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: MICRORNA PANEL IN SERUM REVEALS NOVEL DIAGNOSTIC BIOMARKERS FOR PROSTATE CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is written in a clear and appropriate English and self-contained. The provided information is sufficiently supported by literature references. As far as article structure is concerned, I think that some sections of the article contain repetitive information, for example paragraph 3.1 looks like a repetition of what already explained in section 2.1. Moreover, the methods section about the use of the miRCURY PCR Human panel (lines 121-127) in my opinion does not fit in the “study design” paragraph, I would put it either in the 2.4 section or as a separate paragraph. Figure legends are totally inadequate, they do not contain a description of the graphic elements, such as the reported bars, the sample labels ect…. Moreover, I think that figure 4 and 5 have been exchanged by mistake. In table 2 I do not understand why 4 pools are reported while only three pools are described in the text, in table 3 it is not clear what the combined column stands for.
Experimental design: The study well designed, is performed in a quite large cohort of patients and I appreciate the inclusion of training and testing phases however I think that, in order to understand value of the testing phases, a sample size calculation should be included in the methods. In the materials and methods, I would add more details about blood sample processing. Moreover, in the “external validation in public databases” paragraph I think the authors should indicate if the evaluated databases contain data about tissue of serum miRNA expression.
Validity of the findings: The study is designed to respond to a quite significant issue about prostate cancer diagnosis, however several studies are already available about the possible utility of circulating miRNAs as PC biomerkers (as stated by the authors themselves) so the authors should better argument in the discussion section in what their proposed results are outperforming compared to others. In paragraph 3.6 authors compare their proposed signature with others already available in different databases but I would consider this comparison valid only if it includes databases about circulating miRNAs.
Additional comments: The proposed study is well designed but several inaccuracies should be cleared. Moreover the authors should stress the strength of their proposed biomarkers over others already investigated.


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: MICRORNA PANEL IN SERUM REVEALS NOVEL DIAGNOSTIC BIOMARKERS FOR PROSTATE CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: Zhang et al report a serum diagnostic biomarker signature of miRNA in patients with prostate cancer. The authors use primary tumor samples, bioinformatic analysis of global patients data, exosome and serum profiling to support their findings. The language used is clear with appropriate use and interpretation of the existing literature. The structure and flow of the figures and provided tables is adequate. I find the concept interesting and I would support the publication, if the authors address and expand their model as outlined in the preceding paragraphs.
Experimental design: The experimental design and methods sections are clear. The graphical abstract in figure 1 facilitates the comprehension of the experimental approach by the authors. The techniques used in this study are considered basic but reflect the original experimental question and support the conclusions.
Validity of the findings: The authors present an interesting concept identifying novel miRNA biomarkers in patients with prostate cancer. I have a couple of suggestions in order to strengthen their proposed model

1. Although the miRNA signature is clear, the authors need to experimentally and bioinformatically validate the some known target of their listed miRNAs in the same samples. An established way to screen for potential miRNA targets is the miRDB pipeline (http://mirdb.org). This will go a long way in supporting the whole signaling network affecting prostate cancer prognosis.

2. Furthermore, it would be interesting to plot Kaplan Meier curves from TCGA and other GSE datasets for these miRNA targets and correlate them with poor prognosis.
Additional comments: Zhang et al present an interesting concept for a novel biomarker identification in patients with prostate cancer. If the authors address and expand their experimental podel I would be happy to support he 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: MICRORNA PANEL IN SERUM REVEALS NOVEL DIAGNOSTIC BIOMARKERS FOR PROSTATE CANCER
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, Zhang proposed experimental and bioinformatics analysis for identifying novel diagnostic biomarkers for prostate cancer. MicroRNA panel in serum reveals has been collected and they convinced some important biomarkers of prostate cancer. There are some major points that need to be addressed:

English language should be improved significantly. There are a lot of grammatical errors and typos in this manuscript. The authors should re-check and revise carefully. For example:
- We also compared diagnostic value of the 3-miRNA panel with previously reported biomarkers and verified in four public datasets.
- The diagnostic performance of the identified 3-miRNA signature show stable in the large cohorts of the three public datasets and superior than previously identified miRNA biomarkers.
- ... which could act as a non-invasive serum biomarker to help diagnosing PC.
- ...

Literature reviews are weak. There is a need to provide more literature reviews on bioinformatics-based PC analysis.

"Introduction" is short and does not emphasize the objective/motivation of the study.
Experimental design: A critical issue is that the authors only used a few sample size of data. This amount cannot be considered enough for analysis. Thus, the authors are suggested to have more data if possible.

ROC curve analysis has been used in previous works on bioinformatics such as PMID: 31362508, PMID: 31750297, and PMID: 33539511. Therefore, the authors are suggested to refer to more works in this description.

It is not clear how the authors divided the data into different sets (28, 32, and 26 patients).

The "Methodology" section should be described clearly to help to replicate the methods.
Validity of the findings: It is good to have validation data, but what are the differences in characteristics among training and validation data?

Figure legends should be described clearly. For example, in some figures, I did not understand the information in sub-figures.

The authors uploaded figures as "Tables".

The authors should compare to previous works on the same data/problem.
Additional comments: No comment


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

Input:
Title: MICRORNA PANEL IN SERUM REVEALS NOVEL DIAGNOSTIC BIOMARKERS FOR PROSTATE CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: Zhang et al have provided an extensively revised version of their work that satisfactory addresses all the comments raised in the original revision. The manuscript is easy to follow  and many inaccuracies have been corrected.
Experimental design: The newly added data are well controlled and explained.
Validity of the findings: No comments
Additional comments: Zhang et al have significantly revised their manuscript and proposed model. I support the publication of this concept.


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: MICRORNA PANEL IN SERUM REVEALS NOVEL DIAGNOSTIC BIOMARKERS FOR PROSTATE CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Thanks for addressing my comments. However, some comments still have not been addressed well. Also, some comments have been shown in the rebuttal letter, but the authors did not change in the revised manuscript. Therefore, the authors are suggested to have a minor change before acceptance.
1. More literature review related to bioinformatics analyses should be added in the "Introduction".
2. Did the authors concern about the batch effect removal when merging the data?
3. It is necessary to cite more related works to ROC analyses i.e., PMID: 31362508 and PMID: 31750297.


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: K-MER-BASED MACHINE LEARNING METHOD TO CLASSIFY LTR-RETROTRANSPOSONS IN PLANT GENOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper describes ML methods to detect and classify LTR retrotransposons in plant genomes. They used several ML models, such as SVC. They used k=1 to 6 and precisely classified the LTR sequences from non-LTR sequences. The method they used is conceptually different from the approaches widely used which use sequence alignment to detect and classify transposons (e.g. RepeatMasker), thus this study is an important advance in this field.
Experimental design: Their analyses are mostly acceptable, with a few serious issues in the analysis design as detailed below.

A)	Line 119. It is unclear why they used the negative instances longer than 6 kb. Authors should describe the rationale. Some LTR-transposons, particularly partial LTR-RTs and solo-LTR elements, would be shorter than 6 kb. This difference may influence the classification by ML. Authors should show the distribution of the lengths of sequences used as a training data.

B)	Authors should describe how they calculate k-mer occurrence. In line 230, they show 10 most important features here. Those contains inverse (i.e. reverse complement) k-mers (e.g. AAAAAA and TTTTTTT). This brings me to think that, to calculate k-mers, they only used either forward or reverse strand of the sequences in their dataset. In genomics analyses, it is more common to consider inverse pairs of k-mers because transposons can be inserted at both forward and reverse directions. In such cases, two complementary k-mers should be considered as one k-mer. If they did not consider this property, it may generate potential bias towards one strandness in the machine learning process. The authors should further clarify how they calculated k-mer occurrence.
Validity of the findings: Throughout the manuscript, they used the term “detection” of LTR-RTs. However, what they are doing is the binary classification of sequences into LTR and non-LTR classes. Their current approach cannot determine genome regions or nucleotide-level locations of LTR transposons from the assembled plant genomes, thus “detection” is not an appropriate term to describe this. In the practical setting, the detection will be annotation of LTRs from the assembled genome sequences as described in line 252 and 294. In this context, what needed to say “detection” is annotation of LTR from long assembled plant DNA genomes, not from a set of short sequence instances (i.e. CDS and TEs). Authors should at least rephase the term “detection” or further implement pipeline that allow them to detect/annotate LTR-RTs from assembled genomes. Related to this, the authors should also discuss how the k-mer-based ML model can be applied to the LTR detection/annotation of the assembled plant genomes in future.
Additional comments: Minor points:
Throughout the manuscript, “k” of k-mer should be italic.
Line 249. “The most frequent repeated sequences in plant genomes are LTR retrotransposons.” Here requires references.
Lines 260-262.  Add references.
Fig7. What are the columns and rows of the confusion matrix? Label them.


