph so that it reads large>small scale.
-	In terms of restoration techniques (L54), transplanting adults does enhance recruitment potential. I recommend the author groups the first two techniques together or ranks them i.e 1.(i) technique as (ii) technique b compared to 2. Technique c
I suggest the author revises the reporting here by condensing sentences and having a native English language speaker review your text before next submission.
The author has also included some (not all) of the relevant literature for their topic, however in some cases statements made in the paper were not referenced correctly or sufficiently.  E.g L30-31: “Around thirty-five percent of brown algae species (Fucales and Laminariales; Guiry and Guiry, 2019) contribute to the structure of the coastal landscapes, playing a key role as ecosystem engineers, providing an important value as natural capital.”
Several points are made in this sentence, however only the fact that “35% of brown algal species… is referenced. I suggest the author revises where they have placed the referencing and includes additional references where none are given.
The MS would benefit from expansion of many of the points made so that they include specific examples (and referencing) relevant to the local context. Some examples from the Introduction include:
-	No information is given about when, or why C. amentacea disappeared. Could the authors elaborate more on this in order to justify why they believe restoration is an appropriate measure, given that the population has been replaced by a more tolerant species and they have not mentioned that any of the factors contributing to the decline of these populations have been removed.
-	Could the author elaborate on the use of in situ vs ex-situ techniques and their relationship to dispersal capacity? (L64). Many kelps are thought to have dispersal limited to <10 metres from the parent alga – is this high dispersal capacity compared to C. amantacea?
Reporting of the methods results is also vague and incomplete in many places (specific cases in Methods section are discussed in the Experimental design section below), while some unnecessary information was given instead – eg:
L118 Dates of transplanting and the material of the boat used to transport the tiles are not relevant, but please include temperature and condition information.
 L166 While the reader may look at the figure, the author should be reporting the results (eg, mean and s.e) in the Results section, not just describing trends.
The author should also report the overall presence/absence of the plots following the storm ie at time 5 as rough weather is expected in any field-based restoration attempt.
Experimental design: The authors here report some promising results from the first attempt at restoring Cystoceira amentacea, however they do not explicitly test everything stated in the abstract. A clearer outline of the aims/objectives for this study or including additional data and analyses comparing different methods of transport and grazing could improve this.
There is also an urgent need to include more specific information in the methods - both to improve the reader’s understanding of what was tested and to make it reproducible. Some examples of where more information is necessary include:
-	Recording the GPS points of the sites (L81)
-	The size of the area that they collected algae from (L81)
-	The temperature and conditions of the “environmentally controlled rooms” (L91) you were using in the laboratory
-	The temperature and photoperiod used to culture (L100) (I appreciate this paper builds on Falace et al 2018, however it should include the optimal culture conditions here as a stand-alone document)
-	The statistical packages (and versions) used for data analysis.
Some additional data and analyses could also be presented here without much effort on the author’s behalf, including height/growth measurements of the outplanted juveniles and whether there was a difference between patches.
It is interesting that the author mentions that grazing is one of the predicted barriers to restoration success, and I commend them on trying to associate grazer abundance to restoration success. However, could the author please elaborate on why they collected abundance data from infauna as opposed to other mobile grazers which are often the cause of overgrazing in seaweed populations? Secondly, I suggest that the author includes several more sentences describing how they assessed grazing in the methods as it is not immediately clear how they came to the conclusion that “Grazing did not clearly affect the outplanted juveniles”L189. The authors have valuable data on benthic grazer abundance which they could do more with to support this claim, for example by looking at the relationship between grazer abundance and loss in percent cover in each plot.
Validity of the findings: The general overview of findings presented in the results and discussion includes some elements of subjectivity and also informal comparison to other studies. I suggest that the author moves any value judgements and assessments to the discussion section, but they should also take care not to over-estimate the conclusions that can be made from this study alone. Eg: L204-205: “The new culture medium formula, the antibacterial mixed solution and the enhanced hydrodynamics preserved the culture in good conditions” This was not tested with the study design, therefore I would encourage the author to either adjust this sentence or include experimental data supporting this conclusion in the study.
Some of the conclusions made here are also confusingly reported or contradictory eg: L217 the author discusses cover of juveniles on tiles having increased, but in the results it says the overall number of tiles with juveniles had decreased. If you are to include this result in the discussion, please give more information in the methods and results.
L229 it says that the methods used here “reinforces...the attachment avoiding the possible dislodgement of the clay tiles by wave action" however in the beginning it stated a storm severely affected the tiles...please explain.
Additional comments: The aim of this paper is to report the first ex situ outplanting of the habitat forming seaweed C. amentacea for restoration purposes. The paper describes the methods used to collect wild specimens of C. amentacea, culture juveniles in the lab under pre-defined conditions and provides some estimates of survival and growth over the first 4 months following outplanting in the field.
This is a novel study with some interesting results regarding growth and survival of outplanted C. amentacea in the field, however there are several issues with basic reporting and analysis that are fundamental to the understanding and conclusions drawn in the paper and need to be improved before this MS should be considered for publication. I suggest that the authors revise and resubmit this MS and have included some examples where it could be improved.


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: FIRST EX SITU OUTPLANTING OF THE HABITAT-FORMING SEAWEED CYSTOSEIRA AMENTACEA VAR. STRICTA FROM A RESTORATION PERSPECTIVE
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have improved the manuscript a lot according to my suggestions, In my opinion it is now, with very minor changes (see the pdf file), sitable for publication
Experimental design: ok
Validity of the findings: ok
Additional comments: ok


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors present a cohort of preterm babies followed by longitudinal stool sampling at NICU. Small subset of babies developed NEC (n=4) or LOS (n=3) which are then compared to healthy controls. The presentation of the data, bioinformatic and statistical methods used and English language are currently inadequate for publishing the manuscript.

The introduction is jumping between different topics and many citations are old and appeared in low impact journals (e.g. Matamoros et al.). There is abudance of recent work in infant gut microbiome which should be cited to introduce readers to the field. The importance of sentence on lines 97-100 is unclear. I suggest starting with couple of sentences on post-birth gut microbiome, briefly defining the conditions in focus (NEC and LOS), describing previous gut microbiome work on these diseases and leading up to the motivations and goals of the current study.

