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: APPLICATION OF CARBON DIOXIDE TO THE SKIN AND MUSCLE OXYGENATION OF HUMAN LOWER-LIMB MUSCLE SITES DURING COLD WATER IMMERSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript has numerous grammatical and structural errors and requires proof reading:
-	Some sentences can be written in a more concise manner. For example, Line 29 Abstract, can be revised to “…CO2 hot water immersion results in vasodilation in both cutaneous and muscle tissues”. Line 73 – “vascular constriction” can be replaced with “vasoconstriction”.
-	Define the abbreviation for the spelled-out text at the first mention. For example, skin temperature was mentioned first in Line 32 (Abstract), but the abbreviation “Tsk” was defined in Line 37.
-	Some abbreviations were redefined throughout the manuscript. The authors can consider using the abbreviations after they have been defined at the first mention. For example, Tcore was defined in Line 136, Line 239, and Tsk-cont was defined in Line 139 and Line 249.
-	There was some inconsistency when referring to a parameter, for example, thermal sensation versus thermal score in the figure and VAS in the figure caption.
-	Replace “became significantly increase” with “increased significantly”.
-	Some inconsistency in the format of the abbreviations, for example, core and sk were subscripts in the methods section, but not in Line 239 and Line 241.

Table 1 and Figure 6 are presenting the same variable StO2 for TA and GAS (superficial and muscle), one in absolute, the other in relative change. I suggest the author to include Table 1 as supplementary material only.

Figure 4, 5, 6 – “Time bins and symbols are the same as in Fig. 3”. Define the abbreviation and symbols in each figure.
Experimental design: The water immersion protocol is well-described. The authors can consider using a schematic diagram to help readers to better understand the flow of the protocols and the time points of different measurements, especially for the NIRS measurements. It is also important to indicate if the CCWI and CWI conditions were conducted on separate visits, and how the order of conditions was decided.
Validity of the findings: Throughout the results section, there is some inconsistency and some areas require clarification:
-	Main and interaction effects, and F-values were not reported for HR, Tcore, although these were reported for other variables.
-	Was post-hoc analysis for Tsk-WI, Tsk-cont and thermal sensation performed? As there was a significant time effects, but the exact p values for the mentioned time points were not included.
-	Effect size should only be reported if the p-values approximated 0.05. Additionally, p-values should be presented for Line 269.
-	Include the p values, for example, ∆total[Hb+Mb] (Line 264),  ∆deoxy[Hb+Mb] (Line 269).

Line 331 – Based on the results, there was only a main time effect, but no condition or interaction effect for thermal sensation. Hence the sentences “…a slightly higher thermal score…” an “CCWI facilitated the slightly but significantly lower cold sensation compared to CWI” is not supported by the results.

Line 363 to 411 – A large portion of this section about hydrostatic pressure is informative but has little relevancy. The discussion about the hydrostatic effect is distracting from the main aim of the study, which is to compare between CCWI and CWI, in which hydrostatic pressure is present in both conditions.

Line 377 – Cooling per se may also reduce muscle temperature and metabolic activity, thus resulting in increased oxyHb. Additionally, I recommend the authors to avoid interpreting their results based on effect size only especially when the associated p-value is not close to 0.05.

The changes in the NIRS parameters should be discussed in conjunction with the underlying mechanism of vasodilation during CO2-enriched CWI.
Line 413 to 427 – This paragraph reads like the results section.

Line 418 to 420 – This sentence reads like the authors attributed the difference in the oxyHb between CWI and CCWI to hydrostatic pressure, but the hydrostatic effect is present in both conditions and thus cannot adequately explain the stated difference.

Line 447 to 459 – As only the lower leg is immersed, it is unlikely that HR, MAP and core temperature will be changed drastically. This section has little relevancy to the focus of the study.

The conclusion was not supported by the results. As mentioned in my comments earlier, avoid interpreting the results based on effect size especially when the p-value is not close to 0.05:
-	Figure 5 - OxyHb and deoxyHb were not different between CWI and CCWI, although total Hb increased after CCWI in the TA muscle.
-	Figure 6 – OxyHb was higher in CCWI, while total Hb and deoxyHb was not different in the GAS muscle.
-	Higher StO2 is also indicative of a lower metabolic activity (Ihsan et al., 2013).
Additional comments: This study examines the effect of CO2-enriched CWI on skin and muscle perfusion and oxygenation. In general, the study appears to be carefully performed and well-controlled. They conclude that CO2-riched CWI causes vasodilation and decreases skin temperature to a greater extent compared with CWI. This reviewer finds that the conclusion is not supported by their results.  The basis for this study is that CO2-enriched CWI will induce vasodilation during immersion. However, taken together the changes in total Hb (perfusion) for TA and GAS (both superficial and deep tissues), there was no difference between CWI and CCWI, except for total Hb for TA which appeared to increase post-immersion, but post-hoc analysis was not shown (see below). The results are also in contrast to the findings reported by Nishimura et al. (2002) “Effects of repeated carbon dioxide-rich water bathing on core temperature, cutaneous blood flow and thermal sensation”, and Tanaka et al. (2020) - Body cooling effects of immersion of the forearms in high-concentration artificial carbonic acid water at 25°C. In both studies, skin blood flow as assessed by Laser Doppler increased significantly while thermal sensation was lower in the CO2-enriched CWI:

-	Total Hb for TA superficial as an indicator of cutaneous perfusion was not different between CWI and CCWI and there was no interaction effect (Figure 4, condition effect p = 0.464, time effect p = 0.066, interaction effect p = 0.442).
-	Total Hb for TA muscle as an indicator of muscle perfusion showed that it increased post-immersion in CCWI, as opposed to decrease in CWI (Figure 4, interaction effect p = 0.044, post-hoc analysis not shown).
-	Total Hb for GAS superficial also showed no difference between CWI and CCWI (Figure 5, condition effect p = 0.729, time effect p = 0.027, interaction effect p = 0.091).
-	Total Hb for GAS muscle showed no effect of CCWI or CWI (Figure 5, condition effect p = 0.311, time effect p = 0.294, interaction effect p = 0.334).


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: APPLICATION OF CARBON DIOXIDE TO THE SKIN AND MUSCLE OXYGENATION OF HUMAN LOWER-LIMB MUSCLE SITES DURING COLD WATER IMMERSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors prepared very carefully all comments and suggestions. All criterias are included correctly.
Experimental design: The experimental design is prepared correct, however, I would suggest adding statistical analysis of the sample size - using G Power. The authors recruited 15 participants. It would increase the quality of this work.
Validity of the findings: This point of view is a novel investigation which may find pracctical application is profesional sport.
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: APPLICATION OF CARBON DIOXIDE TO THE SKIN AND MUSCLE OXYGENATION OF HUMAN LOWER-LIMB MUSCLE SITES DURING COLD WATER IMMERSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Congratulations on a vastly improved manuscript and it clarifies the significance of the study. The flow of the introduction and discussion in the paper are much more clearly related to the aims of the study.
Experimental design: Methods:
Line 128 – Thank you for clarifying the blinding process. Just one more question, a double-blind design means that both the subject and the researcher involved were unaware of the condition (CWI or CCWI) during the experiment. Is that the case? You can also consider removing the parenthesis in that sentence and replace the word “insure” with “ensure”.

Statistical analysis:
Line 209 –Thank you for addressing my concerns regarding the use of effect size. Since the effect sizes are not reported in the revised manuscript, consider removing the related section in here.
Validity of the findings: The results are much clearer and the conclusion is in line with supporting data.
Additional comments: Abstract:

Line 51 - Consider removing “despite the lower Tsk-WI values after the CCWI…” since you stated that Tsk-WI remained lower in the CCWI than in CWI later in the same sentence.

Results:

Line 292 – Replace “Alternation” with “alteration” and “under” with “in” – Alterations in O2 saturation in muscles as measured by NIRS

Discussion:

Line 310 – consider remove “blood vessels” in the sentence blood vessels' vasodilation since vasodilation refers to the widening of blood vessels.

The discussion about thermal sensation is a little hard to follow. Line 330 to 331 states that CCWI results in a higher thermal sensation. If based on the scale used (cold = -3, neutral = 0), a higher thermal sensation means that the subjects felt less cold. But in Line 338, CWI results in lower cold sensation. Also, consider removing “significantly” in the sentence “CCWI facilitated the slightly but significantly lower cold sensation”. The absence of a significant difference in thermal sensation between CCWI and CWI may also be related to relative small segment of the body exposed compared with whole body immersion.

Line 369 to 371 - I am not sure if this sentence is necessary here as it is repetitive with the next paragraph.

Line 373 - This sentence is hard to follow. The change in total Hb (muscle) was a result of oxyHb(muscle) increase during CCWI and recovery, which was a result of oxyHb(muscle) and dexoyHb(muscle)? Consider rewriting for clarity.