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

Input:
Title: K-MER-BASED MACHINE LEARNING METHOD TO CLASSIFY LTR-RETROTRANSPOSONS IN PLANT GENOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Figures and tables lack title
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This study compared performance of several common ML models to detect and classify LTRs in plan genomes, and also it conducted feature selection to select some k-mer based features to train models that shows not much difference as using all features. But I recommend rejecting at current version since there are some major problems should be fixed before considering further evaluation.
Major issue
1. This study compared different ML methods and reported their performance. But it does not compare with other tools that are well address to solve the LTR classification.
2. It looks like no independent testing dataset was set. independent testing is important since it can validate the models after the hyper-parameter tunning.
3. The negative datasets may miss some important TE families and sequences such as Helitron as well as Intergenic sequences excluding all TEs, CDS, RNAs.
4. This study only uses F1-score to compare all ML models. Even F1-score may solve data imbalanced issue, more scores such as auPRC and auROC need to be added.
Minor issue
Line 27 change ‘:’ to ‘;’
Line 116 what the composition of the 10,000 genomic feature sequences
Line 119 why keep sequences longer than 6kb not 5kb? Authors may consider checking the average length of LTRs.
Line 134 to 137, when applying k-cross-validation, have authors set independent testing dataset to validate the models?
Line 140, what algorithms used need to be clarified.
Line 221, I am thinking if we loosen threshold to 25 or 20, would the performance increase compared to 95% F1-Score based on 30.


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: CHEMICAL SYNTHESIS OF PEPTIDOGLYCAN MIMETIC–DISACCHARIDE-TETRAPEPTIDE CONJUGATE AND ITS HYDROLYSIS BY BACTERIOPHAGE T5, RB43 AND RB49 L-ALANYL-D-GLUTAMATE PEPTIDASES
Review round: 1
Reviewer: 1

Output:
Basic reporting: V. Azev et al describes the synthesis and the hydrolysis by a set of peptidases of one disaccharide-tetrapeptide. This disaccharide peptide is presented as peptidoglycan mimetic which could be used for hydrolysis study by spectroscopy methods. The English language is clear and unambiguous. The introduction needs to be more documented, especially concerning the structure of peptidoglycan to correctly introduce the choice of the synthesized compound. A review could be found easily on Pubmed concerning PG structures.

Authors claim that “the process of the peptidoglycan binding and hydrolysis by enzymes cannot be studied by the spectral methods”. This assertion must be nuanced [For spectral methods with PG or PG fragments see Bougault et al, J. Struct. Biol. (2019) 206(1):66-72; Forrest et al, J. Biol. Chem. (1991) 266(36):24485-91 or Maya-Martinez et al, Front. Microbiol. (2018), 9:3223].

The authors should explain more clearly the advantage of their methodology. For example, the incorporation of the disaccharide moiety described in the paper seems the most important issue for the obtention of mg of PG analogs.

Figures are relevant even if Fig. 1 and Fig. 3 appear blurry.

Figure 3 must be reorganized: panel C representing the beginning of the reaction must be on the upper part of the figure and renamed panel A. Actual panel A, representing the end of the reaction, must be on the lower part of the figure and must be renamed panel C.

The same modifications must be done for figure 5.
Experimental design: The experiments are well described. Even if the chemistry part is classical, purity analysis performed by TLC  for compound 4 seems insufficient for publication. Nor rp-HPLC chromatograms or HRMS spectra are reported for the final product (in the main text or in the supplementary Data). This point is essential to estimate purity products and need improvement for publication.
Validity of the findings: - Hydrolysis of the amide bond between L-Ala1 and D-iGlu2 is followed by NMR, it seems that an identification of the products could be easily done by MS. These experiments would strengthen the results presented in this article.

- Concerning the disaccharide moiety, it would be interesting to know if this part of the molecule is necessary for the enzymatic activity. Molecule 5 could be used for such a purpose. This study could be introduced in the Discussion section (L253-255)

-  Concentrations of GMTP and protein should be given in molar in the NMR-based analysis of the GMTP degradation to indicate the turnover of the reaction during experiments for proper comparison between different proteins used in this study (concentrations are given only in mg/mL and no indication is given concerning the molecular weight of the proteins).
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: CHEMICAL SYNTHESIS OF PEPTIDOGLYCAN MIMETIC–DISACCHARIDE-TETRAPEPTIDE CONJUGATE AND ITS HYDROLYSIS BY BACTERIOPHAGE T5, RB43 AND RB49 L-ALANYL-D-GLUTAMATE PEPTIDASES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) Background and context: The authors justify the synthesis of the soluble GMTP muropeptide mimetic as follows: “because of the insolubility of peptidoglycans…” they “cannot be studied by spectral methods” (Line 34 and 35). In addition, “It is impossible to study the process of the substrate binding and hydrolysis by enzymes by the spectral methods… since natural peptidoglycans are insoluble” (Line 64 and 65).

These statements are true. However, the authors did not mention that soluble muropeptides can be generated from insoluble peptidoglycan by digestion with mutanolysin or cellosyl, then purified by RP-HPLC (used previously as a source of soluble muropeptides for NMR experiments, e.g. Atrih, et al. Journal of bacteriology, 1999. doi:10.1128/JB.181.13.3956-3966.1999). There are also commercially available soluble muropeptides such as MDP, Mtri-DAP and Mtri-Lysine (such as those produced by Invivogen), which include dibasic amino acids (mesoDAP and Lysine) at position 3 of the peptide stem that are found in naturally occurring peptidoglycan, but absent from the GMTP structure generated in this paper.

The authors should include additional justification for the study. What is the advantage of using GMTP, which has an unusual L-Alanine at position 3, relative to alternatives such as MDP, Mtri-Dap or Mtri-Lysine?

2) Article structure: The article is generally well structured. Regarding the experiment in which GMTP digestion by the 3 enzymes is compared (lines 206-237), the order in which the data are presented is confusing.  EndoRB49 activity is first compared to EndoT5 at pH 7.3 (Panels A and B), then EndoRB49 activity compared at pH 7.3 and pH 4.3 (panel C). Finally, EndoRB43 activity at pH 7.3 is presented (Panel D).

It would be more logical for the authors to present and discuss all of the experiments performed at pH 7.3 together. The activity of EndoRB49 at pH 7.3 and pH 4.3 should be discussed separately. The order of the panels in figure 4 should be altered accordingly. It would aid reading if the enzyme and pH were annotated directly on the relevant panel.

3) “Therefore, the alanine part of GMTP is an “anchor site”…” (Line 250).
This statement is ambiguous. It is unclear whether the authors are referring to all 3 alanine residues, or a specific alanine – if so which one? The authors should clarify this point.

4) Regarding lines 46-47: “…suggesting that EndoRB49 is characterized by the lowest selectivity, and hence the potentially broader spectrum of the peptidoglycan types subjected to hydrolysis.” One of the authors previously published on this topic, in which lysis of bacteria with different peptidoglycan types by the three enzymes was assessed. This citations should be added here. (Shadrin et al. J Appl Microbiol. 2020. doi: 10.1111/jam.14910).

5) Clarity of writing: There are many spelling errors and grammatical issues throughout, particularly in the methods and materials. The paper should undergo a substantial redraft to reach publication quality. There are too many errors to provide a complete list, but examples include:

Line 35: “…studied by the spectral methods” should be “…studied by spectral methods”.
Line 61: “…the L-Ala-D-Glu bond is always exist. It is this bond is the target…” should be “… the L-Ala-D-Glu bond is always present. This bond is the target…”
Line 86: Should read “…to a residual solvent signal.” or “…to residual solvent signals.”
Line 87 & 88:  “standart” should be “standard”
Line 105: “evoparated” should be “evaporated”
Line 103 & 105 “resulted” should be “resulting”
Line 108: “homogenious” should be “homogeneous”
Line 193: The structure is referred to as “GSCP” instead of “GMTP”
Line 115: “(3 2 mL)” should be “(32 mL)” or “(3.2 mL)”
Line 177: “…until the complete completion…” should be “…until completion…”

6) Labelling of Figure 3. The labelling of the spectra is strange, as is the order they are presented in the figure legend. Why does it begin at C and end on A? The order should be changed to A, B, C.
Experimental design: The experiments appear to have been conducted to a high standard, and described in sufficient detail to be replicated.

My concern in terms of experimental design relates to the issue raised in "basic reporting". A justification should be given for using GMTP as an alternative to soluble muropeptides, that contain the dibasic amino acids found in natural peptidoglycan.
Validity of the findings: 1) Regarding lines 46-47, and the work published by Shadrin et al. 2020. (doi: 10.1111/jam.14910). I do not agree the faster activity of EndoRB49 towards GMTP shown in the current study “suggests” it is “characterized by the lowest selectivity”. It would be more correct to say that it is “consistent” with this hypothesis, which was put forward in the paper of Shadrin et al. The authors should rephrase this sentence accordingly.

