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 ENVIRONMENTAL FACTORS ON THE SPREAD OF COVID-19 IN SAUDI ARABIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Zones have to be clarified.
see additional comments
Experimental design: It can be improved with a clear structure.
Zones have to be clarified.
see additional comments
Validity of the findings: It can be imporved in the visual presentation with figures.
Zones have to be clarified.
see additional comments
Additional comments: Investigating the environmental factors that influenced the spread of Covid-19 in Saudi Arabia

The topics of this paper are  interesting, but the structure and content must be revised, and results have to be  better explained by authors before to be reconsidered for publication.

Title has to be shorter. COVID-19 and not Covid-19 in all text.

Abstract has to be shorter focusing on results and environmental and social implications. The presentation of all these zones create confusion…try to clarify that these are climate zones and if the COVID-19 is higher in dry or wet zones, hot or cold zones etc.….

Introduction has to better clarify the research questions of this study and provide more theoretical background. Authors have to better describe the different sources of transmission dynamics of COVID-19 (e.g., mobility, environment, pollution, etc.) and risk factors in society, that can  accelerate  diffusion of this novel coronavirus in this country  (See suggested readings that must be all read and used in the text).

Methods of this study is not clear. The section of Materials and methods must be re-structured with following three sections:
•	Research setting, sample and data
•	Measures of variables
•	Data analysis procedure.

Results. Authors have to avoid subheadings that create fragmentation and confusion. If necessary, can use bullet points.
Table 1 can remove the columns with p, because significance is indicated with *, **. Also, lower and higher 95% can be removed because results can be redundant. Table has to be simple.

Figure 1 has to insert a note clarifying the geoclimatic zones. To explain them in the text is not enough for clearness for readers. Total number should be normalized with total swabs or population within zones under study.

Figure 2 specify on y-axis that it is temperature in °C
For all figures, authors have to insert a note clarifying the geoclimatic zones. As said, to explain them in the text is not enough for clearness for readers.

Discussion has to consider suggested papers to reinforce results on wind speed, air pollution and diffusion of COVID-19.

Conclusion is too short and has no sense….it has not to be a summary, but authors have to focus on manifold limitations of this study and provide suggestions of environmental, health, crisis management  and social policy. In particular, authors have to suggest best responses of prevention and preparedness of next waves of COVID-19 and similar pandemics.

Overall, then, the paper is interesting. Theoretical framework is weak, and some results create confusion… structure of the paper has to be improved; study design, discussion and presentation of results have to be clarified using suggested comments.

If the paper is improved,  by using all comments,  maybe it can be considered.

Suggested readings of relevant papers that have to be read and all inserted in the text and references.

Meo, S.A., Abukhalaf, A.A., Alomar, A.A., (...), Al-Khlaiwi, T., Usmani, A.M.	2020	Effect of temperature and humidity on the dynamics of daily new cases and deaths due to COVID-19 outbreak in Gulf countries in Middle East Region, European Review for Medical and Pharmacological Sciences 24(13), pp. 7524-7533

Zhu Y., Xin J. 2020. Association between ambient temperature and COVID-19 infection in 122 cities from China, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2020.138201

Srivastava, A.	2021. COVID-19 and air pollution and meteorology-an intricate relationship: A review, Chemosphere, 263,128297

Coccia M. 2021. Preparedness of countries to face covid-19 pandemic crisis: Strategic positioning and underlying structural factors to support strategies of prevention of pandemic threats, Environmental Research, n. 111678, https://doi.org/10.1016/j.envres.2021.111678

Bashir, M.F., Bilal, B.M., Komal, B., 2020. Correlation between environmental pollution indicators and COVID-19 pandemic: A brief study in Californian context. Environ. Res. 109652.https://doi.org/10.1016/j.envres.2020.109652

Xu K, Cui K, Young L-H, Hsieh Y-K, Wang Y-F, Zhang J, et al. Impact of the COVID-19 Event on Air Quality in Central China. Aerosol and Air Quality Research 2020b; 20: 915-929.

Coccia M. 2021. The impact of first and second wave of the COVID-19 pandemic: comparative analysis to support control measures to cope with negative effects of future infectious diseases in society. Environmental Research, vol. 197, June, n.  111099, https://doi.org/10.1016/j.envres.2021.111099

Rosario Denes, K.A., Mutz Yhan, S., Bernardes Patricia, C., Conte-Junior Carlos, A., 2020. Relationship between COVID-19 and weather: case study in a tropical country. Int. J. Hyg Environ. Health 229, 113587.

Coccia M. 2021. Comparative Critical Decisions in Management. In: Farazmand A. (eds), Global Encyclopedia of Public Administration, Public Policy, and Governance. Springer Nature, Cham. https://doi.org/10.1007/978-3-319-31816-5_3969-1

Askitas, N., Tatsiramos, K., Verheyden, B. 2021. Estimating worldwide effects of non-pharmaceutical interventions on COVID-19 incidence and population mobility patterns using a multiple-event study (Open Access)(2021) Scientific Reports, 11 (1), art. no. 1972.

Coccia M. 2020. Factors determining the diffusion of COVID-19 and suggested strategy to prevent future accelerated viral infectivity similar to COVID, Science of the Total Environment, Number: 138474. https://doi.org/10.1016/j.scitotenv.2020.138474

Shen, L., Zhao, T., Wang, H., (...), Zhu, Y., Shu, Z.	2021Importance of meteorology in air pollution events during the city lockdown for COVID-19 in Hubei Province, Central China		Science of the Total Environment, 754,142227

Haque, S.E., Rahman, M.	2020. Association between temperature, humidity, and COVID-19 outbreaks in Bangladesh, Environmental Science and Policy, 114, pp. 253-255

Yuan, J., Li, M., Lv, G., Lu, Z.K. 2020. Monitoring transmissibility and mortality of COVID-19 in Europe ((2020) International Journal of Infectious Diseases, 95, pp. 311-315.

Coccia M. 2020. An index to quantify environmental risk of exposure to future epidemics of the COVID-19 and similar viral agents: Theory and Practice. Environmental Research, n. 110155, DOI: 10.1016/j.envres.2020.110155

Sarkodie, S.A., Owusu, P.A.	2020. Impact of meteorological factors on COVID-19 pandemic: Evidence from top 20 countries with confirmed cases. Environmental Research, 191,110101

Coccia M. 2021. The effects of atmospheric stability with low wind speed and of air pollution on the accelerated transmission dynamics of COVID-19. Journal: International Journal of Environmental Studies, vol. 78, n. 1, pp. 1-27, https://doi.org/10.1080/00207233.2020.1802937

Flaxman, S., Mishra, S., Gandy, A., Unwin, H.J.T., Mellan, T.A., Coupland, H., Whittaker, C., (...), Bhatt, S. 2020. Estimating the effects of non-pharmaceutical interventions on COVID-19 in Europe (Open Access), (2020) Nature, 584 (7820), pp. 257-261.

Diao, Y., Kodera, S., Anzai, D., (...), Rashed, E.A., Hirata, A.	2021. Influence of population density, temperature, and absolute humidity on spread and decay durations of COVID-19: A comparative study of scenarios in China, England, Germany, and Japan, One Health, 12,100203

Coccia M. 2021. How do low wind speeds and high levels of air pollution support the spread of COVID-19? Atmospheric Pollution Research, vol. 12, n.1, pp. 437-445., https://doi.org/10.1016/j.apr.2020.10.002.

Rahimi, N.R., Fouladi-Fard, R., Aali, R., (...), Conti Gea, O., Fiore, M.	2021	Bidirectional association between COVID-19 and the environment: A systematic review	Environmental Research, 194,110692

Islam, N., Bukhari, Q., Jameel, Y., (...), Massaro, J.M., D'Agostino, R.B.	2021, COVID-19 and climatic factors: A global analysis. 	Environmental Research, 193,110355

Coccia M. 2020. How (Un)sustainable Environments are Related to the Diffusion of COVID-19: The Relation between Coronavirus Disease 2019, Air Pollution, Wind Resource and Energy. Sustainability 2020, 12, 9709; doi:10.3390/su12229709

Alkahtani, T.A., Alakeel, A., Alakeel, R.A., (...), Abdel-Daim, M.M., Jammah, A.A.	2021. The current reproduction number of COVID-19 in Saudi Arabia: is the disease controlled? Environmental Science and Pollution Research28(33), pp. 44812-44817

Coccia M. 2021. The relation between length of lockdown, numbers of infected people and deaths of Covid-19, and economic growth of countries: Lessons learned to cope with future pandemics similar to Covid-19. Science of The Total Environment, n.  145801. https://doi.org/10.1016/j.scitotenv.2021.145801

Al-Turaiki, I., Almutlaq, F., Alrasheed, H., Alballa, N.	2021. Empirical evaluation of alternative time-series models for covid-19 forecasting in Saudi Arabia, International Journal of Environmental Research and Public Health 18(16),8660

John, M., Shaiba, H.	2021	Correlation between weather and COVID-19 cases: An extensive study covering all provinces in Saudi Arabia2021 International Conference of Women in Data Science at Taif University, WiDSTaif 2021, 9430202

Coccia M. 2021. Effects of the spread of COVID-19 on public health of polluted cities: results of the first wave for explaining the dejà vu in the second wave of COVID-19 pandemic and epidemics of future vital agents. Environmental Science and Pollution Research. 28(15), 19147-19154. https://doi.org/10.1007/s11356-020-11662-7

