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: BLOOD PARASITES IN PASSERIFORMES IN CENTRAL GERMANY: PREVALENCE AND LINEAGE DIVERSITY OF HAEMOSPORIDA (HAEMOPROTEUS, PLASMODIUM AND LEUCOCYTOZOON) IN SIX COMMON SONGBIRDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This descriptive study provides additional information on the haemosporidian parasites prevalence and genetic diversity among six common passerine birds in central Germany. The birds were also investigated for Trypanosoma infections, but such were not found. The authors apply PCR-based and light microscopy methods to diagnose the infections. The manuscript have relatively good English but there are some parts of the text that have to be rephrased or clarified (L 4-6, L 111-112, L 140, L 180-182, L 201-203). There are also some incorrectly used terminology and scientific names. For instance, throughout the majority of the text, the authors used “lineage/s” for addressing the mitochondrial cytochrome b sequences, but in L 209-215 the “lineage” were replaced with “haplotype”. Please unify the terminology along the text or explain what is the difference between both terms in the context of your results. The correct spelling is order “Haemosporida” but not “Haemosporidia”! Please correct it along the text or use noncapital “haemosporidian/s parasites” when appropriate.
The Introduction section is well structured and provides good background related to the results of the study. However, I think that the purpose of collecting material from birds in central Germany is not clarified. What new the authors would expected to find in the avian blood parasites from this particular region? How your work fits to the broader field of knowledge? The statement on L 46-48 is a bit controversial as the prevalence of haemosporidian parasites in nestlings very much depends on the fledging period. When the fledging period is longer the nestlings are exposed longer to the potential vectors and the parasite have enough time to develop and be registered in the peripheral blood. Furthermore it depend if the birds species are hollow breeders and so on. In my opinion, the paragraph have to be extended with more information.
Experimental design: It is questionable why the authors did not use well established nested PCR protocol developed by Waldenstrom et al. 2004 and Hellgren et al. 2004. Nested PCR should increase the sensitivity of the method and in this case perhaps reflect in underestimated prevalence. Furthermore, one of the primer pairs (PaluF-PaluR) amplify shorter fragment of the mitochondrial cyt b gene, which result in an ambiguous identification of the haemosporidian cyt b lineages after BLAST in MalAvi database (Supplementary Table 1). Would you explain why this combination of primers was used?
It is not clear the number of samples screened with different primers. Obviously not all the samples were screened equally (L 86-88) for the potential parasite infection. In a descriptive study as this one, I think this could be a big flaw.
Validity of the findings: The results for the genera Haemoproteus and Plasmodium are expected and comparable with other studies in Europe. The prevalence for genus Leucocytozoon was the highest and it is surprising that none of the parasites were registered on the blood smears. Perhaps the usual light intensity of Leucocytozoon spp. in the wild birds prevent registration of these parasites by microscopic examination. An extensive microscopy would be required in order to confirm most of the infections. There are also indications that PCR-based methods could register sporozoites in the blood stream and abortive development of haemosporidian parasites (see Valkiunas et al. 2009 – doi: 10.1645/GE-2105.1, 2013 – doi: 10.1007/s00436-013-3375-6). I do not think that this is big flaw in the study, but I believe it worth consideration and discussion.
The conclusions (L 342-345) of the study I found quite irrelevant with the main findings. I would recommend that authors comment on how their data fit to the broader filed of knowledge then simply mention that “the impact of the pathogens on the avian fauna is difficult to estimate and future studies are necessary”.
Additional comments: I think that your manuscript might become a nice descriptive study if you use appropriate protocols and obtain good quality sequences. As it is now the identification of haemosporidian parasites is too ambiguous for simply describe the prevalence and the diversity in certain host community in Germany. Otherwise, some sort of hypothesis on the avian host specificity or ecological factors would be required. Please find attached a pdf file with some of the same and additional comments and suggestions on your manuscript.


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

Input:
Title: BLOOD PARASITES IN PASSERIFORMES IN CENTRAL GERMANY: PREVALENCE AND LINEAGE DIVERSITY OF HAEMOSPORIDA (HAEMOPROTEUS, PLASMODIUM AND LEUCOCYTOZOON) IN SIX COMMON SONGBIRDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written in a correct English, and include sufficient background in the introduction using correct literature references.
However the manuscript is ambiguous to understood, it is difficult to follow what samples have been used for in each analyses because some samples were not used due to their length. This fact does that the work seems not to be designed with the objective indicate in the manuscript.
Although the Figures 2, 3, 4 and 5 are appropritately described an labeled, the Figure 1 is not clear. I think that is better if the authors design this one with color.
I think that the table 2 is not necessary.
Experimental design: Different factors indicate a not excellent experimental design of this manuscript.
I do not understand why the author did not check Plasmodium and Haemoproteus in Blue tit nestlings, or why great tit nestlings were not checked for Trypanosoma, or why blood smears of great tits were excluded.
The recollection of great tits samples in 2017 would be excellent to obtain more robust results.

The author could check if the results are similar using the sequences obtained using Primers HaemF and Haem R2 for Plasmodium/Haemoproteus and Primers HaemF2 and HaemR2L for Leucocytozoon and removing the samples of 2015.
Validity of the findings: I think that the authors could do a more novelty manuscript if the authors discuss deeper why is important to study blood parasites in central Germany in comparison with other works about parasites in Germany. They could discuss different factors such as the biology of the vectors in central Germany or land use.

The conclusions are not strong and are not connected with the objective of the work. I suggest re-write the conclusions.
Additional comments: Minor comments.

ABSTRACT:
Results: Blood samples from a total of 281 adult and nestlings...please indicate how many adults and how many nestlings.
Conclusion: The sentence "Currently there are only a few dates... of avian Haemosporidia" is long. I suggest reducing.

INTRODUCTION:
Lines 38-44: I suggest that the authors indicating names of parasites and the birds in the different examples.

MATERIAL AND METHODS:
Lines 78-82: Are correct the primers? Haem F2 and HaemR2L are the primers used for Leucocytozoon.
Lines 86-88: I suggest indicating why Blue tits nestling were only checked for Leucocytozoon and Trypanosoma and why great tits were not checked for T. avium.

RESULTS
Line 163: Is very interesting not detect Leucocytozoon in blood smears with a prevalence of 94.8% by PCR. Could the authors discuss deeper why this result in the discussion section?
Line 165: Why were excluded great tits? I suggest explaining it.
Line 173: How do you calculate the uncorrected p-distance between lineages?
Line 177: What lineages?
Lines 180-184: I suggest remove these sequences from the manuscript and indicate that 86 sequences could not be assigned due a bad quality and were removed from the analyses.

DISCUSSION
Line 217: What extra information gives the blood smears in this study? I suggest removing it because the information of the blood smears is null.
Line: 275: Culicidae.

CONCLUSION
Line 338-339: Because Plasmodium is avian malaria and Haemoproetus avian malaria-like, I suggest that in the line 339 Plasmodium must be indicated before Haemoproteus.


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: BLOOD PARASITES IN PASSERIFORMES IN CENTRAL GERMANY: PREVALENCE AND LINEAGE DIVERSITY OF HAEMOSPORIDA (HAEMOPROTEUS, PLASMODIUM AND LEUCOCYTOZOON) IN SIX COMMON SONGBIRDS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This paper reports on a survey of four genera of hematozoa from central Germany in six species of passerine birds. The rates of infection were generally high (except T. avium). Both microscopy and PCR were used in the survey.

The quality of the paper was generally adequate, though technical aspects of the writing could be improved throughout. Some of the reporting of results was not well directed toward questions being addressed, and questions that could be addressed using this dataset. For example, Figure 1 and Figure 2 were not informative, and the haplotype networks presented in subsequent figures contained much more effective visualizations of the same information. I strongly recommend deleting Figs. 1 and 2.

The most interesting part of the paper is the early Discussion that compares to previous results concerning the same host species elsewhere in Germany or Europe. This Discussion addresses the question of whether central Germany surveys such as this one are consistent with previous knowledge based on surveys of these host species from other locations. This section should heavily focus on the blue tit and great tit, which are the only thoroughly sampled host species in this paper. It should provide sample sizes for the previous studies to put the differences in prevalence in context for the reader. I suggest considering a table to specifically report these comparative results for tits, as this gets to the specific importance of this study.

The results for microscopy are not sufficiently reported or described. These data aren't documented anywhere that I could find. Are there microscopy photos archived somewhere? Why are these results not reported in the Summary_Individuals.xlsx document? Why are there no metadata available for that document? I could not find any information about the MalAvi or Genbank contributions of this paper. Nor could I find where the samples/extracts/smears may be archived.

The section about nestlings should be shortened way down, as it was tangential and failed to add to previous knowledge of the topic. The negative result should have been expected.