Care should be taken, as (unless I'm mistaken) the paper of Shadrin et al. studied lysis of bacteria by the three L-D peptidases, and therefore the presence of other wall polymers such as teichoic acids or capsule could have affected the enzyme activity. To demonstrate conclusively that the enzymes differ in their selectivity toward  peptidoglycan, would require additional experiments performed using purified peptidoglycan or specific muropeptides (which is outside the scope of the current manuscript).

2) In this manuscript, and the paper of Shardin et al, the reactions were performed at pH 7.3 and pH 8 respectively. In this manuscript, EndoRB49 was also tested at pH 4.3, but it seems that EndoT5 and EndoRB43 were only tested at pH 7.3. What is the pH optimum for EndoT5 and EndoRB43? Is a pH of 7.3 close enough to the pH optimum of all three enzymes that it would not to be responsible for the difference in reaction rates? It would be useful to address this point in the manuscript.

3) If I have understood correctly, the authors say that the methyl groups of all 3 alanine residues act as anchor points for protein binding. In this case, what is the biological relevance of binding to the methyl group of the L-Alanine at position 3 of the peptide stem, as this amino acid is not present in naturally occurring muropeptides? The authors should comment on this.

4) Lines 219 to 222. A good negative control is mentioned here (catalytically inactived T5 enzyme) but the data is not shown. The authors should add it to figure 4, or included it as supplementary 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: CHEMICAL SYNTHESIS OF PEPTIDOGLYCAN MIMETIC–DISACCHARIDE-TETRAPEPTIDE CONJUGATE AND ITS HYDROLYSIS BY BACTERIOPHAGE T5, RB43 AND RB49 L-ALANYL-D-GLUTAMATE PEPTIDASES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The requests for corrections to the manuscript by Azev et al have been made.

- The introduction was modified as requested to introduce in a more documented way the molecule synthesized in this study.
- The authors have also discussed the interest of introducing an L-Alanyl moiety at the third position of the synthesized peptide.
- The authors added a discussion part in the body of the text concerning the importance of the disaccharide moiety of their compound.

- All corrections concerning figures are correctly introduced in the text.
Experimental design: -	Some missing characteristics (mass and rpHPLC data) were added in SI as requested.
-	The authors claim that some technical limitation of the LC-MS system they used does not allow identification of the reaction products. They explained that the reaction products are not retained on a C18 column and could not be analyzed by mass spectroscopy. This observation is effectively possible (personal observation on similar molecules). Direct injection on MS could be done after precipitation of proteins but NMR data are sufficiently convincing for publication.
Validity of the findings: no comment
Additional comments: In conclusion, I think this work is suitable for publication.


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

Input:
Title: CHEMICAL SYNTHESIS OF PEPTIDOGLYCAN MIMETIC–DISACCHARIDE-TETRAPEPTIDE CONJUGATE AND ITS HYDROLYSIS BY BACTERIOPHAGE T5, RB43 AND RB49 L-ALANYL-D-GLUTAMATE PEPTIDASES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have addressed all the comments satisfactorily. There is only one issue regarding figure 3, and some minor orthographic/english  errors throughout the manuscript which should be corrected before publication. I have tried to provide a complete list below.

Otherwise the manuscript has been completed to a high standard.

*******

In the uploaded figure files, and review PDF, the figure 3 legend is incorrect. It has been duplicated from figure 2. The correct legend is found in the Word document however. Please ensure the correct legend is included in the published manuscript, particularly as there was a correction made regarding the order in which the data was presented.

The incorrect legend: “Chemical structure if the N-acetylglucosaminyl-β(1-4)-N-acetylmuramyl-l-alanyl-γ-d-glutamyl-l-alanyl-d-alanine (glucosaminyl muramyl tetrapeptide, GMTP).
The numbers correspond to the sequential numbers of the amino acid residues. The peptide bond subjected to hydrolysis is colored in green.”

63: “linked by the interpeptide bridges” should be “linked by interpeptide bridges”

69: “(for example, 1 corresponds to the bridge that does not contain amino acids)” – a little strange to say a bridge with no amino acids. Better so say “(for example, 1 corresponds to direct cross-linking between peptides stems, with no bridging
amino acids).”

71: “…acid, DAP). . The amino acid…” should be “…acid, DAP). The amino acid…”

75: “…the L-Ala-D-Glu bond is always exist.” - “… the L-Ala-D-Glu bond is always present.”

96: Double space.

105: “…specific activity on natural substrate.” Should be “…specific activity on the natural substrate.”

120: “…by the spectral methods” shoud be “by spectral methods”.

214 and 231:  Values are given as (3 1.5 ml) (3 10 mL) (3 10 mL) (3 10 mL). What does the 3 signify? Should it read 3x?

256 and 258: since the units are mM, the “/L” is unnecessary. Alternatively use the units “mmol/L”.

296: “acetylmuramil” should be “acetylemuramyl”

307: “…noted that the addition of a mutant EndoT5 with the point substitution of the catalytic aspartate (EndoT5D130A) to the GMTP solution did not cause the substrate hydrolysis” should be “…noted that the addition of a mutant EndoT5 with a point substitution of the catalytic aspartate (EndoT5D130A) to the GMTP solution did not cause substrate hydrolysis”

312: “Therefore, although pH 7.3 and pH 8.0 that were used in previous work for the analysis of catalytic reactions of EndoT5, EndoRB43, and EndoRB49 (Shadrin et al. 2020) are within this pH optimum range, and pH 4.3 is the value at which enzymes are practically devoid of activity.” Should be “Therefore, pH 7.3 and pH 8.0, that were used in previous work for the analysis of catalytic reactions of EndoT5, EndoRB43 and EndoRB49 (Shadrin et al. 2020), are within this pH optimum range, whilst pH 4.3 is the value at which the enzymes are practically devoid of activity.”

325: “…which is very important for future structural studies of the extremely interesting and promising for biotechnology protein EndoRB49.” Should be “…which is very important for future structural studies of the protein EndoRB49, which is extremely interesting and promising in terms of biotechnological applications.”

380: “Nevertheless, as it was shown, the developed in this study glycopeptide…” should be “Nevertheless, as it was shown, the glycopeptide developed in this study…”

407: “However, there is evidence   that it is so.” Remove the extra spaces.

566: Figure 4 legend, “Black circles show…” should be “Black, filled circles show…” since there are two sets of black circles representing different data.

568: “C. EndoRB49 (pH 4.3); C. EndoRB49 (pH 4.3);” text duplication.
Experimental design: The experimental design and reporting is to a high standard
Validity of the findings: The underlying data is provided in a robust manner. Conclusions and speculative discussion are clear.
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: LIVER PROTEOME ALTERATIONS IN PSYCHOLOGICALLY DISTRESSED RATS AND A NOOTROPIC DRUG
Review round: 1
Reviewer: 1

Output:
Basic reporting: Proteomics was deployed to study the effect of psychological distress and piracetam on the liver proteome. The manuscript was well structured and provided sufficient background and context.
Experimental design: I have a major concern about the experiment design for proteomics: liver samples were pooled for each experimental group, so there is no information about the variation in the group and between groups.
Validity of the findings: Data in Table 1 need to be verified.
Additional comments: Proteomics was deployed to study the effect of psychological distress and piracetam on the liver proteome. The manuscript was well structured and provided sufficient background and context, however, I have a major concern about the experiment design for proteomics: liver samples were pooled for each experimental group, so there is no information about the variation in the group and between groups. Data in Table 1 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: LIVER PROTEOME ALTERATIONS IN PSYCHOLOGICALLY DISTRESSED RATS AND A NOOTROPIC DRUG
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	The professional English used in the article is clear.

-	The introduction and background context of the manuscript is good, but I have the following observations:

-	They emphasize the effect of psychosocial stress and relate it to neuroimmunological changes, highlighting various brain structures, but they will evaluate the effect of the drug on the liver, an organ that should be given more emphasis, since they will not be working with the brain. The authors comment that there is not enough information on this subject, but in the discussion they present a couple of articles that show liver damage after subjecting mice to stress. In addition, in the conclusions in the lines 705-6, they describe that the proteomic analysis of the liver from stressed rats supports the fact that emotional stress causes an imbalance in the metabolism of lipids and antioxidant enzymes.

-	What is the difference between stress and distress? These two terms are used interchangeably in various sections of the article, mainly in the introduction, methodology, discussion and conclusions. It is convenient to handle the terms correctly since they are the parameters of your study. I suggest to review the article by Kiersten et al., 2021. Health Psychol. 2021 Feb;26(2):312-318. doi: 10.1177/1359105318804865.

-	Authors of the manuscript indicate in the line 97 that Piracetam was the first of these drugs to be described and is commonly used as a neuronal enhancer because it mainly modulates cholinergic and  glutamatergic, But Verma et al., 2018, showed that Piracetam is a cyclic derivative of the neurotransmitter γ-aminobutyric acid (GABA) and it is used in clinical practices for the treatment of epilepsy. Therefore, it is not clear if the drug decreases or increases neuronal activity, it is necessary to describe the mechanism of action more precisely.

