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: FREEZE-DRYING CAN REPLACE COLD-CHAINS FOR TRANSPORT AND STORAGE OF FECAL MICROBIOME SAMPLES
Review round: 2
Reviewer: 3

Output:
Basic reporting: ·	I commend the authors for their rapid turnaround and ability to address all reviewer comments.
·	I also commend the authors for their analysis and documentation, which was reproducible. Using information from the methods section, I was able to run the AmpliSeq workflow and produce an ASV table that was nearly identical to that posted on Github. Only minor differences in ASV abundances were noted. Could this be due to the use of a seed number used to generate the ASV table? If so, could this number be included somewhere in the manuscript?
·	I was also able to re-create almost every figure. The only figure I could not re-create required the use of the “pseq.rds” object, which was not available on the Github repository, making the “FDvsfrozen_permanovatable.R” script unable to run. Can this object or the code used to generate this object be included somewhere in the Github repo?
·	Some more minor comments for making the analysis more reproducible: I often had to change the directory for data files from “../data/” to “datafiles/” or “../datafiles/” depending on which script I was using, and the .tree file was in the datafiles folder instead of the data/seff/ folders as was in the scripts. I leave this up to the authors whether they would like to change this or not as it was not a major concern or problem.
·	There are two scripts titled “Freezedried_vs_min80_relab.Rmd” in the repository, one contained in the main folder and one contained in the scripts folder. Is there a difference between these two scripts?
Experimental design: No comment
Validity of the findings: ·	L282 - 284: Though sample preservation treatment only explained 1.5 - 2.1% of variation, this can be a substantial amount depending on the aims of the study. I would suggest changing the wording to reflect that the proportion of variation explained by preservation treatment was minor relative to other factors like sample identity.
·	L320 - 321 and 329 - 331: I am a little confused by the wording here. First the authors state that preservation methods should not be mixed, and then it is said that samples that are currently frozen can be thawed? This could possibly be interpreted as ‘mixing preservation methods’. My understanding was that, from this study, we can conclude that any new samples can be freeze-dried and stored at ambient temperatures, but the authors did not test whether freezing, then thawing + freeze-drying yields similar community composition to samples that are just frozen or just freeze-dried. I think it would be helpful to clarify the final sentence in the conclusion prior to publication.
Additional comments: ·	L291: “origin” should be “originate”
·	L311: please add a comma after “1% mean relative abundance” to improve readability


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: PREDICTING POPULATION: DEVELOPMENT AND VALIDATION OF A NEW PREDICTIVE NOMOGRAM FOR EVALUATING MEDICATION NONADHERENCE RISK IN A TYPE 2 DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a cross sectional study in a Chinese population to develop and validate a medication compliance nomogram for adherence to prescribed therapy in T2D.

Overall, it is well presented and written with clear communication of ideas. There are a few minor errors in the English within the text such as lack of spacing between some words. Care should be taken to proof read and correct this. Otherwise it is written in clear and comprehensible English. The paper is structured appropriately with supporting figures and tables. References are appropriate and used to support arguments/methodology
Experimental design: The authors aim to create and validate a predictive nomogram for use to predict medication compliance in people with T2D in a single centre Chinese population. They include 753 patients from a single centre in their analysis. There is a reasonable breadth of ages and a reasonable balance between sexes. The study was cross-sectional between July to December 2020. The authors use a validated tool for assessment of medication compliance (MMAS-8) and provide appropriate references to support this. Questionnaires were used in conjunction with this to collect basic demographic and medical information. This was then correlated with compliance scores to predict which - if any - features correlate with compliance in this population. Optimisation of features used for the nomogram was derived using LASSO. A prediction model was then created using the appropriate demographic and other features. This model was then assessed for performance using appropriate tools including c-index. This appears to confirm that the model the authors have created is predictive in this population. They then propose using a 12% threshold of likely non-compliance as a target to aim specific interventions. The authors provide suggestions around this but not any great detail.

Overall, the methodology is valid and well described. This would allow replication in other centres.
Validity of the findings: The findings presented are valid and well described and would allow the process to be replicated. There are a number of limitations - most of which the authors address in a succinct limitation section at the end of the manuscript. This is a relatively small cross-sectional study which of course brings limitations. Some of the conclusions - such as compliance being less in single people - are severely limited by the very small number of the total who are single (74 vs 679). The authors could make this limitation clearer. In their conclusion, I would recommend that the authors are more specific in stating that this is only validated in a Chinese population at a single centre as the study cannot yet support wider predictive value for the nomogram. The authors describe some interventions they might use to increase compliance and conclude that future research to determine the clinical validity of the nomogram in a (presumably) prospective study. I think the findings are reasonable, if not entirely novel. I would recommend the authors look at the literature in the area of compliance in multiple daily dosing of diabetes medications again as there is literature examining frequency of medication administration and compliance. I would recommend they remove the line stating that there is no other literature to support this contention, as this is not the case.
Additional comments: Some particular statements are speculative and questionable around the differences in compliance by sex. The authors note that men are perhaps more likely to be non-compliant as they have "busier professional and social lives". Could it also be the case that men are less organised than women? Could there be other health behaviours in men to explain this?  This statement appears slightly clumsy at best; offensive to women at worst - particularly without a reference to support the author's speculation in their specific population. My suggestion would be to remove it.


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: PREDICTING POPULATION: DEVELOPMENT AND VALIDATION OF A NEW PREDICTIVE NOMOGRAM FOR EVALUATING MEDICATION NONADHERENCE RISK IN A TYPE 2 DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written with good grammar and punctuation.

The introduction is quite limited and while it gives context there is not much detail. Nomograms are not mentioned in the introduction, despite being at the core of the paper. They need to be introduced and explained at an early stage, especially given the authors’ claim that this is the first time they have been used in the area of medication adherence of diabetes patients.

The raw data has been shared but is all in Chinese characters. For this to be published in an English language journal I think the data should be in English.

Table 1 has a rogue bracket in the first row.

Table 2 – two decimal places are sufficient for presenting odds ratios and CIs. Three decimal places are sufficient for p-values. I would argue that there is no need for the p-values since the CIs give all of the required information.

The figures are all relevant but are very poorly explained in the text. The results section refers to each figure and table but gives very little or no interpretation of any of them. This makes it difficult for the reader to understand what they show and how it corresponds to the conclusions made, particularly for readers without expert knowledge of the LASSO approach.

The legend for Figure 1B says there is a vertical line drawn at the value selected in the fivefold cross-validation, but there is no line.

Figure 2 needs a clear explanation in the legend and in the text. While this type of visualisation may be understandable to someone in the oncology field who routinely works with nomograms, a reader without this background will not understand what the figure is showing.

The legend for Figure 3 does not explain what the ‘Apparent’ or ‘Bias-corrected’ lines represent.

Figure 4 is not well explained at all. On line 98 the authors mention ‘threshold probabilities’ but do not explain what they are. The text does not make clear where the patient and doctor threshold probabilities come from that are mentioned on line 128. It is not clear what the ‘scheme’ mentioned on line 130 is. It is not clear what the ‘intervention all-patients scheme and the ‘intervention non-scheme’ are.

Lines 76-77 in the ‘Adherence assessment’ section say ‘We categorized adherence as follows: low adherence = <6, medium adherence = 6 - <8, and high adherence = 8.’ Should medium be 6-7 and high be >=8, since it appears that the maximum score can be 11 (7 binary questions plus one Likert scale with a maximum of 4)?

Line 78 in the ‘Adherence assessment’ section: please replace ‘moderate adherence’ with ‘medium adherence,’ assuming that this is corresponding to the ‘medium adherence’ category defined in the previous sentence.
Experimental design: The research question is clear and seems to fill a knowledge gap.

It is not clear why the authors have fitted a logistic regression subsequent to the LASSO model. The latter would be sufficient to provide a model with appropriate odds ratios and confidence intervals. They should clarify why they have done this.

It should be made clearer how the test and validation subsets of the data are used in the analysis and at what stages each are used.
Validity of the findings: The authors give no indication of the potential drawbacks of using a nomogram (Balachandran VP, Gonen M, Smith JJ, DeMatteo RP. Nomograms in oncology: more than meets the eye. Lancet Oncol. 2015;16(4):e173-e180. doi:10.1016/S1470-2045(14)71116-7). These should be discussed.

Most of the discussion focuses on the variables that are found to be associated with nonadherence. The manuscript does not make clear what a nomogram adds to the field beyond just carrying out a logistic regression and this needs to be added. Furthermore, the authors should give examples of how the nomogram might be used in practice for individual patients.

