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: WITHIN-HOST COMPETITION DRIVES ENERGY ALLOCATION TRADE-OFFS IN AN INSECT PARASITOID
Review round: 2
Reviewer: 1

Output:
Basic reporting: OK.
Experimental design: OK.
Validity of the findings: Still some problems with transparency of the statistical treatment. I disagree with some of the interpretations.
Additional comments: I still think that this study has a merit, and I also see various improvements. Nevertheless, the following problems remain.

1. Still, are there any other studies on insects which show how allocation of resources to different parts of body depend on body size? Hard to believe that there aren’t any. If there is something to compare to, please do so explicitly.

2. I still feel uncomfortable with your conclusions about trade-offs in resource allocation. I hope that I am now able to formulate my feelings more clearly. You seem to base your conclusions about the trade-off on what we see in Fig 3b. I am sorry but I would say that this relationship is quite trivial. Let’s consider an analogous example: assume that we cut loafs of bread into two parts (not necessarily equal) and then we study the relationship between relative weights of the left part and the right part. We will get a straight line with the slope equal to -1. We may call this a reflection of a trade-off between the weights of the right parts and the left parts if we wish but, in fact, this is nothing more than a mathematical triviality. Your Fig 3b is not much different: there is some little random noise around the line due to the fact that there is also some allocation to  the rest of the body but as thorax and abdomen constitute by far the largest segments of the body, the line is still close to b=-1 with little residual variance. This is a mathematical inevitability and not a biologically meaningful discovery of a strong trade-off. So I suggest that you remove Fig 3b and all the discussion based on it (Figures 2 and 3a are informative and valuable). If you disagree then please show us how would it be possible to get a different result in this case, i.e. a pattern which you would interpret as no trade-off, or as a weak trade-off.

 3. I rather disagree with your interpretation that the higher relative investment into thoraces in small flies is an adaption to make it possible to escape adverse conditions (line 347). I think that the thing is more simple. I think that it is generally valid for insects and perhaps any organisms that movement-related structures show negative allometries (show less relative variation than the size of the entire body) just because for any given body plan, these structures cannot vary much in size and remain functionality at the same time (if you increase linear measures of a bird 2 times maintaining its body shape it will not be able to fly). That’s why thorax size is more conserved than abdomen size: the thorax is built of the size allowing the insect to fly, and all the rest is allocated into the abdomen. The amount of ‘the rest’ may vary substantially as a function of environmental conditions.

4. You seem to disagree with my criticism of your statistical treatment not being fully transparent. However, for example, in line 277 you say that the best fit was an 2nd-order polynomian: where is this analysis explained? Which functions were compared and how when you decided which non-linear functions best describe the relationships in Figs 2 and 3a? Where are the details?

Minor/specific

line 59. I still insist that ‘body size’ is a too vague term to be used in quantitative statements. Were the flies 6 times larger in terms of body masses or linear measurements of the body? A 6-fold increase in linear measurements corresponds to a 216-fold increase in body mass, given that body proportions do not change.

line 61. I still think that there is insufficient evidence for different life history strategies. I do not
see anything which could not be explained by biomechanically based allometric rules (see above).

line 101. isn’t ‘rules that govern trade-offs’ an oversophisticated expression? Would you be able to give an example of such a rule?

line 119-120. I feel that the sentence ‘Here, …’ is in a wrong place. Does not fit here.

Line 125. Once again: do tachinids really shape plant communities? They are all parasitoids as far as I know.

Line 128. I think that you should explain how do you use the term ’cohort’ here. In population biology, this word has a different meaning (= individuals which were born during a certain period of time), and this may cause confusion.

Line 166. Thank you for explaining the usage of ‘quality’ here, this is essential indeed. However, I feel that this text (starting with ‘Here, we…’) does not fit well here as it stands. As this is in the Methods section, maybe you could start with telling that you measured head capsulae, and then you could smoothly switch to explaining why did you do this. I think that this deserves a separate paragraph if not a separate section. Also, you have somewhat repetitive text starting line 263, may it be possible to combine these texts to avoid repetition?

Line 202. I think that this paragraph is not needed.

Line 220. in there -> if there?

Line 237. I still do not understand ‘population level’ here? Which population?

Once again, please note that in your references list, you sometimes present given names and sometimes not. I did not check it but this is hardly a style acceptable by PeerJ or any journal.