Line 380 – Consider removing “our subjects’”.

Line 383 – Consider including more than one reference since you mentioned several studies have suggested…

Line 385 – Please clarify redistribution of blood flow from where to where?

Line 399 – Even though it may be obvious to some readers, it may be better to clarify why slow-twitch fibers would facilitate the diffuse of CO2?

Conclusion:

Line 450 – Because of the choice of modal verbs used (“could not” and “not able to”), the current sentence reads like the aim of CCWI was to reduce Δdeoxy[Hb+Mb]muscle and to decrease local metabolic demand. Consider rewriting the sentence for clarity.


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: CHEMOTYPE CLASSIFICATION AND BIOMARKER SCREENING OF MALE EUCOMMIA ULMOIDES OLIV. FLOWER CORE COLLECTIONS USING UPLC-QTOF/MS-BASED NON-TARGETED METABOLOMICS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Line 33: Please make this line clearer - “The compositional variations were inferred originate from genetic differences but not from geographical origins.”

Line 160: “306 and 8,616 features were obtained….” – please correct to 11306

Line 294-295: Why do the authors prefer confirmation of annotated compounds via in silico fragmentation instead of using possibly available reference standards and NMR for novel compounds?

Table S4: Correct spelling of “annotation”. Meta data needs to be explained better. Why do some rows in “Adducts” have a “-“ ?
Experimental design: Result: Metabolite annotation – The procedure of how the annotation of candidate metabolites via MS/MS fragmentation matching was implemented needs to explained more elaborately.
Validity of the findings: no comment
Additional comments: The article is well written and explained. I do appreciate the extensive statistical analysis, however I would like to see more emphasis on the identification of interesting metabolite markers.


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: CHEMOTYPE CLASSIFICATION AND BIOMARKER SCREENING OF MALE EUCOMMIA ULMOIDES OLIV. FLOWER CORE COLLECTIONS USING UPLC-QTOF/MS-BASED NON-TARGETED METABOLOMICS
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is clear and unambiguous.
Professional English is  used throughout.
It conforms to professional standards of courtesy and expression..
Sufficient field background/context is provided.
The article includes sufficient introduction.
Relevant prior literature is appropriately referenced.
Professional article structure, figures, tables.

Figures are relevant to the content of the article,
They have sufficient resolution, and are appropriately described and labeled.
Experimental design: Research question well defined, relevant & meaningful.
Validity of the findings: Not all underlying data have been provided;

Results are robust, statistically sound, & controlled.

Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Mayor points
Not all appropriate raw data have been made available.
Please provide data in table format. (ion intensity matrix)

Please discuss in more detail following metabolites: geniposide, geniposidic acid, aucubin, and chlorogenic acid

Please discuss results from earlier studies (Du et al., 2017; Du et al., 2017; Du et al., 2016).
Which outcomes are different?

L184 It should be noted that biological duplications from the six collections SNJ-1, BZ, BJ-3, BJ-1, HZ, and LY were not clustered within a chemotype.
Please explain this problem with more detail.

L204 The classification results were slightly inconsistent with those from the ESI+ mode data
Please address this problem with more detail. The first two components of a PCA plot is not always the best was to discriminate samples.
The chemotype boundaries in the PCA plot were inexplicit for the ESI- mode data.
For how many of the PCA dimensions? The first two or all?
Did you try random forest classification?

Minor points

L61    is the reference (Du et al., 2017) given twice?  Use lettering: Du et al., 2017a; Du et al., 2017b
L63  reference given twice: (Li et al., 2018; Li et al., 2018). Use lettering a and b for all those references across the manuscript.

L150 by using the Elucidean distance matrix  -> Euclidean
Were the data previously scaled/normalized? Euclidean distance is strongly influenced by total ion intensity.

L160 306 and 8,616 features were obtained from the extracted ion chromatograms acquired in ESI+ and ESI- modes.
Only 306 features in positive mode? More features were obtained in negative ion mode than in positive ion mode? Maybe you mean 11,306

L240 E. ulmoides bark has been especially expensive in the market.
Please explain in the introduction if the bark or the male flowers are used for medicine purposes.


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: CHEMOTYPE CLASSIFICATION AND BIOMARKER SCREENING OF MALE EUCOMMIA ULMOIDES OLIV. FLOWER CORE COLLECTIONS USING UPLC-QTOF/MS-BASED NON-TARGETED METABOLOMICS
Review round: 2
Reviewer: 1

Output:
Basic reporting: L205-210: Extremely long sentence. Please simplify!
L264-268: Correct typos and grammar
L275: "Contour"? What is it that the authors want to say here?
L308: Correct spelling of "Phenylpropanoids"
L309: "Glycosides" appears twice
L314: The following study..
L323: "inconsistent" is one word
L324 "On" one hand
L335: phenotyping achieved

This time round I had trouble with the English language.  It makes it hard to understand the authors' points.
Please go through the text with a fine-toothed comb for grammar and typos. The text is missing prepositions.
Experimental design: Original primary research within Aims and Scope of the journal.

Research question well defined, relevant & meaningful. It is stated how research fills an identified knowledge gap.

Rigorous investigation performed to a high technical & ethical standard.

Methods described with sufficient detail & information to replicate.
Validity of the findings: no comment
Additional comments: In general I think the authors have addressed the reviewer's queries. However, the major impediment remains, which is the language and formulation of sentences. Please pay attention to the language.


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: IMPROVING THE PHYSICAL, MECHANICAL AND ENERGETIC PROPERTIES OF QUERCUS SPP. WOOD PELLETS BY ADDING PINE SAWDUST
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clear with professional English language used throughout.
The introduction needs improvements mentioned in the general comments
The Literature review is good and relevant.
The structure is conform to PeerJ standards,
Figures are relevant, high quality, well labelled & described.
Raw data was supplied
Experimental design: The experimental design is correct and follows the standard analysis in this type of study
Validity of the findings: This paper doesn’t present significant novelty, but the results are acceptable. The conclusions are satisfactory. However, the justification for use this type of biomass (oaks) to pellet production is not sufficient.
Additional comments: The aim of this paper is to find better Quercus spp. pellets in their physical, mechanical and energetic properties by adding Pinus durangensis sawdust. The subject is interesting, but paper doesn’t present any novelty. Moreover, the paper mention that the Quercus is the second specie in Mexico, but is the supply (felling, thinning, etc) enough to motivate this study? In terms of Ecology, there’s is not a better utilization for this “residues”? The paper’s structure is good and the and analysis adequate, however, it needs better justification of the purpose of this study. Some detailed considerations are made below.

Detailed comments:
L 20 – “During logging, large amounts of waste are generated”. Please consider review this sentence. Nothing in forest is waste. The remain forest residues are nutrient for the ecosystem.
L 50-52 - “In addition, the use of biomass as fuel reduces greenhouse gas emissions during combustion and is considered neutral for carbon dioxide”. This sentence is controversy. Maybe the authors could include also some reference that claims that biomass is not neutral.
L 69 – “However, several species of Quercus genus which is the second most important forest resource “. Please provide the area occupied and the estimated residues generated by logging, thinning forest operations.
L183 – In order to be more comprehensive, consider write first the results and after comment the statistical results. Do the same in the other results
L183 – Please clarify in the text as well in the Methodology section that the MC is of the samples after “pelletisation” process.
L 185 …”lowest mean content”. Moister content?
L 232 – Please include here the full designation “impact resistance index (IRI)”


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: IMPROVING THE PHYSICAL, MECHANICAL AND ENERGETIC PROPERTIES OF QUERCUS SPP. WOOD PELLETS BY ADDING PINE SAWDUST
Review round: 1
Reviewer: 2