Ben Maatoug, A., Triki, M.B., Fazel, H.	2021How do air pollution and meteorological parameters contribute to the spread of COVID-19 in Saudi Arabia?	Environmental Science and Pollution Research, in press

Alsayedahmed, H.H.	2020	COVID-19 Pandemic’s precautionary measures had hit the reset button of the quality of life at different aspects, Journal of Infection in Developing Countries14(8), pp. 812-816

Coccia M. 2021. Pandemic Prevention: Lessons from COVID-19. Encyclopedia of COVID-19,  1, pp. 433–444. https://doi.org/10.3390/encyclopedia1020036

Alkhowailed, M., Shariq, A., Alqossayir, F., (...), Rasheed, Z., Al Abdulmonem, W.	2020. Impact of meteorological parameters on COVID-19 pandemic: A comprehensive study from Saudi Arabia: Impact of weather on COVID-19, 	Informatics in Medicine Unlocked20,100418

Coccia M. 2021. High health expenditures and low exposure of population to air pollution as critical factors that can reduce fatality rate in COVID-19 pandemic crisis: a global analysis. Environmental Research, vol. 199, Article number 111339, https://doi.org/10.1016/j.envres.2021.111339

Meo, S.A., Abukhalaf, A.A., Alomar, A.A., (...), Usmani, A.M., Akram, J.	2020Climate and COVID-19 pandemic: Effect of heat and humidity on the incidence and mortality in world's top ten hottest and top ten coldest countries,	European Review for Medical and Pharmacological Sciences 24(15), pp. 8232-8238


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 ENVIRONMENTAL FACTORS ON THE SPREAD OF COVID-19 IN SAUDI ARABIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Perhaps the best part of this paper is the introductory review, even if there is a clear mistake in the year of reference of Sanders, put before the outbreak started. Instead the results are not convincing in suppori of the hypothesis of a possible correlation. If a correlation exists it cannot depend on the geographical area, unless this fact finds an explanation.
Experimental design: No comment about the idea of looking for correlations between the diffusion of the pandemic and climatica variable
Validity of the findings: The outbreak in Saudi Arabia is mainly due to two regions, thus the subdivision in six does not pprovide much advantage. The period indicated as one of growing cases (May-August 2021) registered an average of 1100 new cases per day, not much different from the previous fourtyfive days (915) whereas in the two previous months the contaions were significantly less (about 350) and in the following two months even less (about 100). The regions are very similar for what concerns tempperature (with one exception) and similar consoderations are valid for the other variables. There are large differences in thenumber of ontagions between the different regions. This situation makes very doubtful that one can assess the existence of correlations through analysis for different regions. If a correlation exists it should be exhibited  everywhere, unless thee are reasons explaining the difference. This seems not to be the case for the difference, with respect to T of the regions E and F. A possible explanation might be found in the fact that both these region have a much smaller number of cases. making their results less significan than those of recions like C. Similar considerations can be made for the pther variables. The indication of the periods when each variabke reached maximum and minimum values seems irrelevamt to deaw conclusions, that instead seem to have been obtained more on the basis of authors expectations than on scientific results.
Additional comments: No further 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 ENVIRONMENTAL FACTORS ON THE SPREAD OF COVID-19 IN SAUDI ARABIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript subject “Investigating the environmental factors that influenced the spread of Covid-19 in Saudi Arabia” is interesting. However, the manuscript needs improvement. The authors made a considerable effort, but unfortunately, there are severe flaws in the analysis, which affect all results.

•	The role of lockdown and other restrictions also affect the results that are not determined in the manuscript, so the reason for the fluctuation in the Covid-19 outbreak is not just the climate. The periods and locations with the same restrictions (necessity of facemask, lockdown period, etc.) can be compared.
•	Moreover, there would be no correlations between the daily COVID-19 cases and the weather data of the same day, and the achieved results are wrong. The estimated incubation period of COVID-19 is about 2-14 days. The positive cases mean the positive test of the cases with symptoms such as a fever, cough, or shortness of breath, and the symptoms of COVID-19 occur about 3 to 5 days.  Therefore, there is a delay among the weather data and COVID-19 cases. Moreover, there are two other delays, including the laboratory analysis results and the announced time.
•	In addition, the start of the vaccination program is not mentioned in the paper. Until 21 August, was the vaccination zero, or was it an equal rate in all compared zones?
Experimental design: there are severe flaws in the analysis
Validity of the findings: there are severe flaws in the analysis
Additional comments: there are severe flaws in the analysis


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 ENVIRONMENTAL FACTORS ON THE SPREAD OF COVID-19 IN SAUDI ARABIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: It is OK.
Experimental design: It is OK.
Validity of the findings: It is OK.
Additional comments: I have read thoroughly the revised version of paper.

The authors have done considerable additional work, and addressed all concerns and criticisms in the revised manuscript, which I believe has improved substantially in the theoretical framework, study design and discussion of results.

Now, the paper is OK and has a good level to show interesting results to scholars and/or policymakers interested in these topics to solve problems of COVID-19 pandemic crisis.


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: HYALURONIC ACID PLASMA LEVELS DURING HIGH VERSUS LOW TIDAL VOLUME VENTILATION IN A PORCINE SEPSIS MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In general:
The experimental study by Thomas et al. aimed to investigate in a LPS induced porcine sepsis and ARDS model the effect of a lung protective ventilation (LTV) vs a non-protective ventilation (HTV) on endothelial glycocalyx shedding in the systemic, pulmonary and cerebral circulation at baseline (before) and after 3 hours and after 6 h of LPS administration. Parameters of mechanical ventilation, pulmonary gas exchange, hematology and hemodynamics were obtained throughout the study.
They found no differences in hyaluronic acid plasma concentrations (obtained from central venous blood) in the Intervention groups (LPS+LTV and LPS+HTV). They also state they found no differences in hyaluronic acid plasma concentration before and after brain and lung passage, but data is missing for the measurements of hyaluronan in the pulmonary and cerebral circulation (not shown in Figure 2).
The study on this clinically relevant field was performed thoroughly but is far from being mechanistic. The “negative” results are important findings to further assess the role of the endothelial glycocalyx in the pathophysiology of ventilator induced lung injury. Yet I see major weaknesses.
The four major weaknesses I see in this study are:
1.	The manuscript does not make clear how glycocalyx shedding in sepsis is related to glycocalyx shedding in ARDS. Clarification of this detail is essential for the presentation of this study.
2.	Clarity concerning experimental setup. I suggest to make the following more clear throughout the manuscript: Primary Intervention is LPS induced sepsis; this sheds EG as measured by hyaluronan plasma concentrations after 3 and 6 hours in this porcine model. A second hit by HTV does not seem to lead to further shedding of EG. To explore the protective effects of LTV a later time of sacrificed might be more accurate. The choice of model should be discussed in more clarity.
3.	Conclusions from the findings in this study are not well depicted. Clearer Conclusions from the findings are indispensable and would contribute to the scientific quality of this study.
4.	The lack of confirmatory parameters for endothelial glycocalyx shedding. The authors investigated only a single indirect parameter of glycocalyx shedding, the plasma concentration of hyaluronan. They attempted to test heparan sulfate plasma concentrations, but due to technical problems could not provide valid results. I suggest the authors test another parameter to support their data e.g.: Syndecan 1, or MMP9 (Dogné S, Flamion B. Endothelial Glycocalyx Impairment in Disease: Focus on Hyaluronan Shedding. Am J Pathol. 2020 Apr;190(4):768-780. doi: 10.1016/j.ajpath.2019.11.016. Epub 2020 Feb 6. PMID: 32035885.assessment.)
In detail:
The abstract is clear and concise; one remark:
49: sham ventilation might be confusing and the reader could assume another form of LTV. I suggest to name it “no LPS infusion”.
Methods clear and concise;
147: where can the reader find the defined schedule how the ventilator settings were adapted how did they chose PEEP settings? Please provide the used protocol.
Background: a small abstract to shedding of HA in brain and edema would be helpful to put the experimental setup for cerebral measurements and data into context.
Results: Figure 2 lacks the data which is stated in the text (50-51) pre- and post- pulmonary and brain Hyaluronan plasma concentrations.
Discussion
268-274: I would recommend to rewrite this paragraph to make it clearer to the reader that in the study of Coi et al. the mechanisms of action (atelect-trauma ZEEP plus abdominal surgery) differs from that in the underlying study (HTV vs LTV here with PEEP in LPS induced sepsis).
General comments and questions
Alveolar epithelial glycocalyx vs endothelial glycocalyx should be discussed in limitations (Li J, Qi Z, Li D, Huang X, Qi B, Feng J, Qu J, Wang X. Alveolar epithelial glycocalyx shedding aggravates the epithelial barrier and disrupts epithelial tight junctions in acute respiratory distress syndrome. Biomed Pharmacother. 2021 Jan;133:111026. doi: 10.1016/j.biopha.2020.111026. Epub 2020 Nov 24. PMID: 33378942; PMCID: PMC7685063.)
How where compliance data acquired?  Please provide information.
Used much higher LPS concentrations than Lipscey et al.: 160 µg bolus then 16µg/kg/h continuously, as compared to 1µg/kg/h, what was the rationale for this difference? Please clarify.


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: HYALURONIC ACID PLASMA LEVELS DURING HIGH VERSUS LOW TIDAL VOLUME VENTILATION IN A PORCINE SEPSIS MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	The major concerns of this study are the unclear rationale and research question. As the authors stated, it has been reported that sepsis causes endothelial glycocalyx damage, which contributes to acute lung injury (Schmidt et al Nat Med 2012). While it is conceivable that ventilation could cause further damage to pulmonary vasculature after the establishment of ARDS, it is unclear how barotrauma leads to endothelial injury (and therefore, more HA shedding). I think that the manuscript can be strengthen by making a compelling argument in the introduction and providing more detailed background/context. I think this will lead to a clearly defined the study question. Perhaps authors can use references such as Choi et al 2006.