Despite the extensive discussion on which variables are associated with nonadherence, there is no mention of the magnitude of the associations, based on the odds ratios and confidence intervals. Some of the odds ratios are very imprecisely estimated (ie they have wide confidence intervals), but the text in neither the results nor discussion sections give any indication of this. The authors should give clear interpretation in the text and ensure that the reader is aware of the precision with which the odds ratios are estimated.
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: PREDICTING POPULATION: DEVELOPMENT AND VALIDATION OF A NEW PREDICTIVE NOMOGRAM FOR EVALUATING MEDICATION NONADHERENCE RISK IN A TYPE 2 DIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Mainly well reported. Some issues with grammar and could do with a proof-read.

The rebuttal text to point 4 of my first review is better than the corresponding text included in the methods section of the manuscript and should replace it.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I am happy with the authors' responses to my original review.


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

Input:
Title: GEOMORPHICALLY CONTROLLED CORAL DISTRIBUTION IN DEGRADED SHALLOW REEFS OF THE WESTERN CARIBBEAN
Review round: 1
Reviewer: 1

Output:
Basic reporting: I found the paper to be very clearly written and well organised. The figures and tables are useful and clear.
Experimental design: I am not an expert on statistical analysis but from my understanding the methods used are relevant and correctly applied. The research question of the role of geomorphology in controlling coral cover is clear and the formulated hypothesis is testable using the data collected.
Validity of the findings: The fundamental conclusion is that "an accurate analysis of spatial ecological trends in coral reefs requires a detailed geomorphic framework in order to identify subtle changes in communities at large spatial scales". This seems reasonable to me based on the data presented.
Additional comments: There have been very few studies of coral reef geomorphology and this paper fills an important gap in reef research. Note also some comparable work has been done in the Great Barrier Reef:

Harris, P. T., T. C. L. Bridge, R. Beaman, J. Webster, S. Nichol and B. Brooke (2013). "Submerged banks in the Great Barrier Reef, Australia, greatly increase available coral reef habitat." ICES Journal of Marine Science 70(2): 284-293.


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: GEOMORPHICALLY CONTROLLED CORAL DISTRIBUTION IN DEGRADED SHALLOW REEFS OF THE WESTERN CARIBBEAN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is very interesting, it acknowledges the importance of using geomorphologic and ecological processes to understand species distribution on coral reefs across large spatial scales. I also celebrate the idea of testing the role of extrinsic factors that works at large spatial scales to explain why coral assemblages change over and space and time. Therefore, the rationale of the papers is based on solid ecological theory. Another important aspect that authors depicted is the undeniable fact that coral cover alone is a metric that lead us to flunk conclusions when trying to understand trajectories of coral communities. The literature review is good, and the hypothesis is well constructed.

The data is extensive and robust, and therefore is worth to publish. However, there are specific details in the experimental design that need clarification which I explain below. I see errors in the way the factors were declared (fixed, orthogonal, random, or nested) that may hamper the validity of the results. The paper therefore, requires substantial revision before is acceptable for publication.
Experimental design: The experimental design is good in the field, but I think there are conceptual mistakes in the analysis of the data that must be revised. This errors will have an impact on: (1) the outcome of the analysis, (2) the way the pesudo F in the permanova will be constructed and (3) the interpretation of the statistic test for every source of variation.

For example:

Authors declared the factor geomorphologic zone as fixed and nested in wave exposure. By definition a nested factor is random cannot be fixed as declared in the analysis. Is this a typo error or the data was actually analyzed in that way? I would like to see a table with the sources of variation to better undertand the design. Also, for random factors, authors compared between levels of these factors. This is a mistake too. Authors say that Factor A (wave exposure) and B

See below from the manuscript.

The experimental design consisted of 3 factors (Figure 2.C): Factor A: wave exposure (fixed with a = 2 levels: sheltered and exposed), Factor B: Geomorphic zones (fixed, nested in wave exposure with 3 levels nested in sheltered, and 4 levels nested in exposed), and Factor C: Site (random, nested in geomorphic zones, wave exposure) with 95 levels.

Also, authors compared between levels of these 3 factors. This is a mistake too. Authors say that Factor A (wave exposure) and B (Geomorphologic zones) and C (Site) are orthogonal. If one factor is nested to the another, then they cannot be orthogonal.

See below in the manuscript.

The estimates 237 of components of variation (S) for the three orthogonal factors were wave exposure: 178.52; Geo zone nested in wave exposure: 214.98; and site nested in Geo zone nested in wave exposure: 239 875.33.

In any case, I see a lot of contractions along the text, both were the design is explain and the outcomes from these design when results are described.
Validity of the findings: I have to have a better understanding about the design and how the data was analysed. I see inconsistency in the way the design is declared, and therefore, the sources of variation derived from the design might not be the proper ones.
For example, the use of pairwise tests to look at statistical differences between levels of a random factor is incorrect. If there is orthogonality, I see no interactions analysed. If a factor is nested, there cannot be orthogonality.
Another important factor to be included here is beta diversity. This is a measure that is better to discriminate communities across gradients and different habitat types. In this case study, beta diversity would be much more informative than alpha diversity. Beta Part is an R package that can be use for the partition of beta diversity estimated from Sorensen index or any presence-absence index. The Perm disp test is currently interpreted as a prove of statistical changes in beta diversity if the Sorensen index is used.
Additional comments: I think this paper is extremely interesting and the data definitely worth to publish. However, I believe there are errors in the way the experimental design was declared. Please review my comments and if necessary, run the analyses again. I would endorse this paper after substantial review of the current 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: GEOMORPHICALLY CONTROLLED CORAL DISTRIBUTION IN DEGRADED SHALLOW REEFS OF THE WESTERN CARIBBEAN
Review round: 2
Reviewer: 1

Output:
Basic reporting: In this paper a great integration of ecological and geological processes determining reef formation is used as an apporach to evaluate the benthic coral community structure along trhe Mesoamerican reef system. Authors take into account intrinsic and extrinisc factors controlling the structure of these communities. The paper is orginal and represents a great contribution because trend analyses that are guiding decision are based on regional analysis which often ignore the role of extrinsic factors, particularly geological ones, which are highly important.

Literature review is consistent and robust for the scope and goals of the paper. The article is well written, provides a good background and it is easy to follow with figures and table being selfexplainatory and easy to understand. There is transparency as the data is shared.

Results are clear and allows to answer the major questions and hypotheses being tested, likely because experimental design is appropiate for the questions presented. I really enjoyed reading this paper, the multivariate analysis apporach used is robust and the apporach presented is very interesting.
Experimental design: The questions of the paper are presented, they make sense and are clearly presented. The incosporation of geological and ecological processes to understand patterns is a very meaningful apporach.
The experinetal design is clear and well presented in a figure that clearly portrays the structure of the design, type of factors (Fixed and Random) and how thet related to each other (nested) are shown in the figure. The investigation is performed following high technical standards and good knowledge of experimental design and data analysis using semi parametric analysis, ordintation techniques and distance/based linear models that are extremely useful for data with lack of normality and heterogenous dispersions.
Methods are well explained and for me was very easy to follow up results after reading the section of methods, particularly statistical analyses.
Validity of the findings: The findings are meaningful and the approach presented in this paper should be used by managers that are only guided by regional assessments and reports. The replication is extensive, providing strong validity for the results.

Statistical analysis is solid and robust as I mentioned before. However, I would encourage the authors to explore more the beta diversity analysis by incorporating in the discussion more insights about the interpretation of species turnoever or nestedness to explaing changes is species among geomorphological zones across different spatial scales.

Conclussions are adjusted to the rationale of the paper and the hypotheses and questions being addressed.
Additional comments: This is a good paper, I really congratulate the authors. I would apporave the publication of the paper aftern minor changes are included. Particularly, I make two suggestions.

1. Include in the introduction a paragraph to say how global reports are currently being used to inform needs for coral restoration, and how coral restoration ignores the importance of geological processes.

2. Expand in the discussion the partition of beta diversity and what inferences can be made about the relative importance of different proccesses afecting the distribution and abundance of corals across the study sites. Beta diversity is currently thought as the most robust indicator of community change and only a few number of paper have used this community property (see Miyazawa et al. 2020).