Output:
Basic reporting: I suppose that overall quality of the manuscript is very low,  for a relevant journal with a IF 2.35 as Journal of Life and Environmental Sciences is.   First, I think that  the 5.5 pages of references are too much exaggerated for the content of the article. The introduction should mention the potential for negative carbon dioxide budget for biochar from biomass(Line 52). In my view the Introduction is not very  clear:  e.g.,  (i) why the compostion in forest species change from logging methods lines, (lines 53 and 54) (ii) the densification and pelletizing (lines 60-67) are well matured in markets, and thus its description should be shorter and put in the beggining of the Introduction. The Introduction should also focus on the methodologies, for example in mechanical evaluation, used in M&M with same text which is in the Discussion. The obejctives (lines 95-97) with only three lines should be more detailed. The english phrasing should also be improved: for example "narrow"   in line 71 or "reach international standarts" in line 78 are not clear wordings or the Introduction has too many paragraphs for its content.
Experimental design: If the elementary analysis (C,H,N,O,S) was not possible, and only proximate analysis was, a justification should be given, or indeed better that analysys, which is very common for biomass characteerization should be included; Wood or composite hardness should cahacterized through texts with stress values in SI units (e.g. Janka hardness test) under a more precies methodology than drop test is.
Validity of the findings: In the Results section p-values should not given rates of e.g. 4.09*10^(-5). P-values lower than say 0.001, should be enough. Moreover the results should be given in Tables and not only in graphs (Figures 2-5). Besides, some photos of pellet specimens with pine sawdust should be added. In the discussion moisture value should not given the elaboration shown insofar that moisture is controlled by drying(artificial or natural) and thus its values are transient. Oppositelly the eleboration given to vlolatiles and fixed carbon in discussion is very short and should include same considerations related to the structure of the materials. The ash discussion is also too long as it isand should directed to the relations with final use of pelletes: domestic or industrial. The discussion on bulk density is also too lenghty and correlations between bulk density and mechanical beheviour are missing and should of could also be included. In mechanical properties discussion the limitations of drop test should justify a comparision with e.g. Janka hardeness. Also, the absence of Tables turns the presentation of results and discussion as inadequate. Also I don't understand how a decrease in IRI values (line 387) improves mechanical hardness (line 420) in the Conclusions.  Also an evaluation of the interconectedness between bulk density and calorific power should be included, e.g. though correlation analysis, of variables such fuelwood value index or energy density. This would contribute to improve the coherence of the text.
So overall I suppose that this work of improving pellet quality through pine sawdust addtion has potential but the narrative carried out is not enough to demonstrate that potential and I would recommend a major revision for a further review.
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: IMPROVING THE PHYSICAL, MECHANICAL AND ENERGETIC PROPERTIES OF QUERCUS SPP. WOOD PELLETS BY ADDING PINE SAWDUST
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: The experimental design was fair despite the proximate analysis of pellets (important parameter) were nor determined.
Validity of the findings: no comment
Additional comments: I was carefully reading the paper improvements and the authors answered the reviewer’s questions and the paper was significantly improved. Nevertheless, my question about the soundness and global quality of this papers remains. The paper has a low average quality for the high standard quality of PeerJ. However, if the decision is to publish, I do not detect any major inconsistencies in the methodology and results presented.


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

Input:
Title: FURTHER EVALUATION OF DIFFERENTIAL EXPRESSION OF KERATOCONUS CANDIDATE GENES IN HUMAN CORNEAS
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: This study reports the differential expression of transcript genes in normal and keratoconic cornea. Over all, manuscript was well written with good scientific manner.  Interesting to read by clinical scientist, those who working on corneal diseases and pathology. I have few minor corrections below.

1.	Abstract: Results section Line no 39, Among differentially expressed genes were genes involved in collagen synthesis, the TGF-β, Hippo, Wnt signaling and other pathways linked to the extracellular matrix formation. Please rewrite the above sentence for better clarity for readers.
2.	Key words: Please include Keratoconic cornea
3.	Table-s1 – “The clinical characteristics and ophthalmic findings” could you please describe the clinical grades of KC like Amsler-Krumeich classification if not mentioned other method for classification.


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: FURTHER EVALUATION OF DIFFERENTIAL EXPRESSION OF KERATOCONUS CANDIDATE GENES IN HUMAN CORNEAS
Review round: 1
Reviewer: 2

Output:
Basic reporting: - The article has multiple incidences of typographical and grammatical errors.
- It is unclear if the authors' knowledge of signaling pathways are weak or are just mis-identified. e.g. TGFB is called transcription growth factor instead of transforming growth factor.
- Literature review is incomplete in identifying the numerous studies that have identified candidate genes for keratoconus.
- An important candidate gene for keratoconus is LOX and the authors while studying this gene have failed to discuss the importance of the data or lack of thereof.
-
Experimental design: The experimental design is straight forward where it serves to secondary validate expression of genes identified by RNASeq previously. Techniques are apporpriate.
However, it would have been more prudent to identify SNPs for the genes associated which would have bolstered their study design rather than mere RNA expression.
Validity of the findings: The authors tend to over-interpret their data. The authors mention deregulation of multiple signaling pathways but actually do not demonstrate that. They indeed do demonstrate up/down regulation in a few genes that are pertinent to these pathways. However, there is perceived conflict in the data which is not given due discussion: e.g. Why is Smad7 (a negative regulator of TGFß pathway) down yet TGFBIP up? Are the expressions of genes down due to a inhibited activity of stromal cells or are they transcriptionally and translationally altered? It would have helped to correlate changes in gene expression with protein levels either via histochemistry or Western blots.

Authors further claim SLC4A11, Wnt 5a and other genes are not consistently altered within the disease group (see Fig. 1) i.e. large donor variability. Yet the authors talk about up/down regulation within the statistical treatment group which is not true.
The hirearchical clustering is organized haphazardly with not groupings between KTCN and non-KTCN samples. It would benefit with restructuring.
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: FURTHER EVALUATION OF DIFFERENTIAL EXPRESSION OF KERATOCONUS CANDIDATE GENES IN HUMAN CORNEAS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper is written in clear, unambiguous professional English. It uses a professional article structure with relevant figures and tables and the raw data is included as Supplementary data.
The introduction could be improved by expanding the rationale for the study. From the analyses conducted, it appears to be a replication study of previously reported findings (from these authors and others). However, the end of the introduction says the aim is to see if keratoconus patients can be distinguished from controls on the basis of gene expression in the cornea. It is not at all clear why this is the aim. Using corneal tissues to make a diagnosis of keratoconus does not sound useful when there are non-invasive diagnostic techniques that can be applied well before corneal graft is required. I think this study has confirmed some important pathways in keratoconus biology, using a second method to increase confidence in previous findings.
Experimental design: As noted above, the research question is defined, but I am not convinced that it is relevant or meaningful or what knowledge gap the study fills specifically. The manuscript should clarify this for the reader.
More detail is required on the methods for the choice of genes. For example, 23 genes were chosen from the previous RNAseq study, but it is not mentioned what criteria were used for inclusion. Is it a fold change? Top ranked genes? DE genes from specific candidate pathways? Similarly for the additional genes selected from the literature, how was it decided what to include and why?
Validity of the findings: This study reports a replication analysis of previous RNAseq data, using in independent method on the same patient specimens. This is valid and useful and provides increased confidence of the previous findings.
Further clarity could be achieved by indicating which of the genes in Table 1 were from the previous RNAseq study and which ones were added. The results should then be discussed in this context, indicating if any of the findings are novel (i.e. gene expression not previously studied).
The results should also be discussed in relation to the statistical significance. No adjusted p<0.05 was reported, yet it is claimed that all but 1 gene is confirmed as differentially expressed. The validity of this finding appears to be open to interpretation.
Additional comments: In Table 1, please indicate in the table description that these results are from the RTqPCR assay.
In Figure 1, please explain in the legend what the histogram is showing and if possible, clearly indicate which specimens are from keratoconus patients and which are from controls.
Please describe in the methods what range of indications for graft the non-KTCN patients have and discuss that in more detail specific to this study in the limitations section of the discussion.


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: FURTHER EVALUATION OF DIFFERENTIAL EXPRESSION OF KERATOCONUS CANDIDATE GENES IN HUMAN CORNEAS
Review round: 2
Reviewer: 1

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


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

Input:
Title: FURTHER EVALUATION OF DIFFERENTIAL EXPRESSION OF KERATOCONUS CANDIDATE GENES IN HUMAN CORNEAS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No Comment
Validity of the findings: No comment
Additional comments: The authors have responded to all my concerns and the manuscript is much clearer.


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

Input:
Title: THE LONG-TERM OUTCOMES OF DECEASED-DONOR LIVER TRANSPLANTATION FOR PRIMARY BILIARY CIRRHOSIS: A TWO-CENTER STUDY IN CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Chen et al. analyzed the outcome for primary biliary cirrhosis (PBC) up to 5 years after liver transplantation. The authors’ main findings are that a recipient aminotransferase-to-platelet ratio index (APRI) under 2 showed significantly better overall survival and that an age under 50 showed significantly higher risk of PBC recurrence.

I. Introduction
This chapter is well-written, and the references are all pertinent. However, in line 89, “alcoholic cirrhosis” should be changed to alcohol-related or -associated liver cirrhosis. The American Association for the Study of Liver Disease (AASLD) and the European Association for the Study of the Liver (EASL) have updated their guidelines on alcohol-related liver disease (reference, Crabb DW et al. Hepatology. 2020. EASL. J Hepatol. 2018). One of the most significant updates is a change from the term “alcoholic” to “alcohol-related (or-associated).” Even though the name of a disease is a relatively superficial concern, in this case, the word “alcoholic” is very stigmatizing. Therefore, the phrase “alcohol-related” is suggested instead. In addition, the phrase “liver failure” is vague. Please employ a more specific term.

II. Table 1
Information about the full terms abbreviated as AST, ALT, ALP, GGT, T-Bil, and ALB would be helpful.
Experimental design: I. Patients and methods
•	Line 106: Provide details about the type of liver transplantation (living-donor liver transplantation vs. deceased-donor liver transplantation).
•	Patients and Methods: Indicate the formulas for APRI and MELD.