2.	The article formatting is somewhat inconsistent throughout the manuscript. Half the sentences do not end with period which I think is the reference formatting issue. In line 248, references are formatted differently from the rest.

3.	Minor misdefined abbreviation; it should be “hyaluronic acid”=HA, not “hyaluronic acid concentration”.

4.	Heparan sulfate is included as a key word even though it was not measured in this study. It should be removed or replaced with hyaluronic acid to avoid confusion for readers.

5.	In line 134, authors state that the animals that received LPS were randomly divided into 3 groups, while one of the groups did not receive LPS. This sentence needs to be edited.
Experimental design: 1.	In Line 142, it was stated that animals that did not develop ARDS were euthanized. I think that this set of animals could have served as a great control to demonstrate the effect of LPS-induced sepsis alone. HA levels in plasma can be mostly due to LPS-induced sepsis and this group would have demonstrated the contribution of ARDS to this shedding if any. The data from this group would have also clarified how the authors defined ARDS specifically and demonstrated (possible) difference in lung histology.

2.     Although authors stated that 20 animals were bred for this study in method section, line 102, I believe it was actually 26, if the euthanized animals were accounted for.

3.     Please provide number of biological replicates in figure legends.

4.	Authors described their compliance to ethical guidelines with regards to animal transportation and euthanasia well.

5.	I think that the method description for histological analysis could be improved. Although authors cited a study, it was unclear how authored scored the lung injury. It would be helpful for general readers if more description or examples of histological characteristics were shown. It was also unclear how many sections/field were evaluated per animal.
Validity of the findings: 1.	The most concerning point regarding the validity for the study findings is the lack of data that directly addresses the study question #2. Figure 2 shows HA ELISA data from central venous blood samples, but in the main text, it was described that authors collected samples from multiple catheter lines to determine HA levels in pulmonary and cerebral circulations. Was HA measured as described? One of the study questions, as stated in line 93-94 was to determine the extent of endothelial glycocalyx depletion in pulmonary and cerebral circulations for which plasma HA was supposed to be the readout. Lack of this data significantly diminishes the scientific merit of this publication.

2.     Figure 3 can be improved by showing representative histological images.

3.	Authors stated in the discussion (line 264-267) that mechanical ventilation itself can compromise pulmonary integrity. If that is the case, proper control (no mechanical ventilation) should be included to demonstrate the effect of mechanical ventilation to experimental parameters.

4.    The conclusion statement (line 323-325) is not supported by the data provided. As mentioned above, the effect of different ventilation regimens to pulmonary or cerebral circulations was not measured specifically.
Additional comments: I understand that authors acknowledge the lack of direct evidence for EG degradation in their discussion, but I think that additional marker for EG would benefit the manuscript. If heparan sulfate ELISA did not work, perhaps syndecan-1 or chondroitin sulfate could serve as additional marker.


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: HYALURONIC ACID PLASMA LEVELS DURING HIGH VERSUS LOW TIDAL VOLUME VENTILATION IN A PORCINE SEPSIS MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: My major concerns were addressed by authors in this revision.

Minor comments:
1. A reference appears to be missing on line 50, regarding brain edema.
Experimental design: Research questions were relevant and meaningful.
Ethical standard was withheld for the experiments.

Minor comments:
1. Authors responded and provided technical replicates for ELISA, rather than biological replicates. Please include biological replicates (number of animals) used for ELISA in figure legend for Figure 2.
Validity of the findings: Conclusion has been improved with the revision.
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: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSES OF C2H2 ZINC FINGER TRANSCRIPTION FACTORS IN PLEUROTUS OSTREATUS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This MS aims to uncover Genome-wide characterization of the C2H2 zinc finger transcription factors in Pleurotus ostreatus and expression analyses under abiotic stress. In my opinion, it meets the journal's basic requirements.
Experimental design: It would be better to explan the basis of setting heat and cold stress treatment conditions in Introduction. the primordia were cultured at 38 °C for 1 h 3h for heat stress treatment, it is somewhat incomprehensible.
Validity of the findings: All the bioinformation was infered from PC15. While P. ostreatus 3125 was used in this study. There are not the same strain. It would be better to be discussed in the discussion.
Additional comments: Line 118, "Scientific Edible Fungal" ?


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSES OF C2H2 ZINC FINGER TRANSCRIPTION FACTORS IN PLEUROTUS OSTREATUS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this study, author used bioinformatics method to analyze the C2H2-ZFPs of Pleurotus ostreatus. 18 C2H2-ZFs were identified in P. ostreatus genome. The methods and results are acceptable. However, I think your research is too simplistic. I hope you can add some analysis and experiments. As far as writing, there are lots of small problems should be fixed.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: No additional comments


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSES OF C2H2 ZINC FINGER TRANSCRIPTION FACTORS IN PLEUROTUS OSTREATUS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand your text.  Dozens of grammar mistakes in the manuscript should be corrected. Such as Line74-75, 127, 157, 168, 188,190,192, 218 and so on.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I suggest the authors cluster the genes in the heatmap of Fig.3b to make it more visual and helpful for analysis of the similarity of different genes.


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

Input:
Title: GENOME-WIDE IDENTIFICATION AND EXPRESSION ANALYSES OF C2H2 ZINC FINGER TRANSCRIPTION FACTORS IN PLEUROTUS OSTREATUS
Review round: 1
Reviewer: 4

Output:
Basic reporting: Dear Authors,

Thank you for the opportunity to review your manuscript. It is solid and reliable work and I believe it is worth to be published on the pages of PeerJ journal. However, some changes need to be done. First of all, the title doesn't correspond to the content of your article. Basically, you discuss the influence of abiotic (temperature) and biotic (developmental stages, hormones) on the expression of the studied genes. Second, the English language should be considerably improved. I suggest that the manuscript should be corrected by the English native speaker and researcher. I have some concerns regarding the figure legends, namely figures 4 and 5. They reflect the methods used. I would prefer to see the actual finding: visually in your plots and written in the legends. I would also recommend using keywords different from the title, which might increase the "visibility" of your paper for the web search engines, but this is rather a recommendation, not mandatory.

Respectfully,
Reviewer.
Experimental design: well done
Validity of the findings: no 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: EFFECTIVENESS OF ANTIFUNGAL TREATMENTS DURING CHYTRIDIOMYCOSIS EPIZOOTICS IN POPULATIONS OF AN ENDANGERED FROG
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written with a thorough discussion of the results.
Data are clearly presented.
Experimental design: The research question is well defined. The objective of the study was to reduce Bd infection intensity and in doing so alter frog-Bd dynamics and increase the probability of frog population persistence despite ongoing Bd infection. This study yields important insights for future Bd mitigation strategies.
Validity of the findings: This paper describes an interesting study on mitigation of Bd in situ. The objective of the study was to reduce Bd infection intensity and in doing so alter frog-Bd dynamics and increase the probability of frog population persistence despite ongoing Bd infection. This study yields important insights for future Bd mitigation strategies.
Additional comments: Because there are a lot of different treatments I would suggest to add a graphical abstract that presents an overview of the treatments (could be as a supplemental figure).
Miner remarks:
Why is it not possible to determine the bacterial concentration in the J. lividum solution?
Was a necropsy done on the animals that died. Can co-infections, for example with ranavirus, be excluded ?


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: EFFECTIVENESS OF ANTIFUNGAL TREATMENTS DURING CHYTRIDIOMYCOSIS EPIZOOTICS IN POPULATIONS OF AN ENDANGERED FROG
Review round: 1
Reviewer: 2

Output:
Basic reporting: The level of English was excellent and professional throughout with only minor issues in some areas. The literature references seemed good, though some statements (albeit few) require citations. The use of subtitles helped guide the reader and the figures and tables look professional (although there seems to be minor display issues such as Figure 2 where the “Year” label appears to be cut). I suggest some restructuring of the methods/discussion as some aspects of the experiments became confusing rapidly. I have also suggested reducing the number of figures included in the main manuscript to those that describe the most important results. The manuscript seems to be self-contained and does present results relevant to their hypotheses/predictions but these hypotheses/predictions need to be more clearly stated at the end of the introduction. Further details can be found in my general comments.
Experimental design: This research is undoubtedly necessary in the field and represents original primary research within the aims and scope of the journal. Research questions are important but should be more explicitly stated along with predictions/hypotheses. The knowledge gap based on their literature review is important and this study should help fill a part of it. The investigation did appear to be rigorous and performed to a high technical and ethical standard, although some extra groups of controls would have strengthened the results (see additional comments). Finally, although the methods are described with a fair amount of details, they need to be restructured and simplified with the addition of some minor details (see general comments).
Validity of the findings: This study provides excellent and necessary insight on management techniques relating to frog-Bd dynamics. All the data was provided along with metadata, however due to my lack of knowledge when it comes to Bayesian approaches, I cannot evaluate how sound the presented statistics were. The conclusion is very well stated and followed with a promising management approach, although I would have appreciated a comment relating to pulse vs press treatments.
Additional comments: This manuscript was well written throughout, demonstrating a good level of English. I commend the authors for their extensive research and various approaches they investigated. I do believe this is valuable research that needs to be published. I apologize for the lengthy nature of my comments/questions/advice below, though I hope these will help you revise the manuscript. Please note that the manuscript itself is too long and can/should be considerably shortened. Restructuring the methods and ensuing results/discussion should greatly help in this regard. The addition of link sentences between paragraphs will also greatly improve the flow of the proposed narrative.