Lines 326-328: Authors are overselling their study
Experimental design: Authors use an outdated method for processing 16S rRNA gene sequecing data. Independent comparisons operating with mock community data have shown that uparse is prone to false positive OTUs (see e.g. PMID: 27822515). Authors should process the 16s amplicon data using an up-to-date method such as DADA2 (PMID: 27214047) or deblur (PMID: 28289731)
Validity of the findings: Figure 2 legend is missing and any information on statistical test used is missing even though p-values are reported on panel B. Panel B is also mostly unreadable since the relative abundance of most taxa is low and the bars are not scaled accordingly. In Methods, explain the statistical methods used and how do they account for longitudinality in the data.

Authors compare bacterial OTU counts between groups but this is not feasible since the groups differ by size and lacks any statistical assessment. I suggest measure bacterial richness (number of OTUs) or other measures of alpha-diversity per each stool sample and conducting statistical testing between groups. Longitudinal nature of the data (repeated measures from same subjects) needs to be taken in to account in selecting the statistical test used. Similarly, comparing mean relative abundances between groups is not adequate since it doesn't account for repeated measures. I suggest using mixed effects linear models or something similar to conduct statistical testing for relative abundances.
Additional comments: BioProjecgt PRJNA470548 was not found in NCBI SRA.


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 1
Reviewer: 2

Output:
Basic reporting: A small scale study of NEC and LOS in preterm infants, showing the difference in basic metagenomes between controls and NEC/LOS. Clear and unambiguous, professional English used throughout. Sufficient field background/context provided. Professional article structure, figs, tables.

However, some novel data is first presented only in the discussion section.

Raw data (link to SRA) has been shared privately. However, a link to the SRA does not appear to be with the paper text. There needs to be a mention of the SRA ID in the text. Apologies if I missed it.
Experimental design: Very well done experimental design and research question. Methods sufficiently described. Sequencing depth (data generation) and analysis pipeline is appropriate to achieve aims of analysis.

However, results reveal that the design was underpowered for full 16S analysis across the two treatment and one control group. Thus, few significant results. Appropriately powered to generate a hypothesis, seems appropriate for a small journal such as PeerJ.
Validity of the findings: Largely negative/inconclusive results. Small (but significant) differences seen between case and control groups. Conclusions from the data are not overstated. Are reasonable, given the results.

Since NEC average onset was 16 (within the 14-21d range) and LOS was 12 (within the 7-14d range), why is LOS not showing a similar significant trend for the 7-14d range in a similar way to the 14-21d range shows significant differences in NEC?

Lines 285-287: Introducing new data in the discussion section. Why are these figures not reported in the results section?
Additional comments: Line 39: "dysbiotis" should be "dysbiosis"
Line 46: "rest17" should be "the rest (17)"
Line 67: delete "For" and start sentence with Preterm infants, who are prone...
Line 255: "less significant" -- the p value is > 0.05 so it is not a significant finding. Please correct the text.
Line 269: change "little" to "few"
Line 270: change "that" to "those"
Line 279: I believe you mean "7,472,400"
Line 317: "<Fig. S2" remove "<"

Figures:
S1a-c: Order of the legend is confusing. Please re-order the legend so that items in the legend from top to bottom are in order of days (i.e. 0-7 on top, and >28 on bottom)
Fig 2: Since PC1 and PC2 are such a high proportion, there is no need for S2b and S2c. S2a is sufficient to show the trend. Other figures are distracting.


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English is not acceptable (see general comments for some additional information)

No P values are provided in the abstract and so there is no way of knowing if the reported diversity of taxa %s are important.  The same largely applies for the main results.

Sometimes the authors report 3 NEC and 4 LOS, and other times they report 4 NEC and 3 LOS.
Experimental design: With only 24 infants and a total of 192 stool samples, this study falls well short of most studies published in this area over the past years.  As an aside, many of these recent larger studies are not cited in the manuscript.   Additionally, with only 4 NEC and 3 LOS infants, the study is greatly underpowered and fails to add substantially to the current literature.

It is not clear which samples were from the diaper or from the perianal skin surface. Furthermore, the latter represents a unique and none validated sample – is this representative of gut or stool?  I also wonder about the ethics of collecting perianal samples using a spatula from extremely preterm infants.

With read lengths of ~500bp there is likely to be a high error rate in the data. Pat Schloss explains this perfectly in his excellent blog post - http://blog.mothur.org/2014/09/11/Why-such-a-large-distance-matrix/
Validity of the findings: How were the samples normalised?  There is no mention of rarefaction or other?

No P values are reported for the alpha diversity or taxonomic comparisons, with the exception of the random P value on lines 234 and 235.  For the second P value the authors report “weakly significant”, but this P value was 0.11 and is therefore not significant.
Additional comments: The English is generally poor throughout making the manuscript challenging to read.  While I cannot go through the entire text line-by-line and improve the English, from the abstract alone here is a selection of some errors:
-	line 14 I think the authors mean “the remaining 17 were..”, not “the rest17”.
-	Sometimes abbreviations are used and other times they are wrote in full, e.g., line 50 Late-onset sepsis should be LOS as it has already been abbreviated, in the conclusion for both NEC and LOS
-	Line 50-51 “…held the least diversified gut microbiota” is poor English, similarly line 52 “with the control group held the most diversified one” is also poor English.
-	Line 54 “Both two groups”

It is not clear why sometimes rRNA is used and other times rDNA is used.  The authors should be clear this is 16S rRNA gene sequencing and use this phrase throughout (e.g., in place of rDNA).

It is not stated what organism was cultured in the third LOS infant.

Centroids should be added to Figure 4, otherwise it is impossible to see what is going on.


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 2
Reviewer: 1

Output:
Basic reporting: -
Experimental design: -
Validity of the findings: -
Additional comments: Authors have addressed my previous concerns and the manuscript is improved greatly. I have one final comment on the revised manuscript:

lines 255-259: Authors are over-interpreting the increasing trend in the variance explained by principal coordinate 1. This increase only reflects the ability of the PCoA graph to display more variation in the data; i.e. the data is better represented by 2D graph. It does not necessarily tell anything about the separation between the groups. The separation between the groups can to be measured by PERMANOVA (adonis test) R-squared (variance explained by the grouping) or other similar tests. Note that adonis test is not able to account for repeated sampling.