II. Results
•	The authors focused on APRI as a prognostic tool for post-transplantation outcomes. Please clarify the reason why the authors focused only on APRI and not other fibrosis markers.
•	The authors classified patients by APRI and age. However, it is not clear why the cutoff values for these were set to 2 and 50, respectively. Additionally, information about both hazard ratio and the 95% confidence interval in the log-rank tests is missing. Univariate analysis of overall survival for the 62 patients, including gender and incorporating pre-transplant age, Child-Pugh score, MELD score, aspartate aminotransferase, alanine aminotransferase, and platelet count, should be performed.
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: THE LONG-TERM OUTCOMES OF DECEASED-DONOR LIVER TRANSPLANTATION FOR PRIMARY BILIARY CIRRHOSIS: A TWO-CENTER STUDY IN CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: English writing at a high standard and reasonable literature references etc. However major issues include;

1) An abstract which is unstructured.

2) Poor structure: methods section and results section seem to be blended, rather than separated (line 105 to 127 in mostly made up of results, rather than methods).

3) the term "median end-stage liver disease score" is incorrect, it should be "model for End-stage Liver Disease score" and abbreviated as MELD.

4) In Figure 4, it should be clarified if LFTs are mean or median.

5) in line 178, it should be clarified if APRI score is pre-transplant or post-transplant.

6) in line 187, the term "negative indicator" is not a scientific conventional term. Please rephrase.

7) line 203, "The vast majority of infections were microbiologically y bile"- what does "microbiologically y" mean? It should be deleted.

8) line 211-212 should have percentages after "two" and "three"
Experimental design: Major issues include:

1) It is unclear when the 'baseline' blood tests results alluded to in 116 to 124 were taken. In the methods it is stated that blood tests were simply taken "before the transplant". Is that immediately before? 1 day before? 1 week before?

2) It is stated that 2 patients were excluded from LFT analysis post transplant due to "changed liver function as a result of hepatic artery thrombosis". However this is not standard practice to selectively exclude patients just because they had a particular type of complication. Indeed, the authors included many patients who had other complications that could have altered the post-transplant LFTs, and many patients who had IPGF. What is the justification of excluding those with hepatic artery thrombosis? Surely if they had massively abnormal LFTs, it would not matter as the authors are analysing median results, not mean results.

3) It is unclear how many patients ended up having percutaneous liver biopsy. It is stated that 6 had PBC diagnosed via biopsy. How about those who had biopsy but PBC was not diagnosed?

4) The authors found via log-rank testing that APRI >2 (I assume preoperatively) was
associated with poorer overall survival, and that MELD score (I assume preoperatively) was not. However it is not clear from their methods whether they performed log-rank testing for any other variables such as age, gender, Mayo risk score, pre-operative LFTs,  post-operative LFTs, Child Pugh score, and if so what the results were.

5) The authors found via log-rank testing that age <50 was associated with greater disease recurrence.However it is not clear from their methods whether they performed log-rank testing for any other variables such as gender, Mayo risk score, APRI score, pre-operative LFTs, post-operative LFTs, Child Pugh score, MELD score and if so what the results were.

6) Similarly, did the authors consider analysing for any more sophisticated transplantation scores such as the UKELD score, MELD-Na score, iMELD score?
Validity of the findings: 1) In the results, there are no figures showing the overall Kaplan Meyer survival curves for the whole cohort. This would be meaningful to present in addition the curves stratified by pre-operative APRI score.

2) The authors statement that "protocol biopsies are recommended after transplant" and "In future researches, protocol criteria should be recommended in order to detect early recurrence timely" are contentious. Protocol biopsies of patients after transplant for PBC are rarely routine outside of research setting. I do not know of any major transplant centres who perform this in the absence of LFT dysfunction. How can the authors justify performing so many biopsies on asymptomatic post-transplant PBC patients with acceptable LFTs, given the inherent risks and resource utilization inherent in liver biopsy?

3) The authors state in the discussion that "Consistent with our study’s findings, this previous study showed that a high GGT level at day 7 after liver transplantation is associated with increased survival rates within the first 90 days". This is completely wrong. The authors' study did not analyse the relationship between GGT at day 7 and survival. In fact it did not analyse the relationship between any post-operative LFTs and survival: and in fact it is unclear why the authors have therefore spent so much efforts detailing the trajectory of post-operative LFT changes in their cohort. What is the practical value of detailing these trajectories to the reader of this study? How does it change patient prognosis or management?
Additional comments: No additional comments


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

Input:
Title: THE LONG-TERM OUTCOMES OF DECEASED-DONOR LIVER TRANSPLANTATION FOR PRIMARY BILIARY CIRRHOSIS: A TWO-CENTER STUDY IN CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No additional comments.
Experimental design: No additional comments.
Validity of the findings: No additional 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: THE LONG-TERM OUTCOMES OF DECEASED-DONOR LIVER TRANSPLANTATION FOR PRIMARY BILIARY CIRRHOSIS: A TWO-CENTER STUDY IN CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Abstract remains unstructured. By convention, scientific abstracts are separated into sections: aim, methods, results and conclusions.

Abstract states age <50 is a risk factor for PBC recurrence. However now the manuscript states <48 years.

Lines 114-116 do not belong in the methods section. They belong in the results section

"In our study, 26 patients were classified as Child-Pugh grade B; 43 patients, grade C. One patient had a second liver transplant due to thrombosis of the hepatic artery 2 weeks after the first liver transplant."
Experimental design: Nil
Validity of the findings: In the results section, lines 189-192, it is stated that ". Univariate analysis failed to find that age, gender, the MELD score, Child-Pugh score, Mayo risk score , pre- and post- operative liver function were statistically significant in predicting the outcome of liver transplantation."  Firstly, this should be "predicting survival", not "predicting the outcome of liver transplantation. Secondly, what type of univariate analysis was this? Was it univariate logistic regression analysis?

Then in the lines 199-203, it is stated that "In a multivariate Cox regression analyse model adjusted for gender, pre-operative Mayo risk score, 201 APRI score, MELD score, a preoperative age of 48 years or younger remained the only independent risk factor of patient recurrence (hazard ratio 0.028, 95% confidence interval 0.01- 0.71, P = 0.03)". Firstly, this should be "disease recurrence", not "patient recurrence". Why was a multivariate Cox regression analysis performed for disease recurrence, and not for survival as above (where only univariate analysis of some kind was performed)?

The authors have stated in a rebuttal that "In our study,7 patients underwent percutaneous liver biopsy postoperatively. Only 1 patient was diagnosed rejection reaction，and another 6 patients were diagnosed recurrent PBC. " This infromation should be included in the study. In fact, in line 189, it states "Of 69 patients who had abnormal liver function, 6 were diagnosed with rPBC via liver puncture biopsy.". This is incorrect: a total of 69 patients were included in the whole study, but it seems only 7 patients had unexplained abnormal liver function that went on to require biopsy.
Additional comments: No additional comments


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

Input:
Title: THE ANTIFUNGAL ACTIVITY OF VAPOUR PHASE OF ODOURLESS THYMOL DERIVATE
Review round: 1
Reviewer: 1

Output:
Basic reporting: There is a lack of statistical analysis of the experimental results.
Experimental design: no comment
Validity of the findings: no comments
Additional comments: The paper suggests a new idea  and material  to fight against fungal contamination to protect cultural heritage.

I review the manuscript. The paper suggests a new idea about using the vapour of kubicin to fight against fungal contamination to protect cultural heritage. However, I would like to comment on some parts that could increase the readability of the paper.
1.	Page 6, line 67, “The authors conclude that only the use of 68 ethylene oxide fumigation has long-lasting (one year) effect, but this chemical does not remain 69 on treated materials, so the subsequent contamination is possible”. The sentence is inconsistent
2.	Line 139, “To” was removed
3.	There is a lack of statistical analysis of the experimental results. No variability parameter (standard deviation, standard error, etc.) was included in tables. Hope you suppling these data.
4.	In fig 2 and 3, you need to mark a, b, c on the fig and note what they are for every strain under the fig.
5.	In the part of Antifungal effect of kubicin. Is kubicin dissolved in water or in an organic solvent? if it was dissolved in an organic solvent, is there a control group with only added organic solvents?


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

Input:
Title: THE ANTIFUNGAL ACTIVITY OF VAPOUR PHASE OF ODOURLESS THYMOL DERIVATE
Review round: 1
Reviewer: 2

Output:
Basic reporting: - English is so poor. English should be reviewed by native speakers.

- Introduction: Too long. It is necessary to shorten the Introduction section.