L.2-33: The abstract is very well written and structured (but see my comment bellow). I suggest presenting the information in a similar fashion to restructure the methods/discussion sections to improve their clarity.

L.25-29: Your study focuses on two types of treatments performed on different life stages conducted at a fairly restricted time point. Do you think that repeated treatments, treatment of all life stages, and/or the paring of different management efforts (other than your itracanzole + J. lividum trial) may be more beneficial? If you are replicating current strategies or evaluating possible strategies state it clearly and acknowledge that the evaluated strategies did not work but others could. Here you make it sound like treating affected frogs/tadpoles won’t work at all, even though you only covered some strategies. I also think that if space allows, a prompt to investigate other strategies (such as the one described in the conclusion) would close off your abstract well, though this is just a suggestion.

L.34: Good introduction highlighting the importance of studying/managing wildlife diseases.

L.39: Here you write “Diseases of wildlife” which you then switch to “wildlife diseases”. The first formulation is awkward, and I suggest using “wildlife diseases” throughout.

L.42-46: This sentence is a bit long and has some grammatical issues (“many fewer”). I suggest rephrasing it to something like: “As such, the ability to manage wildlife diseases is critically important. However, doing so is often an arduous task considering the lack of information on these diseases, the relatively few intervention measures available, and the inherent difficulty of studying and treating wild animals.”

L.46-47: To say “mostly” would require a citation here considering all the different management strategies for diseases that have come and gone. Otherwise, I suggest rephrasing.

L.48: Add a link to the previous paragraph to improve flow. Something like “One disease that has had worldwide impact is […]”

L.54: I suggest adding a description of the mode of action of this pathogen (how it spreads in the habitat and affect its hosts). It shouldn’t be long, just a few sentences. This is valuable information to the readers and will help them understand points about population density later on. Following this, “In an effort to reduce […]” could be the start of you next paragraph.

L.56: Here you start discussing frogs when just before you were discussing amphibians. I think a link to frogs (maybe something about this group being the most affect, if this is the case) could be made at
L.53-54.

L.59-63: This statement about modeling, though true, does not really fit here. I suggest removing it and moving on to results from laboratory and field studies.

L.63-65: I suggest going a bit further into what these studies found instead of relying on this blanket statement. This sentence also doesn’t really justify your next one since you say that current field tests find limited effectiveness, which does not support your claim of lacking experiments. Perhaps describing the difficulty of evaluating strategies due to the lack of field tests would be a better approach.

L.68: I suggest rephrasing this to improve flow. Something like: "One group of frogs that have been emblematic of amphibian declines caused by Bd are mountain yellow-legged ("MYL") frogs." You need a link with the previous paragraph and following sentence.

L.78: Replace “and” with a comma in “Bd prevalence, infection intensity”

L.79: Remove “epizootic =”

L.86: I think this whole paragraph should be structured differently. The modeling part doesn't add much to point you are making, particularly since you do not develop such models (you are providing experimental evidence). The part about Bd-amphibian dynamics and spread should be higher up when Bd is introduced. If this is done, you could simply refer to the importance of density and load, state it’s backed up by modelling (if needed and without going too much in depth), and briefly describe the potential strategies (L.94-101).

I suggest rephrasing the start of the paragraph with something like this:
"Using empirical data from these rare enzootics and much more common epizootics for modeling revealed that Bd epizootic or enzootic outcomes are governed solely by density-dependent host-pathogen dynamics. This suggests that suppression of Bd loads [...]."

L.90 If kept, “population of frogs”

L.94-99+L.101-111: These parts if combined into a single paragraph highlight very well two strategies which you explore and would be followed up nicely by the third strategy in L.111-119.

L.100-101: The first part of this sentence (up to the comma) is vague and adds nothing to the rest of the sentence. I suggest removing it.

L.199: I would finish this paragraph with a statement on the need for field tests.

L.129: Here you should briefly summarize your predictions/expectations as outlined in your abstract. A brief summary of other field results in the introduction would also help justify these.

L.130-138: I would remove this paragraph altogether as it belongs to the results/discussion and deflates your narrative before it even started.

Methods: I understand that you performed multiple experiments/trials and that concisely summarizing these is challenging, but this section is too long (15 pages long) and must be greatly reduced. Describing general methods as to avoid repetition was a good start, however having these at the start of the methods, without knowing in which context they were performed, lends to confusion. A simple sentence or two at the start of the descriptions summarizing how these methods were applied would help.
You could also start with a general overview of which experiments were performed, how study areas were selected and used (choice of water bodies within basins, etc.) and when they were conducted. This first paragraph will act as anchor point for the rest of the methods and should help guide the reader through these. From there, you could go into general methods, leaving statistical analyses general methods to the end of the methods once all variables have been introduced, along with experiment specific statistic details. Following general methods, you should describe each experiment’s relevant particularities, which would then be followed up by stats and results. Ultimately the format is up to you, but this section needs to be shortened considerably. I have made further suggestions below which may help guide you.

L.144: I would start with a brief sentence of how these surveys are used.

L.144: If you have data on developmental stages of the tadpoles (i.e. Gosner (1960) stage or other; which seems to be the case based on your raw data), I would suggest including these (at least a range of stages). These will be useful in terms of comparisons between studies and to determine how the tadpole's stage may affect its response to treatment.

L.144: Given your definition of an adult vs subadult, this surveying strategy seems arbitrary. Did you measure frogs? Do you have a citation for this way to differentiate developmental stages or is it arbitrary? I believe this requires a citation and a better explanation of how you can assure one frog belonged to one group and not the other.

L.151: Change “During each summer” to “During each experimental summer” or similar wording since you have multiple experiments conducted over multiple years. This statement could be mis-construed as surveying all basins, every year since the first experiment.

L.162-165: I appreciate this statement, but it definitely feels out of place... Maybe include this when appropriate in the results or figure captions? Or perhaps remove the first part of the sentence referring to the figures?

L.170-172: How were subadults and tadpoles ID’ed/ tagged? This would eb a good place to explain this.

L.173-174: At this point I’m wondering which experiments have multi-day treatment periods, which is why a summarizing paragraph at the start might be useful.

L.174-176: What densities of frogs/tadpoles occurred in the pens? We know that tadpoles from certain species of amphibians are affected by population density, with suggestions that this occurs through stress pathways. Could this have affected results? Were pens filled with varying stages/groups or single stage? If multi-stage, could the adults and sub-adults eat the tadpoles opportunistically, which would lower captive/treated tadpole survival? These are questions that could also be avoided if experiments were summarized at the start. I think you should sell the pen for what it is: A way to keep track of wild animals and ensure they receive the proper treatment, reflecting what is done for conservation practices.

L.177: Was this done to prevent resampling of the same individuals? If so, I would state it here as it helps explain the 3-24h range.

L.179-190: Although there is not much you can do about it now, you should have had 2 controls. The first would be your current control, aimed at evaluating how wild frogs react to Bd epizootic when left (mostly) in the wild. The second would have been to verify the effects of the pen (if any) on untreated frogs, to ensure that results aren’t simply dictated by the stress of the pen or other effects it may have. Do you have any citations for the effects of such pens? You should also acknowledge that both forms of controls were feasible and provide an explanation as for why the other wasn’t used (maybe something along the lines of maximizing sample sizes for statistical analyses while also maximizing the number of treated frogs, in light of their imminent Bd related declines). Note that this part feels like it belongs in the discussion too.

L.181-184: The end of this sentence doesn’t make sense given the point you are trying to make.

L.185-186: With the current phrasing this is wrong. If the treatment had no effect, then both groups would be subjected to the same density effects and there would be no treatment bias per se. The bias is in terms of captive controls having lower survival than free controls.

L.189: This is correct, but what about predator-prey dynamics and how they are affected by the pen? Could this cause survival to be higher than controls for instance? Are there predators, and are frogs/tadpoles protected from these while they are in the pens? Is it relevant given the time scale?

L.194-195: I would specify they varied by experiment.

L.197-199: Depending on the experiment or treatment?

L.201: I am not familiar with Bayesian approaches and will thus base my review of this section on comprehension rather than evaluating the statistical approach itself. I would suggest moving all discussions of the statistics to the end of the methods following explanation of the experiments and introduction of variables of interest. This also improves flow from methods to results.

L.211-213: If there is non-independence within/between your treatments (modeled by random effects) it should be accounted for in the full model and can be removed down the line if it doesn't add anything to the model. You need to account for non-independence right away, not "when suggested by the data". This is if sample sizes allow for random effects, which seems to be the case. Of course remember that random effects require at least 5 levels in order to be appropriate and should be fixed effects otherwise.

L.219: There is something wrong with the margins here it seems (in terms of formatting).

L.242-250: This seems like it should be specified in the figure captions rather than the methods.

L.259: Give life stage range if possible.

L.262: Are these populations or sub-populations?

L.265: The prediction should be at the end of your introduction, not in the methods.

L.276: Why the variation? I understand it's the nature of these types of treatments and reflect what is most likely done in the field, but how does this affect results. Are more doses beneficial? Does itraconazole have negative effects if administered too often. How does this variability affect your results? In light of this and other comments, I think your study would benefit from explicitly stating that you are mimicking existing/suggested management strategies (if that is the case), as the design has a large amount of variability that should be controlled for in a classical experiment setup. Then you can easily acknowledge shortcomings as being reflective of current management practices.