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 2
Reviewer: 2

Output:
Basic reporting: I believe this is an impressive level of re-do on the resubmission. I have no further issues with this article.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Congratulations on the vast improvement of this paper.


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

Input:
Title: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 2
Reviewer: 3

Output:
Basic reporting: The English is much improved
Experimental design: I am confused by the new analysis where authors have divided the sampling span into seven time intervals.  It is not clear if repeated measures (i.e., samples from the same individual appear in this analysis)?

I am not clear if the LME adjusted for multiple comparisons?
Validity of the findings: no comment
Additional comments: The issue of repeated measures appears to be outstanding and will skew/inflate the P values.

Figs 2 and 3 compare changes over time within each group, but not between groups.  Therefore, I am unable to see how the authors can make claims about about NEC and LOS showing differences to controls when this direct comparison was not performed.  This is partially addressed in Fig 4, and it is interesting that the Shannon diversity is higher in controls in early disease, but one wonders if this simply reflects the use of antibiotics in the disease groups (which are known to reduce diversity).

My earlier point about adding centroids to the PCoA plots has not been addressed.  Additionally, the authors should add the P values to these plots and inline with the above comments need to ensure they do not have repeated measures in these plots.

The authors have not stated which rarefaction level they used - this needs to be added to the main text.

I wonder if Fig 6 could be improved by making it clear which genera were significant and if they were higher in NEC, LOS, or controls


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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 3
Reviewer: 1

Output:
Basic reporting: Authors have addressed my comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: No 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: PATTERNED PROGRESSION OF GUT MICROBIOTA ASSOCIATED WITH NECROTIZING ENTEROCOLITIS AND LATE ONSET SEPSIS IN PRETERM INFANTS: A PROSPECTIVE STUDY IN A CHINESE NEONATAL INTENSIVE CARE UNIT
Review round: 3
Reviewer: 2

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: EXTINCTION RISK OF NARROWLY DISTRIBUTED SPECIES OF SEED PLANTS IN BRAZIL DUE TO HABITAT LOSS AND CLIMATE CHANGE
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, the paper offers interesting insight into plant threats and conservation in Brazil, and is a timely review of the information found in Rapini´s book (rare plants of Brazil). In terms of balance, I think the 4 maps offered in figure 1 are perhaps not necessary (available elsewhere, not original, not part of their work).

The paper would benefit if the discussion was shortened (I found it overlong and repetitive).

In the introduction (and subsequent discussion) I found that some important literature and statements were missing. For instance, authors expand into IUCN but do not cite:  https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135152
or https://www.nature.com/articles/nplants2015142.
I explain this further in my notes for them (General comments for the authors).

Thus a little more reading on plant extinction would improve the introduction and discussion.

For instance, I missed some reference or mention to the actual extinction in plants (EX, EW), which I presume is not as commonly recorded as with animals would help to provide a fuller picture.

In their methods the authors don´t explain why could they not work with the orchids? A simple search yelded 428 new records (possibly species) described from 2008 until this year. It is a considerable set of data not to use without a full justification.

Discussion:
Nowhere it has been made clear whether the endemic plant areas pointed out in the Rare plants book (Giulietti et al. 2009, of which Rapini is an author) are the same or have changed since.

When the authors write ‘even’ ex-situ conservation techniques… seedbanking it gives the impression they do not have much faith in this techniques, I suggest delete the word ‘even’.

Overall I found the discussion a bit repetitive and overlong – please compress it.

Lastly, I have highlighted the text where I thought it could be improved.
Experimental design: Results:

The biggest problem with reviewing this paper is that, while trying to check an area (by using supplementary material excell table with area data (state, area_name, ecological region) I found no link between that and the map provided (fig 3).

It  would be much more useful if the name of the watershed was given in this table.

The authors mention Serra do Cipó and Diamantina plateau, but these are not named in the table. In order for me to be able to check their results during the review, and because it would make the paper much more useful, the table should also have the name of the taxa that are presumed to be endemic per area, so that this could be used by conservation practitioners.
Validity of the findings: no comment
Additional comments: Review for PEERJ
Extinction risk of narrowly distributed species of seed plants in Brazil due to habitat loss and climate change (#37305)

Overall, the paper offers interesting insight into plant threats and conservation in Brazil, and is a timely review of the information found in Rapini´s book (rare plants of Brazil). In terms of balance, I think the 4 maps offered in figure 1 are perhaps not necessary (biome map is avaliable in Forzza 2012, which they refer to, and again in BFG 2015 etc.), watershed map is very intricate and not very informative as it is presented, could be supplementary material, likewise for habitat loss and climate change maps (available elsewhere, not original).
The paper would benefit if the discussion was shortened (I found it overlong and repetititve).

Introduction:
Lines 89 to 95 when authors expand into IUCN and the situation with plants, I think it is very important to cite papers such as  https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135152
Where the authors provide important insight in the situation of threatened plants worldwide
And also https://www.nature.com/articles/nplants2015142 where they can find out about the particular situation of cacti (many of them are endemic with narrow ranges).

I also think that reference or mention to the actual extinction in plants (EX, EW), which I presume is not as commonly recorded as with animals would help to provide a fuller picture. I think this may be an important area to explore in the introduction.

Lines 101-102 – unclear.

Material and Methods:
Line 119 – the authors don´t explain why could they not work with the orchids? A simple search yelded 428 new records (possibly species) described from 2008 until this year. It is a considerable set of data not to use.

Line 139 – the word Caatinga may not be familiar for all readers and comes without explanation. It is mentioned later (line 141) as one of the domains.

Line 141 – the phytogeographic domains listed here are the same areas referred to as
distinct ecological regions (line 110). Forzza et al. 2012 translate these satisfactorily as biomes, and I suggest this shorter term is used throughout to avoid confusion.

Line 162 – check the figure highlighted

Results:

The biggest problem with reviewing this paper is that, while trying to check an area (by using supplementary material excell table with area data (state, area_name, ecological region) I found no link between that and the map provided (fig 3). It  would be much more useful if the name of the watershed was given in this table. The authors mention Serra do Cipó and Diamantina plateau, but these are not named in the table. In order for me to be able to check their results during the review, and because it would make the paper much more useful, the table should also have the name of the taxa that are presumed to be endemic per area, so that this could be used by conservation practitioners.