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 KEY MICROORGANISMS IN FACULTATIVE STABILIZATION PONDS FROM DAIRY INDUSTRIES, USING METAGENOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article is clearly written and easy to read. The introduction is concise and provide the necessary elements to follow the experiments and the problematic of the paper. Not being involved in research on this type of system, I was not able to assess a possible lack in the background literature.
Experimental design: The research question is well addressed and worth studying. Experimental is straightforward and performed with advanced technology. In particular, shotgun metagenomics is considered as a very powerful technique. The depth of sequencing is high, which is required for such a complex ecosystem. The methods are well described.
Validity of the findings: Overall, the result are interesting and well supported. Nevertheless, we would like authors to consider the following comments:
.
Here, a first exploration is performed with Kraken, a fast procedure, which relies on the availability of a relevant database. In particular it should contain genomes very closely related to those of the ecosystem studied. It seems that it is not the case here, since it assigned only a minor part of the reads. Kaiju, another fast working software would have been probably better, but would have been still limited by the need of reference genomes closely related to those of the pound. It followed that the results presented in Fig. 1 do not reflect the process well, although it is the only easy-to-read figure that provide a picture of the taxonomical composition of the pounds. It also does not provide the value of the amount of unassigned reads (it could be added as a second “small” bar plot ahead showing the part of Assigned/unassigned reads), which is necessary to estimate the representativity of the bar-plots.
.
The authors used a second approach based on assembly, which provided a much better picture of the ecosystem and the main stream of exploitable data (and this fact is stated by the authors). It would be likely relevant to present the taxonomic profiling of the pounds calculated with the MAGs data in a second figure?
.
Discussion around the CAZymes (lines 296-303). Detailing the availably of enzyme capable of degrading saccharides and related compound is a very important issue in many ecosystems. However, in the case of milk-derived products, the main saccharide present should be residual lactose since milk does not contain other saccharides in significant amounts. What else are the authors thinking?
.
Parts “methane production” (lines315-324). Methane is a desirable product when it is recovered for energy production. In the present case, it is probably not desirable because it is released in the atmosphere and could be considered as greenhouse gas (worse than the carbon dioxide). Thus, the fact that methanogenesis is only a minor pathway here might be considered desirable.
.
Last sentence of Chromatiales section (336-8) “Given their unique physiological characteristics, and its conservation in both pond systems, we can conclude that these microorganisms play a key role in the system”. Based on such a type of argument, it may be hypothesized that each group in the ecosystem plays a key role. For example, Patescibacteria also have unique physiological characteristic and several of their MAGs are present in both sets.
.
Line  364: “We observed that the main driver of gene presence or absence in a MAG is its genome size”. What does this mean? It sounds like a truism. May be authors need to find another way to introduce Patescibacteria;
Additional comments: It would have been interesting to further compare and discuss similarities and differences in taxonomical composition and metabolic pathways between AUR and CYC. Based on the (not very relevant) Kraken analysis they appear very different although the MAG analysis shows the presence of several closely related species. May be the MAG-based taxonomic analysis representation would reveal better similarities.
.
Mention of the method should be added in this graph, in addition of the mention of the % of the reads it represent (see ahead)
.
Legend should be added to each supplementary tables and their list presented.


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 KEY MICROORGANISMS IN FACULTATIVE STABILIZATION PONDS FROM DAIRY INDUSTRIES, USING METAGENOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Writing is very good and conveys the ideas well.  Some sections, however, could use a proofread to ensure minor grammar mistakes are corrected.  This was more noticeable in the intro section.  Table S1 is important to set the stage for the environmental conditions present.  Consider placing it in the text if possible.  Literature review was sufficient.  Tables and figures should have standalone captions.  Consider adding addition detail to ensure that a reader has more info to achieve this standalone goal.  Consider adding a map, placing the locations of the study sites.  This is important for global readership.  Supplementary data was good and provided additional information pertinent to the study.
Experimental design: This study presents primary research that will be of interest to researchers involved in understanding the potential for wastewater stabilization improvement for a variety of applications beyond the dairy industry.  The goal of the study is clear and will contribute to global knowledge on microbial community dynamics in wastewater stabilization ponds.  Work appears to follow standards.  Work should be repeatable, except for the sampling protocol.  This can be addressed with the addition of further information and justification of procedures used.

Some considerations.
1.	The experimental design appears sound.  More details would benefit the reader and allow the results to placed in the global setting.  For instance, when were the samples taken (time of year, time of day)?  I found this in Table S1, but in the main document would be beneficial.  Does Santa Fe province experience seasonal influences which might influence the microbial communities present in the ponds?  Your samples are spread apart from April to September.  If not, your date is simply going to describe the collection.  If there are influences, then the date becomes even more important and may explain some of the differences you observed between the two sites.  A suggestion of the authors would be to list the date and time of the sample collection.  If there is a climatic influence based on time of year for sampling, include a discussion point about the affect this might have on the microbial populations present at the sites.
2.	Where were the samples taken at the ponds and how were they taken to ensure they were representative of the pond?  Spatial sampling can sometimes show distinct differences within a wastewater stabilization pond environment.  For example, were samples taken at different depths throughout the pond and combined into a composite sample.  This can be important if there are algal blooms in the pond which will skew results vertically in the water column.  A figure showing the waste stabilization ponds and the location of sampling should be included.  Suggestion to include more details in your sampling methodology.
Validity of the findings: Findings appear to be valid and supported by supplementary material.  Conclusions are linked back to original
Additional comments: It is odd that microalgae were not found in the ponds.  One would expect their presences, particularly in ponds which may have operational issues.  If you specifically excluded algae, please state this and your rationale.


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 KEY MICROORGANISMS IN FACULTATIVE STABILIZATION PONDS FROM DAIRY INDUSTRIES, USING METAGENOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: adequatly revised
Experimental design: adequatly revised
Validity of the findings: adequatly revised
Additional comments: adequatly revised


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 KEY MICROORGANISMS IN FACULTATIVE STABILIZATION PONDS FROM DAIRY INDUSTRIES, USING METAGENOMICS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Their rebuttal and revisions are acceptable.
Experimental design: Their rebuttal and revisions are acceptable.
Validity of the findings: Their rebuttal and revisions are acceptable.
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: PHOSVARDEEP: DEEP-LEARNING BASED PREDICTION OF PHOSPHO-VARIANTS USING SEQUENCE INFORMATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is generally well written. There are a few grammatical issues I noticed

In the title, ‘phospho-variant’ should be the plural ‘phospo-variants’

Line 16 ‘Researches’ should be ‘Researchers’

Line 30 ‘experiment’ should be ‘experimental’

Ensure when referencing authors in the text that the authors’ last name is used.
Experimental design: My main concern is with the positive training and test set used in this study. The authors claim they are taking positive examples of phospho-variants from PhosSNP. However, PhosSNP is not primarily a database of confirmed examples of phospho-variants (these are in the minority), rather it  contains predicted phospho-variants, and predicted on the basis of sequence features. Given that the authors are also training a model on the basis of sequences features, this creates a system of circular logic, where the positive training and test sets have essentially been selected on the basis of sequence features and then trained and evaluated on sequence features. In this regard, it is not a surprise that they see such significant improvement in prediction accuracy over MIMP and PhosphoPICK-SNP.

In the absence of a suitably large positive training set, my question would be whether the authors could take an unsupervised approach to distinguishing potential phospho-variants? There should be plenty of putative pospho-variants available on the PhosphoSitePlus database as well as the high confidence negatives that the authors are already using.