L.275: Have you tried using mesh partitions or more pens to separate individuals by day so that they all receive the same treatment?

L.278: What are the samples sizes of these subsets?

L.295: Why was the sampling so unbalanced and how could this affect your results?

L.299: I believe this section may fit better along with the “general methods” statistical paragraph placed at the end of the methods.

L.317: What does this begin-end range mean?

L.317-319: Doesn’t metamorphosis mean that the stages are no longer separate since an individual may have started as a tadpole and ended up in the subadult category? Shouldn’t stage be excluded from your model since the stages aren’t mutually exclusive anymore? I suggest treating stages as a single group of early development frogs, as your sub-title suggests.

L.359: Beyond the difference in sampling effort, do you think sampling a single treatment for the first days and switching over may have biased your results regarding frog capture opportunity through density effects or by selecting healthier, more active frogs early on (particularly if you say that this is a very small proportion of the available population). I'm not sold on the last statement either (do you have population estimates, perhaps from previous studies which could be cited?)

L.365: Why have the general methods if you are going to summarize methods twice in the same paragraph?

L.386: Just say 3 primary periods with the exception of 2018, since it’s the only exception.

L.397: What is “not recruited” in the context of your study?

L.404: Qualitative results are good, but perhaps this could be moved to supplemental materials considering your manuscript is already quite lengthy.

L.453-455: This should be at the end of the introduction as well.

L.475: a subset of how many? Make sure that samples sizes are explicit throughout.

L.481-482: Did you swab controls?

L.491-494: Why alter the concentrations? Also were all subadults bathed twice or separated between days. If separated between days, then this can bring a bias.

L.506: Non-experimental frogs are introduced but have not been mentioned before.

L.525: There are many tables and figures in both the manuscript and supplemental material. In terms of figures, you may want to reduce the number of figures to the important ones regarding your results. In terms of your results, many of them are supported by tables in the supporting documents, and I was wondering if there was a way you could include an abridged version in your actual manuscript for reference, since you do not present model outputs. Note that Figures don’t appear to be displaying correctly (The bottom of “Year” is cut off in Figure 2 for instance).

L.589: The percentages would be appreciated as well.

L.663: I suggest rephrasing since the surprising effect is not relating to the control group dying more, but rather to the non-experimental group performing better.

L.638: Detection probabilities of what?
Discussion: This section also needs to be shortened and I have made some suggestions on how to do so below.

L.713: Your first 1-2 paragraphs should be a recap of why you did this study and a brief summary of your results, which you then discuss in depth in the paragraphs that follow.

L.716-721: I would reword this sentence because it is too lengthy and feels misleading. You could also add a link to your own study and briefly summarize the results you will be discussing afterwards.
"Complete eradication [...], but can be accomplished through disinfection of aquatic habitats and treatment of all amphibians in simple isolated ecosystems. However, similar methods applied in complex environment failed to achieve long-term eradication (citation), highlighting the importance of further investigating management strategies."

L.718: amphibians, and chemical […]

L.722: Move this paragraph to the introduction or remove entirely. The discussion should be used to discuss your results and not bring in new information or justification.
From the previous paragraph rewording suggested, here you could write: "One strategy involves promoting coexistence between amphibians and the pathogen through various strategies such as [...] though these require field testing. In our various experiments we found [...]"

L.736: This information should be in the introduction and not the discussion unless linked with specific results.

L.747: This should probably be part of the first or second paragraph of your discussion. Though it should be re-arranged to improve flow. Start with the theory, then end with the summary of the results.

L.766-769: What about a more sustained/repeated treatment? Here you discuss a pulse event by treating amphibians, but what about pairing this with treatment over multiple years?

L.789: Remove nonetheless

L.796: How long do these frogs typically live in the absence of Bd? Adult survival should decrease every year, so this statement doesn't tell me much unless frogs are supposed to have much higher survival for this time span. I would remove "and by 2018 few treated frogs remained". This question also relates to L.709 where you say no treated frogs remained in 2019 when the experiment was in 2012.

L.801: A range for comparison would be good.

L.803: Here do you have proportions of each life stage encountered? Having 2434 tadpoles, yet very little recruitment is not very surprising for instance, since tadpole survival tends to be low, but for 2434 subadults it would be surprising. I understand that the point is to show frogs are still reproducing but that no new adults seem to be recruited, but because your sampling dates aren't consistent between years, I'd appreciate a more detailed breakdown of your VES by life stage, or at least the VES for subadults.

L.811: In an enzootic, the population is stable, correct? Then knowing we are now in 2021, and the experiment ended in 2018, do you know what happened to the population afterwards? This is to some extent a question you can answer.

L.814: How many of each group?

L.850-852: Good

L.855: Higher than what? Whenever you use this formulation, the thing you are comparing your subject to, should be specified.

L.856-888: Good discussion

L.915-918: So you say that frog microbiota may be resilient to changes, explaining that concentration of J. lividum return to normal. But at the same time, if control frogs can easily acquire a new bacterium over a short period of time, then how resilient is the actual microbiota? It seems like it can easily be disrupted but some mechanisms allow for it to return to normal. Do you have any more in depth explanation for this?

L.962: Reference numbers? Also, although stated in the author notes, just a reminder that this should be placed in the methods.


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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF N6-METHYLADENOSINE DEMETHYLASE IN SUGAR BEET (BETA VULGARIS L.) UNDER SALT STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors identified 12 sugar beet ALKB family proteins in sugar beet genome-wide database and analyzed its gene structures, chromosome location, physical and chemical properties of protein, motifs, subcellular localization and the phylogenetic tree construction etc, and quantitatively comparing the expression of BvALKB under normal conditions and salt stress. They claimed that these genes were in response to salt stress. However the manuscript suffered several problems. I cannot recommend for further processing.
Main concerns are:
1.	The analysis of the physicochemical propertids of BvALKB proteins is oversimplified. The physicochemical propertid of BvALKB proteins should be validated by complementary experiments in vitro, eg. Prokaryotic expression of BvALKB proteins.
2.	The authors claimed that BvALKB proteins were in response to salt stress. However, there is no other experimental evidence to prove that BvALKB proteins play a role in response to salt stresses except for quantitatively comparing the expression of BvALKB under normal conditions and salt stress.
3.	There is no difference significance analysis was performed in quantitative PCR experiments.
4.	The Fig. 1, 2 and 7 are not very clear.
5.     The analysis of subcellular localization prediction of BvALKB proteins is oversimplified. eg, leaves of Nicotiana benthamiana could be used for transient transformation and subcellular lacalization analysis of BvALKB proteins.

In short, experimental data are missing to prove correlations between BvALKB proteins and salt stress. Thus, I suggest to accepting it for publication after major revisions.


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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF N6-METHYLADENOSINE DEMETHYLASE IN SUGAR BEET (BETA VULGARIS L.) UNDER SALT STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: These requirements are well.
Experimental design: Experimental design is reasonable
Validity of the findings: The findings are novel.
Additional comments: m6A, as a most abundant and highly conserved RNA modification, plays important roles in plant growth, development and response to abiotic stresses. m6A is regulated by Methytransferase, demethylase and readers proteins. Here, Cui et al genome widely identified the genes encoding m6A demethylases from sugar beet, they found out 12 genes and performed bioinformatic analysis and q-PCR experiment for some gene under salt stress. This study provides a theoretical basis for further screening of m6A demethylase in sugar beet, and also lays a foundation for studying the role of ALKB family proteins in growth, development and response to salinity stress. There are some concerns as below:
1.	Please introduce the original and meaning about hush, pkhc,ryeg, et al; the gene need to be named according to homologous from Arabidopsis;
2.	The 10 figures in MS need to be integrated less than 5 figures according to the result description;
3.	The results of Qrt-PCR need provide significant analysis;
4.	Some English writing need to be improved.


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

Input:
Title: GENOME-WIDE SEQUENCE IDENTIFICATION AND EXPRESSION ANALYSIS OF N6-METHYLADENOSINE DEMETHYLASE IN SUGAR BEET (BETA VULGARIS L.) UNDER SALT STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The study was aimed on the N6-methyladenine demethylase gene family in sugar beet. The sugar beet genome database was screened with 2OG-Fe II-oxy conserved domain of the ALKB demethylase family. A total of 12 proteins homologous to ALKB demethylases and the corresponding genes were identified. Several bioinformatic analyzes were performed to provide data on  protein domain distribution, putative cellular localization, phylogenetic relationships within the BvALKB family, gene structure and the chromosomal localization. In my opinion bioinformatic analysis was performed properly and provides  comprehensive characteristic of the ALKB demethylase family in sugar beet. Finally, the effect of salt stress on the transcript level was assessed using RT qPCR approach. It was revealed that one of the BvALKB transcripts, the “uxaj” seems to be strongly up-regulated under salinity treatment in sugar beet leaves.
Experimental design: 1.	The absence of statistical analysis of the gene expression results is a serious problem. Besides, the fold change in gene expression level between the control plants and the salt-treated plants the statistical significance must be determined and shown oh the graphs on Fig. 9. Especially as the Authors say several times that the expression of a given gene is significantly down-or up-regulated with respect to untreated control. The significance has to demonstrated by proper statistical analysis. It also has to be kept in mind that even if the difference is statistically significant the conclusions on the difference in gene expression should be cautious if the fold change is smaller than two.
2.	What is actually shown on Fig. 9? Is it a mean from all three replicates or a representative result for a given experimental variant? Do the error bars show standard deviation or standard error.
3.	 It was said (l. 150-151) that the gene expression experiments were repeated at least three times, but with how many plants per replicate?
4.	The fold change (not “data analysis”, l. 151) was calculated by deltaCt method. This method is applicable when the PCR efficiency of target gene amplification is comparable to the internal control (housekeeping gene) amplification. Otherwise, the Pfaffl’s method with the correction of difference in PCR efficiencies is recommended. Were the PCR efficiencies determined. If so, they should be shown in table 1.
5.	Three different housekeeping genes were used to normalize the gene expression data. Which one was used to calculate the results shown on Fig. 9?
6.	Please correct the l.163-165 by applying correct term to denote the discrete parts of the protein: The aminoacid chain has no “front”, “middle” and the “end”. It has N-terminus (or N-terminal domain), internal domain and C-terminal domain (C-terminus).
Validity of the findings: The study provides some insight into the largely neglected issue of the demethylase in sugar beet and their putative involvement in stress response. However, several topics have to be revised before publication.
Additional comments: 1.	Avoid unjustified speculation, which is not supported by the data such as : “njrf, eskg and uxaj are likely to […] play an important role in the response of sugar beet to salt stress” (l.261-262). This statement should be corroborated by functional study. What is shown here, is the correlation between stress treatment and gene expression.
2.	The English language should be improved, throughout. Several terms are used inappropriately and incorrectly, such as “coping with different environments” (l.41), “The reversibility of m6A […] was confirmed in the paper” (l.55) “structures were analysis” (l.136),  “transcriptional protein”?? (l. 261). Moreover please correct: “identified” instead of “screened” (l.158), “sugar” instead of “suagr” (l. 184),