Discussion:

When mentioning the 437 species listed in the Brazilian Red List, and the 558 that appear in the IUCN, it would be interesting to know whether these species are the same or totally unrelated. The Brazilian Red List had over a thousand species listed as DD back in 2013/14, which reinforces your argument about the conservative approach of their methods.

Line 271 – 272, please read Goettsch et al. (suggested above for introduction), where traditional IUCN methods were used for one family across the board (global assessment), your affirmation that ‘ probably underestimated by using traditional
271 conservation assessments’ does not provide an accurate diagnostic of threat against plants may not hold.  I also think that data present already in Green plants in the Red (2015, also suggested above) are relevant for this part of the discussion.

Line 283 – I think it is both habitat loss and degradation.

Lines 321 to 324 – it is not clear whether the endemic plant areas pointed out in the Rare plants book (Giulietti et al. 2009, of which Rapini is an author) are the same or have changed since.

Line 330 – not sure whether the areas pinpointed harbor thousands of narrowly endemic – from the maps I can see that the majority of the 700 + watersheds include a single micro-endemic

Lines 351-352 – unclear.

Lines 365 – when the authors write ‘even’ ex-situ conservation techniques… seedbanking it gives the impression they do not have much faith in this techniques, I suggest delete the word ‘even’.

Overall I found the discussion a bit repetitive and overlong – please compress it.

Lastly, I have highlighted text where I thought comprehension was difficult or where there were minor mistakes, in the hope that authors correct them.


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: EXTINCTION RISK OF NARROWLY DISTRIBUTED SPECIES OF SEED PLANTS IN BRAZIL DUE TO HABITAT LOSS AND CLIMATE CHANGE
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: this is very straightforward and useful.  well written and clear....only three picky comments.

line 84 I think this is an inappropriate use of the term "orders of magnitude"; better to use a different pgrase
line 359 better "to" rather than "for" design....
line 390: should be irreplaceable not unreplaceable


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: MEASURING THE DISPERSION OF RAINFALL USING BAYESIAN CONFIDENCE INTERVALS FOR COEFFICIENT OF VARIATION OF DELTA-LOGNORMAL DISTRIBUTION: A STUDY FROM THAILAND
Review round: 1
Reviewer: 1

Output:
Basic reporting: See the attached file
Experimental design: See the attached file
Validity of the findings: See the attached file
Additional comments: See 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: MEASURING THE DISPERSION OF RAINFALL USING BAYESIAN CONFIDENCE INTERVALS FOR COEFFICIENT OF VARIATION OF DELTA-LOGNORMAL DISTRIBUTION: A STUDY FROM THAILAND
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting statistical area with a good applciation.
Experimental design: Not applicable.
Validity of the findings: I think the findings are correct as much as I am able to assess this from outside.
Additional comments: I like the paper but have a few suggestions:

It would be good to talk right in the ebginning about the application data. Show that there is a zero-inflation which makes a lot of sense as there are these summer months  Dec to April with almost no rainfall. Then show, for example using the distribution function, that the the delta-log-normal matches very well the EDF.

I have some questions w.r.t. the methodology starting page 3. You derive all posteriors in closed form, so  CIs and HPDs should be easly calculable with a standard function. What is the purpose of the algorithms then? They seem a bit mysterious.

I understand that all Bayesian CIs do good. But why not use clssical confidence intervals as comparisons? I mena those you would get using the SEs derived from the Fisher information matrix?

Fianlly, it would be nice to gibe useres some well-documented R-code that they can use to derive the intervals using their 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: MEASURING THE DISPERSION OF RAINFALL USING BAYESIAN CONFIDENCE INTERVALS FOR COEFFICIENT OF VARIATION OF DELTA-LOGNORMAL DISTRIBUTION: A STUDY FROM THAILAND
Review round: 2
Reviewer: 1

Output:
Basic reporting: None
Experimental design: None
Validity of the findings: None
Additional comments: Thank you for your revision. However, I have a few question as follows.

1.  I(delta', mu, sigma^2) in eq.(9) as you use right now is the Fisher information of the parameters in the delta-LN distribution. It can lead to misunderstanding. So you should change the symbol I() in eq.(9) to represent that it is derived from the Binomial.

2.  Which step is applied the matrix in eq.(8)?

3.  Thank you for giving the R-code. Why you separate the codes for FGCI, HPD, and Equitailed confidence intervals, not compute them using the same simulated data? This is because you need to compare them.


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: MEASURING THE DISPERSION OF RAINFALL USING BAYESIAN CONFIDENCE INTERVALS FOR COEFFICIENT OF VARIATION OF DELTA-LOGNORMAL DISTRIBUTION: A STUDY FROM THAILAND
Review round: 2
Reviewer: 2

Output:
Basic reporting: Revision is fine. However, you responded to my comment on classical CIs as follows:

"For classical confidence intervals, we have previously been applied the Fletcher
method to construct the confidence intervals which can be seen from Yosboonruang et al.
(2018) as references. That method does not work well in term of the coverage probability and
the expected length. Therefore, we do not used it to compare this proposed method."

But I see nothing of this nature in the actual text. It would be good as Motivation to say what are the classical approaches and that they fail (with a reference) and why you did the work present here.
Experimental design: ok
Validity of the findings: ok
Additional comments: see above


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: CONSTRUCTION AND INTEGRATED ANALYSIS OF CROSSTALKING CERNAS NETWORKS IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comments
Experimental design: no comments
Validity of the findings: I appreciate the chance to review this paper, although there is great effort in pre-paring this manuscript, the main drawback is it do not add any information we have already known.
1.	The discussion section is too simple and superficial, the authors must revise it in deep.
2.	In the screened RNA, cytological validation or molecular biology validation in tissues is recommended.
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: CONSTRUCTION AND INTEGRATED ANALYSIS OF CROSSTALKING CERNAS NETWORKS IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, Liu et al. have investigated the functions of lncRNA as ceRNA in LSCC from the TCGA. The results suggested that the potential regulatory roles of different RNAs might be the mechanisms of LSCC tumorigenesis. Overall, the topic is interesting and the data is robust, statistically sound.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. The standard of English is poor and unacceptable, partly so that no sense can be derived from sentences as they stand. There are a lot of grammatical mistakes, omitted words, and phrases not commonly used in English.
2.The authors write “a systematic understanding of how ceRNA network contributes to tumorigenesis and progression of LSCC is still lacking” (lines 83-84). Actually, several studies have already reported the comprehensive analysis of the coding and non-coding RNA in LSCC from tissues before. Other literatures have also reported ceRNA network in recurrence LSCC from TCGA. The paper didn’t have mentioned these relevant prior literatures at all. The novelty of this paper remains to be proved.
3. The section of Abstract needs to be polished. The results are too general and the prognostic significance didn’t have been mentioned in conclusion.
4. The basic clinical information should be provided in detail, such as age, TNM stage, vital status, subdivision.