There are similar issues with the evaluation of methods for use in the PhosFEN part of the model. The difference between the variant and reference site are being used to evaluate the ROC and AUC, but again, these sites have been chosen on the basis of GPS 2.0 predictions so I don’t understand how they can be considered a meaningful test set. As there are dedicated databases of experimentally confirmed phosphorylation sites (e.g. PhosphoSitePlus), surely it would make more sense to make use of these when evaluating what phosphorylation site predictor to use in PhosFEN?
Validity of the findings: As explained in section 3. I feel that the validity of the findings are undermined by an inappropriate positive training and test set.
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: PHOSVARDEEP: DEEP-LEARNING BASED PREDICTION OF PHOSPHO-VARIANTS USING SEQUENCE INFORMATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In the paper “PhosVarDeep: deep-learning based prediction of phospho-variant using sequence information”, the authors proposed a deep-learning based prediction model to predict for phosphor-variant using paired reference and variant sequences as input. In their proposed model, the general phosphorylation sequence features were extracted by an existing pre-trained deep-learning model called DeepPhos. Then a CNN model was introduced to integrate the outputs of the pre-trained model. Finally, a prediction module is used for the final prediction of phosphor-variant. The manuscript was well organized and easy to follow. However, my major concern about this paper is the dataset they used and the performance evaluation. Is there any newer data that can be used for training and evaluation?
Major concern:
1 The authors should clarify the definition of phosphor-variants in their paper. As in the PhosSNP paper, they defined five different types of phosphorylation-related SNPs. But in this manuscript, they didn’t interpret the term of “phosphor-variant” and which type is their target.
2 How they collected the training data is not clear. The authors mentioned that they collected positive examples in PhosSNP and negative sets by Ralph et al. They should introduce these data and how they were collected. Are there any homology sequences between the positive and negative sets?
The phosSNP database also contains predicted phosphor-variants, they should also clarify that.
3 The database phosSNP was published 10 years ago, is there any newer data that can be used for the model training? I know another one published in 2019 in the paper “AWESOME: a database of SNPs that affect protein post-translational modifications”. But I don’t know whether they provide phosphor-variant based on experimental data.
3 One big concern of deep-learning-based methods is the overfitting problem. More complex models tend to obtain better performance in the training data than simple models. In line 124, what does the “strictly non-overlapping training, validation and test sets” mean? Does it mean no exact the same sequences in the training, validation, and test set? If so, it is not enough for biological sequence predictions. A standard approach is to remove similar sequences (>40% sequence similarity) between the training and test set.
Experimental design: no comment
Validity of the findings: More recent data is expected to evaluate the performance. The positive data used in this work was published 10 years ago.
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: PHOSVARDEEP: DEEP-LEARNING BASED PREDICTION OF PHOSPHO-VARIANTS USING SEQUENCE INFORMATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: I appreciate that the authors have improved the definitions and description of the datasets used. Given that the ‘positive’ examples of phosphovariants that the authors are using are in fact phosphorylation sites in the vicinity of variants, rather than confirmed phosphovariants, it would be best to make clear that the positive training set is in fact comprised of potential phosphovariants rather than ‘positive examples of phospho-variants’. I appreciate that in the updated text, the authors refer to potential phospho-variants, however, in the first line of the introduction to the datasets for example, the training set is still referred to as positive examples, which could cause confusion to the reader.
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: PHOSVARDEEP: DEEP-LEARNING BASED PREDICTION OF PHOSPHO-VARIANTS USING SEQUENCE INFORMATION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all my concerns in the revised manuscript. I have no further comments.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: TEXTURAL QUALITY, GROWTH PARAMETERS AND OXIDATIVE RESPONSES IN NILE TILAPIA (OREOCHROMIS NILOTICUS) FED FABA BEAN WATER EXTRACT DIET
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors have conducted a novel and important study worthy of publication; however, a major revision will be recommended with a reconstruction of major parts of the manuscript.
Conceptual issues
The study was focused on formulating the feed mixture with an enhanced antioxidative potentials, but in the entirety of the manuscript, antioxidation was seldom discussed. The introduction has no description of the undesirable effects of oxidation to fish food quality. It might have been expected that the authors will refer to fish oils (such as omega-3-fatty acids) and give few sentences about their unsaturation can cause rancidity.
Furthermore, the studies conducted focused on intestinal organs. The authors did not mention how any oxidative damage to these organs (such as pancreas) could affect the final product, since most of these organs are removed as offal during the evisceration and degutting process. If there is any relationship between these organs and the final fish muscle (majorly eaten by the consumer) that can affect the quality of the latter, this should be well stated in the introduction and discussion aspects.
In addition, the difference between expanded pellet diet (EPD) and pellet diet (PD) were not clearly declared almost in the entire manuscript. Only lines 137 and 138 mention about using an expander. A paragraph in the introduction can be allotted to this where the difference between the two products are clearly stated, and why the pellets are normally expanded.

General correction
The use of the possessive pronoun “our” in line 105 and other parts of this manuscript should be removed. For example, instead of “our previous study…”, it can be “From previous studies…” Line 311 “Our next step…” to “the next phase of study is…”
The technical expression of making conclusions from other studies should be revised:
Line 89: “High stocking density exerted negative impact…” to “High stocking density was reported to exert negative impact…”
Line 91: ” …production did not bring high profit…” to :”…production was reported to not yielding high profit…”

Use of standard deviations instead of the ~ symbol
Lines 145 and 151: 2~3 to something such as 2.5±0.5
Line 151: 23~27 to something such as 25±2
Line 152: 6.5~7.5 to something such as 7.0±0.5

Other grammatical suggestions are shown below:
Line 55: “formulating…” to “formulation”
Line 98: “And its fillets…” to “Its fillets…” since this is a new sentence
Line 155: “On 120d…” to “On the 120th day…”
Discussion structure
The discussion should be decompartmentalized into simpler units of the results obtained with each unit being thoroughly discussed with recent and relevant literature.
Experimental design: Materials and methods reproducibility
The methods used for ROS, H202, and MDA results mentioned in line 260 were not declared in the materials and methods section
Authors did not mention key details to signify the originality and reproducibility of this study. Some questions left unanswered are listed below:
Line 127: “…adjusted pH to 9.0” with what acid (for an initially higher pH) or with what base (for an initially lower pH)?
Line 127: “…ultrasonic processing…” what kind of processing: probe or bath sonication? what are the sonication parameters like amplitude, frequency, and time?
Line 127: “…centrifugation…” what is the revolution speed? And for how long?
Line 128: “…concentrating the supernatant…” Through what separation process?
Line 133: “…dried at 50°C…” for what time duration?
Line 138: “…dried at 120°C…” for what time duration?
Validity of the findings: Study conclusion and recommendation
The authors mentioned in lines 366-370 that “we conclude that EPD… could be an advanced substitute…” thus offering a recommendation for it relative to the PD preparation. However, for textural parameters mentioned in lines 239-240, 301-302 that affect consumers choice and are indirectly the principal objective of the study, the PD preparation was reported to be “significantly higher than EPD”. EPD was marginally higher only for internal organs that might not be correlated with the final product. Besides, EPD requires further processing at a higher temperature of 120°C which could damage the product’s nutrients, cause rancidity, and certainly incur higher processing costs to the food vendor. The conclusion should be thoroughly revised for this.
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: TEXTURAL QUALITY, GROWTH PARAMETERS AND OXIDATIVE RESPONSES IN NILE TILAPIA (OREOCHROMIS NILOTICUS) FED FABA BEAN WATER EXTRACT DIET
Review round: 1
Reviewer: 2

Output:
Basic reporting: The present study formulated the expanded pellet diet (EPD) and pellet diet (PD) containing faba bean (Vicia faba, FB) water extract (FBW), as a potential aquafeed additive to increase flesh texture, conducted a feeding trail, to evaluate the effect of FBW to improve the textural quality of tilapia. This is an interesting study, the findings obtained would be a good reference for the research and industrial practice of assessing product quality and acceptability. On the other hand, there are some aspects needed to be clarified.
Experimental design: 1.	Control diet was called commercial feed (L135), what does it mean? It was bought from certain company or produced based on the requirement of the research team? Is it a kind of EPD? Please make it clear.
2.	It is suggested to add the composition data of FBW in Table 1.
3.	The reason why 8% was added in the diet should be mentioned somewhere.
Validity of the findings: 1.	Since EPD and PD were applied in the present study, it is suggested to cite some references about the comparison of EPD and PD in the discussion part, to discuss the possible influence caused by FBW in the diet or the feed processing method.
Additional comments: 1.	The phrase “Growth parameters” appeared in the title, so in the abstract the growth performance results should be mentioned.
2.	Line 145, why “male” tilapia was emphasized here? Or it should be mentioned earlier?
3.	Line 230, it is better to say that treatment are higher or lower than control, instead of mentioning control first. Same in L237-239, L251, et al.


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: TEXTURAL QUALITY, GROWTH PARAMETERS AND OXIDATIVE RESPONSES IN NILE TILAPIA (OREOCHROMIS NILOTICUS) FED FABA BEAN WATER EXTRACT DIET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been revised and is having a better technical outlook. However, a few minor revisions can increase its presentation profile..
Experimental design: For a novel and technical work as this, a graphical summary figure is suggested as a figure 1. This can be cited in a suitable position in the materials and method section. Other figures can be renumbered accordingly.
Validity of the findings: The authors can state the agricultural and/or industrial relevance and application of the study in the conclusion aspect. One or few sentences regarding this, and also regarding any future prospects of the study can be added.
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: TEXTURAL QUALITY, GROWTH PARAMETERS AND OXIDATIVE RESPONSES IN NILE TILAPIA (OREOCHROMIS NILOTICUS) FED FABA BEAN WATER EXTRACT DIET
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: My previous comments have been well incorporated. There are two more suggestion listed below
1.	Line 373 in revised manuscript (tracked changes), one “of” should be deleted.
2.	The conclusion of abstract and the main body of the manuscript should be consistent. And it is suggested to put the sentence from Line 386-389 to the discussion part, instead of in the conclusion.


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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well written, potentially useful manuscript with a fascinating theme. The introduction is concise with previous findings and rationale included. The methods are appropriate but require a few clarifications. The results and discussion are clear and compelling. Overall, this is a good quality manuscript that has implications for childhood vaccination in Ethiopia. I think some parts of the manuscript can be improved. Below are many suggestions.