The paragraph beginning on Line 299 is vague and speculative and should be cut.

The last paragraph of Discussion, beginning on line 326 is not expressing any new ideas, and is built on a 'straw-man' argument that host-specificity of specific lineages is firmly predicted by the general tendencies of their genus. This paragraph should be deleted. Only the first sentence of it is valid, but that is also redundant with the Conclusions that follow. In the Conclusions, the phrase "currently only a few data..." is not true; though a more nuanced argument could be made that the diversity and geographic variation in haemosporidian communities warrant more study, even in Europe.

Line 275: Culicidae spelled incorrectly.
Line 290: "genera" should be "genus"
Experimental design: Adequate.
Validity of the findings: Valid.
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: BLOOD PARASITES IN PASSERIFORMES IN CENTRAL GERMANY: PREVALENCE AND LINEAGE DIVERSITY OF HAEMOSPORIDA (HAEMOPROTEUS, PLASMODIUM AND LEUCOCYTOZOON) IN SIX COMMON SONGBIRDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment.
Additional comments: Please find attached my few comments in the pdf file.


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

Input:
Title: BLOOD PARASITES IN PASSERIFORMES IN CENTRAL GERMANY: PREVALENCE AND LINEAGE DIVERSITY OF HAEMOSPORIDA (HAEMOPROTEUS, PLASMODIUM AND LEUCOCYTOZOON) IN SIX COMMON SONGBIRDS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for your revisions and reformatting of your paper entitled "Blood parasites in Passeriformes in central Germany: Prevalence and lineage diversity of Haemosporida (Haemoproteus, Plasmodium and Leucocytozoon) in six common songbirds".  You have adequately addressed my comments regarding your paper. I particularly appreciate the changes you made in Material and Methods.


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: GRID-INDEPENDENT MOLECULAR DESCRIPTOR ANALYSIS AND MOLECULAR DOCKING STUDIES TO MIMIC THE BINDING HYPOTHESIS OF Γ-AMINOBUTYRIC ACID TRANSPORTER 1 (GAT1) INHIBITORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written, easy to follow and provides all raw data. Also, the statistics are shown and data (such as score values) are also presented as graphs to get a clearer overview on the data.Comprehensive and up to date literature is cited.

Authors address an important question from the viewpoint of developing GABA transport inhibitors. So far, only Tiagabine is the only FDA-approved drug against epilepsy, with a number of side effects, therefore the search for an effective inhibitor with no or minor side effects is eagerly awaited.
Experimental design: There have been many attempts to develop useful lead molecules or drug candidates as GABA inhibitors. GRID independent modeling is a relatively new approach in the field of SAR therefore, it was reasonable to apply the method for the study of GABA transport inhibitors.
The method was appropriate addressed, and it has been supplemented by molecular modeling and docking studies, as well.

Molecular modelling and docking was carried out appropriately. Modeling was based on the structure of dDAT, which was most probably the closest homologue of hGAT-1 at the time of the writing. (It is to note, that the structure of human DAT and SERT has been already determined, which will probably be even more similar templates than dDAT for further studies). Otherwise, altogether appropriate care was taken to the modeling process.

Concerning the GOLD docking procedure, a relatively large cavity was left for the antagonists to bind. This has the advantage of leaving enough space for the antagonists, however, it might have led to several binding modes in a number of cases.

The number of GA runs and other settings fit to the generally applied standards.
Methods were written carefully enough for reproduction.
.
Validity of the findings: The findings are nicely presented. Table 1 is especially carefully detailed, giving a nice example of how data is to be presented.

Overall, the findings are summarized in Fig. 9. Namely, two hydrogen bond acceptor contours (N1) at a distance of 8.00-8.40 Å from each other. and a hydrogen bond donor (O) at a distance of 5.60-6.00 Å, and finally a hydrophobic group at a distance of 10.40-10.80 Å form the hotspots for hGAT-1 inhibitors, therefore a COOH group at meta position of the piperidine ring may provide an important interaction point within the binding cavity of hGAT1.

This finding is well stated and linked to the original research question. It can be directly  interpreted in terms of developing hGAT-1 - inhibitors in the future.
Additional comments: Overall, it is a well written paper, there are only a few questions remain to be answered.

In row 221 it is stated that the binding site was kept flexible.
However, in row 228 it is said that the side chains of hGAT-1 were kept rigid.
If you mean, they were kept rigid outside the binding site, please state it explicitly.

In supplementary Table 4 the top scoring hits get an unusually high score,(even around and over 70) while the score of the selected poses are much less and they cover a narrower range. Does it mean, that the top pose appeared only once, and there were several other poses? How many times did the "selected poses" appear in the run?

The quality of the figures 5, 6 and 7 should be improved, in terms of sizing. It may be due to the PDF processing, but the figures seem to be "pressed" from the sides.  (It also appears in Table 2).


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: GRID-INDEPENDENT MOLECULAR DESCRIPTOR ANALYSIS AND MOLECULAR DOCKING STUDIES TO MIMIC THE BINDING HYPOTHESIS OF Γ-AMINOBUTYRIC ACID TRANSPORTER 1 (GAT1) INHIBITORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper is well written and very well organized.

About literature references I suggest the authors include some references of 2016, 2017 and 2018. There is only one paper of 2017 (Lutz et al. 2017) and all the other references are 2015 or older. For instance in Introduction, lines 73-88 that describes some related work should be updated as well as the description about new inhibitors for GAT1 (lines 67-72).

Some figures have parts distorted. Figure 6 B,  the close view of class A, B and C are distorted and should be improved. Figure 7 should be in a better quality.
Experimental design: The research question of this paper is well defined and very relevant. The search for new inhibitors for GAT1 is an important topic of research, since according to the authors, there is only one second-generation FDA approved anticonvunsant agent for human sapies GAT1 (Tiagabine). Besides analogues of this drug Tiagabine that have been developed are often associated with off target toxicity and poor ADME-Tox properties (lead to side effects).

Some points of the methodology need to be more detailed to have sufficient information to be reproducible:
- the workflow of the hGAT1 antagonists data preprocessing: it is necessary to discuss about the chosen criterion (molecular weight and IC50) for filter the 375 non duplicated initial antagonists as well as the next steps “assay type” and “cell line” filters. On the text (lines 104-110) these steps are describe different from the figure ( in the text do not appear the number of 152 inhibitors, for instance).
- the validation set should be included on Figure 1.
- Improve the legends of the Figures. For instance the legend of Figure 2. How many molecules are on this figure?

The authors should discuss in Methodology the reasons they chose the applied tools (Pentacle v 1.06, AMANDA algorithm, CLACC algorithm. PSL analysis, GOLD, MOE, Modeller, Procheck and so on). For instance:  for molecular docking, popular docking tools are Vina, GOLD, FleXX,  but the authors opt to use GOLD for what reason?. The same for Modeller and so on.
Validity of the findings: The first point about the Results section is that part of its content should be moved to the Methods Section. I suggest the authors review the first paragraphs of results section because it is confuse. I believe that part of the lines 246-249, 251-255 should be on Section Methods.

Since part of the Results are related with the developed homology model I suggest the authors includes a figure of this model indicating the binding site through highlighting the main amino acids  (for instance the protein in ribbons and these amino acids in lines).

In general the results are well described and discussed.
Additional comments: In this paper the authors present a study where various predictive GRIND models were developed to discover 3D features of hGATA1 inhibitors. Besides, the authors propose a binding hypothesis for the selected GAT1 inhibitors by molecular docking in the binding site of a human GAT1 homology model.
The paper is well written and very well organized however the Section Methods should be detailed to improve the general quality of the paper.


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

Input:
Title: GRID-INDEPENDENT MOLECULAR DESCRIPTOR ANALYSIS AND MOLECULAR DOCKING STUDIES TO MIMIC THE BINDING HYPOTHESIS OF Γ-AMINOBUTYRIC ACID TRANSPORTER 1 (GAT1) INHIBITORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language and all structural formats are acceptable.
Experimental design: The authors have done a good and credible study and they clearly explained the applied methods.
Validity of the findings: Data presentation and the results and discussion are concise and acceptable.
Additional comments: The manuscript organized and written very well. I recommend accepting in its current format.


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: GRID-INDEPENDENT MOLECULAR DESCRIPTOR ANALYSIS AND MOLECULAR DOCKING STUDIES TO MIMIC THE BINDING HYPOTHESIS OF Γ-AMINOBUTYRIC ACID TRANSPORTER 1 (GAT1) INHIBITORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for correcting the points I raised at the first review period. The paper is now ready for publication.