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: BEYOND THE PREY: MALE SPIDERS HIGHLY INVEST IN SILK WHEN PRODUCING WORTHLESS GIFTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Very good. Please double check grammar.
Experimental design: Excellent.
Validity of the findings: Good, but please see comments on the statistical analyses below.
Additional comments: This paper examines some of the costs and benefits of nutritive vs. worthless nuptial gifts in the trechaleid spider Paratrechalea ornata. The authors first ask whether males prefer to present worthless or nutritive gifts to females when both options are available, and whether this behaviour is repeatable. They also compare the investment in silk-wrapping of the two different kinds of gifts, and find that males invest more silk when preparing worthless gifts, whether or not there is a prey item available, and that silk wrapping is related to male size.

I really enjoyed reading this paper! It is very clearly written, the experimental design is very clever (especially the offering of the prey), and the results are interesting, adding to our understanding of the evolution of worthless gifts. The framing of the introduction and the discussion are excellent, and my main suggestions are to do with the data analysis and presentation.

Lines 104-108: I do not follow the reasoning that if the reproductive benefits of worthless and prey gifts are the same, the silk investment must be cost-neutral. I would expect that if the worthless gift has the same benefit as the prey gift, it must be because there is something added (like the silk investment) to cause the female to accept both kinds of gifts equally. I may be missing something, but please explain the reasoning here for others, who also may not follow.

The experimental design exposes males to the same group of females repeatedly. Could there be any effect of male or female experience on type of gift chosen (male) or female receptivity? Can you report whether males who switched tactics did so in the same direction (worthless first->nutritive later or vice versa) or switched back and forth?

I don’t think it’s appropriate to do model selection via AIC *and* then report p-values for specific predictors. My understanding is that one should do *either* model selection to come up with the best model that describes the relationship *or* report p-values from a model that includes the predictors one expects/predicts are influencing the dependent variable (decided a priori). Please see this blog post by Brian McGill for more discussion of this: https://dynamicecology.wordpress.com/2013/10/16/in-praise-of-exploratory-statistics/

I think that the data displayed in figure 1 could be more informative either by switching to a histogram-type graph with the number of males on the y-axis and percentage of nutritive gifts on the x-axis, or, rearranging the individuals in this graph by increasing proportion of grey, so that it is easier to see what proportion of males always gave worthless (or nutritive) gifts, and what proportion gave a mixture.

Please consider adding the actual data points to the box plots (e.g. points in grey behind see-through boxes) or violin plots instead of boxplots so that the reader can more easily see the distribution of the data. More suggestions for the best ways to visualize data here:  https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002128

Minor comments:

Please update figure captions with information about the name of the species and some details of the experimental context, etc. so that each display item can be understood in isolation from the rest of the paper. E.g., for figure 4, “Differences in silk wrapping duration according to body size for worthless gift production by Paratrechalea ornata males. Data are shown as black points, linear predictor in black and the confidence interval in grey. These data are from males (n = xx) used in two experiments, one where they had the option to make a nutritive or worthless gift, and another where they only had the opportunity to produce a worthless gift.”

Lines 20-21: please revise to “…offerING either a nutritive (prey) or worthless (prey leftovers) SILK-wrapped gift to females”

Line 55: “The divergence in an individual’s gift-giving plasticity can lead to the…” I have trouble understanding the meaning of this sentence, please clarify.


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: BEYOND THE PREY: MALE SPIDERS HIGHLY INVEST IN SILK WHEN PRODUCING WORTHLESS GIFTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is an interesting study that has potential to extend our knowledge on gift-giving behavior.
Manuscript structure conforms to PeerJ standards. The text is written in professional English language. Some corrections are needed (see below, in the Section 4. General comments, subsection 4.2 Other comments), but every reader is able to understand the study.
The authors provided all needed context, but the Introduction can be improved. The third paragraph of Introduction is too long (407 words), consequently it is not so easy to understand it. Authors have to reduce it. On the other hand, the fourth paragraph is too short (105 words). Given its content, I suggest adding the fourth paragraph to the fifth one (224). Combining the fourth and the fifth paragraphs will require some adjustments.
Methods are well presented, but some additional information should be provided (see below, in the Section 4. General comments, subsection 4.1 More relevant general comments). Data reporting requires more attention (see below, in the Section 3. VALIDITY OF THE FINDINGS).
The authors cite relevant literature. However, some statements lack references (L59-60; L73-75; L80-82).
Raw data is provided and it is easy to understand all information.
Figures are relevant and well described (but authors need to correct y-axis name in Figure 2B).
Experimental design: This is an original primary research within the scope of PeerJ. The research question is well defined, relevant and meaningful (e.g., do P. ornata males construct worthless gifts when given the opportunity to construct nutritive gifts?). The authors state how the research fills an identified knowledge gap (e.g., what are the implications of worthless gifts and what does it tell us about the evolution of gift construction?).
Authors allowed males to construct nutritive or worthless gifts along five trials. In order to run these trials, authors offered a living fly or exuviae to males and registered which option each male picked. This experimental design allows to: (a) test if males do construct worthless gifts when given the opportunity to construct a nutritive gift; (b) test if males are consistent in their behavior; and (c) compare the resulting nutritive and worthless gifts. In general, authors explain methods in detail (but a few adjustments should be made (see below in Section 4. General comments, subsection 4.1 More relevant general comments). Finally, the investigation seems to have been rigorous and ethical.
Validity of the findings: The authors found that: (a) males do construct worthless gifts when given the opportunity to construct a nutritive gift; (b) males are not consistent in their behavior; and (c) males invest more in terms of silk deposition when constructing worthless gifts.
All underlying data is provided. The reported results are particularly relevant once you acknowledge that the authors did not compare experimental groups according to the frequency of worthless gift construction or according to within-individual variance in gift construction. Appropriately, the authors did not make these comparisons because the experimental procedure is different for each group (in the Nutritive-Worthless group, males were offered a living fly via tweezers) and because the probability of worthless gifts occurrence is higher in the Worthless group from the very beginning. Therefore, the authors restricted the comparison solely to the worthless gifts constructed in each treatment group, circumventing the above-mentioned inappropriate comparisons.
In general, the data is robust and statistically sound. However, two results reported in the main text differ from the information provided in tables. First, Figure 2A and Table 3 show contrasting results. According to Figure 2A, wrapping duration was longer when males constructed worthless gifts. However, according to Table 3, wrapping duration was longer when constructing nutritive gifts (note the negative estimate for worthless gifts). Second, Figure 3 and Table 4 also show contrasting results. According to Figure 3, wrapping duration increased with male size. However, according to Table 4, wrapping duration decreased with male size (note the negative estimate for male size). Checking the data, it seems that the information in the main text is correct, and negative values were erroneously included in Tables 3 and 4. However, authors must revise the analyses that provided these results.
Aside from the comments above, authors need to improve data reporting. First, authors need to inform confidence intervals for estimates in Tables 1, 3, and 4. Second, while authors say that “Silk investment in worthless gifts did not differ between males in the presence (Nutritive-Worthless group) and absence of prey (Worthless group) (Table 4)” (L266-268, also see L270-272), Table 4 does not provide data that leads to this conclusion. To solve this problem, authors need to present estimates for each treatment group and trial number and need to present the AIC values for each model or for the top tested models. Third, a similar problem occurs in Table 3. Once again, authors need to provide AIC values for each model (or at least for the top models) and provide estimates for fixed effects (in this case, male size and trial number). Otherwise, statements made in the main text (L269-270) cannot be verified by the reader.
Finally, conclusions are linked to the original research question and limited to supporting results. Most conclusions are also well stated, but there are exceptions. The most important exception is found in the third paragraph of Discussion (L308-320; for other exceptions, see Section 4. General comments, subsection 4.1 More relevant general comments). In order to explain why males deposit more silk while constructing worthless gifts, the authors mention that silk (1) helps to attract females, (2) may be nutritious for females, and (3) enables males to better hold the gift. However, all these reasons apply for both nutritious and worthless gifts. In fact, avoiding the loss of the gift would be more important when holding a nutritious gift. Therefore, it is not clear why these three factors explain the higher investment in worthless gifts.
Additional comments: For clarity, I below provide suggestions directly to the authors.