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: CONSTRUCTION AND INTEGRATED ANALYSIS OF CROSSTALKING CERNAS NETWORKS IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 1
Reviewer: 3

Output:
Basic reporting: -	Punctuation, spaces issues and capital words in the middle of the sentences throughout the manuscript need to be corrected.
-	Line 380, figure number is missing
-	Acknowledgements were not added
Experimental design: Competitive endogenous RNA network has been used with the same approach in many articles.
-	For figures 4B, 4C and 4D, how did the authors determine the cutoff for low and high expression.
Validity of the findings: The paper would be more relevance by performing an experimental validation to support some of the computational results.The authors need to validate the interaction of the suggested lncRNA-miRNA-mRNA network.
Additional comments: In this manuscript, the authors analyzed expression profiles of lncRNA, miRNA ad mRNA in laryngeal squamous cell carcinoma (LSCC) from the TCGA database. Gene Ontology (GO) as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were analyzed and a ceRNA network was constructed. The authors identified potential regulatory lncRNA, miRNA and mRNA that might be one of the mechanisms of LSCC tumorigenesis. Overall, the work is interesting.


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

Input:
Title: CONSTRUCTION AND INTEGRATED ANALYSIS OF CROSSTALKING CERNAS NETWORKS IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have well revised the manuscript. I think this version is worth to be accepted.


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

Input:
Title: CONSTRUCTION AND INTEGRATED ANALYSIS OF CROSSTALKING CERNAS NETWORKS IN LARYNGEAL SQUAMOUS CELL CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have responded to all concern previously raised by the reviewer and have adequately addressed them.


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: RITAN: RAPID INTEGRATION OF TERM ANNOTATION AND NETWORK RESOURCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Figure 1 legend:
Starting from Line 396 “Adding a study design matrix above ...”, you are already talking about Figure 1C, but this is not indicated. Same for Line 403, you wrote “panel B” but seems to be discussing “panel C”.
Experimental design: Vignette needs update. Please also check other parts.
I ran the “Quick Start” code and found that the parameter “resources” should be “term_sources”.
(https://rdrr.io/bioc/RITAN/f/vignettes/enrichment.Rmd).
Validity of the findings: The authors describe a package (RITAN) that can annotate a list of genes with functions using a large collection of gene function definitions (16 term annotations from public databases). The method is similar to existing ones:  using hypergeometric test to check if the list of input genes overlap significantly with another group of genes with a defined function. RITAN can estimate similarities between functional-related gene groups to avoid including redundant gene sets. RITAN can also handle user defined functional-related gene groups. These functions will greatly save researchers’ effort when trying to use multiple data sources.
Although many of the functions are already provided by published tools such as DAVID https://david.ncifcrf.gov/, RITAN still has its merit:
The programing interface of RITAN makes it a better choice for researchers with R programing skills. It can also easily generate a nice heatmap to visualize the q-values.
Additional comments: 1. Results and discussions are all in one section “Discussion”.   PeerJ does offer a “Results” section, so it is better to separate "results" and "discussion".

2. Figure 1 legend is too long. Many of the content belongs to “Results”.  Please separate them if possible.


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: RITAN: RAPID INTEGRATION OF TERM ANNOTATION AND NETWORK RESOURCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	Basic reporting:

The authors provided examples to explain how RITAN can facilitate hypothesis generation and exploration in research. However, they do not explain the methodology to implement RITAN, which should be part of this manuscript. Is the methodology of RITAN better than other existing resources?

The figures are not well explained. The authors need to be careful about writing/formatting as well. For example:
In the figure legend for 1B fits for 1C.
There is no explanation of figure 1 in the main text. The authors should simplify the figure legend for figure 1 and explain the figure in detail in the main text (line 135).
In Figure 2, parts of the labels are cut off by the margin. In 2B, the labels are overlapping.
The presentation is really sloppy.

There is no result section, which makes little sense, Instead, the result section is integrated in the discussion...WHY?
Experimental design: The purpose of their tool is to assist researchers with the annotation of their gene sets derived from high-throughput studies. It is not obvious who their user target is. Basic biology researchers are not familiar with R programing. Hence, an additional web interface for users would be much more helpful for a broad audience.
Validity of the findings: As the author mentioned, there are already many resources that standardize and codify different databases for gene function from different perspectives. The authors failed to address what  the limitations of these resources are and how RITAN performed better than those. A comparison is important.
Additional comments: It is not user-friendly and cannot be used without using R.  Hence, impact is limited. Also, the presentation is sloppy (e.g. Figure 2B overlapping text; Figure 3A, basal versus basel, 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: RITAN: RAPID INTEGRATION OF TERM ANNOTATION AND NETWORK RESOURCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: All concerns have been addressed in this version.
Experimental design: All concerns have been addressed in this version.
Validity of the findings: All concerns have been addressed in this version.
Additional comments: All concerns have been addressed in this version.