-	The cited literature is adequate and up-to-date. Although the following points must be corrected:

-	The bibliographic citations present errors in the title of the journal, which in some cases appears as abbreviations and in others the complete name, it is necessary to homogenize them. For example the first reference: Abdel-Salam et al., 2011. The Journal appear as Neurochem Res, but in the third reference of Adachi et al, 1993, it says Cancer Research.
-	A reference has an asterisk in the line 972.

-	Missing reference of Jia et al., 2018. Include it.
-
-	Place genus and species in italics and other words in latin language, such as in vitro and et al.

-	Several references contain the title of the article in upper and lower case.

-	The structure of the article conforms to PeerJ standard

-	Figures:
o	For figures 1 and 4 it is recommended to use contrasting colors in the graphs, do not highlight the colors used.

o	The figure legends should highlight the results shown and not be limited to describe the experimental conditions.
Experimental design: -	The research is original and with good impact. The research question is not completely clear.

-	Molecular biology techniques are well described, and the research group has shown their management through several publications.

-	I recommend a flow chart showing the experimental conditions used and a picture of the device they used for distress induction.

-	They used female rats and did not find significant differences in their model, due to the large dispersion of the results in the control group compared to the stress group, which can be attributed largely to the number of animals and variations in their estrous cycle. I suggest documenting this part, since they comment that they have previous studies in males where they did observe differences. It is essential to validate their study model for the results obtained in the quantified proteins.

-	Did you evaluate the estrous cycle of the rats? Since several research groups have identified changes in activity and rest throughout their cycle. In which part of the estrous cycle and the light-dark cycle were the tests performed?. It has been shown that various neurobiological parameters such as stress, sleep, learning, memory and sexual behavior vary according to the phase of the estrous cycle. I recommend reviewing the article by Oyola et al., 2017 (Oyola MG, Handa RJ. Hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes: sex differences in regulation of stress responsivity. Stress. 2017 Sep;20(5):476-494. doi: 10.1080/10253890.2017.1369523) to enrich the discussion of the obtained behavioral data.
-	The statistical analysis performed is adequate.
Validity of the findings: -	The manuscript presents an adequate discussion by sections, but I observe the following drawbacks:

-	In the Introduction they point out the importance of the study performed but in a general way. I suggest highlighting the relevance of the study.

-	In the section of Redox metabolism they point out that: The relationship between psychological stress and the redox status alteration has been previously shown in several studies using different murine models in the liver, brain, pancreas, kidney,lungs, and heart  (Şahin & Gümüşlü, 2007; Jafari et al., 2014; Mejia-Carmona et al., 2014, 2015;López- López et al., 2016; de Sousa Rodrigues et al., 2017). After this paragraph, there is no description of the contribution of your study, it seems that it has already been done.

-	They do not highlight the justification of the work. Therefore, their discussion can be enriched by considering the following aspects:

-	In the previous studies performed by the working group they comment that protein expression changed, but it is convenient to specify which alterations or changes were presented.

-	They also describe that Piracetam decreases the antioxidant level and therefore decreases the oxidative stress and increases the proinflammatory responses (Singh et al., 2011; Liu et al., 2017; Verma et al., 2018). Moreover, this drug may protect the lipids from cellular membranes against oxidative stress due to its ability to scavenge free radicals, in this way avoiding mitochondrial dysfunction caused by such reactive molecules (Keil et al., 2006). In our study, piracetam decreased the GPX and CAT activity in distress conditions due to its ability to reduce the number of oxidant compounds. So, they do not provide new information and only corroborate previously described results?

-	In the Line 693 described: Contrary to our results, CUMS model induced an increase in the activity of ALT and AST in male rats, suggesting liver damage (Jia et al., 2016). Again, gender-associated studies need further investigations. It is not clear, What were your results?

-	The conclusions are general, so they are incomplete to a large extent, it is necessary to highlight the results obtained. They comment that Piracetam had protective effects in the stress group, but not in relation to the controls and that this may be due to the fact that they used female rats, which in comparison with males are more resistant, the great inconvenience is that significant changes were registered only in the stress plus piracetem group in the behavioral test.
Additional comments: -	One of the great advantages of the article is the large number of techniques used to evaluate stress-induced liver damage and the protective effect of Piracetam in rats. These techniques have been developed by the research group in other publications, and they only require to specify some aspects that I pointed out in each of the sections requested in the revision of the manuscript. Besides emphasizing the importance of the study and highlighting the results obtained.


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: LIVER PROTEOME ALTERATIONS IN PSYCHOLOGICALLY DISTRESSED RATS AND A NOOTROPIC DRUG
Review round: 2
Reviewer: 1

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


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

Input:
Title: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 1
Reviewer: 1

Output:
Basic reporting: line 106: Moraes et al. 2016 is missing.

line 217: the use of term “juxta”: according to Niculescu (1972, 1988), the term “juxta” should not be used, once “fultura inferior” was earlier proposed by Petersen (1904), and were replaced for juxta by Pierce (1909, 1914).
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: Dear authors:

The manuscript entitled ‘Description of three new species of Geometridae using species delimitation in an integrative taxonomy approach for a cryptic species complex’ is an important contribution towards the knowledge of the systematics of Neotropical Geometridae.

I made some minor comments and suggestions that should not compromise the acceptance of the manuscript.


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

Input:
Title: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is focused on a very diverse genus of moths from the Neotropics. The authors take up an integrative taxonomy approach in species delineation by combining DNA barcode sequences with ecological information on the larval host plants, as well as a careful examination of relevant morphological characters. The text is clear and very well written with some minor problems that I tried to correct in the Word version of the manuscript (uploaded separately). The authors provided a good background and have cited the relevant works appropriately. The article is overall well structured and the raw data are shared. The illustrations of the genital structures are beautiful as well as very informative and the photographs of the adults are done at a high level, clearly showing all of the relevant features. The tree figure needs to be improved and I provide additional comments on that below.
Experimental design: The research question is clearly stated and defined. It is highly relevant given the accelerated extinctions that are ongoing all around the planet and as has been shown recently, insects are not spared. It is important that alpha taxonomy is carried out and that we document as much as possible of our planet’s biodiversity before it disappears. We can’t do much to protect this diversity if we don’t know that it exists! The methods are described with enough details to replicated the study and the methodology is sound.
Validity of the findings: The authors make a good case that they have discovered new species of the genus Eois in Brazil and the description of these three new species is fully justified. We have evidence from several different sets of characters: DNA barcode, external morphology of the adults and genitalic structures, and host plant specialization. Perhaps the number of reared individuals is quite low to allow for a full assessment of how useful the host plant data is at this level, but it does give additional data that are useful.
Additional comments: Great job overall, but as I stated above, the tree figure needs additional work. Firs, the font is much too small to be readable. It should be 2-3 times larger at the minimum! I understand that most of us these days view papers on our computers, where we can zoom in as necessary, but here, when one zooms in enough to read the text, one can only see a small part of the tree, and given how much empty space there is in this figure overall, you can do a much better job. You can also double or triple the size of the pictures of the moths and larvae, and perhaps somewhat increase the size of the host plant symbols, although they can even remain the same size.

Also, it’s unclear to me whether each of the DNA barcoding clusters represents an already described species or more still unnamed species. For example, odatis clade seem to include several species. It would be helpful if you could label all of these clusters with names of at least the described species. I assume that one of these lineages is Eois odatis.

And the tree figure would overall be easier to read if it were displayed in increasing or decreasing order.


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: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is very well writen; clear and precise.In the introduction of the work, the authors make clear the structure and purpose of the study.
The structure of the article follwes the format of the PeerJ Journal. Table and figures high-quality. Raw data shared.
Experimental design: The manuscript reveals a original and relevant study since contributes to understanding about a complex of cryptic species of neotropical moths.
Moreover, using morphology, host plant use and species delimitation tools proved to be very appropriate for the study.
Validity of the findings: No comment.
Additional comments: Studying neotropical moths is  challenge given its enormous diversity. In their study you describe three new species (Eois genus) and provide a relevant dataset.  The scientific value of the information is excellent.


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: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 2
Reviewer: 1

Output:
Basic reporting: Ok.
Experimental design: Ok.
Validity of the findings: Ok.
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: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 2
Reviewer: 2

Output:
Basic reporting: Looks good.
Experimental design: Looks good.
Validity of the findings: All good.
Additional comments: Looks great! I like how the changes in this version were implemented based on the review 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: DESCRIPTION OF THREE NEW SPECIES OF GEOMETRIDAE (LEPIDOPTERA) USING SPECIES DELIMITATION IN AN INTEGRATIVE TAXONOMY APPROACH FOR A CRYPTIC SPECIES COMPLEX
Review round: 2
Reviewer: 3

Output:
Basic reporting: As a results of the revision (minor revision),  I consider the manuscript has been greatly improved.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I consider your manuscript as a beautiful incentive for future studies of Neotropical species of Macroheterocera. Congratulations!


