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: EVALUATION OF FEMALE AEDES AEGYPTI PROTEOME VIA LC-ESI-MS/MS USING TWO PROTEIN EXTRACTION METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this manuscript, the authors did comparative analysis of two different protein extraction methods in female Aedes aegypti proteome (TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBuster). It is followed by protein identification using LC-ESI-MS/MS. They identified Cytobuster reagent gave highest protein yield, proteome coverage and extraction speed. They substantiated their findings by performing LC-ESI-MS/MS along with functional annotation analysis in these two protein extraction methods.
Experimental design: No comment
Validity of the findings: 1.	Protein extraction reagent CytoBuster has also added advantage for performing Westeren blots and SDS page. Do authors observed this advantage while performing their SDS page gel or they don’t see any advantage between these 2 methods for performing SDS page gel in female Aedes aegypti proteome.
2.	Do they think Cytobuster has more advantage for performing Western blots or 2D gel electrophoresis? They can add some discussion regarding this. They gave some explanation regarding drawbacks attributed to the TCA acetone precipitation extraction method in their discussion.
3.	It would be interesting for the readers to see images of SDS page gel in these 2 methods for comparison. Is there any difference or not? They can show which protein bands or spots are used for in gel digestion.
Additional comments: Overall, in this manuscript the authors supported their findings of CytoBusterTM reagent displayed a superior performance for the extraction of proteins and proteome coverage. The paper is well written and the data supports the observations and conclusions drawn.  The work presented in this manuscript, is a useful tool for future studies in the field of proteomic analysis. I hope my comments will be helpful.


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: EVALUATION OF FEMALE AEDES AEGYPTI PROTEOME VIA LC-ESI-MS/MS USING TWO PROTEIN EXTRACTION METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revision has addressed all the concerns well
Experimental design: The revision has addressed all the concerns well
Validity of the findings: The revision has addressed all the concerns well
Additional comments: The authors have done a good job in addressing all the reviewer comments and revising 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: PUBLICATION RATE AND CITATION COUNTS FOR PREPRINTS RELEASED DURING THE COVID-19 PANDEMIC: THE GOOD, THE BAD AND THE UGLY
Review round: 1
Reviewer: 1

Output:
Basic reporting: A well written original article. The topic is attractive and the results are useful. Aime and the scope of the article are correct.
The text should be written in clear English language, by native speaker or professional translator.
I suggest to place a specific timeline figure where would be described the period of the search, and also the date of search. The title should be more comprehensive, discussion should be shorter and the conclusion should be longer, without any specific and opaque words
Experimental design: Is completely correct with minor suggestions. Methodology is also correct.
Validity of the findings: Is completely correct and in line with journal policy and suggestions with minor suggestions.
Additional comments: A well written original article. The topic is atractive and the results are useful. Aime and the scope of the article are correct.
The text should be written in clear English language, by native speaker or profesional translator.
I suggest to place a specific timeline figure where would be described the period of the search, and also the date of search. The title should be more comprehensive, discusion should be shorter and the conclusion should be longer, without any specific and opaque words

Title:
Line 2: I find the part of the title after „:“ unnecessary (The good, the bad and the ugly), so I suggest to remove it.
Abstract is good structured.
In line 24, and also in the all other text is written: „published on scholarly journals“, but I find more in line with native English language „published in scholarly journals“ to be written. (it should be considered by editor).
Line 28: Te sentence start: „We then determined“. I suggest to be written: „After that we determined“. I find it more in line with native English.
Introduction: in line 52 after the word „report“ should add „of scientific work, “ (full sentence should be: A preprint is a preliminary report of scientific work, that is shared publicly before it has been peer-reviewed.
In line 58 „has been controversial“ should be replaced with „is being disscused“.
In line 77 at the end should delete „that“
In line 82 „works“ should be replaced with „publication papers“ and also afterward in the text.
In „Statistical analysis“ you should describe why you have chosen specific tests for analysis, so it can be clearly understood.
Results: in line 127 -128 is written data of median time from the date of posting to the date of search!? It is more important to publish the median time of posting to the date of publication in a scholarly journals.

In line 132 sentence that with Table 1 is unnecessary because at the end of the line 137 it is correctly declared.
In line 148 should escape word „eventually“ or the results to be more better described.

In line 165 „)“ is missing. Also (Table 3) need to be in separate bracket.

Discussion is too long. It should be shorten.

At the end of the line 174 reference is needed.

Sentence 177-179 is crutial but too long and opaque. It should be reformulated.
Sentence 184 – 187 is too long, and opaque. It should be more in line with english language (even 4x you hve word „that“ in the sentence)

Sentence 193 – 195 is opaque. It should be more understandable and reformulated.

The state in line 216 and 219 need reference if it is from other papers. In case that it is from your work it should be in section „Result“.

Conclusion: It should be longer. The last sentence (line 268 – 269) is controversal, and should be deleted. Also in conclusion should be more stated to justify or not the use  of the preprint servers in future and to publish or not IN PREPRINT SERVERS in line with your results.

Table 1. should be added on the left side with column in which will be explained data in the table
Table 2. should be added on the left side with column in which will be explained data in the table

Also should be considered to add separate analysis for each preprint server (medRxiv, bioRxiv and RS) in Table 1 and 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: PUBLICATION RATE AND CITATION COUNTS FOR PREPRINTS RELEASED DURING THE COVID-19 PANDEMIC: THE GOOD, THE BAD AND THE UGLY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Adequate
Experimental design: Adequate
Validity of the findings: Adequate
Additional comments: The authors have revised the manuscript adequately


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

Input:
Title: ITRAQ-BASED COMPARATIVE PROTEOME ANALYSES OF DIFFERENT GROWTH STAGES REVEALING THE REGULATORY ROLE OF REACTIVE OXYGEN SPECIES IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript presents an iTRAQ based proteomic analysis of three developmental stages of the medicinal enthomopathogenic fungus Ophiocordyceps sinensis, focusing on the role of ROS in fruiting body development.
The English of the manuscript is understandable but definitely needs improvement in order to be correct and precise. Some remarks have been posted in General comments.
Background. The Introduction  is concise and well-focused but in my opinion more information should be given about the biology and artificial cultivation of Cordyceps sinensis, as well as on omics research concerning  this fungus.
The structure of the manuscript is adequate and conforms to PeerJ standards but some improvement in data presentation  is necessary (see remarks on figures).
Raw data are supplied as supplementary files.
Literature used is relevant but some important sources are not included. For example, about biology of Cordyceps sinensis - Li, M., Meng, Q., Zhang, H., (...), Qin, Q., Zhang, J. Vegetative development and host immune interaction of Ophiocordyceps sinensis within the hemocoel of the ghost moth larva, Thitarodes xiaojinensis. 2020 Journal of Invertebrate Pathology 170,107331; Liu, G., Han, R., Cao, L., Schmidt-Jeffris, R. Artificial Cultivation of the Chinese Cordyceps from Injected Ghost Moth Larvae 2019 Environmental Entomology 48(5), pp. 1088-1094. For participation of ROS in fruity body development see also Liu, Q., Zhao, Z., Dong, H., Dong, C. Reactive oxygen species induce sclerotial formation in Morchella importuna 2018 Applied Microbiology and Biotechnology 102(18), pp. 7997-8009.
Experimental design: This is an Original primary research within Aims and Scope of the journal. The Research question is relevant and meaningful but could be better defined to emphasize how research fills this knowledge gap. The methods are not described with sufficient detail & information to replicate (see General comments).
Validity of the findings: no comment
Additional comments: In my opinion, the manuscript needs a major revision in order to be published.
Major remarks
Abstract
Line 17 – “its potential mechanisms” – unclear, the meaning is lost
Line 18 – the statement “protein patterns between PR and MF were more similar than ST” is in concert with the main text but it is not in concert with the next sentence about the numbers of DAPs. In the text, there are 510, 173 and 514 DAPs detected in the comparisons of ST vs. PR, PR vs. MF, and ST vs. MF, respectively.
Line 21 – “enriched in terms of…” did you mean enriched in proteins related to…
Line 22 – “enriched in pathways of..” more convenient is here enriched in terms…. (pathways of peroxidase does not sound well)
Line 25 – “R package” – not clear
Line 29 – I wonder about electron transport protein (T5AHD1) and  histidine phosphotransferase (HPt, T5A9Z5)  - are they  ROS-related proteins? Especially histidine phosphotransferase is a component of signal transduction system – see Front. Chem., 19 May 2017 | https://doi.org/10.3389/fchem.2017.000 Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae
The conclusion in the abstract : This findings firstly offer  the perspective of proteome to understand the biology of fruiting body development and highland adaptation, which inform the future large-scale artificially breading of O. sinensis. – needs better elaboration.