Figures. It is great to know that most readers will read your paper online but, nevertheless, please change your colour scheme so that there would be a slight difference also in the darkness of different symbols, not only in the wavelength. This is easy to do and would not hurt your online readers in any way. Please.

Figure 2.  Once again, is ‘fly weight’ (horizontal axis) and ‘body weight’ (vertical axis) the same thing or not? If yes, please use identical terms on both axes. If not, please explain the difference.
*******************************************


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: DATA-DRIVEN CLASSIFICATION OF THE CERTAINTY OF SCHOLARLY ASSERTIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The theme of the article is relevant and marks a continuity of research reported in previous articles. Language is clear, professional and unambiguous. Raw data is supplied that enables checking and validating the results.
Experimental design: The article is within the scope of PeerJ, considering this is a research article with focus on rhetoric and methodological issues concerning scientific communication in the biomedical domain.
The research problem is clearly stated. Possible benefits of the research are shown. Methods are adequate and described in detail. The intended audience and beneficiaries are clearly stated, line 405.
Validity of the findings: Maybe the Conclusion, line 441, is too general. As authors themselves suggest, line 51, “As a result, biology papers contain is a wide range of 52 argumentational structures that express varying degrees of confidence or certainty.”
Maybe the Conclusion is valid only within the biomedical domain. No evidence is presented of its general validity or in other domains of knowledge.
Additional comments: Improve the abstract for clarity; on page 6, line 30, Abstract, for example, insert a sentence to make clear the relation of the degree of certainty of scientific claim with text-mining, e.g. , the sentence on page 7, line 65, "This problem is worsened in the context of text mining algorithms that may miss the nuance of a scholarly assertion when extracting the entity-relationships that make up the claim."


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: DATA-DRIVEN CLASSIFICATION OF THE CERTAINTY OF SCHOLARLY ASSERTIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text needs some revisions in terms of English expressions etc. It seems that the submission has been rushed a bit, since there are several typos, repeated text etc that should be corrected.

More specifically:

Line 78: “[e]ach [sentence] fragment conveys a degree of certainty about the" :
I think the use of [] here is erroneous especially in the [e]ach part.

Line 123: "... to or a numerical scale." This is either a typo or an unfinished sentence. Please rephrase

Line 132: "Principle Component Analysis" : I assume you mean Principal Component Analysis but even so, you can use the acronym presented in line 129.

Lines 156-163: This whole part is repeated from lines 147-154

Line 165: Seems as if the beginning of the sentence has been cut off?

Overall, the document would benefit from a thorough revision as currently it seems a bit rushed and hasty and this renders the understanding and appreciation of the presented results difficult.

The literature review is quite limited. The article neglects related work in the field and especially work related to uncertainty on scientific and bio-medical corpora which is the focus of this article.

In terms of older work, some reference to the work of Hyland would be reasonable. And maybe Light et al. (2004) since they also worked on 2 and 3 level uncertainty. Also I would like to see some reference and maybe comparison with the annotations in the BioScope and/or GENIA-MK corpora, or even the CoNLL 2010 shared tasks. How are the selected sentences different if at all to other work?

In terms of user-based surveys:

Malhotra et al. (2013), also uses results of user-based questionnaires

Zerva et al. (2017) also  discuss the annotations of uncertainty on a scale of 1-5 among two groups of researchers in biomedicine.

Some discussion and comparison with the conclusions of the aforementioned articles would definitely benefit the paper.

On the conceptual level, other work such as the ones of Szarvas and Vincze is also relevant, and maybe some reference to factuality as well. The work of Soldatova and Rzhetsky might also be of interest in addition to ORCA. Overall the related work is in needs of expansion and more thorough review of recent literature.

In terms of the selection and curation of the 45 statements of the corpus. The source and the selection process for the statements is unclear, and the linked protocol does not seem to have any related guidelines.

In terms of the presentation of the surveys: The actual annotation scale and differences for each survey is not presented clearly in any part of the document and has to be inferred. This is a crucial point for the experiments and result interpretation and should be presented clearly in the beginning of the Materials and Methods section. Also, the statistics of the coverage for each statement of the corpus are not presented very clearly. It was hard to understand what the presented numbers correspond to. More detailed statistics on the coverage for each statement in each survey would be useful.