Introduction
[1] Lines 52-58 talk about recommended vaccines by WHO, but the authors did not elaborate on the vaccination programme in Ethiopia. The authors did not make clear what the EPI is and what it entails.
[2] EPI ends at nine months so, why was your study group aged up to 23 months? Moreover, are you considering only vaccines on EPI? Then it would help if you made it known.
[3] Authors need to give more epidemiological statistics on vaccination issues in Ethiopia; for example, the rate of fully, partially, and unvaccinated children in Ethiopia; the prevalence of vaccine-preventable disease; how many still die from vaccine-preventable disease or suffer from chronic morbidity related to these diseases in Ethiopia.
[4] Lines 69-70 mentioned the vaccination rate of 39 to 43%. Does it stand for fully or partially vaccinated children?
Experimental design: Methods
[5] In line 99, why did you use the prevalence of full immunisation from a single study by Etana and Deressa in 2012 (N= 536) instead of the pooled estimate from recent systematic reviews and meta-analyses? Also, I think it is good to include these recent systematic reviews in your paper.
-  https://doi.org/10.1186/s12889-020-09118-1
- https://doi.org/10.1016/j.vaccine.2017.04.034

[6] Line 108: what do the authors mean by total sample size was divided by the size of all households? Is it the number of house occupants? If yes, does it mean that your denominator contained participants not eligible for the study?
[7] Lines 109-110: what do authors mean here?
"Then, children were proportionally allocated according to the number of children they had".
[8] Your sampling technique needs to be revised. Is it not better to use a cluster sampling technique?
[9] Did a qualified translator translate the questionnaire? If no, did the translation have the same meaning?
[10] Lines 117 – 118, you mentioned that the questionnaire was developed from the literature. How was this developed questionnaire assessed for validity and reliability?
[11] In lines 126 – 127, why was pretesting performed on 5% of the sample size? Please mention a reference for this.
[12] In lines 30 – 34, it is important to mention a reference for the definitions of fully vaccinated, partially vaccinated, and unvaccinated.
[13] In lines 135-138, were you quantifying the knowledge and attitudes? If yes, is this a valid knowledge score and attitude score? A reference should be included.
[14] Lines 120-121 talk about training, but the training was given to who? By who? And what for? Is it the same training as in line 125?
[15] Why were interviewer-administered questionnaires used instead of self-completed questionnaires (mothers asked to show vaccination cards)? Would this introduce social-desirability bias?
[16] In lines 146 – 152, what was the dependent variable in the bivariate and multivariate analysis? In a previous study by Etana and Deressa, "full immunisation status of the children (card plus mothers recall) was included in the logistic regression model as a dependent variable".
[17] Was a chi-square test not necessary in your analysis?
Validity of the findings: Results
[18] I think the authors should include the relevant responses for the questions in lines 185-187.
[19] The knowledge and attitude score in lines 187-189. More details need to be provided. See comment [13].
[20] The statement in lines 192-193 is contradictory to lines 87-89 which states that EPI service (where I suppose vaccination is done) was provided only by governmental health facilities and not health posts. Are health posts also government health facilities.  Why is the EPI service provided only by governmental health facilities?
[21] Lines 197 – 198, you stated that "No child had a vaccination card, and all data were collected by recall alone". Why? Is it standard practice in this area? Are vaccination cards kept in the government health facilities?
Etane and Deressa showed a discrepancy between full vaccination by card only (27.8%) and full vaccination by card and recall (35.6%). How reliable is your finding "47.0% were fully vaccinated or completed all the recommended vaccines before celebrating their first birthday"?  Lines 274 – 275, you stated that "recall bias of mothers could affect the study findings dispite the efforts made to minimise them". What were the efforts made?
[22] Lines 229-230 and lines 238-239: only general reasons are given for the differences between your results and the one in other studies. Authors need to provide precise and concise explanations for that.
[23] In lines 240-244, why was there a difference between your study and others?
Additional comments: Conclusion
[24] The conclusion needs to be summarised and must be based on the results

General
[25] The English language and the flow would need to be verified.


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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Vaccination is the administration of a biological substance in (line 43) might not be appropriate since we have lots of biological substances with classifications. I would advise you to use simple terms “administering vaccines” or you could say “vaccination is the efficient way to protect people from infectious diseases”.
In line 45 you could go straight to the point by stating “vaccination has reduced and eliminated the six childhood killer diseases”. I do not think it is necessary to mention all the diseases.
Again, in line (48-50), you could be more concise. “Global vaccination coverage has increased, contributing to decreases in child mortality from 9.6 million in 2000 to 5.9 million in 2015, especially low and middle-income countries”.
Have a look at line (57-60) “Expanded Program on Immunization (EPI) agenda plans to give the primary vaccination series to at least 90% of children”.  I suggest you put a full stop here. Start another sentence with “Despite these efforts, the target has still to be attained in many LMICs”. You can now continue to write “For instance”, “Over 20,000 children…….”. I will suggest you write the word twenty Thousand in line 59 in figures 20,000. It conveys a message, and it stands out.
Also, you could remove “only in only ten countries” in line 66. Just write “in only 10 countries including Ethiopia”.
Line 68-70 need referencing
Can you be more formal in lines 70-71? I suggest you write “reported to be low (15%) in Afar and High (89%) in Addis Ababa”. The use of “reported to be as low as 15% in Afar and as high as 89% in Addis Ababa” seems inappropriate.
Experimental design: Since the questionnaire was developed by the authors, it means that it has not been applied elsewhere, How did you check for validity and reliability of the questionnaire during pre-testing
Under the Study design, I suggest you explain why you chose Demba Goaf District for the study. Are there frequent outbreaks in this district? Have reviewed records shown that this district is trailing under the EPI program? Tell us the gap you are going to fill in this community.
Also, give reasons why you chose to work on the age group 12-23 months.
Why do you choose to exclude mothers who are mentally disabled/critically ill and caretakers? What are the reasons for excluding them?
What is the difference between caregivers and caretakers? Because caretakers ware in your exclusion criteria and caregivers have been mentioned in lines 121-123 during data collection
Validity of the findings: In lines, 88-89 Is it only government institutions that provide EPI in Ethiopia? No other facilities provide EPI programs.
How did you come by Pre-testing of 5%? Is it standard?
In lines 197-198 how come all 657 children had no vaccination cards? Is it possible that all these populations of children did not have vaccination records? I doubt the validity of the results with only recall.
How did you check for recall bias since you stated this in lines 274-275. Could you explain the measures put in place to check recall bias?
Additional comments: Overall the study is important


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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comment
Experimental design: No additional comment
Validity of the findings: No additional comment
Additional comments: Data analysis and results should be reviewed by a biostatistician before publication.
You have adequately addressed the concerns and the information will add to the general literature.


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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: I commend the authors for making the necessary changes to clear all ambiguity.
Experimental design: Thank you for addressing the necessary concerns. However, since the vaccination cards serve as a form of reminder to many mothers and caregivers, and one of the reasons for this study, it will be appropriate to suggest it as your first policy recommendation.
Validity of the findings: No comments
Additional comments: Overall, the concerns raised have been 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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: I have no further comments.
Experimental design: I have no further comments.
Validity of the findings: I have no further comments.
Additional comments: The authors have responded to reviewers comments and the study is clear and
addresses an important research question.
The English language should be improved to ensure that an international audience can clearly understand your text. An audience might find some of these sentences hard to read. For example
Lines 58-63
Lines 92-95
Lines 136-142
Lines 222-228


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: FULL VACCINATION COVERAGE AND ASSOCIATED FACTORS AMONG CHILDREN AGED 12 TO 23 MONTHS IN REMOTE RURAL AREA OF DEMBA GOFA DISTRICT, SOUTHERN ETHIOPIA
Review round: 3
Reviewer: 2

Output:
Basic reporting: No 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: THE APPLICATION OF SUDOSCAN FOR SCREENING MICROVASCULAR COMPLICATIONS IN PATIENTS WITH TYPE 2 DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: See additional comments
Experimental design: See additional comments
Validity of the findings: See additional comments
Additional comments: The authors submitted a paper in which they evaluated the performance of the Electrochemical Skin Conductance, measured with a Sudoscan device, as a potential marker of different microvascular complications in type 2 diabetes patients, namely diabetic neuropathy (DPN), diabetic kidney disease (DKD) and diabetic retinopathy (DR).
Additionally, the authors analyzed a derived score given by the device (SUDOSCAN modification of diabetic renal disease) also as a potential marker of DKD.
Each condition was determined according to standard international accepted criteria.
Subjects were divided into 2 groups according to ESC results (normal > 60 µS; abnormal < 60 µS).
The authors reported significant correlations between hands and feet ESC and several clinical variables.
The authors then performed a binary logistic regression analysis and found that a mean of the ESC from the 4 limbs was a significant independent predictor for DPN and DR. The authors also reported that the score SUDOSCAN-MDRD was also a significant independent predictor for DKD.
Finally, the authors performed a ROC curve analysis of the diagnostic value of hands and feet ESC for DPN and DR as well as SUDOSCAN-MDRD for DKD.
The authors then concluded that ESC, as measured by the Sudoscan device, has an effective, but limited, value as a diagnostic tool for diabetic microvascular complications.