The Introduction needs improvement. I find some contradiction in the statement “Till now, It has not yet commercially cultivated” (line 49) and the cited reference Li et al in 2019, Critical Reviews in Biotechnology 39(2), pp. 181-191 “ omics studies, including genomic, transcriptomic, proteomic, and metabolomic studies, have helped to understand the biology of the fungus, the success of the artificial cultivation of the Chinese cordyceps is clearly a milestone “
More info should be added in the first paragraph on the biology and medical importance of this fungus and the problems of natural exhaustion of these resources and difficulties in cultivation for commercial purposes, as well as the scarcity of data for understanding the biology of development of the fruiting body.
Recent omics research (genomic, transcriptomic, proteomic) on this species should be briefly stated. Some insights from transcriptomic studies (more references should be used) - refs not cited: Zhao, Y., Zhang, J., Meng, Q., (...), Chen, C., Qin, Q. Transcriptomic analysis of the orchestrated molecular mechanisms underlying fruiting body initiation in Chinese cordyceps. Gene 2020, 763, 145061.  Proteome analyses up to date should be summarized -  for ex. Zhang, X., Liu, Q., Zhou, W., (...), Qi, L.-W., Yin, X. A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis. 2018 Journal of Proteomics 181, pp. 24-35. Other proteomic studies - the cited as ref  32. Feng K , Wang L Y , Liao D J , et al. (2017). "Potential molecular mechanisms for fruiting body formation of Cordyceps illustrated in the case of Cordyceps sinensis. " Mycology, 8(4):1-28; the non-cited Dong, Y.-Z., Zhang, L.-J., Wu, Z.-M., (...), Ni, L., Zhu, J.-S. Altered proteomic polymorphisms in the caterpillar body and stroma of natural Cordyceps sinensis during maturation. 2014 PLoS ONE 9(10),e109083.
 Lines  49-51 – the sentence “The  understanding of biology of the fruiting body development not only helps to realize the large-scale industry of this fungus but also protect this precious resources for sustainable usage” should be rephrased, it is not very convincing in the above content. Something like: This comparative proteomic study fills the gap in knowledge and provides useful information for sustainable usage and possible cultivation of this fungus in a large scale industry.
Minor remarks in Introduction:
Line 50 – realize=become aware, other meanings of realize=implement, fulfill, materialize, accomplish, achieve. Please chose more appropriate synonym.
Line 54 – “produce the ROS antioxidant defense” – please replace “produce…” with “develop an adequate ROS defense”
Line 55 – “precious” or previous?
Line 57 - conditions have/ condition has
Line 63 – “genes involved in peroxidases” - please replace with “genes coding for peroxidases”
Lines 70,71 – sentences beginning with numbers (12, 18) – should be written in letters (Twelve, eighteen)
Line 70 – DEGs should be explained at first mentioning
Line 76 – based on the above evidence…
Line 77 – with – unclear. Do you mean along with? guided by?
Line 81 - proteome was/ proteomes were

MMs  - several small points of incorrectness
Collection of O. sinensis samples – How was mycelia separated from the dead larvae? Could you identify some animal proteins in ST samples? Did you separate ascospores from the mycelium at MF stage?
line 91 – “Traditional Chinses Medicine” – Chinese?
Protein preparation – line 97 – cocktail of what? IAA – defined only at line 104 with abbreviation IAM; TEAB - Triethylammonium Bicarbonate – defined only at line 110, first mentioning line 100. All abbreviations should be defined at first mentioning. Line  101 “Bradford Protein Assay Kit according to the manufacturer’s instructions” - which was the manufacturer? Specify the way it was done for trypsin. Line 108 – peptides; line 114 – “on Q Extractive” or Q Exactive? ACN – acetonitrile? FA – formamide? Line 147-148 – unclear sentence, the second werb “was” – not necessary.
Lines 160 -167 Reactive oxygen species (ROS) measurement – missing reference.
Line 169 – principle of methods for CAT and SOD activities should be given in brief.
Line 176 – root samples?
How many biological repeats were included – three (lines 186,189) or four (lines 144-145, 198)?
Lines 158-159 – R package – unclear.

Results and discussion:
Line  196 “Quality proteomic analysis” – unclear. An overview?
Line 210 – biological replicates are explained in MMs, not necessary to repeat.
Line 215 – “significant proteins” – unclear. DAPs?
Line 216 –“.And” – do not begin sentence with an “and”
Line 230 – omit “serial”
Line 231 – “molecular function (MF)” but MF is abbreviation for mature fruiting body
Line 241 – “paly” or play?
Line 242 –especially in the response
Line 247 – omit “that”
Line 272 – “into activate varieties of amino acid” – unclear
Line 279 – “has defects in a sterile” – please reformulate
Line 284 – this sentence is highly speculative
Line 400 - ROS signaling pathway
Line 402 –403 - a reference is necessary.
Line 404 - were enriched in
Line 405-406 –“ SOD and CAT are important antioxidant enzymes to eliminate harmful free radicals – please add more details – SOD converts superoxide radicals to hydrogen peroxide whereas CAT detoxifies hydrogen peroxide – different functions. Depending on SOD/CAT ratio different forms of ROS will be more abundant
Line 410 – “SOD has an increase tendency on protein level “SOD has a tendency to  increase at the protein level

Conclusion
Line 423 – “investigate the genetic basis” – unclear, this is a proteomic study
Lines 431-434 How the authors could explain “a stronger ROS  accumulation was detected at the stage of MF compared to stages of ST and PR. Meanwhile, SOD activity  significantly increased across the three stages and the activity of CAT significantly increased in MF compared  to the other two stages”? – both ROS increased and their scavengers also increased at MF. ROS gradient should be confirmed histochemically with specific staining for ROS forms.

Figure 2B – “The number of DAPs is shown on the top of histograms” – not seen
Figure 3  - enriched GO functional classification of DAPs. The green bars represent cellular components; The orange represent biological processes; the blue bars represent molecular functions. – Usually cellular components, biological processes and  molecular functions are given in separate charts, not mixed.
DEGs –please define.
Figure 6 – black bars, white bars  - but  bars are in colours.
Figure 7 – Y axis – the title should be more exact (not – ROS fluoresncence)
Table S5 - the enriched KEGG pathway in the comparisons – alcoholism or alcohol metabolism?


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: ITRAQ-BASED COMPARATIVE PROTEOME ANALYSES OF DIFFERENT GROWTH STAGES REVEALING THE REGULATORY ROLE OF REACTIVE OXYGEN SPECIES IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Authors are advised to proof read the manuscript to identify the grammatical errors including sentence construction and word usage.
Experimental design: no comment
Validity of the findings: The current study provides proteomic analysis of the developmental stages of O. sinensis with specific focus on the fruiting body as a follow up to the previous publication by the authors (Tong X et al; Peer J; 2020) which analyzed the transcriptomic differences in the same. The current study provides data to suggest involvement of ROS signal transduction in the development stages of the fungi. The authors provide sufficient literature to corroborate their data.

However, the authors are advised to assess the novelty of the study in greater detail and add the lacunae in the field to emphasize the need to undertake the study.

Major Comments:
•	The aim of the paper; as stated by the authors is to discuss the underlying biology of the development of fungus towards the overarching goal of providing better understanding for  eventual commercial cultivation. In view of this, it would be pertinent to also discuss the DAPs and associated pathways (if any) exclusive to each development stage.
•	The authors provide data to implicate ROS and antioxidant (CAT/SOD) levels as major regulatory factors in development of O. sinensis. It would also be pertinent to explore and demonstrate experimentally whether perturbations in the two could actually result in developmental delay/abnormalities.
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: ITRAQ-BASED COMPARATIVE PROTEOME ANALYSES OF DIFFERENT GROWTH STAGES REVEALING THE REGULATORY ROLE OF REACTIVE OXYGEN SPECIES IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised ms has been substantially improved, however some small mistakes still remain.
Line 106 – “containing of urea lysis buffer containing 8M Urea” – repetition, plrase omit the first “containing of”
Line 107 – are you sure that “ultrasonic pyrolysis” is pyrolysis? Please check.
Line 112 – then samples were kept
Line 116 – to obtain urea concentrations
Line 130 – followed by what? Unclear
Line 135 – 136% solvent A – are you sure?
Line 247 – were oxidoreductases
Line 248 - functions in carbohydrate transport
Line 315 – harmless particles
References should be written according to the requirements of the journal. At some places, italics is not used for Latin names of species.
Figure 4 – ST vs PR – ordinate axis – as far as I know “Systemic lupus erythematosus” is an human disease


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: ITRAQ-BASED COMPARATIVE PROTEOME ANALYSES OF DIFFERENT GROWTH STAGES REVEALING THE REGULATORY ROLE OF REACTIVE OXYGEN SPECIES IN THE FRUITING BODY DEVELOPMENT OF OPHIOCORDYCEPS SINENSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript now conforms to PeerJ standards of clear and unambiguous reporting with relevant literature cited and professional English.
The authors have acknowledged the comments and incorporated the data in the revised manuscript addressing the comparison of DAPs at each stage of the fruiting body development of O. sinensis and have provided tabulated data for clarity. They have also tried to assess the ROS generation experimentally to clarify the importance of SOD/CAT in the hyphae and fruiting body while acknowledging and clearly stating future studies to be undertaken for mechanistic advancement.
Experimental design: Additions to the material and method section now provide sufficient details to replicate the study.
Validity of the findings: The authors have now addressed the novelty of study in the introduction with sufficient literature citation.
The conclusions are accompanied by valid speculations and limitation of the study.
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: INFLUENCE OF PLASTIC FILM MULCH WITH BIOCHAR APPLICATION ON CROP YIELD, EVAPOTRANSPIRATION, AND WATER USE EFFICIENCY IN NORTHERN CHINA: A META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Manuscript is interesting, but needs a lot of clarifications. Title seems to be experimental manuscript... How you decide about use just only this References. Yes, 2 people search and one decide - l. 109 - 112. Why the criterium about crop yield and WUE, ET is last...