- Discussion: Too short and few references. No deep discussion about their finding. No discussion about prior studies.
Experimental design: 1. Materials & Methods
- All chemicals: Purity should be described.
- Synthesis of kubicin: Mass data?
- Antifungal activity: Only figure (Fig. 2 and Fig. 3) is shown. Where is data?
                               It is necessary to describe fungicidal activity as % or length of inhibition
                               zone. No statistical analysis. Only one concentration of kubicin was
                               tested for contact and vapor phase antifungal activity (FIg. 2 and Fig. 3)
- Data for GC-O analysis is only described as text. No figure or table. It is necessary to describe mean and standard deviation (or error).
- Table 1: Only one replication? Describe as mean and standard deviation (or error).
-  Table 2: what is unit? uL/mL air?
Validity of the findings: - Interesting finding but lack of data and poor presentation of their result.
Additional comments: This manuscript is about the antifungal activity of kubicin.

Experimental design and presentation of results are not logic.

This manuscript should be redesigned and rewritten for publication.


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

Input:
Title: THE ANTIFUNGAL ACTIVITY OF VAPOUR PHASE OF ODOURLESS THYMOL DERIVATE
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Fig 4 % inhibition change to inhibition (%)


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

Input:
Title: COMPARATIVE ASSESSMENT OF SINGLE-STAGE AND TWO-STAGE ANAEROBIC DIGESTION FOR BIOGAS PRODUCTION FROM HIGH MOISTURE MUNICIPAL SOLID WASTE
Review round: 1
Reviewer: 1

Output:
Basic reporting: It contains what is necessary to be published.
Experimental design: On line 110 with respect to IFA 0.5% and 1%, is it related to the VS load? or even the pH adjustment? or by weight ?, explain.
In the results appears the aforementioned relationship line 184 (w / v)
Doesn't it explain what type of reactor it is, agitated tank ?, drained bed? in batch, etc. is very important.
Validity of the findings: In general it is a good article that shows the incorporation of elements to buffer pH, in addition to evaluating the microbial populations that are in each stage and explains their influence on the process.
Additional comments: On line 110 with respect to IFA 0.5% and 1%, is it related to the VS load? or even the pH adjustment? or by weight ?, explain.
In the results appears the aforementioned relationship line 184 (w / v)
Doesn't it explain what type of reactor it is, agitated tank ?, drained bed? in batch, etc.
in line 125 it mentions the alkalinity range in mg-CaCO3 and in the discussion it does not compare with the appropriate ones.

In general it is a good article that shows the incorporation of elements to buffer pH, in addition to evaluating the microbial populations that are in each stage and explains their influence on the 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: COMPARATIVE ASSESSMENT OF SINGLE-STAGE AND TWO-STAGE ANAEROBIC DIGESTION FOR BIOGAS PRODUCTION FROM HIGH MOISTURE MUNICIPAL SOLID WASTE
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. The authors should define the uncommon abbreviations at their first mention. For example, L27: VS, L31: AD.
2. The definition of abbreviation IFA was needed at first mention and authors defined it twice at lines 91 and 93. Same problem of the abbreviation VS (volatile solids) was found at line 95 and 103.
3. Lines 110-111, the sentence “The 0.5% and 1% of IFA were added for pH adjustment in all initial VS loading” could be improved- the current phrasing makes comprehension difficult.
Experimental design: 1. The procedure of PCR should be provided. And more details or the references of DGGE should be added.
Validity of the findings: no comment
Additional comments: 1. Lines 109 and 121, Why the initial VS loading of 9, 17, 26, 35, and 43 g-VS L-1 was mixed with different v/v ratio by single-stage AD (30%) and two-stage AD (20%)?
2. The substrate to inoculum ratio (S:I) of 20:1 was used at two-stage AD process and the S:I was 2:1 at single-stage AD process. The authors should provide more details or reference about the difference.
3.  Lines 277-278, I suggest that authors should provide the references for the reason of the suitable VS loading was <26 gVS L-1.


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

Input:
Title: COMPARATIVE ASSESSMENT OF SINGLE-STAGE AND TWO-STAGE ANAEROBIC DIGESTION FOR BIOGAS PRODUCTION FROM HIGH MOISTURE MUNICIPAL SOLID WASTE
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have focussed on the biogas production from high moisture containing MSW using single and two AD. However, the authors have not much focussed on the volatile fatty acids production during two stage AD. Quantifying the VFA production would be a value added product apart from biogas if two stage AD is used.
The authors may try to add VFA production profile if the data is available
The authors did not define "IFA" for the first time in abstract. So it is not clear what actually is IFA meant for...
In the abstract, AD has been defined, however it is not used as AD further. For instance, however, anaerobic digestion...... this should be AD
So, the authors may thoroughly check the grammatical errors, typos, and the sentence formation and abbreviations
Experimental design: No Comment
Validity of the findings: No Comment
Additional comments: The language in the manuscript still needs further improvement. The authors may check the manuscript for grammatical errors, typos, and sentence formation.
VFA production may also be taken into consideration during two-stage AD of MSW.


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

Input:
Title: COMPARATIVE ASSESSMENT OF SINGLE-STAGE AND TWO-STAGE ANAEROBIC DIGESTION FOR BIOGAS PRODUCTION FROM HIGH MOISTURE MUNICIPAL SOLID WASTE
Review round: 2
Reviewer: 2

Output:
Basic reporting: - Please emphasize the significant of the current study;
- The introduction section should refer to the recent reports (5 years);
- What is the significant of microbial analysis?
- Two-stage AD requires higher energy consumption comparing to single-stage AD, thus, It would be more convinced if the authors calculate the energy balance for both cases;
- It seems there was a positive correlation between organic loading and alkalinity at IFA 1%, but not with IFA 0.5%. Can you explain that?
- Please provide the high quality figures (≥300 dpi);
- Please provide the initial pH of the tests;
- Please provide the characteristics of inoculum;
- What are the components of the high moisture MSW in the current study?
Experimental design: - Experimental design should be complied in accordance with a standard test method such as ASTM D5511-12;

- High organic loading points may require higher ash addition to attain the suitable initial pH range, therefore, the ash addition at higher percentages should be investigated.
Validity of the findings: no comment
Additional comments: The present study falls within the scope of PeerJ and covers an interesting and important area of research. The manuscript has clear content and tight structure. However, there are some weak points that make the current manuscript unsuitable for publication.


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

Input:
Title: MUTATION DENSITY CHANGES IN SARS-COV-2 ARE RELATED TO THE PANDEMIC STAGE BUT TO A LESSER EXTENT IN THE DOMINANT STRAIN WITH MUTATIONS IN SPIKE AND RDRP
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors describe the importance of the mutation density of SARS-COV-2 in the current pandemic related to RdRp and the Spike gene. These are related to the evolution of virus variants according to mutation density. The description of this molecular mechanism in mutations analyzed is essential to understand how SARS-CoV-2 is spreading around the world and opening perspectives on possible drug developments used. The manuscript is mostly well written and referenced. The professional language in English is consistent, as are the results presented through graphs and tables. The hypothesis discussed is in accordance with the data presented and with the literature provided in the manuscript.
Experimental design: The research data is within the scope of the paper and well defined in the materials and methods. The research theme is very actual, relevant, and interesting, also described with sufficient details for understanding and replication.
Validity of the findings: It seems to me that the authors performed a robust analysis and that their fundamental conclusions are consistently based on the generated results. So, there is no need to repeat the experiments, being well analyzed according to the existing literature. The conclusions are in line with the discussion and linked to the central research of the work.
Additional comments: I consider that the manuscript entitled “Mutation density changes in SARS-CoV-2 are related to the pandemic stage but to a lesser extent in the dominant strain with mutations in spike and RdRp ” contains important and relevant new information, which will have impact on the understating of SARS-CoV-2 worldwide spreading and adapting. Despite a scarce literature, it is noted the importance of genomic studies about COVID-19 virus.
The authors made good and extensive use of public available sequence data on SARS-CoV-2, constructed an interesting hypothesis on the relationship between occurrence of RdRp and Spike mutations density.


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: MUTATION DENSITY CHANGES IN SARS-COV-2 ARE RELATED TO THE PANDEMIC STAGE BUT TO A LESSER EXTENT IN THE DOMINANT STRAIN WITH MUTATIONS IN SPIKE AND RDRP
Review round: 1
Reviewer: 2

Output:
Basic reporting: In their work, Eskier et al. presented a study based on changes in the density of mutations in sars-cov-2. The objective of this work was to understand possible differences between the time-dependent variations of the mutation densities located in the RdRp and S protein.

In the introduction, I believe it is interesting to point out where these two mutations to which the work refers, are located and related to which activities. P323L in the RdRp which is involved in replication of the viral genome and the change of D614G in the Spike glycoprotein which is essential for the entry of the virus in the host cell by binding to the recipient ACE2.