4.1 More relevant general comments
(L38-39; L301-304) These sentences sound finalist/teleological. Why not try ‘In some invertebrate species, individuals (usually males) offer nuptial gifts to potential mates (usually females), increasing their reproductive success’ and ‘Thus, the fluctuating climatic conditions likely favour the evolution and maintenance of flexibility in the nuptial gift production, optimizing males reproductive success’?
(L55) What does “divergence in an individual’s gift-giving plasticity” stand for? This sentence could be clearer.
(L59) Here, you say that alternative tactics are less successful than the dominant strategy. However, this statement contradicts other statements in your text (e.g., L88-89; L94-97; L287-289).
(L107-108) Why does this rationale require that silk investment is cost neutral? You should clarify this part.
(L114-125) This paragraph should be improved in order to make predictions clearer. First, you present your hypothesis (L114). However, in the next sentences, you present scenarios that go against your hypothesis (L114-116; L116-118) or give support to your hypothesis (L120-123). It is interesting to show these contrasting scenarios, but it would be better to present them in a way that leads to your hypothesis.
(L118-120) You need to explain how this result can “indirectly demonstrate that males have no restrictions on their reproductive benefits in relation to the gift type”.
(L124) No reason is given for this comparison. Why is it interesting to contrast the silk investment in presence and absence of prey?
(L125) No reason is given for this prediction. Why do you expect no differences between presence and absence of prey?
(L184-185) Did you offer a live housefly to all males in the Nutritive-Worthless group? Or did one or more males capture an exuviae before you could offer the fly? In case you were able to offer a live housefly to all males, inform this in the text. Otherwise, explain why you included in your analyses males that captured an exuviae before having the opportunity to capture a fly.
(L192-194) It is not clear what ‘ending a silk wrapping’ stands for. How did you define the exact moment in which each male “ended the silk wrapping”? Please inform this in the text.
(L200-201) Please define a silk wrapping bout. Otherwise, one cannot completely follow your study protocol.
(L206-207) Because males in each treatment group differed in the type of gift they could produce, you did not exactly follow the same experimental protocol in both groups. I think you should inform that treatment groups differed in one single aspect (males in one group received an item via tweezers) and make implicit that you see no reason to expect that this difference affected male silk investment in worthless gifts. For example, you can say: Aside from offering a living fly via tweezers, “we followed the same experimental protocol described above, so males in both treatment groups had comparable courting opportunities but differed in the type of gift they could produce”.
(L218-220) It is not clear how the response variable was calculated. Is it the ratio between worthless gifts and nutritive gifts per male?
(L222-224) Male 2891 only constructed one gift. Is he included in this analysis? If yes, why?
(L308-310) A recent study tested if female quality influences male mating and reproductive effort (including investment in the wrapping procedure). See: Solano‑Brenes et al. Differential allocation in a gift‑giving spider: males adjust their reproductive investment in response to female condition BMC Ecol Evo (2021) 21:140, https://doi.org/10.1186/s12862-021-01870-1.
(L311-313) You say that you “found that males spent more time and deposited more silk during worthless gift production than when the gift was nutritive, and that large males can wrap the item better as they are able to add more silk to the gift”. Actually, you did not demonstrate that large males can better wrap gifts because they are able to add more silk to the gift. Therefore, this last part of the sentence “as they are able to add more silk to the gift” should be clearly presented as a hypothesis.
(L320-323) This sentence is disconnected to the previous one. However, the argument presented in the following sentences is convincing. Please, connect these sentences to the first part of the paragraph.

4.2 Other comments
In authors’ affiliations, some corrections are required. “Instittuto de investigaciones Biológicas Clemente Estable” should be “Instituto de Investigaciones Biológicas Clemente Estable”. Also, ‘montevideo’ lacks an initial capital letter.
Abstract is interesting and clear.
Throughout the text, sometimes you say trial, sometimes you say experiment. ‘Trial’ is more appropriate because you have one experiment and each male is used in five trials. Therefore, replace “experiment number” with “trial number”.
(L46) There should be no space between ‘e.’ and ‘g.’.
(L61; L66; L71; L72; L197; L267; L268) Please, include ‘i.e.,’.
(L69) It is not clear what ‘this’ stands for in the sentence “This rises because...”.
(L69) “for a female to consume complete the gift”. Please, revise this sentence.
(L79) First, it is likely worthless that
(L81) Early studies confirm that
(L90) quality, leading (include comma).
(L91) "preferences for the gift" is not clear enough.
(L104) To make clear that you are still talking about P. ornata, please say: “Based on the assumption that when a P. ornata male offers a worthless gift”.
(L105) Maybe replace ‘choose’ with ‘choosing’.
(L109) Consider saying ‘quality indicator’.
(L127) Materials & Methods
(L147) 21:00 and 23:00
(L150) Remove ‘were’.
(L154) field, with
(L166) co-occur, we
(L191) captured the fly
(L203) of prey, we
(L204) group, we
(L230-233) At first, R is in italic and in capital letter (L230), then, it is solely in lower case (L232-233). Please standardize it.
(L237) distributions
(L244) effects
(L251) ‘remaining’ is not appropriate because there are also the 19 trials in which males did not construct a gift.
(L259) (using nutritive or worthless gifts)
(L260) ‘also’? This is the only experimental group in which you tested repeatability.
(L261) bouts, differed
(L264-265) Consider saying: Comparing silk investment between both gift types by using the Nutritive-Worthless group showed that males invested...
(L266) No comma.
(L267-268) or in the absence of prey
(L271) from both groups, we found
(L275-276) Consider saying that ‘Here, we demonstrated that, even when P. ornata males had the opportunity to produce a nutritive gift, 20% of males chose to offer worthless gifts’.
(L287) Consider saying ‘(see Albo et al., 2014)’.
(L315) Replace ‘It’ with ‘Silk’.
(Figure 2B; Table 3; Table 4) Replace ‘number silk wrapping bouts’ with ‘number of silk wrapping bouts’.
(Figure 2) Figure 2A was uploaded in the online system as Figure 2, Figure 2B was uploaded as Figure 3, and Figure 3 as Figure 4.
(Table 2) Confidence intervals should be presented before the p value (i.e., in the second column).
(Table 4) produced by males in the Nutritive-Worthless group compared to the Worthless group.


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: BEYOND THE PREY: MALE SPIDERS HIGHLY INVEST IN SILK WHEN PRODUCING WORTHLESS GIFTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Good!
Experimental design: Very good!
Validity of the findings: Good.
Additional comments: I thank the authors for their careful consideration of my comments and those of the other reviewer. I think that they have addressed all of the comments adequately and I appreciate the updating of the figures. There are still a few places where the grammar could be cleaned up, a couple of examples below, but the writing is very good overall.

line 63: empidid should not be capitalized unless at the beginning of a sentence
line 107: change "within individual" to "within-individual"
line 133: should be "usually wraps..."

Also please note that the revised versions of the tables are in the tracked-changes version of the manuscript but not the pdf.


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

Input:
Title: BEYOND THE PREY: MALE SPIDERS HIGHLY INVEST IN SILK WHEN PRODUCING WORTHLESS GIFTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: First, I congratulate the authors for this new version of the manuscript. As previously indicated, I think this study is a valuable contribution and I hope to see it published after a few adjustments. In the following, I present a few additional comments.
Manuscript structure and content in this new version conform to PeerJ standards. Once again, raw data is properly shared.
Introduction’s quality was undoubtedly improved. However, one sentence (L119-120) is disconnected from the previous ones. Thus, it is not clear how the inclusion of a second experimental group helps us to better understand male mating effort in this spider species. In my opinion, authors need to better connect L119-121 to the rest of the paragraph. Otherwise, Introduction is abruptly finished. OBS: small changes in the text suffice to solve this problem.
Experimental design: Methods are better described in this second version. Experiment design is appropriate.
Validity of the findings: As mentioned before, findings are meaningful.
Authors improved the quality of data reporting in several ways. However, it is crucial to correct a small (but highly important) mistake. In the Word document with tracked changes, authors correctly included the new versions of Tables 1, 2, 3, and 4. However, in order to build the pdf file containing all material, authors uploaded the old (and incorrect) versions of these four tables.
Authors also improved the quality of Discussion.
Additional comments: 4.1 Most relevant general comments

(L56-62) I am not sure whether these statements on alternative mating tactics are accurate. In case they are accurate, maybe they should be clearer. For example, is it appropriate to say that “By definition, AMTs are a conditional strategy”? According to the same book chapter cited after this statement (i.e. Taborsky et al., 2008) “The relative importance of genetic monomorphism with conditional responses as opposed to genetic polymorphism for the evolution of ARTs has been extensively debated (Pienaar and Greeff 2003; see Gross 1996, Shuster and Wade 2003 for review). The vast majority of described cases of ARTs involves some conditional responses of reproductive competitors (Gross 1996, Lank et al. 1999). Because a conditional choice of tactics has been associated with genetic monomorphism, it has been argued that genetic polymorphisms play only a minor role in the causation of ARTs (Gross 1996, Gross and Repka 1998a). This view has been challenged (Shuster and Wade 2003)” (...) “Both a genetic polymorphism and a conditional expression of tactics with strong environmental influence during development have been found in different species of this group”. Additionally, is it appropriate to say that “By definition” (...) “the alternative tactic is less successful than the dominant strategy and therefore, typically occurs at a lower frequency within the population”? According to Taborsky et al. (2008): “In this case, genotype frequencies underlying ARTs are believed to be balanced by frequency-dependent selection, leading to equal lifetime fitness expectations of individuals using different tactics (Shuster and Wade 1991, Ryan et al. 1992, Repka and Gross 1995; but see Boxes 1.2 and 1.3 ; see also Chapter 2 of this book)”. I suggest that authors review this part of the manuscript (i.e., L56-62), which may lead to some corrections and/or clarifications, depending on their evaluation.