Current publications are missing. I recommend adding: doi.org/10.3390/plants9010043;  DOI: 10.1016/j.scitotenv.2018.12.047;  DOI: 10.3389/fpls.2016.01111;  DOI: 10.3390/su12176779;  DOI: 10.3390/agronomy10101513; 10.1016/j.resconrec.2018.04.022

Add more information on the molecular and physiological flexibility of plants (potato, wheat and maize) under biochar application. The discussion section needs to be revised. Arguments require a clearer and more accurate presentation. The understanding of physiological mechanisms is limited because it is limited to works that have a specific view and deliberately ignore alternatives, and do not represent a balanced view of the evidence.
Experimental design: Meta-analysis have big variance...How you measure (calculate) statistical importance of studies? (l. 127)

Do you used cross searching in databases? More connections together? For example: plastic mulch AND biochar AND WUE? More cleary - l. 108

l. 112 - 556 publications on WOS and Scopus or from all sources... Google scholar counted textbooks etc... Needs clarification and using only peer-reviewed papers!
Validity of the findings: It is very hard to check validity - because in Methods is about 556 publications sources, but Table 1 - just only 21 References. We needs to see whole list of references and their impact.

Findings: only indication... it not could be statistically significant..
Additional comments: In my opinion, needs this meta-analysis study more clarifications, cleary results with valuable and actual worldwide (now is focused to the local sources) disscusion (missing mainly in case of physiological parameters WUE, ET, now is very general not pointed).


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: INFLUENCE OF PLASTIC FILM MULCH WITH BIOCHAR APPLICATION ON CROP YIELD, EVAPOTRANSPIRATION, AND WATER USE EFFICIENCY IN NORTHERN CHINA: A META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Required more information and review of previous studies. Also, need to check the consistency of each sentence.
Experimental design: No explanation about experiment design. Further methodology discussions are needed.
Validity of the findings: The result should discuss specific aspects and data needed in conclusions.
Additional comments: Overall comments:
The manuscript describes the film mulching effects on ET, crop productivity in China which have a significant impact on water-saving agriculture. This paper is relevant and within the scope of this journal. However, the structure of the manuscript is well while I found several grammatical mistakes in sentences where a few terms are repeated.
Furthermore, the abstract need to be revised by considering the importance of the study, appropriate objectives,  and specific outcomes. The introduction is not much informative and has been found in many misleading sentences which should be discussed clearly.  Therefore, the manuscript needs to significant revision and tremendous efforts to be publishing PeerJ journal.
I made several major and minor comments marked by yellow highlighted colors in the attached pdf file. Thank you.


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: INFLUENCE OF PLASTIC FILM MULCH WITH BIOCHAR APPLICATION ON CROP YIELD, EVAPOTRANSPIRATION, AND WATER USE EFFICIENCY IN NORTHERN CHINA: A META-ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Dear Authors,
thank you for your deep revision. Now is meta-analysis manuscript sufficient and clearly readable.
Experimental design: In revised version is clearly described whole meta-analysis design.
Validity of the findings: Completely revised Discussion and clearly Results.
Additional comments: Thank you for improving 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: INHIBITORY EFFECTS OF Α-MANGOSTIN ON T CELL CYTOKINE SECRETION VIA ORAI1 CALCIUM CHANNEL AND K+ CHANNELS INHIBITION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this manuscript, Hyan Jong Kim et al., investigated the effects of  - Mangostin on ORAI1 calcium channels and potassium channels of immune cells. Using electrophysiology, they find that  - Mangostin inhibits ORAI1 calcium channels at IC50 of sub-micromolar concentration using both HEK293T and Jurkat T cell lines. They also find that  - Mangostin inhibits both voltage gated potassium channels Kv 1.3, and calcium-activated conductance channels KCa 3.1 that are essential for regulating membrane potential and for calcium signaling homeostasis. The authors looked at downstream effects of inhibition of calcium signaling on cytokine secretion and they find that IL-2 cytokine secretion was inhibited in Jurkat T cells. Thus, the authors concluded that inhibiting calcium channels and potassium channels is an underlying mechanism for anti-inflammatory effects of   - Mangostin. This is altogether a nice paper as authors provide direct evidence of  - Mangostin effects on ion channels in immune cells for the first-time using electrophysiology.

1.	Background part of the paper requires rewriting.

2.	I have annotated few sentences on a PDF file that needs to be rephrased or rewritten.
Experimental design: 1.Please elaborate on Fura-2 Ca2+ imaging method section especially on how you use 340/380 ratio to calculate calcium concentration.
Validity of the findings: 1. In Figure 1A, authors should provide a control trace for the entire length of the experiment. Also, in their Figure 1 legend, concentrations of  - Mangostin are in millimolar concentration, is it supposed to be milli molar or micro molar?

2. Cell viablity of Jurkat T cells went down by 90% at 10 M concentration (Figure 5).  The authors look at ORAI1 channel inhibition at different concentration of  - Mangostin from 0.1-10 M, what about cell viability of HEK293T cells?

3.In Figure 4, if HEK293T cells are viable till 10 uM concentration, they should include I-V plot for other concentrations as in Figure 1 and determine IC50.
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: INHIBITORY EFFECTS OF Α-MANGOSTIN ON T CELL CYTOKINE SECRETION VIA ORAI1 CALCIUM CHANNEL AND K+ CHANNELS INHIBITION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study by Kim et al – Inhibitory effects of α-Mangostin on T cell cytokine secretion via ORAI1 calcium channel and K+ channels inhibition. The authors analyzed the inhibitory effect of α-mangostin on immune cell activity via inhibition of calcium and potassium ion channels expressed in immune cells and demonstrated the anti- inflammatory effect of α-mangostin through the regulation of calcium signals.
Experimental design: The objective of the study is reasonable. The methodology is well described, and experiments are performed with appropriate controls.
Validity of the findings: The data looks significant and conclusions are well drawn with statistical significance.
Additional comments: The authors use Jurkat T cells activated with anti CD3/CD28 antibodies and monitor the IL2 release following treatment with α-Mangostin. It would have been more convincing if authors would have monitored the primary T cell activation using CD3+T cells isolated from PBMCs and treated with α-Mangostin. It would be interesting, if authors can include in the discussion section, the speculative mechanism of α-Mangostin in modulating calcium signals in T cells.


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: INHIBITORY EFFECTS OF Α-MANGOSTIN ON T CELL CYTOKINE SECRETION VIA ORAI1 CALCIUM CHANNEL AND K+ CHANNELS INHIBITION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I appreciate Hyan Jong Kim et al., for addressing questions I raised. I also thank them for considering my suggestions and making necessary changes to the revised manuscript. it is well written now.
Experimental design: The methods described with sufficient detail.
Validity of the findings: Overall, it is a nice work on providing direct evidence of -Mangostin effects on ion channels in immune cells  with carefully designed experiments.
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: INHIBITORY EFFECTS OF Α-MANGOSTIN ON T CELL CYTOKINE SECRETION VIA ORAI1 CALCIUM CHANNEL AND K+ CHANNELS INHIBITION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The study by Kim et al – Inhibitory effects of α-Mangostin on T cell cytokine secretion via ORAI1 calcium channel and K+ channels inhibition. The authors analyzed the inhibitory effect of α-mangostin on immune cell activity via inhibition of calcium and potassium ion channels expressed in immune cells and demonstrated the anti- inflammatory effect of α-mangostin through the regulation of calcium signals.
Experimental design: Well described.
Validity of the findings: The authors provided the answers to the comments.
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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS REVEALS THE COLD ACCLIMATION DURING CHILLING STRESS IN SENSITIVE AND RESISTANT PASSION FRUIT (PASSIFLORA EDULIS) CULTIVARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The current manuscript entitled “Comparative transcriptomic analysis identifies key genes and regulatory mechanisms of Passiflora edulis in response to chilling stress” is well organized and the findings of this study will provide necessary information for the development of passion fruit with enhanced chilling tolerance. Although, similar results have been reported in a huge number of MS regarding cold tolerance and it is well known that sugars, hormones, and other signaling pathways, etc. play a vital a necessary role in cold tolerance in several plant species. Notably, this study will add additional information to the present knowledge and help passion fruit researchers to continue work at the molecular level. However, this manuscript needs great improvement. Below I have mentioned some minor and major concerns which will help authors to improve the presentation and scientific value of their work.
Experimental design: Check the below comments
Validity of the findings: Check the below comments
Additional comments: Introduction
	Line 56-59, add 1 or more references for these statements.
	Line 59, define LT. Defines abbreviations the first time they appeared in the text. Check the entire document for this issue. Line, 69, 74, 75, 76, etc.
	Line 64, define ICE1-CBF-COR as they appear first time here.
	Line 60-63, expand this paragraph. How cold stress mainly chilling temp affects plant growth and production; and how plant response and adapt to chilling stress concerning morphological, physiological, biochemical mechanisms.
	Further, the authors should discuss the importance of a transcriptome approach with vital experimental proofs.
	Line 79, in this study….during cold stress….. it should be chilling stress. Please be specific while using this term. Cold include both freezing and chilling. Better to use chilling stress in the entire manuscript, figures, tables, captions, etc. Carefully check the whole manuscript and fix this issue.
	Line 80, mention the full name of passion fruit varieties as Huangjinguo (HJG; cold-sensitive) and Tainong1 (TN1; cold-tolerant). After that, authors can use HJG and TN1 in the entire manuscript.
	Line 82, define DEGs.
	Overall, the introduction needs to be rewritten. The authors should emphasize on the objectives of their work and discuss the background accordingly. For instance, low temperature includes both chilling and freezing. According to the title and temperature used in this study (7 oC) is chilling stress. So, the authors should be consistent using this term. Please emphasized on chilling term instead of LT. However, you need to define it as a part of cold stress, after this, stay focused only on chilling stress. LT term must be replaced with CT/CS (chilling temp/chilling stress) in the entire manuscript including abstract, introduction, material and methods, results, discussion, figures, tables, and captions.
Material and methods
	Line 96, replace LT with CT or CS. You can choose any of these two terms and be consistent with it throughout the text.
	Line 86, in plant materials, authors should mention the source of these two genotypes.
	Line 103, A single library requires 1 ug of RNA… Replace “ug” with the symbol of micro “μg”.
	I suggest authors combine RNA extraction and transcriptome sequencing section and concise the detailed information of sequencing.
	Line 126-127, define abbreviations wherever applicable and available.
	Cite a suitable and recent reference for HMMER.
	Line 130, analysis of DEGs. It should be Enrichment analysis of DEGs. Some sentences are poorly written. Please rewrite them in a well-organized style.
	DESeq2, define it as the package of Bioconductor/R.
	Keep the abbreviations inside the parenthesis, not the elaborated form, e.g., TPM, DFR, FC, etc. Check the entire manuscript for this issue.
	Line 146, ddH2O should be ddH2O.
	Line 153, correct the abbreviation within parenthesis (WGCNA).
	Line 157, mention the version of Cytoscape.
	Line 141, section quantitative real-time PCR (142-150) should be transferred to the validation of the cold acclimation-related genes. Combine these two sections and update the title as “validation of the cold acclimation-related genes using qRT-PCR”.
Results and discussion
	Merge Fig 1-3 in one figure and present them as Fig 1A/B/C. Same goes for Fig. 4 and 5. Merge Fig 4 and 5 in one figure and present them as Fig 2A/B/C.
	Line 185, cell composition (CC). It should be cellular component (CC). Check the entire manuscript.
	Line 213, DESeq2 is a package, not a software.
	Merge Fig 8 and 9 in one figure and present them as Fig A/B.
	Line 343-381, authors should relate this part with their findings. Currently, it is just a piece of general information for cold tolerance and accumulation, and the connection is not clear with the results of this study. Similarly goes for Line 382-408, the connection is not clear. The authors should discuss their findings with previous reports. Do not just briefly explain the cold-related mechanisms. Discuss them to support your results.
	Overall, the discussion needs serious improvement to support the authors' findings.