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

Input:
Title: GRID-INDEPENDENT MOLECULAR DESCRIPTOR ANALYSIS AND MOLECULAR DOCKING STUDIES TO MIMIC THE BINDING HYPOTHESIS OF Γ-AMINOBUTYRIC ACID TRANSPORTER 1 (GAT1) INHIBITORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The literature references were improved on the reviewed version.
The authors also modified the Figures 6 and 7 as suggested.
Experimental design: The authors addressed some points indicated to improve on the Methods section.
Validity of the findings: The results are now well described.
Additional comments: The authors addresses almost all the points  indicated on the first round of Review.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NEW CYTOKININ METABOLIC GENES IN BREAD WHEAT (TRITICUM AESTIVUM L.)
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: Detailed information for experiment design are needed.
Validity of the findings: no comment
Additional comments: This manuscript reports the identification and characterization of IPT, ZOG, GLU and CKX gene families that are involved in the synthesis and metabolism of cytokinins. Using bioinformatic tools, the authors have predicted 4 new TaZOG, 4 new TaIPT and 25 new TaGLU genes in wheat; and further evaluated their sensitivity towards phyto-hormones. Considering the importance of CKs in regulating vegetative and reproductive development of the plants, the results may provide useful information for the functional characterization of the mentioned gene families in wheat. But there are some points to be considered before publication.

1.	Detailed information for experiment design are needed in the M&M section. For example, did the experiment have any replications? Were there any biological replicates of each treatment? How many seedlings were used for each treatment? Were the tissues for each treatment collected at the same time of the day? What is the composition of the Hoagland solution used in the experiment? At least a reference is needed.
2.	The English language of the manuscript needs improving. The abstract and discussion sections are well written. But the result section needs to be revised carefully. Too many results were listed in the manuscript, which needs simplification. I encourage the authors to use some summative language instead of simply repeat the information in Tables and Figures. The sentence structure should also be considered for revision. Some detailed information can be found in the attached file.
3.	Not much references were referred across the M&M section. Representative references are encouraged to be added for the methods used in the experiments.
4.	For references, the full name of journals should be used instead of abbreviations.
5.	The manuscript has some careless mistakes. For example,
a)	The website supplied for wheat database indicated to an error result. I guess the authors intended to supply this: https://urgi.versailles.inra.fr/blast_iwgsc/blast.php.
b)	In the section of “In-silico promoter analysis”, “2000bp” should be “2000 bp”
c)	Fig.1, “9 TaIPT genes” instead of “10 TaIPT genes”
Other minor points to be considered can be found in the attached file.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NEW CYTOKININ METABOLIC GENES IN BREAD WHEAT (TRITICUM AESTIVUM L.)
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language and typo should be improved for clearly understand the manuscript.
Experimental design: The experimental design requires some proper controls or references.
Validity of the findings: Most of the results do no have proper statistic analysis.
Additional comments: The manuscript describes the identification and expression of cytokinin metabolic genes in wheat. The levels of cytokinins have been shown to regulate grain yield, which can be regulated by IPT, ZOG, GLU and CKX. In this study, the authors identified new IPTs, ZOGs and GLUs genes from bread wheat with bioinformatics tools. Most of these genes are up-regulated the transcript levels in response to different phytohormone treatments. The cis-regulatory elements of these gene families may also link to ABA, auxin, SA, Sulphur, drought and light responses. However, several points are need to be addressed before further process.

1. The readability of the manuscript is needed to significant improvement. For example, Page 9. “Except TaIPT2, TaIPT3 and TaIPT5 cis-regulatory elements related to low temperature were present in all promoter sequences (Table 7).” There is no TaIPT5 in Table 7 and not clear about “all promoter sequences”. In addition, the cold response element also does not present in TaIPT4.
2. In Table 1, based on the results from Song et al., 2012 and Lu et al., 2015, the TaCKX6 gene did not present in this study. Is TaCKX6 same and TaCKX5 or else? Please clarify and consist with the name in all gene families (Table 1-4).
3. In Table 5-8, the number of cis-regulatory elements in the promoter region of cytokinin metabolic gene families may need to compare with the up-stream region of other none related genes. The comparison can be used as a reference for the frequency of each cis-regulatory element.
4. The description of promoters may be more suitable for 2kb upstream region of the translation sites.
5. In Figure 5-7, the relative expression levels of each gene are very different. It would be necessary to describe the amount of total RNA input or the reference gene Ta4045- Otherwise it would be difficult to compare between different genes. Please also add the statistical analysis to the results.
6. Please address the issues why only analyzed portion of the cytokinin metabolic genes (Figure 5, 6, 7). Some the gene expression in response to the phytohormones had been studied prevouly. It would be more meaningful to address the expression of those newly discovered genes or the whole gene family.
7. Thank you for providing the raw data of qRT-PCR, however the calculation of expression ratio is hard to understand. Why the equations for relative expression level needs to *1000 and the standard deviation needs to *LN(2) ? Some of the samples only present two repeats may not sufficient for statistic assay.
8. In abstract, the following statement may need to reconsideration- “Maximum fold change was observed for TaGLU gene family conferring that reactivation of dormant CK isoform is the quickest way to counter the external stress.” The relative expression level of TaGLU7 in the leaf and TaGLU14 in the root after BA treatment are the highest relative to other genes. However, the expression levels only 4-fold and 3-fold different respectively from the control-treatment.
9. Please add the detail statistical analysis including sample number and significant test.
10. Please provide more detail description for each figure/ table legend.
11. The quality of figures (Figure 5-7) is needed to improve, including the data arrangement.
12. It is highly recommended that the number 9 and numbers under 10 should be spelled out.
13. All the gene name should be in italics.
14. The English language and typo should be improved for clearly understand the manuscript. Page 6, Line 5- the RNA quality was check using “1% agarose gel electroporation”- should be “gel electrophoresis”. Page 9, Line 12- “Conversely, cis-elements related to gibberellic acid were confined to TaZOG4; and low temperature responsive elements were bound to TacisZOG1 only.”- not sure the meaning of “confine” and “bind” in the description.


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NEW CYTOKININ METABOLIC GENES IN BREAD WHEAT (TRITICUM AESTIVUM L.)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript has been well improved. I have gone through the revised manuscript and find that most of the suggestions, comments and corrections by the reviewers have been incorporated. I suggest the manuscript be accepted for publication after the consideration of the suggested issues.

Line 140, “Immediately after treatment” contradicts with the “3 h treatment” mentioned above.

The captions of Figs 5, 6 and 7 indicated four (A B C D) parts for each figure. Whereas, only A and B parts are found in the figures. Furthermore, it may look better if the figures 5, 6 and 7 are combined together, so do Tables 2-5 and Tables 6-9.

Line 567 and Page 45, “7 TaZOG (B), 9 TaIPT (C),” should be “seven TaZOG (B), nine TaIPT (C),”.

Some references in the reference list are not referred in the manuscript, such as Miyawaki et al. 2004, Tsai et al. 2012, and Zalewski et al., 2003. The authors should check the manuscript again to make sure the correspondence.


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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSIS OF NEW CYTOKININ METABOLIC GENES IN BREAD WHEAT (TRITICUM AESTIVUM L.)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Please be consistent with the terminology throughout the manuscript.
Table 6-8, line138, 278, 279, 337: "GA3" or "GA[subscript]3".
Figure 5-7, line 345-349, The abbreviation of Triticum urartu is "Ta" or "Tu"?

The image quality of Figure 4 is too low.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors had significantly improved the manuscript from the previous version.
A few minor points are still needed to fix before the following processes.


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: TRANSCRIPTOME-WIDE CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF MATE TRANSPORTERS IN RESPONSE TO ALUMINUM TOXICITY IN MEDICAGO SATIVA L.
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The study has identified 88 non-redundant MATEs in Alfalfa (Medicago sativa L.) based on BLAST analysis and characterized their gene structures, phylogenetic relationship, and expression patterns in different tissues. Ten MsMATEs considered as Al-stress related were furthered validated their expression profiles under Al stress treatment. The manuscript is generally well-written and easy to read for the materials and methods and results sections. But there are gramma or spelling mistakes in the introduction and discussion sections, and many sentences in the discussion section are hard to read and understand. The representation of the materials and methods is clear, but some information is missing, such as the transcriptome data used for expression patterns of MsMATEs. The result section has included the text for data interpretation, while the discussion section contained some wrong description of the results and has many sentences not easy to read. For these reasons, the present MS certainly warrants thorough revision before consideration of publication in Peer J.