Despite not being the first study to investigate this relationship, the article is interesting and has some strong points in its favor, in particular, the large number of patients studied, with a wide range of ages.
However, there are some points that need clarification before a publication endorsement.
Concerns:
1-	The article would benefit from a thorough revision regarding the language used.
2-	Across the article, the terms Sudoscan and ESC are used interchangeable. This should be avoided. The technique used is the Electrochemical Skin Conductance. The device used for this is called Sudoscan. The name of the device should not be used as a technique. Please clarify this throughout the text
3-	Clarification regarding SUDOSCAN-MDRD is needed. The authors do not explain this score or reference any publication in the Methods section. Why was this score used in the ROC analysis instead of the ESC values?
4-	In the results section, the authors stated that FESC was lower in males when compared to females (59.53 ± 21.66 vs 64.28 ± 18.89, P = 0.002). However, when verifying this values with the raw data supplied, the p value obtained is not consistent with the one reported.

As can be seen in the picture above, the p value of 0.002 refers to the Levene Test For the Equality of Variances. The correct p value to be reported should be p = 0.008, since the Levene test suggests that the homogeneity of variances assumption is violated.
This is not an overly important mistake but should warn the authors to double check their results interpretation.

5-	Regarding the binary logistic regression analysis, the results are somewhat confusing. What are the results of each model? Where all variables considered for the final models? Looking at Table 3, it appears that all variables were considered for the 3 models analyzed. If so, what is the clinical rationale to consider BMI, hypertension or eGFR for the DPN model? Or the clinical rationale to consider VPT for the DKD or DR models? This needs to be clarified.
6-	Another point in the binary logistic regression is the choice of using the mean of the ESC from the 4 limbs. Since the authors chose to use the cut-off of 60 µS to define normal vs abnormal, this dichotomic variable should be used in the logistic regression.
7-	The ROC analysis showed interesting results for the diagnostic value of the ESC, in particular regarding DPN, as expected. However, I do not understand the choice of comparing the AUC of ESC with other variables. Since the variables chosen for comparison where the ones used as goal standard for the definition of each condition, ESC would always shown poorer results. Also, it is not clear which cut-off value was used. 60µS? Other?
8-	Additionally to the ROC analysis, the authors should clearly present sensitivity, specificity, PPV and NPV for ESC in each condition, in the results section, and not throughout the discussion.
9-	In the beginning of the Discussion section, the authors state that “patients with a lower ESC (<60μS) had 2.1-fold increased likelihood of having DPN,  2.4-fold increased likelihood of having DKD, 1.2-fold increased likelihood of having DR, than those with a higher ESC.” However, there is no reference in the results to this analysis. It is not clear where do these results come from.
10-	The discussion should be shortened around the main findings of the present article. Results of the study should be clearly stated in the Results section and not scattered though the discussion.
11-	The authors state that “it is the first study to report the relationship between sudoscan and DR in T2DM”. However, Camion et al. 2019 (https://doi.org/10.2337/dc18-2202), reported lower feet ESC in patients with severe diabetic retinopathy.
12-	The authors suggest that one possible explanation for the lower FESC in male, when compared to females could be “the fact that men are more likely to have bad habits such as smoking”. However, when looking at table 2, one notes that HESC had a higher and stronger correlation with smoking than FESC (-0.134 p = 0.002 vs -0.095 p = 0.031). With the current data, the authors cannot back up this statement.


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

Input:
Title: THE APPLICATION OF SUDOSCAN FOR SCREENING MICROVASCULAR COMPLICATIONS IN PATIENTS WITH TYPE 2 DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study by Lin et. al. evaluates the relationship between parameters of an electrical skin conductance measurement device called SUDOSCAN and microvascular complications in T2DM. It is a well-designed clinical study involving 515 patients with T2DM in China. The methods are appropriately described, results clearly mentioned and conclusions properly drawn. However, there are some concerns that need to be addressed before the manuscript can be considered for publication.
Experimental design: •	What was the percentage of males vs. females used for the study? Table-1 describes only males. Please correct accordingly and include this under ‘materials and methods’ section
•	How is the value of SUDOSCAN-MDRD calculated? Please include details on how this and other relevant parameters are calculated
•	One of the major shortcomings of this manuscript is the absence of an appropriate non-diabetic control group. Are the authors aware of any prior studies done in non-diabetic individuals with measurement of similar parameters? The authors are encouraged to make use of that data and compare it to their own data, with appropriate referencing.
•	It would add to the strength of the manuscript if the authors can categorize results under separate subtitles highlighting each finding.
•	When citing prior work, please include ‘et. al.’ after author’s name. For example, refer to lines 170, 171, 172. Please change in entire text accordingly.
•	Results line 121-122: Please include data values (Mean+/-SEM, P) in text for significant differences in CHD, DPN, DKD and DR
Validity of the findings: •	The SUDOSCAN device measures skin conductance based on activity of the sweat glands which can be significantly altered under conditions of physical activity, stress, and blood pressure medications. Were these parameters considered to normalize ESC data?
Additional comments: No additional comments


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

Input:
Title: THE APPLICATION OF SUDOSCAN FOR SCREENING MICROVASCULAR COMPLICATIONS IN PATIENTS WITH TYPE 2 DIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: See additional comments
Experimental design: See additional comments
Validity of the findings: See additional comments
Additional comments: The authors submitted a revised version of the original paper, with point by point responses to the questions asked initially.
While there were some changes that improved the manuscript quality, there are still some points that need to be addressed before a publication recommendation.

2- Across the article, the terms Sudoscan and ESC are used interchangeable. This should be avoided. The technique used is the Electrochemical Skin Conductance. The device used for this is called Sudoscan. The name of the device should not be used as a technique. Please clarify this throughout the text

Response: Thanks for your comment. We have checked the whole article and made changes.( line 199, 219, 221, 227 )
While the changes made were adequate, they were clearly insufficient. Some examples of the incorrect use of the term Sudoscan as a technique and not a device:
Line 24 “SUDOSCAN was tested and evaluated with electrochemical skin conductance”
Line 58 “Sudoscan is a method”
Line 200 “The results suggested that sudoscan can be used as a clinical screening tool for diabetic neuropathy”
3- Clarification regarding SUDOSCAN-MDRD is needed. The authors do not explain this score or reference any publication in the Methods section. Why was this score used in the ROC analysis instead of the ESC values?
I have some reservations in using algorithms that are unknown to the authors, since they are proprietary of the firm that sells this specific device and, for that matter, impossible to scrutinize. However, the authors clearly state in the text this particular point (line 89) so readers can be aware of the fact.
6- Another point in the binary logistic regression is the choice of using the mean of the ESC from the 4 limbs. Since the authors chose to use the cut-off of 60 µS to define normal vs abnormal, this dichotomic variable should be used in the logistic regression.
Response: This is a good comment. The main reason we used the average value of ESC instead of the binary classification value is that the registration data will reduce the statistical efficiency compared with the measurement data.
For example, one patient ESC11 and another patient ESC 59. Both are ESC abnormal, their binary classification value are the same, but there are great differences in clinical severity. Therefore, we choose original measurement data.
I have to disagree with the choice to use the original measurement data. The objective of this paper is to assess the potential role of ESC as a biomarker of microvascular diabetic complications. For that matter, the work should focus on cut-off values and their respective diagnostic value.

9- In the beginning of the Discussion section, the authors state that “patients with a lower ESC (<60μS) had 2.1-fold increased likelihood of having DPN, 2.4-fold increased likelihood of having DKD, 1.2-fold increased likelihood of having DR, than those with a higher ESC.” However, there is no reference in the results to this analysis. It is not clear where do these results come from.

Response: This statement is based on table 1. There were significant differences in presence of DPN (88.99% vs 42.86%, P < 0.001), presence of DKD (39.76% vs 16.40%, P < 0.001) and presence of DR (30.58% vs 25.53%, P = 0.012) between both groups. We have realized that this statement was not rigorous. So we changed this sentence to " patients in ESC normal (<60μS) group had 2.1-fold increased likelihood of having DPN,  2.4-fold increased likelihood of having DKD, 1.2-fold increased likelihood of having DR, than those in ESC abnormal (≥60μS) group. "  in the text. (line 179-182)
This analysis is methodological incorrect. The fact that in this particular group of patients there was 2.1 more subjects with DPN in the abnormal ESC group, does not mean that subjects with abnormal ESC have 2.1-fold increase in likelihood of having DPN. The same is valid for the other statements.
In order to assess this kind of relationship, the authors should use the binomial logistical regression, with a dichotomic variable of normal/abnormal ESC. The analysis of the odds ratios is what would allow for the authors to make this kind of statements.