The English language needs great improvement. Please revise the language from a native speaker.


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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS REVEALS THE COLD ACCLIMATION DURING CHILLING STRESS IN SENSITIVE AND RESISTANT PASSION FRUIT (PASSIFLORA EDULIS) CULTIVARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The structure of the article is according to the required format by PeerJ.
The introduction includes sufficient background although it is somewhat inconsistent with the rest of the manuscript (see below). It must show more clearly the context.
Figures are relevant, high quality and appropriately labelled and described. Tables are appropriately labelled.
Experimental design: The research question is well defined and relevant, it is stated how the research fills an identified knowledge gap. The methods are described with sufficient detail and information to replicate, however, a statistical analysis is missing (Figure 11, for example) or not clearly indicated.
Validity of the findings: The statistical analysis of the data must be appropriately described in the “Materials and Methods” section and clearly showed in figures and tables. The conclusions seem to be a small summary of the results rather than an analysis and conclusion of the data found. I recommend rewriting this section, which have to be linked to original research question and limited to supporting results.
Additional comments: The manuscript entitled “Comparative transcriptomic analysis identifies key genes and regulatory mechanisms of Passiflora edulis in response to chilling stress ” by Wu et al. addresses a very interesting topic. The authors performed RNA-seq of two passion fruit varieties HJG and TN1. The first one is considers cold sensitive and the second one is cold resistant. Both varieties were study in two different season when the temperatures were 25ºC (NT, Normal Temperature) and 7ºC (LT, Low Temperature). They made a comparative analysis of the differentially expressed genes (DEGs) between the passion fruit varieties under NT and LT conditions. The RNA-seq data were deposited to National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) by authors. Using different databases as GO, KOG and KEGG, authors acquired annotation information of the unigenes and divided them into hypothetical functional categories.

This study provides an interesting but limited catalog of gene expression profiles. The data about the DEGs under different temperature conditions are also interesting and possibly useful for further research, however, I consider that the analysis that authors did is too much general. Analytic tools as GO, KOG and KEGG give only putative and very general functions of genes, it data can be uninformative and confusing (for example, are the protein phosphorylation and phosphorylation categories actually different as indicated in line 381? or why the catabolic process category is not included into the metabolic process category? line 212-214). It is difficult to obtain conclusive data based on this type of general analysis.

I consider that the authors should make a better analysis of the RNA-seq data focusing on genes with known functions in the regulation of cold tolerance such as genes of the ICE1-CBF-COR pathway. What about the CBP and COR gene expression? In the “Introduction” and “Discussion” sections, they mentioned some of these genes but did not analyze them, they only speculate about the possible roles of the signaling pathways and biological process deduced by GO or KEGG analysis in the cold tolerance phenomenon. Curiously, more than half of the “Introduction” section is about the ICE1-CBF-COR pathway but it is never mentioned in the “Results” section. I think that the lack of a more specific analysis makes the manuscript very speculative.

Additional comments:

I think that a comparative transcriptomic analysis is not sufficient to identify key genes and regulatory mechanisms, it analysis only can identify changes in gene expression, other experiments must be performed to demonstrate whether these changes have a functional impact. I consider that the title and some parts of the manuscript (lines 45-48 and 76-78) should be modified so that it could be in according with the results.
In the “Results” section, the subtitle “Gene expression analysis” is not according with the content of this section, actually the authors are not talking about gene expression analysis.

Minor comments:

A1, A2, B1 and B2 first appear in the Abstract, but their meaning is specified later, in the “Materials and Methods” section. It should be specified the first time it appears in the manuscript.
Major and secondary classification terms of GO, KOG and KEGG should be clearly differentiated from the main text, using italics or quotation marks for example.
Line 112. Change “sequnces” to “sequences”.
Line 135. Change “Tripricates” to “Triplicates”.
Line 370. Change “reduce the damage caused by cold damage” to “reduce the damage caused by cold”
Line 410. Change “collectd” to “collected”


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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS REVEALS THE COLD ACCLIMATION DURING CHILLING STRESS IN SENSITIVE AND RESISTANT PASSION FRUIT (PASSIFLORA EDULIS) CULTIVARS
Review round: 2
Reviewer: 1

Output:
Basic reporting: First of all, I would like to say thanks to the authors for revising the MS according to the proposed comments and suggestions. Notably, the MS has been improved, however, only a few corrections, as mentioned below. Please refer to the track changes file for the line number.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: 	Line 65, add a space between to and cold.
	Line 91-92, keep the abbreviations within the parenthesis, not the elaborated form. Check the entire manuscript.
	Line 313, Fig. 42A. It should be Fig. 2A.


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: COMPARATIVE TRANSCRIPTOMIC ANALYSIS REVEALS THE COLD ACCLIMATION DURING CHILLING STRESS IN SENSITIVE AND RESISTANT PASSION FRUIT (PASSIFLORA EDULIS) CULTIVARS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comments.
Experimental design: No comments.
Validity of the findings: No comments.
Additional comments: The authors have adequately addressed most of the reviewers comments. However, the manuscript still contains some conclusions not completely supported by the results. A comparative transcriptomic analysis reveals changes in gene expression between two or more conditions, showing which genes change significantly. This type of analysis also allows us to suggest whether a gene or genes could have a function in the biological process of interest (chilling tolerance) but it is not enough to determine whether these genes really have a function or not in this particular phenomenon. Additional studies must be carried out to determine the function of the genes and the molecular mechanisms involved. These inconsistent conclusions are found in: line 43-44 “This work was the first systematic study of the molecular mechanism of chilling tolerance in passion fruit”; line 293-294 “To reveal the molecular mechanisms of chilling acclimation of passion fruits under CS, RNA-seq and analysis were performed”; and line 356-358 “32 hub unigenes were assigned to modules and played a regulatory role in the chilling acclimation of passion fruit”. The authors should change theses sentences for others more appropriate to the type of study carried out.

The manuscript contains many typographic and drafting errors: line 31-32 “we still know little is known about how the passion fruit…” instead of “little is known about how the passion fruit…” ; line 52 “cold sress” instead of “cold stress” ; line 177 “(BP),, cellular” instead of “(BP), cellular” ; line 235 “Fianlly, and obtained” instead of “Finally, we obtained” ; line 256 “annlysis” instead of “analysis” ; line 311 “tempreture” instead of “temperature” ; line 352 “omparative” instead of “comparative”. In many cases, space between words is lost.