Major comments:
1)	In the introduction section, the phrase “Al ability” has appeared many times, but it is misleading. Does it mean antioxidant ability against aluminum toxicity? In L54, should the phrase “no floxacin” be corrected as ”nofloxacin”? In L57 to 59, five species have been listed, but only four MATE transporters gene numbers have been shown. L94 to 96, the last sentence of this paragraph is hard to read.
2)	In the Materials and methods section, there are missing data needed. In L99, the alfalfa cultivar name in incomplete. In L101, full name for MS should be provided in its first appearance in the text. In L103, is there missing information for temperature in the brackets? In L108, do you mean that e-value cutoff is 1e-5? According to the text in the result section (subtitle: Expression profiles of MsMATE genes in different tissues), the datasets used for RNA-seq analysis was not mentioned in the Materials and Methods section.
3)	In the Result section, the text for data interpretation is included in this section, which is not suitable because a separate discussion section is existed in the manuscript. For Figure 1 and Figure 2, no bootstrap values have been displayed. Under the subtitle “Functional identification of the MsMATE transporters”, the text is not informative and litter information has been provided in Figure 4. It seems to me that this part of result is no different from gene annotation and the gene network for rice is not suitable to display here. Maybe, an improved version of this figure could be listed as a supplementary figure. In L260 to 261, the sentence “Increasing of the evidence….” should be rewrote and the whole paragraph should be moved to the discussion section. In L269 to 270, the gene names “MsMATE05” and “MsMATE09” were not in italics.
4)	In the discussion section, many sentences are hard to read. In L278, the sentence “…, that can be act as phytotoxic…” should be rewrote; In L353, the mistake in the sentence “it’s been urged…”should be corrected. In L294 to 295, the sentence is hard to read. In L295, “the genome of alfalfa….” should be corrected as “the genome sequence of alfalfa….”. In L355 to 356, It is hard to conclude that five MsMATE genes is Al stress related. As we seen from RT-qPCR results, all genes were responsive to Al stress treatments. It will be great if homologous gene related with Al stress in model plants or reported researches could be discussed here and comparing with the results of this study. The whole discussion section should be carefully revised.


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

Input:
Title: TRANSCRIPTOME-WIDE CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF MATE TRANSPORTERS IN RESPONSE TO ALUMINUM TOXICITY IN MEDICAGO SATIVA L.
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language of the manuscript should be improved and also use proper scientific words.
Excellent background information has been provided by citing correct references.
Article can be improved on result part especially when talks about conserved motifs where readers fail to get clarity.
What was the criteria for selecting a subset of MATE genes for Al responsive expression analysis? A more clear explanation can be given here.
Experimental design: Methods have been described in detail.
Validity of the findings: Data is good but a more concrete conclusion and future prospects need to be done.
Additional comments: A Well executed experiment with proper documentation. But try to put your findings in a more interesting scientific language by reducing text and concentrating in giving accurate well defined point based explanation


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: TRANSCRIPTOME-WIDE CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF MATE TRANSPORTERS IN RESPONSE TO ALUMINUM TOXICITY IN MEDICAGO SATIVA L.
Review round: 1
Reviewer: 3

Output:
Basic reporting: Could you confirm citation in your paper

Line 53-57; A MATE gene (Norm) was cloned in Vibrio parahaemolyticus, which possesses an energy-dependent system that effluxes no floxacin and other antimicrobial agents outside of the cells by Na+/drug antiport (Morita et al. 1998; Yokosho et al. 2016)
This reference is correct ? I think Yokosho et al.,2016 described rice MATE transporter.

Line 180-181; To similarly predict and distinguish the feature of identified MsMATE genes in this study, an NJ tree was constructed using 38 well-known plant MATE transporters (Eisen 1998; Takanashi et al. 2015; Tiwari et al. 2014) and 88 MsMATE proteins (Fig. 2, Table S5).
And I think “Eisen 1998” this citation is not necessary.

Line 235-238 The Nramp aluminum transporter (NRAT1) encodes a transporter involved in Al uptake from the root tip cell wall into the cell and plays a key function in rice aluminum tolerance (Li et al. 2014).  I think Xia et al., 2011 also add citation.
The Al-induced gene STAR1 encodes an ATP-binding protein and a transmembrane domain protein, which are required for Al tolerance (Arenhart et al. 2014).
Arenhart et al., 2014 is not described function of STAR1. Author should be cite Huang et al., 2009
Experimental design: I have some question in experimental design and suggestion for improve expression analysis part.

Line 230-231; In this study, ten MsMATE proteins predicted to participate in Al detoxification were used to determine the functional and physical relationship through a rice association model using the STRING software (Fig. 4). Why author used rice association model. Since Alfalfa is dicot plants, I think it is better to Arabidopsis data.

Line 180-181; To similarly predict and distinguish the feature of identified MsMATE genes in this study, an NJ tree was constructed using 38 well-known plant MATE transporters (Eisen 1998; Takanashi et al. 2015; Tiwari et al. 2014) and 88 MsMATE proteins (Fig. 2, Table S5). Why author select 38 genes and what do you mean “well-known plant MATE” At least Takanashi et al., 2015 (2014?) mentioned 44 genes.

I think author should be more detail expression analysis for characterization of these MATE genes. 1) To confirm Al specific expression, author should be check to response in different pH, other metal stress. Because Arabidopsis AtMATE1 response to Al and low pH. 2) To determine highest expressed gene in root tip, author compare absolute expression level in 10 candidate genes. 3) Comparison of different root segment. In this paper Author determined expression at only root tip. I think Al tolerance citrate transporter expressed in root tip. On the other hand citrate transporter for iron translocation are expressed in basal root region.  So this is more information for understanding of 10 MATE gene function.
Validity of the findings: Figure 2; Author described G1; Al detoxification/iron translocation. It should be citrate transporter. G3; Efflux of various compounds. It is also incorrect caption. Because almost all MATE is efflux transporter. And G3 group including NtJAT1. This protein tansport nicotine, which is one of the alkaloid. Could you consider caption of G1,G2, G3, G4 in Fig 2.

Line 57-59; To date, 56, 53, 117 and 70 MATE transporters have been identified from Arabidopsis thaliana (Li et al. 2002), Oryza sativa (Tiwari et al. 2014), Glycine max (Liu et al. 2016), Medicago truncatula (Wang et al. 2017), and Zea mays (Zhu et al. 2016), respectively. This number is different in Line 113-114. And the number of Zea mays is including ?

Line 164-166; The ORFs of these genes 165 varied in length from 321 (MsMATE80) to 1788 (MsMATE10) bp. This length is different from your data. Supplemental data showed that MsMATE80 is 1485bp, MsMATE10 is 1449 (Sup data1).
Additional comments: This paper re-construct genome-wide analysis for identify MATE transporter in Medicago sativa L , which is an important crops, but sensitive to acidic soil condition. Author identified 88 MATE genes, which were divided 4 subfamilies comprising 11 subgroups. As a result of expression analysis by RT-PCR, they found 10 candidate MATE genes for Al tolerance mechanism in Alfalfa.
But I think more detail expression analysis is improve your speculation (described above).
And this author has some misunderstanding for reference paper.  Author should be more carefully cite reference paper.

Other minor point

Line 262 (Nobukazu Shitan 2014; Tovkach et al., 2013) should be Shitan et al., 2014
Line 103 (, light intensity 120 μmol) remove “,”.
Table S4 MsMATE33 (Cell H35) should be “plas”


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: TRANSCRIPTOME-WIDE CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF MATE TRANSPORTERS IN RESPONSE TO ALUMINUM TOXICITY IN MEDICAGO SATIVA L.
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have performed the suggested revsions in the materialas and methods section, results section and discussion section. Since the authors have addressed all major concern, the present MS is acceptable in my opinion.


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: TRANSCRIPTOME-WIDE CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF MATE TRANSPORTERS IN RESPONSE TO ALUMINUM TOXICITY IN MEDICAGO SATIVA L.
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This paper re-construct genome-wide analysis for identify MATE transporter in Medicago sativa L , which is an important crops, but sensitive to acidic soil condition. Author identified 88 MATE genes, which were divided 4 subfamilies comprising 11 subgroups. As a result of expression analysis by RT-PCR, they found 10 candidate MATE genes for Al tolerance mechanism in Alfalfa. I think this is beneficial data for understanding of molecular Al tolerance mechanism in Medicago sativa in future.


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: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 1
Reviewer: 1

Output:
Basic reporting: the author described carefully review of this caption in order to easily understand the readers.
Experimental design: if possible use any indexing abstract except google scholar and Scopus such as EBSCO, pubmed etc for collecting references of mangosteen related to health and functional compound
Validity of the findings: no comment
Additional comments: The authors should be adding more references about how to increase the functional compounds by agronomical/breeding/etc technicals in their part of the review.