7- The ROC analysis showed interesting results for the diagnostic value of the ESC, in particular regarding DPN, as expected. However, I do not understand the choice of comparing the AUC of ESC with other variables. Since the variables chosen for comparison where the ones used as goal standard for the definition of each condition, ESC would always shown poorer results. Also, it is not clear which cut-off value was used. 60µS? Other?

Response: In this study, we compared AUC of ESC with other variables because we want to explored the diagnostic value of ESC. In particular, we wanted to understand the gap between ESC and other gold indicators, and whether it can play an auxiliary or alternative role in clinical diagnosis. Cut-off value was showed in table 4.

The Results sub-section “ROC curve analysis：effective diagnostic value of ESC in diagnosing DPN” (Line 161) repeats data that is given in table 4.
I would advise to cut the text, since the results are well described in table 4 and figure 1.


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

Input:
Title: THE APPLICATION OF SUDOSCAN FOR SCREENING MICROVASCULAR COMPLICATIONS IN PATIENTS WITH TYPE 2 DIABETES
Review round: 2
Reviewer: 2

Output:
Basic reporting: All the questions have been satisfactorily answered, I have no further comments.
Experimental design: None
Validity of the findings: None
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: THE APPLICATION OF SUDOSCAN FOR SCREENING MICROVASCULAR COMPLICATIONS IN PATIENTS WITH TYPE 2 DIABETES
Review round: 3
Reviewer: 1

Output:
Basic reporting: See additional comments
Experimental design: See additional comments
Validity of the findings: See additional comments
Additional comments: The authors incorporated the suggestions given.
It is the opinion of this reviewer that this work now meets publication criteria.

There are only very small details, not pertinent to study design or quality.

Abstract - Line 25, The word SUDOSCAN is repeated, and should be deleted.
The word "Sudoscan" should be written in a consistent fashion throughout the text. It is written either in all caps, with a capital first letter or always in lowercase. Please revise this.


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: INTEGRATION OF THE METABOLOME AND TRANSCRIPTOME REVEALS INDIGO BIOSYNTHESIS IN PHAIUS FLAVUS FLOWERS UNDER FREEZING TREATMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Needs significant correction in text at various places, grammars and updating the literature.
Experimental design: - Manuscript has tried to make some advancement in indigo biosynthesis in plants specifically in Phaius flavus a monocot.
- Materials & Methods: Plant material - This section is rather more ambiguous. Needs justification for experimental setup and starting material for downstream analysis.
Validity of the findings: - Data values needs to be re-check and verified including in supplementary tables.
- Speculation about the experimental treatment in conclusion section needs to be revised.
Additional comments: 	Manuscript entitled “Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment (#55602)” has tried to make some advancement in indigo biosynthesis in plants specifically in Phaius flavus, a monocot.

	Overall manuscript and data presentation is satisfactory and needs correction in text at various places, grammars and updating the literature, points needs attention are listed below (also find in attached PDF file);

1 	Introduction; line 62 “plants causes blue-changing in short time.”
2 	Line 72 “ protection of its”
3 	Line 75 “(Daykin, 2011) When the” needs full stop
4 	Line 81 “indigo, indigo” delete repeats
5 	Line 87-90 “Re-write the sentence as follows - The transcriptomics approach has been used to analyze the global expression of genes related to indigo biosynthesis. Further, we used the real-time quantitative polymerase chain reaction (qPCR) to verify the key gene expression patterns of indigo biosynthesis before and after the freezing treatment.”
6 	Line 95-104 Materials & Methods: Plant material - This section is rather more ambiguous. Re-write this section to make it clear to justify the experimental setup and starting material for downstream analysis.
7 	Line 98-99 “full blooming period - mention the time (date/ months)”
8 	Line 100-102 experimental plan of freezing treatment is not realistically justified. Why not the flowers were sampled from plants during the lowest temperature condition OR kept inside the cold room like facility at 4-7 degree C (fridge compartments). Treatment with liquid nitrogen does not resemble the natural chilling/freezing conditions.
9 	Line 104 “each plant part” - Flower is the only plant part was taken for analysis and presented in Fig. 1. What is the meaning of "each plant part"?
10 	Line 104 “Add your materials and methods here” delete the sentence.
11 	Line 105 correct metabolomics to metabolites
12 	Line 105 correct metabolomics to metabolomic
13 	Line 117 check the grammar “was used” instead of “are”
14 	Line 119-120 what is “B” after percent unit
15 	Line 133 correct “come” to “were procured/ obtained”
16 	Line 138 “P. flavus powder” not the plant powder but the “P. flavus flower powder”
17 	Line 139-140 “Dry 1 ml ----- methanol solution” Write this sentence in past tense.
18 	Line 143 correct the “indicant”
19 	Line 146-147 what is “B” after percent unit
20 	Line 150 correct the “indicam”
21 	Line 152-153 (Bai et al., 2019)[40]”, line 178-179 (Bryant et al., 2017)[41], line 200, 211 ---- etc., follow the journals guideline of citations.
22 	Line 154-155 Correct “Before injecting the sample for GCMS-QQQ analysis, it was passed through a 0.45μm membrane filter.”
23 	Line 157 “split injection split ratio” not clear
24 	Line 158 at “the” rate of
25 	Line 164 correct “Extraction of RNA was carried out from P. flavus flowers”
26 	Line 168-170 What is the difference between RNA concentration 100 ng/ul and total amount 5ug. Re-write this section.
27 	Line 195 “P. flavus flower,”
28 	Line 199 “edgeR” Original author for edgeR package is "Robinson MD, McCarthy DJ, Smyth GK (2010). “edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.” Bioinformatics, 26(1), 139-140. doi: 10.1093/bioinformatics/btp616"
29 	Line 208-209 Re-write “The specificity of ------ total RNA”
30 	Line 230 “flavonoids” remove the hyperlink
31 	Line 244 “freezing treatment. (P < 0.05).” remove full stop
32 	Line 247 “synthesis” change to “biosynthesis”
33 	Line 260-261 “which 121,802 (53.12%) unigenes were annotated in the NR database;” Re-check the value against respective database and table 1.
34 	Line 269-272 Recheck the values. Number of unigenes annotated against each category (Cellular processes - 65264; metabolic processes - 59455 and biological regulation - 23744), do not corroborate with total number of unigenes under GO functional annotation.
35 	Line 286 (Fig. 4A) What is the difference between Fig. 4A and supplementary figure 1, in terms of sequencing analysis and ontological distribution?
36 	Line 293-297 Number of unigenes mapped against KEGG database; do match with total number of gene count in Table S1.
37 	Line 310-311 (Fig. 5 and supplementary table S3) Delete.
38 	Line 312-315, 363-364 Arrange the citations as per journals guideline.
39 	Line 320 & 321 what was the basis for selection of unigenes [CYP71A4 (Cluster-38184.135548), GLU (Cluster-38184.103035)] for qRT-PCR analysis ?
40 	Line 367 “identify” change to “identified”
41 	Line 375 Check for relevance of the supplementary table to the sentence.
42 	Line 397 Supplementary Table S2 is listed the primers detail. Check for relevance of the supplementary table to the sentence.
43 	Conclusion section: Line 422-423 “Therefore, freezing treatment ------ indigo-related compounds.” In general, physical (mechanical ) and biological (fermentation) treatment is given to break cells to release the cell content, feasibility of freezing treatment/injury to tons of biomass seems to be costly affair.
44 	References Section: In most of the references, list of authors is incomplete. Last author separated by "and", missing in other references. Follow the journal guidelines.

Check all references for correctness; few examples are given below;

45 	Line 429 Adachi, J., 2001. Line 440, 443, 472 Incomplete author list. Follow the journal guidelines.
46 	Line 432 Hubei Agric Sci. 58(09), 107-109 ”+113”???.
47 	Line 450-452 Re-check the reference for correctness.
48 	Line 456-457 Volume number is missing
49 	Line 461 and 467 Huang et al., References not verifiable.
50 	Line 464 Correct either “and” or “&” and follow the same consistency throughout the manuscript and reference list.
51 	Line 497-498 remove hyperlink from authors name
52 	Line 524 Check the reference for correctness.

Summarizing, manuscript needs major correction prior to acceptance for publication.


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

Input:
Title: INTEGRATION OF THE METABOLOME AND TRANSCRIPTOME REVEALS INDIGO BIOSYNTHESIS IN PHAIUS FLAVUS FLOWERS UNDER FREEZING TREATMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: In Line 103 -104, “ Three samples were prepared for each plant part as replicates (Fig. 1)Add your materials and methods here.”
There is a mistake in the sentence.

In Line 140, “…was performed using the indigo, indirubin, indicant, and the indole quantitative reference…”          indicant → indican

In Line 150, “….263.0815→219; indicam 296.1129→134.”
indicam → indican

In Line 394, “eventually forming indigo (Epstein et al., 1967; Minami et al., 1999). Studies”
Please enter "." after "...1999)"

In Line 421, “such as indole, indigo and indirubin in P. flavus were….”  Please enter "," after "...indole".