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: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The Manuscript is written well.
The structure conforms to PeerJ standards
Experimental design: Article content is within the Aims and
Scope of the journal.
Yes
Rigorous investigation performed to a
high technical & ethical standard.
yes
Methods described with sufficient detail &
information to replicate.
NO
Is the Survey Methodology consistent with
comprehensive, unbiased coverage of
the subject? If not, what is missing?
No, the difference between alpha and beta-amylase is missing, the mode of action is missing.
Are sources adequately cited? Quoted or
paraphrased as appropriate?
Yes
Is the review organized logically into
coherent paragraphs/subsections?
No
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale & benefit to literature is clearly stated.
Yes
Conclusions are well stated, linked to original research question & limited to supporting results.
No

Is there a well developed and supported argument that meets the goals set out in the Introduction?
No

Does the Conclusion identify unresolved questions / gaps / future directions?
No
Additional comments: The Manuscript is written well, but there are some comments in the body of the manuscript that need to be improved.
The authors must explain the mode of action of a-amylase.
Explain what the difference between alpha and beta-amylase in the mechanism of catalysis.
 The abstract is descriptive and qualitative. Normally an abstract should state briefly the purpose of the study undertaken and meaningful conclusions based on the obtained results. Hence, this needs rewriting. I would expect a brief, yet concise, quantitative data description of the results in the abstract.
The conclusion should contain more specific values to reflect the significance of the work.


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: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: On the whole, the manuscript reviews the reports concerning producing native or engineered microbial α amylases for application quite thoroughly, thus it will be a good guideline literature for the researchers of the area. However, before publication, several issues listed below are suggested to be addressed.

1.	In the most situations in the paper, “engineering” seems to denote “changing amino acid sequence by mutation including substitution, truncation, insertion or fusion”, then “…engineering and mutations…” in the line 74 should be corrected to avoid confusion.
2.	Since the paper includes many examples of producing native or recombinant α amylases by homologous or heterologous expression without changing the amino acid sequences (engineering), it would better be titled “Native to designed: microbial α amylases for industrial applications”.
3.	Although the authors state the desirable traits of α amylase for every specific applications, it would be better to summarize all the desirable traits and corresponding applications in a table for clearness.
4.	In section “Structure Properties if Microbial α amylase”, another figure that diagrams the general architecture of α amylases including domain A, B, C and CBM/SBD would be better for readers understanding their common features. In addition, more detailed structural characteristics such as hydrophobic residues on the surface of SBS and SBD should be provided in the text to help the readers to understand the “rationale” of “rational design”.
5.	Figures showing the specifically engineered structure are favorable for understanding the examples in “Engineered microbial α Amylase with Altered Substrate and Product Specificities”.


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: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is well designed. However, it does not take into account cold-adapted alpha-Amylases and, most importantly, from protists. These enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various industrial applications as it implies a reduction in energy consumption during the catalyzed reaction.
Therefore, I suggest adding to the review some results reported in the two following papers:
1: Yang G, Yao H, Mozzicafreddo M, Ballarini P, Pucciarelli S, Miceli C.
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate
Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic
Activity. Appl Environ Microbiol. 2017 Jun 16;83(13):e00449-17. doi:
10.1128/AEM.00449-17. PMID: 28455329; PMCID: PMC5478988.

2: Yang G, Yang G, Aprile L, Turturo V, Pucciarelli S, Pucciarelli S, Miceli C.
Characterization and comparative analysis of psychrophilic and mesophilic alpha-
amylases from Euplotes species: a contribution to the understanding of enzyme
thermal adaptation. Biochem Biophys Res Commun. 2013 Sep 6;438(4):715-20. doi:
10.1016/j.bbrc.2013.07.113. Epub 2013 Aug 2. PMID: 23916704.

which report the comparison of alpha-amylases from two congeneric species adapted to two different environments. This comparison allows identifying residues to be changed with rational design by site-directed mutagenesis.

I suggest adding more figures that represent the important sites of the alpha-amylases.

In my opinion, the paper is acceptable after these minor modifications.
Experimental design: The study is well designed. However, I would check better the results obtained from the bibliography databases. For example, I would add extremophiles, microbial and cold stability.
Validity of the findings: The findings are well described. In my opinion, there is a lack of more information on the structure and amino acid sequence of the alpha-amylase.
Additional comments: The paper is well designed. However, it does not take into account cold-adapted alpha-Amylases and, most importantly, from protists. These enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various industrial applications as it implies a reduction in energy consumption during the catalyzed reaction.
Therefore, I suggest adding to the review some results reported in the two following papers:
1: Yang G, Yao H, Mozzicafreddo M, Ballarini P, Pucciarelli S, Miceli C.
Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate
Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic
Activity. Appl Environ Microbiol. 2017 Jun 16;83(13):e00449-17. doi:
10.1128/AEM.00449-17. PMID: 28455329; PMCID: PMC5478988.

2: Yang G, Yang G, Aprile L, Turturo V, Pucciarelli S, Pucciarelli S, Miceli C.
Characterization and comparative analysis of psychrophilic and mesophilic alpha-
amylases from Euplotes species: a contribution to the understanding of enzyme
thermal adaptation. Biochem Biophys Res Commun. 2013 Sep 6;438(4):715-20. doi:
10.1016/j.bbrc.2013.07.113. Epub 2013 Aug 2. PMID: 23916704.

which report the comparison of alpha-amylases from two congeneric species adapted to two different environments. This comparison allows identifying residues to be changed with rational design by site-directed mutagenesis.

I suggest adding more figures that represent the important sites of the alpha-amylases. There is a lack of more information on the structure and amino acid sequence of the alpha-amylase.

Furthermore, I would check better the results obtained from the bibliography databases. For example, I would add extremophiles, microbial and cold stability.


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: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: acceptable
Experimental design: acceptable
Validity of the findings: acceptable
Additional comments: The authors made all modifications and therefore I recommended that the manuscript be accepted as it is


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: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 2
Reviewer: 2

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


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

Input:
Title: NATIVE TO DESIGNED: MICROBIAL Α-AMYLASES FOR INDUSTRIAL APPLICATIONS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The paper has been improved. The authors replied to all reviewer's comments. The paper now is ready for acceptance.
Experimental design: The study design has been improved. The authors added more information about the topic and the methodology.
Validity of the findings: Now the findings are real valid and supported.
Additional comments: The paper can be accepted in this form.


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

Input:
Title: HIGH GLUCOSE TREATMENT PROMOTES EXTRACELLULAR MATRIX PROTEOME REMODELING IN MÜLLER GLIAL CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very well-written and structured manuscript.
Experimental design: Acceptable.
Validity of the findings: Acceptable.
Additional comments: This is a very-well written manuscript describing a proteomic study on pig primary retinal Müller cells (RMCs) or explants cultured under high glucose conditions. The manuscript does provide new information in the field: potential protein alteration pattern in RMCs in diabetic retina and new information about osteopontin under similar conditions. However, following changes may be necessary/helpful:
1.	As there are several proteomic studies related to RMCs, the authors should provide a short discussion on the differences (distinctive features) of their results vs that in previous studies.
2.	No culture conditions will be completely identical to that in real diabetes. Therefore, an accurate description of experimental procedures/results under diabetes-mimic condition should use something like diabetes-like or high glucose condition. Hyperglycemic is not the correct term.
3.	Figure legends should provide sufficient details, eg. Fig 2 legend 1st sentence: “Differential protein abundances in hyperglycemic (a,b) or glycolysis inhibition (c,d) compared to normoglycemic treatment group..” should at least provide info about the source of protein, such as primary cultures, etc.
4.	For retinal Müller glial cells, the conventional abbreviation should be RMGCs. If the Müller cell cultures are the experimental focus of this study, the term retinal Müller cells (RMCs) may be a better term to use.
5.	Conclusion should add a sentence or two for other major findings, such as significant changes in ECM remodeling—related proteins.


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

Input:
Title: HIGH GLUCOSE TREATMENT PROMOTES EXTRACELLULAR MATRIX PROTEOME REMODELING IN MÜLLER GLIAL CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is generally well written and presented clearly.
Experimental design: The experimental design and methods to elucidate the hypothesis were adequate.
Validity of the findings: The presentation of the results was clear too. However, some aspects should be revised and the conclusions softened.
Additional comments: The study by Sagmeister et al. examined the effect of short term hyperglycemic as well as glycolysis inhibition on retinal Müller glial cells (MGCs) proteome. The results revealed significant changes in RMG proteome primarily in proteins building the extracellular matrix (ECM) indicating fundamental remodeling processes of ECM as novel rapid response to hyperglycemia. Osteopontin (SPP1) as well as its interacting integrins were significantly downregulated and organotypic retinal explant culture confirmed the selective loss of SPP1 in RMG upon treatment.
The manuscript is generally well written and presented clearly. The experimental design and methods to elucidate the hypothesis were adequate. The presentation of the results was clear too. However, some aspects should be revised and the conclusions softened.