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

Input:
Title: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Well written in English.
Literature nicely referenced.
Figs. can be improved-as commented below.
Experimental design: Article content-nicely investigated with complete details.
Validity of the findings: Well summarized results in a nice table.
Additional comments: Comments on manuscript # 30985 " Recent updates on metabolite composition and medicinal benefits of mangosteen plant" by Aizat et. al.

The author compiled the updated literature about the Mangosteen plant’s medicinal and other research based benefits in a review-nice compilation of all the data at one place.

Following things need to be addressed in the MS.
1.	Best thing about this review-The literature was nicely summarized with respect to all the extracts or compounds extracted from the Mangosteen plant up to date. But, the Figure 2-publication record over the years is making no scientific knowledge improvement. I would suggest, author should make a figure showing the different forms of extracts obtained from the Mangoosteen (plant or a fruit) and then all the well characterized compounds from each extract, if any.
2.	Related to comment 1, also author can give another figure- very well characterized compounds, e.g., alpha-, beta- , gamma-mangostin, and Xanthones- their structures, if known?

3.	Table 1: Author summarized the studies as different categories like anti-cancer, anti-diabetic… excellent job. To make it more effective for all the researchers working in the related area- separate table for in-vivo studies could be even more effective in highlighting the importance of the extracts/compounds for a particular purpose with known dose of the compound/extract used.

The paper is technically sound and well written in nice English. Comments above- will make the MS better.


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: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 1
Reviewer: 3

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 17- October-2018
Journal: Peer J

Manuscript ID:
Title: Recent updates on metabolite composition and medicinal benefits of mangosteen plant

Authors:  Wan Mohd Aizat, Faridda Hannim Ahmad-Hashim, Normah Mohd Noor

Dear Editor:

The authors have investigated the chemical compositions and medicinal benefits of mangosteen plant. The authors present a sufficient interesting review and thus I accept this review for publication after minor revision.
  .
Hesham El-Seedi, Professor,
Division of Pharmacognosy
Department of Medicinal Chemistry
Uppsala University, Biomedical Centre
Box 574, SE-75 123, Uppsala, Sweden
Tel: +46-18-4714207
Fax: +46-18-509101
E-mail:  hesham.el-seedi@fkog.uu.se

Comments to Author:
List of authors:
1-the authors would add the affiliations of co-authors.
Abstract:
1-	The authors would add some examples of the uses of the plant in folk medicines
2-	The authors could show the known medicinal benefits.

Introduction:
1-	 The authors would add references for line 41:45, 48:50 and 51:54 in page 7.
2-	The authors would clarify effect of this plant that used in folk medicines compared to other plants or solely.
3-	The authors would clarify this sentence (Such claim is not without experimental basis as shown in various studies to characterise the fruit usages as anti-oxidant, anti-inflammatory, anti-cancer and anti-hyperglycemic substance).
Metabolite composition of mangosteen:
1-	The authors would check again the statements without references.
2-	The authors would show pharmaceutical properties of the compounds.
       3-If the authors would need more information about xanthones, take benefit from the below mentioned reviews:
1-	El-Seedi, H.R., El-Barbary, M.A., El-Ghorab, D.M.H., Bohlin, L., Borg Karlson, A.-K., Göransson, U., Verpoorte, R. (2010). Recent insights into the biosynthesis and biological activities of natural xanthones. Current Medicinal Chemistry 17, 854-901.
2-	El-Seedi, H.R., El-Ghorab, D.M.H., El-Barbary, M.A., Zayed, M.F., Göransson, U., Larsson, S., Verpoorte, R. (2009). Naturally occurring xanthones; latest investigations: isolation, structure elucidation and chemosystematic significance. Current Medicinal Chemistry 16, 2581-2626.
  4- The authors could put the ratio of chemical compounds in figure and general     chemical structures.
5-the authors could use (anti-microbial) instead of (Anti-bacteria, anti-fungi and anti-virus)
Conclusions:
Conclusion should follow the instructions of the journal.
References:
The authors should follow the instructions of the journal and add DOI and link of journal.


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: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 2
Reviewer: 1

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: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 2
Reviewer: 2

Output:
Basic reporting: Standard English, well cited literature and nice figures/tables reported.
Experimental design: Well defined methods and content.
Validity of the findings: Nice conclusions and well written summary.
Additional comments: The author included the comments of all reviewers nicely in their revised MS and thus the MS looks better now.


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: RECENT UPDATES ON METABOLITE COMPOSITION AND MEDICINAL BENEFITS OF MANGOSTEEN PLANT
Review round: 2
Reviewer: 3

Output:
Basic reporting: Yes, it has been modified according to our suggestions.
I strongly recommend the paper for publication.

Kindest regards, Hesham
Experimental design: Yes, it has been modified according to our suggestions.
I strongly recommend the paper for publication.

Kindest regards, Hesham
Validity of the findings: Yes, it has been modified according to our suggestions.
I strongly recommend the paper for publication.

Kindest regards, Hesham
Additional comments: Yes, it has been modified according to our suggestions.
I strongly recommend the paper for publication.

Kindest regards, Hesham


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

Input:
Title: A NEW SPECIES OF ESCALLONIA (ESCALLONIACEAE) FROM THE INTER-ANDEAN TROPICAL DRY FORESTS OF BOLIVIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I have had the pleasure to review the manuscript ‘A new species of Escallonia (Escalloniaceae) from the inter-Andean tropical dry forests of Bolivia’.

The major highlights of this contribution are the number of specimens analyzed and the interesting statistical approach they have employed to find morphological discontinuities among species and hence hypothesize species limits. The manuscript is well written, well referenced and results are supported by the analysis and discussed correctly.

I have a few suggestions in order to improve this contribution:
1- Materials and Methods
Morphological measurements
 I think authors should clarify why they chose leaf length and width and the number of flowers from among 6 vegetative and 21 reproductive characters used by Zapata & Jiménez (2012).

2- Results
Support for the new species boundary
	Taking into account that the published phylogenies show high support for a geographical structuring of the groups, I would add a phrase hypothesizing about the probable position of E. harrisii.

Lack of support for morphological discontinuities, differences in habit and habitat, and alternative explanations
In the same way you added the characters “revolute, tomentulose leaves” to E. polifolia, I think you can add other relevant characters to differentiate the remaing species.

After “Taxonomic treatment”, and to conclude, I suggest including a short key to differentiate E. harrisii from the remaining species of the region (Bolivia and northern Argentina), using discontinuities in leaf morphology and flower number, and also some other characters / habitat, etc…

Finally, I have annotated the pdf file; place special attention to Table and Figure legends.
It was difficult to get to the additional information; please take a look at the links.


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

Input:
Title: A NEW SPECIES OF ESCALLONIA (ESCALLONIACEAE) FROM THE INTER-ANDEAN TROPICAL DRY FORESTS OF BOLIVIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The present paper describes a new species of plants from Bolivia. Authors incorporate recent methods for species discrimination to validate their proposal. This study is thorough, scientifically, and exemplar; to my knowledge is one of the best species descriptions out in the literature because it incorporates statistical comparison with other species in the genus. I believe this study would become an example of great contemporary taxonomy, reason why the timely publication of this study is urgent.

The study is clear, professional, and well written from title to references. The introduction is appropriate, methods are well thought, results are well explained, and the interpretations are logical. I praise that authors identify and explain the weaknesses of their analyses.

Things to improve:
-	Authors should mention the new species name in all the figures, that way every figure is completely self-explained and understandable.
-	Proportion plots (fig 2) are neither explained in the text nor in the figure. As a reader I was not able to interpret those. An explanation of these plots should be included in the text.
- See minor edits in the pdf attached.


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: SUBJECTIVE ASSESSMENT FOR SUPER RECOGNITION: AN EVALUATION OF SELF-REPORT METHODS IN CIVILIAN AND POLICE PARTICIPANTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: See General comments
Experimental design: See General comments
Validity of the findings: See General comments
Additional comments: This paper describes two correlational studies examining the relationship between self-report measures and face identification ability. The first tests a group of people that self-identified as having high levels of face identification ability, the second recruits a cohort of police officers. Overall, the authors report mild-to-moderate correlations between a new self report measure called the SRU (developed by the authors) and performance in the face tests (CFMT, PMT), which is consistent with previous work on this topic.

The difference between the SRQ and other measures is that the authors specifically specifically designed it to discriminate between high performers. The authors find a statistically significant correlation between SRQ and face identification the first study (where the group mean performance is higher than an average group), but not it the second study using police officers.  Because the police officers had normal levels of CFMT performance as a group, the authors conclude that the measure is effective in discriminating between high performers but not the general population.

Overall, this study addresses an important question which has clear practical and theoretical motivation. The use of a professional cohort in this study is a novel aspect of the design, and a real strength of the paper. Nevertheless, the evidence presented is somewhat ambiguous with respect to whether the SRQ is indeed better at discriminating between high performers. This question is central to the paper, and I suspect further data collection may be necessary to provide clarity on this question.