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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper "Estimating species sensitivity distributions on the basis of readily obtainable descriptors and toxicity data for three species of algae, crustaceans, and fish" presents linear regressions of HC5 and SDs vs Kow.
Introduction: The 1st and 2nd objectives cover all and three species respectively, more or less repeating the Hoondert and Douziech studies. The 3rd objective appears to be to compare the two but that is not clear from the wording. A scheme in the Methods might helpt to explain this.
Experimental design: Desk study
Validity of the findings: The comparisons between 3 and all species, with/without descriptors, are confusing and the tables and figures do not help:
Fig. 1 Caption incomplete text. Legend of different dots (chemical classes? not given. Wording confusing, "estimates based on measured" appears to be just "measured", and "estimates based on developed models" just "modelled". "Only descriptors" should be "Only descriptors mean / SD" to be consistent with the other panels
Fig 2. Now all dots are the same. Yet, for the interpretation is crucial the the MoA is known.
Table 2 I do not understand how to interpret the values, appear to be coefficients of a regression formula not given in the caption.
Additional comments: I can see the added value of comparing 3 vs. all species, based on data only and on regressions with descriptors. Yet, after reading an hour, I still find it hard to understand what is compared where, in particular in the lower panels where models are based on both descriptors and 3 species data, which appears rather unlikely to be applied in regulation. Hence, I suggest to illustrate the calculation by a scheme, simplify the results presented, use consistent legend etc. to get readers like me on board.


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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the article is well written, clear, and easy to follow. Here and there some sentences are very long and could be shortened as suggested in the next comments. The argumentation in the introduction is sometimes not clear enough (LL44-57). The structure is adequate, with some bits that could be moved as suggested in the comments below. The figures and tables are relevant, the raw data supplied.

Abstract: very long sentence staring with “Here,” on L16-20 please shorten and rephrase. Also mention in the abstract that 45 chemicals were used for model fitting and 15 for validation.
Introduction: LL44.57: the line of thought is difficult to follow. I would suggest the author explain the findings in Hoondert et al 2019 first, highlighting the need for models predicting the mean and SD of SSDs separately and the difficulty of accurately modelling SDs using only chemical-specific data (given the argument on L54). The sentence on LL65-69 can be used here.
LL70-72 is too much focused on the methods to be included in the introduction.
Experimental design: The study presents a very interesting and promising approach to derive toxicological information on a chemical in case few data is available. However, the chemical dataset used by the authors is too small in my opinion to ensure a good coverage of the large variability of chemicals encountered. I ask for a major revision of the paper considering (1) the use of a more extended database like the one published in Posthuma et al 2019, (2) the consideration of using experimentally-derived physico-chemical properties instead of estimated ones.
(1)	Posthuma et al., 2019 published an extensive database available upon request with more than 12 000 chemicals, which the authors already cite. I suggest the authors investigate this database and provide a clear explanation why they did not test their suggested approach on this extended dataset. In case such explanation cannot be found, I suggest the authors apply their methodology on this large set of data since it would greatly increase the relevance of their proposed method and its potential use for future research.
(2)	Physico-chemical property predictions are not always accurate so I suggest the authors look for experimental values for the predictors used. This could increase the reliability of their model.
In addition, the following comments should be considered to make the methods clearer:
•	L136: were the means and SDs of the toxicological data of three species calculated per chemical? And the 3000 bootstrapped samples were per chemical, a random combination of three species from algae, crustacean, and fish? I would suggest to rephrase this because this part is very difficult to understand.
•	Did the authors ensure that during bootstrapping, the 3000 sample sets randomly drawn were not the same?
•	LL176-178: belongs to the methods in my opinion
•	Could Table 1 be extended to describe more the chemicals included. Potentially by classifying all chemicals into the Verhaar scheme? Or using the same classification as reported in Hoondert et al 2019.
•	L156: were the models (3) fitted using each time (so for SD and mean) the mean and SD of the three species?
Validity of the findings: LL234-237: I do not agree with this statement: indeed it is difficult to clearly understand the driving factors, but Hoondert et al 2019 do account for the quality of the data (see Figure 1 of the paper) and also test the models on a much larger dataset used (around 300 data points).
L245-256: I understand the point the authors are trying to make here, but the argumentation is not clearly articulated in my opinion. I suggest a rephrasing starting with the sentence on LL249-253, and then explaining that Douziech et al 2020 showed a large uncertainty of estimated HC5 values based on three species because of the lack of data (sampling uncertainty, described in the SI Equations 4-6) and finishing with LL253-256.  At this point, the authors could also mention the uncertainty in their model coming from the fact that the predictors relied on estimated values.
LL282-286: very long sentence, please shorten.
LL288-290: if the authors choose not to extend their database to more chemicals, I recommend to reflect on the number of datapoints used to derive the model in the conclusion too.
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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 1
Reviewer: 3

Output:
Basic reporting: Sufficient field background is provided. I suggest some additional references in General comments. The structure of article is fine. The unit of the paper is self-contained.
Experimental design: The content meets the Aims and Scope of PeerJ. Research question is well defined. I suggest Method section should be more detailed (See General comments).
Validity of the findings: All underlying data have been provided as Supplemental Data. Conclusions are well stated and linked to original research question.
Additional comments: This paper demonstrated the predictive ability of the species sensitivity distribution (SSD) using limited ecotoxicological data. SSD approach is highly useful for the derivation of water quality benchmarks and quantitative ecological risk assessment of chemical substances. However, the quality and quantity of available toxicity data varied markedly among substances, and therefore, estimation of SSD using limited data would promote the practical use of SSD.

In the present study, the predictive ability of SSD was improved using 3-species mean and SD of acute toxicity, as well as previous descriptors (physico-chemical properties and information on mode of action). The analysis is reasonable and well described in the paper. Therefore, I recommend accepting for PeerJ. Minor comments are listed below and should be addressed before being accepted.

1. line 97-99
Categorization of reliability into four classes complies with MANUAL FOR INVESTIGATION OF HPV CHEMICALS: CHAPTER 3 DATA EVALUATION by OECD and Klimisch HJ, Andreae E and Tillmann U (1997) A systematic approach for evaluating the quality of experimental and ecotoxicological data. Reg.Tox. Pharm. 25:1-5. The references should be provided.

2. line 106-110
The references of these criteria should be provided.

3. line 112-114
"the means and SDs" should be logarithmic means and logarithmic SDs. The description should be revised for accuracy throughout the paper. Moreover, the method of SSD analysis (there are multiple ways for determination of mean and SD) should be described. See "Aldenberg and Jaworska (2000) Ecotoxicol. Environ. Saf. 46:1-18".

4. line 114-116
This part should be moved to "Model evaluation (line 152-)".

5. line 119-124
When these descriptors are all estimated values (not measured values), the uncertainty of these estimations would affect the SSD estimation model. The description about this uncertainty should be included in Discussion part.

6. line 127-131
As with comment 5, the uncertainty in estimating mode-of-action classification should be described in Discussion part.

7. line 139
the three species -> each one species from three biological groups?

8. line 142-144
I couldn't figure out what the authors did. More detailed information is necessary.

9. line 265-271
I suggest it is helpful to indicate the difference in the predictive ability of each chemical class.

10. Figure2
More information on deriving 95% confidence intervals is necessary (preferably in Method section, not in the figure caption). Was parametric bootstrapping (n = 3000) applied to the best estimation model? It is confusing with line 138 (produced 3000 bootstrapped sample). Moreover, HC5 should be Log10 HC5 (if correct).


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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 2
Reviewer: 1

Output:
Basic reporting: Presuming that the authors are eager to get their message across clearly, I did not check improvement of wording of the revision.
Experimental design: Though simple, the new figure on the Methods is helpful to quickly confirm whether one's interpretation of the text is correct.
Validity of the findings: I now understand the results. I asked for figures expressing the MoA in the symbols but the authors choose to stick to the train vs test set subdivision. So, I turned to the table to learn that only 1 chemical with a specific MoA was included in the test set. As toxicities of specific MoAs (class 4) and to a somewhat lesser extend reactive MoAs (class 3) are most difficult to predict, the outcomes of the paper are largely restricted to (non-)polar narcosis (class 1-2). Hence, I suggest to include this in the conclusions and abstract. Nevertheless, showing that non-polar narcosis is better predicted by 3 species data rather than overall QSARs is still interesting.
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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors carefully reviewed their manuscript and provided clear and justified answers to each point I raised in the first review. The manuscript now reads better and I believe that all the sentences are in their right section.
Experimental design: I understand the comment of the authors justifying their choice of a smaller database. In fact, the study deserves publication as is, and I appreciate the efforts to state more clearly and often on how many data points the model relies. I really hope the authors test the validity of their approach on a larger dataset in a future study.
I just spotted one mistake in my opinion on LL128 where the authors mention 31 SSDs with information for at least 10 species. Looking at the SI it should be 40, I believe. (Also to reach the 60 chemicals).
Validity of the findings: LL280-281: I still do not agree with this statement: “however, they did not take the quality of each of the toxicity values into consideration.” This is not true since in Figure 1 of Hoondert et al 2019 they do differentiate high and moderate quality data, they therefore excluded low quality data from the analysis. Please rephrase this statement.
Additional comments: Overall, after considering the two minor comments stated above, I believe that this manuscript is 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: ESTIMATING SPECIES SENSITIVITY DISTRIBUTIONS ON THE BASIS OF READILY OBTAINABLE DESCRIPTORS AND TOXICITY DATA FOR THREE SPECIES OF ALGAE, CRUSTACEANS, AND FISH
Review round: 2
Reviewer: 3

Output:
Basic reporting: I evaluated in the first review.
Experimental design: I evaluated in the first review.
Validity of the findings: I evaluated in the first review.
Additional comments: I checked that my first review comments were properly addressed in the revised 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: INFLUENCE OF WATER AVAILABILITY AND TEMPERATURE ON ESTIMATES OF MICROBIAL EXTRACELLULAR ENZYME ACTIVITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Generally well written, and very well referenced.
Experimental design: See below
Validity of the findings: General Point
The authors appear to have ignored the fact that the three enzymes chosen are all hydrolases (requiring water for their hydrolytic reactions).  Reductions in catalytic rates may therefore be the consequence of substrate limitations (at some point), accepting that [H2O] substrate is 55M at Aw=1. This issue could be addressed by expanding the enzyme range to include some non-hydrolytic enzymes.