Regarding the D614G change in protein S, which seems to make the virus more infectious, it is important to make it clear that the experiments carried out so far have not been carried out on human cells and that the increase in infectivity should be considered with caution, especially when related this with the increase in transmission that occurs in humans.
Experimental design: No comment
Validity of the findings: About the results, it could have been better discussed with reference to articles that have already demonstrated the relationship between these mutations. For example, In the article, "The hot spots of the emerging SARS-CoV-2 mutation include a new variant of RNA-dependent polymerase" (Pachetti et al.,), the authors showed that new viral variants were spreading across countries.
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: MUTATION DENSITY CHANGES IN SARS-COV-2 ARE RELATED TO THE PANDEMIC STAGE BUT TO A LESSER EXTENT IN THE DOMINANT STRAIN WITH MUTATIONS IN SPIKE AND RDRP
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Eskier and coworkes performed an analysis of mutation density (MDe) in SARS-CoV2 genomes over time, showing time-dependent changes related to pandemic stage. In their analyses, they compared the MDe dynamics of synonymous and non-synonymous substitutions in both  "wild-type" (WT) and "mutant" (MT, RdRp 14408 C>T and S 23403 A>G) SARS-CoV2 sampled in two different countries (US and UK). MDe where analyzed temporally (in terms of pandemic stage) and spatially (in terms of genome distribution).

Overall I find the study interesting. The paper is well organized. Results are clearly presented and discussed. I have only minor concerns.

1- The authors clearly described and justified the criteria for the SARS-CoV2 genome selection. According to these criteria they reached a dataset of 19705 genomes. In the first paragraph of Results&Discussion is described the number of sites and variants of this dataset, but all the analyses presented in the work are done on a subset of this original dataset, which represents just over half of it (58.76%, including sample from UK and US). I strongly suggest to briefly clarify this point and to deepen the description of this subset (i.e.: sample size for each category; number of polymorphic sites and of potential variants, etc...). Are sample sizes of the two groups (UK and US) comparable? Again, how many genomes constitute each of the four categories analyzed (UK-WT, UK-MT, US-WT and US-MT). Which is the relative proportion of MT and WT samples in the four period analyzed?  Please add some comments.

2- Where the analyses of mutation distribution for S and RdRp performed using UK and US genomes jointly? Please clarify. Which is the frequency of 14805 C>T synomymous mutation in the dataset? (Please add the value in the text).

Minor remarks:

I thank you for providing all the raw data, but I think that add also the GISAID EpiCoV database accession date in the main text could be useful for future readers.

Some references are missing  in the Introduction Section:
-line 49:
"Therefore, despite the disease having a current mortality rate of <6%, lower than similar diseases caused by betacoronaviruses such as SARS or MERS, a fuller understanding of and cure for the underlying pathogen is a high priority for researchers and clinicians everywhere".
-line 60:
"Although these and other studies provided suggestive evidence that indeed this new strain could have altered phenotypic characteristics, only after mechanistic studies are performed in cell culture and animal models, we will be able to know whether these inferences are true".

line 193: MDE -> MDe


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: EVOLUTION OF THE PATELLA AND PATELLOID IN MARSUPIAL MAMMALS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I commend to the authors because, through MS "Evolution of the patella and patelloid in marsupial mammals" they identify and address the information gaps in the subject of comparative morphology and evolution of sesamoids, in particular of patella in marsupial mammals.
I recommend that the article be published. I only suggest minor changes, which I believe will help to improve the presentation of the results
See pdf attached
Experimental design: In order for the reader to have a concrete idea of the representativeness of sampling in optimizations, I suggest placing the percentage of species included in relation to the total species of the analyzed group. Also note if all families had representatives analyzed.

In the edition of the cladogram I suggest adding in the corresponding nodes the names of the big clades so that the follow-up with the reading of the text is more fluid, especially for those who are not familiar with the classification of the marsupials.

I suggest to show the optimizations not included in the presented figure (e.g. maximum parsimony analysis with ordered character states, or switching state “0” (no patella) coded fossil taxa to state “0/1” (patelloid possible)) in the appendix section.

In the line 222-223, the finding of bilateral patella is not very clear. Could you clarify the brief description that you refer to as "bilateral"? Do the 3 D and E figures correspond to the two hindlimbs?

Other suggestions about the edition of the figures are indicated in the attached pdf
Validity of the findings: The issue is addressed in a synthetic and direct way, the main problem is clearly raised. The discussion does not give rise to speculation, except for minims that are perfectly clarified and identified by the authors.

Its most important contribution is the exhaustive anatomical analysis of marsupial species not reviewed so far in the framework of this topic (sesamoid evolution)
Additional comments: The section on the functionality of a fibrocartilaginous patella could be enriched by discussion with the work of Abdala et al (2017 On the Presence of the Patella in Frogs). It would be interesting to analyze / discuss what happens specifically with the patella of groups "jumpers" like frogs and kangaroos, the latter analyzed here. While this discussion can be speculative with the information available so far, I think the topic is interesting to be raised and will give a broad context to your results.


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: EVOLUTION OF THE PATELLA AND PATELLOID IN MARSUPIAL MAMMALS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper was clearly written and sufficiently referenced. The article did not fail in this category.
Experimental design: It is an ambitious study that adds much needed knowledge to the literature, not only pertinent to the patella but the development of sesamoid bones and bones in general.
Validity of the findings: All seem valid but results could be delved into deeper, particularly in understanding the lack of congruency of the evolutionary analyses.
Additional comments: Basic reporting
In this manuscript on the evolution of the patella and patelloid in marsupials, researchers examine patella/patelloid presence/absence and found novel evidence of ossified patellae in some marsupials. Through phylogenetic reconstructions, they identified a complex history of the patella: one that does not seem to have converge onto a single evolutionary model.  This points towards substantial homoplasy in marsupial patellae, and a fascinating evolutionary history of this (and potentially, other) sesamoid bones in this clade.

Materials and Methods
Survey of osteological museum specimens
More detail needs to be added here. From my work with the fabella, I am aware of the difficulties in determining sesamoid presence in osteological collections as it is possible these small bones were lost, making it difficult to have confidence that the bones are truly “absent” when not present. (I imagine it would be even more difficult in the case of cartilaginous bones.) What would qualify as adequate QF preservation and absence of the patella/patelloid?
What would help is a figure showing several pictures of osteological specimens that were adequately preserved and some that were not adequately preserved. Perhaps this was meant to be Figure 3a, but it is difficult to tell from the photograph

General
Were zoo, wild, or a mix of specimens used? Likely does not affect the results, but could be interesting to consider abnormal loads (esp in older zoo specimens) vs. physiologically normal loads.

Results
Observations and Imaging of Osteological Specimens
L226 I am confused – what was its structure then and how did it differ?

Evolutionary reconstructions
L285 – double period

General
The results section is quite long, which is not unexpected given the several different methods employed here, but it could be made shorter, and there are sections that would be more appropriate in the discussion section (e.g., L276-279 and L286-288). Please shorten the results and move more to the discussion.

Discussion
L327-328: Given your results, and the intriguing dissection of M. rufogriseus, the discussion on the development of the patella is critical to the discussion of its evolution. Does the patella develop similarly in marsupial and placental mammals? If so, is it a portion of the femur mechanically removed during growth (as observed by Eyal et al., 2015; Eyal et al., 2019)? What would cause one patella to ossify and the other to not? If most or all marsupial species have the potential for ossified patellae, why does ossification only occur in a relatively small number of individuals? And how certain can one be of the evolutionary analyses done here if all species with patelloids could easily be coded as havine patellae?
Not all these questions need be addressed, but if some could be, it would greatly improve the paper in understanding and interpreting the evolutionary results.

L337: Sarin et al., 1999 found os peroneum prevalence more or less constant in the hominoids, and that fabella prevalence decreased into the hominoids and increased in Homo, so it is not a decline towards Homo for either bone. Decline towards hominoids for the os peroneum, but not the fabella, where some prevalence rates in modern humans exceed the estimate for the lesser apes.

L349-350: I stated something similar in a paper that was under review, and Abdala pointed towards my error. She states “Please note that the dynamic model advanced in Abdala et al. (2019) does not propose a third origin to sesamoids but a source to new skeletal morphologies.”

L353: Surely you could account for time to hind limb development as a factor in predicting patella vs patelloid presence?

L395: This is also supported by fabella prevalence rates in Homo (see Berthaume and Bull, 2020, J of Anat).

Figures
Figure 1: I would suggest standardizing the scale on the abscissa. There is almost 100 my between the divergence of marsupial and placental mammals and the Cenozoic, but it looks to be the same as the 17 mya between the divergence on the Monotremes and Therian mammals.

Figure 6: please label the patella and tendon/surrounding structures if applicable


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: EVOLUTION OF THE PATELLA AND PATELLOID IN MARSUPIAL MAMMALS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is written in clear and technical English. The background (literature) is enough. Figures and Tables (including raw data at the supplementary material) are clear and informative. Finally, the manuscript is "self-contained".
Experimental design: The manuscript constitutes original research, that deepens in a previous study (Samuels et al., 2017), and is relevant and meaningful for the evolutionary studies on mammals anatomy (specifically for marsupials).