MAJOR POINT

The authors conclude that the SRQ is effective in discriminating between high performers but not the general population. However, based on the current data it is not clear whether the difference in SRU-CFMT correlation in Exp 1 and Exp 2 is caused by the fact that there are differences in the accuracy of these different groups or whether it has something to do with the professional experience/ context of the police officers. As the authors discuss (e.g. p5, para 2), there are many possible explanations for why this difference may emerge -- e.g., perhaps better performers have more insight into their abilities? Perhaps q'naires for professional cohorts need to ask about the tasks performed in their job rather than everyday life?. So the data as they stand are not especially informative with respect to this important question.

This could be addressed, either by stating more explicitly in the conclusion the need for future work to clarify this important question, or by doing that work and including in this paper.

Running a web-based study of individuals that are recruited through M-Turk or similar would enable the authors to examine the correlation between SRQ, PMT and CFMT+ in the general population. That would provide normative scores of the test in normal population, a group that have presented to the experimenters pertaining to have high levels of ability, and a professional cohort, which would strengthen the paper considerably regardless of the outcome.

MINOR

- Please provide summary descriptive statistics for the CFMT and PMT in Exp 1 and 2

- p4, Stolhoff et al citation: insert year

- p8/9: 'technical errors were monitored by the website' I wasn't sure what that meant

- p10: 'the sample all self-referred for super-recognition'. Please clarify what this means - e.g. did they visit the website following media about SRs? or did they e-mail in response to a recruitment ad? Please specify.

- p13 last sentence 'Unsurprisingly' ... why is this unsurprising?

- p14 section 'Subjective assessment for super recognition': clearer labelling of this section would help the reader. For example '19.3% of the variance' -- variance in what? Overall accuracy scores? In general I found this section quite confusing, and it would have helped if the predictor variables and the variables to-be-predicted were more explicitly stated.

- Table 5. Were these corrected for multiple comparison? It struck me that the authors devote a large section of the discussion to a correlation of .21 here (the relationship between time in police and target absent accuracy), and there is a very high risk of a false positive with an r value that low. I agree that this is a potentially interesting result but think the discussion should be prefaced by acknowledgment of this risk.

================
David White
UNSW Sydney


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: SUBJECTIVE ASSESSMENT FOR SUPER RECOGNITION: AN EVALUATION OF SELF-REPORT METHODS IN CIVILIAN AND POLICE PARTICIPANTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well-written and easy to follow. The introduction in particular was a pleasure to read. The article includes sufficient background information and references to relevant literature. The article is structured in a logical way and figures are useful.

I will however note that the text refers to effect sizes in the tables that appear to be missing.
Experimental design: The article represents original, primary research. The research question is well defined, but in my opinion illogical (see General comments). The experimental design is robust, and the authors should be commended on their large police officer sample. Methods are reported in sufficient detail.
Validity of the findings: The conclusions drawn from the data and implications are not well-founded (see General comments).
Additional comments: MAJOR POINTS
The logic of the research question doesn’t make sense. The authors note that screening potential SRs with standardised tests can be very time consuming, and so it would be useful to have a quick way of detecting potential SRs. The authors then discuss how single-item self-report measures typically have very low predictive power and so while quick, don’t offer much useful information. I’m completely onboard up to this point. What I don’t understand is why, in response to this need for a quick and informative screening tool, the authors developed a 20-item self-report questionnaire. All the research points to people having poor insight into their face recognition abilities. Why not develop a screening tool that directly tests their face recognition ability? Why use a proxy for a skill you can actually test? I realise that the authors were trying to develop a quick screening tool, but in the time it takes to answer 20 questions on a 5-point scale participants could just as easily make multiple face identification decisions. If these comparisons were carefully selected based on performance data they would provide a reasonable basis upon which to screen for SRs. In fact, this approach is already used by at least 2 online SR tests. Given that the SRQ has almost no ability to screen SRs from ‘normal’ performers in civilian and police samples, I am at a loss as to why the authors are attempting to publish the SRQ at all. Surely the next logical (and necessary) step in this line of research is to develop a SR screening tool with the ability to predict SRs to a reasonable degree. I see the research reported in this paper as a first step in that line of work, and not necessarily worthy of publication in itself.

Could the authors please comment on the possibility that part of the reason why the SRQ had poor predictive power for the police officers was because they responded in a socially desirable way? It would be very easy for a participant to answer the SRQ as if they have very good face recognition ability even if they don’t. This is a critical issue with self-report measures and another reason to avoid using a proxy measure rather than measuring face recognition ability itself.

The general discussion largely ignores the fact the data show the SRQ has almost no discriminatory power for screening SRs, and makes claims as if the SRQ could be useful in applied settings. This sidesteps the data. It is not until the final sentence of the discussion (line 434-437) that the authors acknowledge that the SRQ is unlikely to be useful.

MINOR POINTS
Pg 3 line 70 – Phillips et al. paper is now published.
Pg 4 line 90 – Stollhoff citation missing year.
Pg 4 line 92 – This sentence is unclear. What values? What multiple validation studies?
Pg 5 line 114 – I don’t follow the authors’ argument that police officers could have more opportunities for feedback than civillians. In forensic situations the ground truth is often unknown and so feedback is rare. The type of feedback police officers receive often comes in the form of convictions, or peer-review, neither of which is veridical. As a percentage of the total number of identifications made, I would argue that civilians get more feedback.
Pg 8 line 174-177 – The description of the CFMT+ is very brief and not overly clear. The PMT was described in much more detail, even though a full description of both tests is available elsewhere.
Pg 10 line 224 (and elsewhere) – In a couple of places the authors write “…statistically significant difference in self-report between….”. Please help the reader by being clearer – self-report what?
Pg 10 line 238 – please clarify if 2SDs away from the SR mean in this context means ‘above’ or ‘below’.
Pg 10-11 – Is the SRQ only a significant predictor of the PMT when it’s broken down into target-present and absent trials?
Pg 11 line 257-259 – where are the effect sizes in table 3?
Pg 11 line 269 to Pg 12 line 274 – The summary overstates the predictive value of the SRQ.
Pg 14 line 342 to Pg 347 – The summary doesn’t provide important caveats on the claims, such as that some findings were only applicable to typicals, or SRs, or that the relationship with length of time as an officer was negative for SRs.
Figure 2 – panel A is missing the legend.


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: SUBJECTIVE ASSESSMENT FOR SUPER RECOGNITION: AN EVALUATION OF SELF-REPORT METHODS IN CIVILIAN AND POLICE PARTICIPANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Please see general comments.
Experimental design: Please see general comments.
Validity of the findings: Please see general comments.
Additional comments: The authors have addressed all my comments satisfactorily and the additional experiment strengthens the paper considerably. I recommend 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: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Grammar errors and small mistakes can be found over all over the manuscript and can sometimes hinder our understanding. Some large omissions and errors can be found and give the feeling the manuscript was rushed.e.g.: line 187, authors defined DAF 2 but forgot to define DAF 15 just after even though they refer to it later.
The authors should then spend more time making sure everything is perfectly defined and well written.

Overall the clarity of the article could be improved by using well defined terminology. For example, the authors refer several times to “transmission days” without ever defining what this means and to this day I still don’t understand what this refers to… The manuscript could also be easier to read and understand if the authors replaced some expressions such as “area of reception” (could be replaced by “site”) or “motion-controlled tests” which is vague and most of the time useless and could just be referred to as “tests” since no other tests were performed.

Background is clear and well documented even though a quick overview of how the Argos system works could be of interest for uneducated readers.

Figures are well made even though Figure 2 could be smoothed a little.
There is redundancy in figures and tables though, and some could be removed easily or move in supplementary material without harm.

The discussion section of the abstract is very vague and short and could be a little more substantial.
Experimental design: Methods should in my opinion be separated in named subsections (e.g: study sites, statistical analysis) because as it is it can be really confusing. Overall, the whole method section could be clearer.
The methods seem quite exhaustive except for the part about mixed models where the variables used were never cited nor described. A quick listing or a table with the different models used is required.
I believe the article would also gain to test different values of MAXREDUN. The authors test the filters for 2 and 15 km but this leaves a huge gap between the two. Different filters could be made to examine the effect of MAXREDUN on data accuracy. Maybe there is an optimal value that offers the best compromise between loss of data and accuracy gain.
Also the authors note that three different filters are offered by Movebank (line 182-183) yet they only use one without justifying why. Maybe a comparison of the type of filters could have been nice, especially since this is one of the objectives of the paper.
Validity of the findings: The results seem solid but clarity could be improved (see previous comments on figures and terminology and the use of subsections).
The conclusion section essentially just repeats what was said in the discussion section and is therefore quite useless as is.
Additional comments: While the science behind the article seem sound and it appears the authors know what they are talking about, the form could greatly be improved for a better understanding of the paper.
The article provides so many figures and tables that it is sometimes hard to make sense of it. This is not help by grammar errors and omissions all over the paper.
I recommend spending more time trying to make the paper more accessible and make sure every variable is well defined.
Additional filters could also be added to the study to improve its added value.