Methodology
Line 169 onward.  Having spent some time explaining that soils are highly heterogeneous, and soil microbes are found in a wide range of microenvironments in fact, most in biofilm assemblages within the interstices of soil conglomerates and particles), the authors then use a protocol which involves the supplementation of soils with pure microbial cultures.  These cells are likely to mostly be adsorbed to soil particle surfaces, will not be in biofilms, and are unlikely to represent the ‘native state’.
Given this concern, the authors would be advised to provide more basic information on the mineralogy of their two samples (sand/clay/silt content; particle size range).

Line 199.  2M phosphate buffer – this is an extraordinarily high buffer concentration, and would certainly affect the ionic environment of the enzyme.

Line 209 – specify the substrates (for those readers who don’t have instant access to Gonzalez et al 2015).

Line 207 onward.  This is a complicated protocol, with many points at which the fluorogenic substrates (which are known to be reasonably labile) might be autolysed non-enzymatically.  I don’t have the references to hand, but I do remember old literature which shows that some soils (clays?) are quite potent catalytic surfaces. I just don’t see the evidence for the negative controls.
I am particularly concerned about the possible abiotic effects on the pH adjustment (pH 11, line 225), since many of the chromogenic and fluorigenic substrates are VERY subject to alkaline hydrolysis.
I also have some concerns about the freeze-drying process for establishing the ‘desired’ water activity. It is not clear how the ‘desired’ water activity was reached – was freeze-drying terminated at intervals, the sample thawed and tested with the Rotronic probe, then f-d some more?  Aw measurements with the probe could only be done on thawed samples (at constant temperature).

Results
I am struggling to understand, mechanistically, how enzyme activities can reduce at high Aw levels (Fig 3).  These are all hydrolytic enzymes, so water is involved in the enzymic reactions, and reduced rates at lower aw levels are therefore entirely to be expected (apart from the effects of low Aw on the enzyme structures).
I can’t help feeling that the reducing ‘activities’ at high Aw levels (Figs 3A, 3B) are the product of some uncharacterised artefact.  The authors attempt to interpret these results in an ecological context (hot dry southern Spain vs cool wet northern Spain: that the microbial communities, through their enzymes, adjust their physiology to the environment (differential adaptation patterns) are not wholly convincing. At the very least, the authors should include macro/microenvironmental data from the locations to support these statements.
There are also some other data that make me very worried that the data are largely artifactual – for example, Fig 3C (pale squares).  I find it quite inexplicable that enzyme activity would increase so dramatically from Aw 0.9 to Aw 0.95 (which represents a rather small change in water availability). Basically, I think that there is more going on here than can be explained.

I would want to see substantive supporting data before I could accept this rather ‘circumstantial’ ecological correlation. I just don’t think that the data are strong enough, particularly in light of the complexity and uncertainty of the assay systems (see comments above), for such broad fundamental conclusions.

Minor issues
Line 308 and elsewhere – the term ‘soggy’ is a colloquialism!
Additional comments: Despite the fact that there could be something very interesting here, my view is that there are just too many concerns and unanswered issues in this piece of work. Even if the assay results are accurate and valid, the attempt to place them in a macroecological and evolutionary context is just a 'step too far'.


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: INFLUENCE OF WATER AVAILABILITY AND TEMPERATURE ON ESTIMATES OF MICROBIAL EXTRACELLULAR ENZYME ACTIVITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall this manuscript is well-written and concise. I listed a few sections below that you may consider revising.

Title: It is a bit strange/confusing that it has a period in the middle.

Lines 111-115: Awkward sentence.

Lines 151-154: Awkward sentence.

Lines 384-387: Awkward sentence.

Line 389: You only list one study here, but mention previous "reports".

Figure 1: Can you indicate which point corresponds to which enzyme and make the error bars more clear? Additionally, please indicate that the axes are in the log scale and add the correlation coefficient to the plot.
Experimental design: This study presents a new approach to measuring extracellular enzyme activity in soil and explores the effect of water availability on extracellular enzyme activity. Soil moisture level is integral part of microbial ecosystem response, yet the assays for measuring extracellular enzyme activity rely on an aqueous solution. Thus, this study is a very exciting contribution to the field of soil microbial ecology. However, I have a few concerns/questions about the methodology that should be clarified or explained. Particularly, how do you know that the measured enzyme activity is not just a reflection of how well you mixed the substrate with the soil? I understand the purpose of the procedure is to better replicate in situ conditions, but with such short incubation time, it seems like this enzyme activity would be highly based on mixing success.

Other minor comments:
Lines 170-173: I’m assuming these bacterial strains were isolated from soil at these sites, but I didn’t see that explicitly stated in the manuscript.

Lines 177-179: What was the water activity or capacity of the soil when it was autoclaved? Was the water activity different for the different soils/treatments? The amount of water can affect how well the soil was autoclaved.

Lines 207-234: Can you add a photo or figure detailing this procedure? I don’t quite understand how you added the substrate in a non-aqueous solution, which is important since this is a major focus of your manuscript.

Lines 220-221: Can you show an example kinetic curve from this study? I think it may be helpful since this is a new method and readers might have questions about incubation times and substrate saturation when conducting their own experiments and to show that you met these criteria as well.
Validity of the findings: With additional clarification of the methods, I think the findings will be better supported. However, overall I found the conclusions reasonable based on the evidence provided. I have also listed some other minor concerns below.

Other minor comments:
- There seems to be an underlying assumption throughout the manuscript that optimum growth temperature, optimum temperature of enzyme activity, and mean environmental temperature are the same. However, this is likely not true (see, Prentice et al., Biochemistry 2020 and Alster et al. 2018 Glob. Chang. Biol.). I would make sure to clarify this throughout the text.

Line 256: If the results are equivalent, what is the advantage of using this new method?

Lines 366-368: This is a strong claim. Did you provide statistical evidence for each of these factors in the manuscript?
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: INFLUENCE OF WATER AVAILABILITY AND TEMPERATURE ON ESTIMATES OF MICROBIAL EXTRACELLULAR ENZYME ACTIVITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors did a great job revising this manuscript and incorporating the relevant comments from myself and the other reviewer.

Minor comments:
1. Lines 173-178: I appreciate that you added this sentence, but it is very long and therefore confusing to read.
2. Line 198: Is there a typo here? Do you mean “scheme” instead of “schee”?
3. Figure 3: Are the values in the log scale or are the axes? The way this has been changed from the prior draft is confusing.
4. Line 143: Change “on” to “of”
Experimental design: The addition of Figure 1 was very helpful in clarifying the methods. Thank you for adding that. However, I still have some questions about the incubation times. In particular:

1. Lines 215-222 and 253-255: Was the total incubation time only 10 minutes for all of the different temperatures and moistures? It would be helpful if you could clarify this specifically in the methods. That seems on the shorter side for the lower temperature incubations.
2. How do you know that when you did the drying step you didn’t remove some of the florescent substrate? Maybe you explained this somewhere and I didn’t understand, but I think this is important to clearly demonstrate or note. I think that is maybe what you are showing in Fig. 2, but you only show the high moisture level.
Validity of the findings: The authors narrowed the scope of the discussion from its prior version and it now better represents the findings.
Additional comments: I would like to see how the authors address the substrate saturation and incubation time questions, but overall this seems to be an exciting new method that I would be eager to try in my own experiments. Also, interesting result of peak EEA at lower moisture levels for some of the species. Very relevant to the field - Nice job!


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: CRANIAL ORNAMENTATION IN THE LATE CRETACEOUS NODOSAURID ANKYLOSAUR HUNGAROSAURUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: An excellent description of the cranial elements of Hungarosaurus.

The only glaring weakness is the lack of comparisons with other European nodosaurids such as Europelta and Struthiosaurus for which cranial material is known.

Figures are well done.

The supplemental rotating skull models are exceptionally informative.

It should be noted that the holotype skull of Europelta also has unfused nasals (Kirkland et al., 2013),
Experimental design: Wonderful no comment!
Validity of the findings: In full agreement the conclusions regarding the origin of the cranial ornamentation.
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: CRANIAL ORNAMENTATION IN THE LATE CRETACEOUS NODOSAURID ANKYLOSAUR HUNGAROSAURUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clear and unambiguous. Most issues with the manuscript were grammatical, particularly with sentence structure and the correct use of verbs/pronouns etc.
Literature review is thorough, however, a couple of issues arose – see suggestions below and in text. Tables of data are excellent and thorough.
Structure conforms to industry norms
Figures are also good, although I have made some suggestions below.
Experimental design: Original research in terms of the group nodosaurids, but based on previous work for the sister group ankylosaurids. Nice to see it finally happen!
Research question is well defined and relevant. ‘Gap’ in the literature answered. Would like to see mention of future work for other nodosaurids. Investigation was thorough and methods sufficiently detailed.
Validity of the findings: All data has been provided, and arguments and inferred conclusions are sound. I would like to see a sentence or two about potential future work and the what the conclusions infer for nodosaurids in general.
Additional comments: Corrections/Suggestions
Main text
I have also made minor corrections/suggestions within the text document – see the manuscript document in conjunction with comments below

no need for a comma after using – e.g.    see lines 44, 53, 54, 61 etc.
no need for a comma after using – i.e. see line 75

Line 48-49: Here you list ‘cranial elaboration’ first, and ‘fusion of osteoderms’ second, however in the main text your information and arguments discuss ‘fusion of osteoderms’ first and ‘cranial elaboration’ second. To maintain flow throughout the text, consider swapping how you first list the options.