Major concerns:

1. In lines 65 and 67 the authors refer to diabetes but they do not reference the type. Clarify this important point. In addition, in line 118 they state that the 2-DG uptake competes with glucose, as it is transferred by the same transporters. Define which transporters the authors refer to.

2. Usually, the glucose concentration used in culture of RMG under hypeglycemia conditions is 25 mM. Please provide information and references about the selected concentration of 35 mM of glucosa (line 112). Was this concentration related with DR in humans?

3. In the same way (line 233), authors should provide more detailed information and references related with the hyperglycemic explants treatment. Was this concentration (70 mM) toxic? How was the viability of the explants affected after 72 h of treatment?

4. In line 412, the autors indicated that they confirmed the findingsobtained in RMG in a model that more closely resembling the in vivo conditions. What exact culture medium was used in the porcine neuroretinal organotypic explant cultures?  Did the medium contain hormones, growth factors etc? Usually, the culture medium used is Neurobasal medium (Kuehn 2016, 2017). Please, clarify this important point.

5. According to Pereiro et al., (2020) primary porcine RMG cultures reach confluence after 7 days in vitro. This time point was stablished as the best, since it has been previously demonstrated a possible transdifferentiation of Müller cells after 14 days in vitro (Guidry, 1996; Hauck et al., 2003). How do the authors justify the completion of their studies after the 14days?

6. Due to the neuroprotective function attributed to SPP1, it would be interesting to determine the cell death in retinal explants. With this experiment, the conclusion of the manuscript and abstract will be justified. On the contrary, this paragraph related to SPP1 is overstated and need to be moderated. Please consider this when re-writing this sentence and revisiting your abstract and conclusions.
In addition, in Figure 5, could the authors add a Western blot assay with quantification of RMG under hyperglycemia or glycolisis inhibition also showing the reduction of SPP1 levels? This assay would help to complete the results.

7. In line 437, the authors refer to another protein, ITGB1, which was downregulated under diabetic conditions and its posible effect on the gliosis. In this sense, the authors should include the complementary analysis of GFAP in retinal organotypic explants, which be helpful to add more evidence to the discusión of this manuscript.

Minor concerns:

Line 46: authors should check for punctuation marks

Figure 1 Legend: improve the writing of this paragraph. Pay special attention to… after lysis, cells were lysed and …


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

Input:
Title: HIGH GLUCOSE TREATMENT PROMOTES EXTRACELLULAR MATRIX PROTEOME REMODELING IN MÜLLER GLIAL CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This is a very well-written and structured manuscript.
Experimental design: Acceptable.
Validity of the findings: Acceptable.
Additional comments: The authors have a dressed most of my concerns. The remaining part(s) can be considered as difference in opinion that I have no objection.


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

Input:
Title: HIGH GLUCOSE TREATMENT PROMOTES EXTRACELLULAR MATRIX PROTEOME REMODELING IN MÜLLER GLIAL CELLS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This is a very well-written and structured manuscript.
Experimental design: Acceptable
Validity of the findings: Acceptable
Additional comments: No more 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: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is very well written. There are a few minor places where there is some syntax/grammar issues, but these can easily be edited.

The literature background is extensive and well covered.

The figure and table are appropriate.  However, I do not see any reference to the raw data.
Experimental design: I am not an expert on the calculations related to RMR and DEE, I will assume, based on the supportive literature, that the analytical steps taken are the most appropriate at this time.
Validity of the findings: No raw data presented.
Additional comments: Some minor comments, mostly by line:

Line 91 – grammatical error…

Curious – were birds banded so that they were known to have participated in this experiment?

 Is there any speculation on what happened to the other 7 birds that were used for the DLW field experiment?

316-317..something about the change/transition between sentences is awkward

321: Period after Elliot 2013a. ??

333:  Edit the sentence:  "..but common murres likely spend less time on the water as they have more frequent feeding return trips.”  Or some such slight modification.

333+ This sentence is awkward:  "The feeding behavior of auklets could make them to stay longer time to digest food following diving bouts (Elliott et al., 2014) and then to increase their metabolic rate for the obligatory component of the heat increment of feeding (Hawkins et al., 1997)."

The last paragraph starting on page 325 needs better formatting of the ideas.  Perhaps it can be divided into two or more smaller paragraphs?  Discuss thermoregulatory issues, and then the issues of timing to deliver food back to the colony (one trip per day vs multiple trips)?  Maybe a new paragraph starting on line 336????

In the conclusion:  perhaps the last sentence might better say something that this high energetic cost (26.6%) may be a significant energetic concern for some seabirds, especially when they are in waters below their LCT.  The point is that this time, when historically has been ignored as not energetically costly (like flying or diving), may indeed, be significant.

Table 1:  I would add the genus and species name to the table.  I would also italicize the data from THIS study


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

Input:
Title: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is clearly written and presented.
Experimental design: The experimental design is suffiicent to answer the question they are asking (see direct comments below).
Validity of the findings: Underlying conclusion is robust.
Additional comments: Seabirds, especially auks, spend a lot of their life sitting on the water, but this is seldom thought to be an important component of their daily energy expenditure--although clearly an activity that takes considerable time will contribute significantly to energy budgets even if it isn't particularly costly. The authors make detailed measurements of daily energy expenditure and resting metabolic rate, and illustrating that sitting in cold water can be a significant contributor to energy budgets. These conductivity curves and daily energy expenditure measurements are technically challenging; kudos to the authors for completing them.

I have only a few minor suggestions:

L36 Suggest "on thermoregulation" rather than "for thermoregulation".

L60. Suggest adding "The" before "rhinoceros".

L. 106. Was CO2 production measured? It was be nice to have a robust estimate of RQ for DLW.

L 165. Could there be differential partitioning of isotopes into RBC water and plasma water? Usually water from whole blood is used.

L 217. The authors have validated their DLW measurements with direct respirometery. This is an important step that is seldom done, and provides great confidence in the results.

L. 313. A number of authors have tried to use similar analyses to your own to estimate energy costs year-round (Fort et al. 2009; Dunn et al. 2020). For example, Fort et al. suggest that the cold water in late winter is an energetic bottleneck and that birds switch between warm and cold water of the Golf Stream for that reason. I think it would be worth commenting on the use of your data for estimating energy costs year-round--as others are likely to do so.

Fort, J., Porter, W.P. and Grémillet, D., 2009. Thermodynamic modelling predicts energetic bottleneck for seabirds wintering in the northwest Atlantic. Journal of Experimental Biology, 212(15), pp.2483-2490.

Dunn, R.E., Wanless, S., Daunt, F., Harris, M.P. and Green, J.A., 2020. A year in the life of a north Atlantic seabird: behavioural and energetic adjustments during the annual cycle. Scientific reports, 10(1), pp.1-11.

L. 333. When capelin is present, common murres at Witless Bay have access to very abundant food, and can forage very close to the colony (~10 km, compared to ~50 km for Coats and Hornoya; Regular et al. 2013). This likely explains why they can spend so much time resting on the water and very little flying.

Regular, P.M., Hedd, A. and Montevecchi, W.A., 2013. Must marine predators always follow scaling laws? Memory guides the foraging decisions of a pursuit-diving seabird. Animal Behaviour, 86(3), pp.545-552.