Indicative references discussed earlier:
Soldatova, L. N., Rzhetsky, A., De Grave, K., & King, R. D. (2013, April). Representation of probabilistic scientific knowledge. In Journal of biomedical semantics (Vol. 4, No. 1, p. S7). BioMed Central.
Light, M., Qiu, X. Y., & Srinivasan, P. (2004). The language of bioscience: Facts, speculations, and statements in between. In HLT-NAACL 2004 Workshop: Linking Biological Literature, Ontologies and Databases.
Malhotra, A., Younesi, E., Gurulingappa, H., & Hofmann-Apitius, M. (2013). ‘HypothesisFinder:’a strategy for the detection of speculative statements in scientific text. PLoS computational biology, 9(7), e1003117.
Zerva, C., Batista-Navarro, R., Day, P., & Ananiadou, S. (2017). Using uncertainty to link and rank evidence from biomedical literature for model curation. Bioinformatics, 33(23), 3784-3792.
Hyland, K. (1996). Writing without conviction? Hedging in science research articles. Applied linguistics, 17(4), 433-454.
Vincze, V., Szarvas, G., Farkas, R., Móra, G., & Csirik, J. (2008). The BioScope corpus: biomedical texts annotated for uncertainty, negation and their scopes. BMC bioinformatics, 9(11), S9.
Szarvas, G., Vincze, V., Farkas, R., Móra, G., & Gurevych, I. (2012). Cross-genre and cross-domain detection of semantic uncertainty. Computational Linguistics, 38(2), 335-367.
Vincze, V. (2015). Uncertainty detection in natural language texts (Doctoral dissertation, szte).
Experimental design: The research question is well defined and interesting.

The rationale for the survey design is reasonable, although I would find the results more convincing if there was better correspondence between the qualitative and quantitative surveys. As it is, S1 and S2 are 4-level and 2-level respectively and both qualitative with different labels for each level/class of uncertainty. S3 is quantitative (i.e., there is no qualitative description of each certainty level/class), but is 3-level. As such, it is rather hard to infer whether the differences observed in the resulting annotations and inter-annotator agreement patterns, are due to the quantitative scale or the 3-level class. This in my opinion somehow  weakens the conclusions about the distinguishability of the three underlying certainty categories.

In terms of the clustering it is a bit unclear what was the PCA and clustering applied on. Was each statement clustered using the choice of annotators as data points/features? And the same for PCA? How was the potential difference in the number of annotators per statement handled in that case? Also, if this is the case, in the case of S2 aren't the results limited to a maximum of 4 clusters/components by definition? The Spearman Rank Correlation in Fig. 3 is interesting but rather intuitive and I am not sure it is sufficient to draw conclusions.

Overall, is this the typical analysis statistics for such surveys in the field? I would like some more lengthy explanation of the survey analysis and the rationale for choosing the presented methods and metrics.
Validity of the findings: I would appreciate some more detailed explanation of the rationale for the presented metrics and some more careful discussion of the concluded statements, as I explained in the previous section.

Moreover, I would like to see some qualitative analysis of the findings as well. Based on the examples, the researchers were asked to judge the certainty level of the statements based on the textual information solely. Hence, since it is proposed that there is sufficient evidence supporting a three-level categorisation of uncertainty for scientific statements, it would be very interesting to see some textual analysis of the statements with respect to the attributed categories. Are there some textual patterns supporting the three-level categorisation? Such a finding would further support the proposed application for automated application of the categorisation system.

On a more specific point, in line 357 it is mentioned: "The studies that preceded this one have all suggested four categories of certainty. In this study, we identify only three." This si a very strong statement, and rather unfounded. Which studies specifically have all suggested 4 categories? To the best of my knowledge most studies conclude that multi-class categorisation of uncertainty is rather unfounded (see the experiments of Rubin et al. (2007), Zerva et al. (2017) etc. ). Also most biomedical corpora annotations opt for binary or 3-level annotations of certainty/speculation (see BioNLP ST, Genia-metaknowledge corpus, FactBank corpora, BioScope etc). I have included some references at the end but overall, reconsideration of related studies and categorisation proposals is essential here.