Line 63: (as well as throughout text and in reference list)  Maryańska not Maryanska

Line 73: Molnar (1996) provides a description only of the cranial ornamentation. He goes into further detail in Molnar (2001 – within Carpenter’s The Armoured Dinosaurs). In either texts, he does not discuss the ontogenetic origin of the cranial ornamentation. It is discussed in Leahey et al. (2015) Cranial osteology of Kunbarrasaurus ieversi (see pgs 36-7). Furthermore I would not consider Kunbarrasaurus an ankylosaurid, the most recent published phylogenetic analyses and my own work on it place it as a basal ankylosaurian.

Line 75-9: Sentence is very convoluted, consider revising to:
Based on the investigation of multiple specimens of the ankylosaurids Euoplocephalus and Pinacosaurus, including material attributed to subadult (i.e., not skeletally mature) individuals, the cranial ornamentation of these forms are interpreted involving both the coosification of osteoderms with the skull and the exaggerated outgrowth of individual cranial elements (Vickaryous, Russell & Currie, 2001; Hill, Witmer & Norell, 2003; although see Carpenter et al., 2001).
* This is also the process of cranial ornamentation in Kunbarrasaurus ieversi  (Leahey et al 2015)
Line 80: Basal ankylosaurian Kunbarrasaurus ieversi  (Leahey et al 2015) has both osteoderms and cranial elaboration

Line 82-4: I’m not sure if this is the best example of a nodosaurid to discuss the group in general. The nodosaurid noted (SMU 7244 – Jacobs et al, 1994). Is a VERY juvenile specimen, and thus osteoderms may not yet have formed (i.e. similar to extant crocs – osteoderms do not develop very early). Also, the specimen is very fragmentary, and osteoderms if present may not have preserved. I think that this needs mentioning at least.

Line 90: I think here would be a good place to introduce your use of extant forms to help in your understanding of cranial ornamentation. I was a bit confused when you went straight into extnant forms from ankylosaurs. Just a sentence or 2 is needed. - e.g. Herein we investigate and utilize extant forms of sauropsids to better understand the processes of ontogenetic development in ankylosaurians.

Line 111: maybe add...Thus fusion of osteoderms to the underlying cranial elements is species-specific process????

Line 112: it is unclear to which skulls you are referring to

Line 175-6: Consider rephrasing:
…..from that of the holotype of Hungarosaurus. Herein we interpret this difference as either a factor of ontogeny or sexual dimorphism (see discussion below).

Line 230: longitudinal is an unclear direction, use anatomical directions e.g. lateral. Also, here you use ‘profile’ but you have used ‘view’ throughout you text, best to be consistent throughout

Line 238-41: It is unclear to which specimen you are referring to here, and similarly in the next paragraph (Lines 242-246), always good to use the specimen numbers to be ultra clear

Line 258: grooves are they 1-5mm wide? Long?

Line 263: do these channels reach the surface or end within the compact bone?

Line 278-9: Consider revising, to make clear:
The postorbital elements that underwent microCT (Fig. 4D-F), all reveal cancellous bone…..

Line 280-2: Consider swapping the sequence of the first two sentences – it will help with flow

Line 338: possibly add at the end of the sentence: (discussed further below)

Line 343-4: “co-osification of multiple elements” I’m a little confused by this term. Are you referring to the coossification of dermal elements or cranial elements? i.e. for the latter, as is the case for Pinacosaurus grangeri; multiple supraorbitals
Need to define which elements are coosifying: dermal or cranial elements

Line 349: At no point in this manuscript or in the supplement can I find any reference/description/discussion on ‘cornified sheath’. This either needs to be expanded upon as to why you interpret this or remove from the manuscript.

Line 359: ‘non-ankylosaur ankylosauromorphs’ I would hesitant in using this term as it has yet to be confirmed with recent phylogenetics. And the phylogenetics that Norman refers to ‘Carpenter 2001’ is consider unconventional. Probably safer using the term ‘non-eurypodan thyreophoran’
Also, the reference should be Norman 2020: the postcranial description NOT the cranial description as listed here and in the reference list.

Line 336-362: This form of cranial ornamentation also occurs in the basal ankylosaurian Kunbarrasaurus ieversi (Leahey et al., 2015)

Line 411: It would be great to see a sentence or two about potential futurework and the what the conclusions infer for nodosaurids and ankylosaurians in general.

Figures
General comments to consider
Place scale bar in areas where it is not so busy
Some of the microCT images are a bit fuzzy, consider increasing the black/white contrast to exaggerate the differences in fusion
Figure 1:
1. This image was a little hard to follow. When you have multiple groups or related images within one figure consider using ‘Ai, ii, iii; Bi, ii iii’ instead of A, B, C, D etc. Then the reader knows the relationships without having to search through the figure caption multiple times
2. For the focused/magnified images B, D, F & H. Consider placing a small box shape on the corresponding whole cranium photo (A, C, E, G) to show the area of magnification OR label the corresponding bones on the whole cranium photo that are labelled in the magnified image.

Figure 2: put a scale in for the basicranium only

Figure 4: (line 780) depressed “lip”

Supplementary data notes
1. It is unclear to which fused sutural contacts you are referring to: pmx-pmx, nas-nas, or pmx-nas?
2. ‘shows some of’ - as your previous sentence states otherwise.
3. ‘large’ is an ambiguous term, large relative to what?
4. Add ‘with an’
5. consider revising to: All these are diagnostic features of Hungarosaurus (ref), and are present on MTM PAL 2020.31.1.
6. need to label on figure
7. Why does the blood vessel only supply ornamentation? If it is an ontogenetic feature than the bone element itself would be growing as well (?).
8. This dimension is unclear.
9. Consider revising this sentence to: The outer surface is strongly weathered (and relatively thin, max. thickness of 4-6mm) and thus not overly informative, but …..
10. Are the sutures preserved in other specimens of Hungarosaurus?
11. Reconsider revising to:
The nasals are at least twice as anteroposteriorly long than wide (although anteriorly they are not completely preserved), dorsally highly ornamented (for details see main text), trapezoid elements. At the nasal-frontal contact, the nasals overlap the anterior process of the frontals (Suppl. Fig. 2A, B).
12. Needs to be labelled on figure
13. Reconsider revising to:
In dorsal view, the squamosal is laterally- posterolaterally oriented and bears no significant ornamentation. Only some rugose texture can be seen on its mediodorsal surface (Suppl. Fig. 2E), extending towards the exoccipital. This may have been served for the attachment of the dorsal neck muscles.
14. Reconsider revising to:
Based on the diagnostic characters of Hungarosaurus tormai; the postorbital bearing a high and anterodorsal–posteroventrally elongated crest, and the mandibular quadrate condyle having rhomboidal articular surface (Ősi et al. 2019), we herein assign MTM PAL 2020.32.1 to Hungarosaurus. However the very slight differences in the morphology of these characters in conjunction with the relatively large orbits, unfused postorbital bones and the relatively small size of the skull suggests a subadult ontogenetic stage. Thus herein MTM PAL 2020.32.1. is consider an ontogenetically immature form of Hungarosaurus tormai.


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: CRANIAL ORNAMENTATION IN THE LATE CRETACEOUS NODOSAURID ANKYLOSAUR HUNGAROSAURUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Introduction:
The introduction section is rather disjointed and does not really ties the topics together with regard to nodosaur cranial ornamentation and the lack of data regarding cranial osteoderm ontogeny, sexual dimorphism and taxonomic diversity. The introduction steers away from the central topic of this manuscript, which focuses chiefly on the cranial ornamentation of Hungarosaurus. The authors briefly state that they describe fragmentary skulls and elements that potentially represent different ontogenetic stages or sexually dimorphic individuals, but the introduction fails to clearly mention what research questions or problems the authors are aiming to answer with these specimens in the broader (taxonomic) sense and why this topic is scientifically relevant to ankylosaurian dinosaurs, and nodosaurids in particular.
Also, the cranial ornamentation in ankylosaurian dinosaurs is discussed very generally and briefly in two short paragraphs and would benefit from being expanded upon. Instead, the focus shifts heavily towards more extant archosaur and lepidosaur taxa cranial osteoderm morphology, ontogeny, and diversity. I understand what the authors are attempting to demonstrate with this comparative approach, but it loses the focus on ankylosauria, and nodosaurids in particular, entirely. The introduction would benefit from restructuring, focusing on the lack of cranial osteoderm ontogenetic and sexually dimorphic data across a wide range of ankylosaur taxa and how their data has the potential to elucidate some of these problems in extinct forms. Include the extant data because it is quite informative with regard to said topics, but it needs to be tied back together better to the central topic of the manuscript: Hungarosaurus and Ankylosauria more generally or nodosaurids more specifically. In its current state, the paragraphs on extant data are standalone and appear as separate sections that don't properly contextualize with Hungarosaurus.