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: RITAN: RAPID INTEGRATION OF TERM ANNOTATION AND NETWORK RESOURCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have made proper corrections to address our concerns.
One minor issue: Figure 2A, the color scale is missing when the file is visualizing using Preview. The figure looks fine with Adobe reader though.
Experimental design: The authors have separated the result and discussion section that is much clearer now.
Validity of the findings: None
Additional comments: No additional comments


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

Input:
Title: IDENTIFICATION OF PRIMARY GENES IN GLOMERULI COMPARTMENT OF IMMUNOGLOBULIN A NEPHROPATHY BY BIOINFORMATIC ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript submitted by Miraji A. et. al. reported the gene expression profiling data performed on the glomeruli of patients with IgA nephropathy and normal controls using the Gene expression Omnibus database system. Several studies earlier reported the association of these differentially expressed genes including CX3CR1 and fibronectin in case of IgA nephropathy (Romgnani, 1999, JASN). So, the gene expression profiling and the reported genes in case of IgA nephropathy may not be novel but still important at this juncture. I have a few comments.
1.	They reported that total 28 genes were upregulated and 12 genes were downregulated. But the authors briefly discussed fibronectin, CD44, CX3CR1, and CCL4. Also, it is well reported these genes related to chemokines CX3CR1 and CCL4 involve in the signaling pathways. It would be interesting if they discuss and specify the name of the signaling pathway and their role(s) in the progression of IgA nephropathy. Also, they should support their findings by citing earlier published reports. For. e.g a study by Berthous F.C, 2006, Kidney Int. (not cited in the paper) reported the genetic polymorphism in the CCR5 (chemokine receptor for CCL4) in patients with IgA nephropathy and showed the genotype-phenotype correlation.
2.	It is advised to explain the details of the database selection criteria used for the selection of glomeruli samples from patients with IgA nephropathy and healthy controls in the method section.
3.	It is recommended to revise the language and make it crisper. For example line 130, it is mentioned: “Cellular components analysis showed that up-regulated DEGs were mainly involved in the extracellular region, extracellular space and integral component of plasma membrane”. There is no difference in the meaning of “extracellular region” and “extracellular space”. Line 134, it is mentioned, “Down-regulated DEGs were mainly involved in sequence-specific DNA binding, transcriptional activator activity and DNA binding”. “DNA binding” reads repetitious!
4.	A careful proof of the manuscript is recommended to correct grammatical and typographical errors. For e.g, line 122, “The DEGs was annotated...”
5.	References: be consistent throughout the m/s. a)	At most of the places references are cited with the name of the authors except ref.14, line 102. b) The font style of some of the references (line 135, 318, 364) is different from others.
Experimental design: The detailed comments are mentioned in the basic reporting section. They need to specify the selection criteria used for selecting the databases for glomeruli samples from patients with IgA nephropathy and healthy controls in the method section.
Validity of the findings: See general comments for the authors.
Additional comments: The manuscript submitted by Miraji A. et. al. reported the gene expression profiling data performed on the glomeruli of patients with IgA nephropathy and normal controls using the Gene expression Omnibus database system. Several studies earlier reported the association of these differentially expressed genes including CX3CR1 and fibronectin in case of IgA nephropathy (Romgnani, 1999, JASN). So, the gene expression profiling and the reported genes in case of IgA nephropathy may not be novel but still important at this juncture. I have a few comments.
1.	They reported that total 28 genes were upregulated and 12 genes were downregulated. But the authors briefly discussed fibronectin, CD44, CX3CR1, and CCL4. Also, it is well reported these genes related to chemokines CX3CR1 and CCL4 involve in the signaling pathways. It would be interesting if they discuss and specify the name of the signaling pathway and their role(s) in the progression of IgA nephropathy. Also, they should support their findings by citing earlier published reports. For. e.g a study by Berthous F.C, 2006, Kidney Int. (not cited in the paper) reported the genetic polymorphism in the CCR5 (chemokine receptor for CCL4) in patients with IgA nephropathy and showed the genotype-phenotype correlation.
2.	It is advised to explain the details of the database selection criteria used for the selection of glomeruli samples from patients with IgA nephropathy and healthy controls in the method section.
3.	It is recommended to revise the language and make it crisper. For example line 130, it is mentioned: “Cellular components analysis showed that up-regulated DEGs were mainly involved in the extracellular region, extracellular space and integral component of plasma membrane”. There is no difference in the meaning of “extracellular region” and “extracellular space”. Line 134, it is mentioned, “Down-regulated DEGs were mainly involved in sequence-specific DNA binding, transcriptional activator activity and DNA binding”. “DNA binding” reads repetitious!
4.	A careful proof of the manuscript is recommended to correct grammatical and typographical errors. For e.g, line 122, “The DEGs was annotated...”
5.	References: be consistent throughout the m/s. a)	At most of the places references are cited with the name of the authors except ref.14, line 102. b) The font style of some of the references (line 135, 318, 364) is different from others.


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

Input:
Title: IDENTIFICATION OF PRIMARY GENES IN GLOMERULI COMPARTMENT OF IMMUNOGLOBULIN A NEPHROPATHY BY BIOINFORMATIC ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In this manuscript, the authors integrated mRNA profiling studies to identify differentially expressed genes in IgAN glomeruli samples. There are some concerns before the manuscript could be published.
1.	The criteria of selecting mRNA studies deposited in GEO database should be more carefully checked. As far as I am concerned, there are some additional datasets containing IgAN with more larger sample size.
2.	For multiple correction in statistics, proper method should be adopted.
3.	In expression data, there would be more than one probes designed for different exons in a single gene. The issue should be carefully checked for expression in a single study and in a combined analysis from multiple studies.
4.	I would recommend to conduct a meta-analysis with these data instead of just checking the overlapping, and a strictly quality control should be taken before further statistics.
5.	The introductions of the PPI in the METHODS section should be moved to the INTRODUCTION section.
6.	It is confused that both the positive regulation of transcription from RNA polymerase II promoter and the negative regulation of transcription from RNA polymerase II promoter exist in down-regulated DEGs.
7.	There are some overstatements. i.e. “Mesangial cells proliferation is a pathological feature in IgAN, thus FN1 is directly associated with the occurrence and development of primary IgAN”
8.	The references should be updated, especially in the DISCUSSION section.
9.	The limitations of the study design and the methodology should be discussed.