References:
Vincze, V., Szarvas, G., Farkas, R., Móra, G., & Csirik, J. (2008). The BioScope corpus: biomedical texts annotated for uncertainty, negation and their scopes. BMC bioinformatics, 9(11), S9.
Rubin, V. L. (2007, April). Stating with certainty or stating with doubt: Intercoder reliability results for manual annotation of epistemically modalized statements. In Human Language Technologies 2007: The Conference of the North American Chapter of the Association for Computational Linguistics; Companion Volume, Short Papers (pp. 141-144). Association for Computational Linguistics.
Saurí, R., & Pustejovsky, J. (2009). FactBank: a corpus annotated with event factuality. Language resources and evaluation, 43(3), 227.
Zerva, C., Batista-Navarro, R., Day, P., & Ananiadou, S. (2017). Using uncertainty to link and rank evidence from biomedical literature for model curation. Bioinformatics, 33(23), 3784-3792.
Nédellec, C., Bossy, R., Kim, J. D., Kim, J. J., Ohta, T., Pyysalo, S., & Zweigenbaum, P. (2013). Overview of BioNLP shared task 2013. In Proceedings of the BioNLP Shared Task 2013 Workshop (pp. 1-7).
Thompson, P., Nawaz, R., McNaught, J., & Ananiadou, S. (2011). Enriching a biomedical event corpus with meta-knowledge annotation. BMC bioinformatics, 12(1), 393.
Additional comments: I strongly believe that a thorough revision in terms of the use of English, the  and the explanation of the surveys and methods would significantly benefit the document.


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: DATA-DRIVEN CLASSIFICATION OF THE CERTAINTY OF SCHOLARLY ASSERTIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This version of the paper is now more robust and concise. The questions relating to the clarity and precision of the conclusions we have raised were considered
Experimental design: The methods are described in detail.
Validity of the findings: The paper clearly demostrates the validity of the findings
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: DATA-DRIVEN CLASSIFICATION OF THE CERTAINTY OF SCHOLARLY ASSERTIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The text and use of English have been greatly improved.

I have some minor observations:

lines 33-35: The terms author-annotated corpus and publicly-annotated corpus initially confused me. Since you are using this distinction (or any other term you decide), it would be good to mention it in the main text as well, and use it consistently, so that we always know which of the two corpora you are referring to.

lines 158-161: It would be better to specify in which of the aforementioned publications this is the case, otherwise the statement is a bit dubious unless it holds for all annotations.

line 172: I am a bit confused with the term "fidelity". does it mean consistency, or something else?

lines 209-211. Perhaps here is a good point to clarify whether this is the "author-annotated" or "public-annotated" corpus, introduce the naming convention properly and stick to that name henceforth.

lines 291-292: I would avoid the repetition of the word "represent" in this sentence, it distorts the meaning. Perhaps the first instance could be replaced by "constitute/are/" or "depict/exhibit/illustrate" (I think the former are closer to the intended meaning)

line 295: Spearman’s formula rank  --> Spearman’s formula ranks

lines 438-439: Perhaps "...was used to build a neural network model" should be replaced with "...was used to train a 5 layer NN model"

line 441: Is there a comma needed after "Survey 3"?

lines 464-465: after the comma, the sentence does not make sense. what does "this in these " refer to? and "more detailed arguments"? Is there a verb missing? Also, please clarify which corpus you refer to since you have mentioned two versions.

line 506-507: It would be better to use consistently either numbers or words to represent numbers especially if referring to the same thing (11 indices versus six indices)

General comments:
Shouldn't equations be numbered?
There is inconsistent use of quotation marks when quoting other authors (e.g., compare line 277 to line 147.
Experimental design: I am satisfied with the explanations, evidence, and support for the experimental design. Just some observations:

lines 195-227: In the light of the new text added, I am wondering about the reasons/motivation the TAC dataset was chosen (as opposed to a collection of abstract for example). Since TAC was generated for citation identification and summarisation, did you use any such information to select the sentences?

lines 346-347: The typical reporting of performance results when it comes to Precision/Recall typically includes F-score, unless for some specific reason this cannot be calculated. But in this case, I do not understand why F-scores are not presented, especially since that sentence is supported by some provided references, which, as far as I could see, do use the typical Precision-Recall-Fscore schema. F-score can be calculated from the Precision/Recall scores you provide, so I would like to see it in the tables and evaluation methods.
That said, I am not sure why these references are provided at that part of the text. In my understanding, there is no direct/indirect comparison to their results. Are they cited just because they used a similar evaluation schema? I think the role of this citation could be clarified (or the citation removed if they do not contribute something).