Materials & Methods:
The authors utilized extant taxa as well for comparative purposes. These specimens need to be listed in this section as well, including the purpose of CT scanning (clearly for comparative analysis), equipment used and slice thickness for each specimen. State how many individuals of each species where scanned, where the specimens are from and housed, including specimen numbers.

Discussion:
The discussion section overall doesn't flow very well and does not really add much in terms of context or putting the data together. What does it all mean, and how can we utilize/implement the findings when continuing studies on ankylosaur cranial ornamentation? The discussion repeats largely what has already been mentioned in the Introduction section and thus feels repetitive and therefore redundant. The discussion needs to be re-written and focus clearly on the characteristics of these cranial features in the Hungarosaurus specimens, supported by comparisons made with the extant taxa. The authors have great CT data for both extant and extinct taxa and I think if you combine the similarities and differences (aided by an extra figure or two to highlight these difference/similarities) between extant taxa and Hungarosaurus, the authors can discuss the implications of these multiple Hungarosaurus specimens and cranial osteoderm variety in a much more focused sense.
Also, the authors emphasize dimorphism and cranial outgrowth in birds, osteoderm presence and coosification in squamates etc, based on CT data analysis. However, I would stress to also discuss and observe the possibility of interspecific variation in the taxa observed and discuss the possibility of this also (partially) being the case for Hungarosaurus. Phenotypic plasticity is very common in extant taxa, and has been well documented (especially in lizards, anoles, bats, etc), but it is surprisingly often overlooked in extinct taxa (even in cases where large numbers of specimens from a single population are available for study). I think it would be beneficial for the manuscript and the hypotheses stated to strengthen the arguments by:
1) including more extant specimens of the same species studied to observe the level of variation of osteoderms or bone outgrowth in adult males, females, males and females and juveniles. Compare for intraspecific and ontogenetic variation of cranial ornamentation and highlight the major differences/similarities.
2) compare these results with the CT data and qualitative data of Hungarosaurus ornamentation to better discuss the hypotheses of excessive outgrowth, potential sexual dimorphism and ontogeny in Hungarosaurus. This will result in a much stronger and more focused, to-the-point discussion.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: First of all, I would like to thank the Editors for letting me review the manuscript titled: "Cranial ornamentation in the Late Cretaceous nodosaurid ankylosaur Hungarosaurus". I very much enjoyed reading the manuscript, and it is overall well written and provides important new data regarding the ontogenetic stages of cranial ornamentation for ankylosaurian dinosaurs; a topic that is in continuous need of study for this generally poorly understood group of dinosaurs.
I really like the figures; they are very clearly illustrated and professional. I made a few minor suggestions in the pdf but overall, they are effective and well done.
There are a few issues in this manuscript that should be addressed before the paper can be accepted for publication, and I highlighted these in the Basic Reporting section. Additionally, I provided an annotated pdf with minor comments, consisting mostly of typo corrections and some grammatical issues and some suggested revisions.
The paper has a lot of potential and is important for thyreophoran research, but in its current state it reads more like a descriptive paper rather than really focusing on the implications of the different states and stages of cranial ornamentation in the discussed Hungarosaurus specimens. It misses comparison with the extant CT data to support and contextualize the authors' arguments. This is unfortunate, because there is merit in this paper and the topic of ankylosaur cranial ontogeny and potential sexual dimorphism in cranial ornamentation is heavily understudied, making this manuscript a very much needed addition to this field of study.
I think the authors should think about adding a little more data (see suggestions above) and re-write the aforementioned sections to strengthen the arguments more convincingly. I would like to see these suggestions implemented and corrections addressed before the paper can be accepted for publication.
I am looking forward to read the published work.


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

Input:
Title: COMPARISON OF CIRCULATING TUMOR CELL (CTC) DETECTION RATES WITH EPITHELIAL CELL ADHESION MOLECULE (EPCAM) AND CELL SURFACE VIMENTIN (CSV) ANTIBODIES IN DIFFERENT SOLID TUMORS: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments:
This is an interesting study investigating the CTC detection rates with EpCAM and CSV capture antibodies in different solid tumors. The findings provide evidence that EpCAM can be used in CRC, LC, GC, BCa, EC, HNSCC, CC and PCa, but not suitable in PDAC, HCC and OC, while CSV can be used in most solid tumors for CTC detection.
The manuscript is well designed and the analyses are sufficiently performed. I have some comments to mention:
Question 1: The method and result part are not clear enough to read and to understand. The authors should revise these parts. It should be easily to read and give the reader the chance to understand.
Question 2: The authors should describe clearly that how many patients were captured CTCs with EpCAM and CSV antibodies separately and both? The numbers in the manuscript are very confusing. Please make it clear.
Question 3: The patients’ clinic-pathological characteristics are described poorly. The authors should give an exact overview of disease histology, grade, stage, the types of surgery, and IHC staining data and disease status.
Question 4: The authors should provide the patients’ status at the time of blood draw, pre- or post-surgery, pre- or post-chemotherapy or immune therapy, first-line or second line therapy, et al.
Question 5: The authors should describe the method of data collection in method and material parts, including clinical data collection and blood sample collection.
Question 6: The authors should provide the inclusion and exclusion criteria of enrolled patients and healthy donors in methods and materials part.
Question 7: Typing error: the authors should double check the number of enrolled patients. In abstract part, the total number is 695, but in method part, the number is 690.


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: COMPARISON OF CIRCULATING TUMOR CELL (CTC) DETECTION RATES WITH EPITHELIAL CELL ADHESION MOLECULE (EPCAM) AND CELL SURFACE VIMENTIN (CSV) ANTIBODIES IN DIFFERENT SOLID TUMORS: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors describe and compare detection of CTCs with classical EpCAM-based approach and CSV-based method in order to capture more mesenchymal CTCs.
The use blood of different cancer patients and of healthy persons.
For a small subset they test both antibodies in the the same blood sample. The observe different positivity rates for both approaches in different tumor entities with CSV slightly outperforming EpCAM.

- language good: only small mistakes
- literature: sufficient
- structure: good, tables are large and complicated and may be placed in a supplement, Figures are small and hard to read.
- discussion is a bit redundant to introduction
- please change the abstract. too many details
Experimental design: - is within scope
- question is well defined
- instead of discussing the potential reason of false-positive "CTCs" the authors could have verified tumor origin by single cell aCGH or low pass sequencing. It would anyway be good to have executed this on a few EpCAM and CSV-positive CTCs. In addition, comparison of such cells from the same patient may point towards different origin of EpCAM and CSV-positve cells.
- antibody clones should be provided
Validity of the findings: - see above (aCGH and low pass)
- there is hardly any statistically significant outcome (it should be marked in the figure)
-
Additional comments: I have made a few comments in the PDF.
- please change the abstract. too many details


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: COMPARISON OF CIRCULATING TUMOR CELL (CTC) DETECTION RATES WITH EPITHELIAL CELL ADHESION MOLECULE (EPCAM) AND CELL SURFACE VIMENTIN (CSV) ANTIBODIES IN DIFFERENT SOLID TUMORS: A RETROSPECTIVE STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English in the manuscript is proficient and professional. It is unambiguous for most part except a few lines (line 74, Available should be replaced with discovered/found, line 87-88) Never use etc. (line 63) in a manuscript. Be definite about what you are stating.

Introduction needs more detail. It focuses about EpCAM and CSV, it would be pertinent for authors to describe other CTC markers that are under study and describe why did they chose to focus on these particular markers.

Last section of introduction focuses more on the CytoSorter and that should be a part of the methods section and not introduction.

Figure quality is adequate. In Fig 1, the merged field should always be on the right (last image) by convention and not the first. In Fig 2, the authors need to mention what does the red line signify in Fig 2A and B. Fig D is too small to appreciate the data!
Experimental design: 1] The biggest concern I have is that the authors are using a CytoSorter system and for monitoring studies like this a flow based approach is highly adopted for you run a high number of cells to give a high power for statistical significance.  Stating number of CTCs, in each sample lacks context. How many cells were actually analysed, what is the percentage in those cells etc.

2] Staining for CSV and the EpCAM should have been performed on the same aliquot together so that would have answered:
a] Are these markers exclusively expressed on cells or have a concurrent expression? Figure 2D would have made more sense if they would have done a flow and then measured it.
Why flow wasn’t used for this study?

3] Authors should have first established the EpCAM and CSV expression in different cancers through TCGA. ( A figure pertaining to it would be nice). That builds up the base for the manuscript, which it currently lacks apart from the explanation in the discussion section. Is there a correlation with EpCAM and CSV expression ?
Validity of the findings: Line 159-162 and 173 – 174 does not inspire confidence in me. “Healthy patients have less than 1 and Cancer patients usually have more than 2 CTCs detected”. I infer that many patients have also 0 or 1 CTC detected the same as healthy! There is not much of a difference between the healthy and cancer. Since, the average EpCAM detection rate is ~62% and ~70% for CSV , the authors wouldn’t be able to confidently tell the healthy and patient apart using this methodology if given a sample blind folded. Again, this would have been overcome if a flow based approached would have been used.

Line 167-168 has data not shown! To be open to the scientific community, I urge the authors to share the data.
Additional comments: It is an extensive study with many different tumor types and numbers.