The research question is clear, and correctly developed throughout clear technical methods, filling a knowledge gap on sesamoids evolution in marsupials. The experimental design is replicable.
Validity of the findings: The findings recorded in the manuscript include novelty results, improving our knowledge of mammals anatomy evolution. However, it would be interesting if the authors delved a little more about the possible evolutionary implications of plasticity in the presence/absence of sesamoids in marsupials (traction epiphysis; see comments on the manuscript pdf). Conclusions are well stated, linked to original research question & limited to supporting results. When assumptions are made, these are prudently exposed.
Additional comments: This research constitutes an important contribution to our knowledge of the evolution of mammals anatomy, specifically of marsupials. This is a necessary deepening of previous works on the evolution of sesamoids in mammals. I congratulate the authors for this initiative since evolutionary investigations of anatomy in mammals such as this one (with large taxonomic samples) constitute the basis for future research in fields such as genetics, functional morphology, and others. The raw data is very useful and the experimental design is adequate. The interpretation of the findings is very adequate and prudent.


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: EVOLUTION OF THE PATELLA AND PATELLOID IN MARSUPIAL MAMMALS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This second version of the manuscript has improved, and the results and discussion are very clear. I congratulate the authors for their predisposition to incorporate and answer all the suggestions of the reviewers.
The work is a great contribution to the study of the anatomy and evolution of sesamoids, and marsupials
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: EVOLUTION OF THE PATELLA AND PATELLOID IN MARSUPIAL MAMMALS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I have read through the response to reviewers and the new manuscript. I am happy to give my support to the authors for publication of this excellent manuscript.
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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the manuscript entitled “Exploring the classification of cancer cell lines from multipleomics view”, Yang et al. addressed the multiomics-based, pancancer-scaled molecular taxonomy of cancer celllines. The study is based on the public, CCLE multiomics data encompassing mRNA/miRNA/SCNA/methylation/RPPA data. As clearly stated in Introduction, the value of multiomics datasets of cancer celllines has long been proposed and this study demonstrates the feasibility as well as some novel molecular taxons and interdependency among the datasets. I believe their results are of potential interests to general cancer biologists and clinicians, but I have a number of comments as the following.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Major comments:

1. Their results can be compared with similar work based on PanCancer-TCGA in terms of Fig.2B and Fig.2C. I believe that the most distinctive features of celllines compared to primary/TCGA tumors are high purity (i.e., the lack of intratumoral heterogeneity and contamination of normal tissues) and I am very curious how this features affect the PanCancer clustering.

2. One important features of CCLE database is that they include the data representing drug sensitivity. Fig. 4 can be further revised to demonstrate the relationship between the drug susceptibility or resistance and their pan-cancer clustering.

Minor comments
- It is hard to read the fonts in the main Figures, esp. Fig. 3 and also Fig. 4.
- In Abstract, “Results. 18 molecular clusters” should be revised into “Eighteen molecular clusters”.


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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Review Report
“Exploring the classification of cancer cell lines from multiple omic view”
Yang et al.

The authors classified single omics data including mRNA, miRNA, CNV, Methylation and  Reverse Phase Protein Array from cell lines of the Cancer Cell Line Encyclopedia (CCLE) consortium and integrated multi omics data.  Subsequently, the authors visualized the single and integrated data using the TumorMap tool to show the landscape of cancer cell lines based on molecular data.
Based on their analysis they identified  18 distinct clusters which consist of 3 pan-cancer clusters and one pan squamous cluster. Moreover, they showed that integrated omics analysis has an added value over single omics data analysis and that the mRNA and proteomics data contribute mostly to the distinct clusters.
Although there approach of integrated CCLE has added value I have some questions regarding the current manuscript.

Major comments.

    • The description of the methods is very general and the results are likely very hard to reproduce based on the M&M section. Please elaborate more on the analysis methods and the parameter settings.
    • The main message of the manuscript is not clear. Is it focused on the bioinformatics analyses methods or did the author wish to show new cancer biology? I strongly suggest to focus on the bioinformatics part, since the current added-value for cancer biology is limited as new testable cancer hypotheses are generated.
    • Limma is used for differentially expression analysis, but this method is less suitable for Next Generation Sequencing data analysis. Why did the authors not use DESeq2 or EdgeR as analysis method. Please clarify.
    • All the main figures and supplementary figures have very limited legend text. Therefore they are hard to impossible to interpret. In figure 1A the legend colors go from 0 -10. What unit is used? It seems that colors reflect just an on and off situation of a gene. Most dots are very blue for instance. So could a binary clustering better reflect the biology? Please clarify. Moreover, the first track is not clear. Maybe the authors could add a dendrogram to show the different groups more clearly. In addition the number of samples differ hugely between the groups. In 1A and 1B the dark purple TCGA group has about a 3-fold difference. Can the authors explain this difference in more detail by for instance add the number of samples in the legend text.
    • Line 134 -190 discuss the clusters with labels such as C1 C9 etc. However, the reader cannot find these labels back in the manuscript. Therefore, the description of the results are very unclear.
    • Please correct the clustering analysis for gender or show that the clusters are not a reflection of gender, since expression data is strongly biased to gender and might explain the pan-gynecological group.
    • Line 197: The authors emphasize that the BRCA cluster 	C7 covers three prostate adenocarcinoma cell lines. This is a huge surprise since BRCA cell lines are mostly if not all female derived and prostate cancer cell lines are all male derived. Please show that this result is not an artifact of your analysis by selecting the features of the BRCA and the three prostate cancer cell lines that they share and give a biological explanation.
    • Figure 4B shows that the BRCA and PRAD group is similar. This is very unlikely and the authors should provide much more evidence that Breast cancer and prostate cancer has very similar molecular features

Minor comments.

    • Line 28: In the abstract transcriptomics is indicated as important factor to the integrated clustering, but this is mRNA and not miRNA. Please change it.
    • Line 82: Could the authors be more specific what they used as input for the CNV analysis? Is this the Methylation data, SNP-array data or the DNA-seq data? Please clarify and show more details of the analysis.
    • Line 88: The RNA data is normalized by the authors.
The method of normalization is not described and it is not clear why the authors did not use the normalized RNA data used and shared by CCLE (indicated with RSEM values).
    • Please add the versions of all software packages that were used.
    • Four single omics data clustered were built with 1-Pearson’s correlation coefficient distance metrics. Euclidean distance would reflect the amplitude of the data as well. Why did the authors not used the Euclidean distance as distance metrics for the clustering? Is this based on the output of NbClust? If so please provide the results.
Experimental design: see Basic reporting
Validity of the findings: see basic reporting
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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Although heatmaps like those shown in figure 1 are very popular, they do not really convey much information to the reader. Moreover, in this particular instance, clusters –that are the focus of the message- are barely distinguishable from the small color-coded boxes on the top of each panel. Color codes are good when dealing up to 6 or 7 entities, but they become confusing with larger sets. Numbering or lettering should be used instead. Moreover, each panel has a double color code (for instance, in Fig1A , "mRNA group" and "TCGA disease"), but only the latter has a legend; for the mRNA clusters there is not a color-key legend.
Overall, this kind of figures is not really informative and I strongly recommend swapping Figure 1 with Supplemental figure S1 (or something similar to it), where the relationship between the clusters found with the different techniques and the original classification is more evident. Before this, it is however mandatory a clear explanation of what the numbers in Supplemental Fig 1 exactly represents, and to omit all the instances of "-0.00", turning them in blank cells.
While in general the language is adequate, unambiguous and clear, some key definitions are missing (or well hidden). For example, it is not clear the definition of "domination" of a cancer type in a cluster. Cluster C6 (from integrated clustering analysis) is considered to be dominated by a single cancer type (SARC), while C5 by two types (HNSC-ESCA). However, by judging from the proportion of cancer lines in the pie charts in Fig2B, the two clusters seem quite similar in term of heterogeneity. The criterion for "domination" should be better explained.
Minor points about basic reporting :
Page 11 line 143: when reporting about GO enrichment, expressions like: "…these cell lines were upregulated in reproductive system development and morphogenesis of embryonic epithelium.", although understandable in the context, are a rather odd, and should be rephrased.
Page 13 Line 209: it is not clear how the high level of CDH1 in C3 cluster or the levels of VAV1 and STAT5 represent "an example" of what is stated in the previous sentence about the proportion of cancer type in two-cancer type dominated clusters.
Page 13 Line 212 "dominated type" should be "dominant type".
Experimental design: The main issue of the manuscript is about the scope of the study. Thorough multiplatform analyses on different cancer cell types have been already performed on patients samples, and to "extend" this kind of analysis to cell lines would not add much information about the biology of cancer by itself. Especially since many doubts about the reliability of cell lines have been casted repeatedly in the last decade.
The present study, however, could represent a valuable analysis for the reliability of available cell-lines as model of cancer. Rather than focusing on conclusion that have been already drawn by others researchers on more biologically relevant data, authors should focus more extensively on the comparison between the results of their analysis on cell lines and those obtained from patients. It would be useful, for example, to determine at which extent the cell lines resemble their (putatively) respective counterparts.  In parallel, it would be of great interest to identify some "pan-cell-culture" artifacts, due to exposure to FCS or other tissue-culture media. This would be of great help in interpreting results from many studies performed on cell lines.
Validity of the findings: In general, the methods employed for the analysis are sound.  It is rather surprising that all SKCM cell lines are strictly clustered together by many different kind of analysis, when melanoma is known to harbor a large variability. This aspect should be carefully discussed.
Additional comments: The manuscript from Yang and colleagues describes a meta-analysis of data derived from multiple high throughput techniques, on several cancer cell lines. Although the analysis has several interesting aspects, the manuscript should be modified to be suitable for publication. As detailed above, the major issues are two: the overall scope of the study, that should be refocused and the selection of the kind of figures to show in order to maximize the information conveyed.