Line 438: How was this corpus generated? I am afraid I am a bit confused with the two versions of the corpus. Aso, please stick to the same names used.
Validity of the findings: Lines 410-413: Is this X indices out of 30 that were available in NbClist? Please specify this.

Table 7: Please add F-scores, so that the results are complete and aligned with the standardised way of presenting machine learning results in such NLP/text mining tasks. Also, it would be nice to have the confusion matrices as well, if there are no space concerns.

I understand the limitation of the authors in terms of annotations for the surveys and sympathise with the difficulties involved with carrying out lengthy annotation surveys". I appreciate their response to my previous question and the explanation on the quantitative/qualitative nature of S3. My point is that while I agree on the potential bias introduced by the "named" labels as opposed to the enumerated ones, in order to properly examine/remove the bias, we would need either exclusively enumerated labels, or an experiment with enumerated/named labels on the same number of classes. E.g.,: Sx = {1,2,3,4} and Sy = {high, medium-high, medium-low, low}.
However, since the relevant claims were softened and the limitations explained in detail, I do not believe that this discrepancy should interfere with the acceptance of this paper, but perhaps the issue could be addressed in future work. With the explanation in lines 529-543, the reader gets a very clear view of the experiments, findings and related implications.
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: DATA-DRIVEN CLASSIFICATION OF THE CERTAINTY OF SCHOLARLY ASSERTIONS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No major comments, I am satisfied with the response of the authors. Just two typos:

-lines 375 and 376: Extra parentheses in the Recall and F-score formulas
-line 473: Full-stop at the end of the sentence missing
Experimental design: No more comments, I am satisfied with the response of the authors.
Validity of the findings: No more comments, I am satisfied with the response of the authors.
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: RNA SEQUENCING OF CD4 T-CELLS REVEALS THE RELATIONSHIPS BETWEEN LNCRNA-MRNA CO-EXPRESSION IN ELITE CONTROLLER VS. HIV-POSITIVE INFECTED PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The author described the relationships between lncRNA-mRNA co-expression in elite controller vs. normal-process HIV patients. They performed bioinformatics and RNA-seq to identify differentially expressed mRNAs and lncRNAs from CD4+ T cells from ECs, NPs, and HCs, and construct a PPI network. Finally, they obtained some hub genes associated with viral infections and immune responses. However，the manuscript had some writing and logic mistakes and should be revised (e.g. the rather than th).
Experimental design: Why authors just collected CD4 T-cell samples from two ECs, two NPs, and two HCs to perform second-generation transcriptome sequencing? I think the sample size is too small to be statistical significance.
Validity of the findings: The article focused on bioinformatics and was lack of in vivo and in vitro experiments to confirm the final conclusions.
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: RNA SEQUENCING OF CD4 T-CELLS REVEALS THE RELATIONSHIPS BETWEEN LNCRNA-MRNA CO-EXPRESSION IN ELITE CONTROLLER VS. HIV-POSITIVE INFECTED PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript by Chen et al titled 'RNA sequencing of CD4+ T cell reveals the relationshis between lncRNA-mRNA co-expression in elite controller vs normal-rocess HIV patients' reports differential expression of lncRNAs and protein-coding mRNAs in CD4+ T cells from elite controllers, HIV-patients (NP) and Healthy controls (HC). Two individuals, one male and one female are included in each group.
Major concern:
1. Given the inter-individual variability in gene expression, the small number of samples are insufficient for such analyses.
2. Just one male and female in each group are not sufficient account for gender-based differences in transcriptome.
Experimental design: Major concerns
1. Authors describe 'corrected p value' but the correction is not explained in the methodology. it is unclear if the corrected p value takes false discovery rate (FDR) into consideration.
2. it is unclear if
Validity of the findings: 1. In the raw data files, please mention genomic co-ordinates of potentially novel lncRNAs for which there are no gene or transcript identifiers
2.A gene may make multiple transcript isoforms. If some of the transcript isoforms are differentially expressed as in "Differentially_Expressed_Transcripts", the sum of all the transcript isoforms for that gene may not be deferentially expressed (statistically significant). Authors seem to have considered only the statistically significant transcript
 (line 211-213). This is unconventional and inaccurate unless the expression or function of the gene is driven by the differentially  expressed isoform.
Additional comments: Minor
grammatical and typographical errors in the manuscript