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

Input:
Title: IDENTIFICATION OF PRIMARY GENES IN GLOMERULI COMPARTMENT OF IMMUNOGLOBULIN A NEPHROPATHY BY BIOINFORMATIC ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The author added a brief description of the potential signaling pathways associated with the genes that were upregulated in patients with IgA nephropathy. They did not propose the potential/possible pathway(s) related to the downregulated genes and mentioned that there was no significant pathway involved with the down-regulated genes. There are still several typographical and grammatical errors in the manuscript. For example:

a.	Line 32 replace “Therefore, IgAN has been a healthy concern throughout the world” with “Therefore, IgAN has been a health concern throughout the world”
b.	Line 65, replace “(AQCg and AQCp) and consistency quality control (CQCg and CQCp)” with “(AQCg and AQCp) and consistent quality control (CQCg and CQCp)”.
c.	Line 138, replace “fibronetin” with “fibronectin”.
d.	Line 142, correct “Therefore, the expression of FN1 gene may also influeced the..” with “Therefore, the expression of FN1 gene may also influence the..”
e.	Line 154, replace “CX3CL1/CX3CR1 axis can up-regulates..” with “CX3CL1/CX3CR1 axis can up-regulate..”
f.	Line 163, correct “end stage real disease” with end-stage renal disease”.
g.	Line 167, correct spelling of mechanism as written “mechanism”
h.	Line 1. Title replace “a nephropathy” with “A nephropathy”
i.	Minor, Line 7, correct spelling of author as written “aurthor”
Experimental design: We have earlier discussed this m/s. The comments on the revised version are discussed in the basic reporting.
Validity of the findings: We have earlier discussed this m/s. The comments on the revised version are discussed in the basic reporting.
Additional comments: The author added a brief description of the potential signaling pathways associated with the genes that were upregulated in patients with IgA nephropathy. They did not propose the potential/possible pathway(s) related to the downregulated genes and mentioned that there was no significant pathway involved with the down-regulated genes. There are still several typographical and grammatical errors in the manuscript. For example:

a.	Line 32 replace “Therefore, IgAN has been a healthy concern throughout the world” with “Therefore, IgAN has been a health concern throughout the world”
b.	Line 65, replace “(AQCg and AQCp) and consistency quality control (CQCg and CQCp)” with “(AQCg and AQCp) and consistent quality control (CQCg and CQCp)”.
c.	Line 138, replace “fibronetin” with “fibronectin”.
d.	Line 142, correct “Therefore, the expression of FN1 gene may also influeced the..” with “Therefore, the expression of FN1 gene may also influence the..”
e.	Line 154, replace “CX3CL1/CX3CR1 axis can up-regulates..” with “CX3CL1/CX3CR1 axis can up-regulate..”
f.	Line 163, correct “end stage real disease” with end-stage renal disease”.
g.	Line 167, correct spelling of mechanism as written “mechanism”
h.	Line 1. Title replace “a nephropathy” with “A nephropathy”
i.	Minor, Line 7, correct spelling of author as written “aurthor”


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

Input:
Title: IDENTIFICATION OF PRIMARY GENES IN GLOMERULI COMPARTMENT OF IMMUNOGLOBULIN A NEPHROPATHY BY BIOINFORMATIC ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors significantly addressed the raised issues.
However, there were still some typos and grammar errors. I would like to suggest a language editing by native speakers.


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: EVALUATING THE OESTROGENIC ACTIVITIES OF AQUEOUS ROOT EXTRACT OF ASPARAGUS AFRICANUS LAM IN FEMALE SPRAGUE-DAWLEY RATS AND ITS PHYTOCHEMICAL SCREENING USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.1 English is clear and professional english.
1.2 References in the main text are numbered but the reference section is just alphabetical. Which makes a detailed review impossible.
1.3 Introduction: Add sentence for what specifically asparagus africanus is used for in traditional medicine?
1.5 I am lacking some more discussion and examples of other plants which are known to be endocrine disruptors and their possible use and interaction effects in medicine. E.g. Female hop (Lupuli flos) ploughers can have varying menstrual cycles and decreased fertility rates.
1.6 line 93: Weight of the extract after drying should be noted in the manuscript.
1.7 Line 254: “This dose …” Unclear about what dose the authors talk?
1.8 Line 257: Abbreviation “SERM” not introduced in the manuscript and meaning is unclear.
1.9 Figures are relevant, high quality, well labelled & described. Raw data is attached which helps with the review.
Experimental design: 2.1 The primary research is within the scope of the journal.
2.2 Research question well defined, relevant & meaningful. It is stated how the research fills an identified knowledge gap.
2.3 Line 91: Has the extraction been performed under light or no light?
2.4 Otherwise experimental details and results are well described and acceptable for publication.
Validity of the findings: 3.1 Several of the author conclusions are well stated but I am unable to link them to the supporting results. In general, the results give more questions than answers and I would like to explain and discuss that with the next points. In general, I m lacking to see dose-response curve like effects between the 50, 200 and 800 mg/kgBW groups for the various measured endpoints. It looks a little bit like that for the highest dose effects are different compared to the other two groups (downturn effect at too high doses). Dose-response effects have not been proper identified in the manuscript or discussed: The authors have not performed any form of trend test or dose-response models to confirm their conclusions (just ANOVA plus posthoc which tests nominal differences and not ordinal trends). This makes many of the conclusion’s speculations.
3.1.1 The interpretation of figure one is already very difficult.
In the group without ER receptor antagonist the weight gain is obviously the smallest in the lowest concentration groups comparable to EE, however, it increases dose dependent and is absent in the highest dose group compared to negative controls. Which gives the impression the effect decreases with the concentration of AEAA.
In the group with ER antagonist the weight gain is seemingly dose dependent decreasing compared to negative control. It looks like there is a positive synergy between AEAA and the ER antagonist.
3.1.2 Figure 2 and 3: The authors claim that “… the AEAA demonstrated an increase in the level of estrogen and a severe reduction in the progesterone in a dose-dependent manner.” (line 236f as well in the abstract). However, I can not see a dose dependent increase or decrease of any of the two in the group without ER receptor antagonist.
3.1.3 The authors implicitly claim that the phytochemicals they have identified are responsible for the effects they have observed. However, also other pharmacological interactions are possible and not discussed, mentioned or tested for. E.g. high doses of St John's-wort (Hypericum perforatum) induces CYP3A4 which is also responsible for the metabolization of several hormones and can lower the effectiveness of e.g. hormonal contraception therapies. The authors have not supported their speculative conclusions by additional experiments (e.g. repeating experiments with the identified pure phytochemicals.
3.2 For GC/MS how the metabolites are identified is unclear. Has the main fragment m/z just been confirmed to a reference spectrum from the literature or has the entire fragments spectra been compared to pure reference substances spiked in AEAA or pure? Some more explanations are needed here.
Additional comments: none