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: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1.	The background and introduction to the paper set the study up to focus on the high energetic costs of time spent on the sea surface for seabirds, species which must deal with the contrasting thermoregulatory challenges of life in the water, the air and the land. However as noted elsewhere in my review, I do not think that this is a very successful or helpful angle for this dataset. The data do not particularly support the thesis and as such this background is not very helpful to introduce what the data could instead be used to show, which is a more general exploration of the energetics of this species, which would be valuable contribution to the literature.
2.	The paper is well written enough to be understood for the most part, though the use of the word ‘could’ at times is not easy to understand and it is possible that this word is being applying incorrectly. ‘Could’ would normally be used in circumstances were an inference or interpretation is not certain or speculative, whereas at times in the paper it appears that it is used to be more certain or positive that something is happening. This is distracting and some messaging is lost.
3.	The word ‘may’ is also used out of context. For example in Line 43 – I think it is clear that seabirds have many adaptations for thermoregulation in water, as the paragraph goes on to describe. So the word ‘may’ is unnecessary.
4.	The figure is too small to view and assess properly. The table is fine.
5.	The Introduction of your paper would benefit from a much stronger final sentence or two which clearly lays out the specific aims, questions or hypotheses that your study addresses. For example this would help the reader understand why there is a section in the Methods and Results on ‘Data obtained from the literature’. As written, this comes as a surprise to the reader. As a reader, it really helps to know as early as possible what to expect and why. If there is objective in your Introduction to make an explicit comparison to existing data then I will be expecting this section later on.
6.	Similarly, your Discussion would benefit by starting with a restatement of the main findings, in consideration of the aims, questions or hypotheses set at the end of your Introduction. Jumping straight into literature and allometric comparisons of RMR and LCT is not a compelling way to start this section.
Experimental design: 1.	In methodological terms, everything seems to have been conducted sensibly and appropriately. Respirometry and DLW techniques have been applied appropriately though some details could be clarified as listed in the following comments.
2.	Line 92. Specify how many birds were captured each year and how many for in air and how many for on water measurements.
3.	Line 92. Do auklets carry prey in their bills or in their stomachs? Did you consider them to be post-absorptive? How might digestive status have affected your measurements and how might this affect the in air and on water measurements differently?
4.	Lines 107-109. Specify how many individuals were tested at each temperature in both air and on water. Clarify how temperatures were selected and that there were discrete temperatures used for both air and water (as suggested by Figure 1). Clarify whether each bird was only used at one temperature and only used for in air or on water (or both).
5.	Lines 115-117. If you saw an appreciable mass loss overnight, did you consider using this to estimate energy expenditure?
6.	Line 154. Please clarify what time of day the initial enrichment occurred for the auklets. They were recaptured at night and the Results suggest approximately a 24 hour period until recapture, but this should be clearer.
7.	Lines 227-230. This is confusing. These equations suggest that there is no thermoneutrality in water in these species and that these equations would apply at any water temperature. However as noted later on, the two guillemot species do appear to have an LCT in water (Croll & McLaren 1993).
8.	Lines 230/231. If units are being converted from W (which is a rate, J/s) then there needs to be a time component to the converted rate. Joules per what unit time?

Croll, D.A. & McLaren, E. (1993) Diving metabolism and thermoregulation in common and thick-billed murres. Journal of Comparative Physiology, 163, 160-166.
Validity of the findings: 1.	My major concern with the paper is the conclusion that time spent on water is a large component of DEE for this species. The data presented do not demonstrate this point. The auklets spend ~50% of their daily time-budget on water but only 25% of their energy budget. To me this suggests that time on water represents a considerable energy saving when compared to flying most obviously. Furthermore this ratio of time to energy is nearly identical to the two other species and three other studies presented in the paper. As such, the finding is in no way remarkable and certainly not different to the other species presented. This is not a query about novelty, but an observation that the data do not support the conclusion for this species which underlies the current thesis of the paper.
2.	Another important point is the way in which the total energy expenditure of time on water during free-ranging is calculated. The animals dosed with DLW did not have their time-budget or location recorded. Thus the estimate of total energy expenditure on the water comes from an assumption both about the time spent and the temperature, both of which will influence this sum. Thus to make this the main finding when based on two estimates seems odd. Better to focus on the difference between air and water measured in the laboratory, which you can be much more sure of and compare to other seabirds. I recommend separately reporting the DEE from DLW, a value which is always welcomed for a new species and new study sytem.
3.	The findings of the study seem valid in terms of the values that are produced. As noted in the manuscript, the values are consistent with similar species which gives confidence in the results.
4.	Only 4 of 11 auklets injected with DLW were recovered. This is a low return rate and one of the four birds did not return for three days, which seems like quite a long time. This species is known to be prone to investigator disburbance (Sun et al. 2020). Please comment on the return rate and what implications this might have for your study findings. In particular, how representative might the behaviour of your birds be?
5.	Line 275. Did you consider comparing your DEE measurement to predictions from the Seabird FMR Calculator (https://ruthedunn.shinyapps.io/seabird_fmr_calculator/) and accompanying paper (Dunn, White & Green 2018) which is the most up to date meta-analysis of seabird DEE?

Dunn, R.E., White, C.R. & Green, J.A. (2018) A model to estimate seabird field metabolic rates. Biology Letters, 14, 20180190.
Elliott, K.H., Ricklefs, R.E., Gaston, A.J., Hatch, S.A., Speakman, J.R. & Davoren, G.K. (2013) High flight costs, but low dive costs, in auks support the biomechanical hypothesis for flightlessness in penguins. Proceedings of the National Academy of Sciences, 110, 9380-9384.
Sun, A., Whelan, S., Hatch, S.A. & Elliott, K.H. (2020) Tags below three percent of body mass increase nest abandonment by rhinoceros auklets, but handling impacts decline as breeding progresses. Marine Ecology Progress Series, 643, 173-181.
Additional comments: 1.	At times the paper talks about the conductivity of ‘salt water’ but the on-water respirometry work was conducted in fresh water so probably safest to just talk about conductivity and time spent on ‘water’.
2.	Line 81-83. This is an odd statement and seems out of place here in the final setup of the paper.
3.	Line 91. Repetition of ‘in air and on water’.
4.	Lines 278297. This extensive discussion of LCT in polar seabirds is interesting, but not really relevant to the goals of your paper, especially since you did not specifically determine LCT for your study animals. This could be helped by restricting the Introduction and Discussion as recommended above.
5.	Lines 288-301. Similarly, this discussion is interesting, but barely relevant to your work as you did not look at metabolic costs during flight and did not fully explore LCT or UCT of your birds in your experiments. However you could have attempted to explore flight costs, using the a modified approach at the population level developed in previous studies, which would be a very valuable addition to your work (Elliott et al. 2013). As noted above, because of the low cost of time on water compared to high time investment, flight costs must be very high. Since you can estimate the costs on land and in water and estimate time in all activities from the previous study, you could also estimate the energy cost of flight and compare to published models and estimates and measurements from other alcids.
6.	Lines 316-324. You seem to be suggesting that your auklets and common guillemots from Witless Bay spend more time at sea because they are in water that is inside their TNZ. This appears to be what you think is your major finding, but as noted, it is so well hidden inside the paper that it is hard to extract this. I am also not convinced of this conclusion since the temperature experienced by the Witless Bay guillemots is still less than the LCT reported for this species (Croll & McLaren 1993). I am also not at all convinced by this argument since if you divide the %time on water by %energy spent on water, the ratios are nearly identical across the four studies and the top two are certainly not better than the bottom two from Table 1. This implies that as time on water increases the proportion of energy expended on water increases at broadly the same rate, across all four studies. Indeed compared to flight, as with the other studies of auks, it could be argued that the auklets save energy by staying on the water. This is indeed consistent with a nocturnal strategy found in this species.
7.	Lines 341-353. I found this element of the Discussion not that easy to follow and not well connected to the data that you collected.
8.	Lines 356-361. Overall while this statement is correct, as I have attempted to explain elsewhere, this is not the most interesting or easy to reach conclusion that could be explored with these data. As noted, the amounts are not remarkable compared to other alcids and so I do not think that structuring your paper around this idea has served you or your data very well.

Croll, D.A. & McLaren, E. (1993) Diving metabolism and thermoregulation in common and thick-billed murres. Journal of Comparative Physiology, 163, 160-166.

Elliott, K.H., Ricklefs, R.E., Gaston, A.J., Hatch, S.A., Speakman, J.R. & Davoren, G.K. (2013) High flight costs, but low dive costs, in auks support the biomechanical hypothesis for flightlessness in penguins. Proceedings of the National Academy of Sciences, 110, 9380-9384.


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: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all of my concerns in their rebuttal.
Experimental design: The authors have addressed all of my concerns in their rebuttal.
Validity of the findings: The authors have addressed all of my concerns in their rebuttal.
Additional comments: The authors have addressed all of my concerns in their rebuttal.


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: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I am concerned that the authors failed to pick up my major criticism of the previous version due to this review format. Therefore I have attached a more conventional review this time.
Experimental design: I am concerned that the authors failed to pick up my major criticism of the previous version due to this review format. Therefore I have attached a more conventional review this time.
Validity of the findings: I am concerned that the authors failed to pick up my major criticism of the previous version due to this review format. Therefore I have attached a more conventional review this time.
Additional comments: I am concerned that the authors failed to pick up my major criticism of the previous version due to this review format. Therefore I have attached a more conventional review this time.


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: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The authors have carefully considered the comments provided by both myself and the other reviewer and altered their paper. Perhaps the main result that the proportion of the daily energy budget associated with resting is proportional to time spent resting isn't particularly surprising, but I believe these are nonetheless challenging measurements to make and are worth reporting. I have no issue with their actual measurements (and I don't believe the other reviewer had substantial concerns either; it was mostly about the interpretation of those measurements). The calculation of DEE from DLW can vary by +/- 50% depending on the equation used (see Shaffer below), and RMR in the wild for a burrow-nesting auk is likely to be substantially lower due to the absence of stress combined with metabolic suppression in the burrow. Thus, there is considerable uncertainty in what these measurements actually mean and how to compare them. Nonetheless, they're the best we have, and well worth reporting.