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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has been revised thoroughly and improved significantly. Moreover, many reviewer questions have been properly addressed in the newest version of the manuscript. However, I have still some concerns regarding the current manuscript.

Major comments:

    1. In the revised manuscript the figure legend are still very limited. For instance Figure 1 is not interpretable if you only read the figure legend.
    2. In am still not convinced that the three PRAD cell lines ought to cluster with the BRCA cell lines.  Although in both cancers drivers of genes in the Homologous repair system have been identified, such as BRCA2, their physiology is very different. In my opinion the number of PRAD cell lines is too limited and I expect when more PRAD cell lines could have been incorporated a distinct PRAD cluster would popup. Therefore, the conclusions in the manuscript based on the clustering of multiple tissues in a single cluster may be premature and based on underpowered data. Now, it is still hard to assess how reliable the clusters are.

Minor comments

    1. line 145  “dada” should be changed to “data”
Experimental design: -
Validity of the findings: -
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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment (see below)
Experimental design: No comment (see below)
Validity of the findings: No comment (see below)
Additional comments: In my first report, the rigid form of PeerJ forced me to start my comments by writing about basic reporting, experimental design and validity of the findings, while -in this case- my main concerns were about the overall structure and focus of the manuscript (that I inserted in general comments). My impression is that this "unnatural" structure of the report prompted the authors to almost overlook what was my main concern: namely the scope of the study. While I acknowledge they modified their manuscript improving the definition of key concepts and improving the quality and the information conveyed by the pictures, however, the structure of the manuscript remains substantially unchanged, as clearly underlined by the abstract and the introduction. Therefore, I can't omit to repeat my previous comment:

"The main issue of the manuscript is about the scope of the study. Thorough multiplatform analyses on different cancer cell types have been already performed on patients samples, and to "extend" this kind of analysis to cell lines would not add much information about the biology of cancer by itself. Especially since many doubts about the reliability of cell lines have been casted repeatedly in the last decade.
The present study, however, could represent a valuable analysis for the reliability of available cell-lines as model of cancer. Rather than focusing on conclusion that have been already drawn by others researchers on more biologically relevant data, authors should focus more extensively on the comparison between the results of their analysis on cell lines and those obtained from patients. It would be useful, for example, to determine at which extent the cell lines resemble their (putatively) respective counterparts."


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: EXPLORING THE CLASSIFICATION OF CANCER CELL LINES FROM MULTIPLE OMIC VIEWS
Review round: 3
Reviewer: 1

Output:
Basic reporting: - As my major comments on the power of the analysis and the unclear figures are solved I have no additional comments or remarks
Experimental design: NA
Validity of the findings: NA
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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a very interesting paper.  I have flagged up a couple of anomalies in terms of what has been reported within it from existing literature, but these should be relatively easy to address.
Experimental design: Generally a good and solid approach.  As I have mentioned, I am rather surprised that the authors did not take the opportunity to analyse the caecal contents in this work, but nevertheless the data from faecal samples is novel and interesting.
Validity of the findings: One question about figure 5 but otherwise no issues.
Additional comments: A few typos which could dramatically change the course of the content.  Otherwise nothing to add.


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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: I only suggest to improve discussion in following lines:
In lines (407-408), authors should improve the evidence to support the idea that  Nanopore platform is the technology of choice for metagenomics studies,  because I think before that, they should correct nanopore reads before the taxonomic assignation, to make that statement.
In lines 385-386.  I suggest that authors should add the geographic difference and the kind of plants that they have in the area to  to support  the idea of "absence Spirochetes in hares is a reflection of geographical differences".
Additional comments: Manuscript is well writing and it has a novel informations, but I suggest to improve discussion in following lines:
In lines (407-408), authors should improve the evidence to support the idea that  Nanopore platform is the technology of choice for metagenomics studies,  because I think before that, they should correct nanopore reads before the taxonomic assignation, to make that statement.
In lines 385-386.  I suggest that authors should add the geographic difference and the kind of plants that they have in the area to  to support  the idea of "absence Spirochetes in hares is a reflection of geographical differences".


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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript submitted by Shanmuganandam et al studied microbial community structure of European hares and rabbits in Australia. The authors found significant difference in microbial composition between hare and rabbit in terms of beta diversity and taxonomic structure. Also, authors compared microbiome in hares in Australia and those in Europe and found some differences in taxonomy, especially absence and presence of the phylum Spirochaetes. They also stated the similarity of community structure of hares/rabbits fecal microbiome analyzed by difference sequencing platforms, Illumina and Nanopore.
Generally, this experiment was precisely conducted and clearly documented. I would recommend it for acceptance after a few minor concerns listed below are addressed.

L152, In this study, the raw sequence reads were converted to ASVs. Authors should clarify the cut-off similarity for "OTU" in this manuscript.

L277-279, There was DNA contamination in ROCs, and 40-90% of ROCs were assigned as Firmicutes or Bacteroidetes. Authors should document how to exclude the effect of contaminated DNA in Hare and Rabbit samples.


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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have dealt with the comments I made in an appropriate manner.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have dealt with the comments I made in an appropriate manner.


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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The manuscript is well written and shows great information.
I have just found a minor correction:
Line 321 Add “)”  after 19 in (14-17, 19


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: UNCOVERING THE MICROBIOME OF INVASIVE SYMPATRIC EUROPEAN BROWN HARES AND EUROPEAN RABBITS IN AUSTRALIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: L154, My previous comment meant I wonder this part should be "observed ASV", rather than "observed OTU".


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: TRANSCRIPTOMIC ANALYSIS REVEALS ETHYLENE SIGNAL TRANSDUCTION GENES INVOLVED IN PISTIL DEVELOPMENT OF PUMPKIN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript by Li et al. reports on an RNA-seq study related to pistil development in pumpkin. The authors find that especially genes related to ethylene signaling are enriched, when comparing pistils and aborted pistils. A few genes are verified by qRT-PCR. A drawback is that the RNA-seq is only performed in duplicate and not triplicate, which is the current standard.
It has been known that ethylene affects flower and fruit development. For instance, many studies reported on the effect of ethylene in cucumber, which the authors also cite.
The manuscript reads well and the figures are clear. The data will be interesting for people working with pumpkin.
Experimental design: In general good, however, a drawback is that the RNA-seq is only performed in duplicate and not triplicate, which is the current standard.
Validity of the findings: No comment.
Additional comments: Comments to the authors:
1. In pumpkin, a recent study reported also an RNA-seq study (Wang et al. 2019, doi:10.3390/ijms20133185) and conclude that ethylene and auxin signaling is enriched, and that this affects sex termination of the flowers. This study has not been cited, though the study is not the same but related, and the authors should cite this study and discuss their results in the light of the published data.
2. It would have been nice to add some experiments applying for instance ethylene to developing plants and observe the effect on flower and pistil development.
3. The authors could add some more visualization of their data (eg. heatmap) related to the ethylene pathway.
4. Fig. 4 The authors write that Pearson correlation was calculated, but it would be good to see per gene the correlation between qRT-PCR and RNA-seq in the same figure.
5. Suppl. Table 2. Add accession numbers of the homologues genes in the column ´Gene annotation´.
6. In general, the authors report DEGs, but lists are missing with the actual gene identities. Please add these tables (can be suppl. data) .
For instance: ´Finally, 3817 DEGs were identified, 186 including 1341 up-regulated and 2476 down-regulated genes, in the aborted pistils compared 187 with the normal pistils (Fig. 1B, C).´
´….84 DEGs were enriched in the plant hormone signal transduction pathway….´
´…..we found that 68 DEGs were enriched in phenylpropanoid biosynthesis….´
Where are the tables?