In Line 389-390 & 390-391, “the gene expression level of indican…”
Indican is not gene.
Experimental design: 1. How long perfom you the freezing treatment?  Just flash by liquid nitrogen?
How treatment method with liquid nitrogen did the authors perform?
Immerse? Flash?
You should describe a more detailed method. It is crucial.

 2. After the freezing treatment, the cells were alive? The cells were frozen?
If the cells died, it is a normal phenomenon to form indigo.
You should describe the condition of cells.

 3. If the cells died…   After the freezing treatment, is it possible that the cells can biosynthesize the metabolites? It seems to be impossible?
Please explain why you think the metabolites are biosynthesized.
Validity of the findings: You said that the increase of indole resulted from the promotion of metabolism by some enzymes such as TSA (in Line 366-370).
It is thought a possibility of degradation of indole derivatives such as indican?
Please explain why you think that indole increased by the metabolism.

In Line 382, “The reason is that indoxyl has a toxic effect on plants.”
Indoxyl is a toxic. It is true?
Indigo forming from indoxyl is precipitate, so indigo is any problem in the cells.
Additional comments: The data of transcriptome and metabolomics are well quantified, organized, and described. However, the condition of cells after freezing was not explained. At present, I cannot tell whether the freezing treatment is reasonable or not. Please describe more detail of the method.

I think that you need to describe the purpose of this experiment in more detail.


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

Input:
Title: INTEGRATION OF THE METABOLOME AND TRANSCRIPTOME REVEALS INDIGO BIOSYNTHESIS IN PHAIUS FLAVUS FLOWERS UNDER FREEZING TREATMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: Most of the comments have been replied/ resolved satisfactorily. And accordingly the MS is improved.
Experimental design: Material and method parts needs revision which is suggested in comments. Most of the comments related to experimental design and sample treatments have been replied.
Validity of the findings: No comments
Additional comments: Manuscript entitled “Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment (#55602-v1)”

	MS now seems to be improved with some clarity, however it still needs minor corrections as listed below (also find in attached PDF file);

1 	Materials & Methods: Plant material - Line 103-104 Re-write as follows “The flowers without treatment were used as the control group (CK).”
2 	Line 105 replace “RNA sequencing” with “RNA isolation”
3 	Line 168-176 rewrite paragraph as follows “Extraction of RNA were carried out from treated and control P. flavus flowers using the plant RNA rapid extraction kit (Beijing Adler Biotechnology Co., Ltd., Beijing, China; RN38 EASYspin PLUS) as per the manufacturer’s instructions. The concentration and purity of the RNA were measured with the Nanodrop 2000 spectrophotometer, and the Agilent 2100 instrument was used to assess RNA integrity. Samples with RNA integrity number (RIN) > 7 and concentrations ≥ 100 ng/μL were taken for cDNA library construction. cDNA library construction was performed using 5µg total RNA and NEB Next Ultra RNA Library Prep Kit from Illumina. The cDNA library was checked for quantity and quality with Qubit 2.0 (Life Technologies, USA) and Agilent Bioanalyzer 2100 system respectively. Then sequencing was carried out using Illumina HiSeq 2500 platform.”
4 	Line 214-215 Re-write as follows “Gene-specific primer pairs for 150-250 bp regions were designed for different genes (Supplementary Table S1).”

Check all references for consistency; few examples are given below;

1 	Line 436-438, Line 470,... etc. Check for space in name initials. Check throughout the reference list.
2 	Line 461 “Garcia-Macias, P., and John, P., 2004.” use of "," and along with full stop, "and" delete
3 	Line 471-472 Abbreviate the journal name to "J. Mol. Catal. B Enzym."
4 	Line 478 correct the authors name “Thul S T, Sarangi B K,”
5 	Line 493 and 495 Check the style of mentioning authors (See PDF).
6 	Line 505 “Plant Cell Physiol”
7 	Line 521, 532, 556 write journal names in italics.


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: INTEGRATION OF THE METABOLOME AND TRANSCRIPTOME REVEALS INDIGO BIOSYNTHESIS IN PHAIUS FLAVUS FLOWERS UNDER FREEZING TREATMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The sentences added by the authors are reasonable and include the needed information.
Experimental design: The same comment with “Basic reporting”.
Validity of the findings: The same comment with “Basic reporting”.
Additional comments: I have understood your answers to my question.
However, I think that the experimental design of frostbite may be hard to understand.
I recommend that you should add some more explanation in the discussion or other sections. For example, as you said in your answer, “after being frozen for 2 seconds, the P. flavus flowers cells damaged due to frost injury which may disrupt the lipid…………, but the DNA, RNA, and protein were all alive, which could express……”.


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: INTEGRATION OF THE METABOLOME AND TRANSCRIPTOME REVEALS INDIGO BIOSYNTHESIS IN PHAIUS FLAVUS FLOWERS UNDER FREEZING TREATMENT
Review round: 3
Reviewer: 1

Output:
Basic reporting: The sentences added by the authors are reasonable and include the needed information.
Experimental design: The same comment with “Basic reporting”.
Validity of the findings: The same comment with “Basic reporting”.
Additional comments: This article is well revised, but I recommend a little more modification for a better one.

1. The content of line 101~103 may not fit in “Material & Methods”.
Therefore, the sentence should be added in the last paragraph of “Introduction” or the “Discussion”.

2. Line 73~74 in “Introduction”
The authors described the enzyme to form indoxyl as CYP450. Recently, Inoue et al. reported the possibility that Flavin-monooxygenase may catalyze the hydroxylation of indole. Therefore, I recommend touching on the possibility in the sentence. (An indigo-producing plant, Polygonum tinctorium, possesses a flavin-containing monooxygenase capable of oxidizing indole. Biochem. Biophys. Res. Commun. 534, 199-205 (2021))


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

Input:
Title: A REVIEW OF THE OPPORTUNITIES TO SUPPORT POLLINATOR POPULATIONS IN SOUTH AFRICAN CITIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: I think the literature is nearly sufficiently reflected, in light of being South African specific I find the literature review in respect to the data available from South Africa rather weak and lacking depth.
The authors data is shared but it is not really clear to me why they did the literature search if they do not use the results thereof.
Experimental design: It brings me to the point that I am not convinced that they showed that one can use the data from the global north on urban data and compare that to the agricultural setup in South Africa and then conclude onto the South African urban set up.
Validity of the findings: It is one of the first reviews putting it into a Southern African context, so I am not disagreeing with the conclusions but the way how they get to them should be more detailed and focused.
Additional comments: Dear authors,

I find the review well written, however, the title is misleading since I seems to have missed the opportunities for South Africa. Moreover, the link / transition of agriculture vs urban land-use to being able to add the international studies is rather weak in my opinion and should be one of the more detailed aspects, convincingly showing that one can interchange agri and urban, especially looking at line  505 “… structural differences between agriculture and urban…”

Since the review wants to have a more South African perspective I am surprised that the authors only relied on published papers from South Africa and not included thesis work or conference proceedings. I am sure Anderson is doing work on that topic for a while and there is more data available.

I suggest that either broaden it and delete “South African” from the title, or find more South African data and show that one can actually make conclusion based on agricultural studies to an urban setting.
Another option would be by expanding it to “African” cities and include the data from Africa, rather than South Africa.

What about the Ruan Veldtman’s work and the FAO project - I miss some of their publications

Why limited the agricultural aspect to indigenous plants - what about citrus industry?
Why only Eucalyptus, that seems only important in the Western Cape. What about the rest of the country.
Why did you actually do the literature search if you do not really use it and analysis it. Table 1 is a selective summary of the literature

Also, I miss the importance of Apis for Fynbos
Several places were Apis mellifera is misspelled

Line 276 could have more South African Examples see below

A summary graph would be helpful to better capture the results in the supp

Spelling line 181,

Archer, C.R., Pirk, C.W.W., Carvalheiro, L.G., and Nicolson, S.W. (2014). Economic and ecological implications of geographic bias in pollinator ecology in the light of pollinator declines. OIKOS 123, 401-407.10.1111/j.1600-0706.2013.00949.x
Carvalheiro, L.G., Seymour, C.L., Veldtman, R., and Nicolson, S.W. (2010). Pollination services decline with distance from natural habitat even in biodiversity-rich areas. Journal of Applied Ecology 47, 810-820.10.1111/j.1365-2664.2010.01829.x
Carvalheiro, L.G., Veldtman, R., Shenkute, A.G., Tesfay, G.B., Pirk, C.W.W., Donaldson, J.S., and Nicolson, S.W. (2011). Natural and within-farmland biodiversity enhances crop productivity. Ecology Lett. 14, 251-259.10.1111/j.1461-0248.2010.01579.x