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: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language needs checking by a native English speaker

I have suggested literature that should be cited in the text.

The structure, figures, table were all clear.

It is clear and self contained

Items to amend
Line 51, here cite key papers, e.g.
Block et al. 2012. Tracking apex marine predator movements in a dynamic ocean. Nature volume 475, pages 86–90
Sequeria et al 2018. Convergence of marine megafauna movement patterns in coastal and open oceans. Proceedings of the National Academy of Sciences https://doi.org/10.1073/pnas.1716137115

Line 55, also cite these papers here:
Pendoley et al (2014). Multi-species benefits of a coastal marine turtle migratory corridor connecting Australian MPAs. Marine Biology 161, 1455-1466. DOI 10.1007/s00227-014-2433-7
Schofield et al (2013). Evidence based marine protected area planning for a highly mobile endangered marine vertebrate. Biological Conservation 161, 101-109 doi.org/10.1016/j.biocon.2013.03.004.

Line 130. The wording does not flow here: “were glued on stake 20 cm each other”
Experimental design: All of the items are covered adequately.
Validity of the findings: All of the items are covered adequately.
Additional comments: General
This is a useful paper investigating Argos doppler location errors in Romania. I have made some suggestions on citations that need to be included. I would also advise that the English language is proof read by a native English speaker. Otherwise the structure is good and the tables and figures are presented well. This paper should make a useful contribution to the journal.


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: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: See annotated .pdf
Experimental design: See annotated .pdf
Validity of the findings: See annotated .pdf
Additional comments: See annotated .pdf


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

Input:
Title: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The readability of the article has really been improved through the changes to the text.

Only 2 minor concerns:

line 154: I would add a brief explanation of what transmission day or transmission hour means in the real world for the uneducated people

line 274: I think you mean to refer to figure 5.

A figure is not cited in the text, either figure 4 or 5 depending on the result of the previous comment.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I commend the authrs for the improvement on the readability of the text. It is now much more clear to read  and understand.

I only suggest minor revision because a figure is never cited in the text but would not recommend further revision.


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: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and unambiguous. Overall the English language is clear throughout; though there are some formatting issues - paragraph presentations, extra spaces, mix of US and UK English style that need fixing. The article structure is good. The manuscript is self contained.
Experimental design: The experimental design is clearly explained and in sufficient detail, with god rigour.
Validity of the findings: The findings are valid, with clear supporting rationale.

All listed points have been addressed.
Additional comments: The authors have addressed the comments of the reviewers, with the manuscript being greatly improved.


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

Input:
Title: EMPIRICAL ANALYSIS AND MODELING OF ARGOS DOPPLER LOCATION ERRORS IN ROMANIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors addressed my concerns.
Experimental design: The authors addressed my concerns.
Validity of the findings: The authors addressed my concerns.
Additional comments: Thank you for addressing my edits and 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: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this study, authors aim to evaluate the protective effects of Taxifolin in myocardial ischemia/reperfusion in rat model. Authors describe that the compound “Tax” modulates
mitochondrial apoptosis pathway to improve the overall cardiac function. This study provides a new insight into the mechanism of this compound on inducing apoptosis.
Experimental design: Well designed, except for the lack of standard Annexin-PI assay for cellular apoptosis.
Validity of the findings: Valid data set
Additional comments: Minor Essential Revisions:
Line 79- recent studies- please provide additional references to support the statement.
Line 117- The hearts was-should be “The hearts were”. (+dp
Line 136- Please define briefly - dp /dtmax and -dp/dtmin), as this is one of the critical parameters of the study.
Line 141-142- “After two hours, the flat tissue blocks were embedded in paraffin µm to make it blocks and then cut into thick tissue sections and stained with H&E”- Poor English need editing.
Line 150- “63 min”, unusual time point, Can authors provide an explanation?
Line 153- what do authors mean by “Lysate? Is it Lysis buffer?”
Line 155- elisa should be “ELISA”.
Line 162- define “specific lysis buffer”
Line 200- heart functional recovery; please spell out the parameters of heart functional recovery here.
Why did the authors ignore the standard qualitative/qualitative assay for cellular Annexin-PI staining of cardiomyocytes in this study?
Figure 4- Arrows indicating the site of tissue damage and edema would add more light to the histological images.


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: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language of the manuscript lacks clarity, at points it creates ambiguity bordering with nonsense and some phrasing is odd. To give a few examples: Line 46: The sentence “However, blood supply…” lacks a subject and it is not clear what “..will cause additional damage to the heart.” Similarly on line 80: “…TAX involved in the amelioration of cardiac disorders associated with against hypertrophy…” the sentence is incomplete. What is the associated condition and why does it go against hypertrophy? Line 93: “…fed common feedstuff and drank tap water freely …”. What the authors meant is probably feeding the rats with common lab chow and allowing the animals to drink water ad libitum. Certainly using the word “whittled” (line 315) in association with the effect of TAX on apoptosis is very odd. Not only needs the manuscript re-writing, it needs thorough editing by a native speaker. There are too many lines affected by language errors to list.
Setting the language problems aside, the intro provides sufficient background for the study. Although it is not clear, why post-conditioning was used. This is not explained anywhere in the manuscript. Therefore the reader is left wondering why this approach was selected.
Overall structure of the manuscript conforms to standards within the field.
Figures are relevant and of acceptable quality. Labelling is fine, figure legends give redundant information in that they describe what is already apparent from the figure itself.
Raw data are supplied.
Experimental design: The manuscript describes primary original research.
Due to language problems the research question is not explicit besides testing what could be the effect on ischemia/reperfusion damage.
There are several important questions regarding the experimental design and how was the research conducted. Why were the two concentrations of TAX selected? How was the medium containing TAX prepared? Was TAX dissolved in aqueous media directly? If not and stock solution of TAX dissolved in another solvent was used did the controls contain the solvent? Why was post-conditioning used? If selection criteria were applied for the isolated hearts what was the fail rate, i.e. how many hearts failed to meet the criteria, if eight animals were selected per experimental group? At what time point were collected tissue samples that were used for enzyme activity test and immunoblotting? There is no mention of performing subcellular fractionation. Were the proteins, cytochrome c in particular, tested for in total cell lysate? The kits indicated in Materials and Methods generate protein level data in pg/ml, but the corresponding figure gives ng/mg protein. What was the conversion used? The data in Figure 1 show no error bars and do not indicate whether the points shown are mean or median. What was the standard deviation for data shown in Figure 1?
There is no sufficient detail of Methods to allow replication.
Validity of the findings: It is not clear from the manuscript whether the data are robust. That should be made clear in several instances (Methods, Results, Figure legends). The only mention is that there were eight animals per experimental group. Does it mean that each and every heart was subjected to the ischemia protocol, histopathological examination, antioxidant enzymes activity and protein expression? It is possible, however, it is not clearly stated. In any case, the number of samples in each tested group should be given.
Conclusions are based on the data presented and are stated well within the limits of the language used.
Additional comments: Lot of the questions were put forth above. The idea is not irrelevant, however, the manuscript overall is poorly written with insufficient detail given.
For better clarity the main concerns (questions) are repeated here.
1. Why were the two concentrations of TAX selected?
2. How was the medium containing TAX prepared? Was TAX dissolved in aqueous media directly? If not and stock solution of TAX dissolved in another solvent was used did the controls contain the solvent?
3. Why was post-conditioning used?
4. If selection criteria were applied for the isolated hearts what was the fail rate, i.e. how many hearts failed to meet the criteria, if eight animals were selected per experimental group?
5. At what time point were collected tissue samples that were used for enzyme activity test and immunoblotting?
6. There is no mention of performing subcellular fractionation. Were the proteins, cytochrome c in particular, tested for in total cell lysate?
7. The kits indicated in Materials and Methods generate protein level data in pg/ml, but the corresponding figure gives ng/mg protein. What was the conversion used?
8. The data in Figure 1 show no error bars and do not indicate whether the points shown are mean or median. What was the standard deviation for data shown in Figure 1?