(L260-261) Values are not correct here. Instead of 55%, it should be 50%; instead of 6%, it should be 11% (as reported in Table 1).

4.2 Other general comments
(L51) It is written BussiÈre. Change it to Bussière.
(L195) Consider writing “(i.e., the last silk wrapping bout)”.
(L267) It should be “by using the Nutritive-Worthless group”.
(L278-279) This sentence starts in the past tense “even when P. ornata males had the opportunity to produce a nutritive gift” and continues in the present form “20% of them choose to offer worthless gifts”.
(L320) ‘its content’ instead of ‘it content’.
(L324) ‘receive’ instead of ‘received’.
(L328) It would probably be better to start a new paragraph here (in this current version, this paragraph is particularly long). The new paragraph would start with “Overall, research indicates”.
(Figure 1) Numbers in x-axis are not aligned with respective columns.
(Figure 2) Please, inform the meaning of ‘a’ and ‘b’.
(Figure 3) ‘Differences in silk wrapping duration according to Paratrechalea ornata male size for worthless gift production’ is not simple to understand. But more importantly, this figure does not exactly present differences in silk wrapping duration once there is no comparison between males and/or experimental groups. Instead, this figure shows how silk wrapping duration varies according to male size in Paratrechalea ornata.
(Table 1) Authors included confidence intervals for estimates in Tables 3 and 4, but not for estimates in Table 1. CIs or SE should be provided.
(Tables 1, 3, and 4) Authors forgot to substitute the term ‘Experiment’ for the term ‘Trial’.
(Tables 3 and 4) Sentences are duplicated.
(Tables 3 and 4) It would be better to report delta AIC values, setting the lowest AIC value to zero.
(Table 3; Silk wrapping duration; Model 1) Please, check the estimate value for gift type. Is it really a negative estimate?
(Table 4; Silk wrapping duration; Model 4) Please, include a space between -0.24 and (0.71).


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: METABOLOMICS OF A CELL LINE-DERIVED XENOGRAFT MODEL REVEALS CIRCULATING METABOLIC SIGNATURES FOR MALIGNANT MESOTHELIOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Gao et al propose a CDX model using metabolomics studies to identify drivers of mesothelioma. I find the concept very interesting and approach of the authors unbiased. I would have to propose some minor changes and additional experiments before the acceptance of this interesting work.

1. The language used through the paper is clear, but harsh in some places. The authors analyze the excising literature.

2. The structure of the figures is adequate.
Experimental design: I appreciate the fact that the authors reach these conclusions using an unbiased approach.  Their question is well defined and the methods sufficiently detailed.

I have a couple of additional experiments/analyses that would support the proposed model.
Validity of the findings: 1. The authors could also analyze the DepMap dependency map for their proposed genes https://depmap.org/portal/achilles/. The shown effect of the SLC genes in Mesothelioma cell Iines, will go a long way on supporting the proposed model.

2. The fact that some specific metabolites are differentially present in the proposed group, does not provide a direct link with their transporters. The authors should validate the expression of the proposed transporters in the CDX derived tumors.

3. It would be interesting to show the top differentially expressed genes in MM patients with high or low SLC signature. The analysis of one of the proposed genes is enough.
Additional comments: Gao et al propose a well supported model for markers of mesothelioma development. I would be happy to review a revised version of the manuscript. Good luck with the revision process.


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: METABOLOMICS OF A CELL LINE-DERIVED XENOGRAFT MODEL REVEALS CIRCULATING METABOLIC SIGNATURES FOR MALIGNANT MESOTHELIOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: see general comments
Validity of the findings: see general comments
Additional comments: The study of Gao and colleagues is focused on the identification of blood metabolites potentially associated with mesothelioma (MPM). They have applied mass spectrometry based metabolomics to investigate plasma samples from 10 xenograft mouse injected with tumor cells from the Ren MPM cell line and compared with 10 control mouse with no cancer. In general, the manuscript is well written and the methods are clearly described. Still, there are points which I would suggest to address for further clarification and cohesion of the message:
-	Methodology:
o	Looking into the literature, it seems that the original report about the MPM Ren cell line is from Smythe W.R. et al. (Smythe W.R., Hwang H.C., Amin K.M., Eck S.L., Davidson B.L., Wilson J.M., Kaiser L.R., Albelda S.M. Use of recombinant adenovirus to transfer the herpes simplex virus thymidine kinase (HSVtk) gene to thoracic neoplasms: an effective in vitro drug sensitization system. Cancer Res. 54:2055-2059, 1994)
o	Line 196: what is the reason for reporting/mentioning about adenocarcinoma?
o	Line 141, sample preparation: were the plasma samples pooled up to 50ul total volume or, rather, each sample was analyzed individually? How was normalization across the samples performed? What was the purpose of using an internal standard? Was this applied for normalization or for quantification or for others? What was the injected sample volume and were replicate injection used?
o	How has been pathway enrichment analysis performed, resp. how is this done in the online platform https://www.metaboanalyst.ca/ ?
o	Line 219, Figure 4a: what is the figure reporting? Is this a correlation based on circulating levels across all the samples? Relative circulation level of metabolite is relative to what? What is reported in the box-plots of figure 4?
o	Line 180, bioinformatics analysis: what is the meaning of high and low rank samples (line 183)?
-	Results:
o	Line 226, dysregulated amino acid metabolism: what is the link of the amino acid results and the genes selected from the TCGA data? Why did the authors focus and select these genes?

-	Discussion: the discussion section is verbous and could be shorter. More important, speculations are reported which are poorly supported by the results of the study or from literature evidence (e.g. line 253 hormons are unlikely the cause of cancer, line 261 “we suspect that …, line 286 “we suspect that the decrease in isoleucine…, etc. etc.). It would be helpful to report more evidence (from the data of the manuscript of from the literature) to provide evidence for the conclusions/interpretation

-	Abstract: I would suggest to smooth the statement that MPM is not well studied because it does not reflect the major scientific efforts in the field


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

Input:
Title: METABOLOMICS OF A CELL LINE-DERIVED XENOGRAFT MODEL REVEALS CIRCULATING METABOLIC SIGNATURES FOR MALIGNANT MESOTHELIOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: Gao et al., return a significantly improved version of their already well controlled study.
I find the additional materials of high clarity and importance, overall improving the impact of the proposed work.
The English language has been significantly improved.
I would fully support the publication of this interesting work.
Experimental design: All the newly added experiments, are well controlled and adequately presented in the paper. No additional comments.
Validity of the findings: No comments, all the experiments are well controlled and supported by a combination of preclinical in vivo and clinical dataset models.
Additional comments: Congratulations to the authors for their effort.


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: METABOLOMICS OF A CELL LINE-DERIVED XENOGRAFT MODEL REVEALS CIRCULATING METABOLIC SIGNATURES FOR MALIGNANT MESOTHELIOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: The references should be reported at the right place in the manuscript
Experimental design: see additional comments
Validity of the findings: see additional comments
Additional comments: The authors have provided revisions on their manuscript. There still are major methodological as well as conceptual points to be addressed in order to support the data and the results, among them:
•	Report in the method how normalization across samples was done.
•	Report in the methods how quantitation of the metabolites was done and which strategy was used.
•	Report in the methods if this was a pooled sample analysis (xenograft vs control) or if single animals were investigated.
•	If single animals were investigated, report in the methods how data for each metabolites from each animal where handled and used for the analysis: average signal in xenograft-controls? Median? Ratio to internal controls? Ratio between groups? Etc. etc. .
•	That following, report in the methods what was used for PCA etc..and what FC represents in the volcano plot and fold change of what.
•	Explain in the manuscript how the data were retrieved from the XENA platform and which dataset was used and how these were combined with the dataset from the GEO database.
•	Please report within the manuscript the definition of high/low rank samples.
•	Figure 4 remains not clear. A: Is this a pearson correlation as reported in the method or a spearman as in the results? What do they correlate? Average or median values of the metabolite over the entire cohort (xenograft and controls)? Circulating levels are relative to what? Please explain in the text, within the method and in the results/figures. B-G: Please explain in the figure legend what the abbreviations are (e.g. FC), on what is the fold change based in the figure (average?median?others?), what are the whiskers and what are the bars. Please report in the methods how the P values are calculated.
•	Please report the many prognosis-related genes from the bioinformatic analysis.
•	What level of confidence has been used in table 3?
•	Again, the link to why the authors have decided to focus on the SLC transporter genes is not provided. There is no experimental evidence within the manuscript to support the hypothesis that the levels of amino acid observed in the blood of the animal models may be related to the SLC transporter genes. The authors have decided to follow this hypothesis and explore a potential prognostic effect of their hypothesis, but they should guide the reader to what is the motivation of doing so and provide evidence for that. The “referenced paper” should be reported and referenced at the right place. They should also discuss this hypothesis in the discussion section. There is no evidence reported in the manuscript that these genes may be therapeutic targets.