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

Input:
Title: EVALUATING THE OESTROGENIC ACTIVITIES OF AQUEOUS ROOT EXTRACT OF ASPARAGUS AFRICANUS LAM IN FEMALE SPRAGUE-DAWLEY RATS AND ITS PHYTOCHEMICAL SCREENING USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the article is well written, provides sufficient background information, figures and literature. However, the manuscript needs some editing. Throughout the manuscript spaces are missing between some words (see lines 20, 48, 62, 117,119, 131, 189, 280, 335, 339). Also in the literature section there should be either a free line or no free line between all the references. All references should be also numbered as cited in the manuscript. Some references miss complete publication details and only part of the cited papers have their doi's mentioned.
Experimental design: In general the experimental design is very good and the original primary research is within the aims and scope of the journal. The research questions are well defined, relevant and meaningful. The GC/MS section however needs some improvements. First of all, the GC/MS manufacturer should be mentioned to be consistent with other methodological parts of the manuscript. The reference 24 mentioned is a corporate website without detailed method information. Please provide a different reference paper. Furthermore, references 25 and 26 mentioned in the GC/MS method sections do not contain any GC/MS experimental data. Please replace with proper references. Please also mention whether this instrument is using electron impact ionization and that no derivatization of the sample was necessary. Also missing is the information how peak identification was performed. Where spectra libraries used or chemical standards? The sentence in lines 175/176 starting with "during sample analysis...." can be deleted. In line 174 please mention precisely at what time in the gradient the 10C/minute increase was started. Also in line 174 break up the long sentence after … per minute to 300C and then continue with "This temperature was maintained for 30 minutes.
Validity of the findings: The authors used various methods to characterize potential oestrogenic activities of an aqueous root extract of Asparagus africanus Lam in rats. The data generated by gene expression and hormonal analysis show a clear impact by the root extract. GC/MS analysis supports the finding by the identification of two steroidal saponins. The only problem I can see with this manuscript is that the entire findings are based on a single root sample hence missing statistically relevant data. It would have been great, if the authors would have performed GC/MS analysis of several more root samples to see whether similar amounts of steroidal saponins are present in individual root samples.
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: EVALUATING THE OESTROGENIC ACTIVITIES OF AQUEOUS ROOT EXTRACT OF ASPARAGUS AFRICANUS LAM IN FEMALE SPRAGUE-DAWLEY RATS AND ITS PHYTOCHEMICAL SCREENING USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: The basic language should be improved to ensure that an international audience can clearly understand your text. While there is a certificate that states that a native English-speaking colleague reviewed this manuscript, there are still many places where the language could be improved. Some examples of this include the abstract. Please mention the use of this plant as a contraceptive in the opening sentence, then mention that this use prompted research into what mechanisms may be involved. This would also improve the introduction.
Another example of where the language could be improved is found in lines 62-65. It is unclear what is being said. Is the intent to introduce the uterotrophic assay with something along the lines of “A model used to evaluate the effect of chemicals on estrogen receptors is the uterotrophic assay in which removal of the ovaries reduces endogenous estrogens, causing the uterus to shrink. Replacement of these hormones with external sources of estrogen causes a trophic response of the uterus. ”
Please correct the references. Citations in-line are numbered, but the reference section appears to be sorted alphabetically, making it difficult to determine whether the citations used are appropriate.
Experimental design: The experimental design is generally well reported, but there are some areas where the rationale for the design and the components used could be clarified. For example in line 103, the immature rat version of the uterotrophic assay was developed because immature rats already have a uterus that is highly responsive to exogenous estrogens. Why where the immature rats used for this study also ovariectomized? Another example from line 103 includes the diet, which ought to be specified, and a statement made about whether the diet is low in phytoestrogens or phytoestrogen-free, as diet has been identified in the OECD test guideline for uterotrophic assays as a potential confound in uterotrophic studies.
In line 107, please indicate why the extract was injected twice daily. Was there a concern that some of the polyphenols might have a short half life? Also please clarify whether 800 mg/kgBW was injected twice per day for a total dose of 1600 mg/kgBW/day, or 400 mg/kgBW was injected twice per day for a total dose of 800 mg/kgBW/day.
In all of the supplemental tables, please use color consistently. For example, both of the graphs in the BW WO and BW WA tabs use blue columns, then in the BW combined tab, BW WA is colored orange. Please make the columns in all of the WA tabs orange as well, to facilitate comparisons. It would also be helpful if the axes were consistent for all graphs for the same endpoint. For example, the Y axis for ER WO runs to 125, ER WA to 100, and ER combined to 140. This makes it difficult to compare the magnitude of response between the graphs.
In the supplemental tables, for the weight reports, please check that the correct values have been graphed and in the correct order. In the BW WO tab, the 50 mg/kg body weights are near controls, then there is a decrease at 200 mg/kg. In Figure A in the BW combined, the 50 mg/kg body weights are very low, then increase dose-dependently until at 800 mg/kg, the weights are close to controls.
On the BW Combined Figure A, please also indicate what ## refers to. Is it statistically significant compared to controls pre-treated with antagonist?
In the supplemental tables on the ER combined page, there are two version of the same graph, which reports estrogen levels for rats with and without antagonist pre-treatment. Please remove one graph. Please also remove the extraneous ##, **, and * in columns A and B.

In the Prg combined tab, please remove the extraneous ## and # in columns O-R.
Please be consistent about indicating statistical significance within this supplemental data. The progesterone WO is indicated in the graphs in the combined tab as being statistically significant for all treatments, but the graph in the PRG WO tab does not indicate any statistically significant effects.
Validity of the findings: The conclusions appear to be appropriate, given the findings.
Additional comments: Please see the annotated PDF for additional comments relating to data reporting and experimental design.


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: EVALUATING THE OESTROGENIC ACTIVITIES OF AQUEOUS ROOT EXTRACT OF ASPARAGUS AFRICANUS LAM IN FEMALE SPRAGUE-DAWLEY RATS AND ITS PHYTOCHEMICAL SCREENING USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC/MS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: I can confirm that all comments by all three reviewers have been sufficiently addressed.

No further comments.
Experimental design: I can confirm that all comments by all three reviewers have been sufficiently addressed.

No further comments.
Validity of the findings: I can confirm that all comments by all three reviewers have been sufficiently addressed, except my comment 3.1 (comment #8).
I would strongly suggest that the authors implement the first paragraph "The dose response curve...negative feedback response." of their response somewhere in the discussion section of the manuscript.
These comments are of vital importance to interpret the data.

No further comments.
Additional comments: The manuscript has significantly improved. It has reached a level to be ready for publication.