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

Input:
Title: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, Tang Z et al., aim to demonstrate a function for Taxifolin in protecting against myocardial ischemia/reperfusion injury. Overall, the manuscript is well structured, and provides sufficient evidence to suggest a potential role for Taxifolin in this process. A few minor revisions could help strengthen the quality of the manuscript:

- The legend to figure 1 is missing. A few sentences describing the protocol would be helpful

- The graph in figure 5B is titled MAD, when it’s likely meant to be MDA

- There are several instances of grammatically incorrect sentences. For example, line 57 (‘in the I/R injury”, “cardiomyoctes were observed”), line 81 (“quercetin potentially beneficial”), line 84 (“in present study”), line 226 (“few cells dissolve and degeneration”), line 247 (“may protect mitochondria attenuate apoptosis”), line 252 (“proteins expressions”) , etc.  These are not exhaustive, and significantly affect the readability of the manuscript. Perhaps having the text reviewed by a native English speaker would help.
Experimental design: In figure 6, the authors aim to measure the expression of several apoptosis-related proteins. A few concerns regarding this experiment:
- It is unclear from reading the materials section if the commercially available kits used in this experiment are directly measuring protein level (through an ELISA-type method) or if a western-blotting type quantification is used. The authors should clarify the precise method used here
- In panel 6A, the authors claim to be specifically measuring levels of cytosolic-Cytochrome C. However, it is unclear if a specific nuclear-cytosolic fractionation of cell lysate was employed. If so, measuring levels of control proteins that are found only in the cytosol or nucleus will provide confidence in the integrity of the fractionation method
Validity of the findings: Overall, I believe the data presented in this manuscript support the conclusions made by the authors. One point to note however is that I/R-associated injury has been well documented to involve multiple forms of cell death including apoptosis, ferroptosis, and necroptosis. It appears that Taxifolin mediates its protective effect through apoptosis. But the role of other forms of cell death cannot be discounted. The authors should consider adding a brief speculative discussion addressing this point.
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: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 2
Reviewer: 1

Output:
Basic reporting: Revised
Experimental design: revised
Validity of the findings: revised
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: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Language is still a major issue. The errors and inconsistencies pointed out in my first review were by no means exhaustive. However, the authors apparently thought so and amended only those. It is not my role to list here every and all errors, therefore just few more examples are given here. Line 53 “…which often cause to death.” Select another verb or re-phrase; line 87 “quercetin has been demonstrated improves post ischemic….” Past perfect or present tense? Cannot be both side by side. Line 94 “..involved in…disorders associated with against cardiac hypertrophy..” Associated with what (an effect? Substance? Protein?) that apparently acts against cardiac hypertrophy. Either complete the sentence or re-phrase it. Line 200 “…homogenized them using …extraction kit…..with a glass homogenizer on ice.” So what was the tool: the kit or the homogenizer? Line 233 “…rehydrated in graded concentration of ethanol.” Did you mean concentration gradient? Or was it a defined concentration of ethanol? Line 265 “At different time points of perfusion, the heart effluents were perfusate.” No clue what information is hidden in this sentence because the heart effluent is in fact the perfusate at all times of the perfusion. What were the authors trying to say here? Line 271 “…CK-MB level was not significantly altered in different group in baseline.” Once again, it is not clear what information is imparted in this sentence.
Let me stress again: this is not an exhaustive list of all unclear statements and it is not my task to find and list all that occur in the manuscript. My persistency in forcing the authors to thoroughly read and correct all unclear and ambiguous language in the manuscript is for the sake of readers of PeerJ.
Experimental design: Methods still lack information to allow replication of the data. Please see also part 4 General comments.
Validity of the findings: no comment
Additional comments: I do understand the answers given by authors to my previous questions and comments. However, to my disappointment only scant information given in the answers actually appears in the manuscript.
Example: Preparation of TAX. Clearly explained but the reader is not told in the manuscript. Even if this communication is posted for readers of PeerJ, I doubt they would look for it. And seek here answers to their questions. So I urge the authors to incorporate these answers into the manuscript. All of them.
I am happy with the explanation of why post-conditioning was employed. It is incorporated in the manuscript.
Selection criteria for animals. Again, please incorporate your answer in the manuscript. The reader should know there were actually 12 animals per group but only eight were used because these met the selection criteria. And of these only some were used for certain post reperfusion testing.
The subcellular fractionation is not sufficiently described in the Methods. I doubt it is one step procedure. What was the g-force used in the centrifugation? Time alone is useless.
Data in figure 2 are now improved. Addition of the error bars raised a question whether there is a significant effect of TAX. The error bars overlap so much that even with reasonable group size (eight) one wonders on the statistics.


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: TAXIFOLIN PROTECTS RAT AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY BY MODULATING THE MITOCHONDRIAL APOPTOSIS PATHWAY
Review round: 2
Reviewer: 3

Output:
Basic reporting: It is clear that the authors have taken the reviewer's comments constructively. I have no further suggestions, and believe the manuscript is adequate for publication
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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN POST-HARVEST LEAVES OF TEA PLANT (CAMELLIA SINENSIS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1- The English writing needs to revise. Also, some correction should be done for some incorrect words inside the text.
2- In Abstract section - lines 54 and 55: while CsTBP, CsPCS1, CsPPA2, CselF-4α, and
CsACT were the four best reference genes in the witheringgroup.
They are 5 and not 4. Please correct.
3- 18S rRNA and something like this should not be written in italic. In Discussion section, they are italic. Please correct them.
4- In Material and Methods - lines 141 and 142 has been included the results. Please put the numbers regarding to results inside the Results section.
5- Please explain that why did use agarose gel electrophoresis in 1.2%?
6-The current more that 50 mA could make heating inside the gel that could degrade RNA. Please rewrite this section (line 144).
7-In title of "Quantitative Real Time assay", line 161, agarose gele electrophoresis is not considered as quantitative analysis. Please insert the results of standard curves that mentioned inside the text.
8-Inside the Table 1, three genes has been shown but in its description has been written 4 genes. Which of them is correct?
9-Inside the Table regarding to primer pairs, The amplicons more than 180 to 200 bp is not suitable for Real time analysis. They are acceptable for publishing. The primer designing should be done again and this section should be repeated.
Experimental design: The primer pairs with amplification of more than 200 bp should be repeated in order to re-designing the primers and then do qRT-PCR.
The standard curve should be added and discussed more.
Validity of the findings: The authors should explain why the previous reference genes are not suitable and why they chose their interested genes?
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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN POST-HARVEST LEAVES OF TEA PLANT (CAMELLIA SINENSIS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.
Validity of the findings: Data is robust, statistically sound, & controlled.
Additional comments: The manuscript "Validation of reference genes for gene expression studies in post-harvest leaves of tea plant" by ZhOU et al. sumamrizes a good and solid research endeavor that tested the reference genes suitable for the quantitative real-time PCR analysis of gene expression in post-harvest leaves of tea plant.

In a word, the manuscript was well-prepared, especially it is written in professional English, which makes understanding easier for readers. The manuscript is technically sound, and the data supports the conclusions. Besides, the statistical analysis been performed appropriately and rigorously.

0verall, the present version meet the criteria of Peer J, however the revision include but not limit to the following should be needed before it can be reconsidered for publication (For detailed information, please see the attached review commented file). Therefore, I recommend the publication with minor revision.

1.	When you describe the plant material, what do you mean by "second"? From the top or from the bottom? And you also need to confirm that it is a scientific and reasonable description on the leaf position.
2.	In the parts of "Materials & Methods", you should not describe know the results, such as “The concentration (≥ 500142 ng/μL) and A260–A280 ratios (1.90-2.10) of total RNA were evaluated by a spectrometer 143 (Thermo USA) to detect the purity.” In lines 141-143.
3.	How many candidate reference genes have you selected? 15 or 16 (8=8)? If CsTIP41 is redundant? If so, you should deleted all its related information, including in the Table.
4.	Regarding the “Validation of Selected Reference Genes”, you are suggested to highlight how the results of CsLOX1 gene expression normalized by the selected reference genes accord with what it is assumed to be, which is the most direct evidence on the feasibility of the selected reference genes.
5.	Several references are missing, and you need to add the corresponding references accordingly.
6.	Please keep in mind that, in the Parts of Discussion, de-emphasize the findings that have been previously reported by others and the differences between this research and previous researches. Especially, perform a critical discussion of the main results and novel contributions of this article, and how this adds to the current knowledge status in the field.


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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN POST-HARVEST LEAVES OF TEA PLANT (CAMELLIA SINENSIS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: All the changes are acceptable.


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: VALIDATION OF REFERENCE GENES FOR GENE EXPRESSION STUDIES IN POST-HARVEST LEAVES OF TEA PLANT (CAMELLIA SINENSIS)
Review round: 2
Reviewer: 2

Output:
Basic reporting: Good article
Experimental design: Correct
Validity of the findings: Valid
Additional comments: No comments