Shaffer, S.A., 2011. A review of seabird energetics using the doubly labeled water method. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 158(3), pp.315-322.

The one point that is still missing is to fully acknowledge the potential for bias due to capture. I suggest a line in the methods such as "Nonetheless, we acknowledge that capture may cause a stress response whereby birds spend considerable time preening on the water leading to a low DEE (Schultner et al., 2010), especially for auklets, which are known to be particularly sensitive to disturbance (Sun et al. 2020) as shown by our low recapture rate." Or something similar.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No comment.


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

Input:
Title: FIELD AND LABORATORY METABOLISM AND THERMOREGULATION IN RHINOCEROS AUKLETS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The manuscript is acceptably structured and written. In some sections, particularly new material, the standard of English is lower. I have tried to indicate these sections on the annotated pdf to help make final improvements.
Experimental design: All previous concerns have been attended to. Methods are adequately and repeatably described. The research question is now simply defined and for the most part answered. However the final message of the study could be clearer, as indicated on the annotated pdf.
Validity of the findings: The authors have restructured the paper in line with my suggestions and the findings are now valid in terms of how the measurements were made and what they might mean. I have made some suggestions on the annotated pdf for final improvements in this respect.
Additional comments: Thank you to the authors for rewriting the paper. I have made some final suggestions to help expression and aid in the readability of your work.


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: AN INTEGRATIVE MONOGRAPH OF CAREX SECTION SCHOENOXIPHIUM (CYPERACEAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is very strong and detailed.
Experimental design: This is appropriate for the nature of the study. The research objectives are effectively stated.

It is a sound piece of work that involves a rigorous investigation of this group of plants. The Methods are described in appropriate details.
Validity of the findings: The findings are valid and robust given the many types of data considered.

The data has been provided, but additional captions would improve their usefulness for others in the future.
Additional comments: General comments
This integrative monograph is an exemplar work in the discipline that uses several types of data to support the species circumscriptions. This work should be referred to for many years to come, as the species are well-supported and described. It is also evident that this is the culmination of several years of detailed work.

My suggestions are mostly minor. I noticed a few grammatical/spelling errors throughout. I also think that a few figures could be slightly improved and many captions expanded.

Specific comments

Abstract
- Lines 37-38: I recommend to not start a new sentence with a number when using formal english.

Introduction

This is an excellent, detailed Introduction that is well-written and sufficiently justifies the study.

Results
- Line 336: I recommend to write out number when starting a sentence in formal english.

Discussion
- Line 403: I recommend to write out number when starting a sentence in formal english.

Key

It might make it a little easier for the reader if you cite/refer to your figures in the Key couplets. For example, for C. ackocksii, you could refer to Fig. 7 directly in the key.

I say this because I found the drawings very useful for understanding exactly what you mean in your leads.

Descriptions

- Line 920: I am not quite sure what you mean by CW here.

- Line 1217: Do you mean "to the apex"?

- Line 1271: "Similar ones" might be more appropriate than "close ones" here.

- Line 1321: Should this be very "abruptly"?

- Line 1346: Charles Darwin?

- Line 1513: I think that "as it is shown in the phylogeny of Figure S2" would be better here.

- Line 1765: Do you mean 'longer than the inflorescence' here?

- Line 1889: Change from 2,2 to 2.2

- Line 2149: Do you mean C. spartea or C. esenbeckiana here?

- Lines 2164-2166: I think the wording of this paragraph could be reworked. Unlike most of the other parts of the manuscript, I had to read it a few times to understand what you are trying to state here.

Table 1:
- Remove italics for authrity of S. lanceum

Table 2:
- I think that you should spell out in a little more detail the information you have about the populations of origen. For example, I assume that the bold code is a collection number, but I am not sure.

Figures

Figure 3 caption
- I think that it should read "countries are colored to reflect their Schoenoxiphium species richness"

Figure 3
- I find the legends and axes very hard to read, since they are relatively small. Would it be possible to enlarge them?
- the use of colors in Figure 3B is very difficult to follow since there are so many colors here (i.e. it is too busy). A suggestion is to divide this into separate maps for each clade.

Figure 5 caption
- I am not quite sure what the first number in brackets before the count number represents. I am assuming this is the collection number (e.g. MM4544), but I think that this needs to be stated explicitly in the caption.

Figure 15
- the clear circles are very difficult to see in this figure. I would suggest replacing them with another symbol.

Figures S1 and S2:

- I don't see a caption here. Would it be possible to include one with these .pdf's so that they are stand-alone documents?

Table S1
- this Table is also missing a caption. I quickly browsed through it and noticed that there are many spelling mistakes. I suggest spending some time to clean it up a bit before submitting it for publication so that it looks more professional.
- are these all of the specimens that were measured, or were only a subset of these specimens measured?

Table S2
- I am assuming that these were the new Sanger sequences produced for this study, but I am not sure if this since there is no Table caption.

Tables S3 and S4
- a caption would be useful here.


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: AN INTEGRATIVE MONOGRAPH OF CAREX SECTION SCHOENOXIPHIUM (CYPERACEAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript presents complete, nice piece of work, technically very sound and written in strong professional language.
Background of the taxonomically most complicated section has well discussed in the introduction part.
Authors described one new species Carex gordon-grayii on the basis of three older collections, which is well described and nicely illustrated. Beside these, all the illustrations are well drawn; photographs are also very good and publishable resolution. Key provided here in the monograph is well constructed.
The new species (Carex gordon-grayii) described by the authors diagnose well in comparative manner with nearest species.
Authors have first time reported chromosome numbers with cytogenetic counts on 44 populations from 15 species of the section Schoenoxiphium and one hybrid, which are really worth mentioning to understand the evolution of genome size of the taxa. Reporting of Carex gordon-grayii chromosome number would have been better, though has not been performed probably because unavailability of live specimens.
References are very accurate and updated. To me, the manuscript warrants publication in PeerJ after very few minor corrections.
Experimental design: The methodologies are well described in the materials and methods section.  Authors also critically performed nomenclatural revision and typified 19 names of the section as per ICN rules and recommendations. Collections of the samples were made with proper protocol required for Rare, Endemic and Endangered species.  Finally, authors have performed everything required for a monographic study.
Validity of the findings: no comment
Additional comments: Lines: 37-38: “985 herbarium specimens were examined and the majority of the species were studied in the field”. Specimen serial number according to Table S1 is 885. Other 100 specimens probably included within these. If possible, please insert a column in Table S1, after the column "Voucher" to indicate total number of sheets/specimens.
Line137 ... “i) degeneration of three nuclei during pollen formation (pseudomonads)...” though http://www.mobot.org/MOBOT/research/APweb/ mentioned “pollen as pseudomonads, (grains 2-celled)”. Is the second condition found in other taxa of Cyperaceae?
Both for cytological reporting and images, authors mentioned superscripts III+II+I etc. Can you please give a note of few words for readers about these superscripts?
Can you please include few more photographic images (if you have, as you have sampled in-situ or of the original specimens; though not mandatory) as one more Figure for newly described species corresponding to Figure 19 (i.e. C. gordon-grayii) as done for other species under “Morphology of different species of Carex section Schoenoxiphium”?
In all the illustrations of Carex spp., authors represented either (A) General aspect or (A) General aspect and inflorescence. Can these be specified further?

In Figure 4A Phylogenetic relationships within Carex section Schoenoxiphium, in the tree C. gordon-grayii is spelled as C. gordon-grayi. Please check and correct it.

Lines 878-879: “...(lectotype: K-000693810 digital image!, here designated; iso: BOL-70348 digital image!). Syntype: Natal, Buchanan 328 (K-000693809 879 digital image!)”. Lectotype of the name correctly designated here.  Then BOL-70348 mentioned as iso to mean isotype or isolectotype? If the specimen is isotype then it is all right but if it is isolectotype, it is sometimes written as “isolecto”. Please check in other species, where names are lectotypified. Secondly, Buchanan 328 (K-000693809) considered as syntype of the name, but in typification note (line 948) considered as paratype.

Lines 1368-1370:  “Paratypes: South Africa, Eastern Cape, Hagga Hagga district, marsh, 7-II-1995, B. Sonnenberg 446 (NU-s.n.!). Ibidem, Kentani, 32º30’7.628”S 28º20’9.499”E, edge of stream in forest, 4-IV-1904, A.M. Pegler 1097 (BOL-4655 digital image!)”. Please include notes on types for the readers if possible.
Lines 2395-2396:  “Vegetti AC. 2003. Synflorescence typology in Cyperaceae. Annales Botanici Fennici 40(1):35- 46 ISSN 0003-3847”. ISSN 0003-3847 is not required.
In caption of Figures 18 and 19: (A) General aspect vs.  (A) General aspect and Inflorescence. But in both the illustrations inflorescence are drawn in detached condition from the mother plant. It is also found in Figures 21, 22 and 32.