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

Input:
Title: RNA SEQUENCING OF CD4 T-CELLS REVEALS THE RELATIONSHIPS BETWEEN LNCRNA-MRNA CO-EXPRESSION IN ELITE CONTROLLER VS. HIV-POSITIVE INFECTED PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript needs careful editing for English language (including many spelling mistakes), but also for correct use of terminology (e.g. what is normal-process HIV patient?) and consistency in terminology (for the same therapy the authors use anti-viral therapy, antiretroviral therapy (ART) and highly active antiretroviral therapy (HAART)). Please also focus on improving the title and abstract. The results in paragraph under "Immune regulation and viral infection associated lncRNA-mRNA networks" should be moved/written in a form of the Table for better clarity. The Discussion section is too long.
Experimental design: The authors investigate the relationship between lncRNA and mRNA in CD4 T cells from 6 donors (two per each patient group) to better understand the natural ability of elite controllers to repress HIV replication.
As HIV infected individuals from the same patient group can vary greatly in their clinical characteristics (also holds true for elite controllers as they likely employ different mechanisms for repression of HIV replication), sequencing transcriptome of two patients per group is not enough to make any relevant comparisons and statements. The authors should include more patients per group OR they should validate their sequencing/analysis outcomes on a larger group of patients by qPCR for few target lncRNA/genes. As 196 patients were included in the study that shouldn't be a problem.
The authors should also better explain the differences between patients under treatment and untreated normal process patients. Are normal process patients infected but untreated patients with high viral load? If so, it would make much more sense to compare ART treated infected individuals vs. elite controllers to make any conclusions on how elite controllers repress HIV replication.
The authors should also include in the Method section how the data was normalized to enable comparisons between patients and between groups.
Please add and discuss the paper on lncRNA HEAL regulation of HIV replication (PMID:     31551335).
Validity of the findings: No further comment beside the comment under Experimental design.
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: RNA SEQUENCING OF CD4 T-CELLS REVEALS THE RELATIONSHIPS BETWEEN LNCRNA-MRNA CO-EXPRESSION IN ELITE CONTROLLER VS. HIV-POSITIVE INFECTED PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The discussion part is still too long and not closely related to the topic. The author should pay attention to highlight the key points.
Experimental design: Although the author replied that biological repeats did not seem to be necessary in some earlier studies, the examples are research about plants. As is known, plants are more biologically conservative than animals, so I think you should also expand the sample size. Considering the limited sample size, maybe you can use bioinformatics to mine published data.
Validity of the findings: no comment
Additional comments: The author described the relationships of lncRNA-mRNA in elite controller vs. HIV-positive infected patients. They performed bioinformatics and RNA-seq to identify differentially expressed mRNAs and lncRNAs from CD4+ T cells from ECs, HPs, and HCs, and construct a PPI network. Finally, they obtained some hub genes associated with viral infections and immune responses. However, there are still some parts to be revised.


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

Input:
Title: EXPRESSION OF 1B CAPSID PROTEIN OF FOOT-AND-MOUTH DISEASE VIRUS (FMDV) USING BACULOVIRUS EXPRESSION SYSTEM AND ITS VALIDATION IN DETECTING SAT 2- SPECIFIC ANTISERA
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I have a comment for studying the cross-reactivity of the developed protein in insect cells with seven serotypes
Validity of the findings: no comment
Additional comments: This article has described the procedure of expression of the 1B capsid protein of foot-and-mouth disease virus (FMDV) using baculovirus expression system and its validation in detecting SAT 2- specific antisera. I think the paper is excellent and written well (some minor things should be revised) and includes detailed steps of expression of 1B protein of FMDV based on baulovirus system. I have only minor comments, which are listed below:
1. The authors should add an experiment to illustrate the cross-reactivity of the developed 1B capsid protein of FMDV with several sera raised against several serotypes.
2. In ELISA, please change the dilution (1:1 to 1:32) to be a protein content for each dilution (µg)
3. It would be nice if you have another photo for western blot because the current photo has an air bubble above 1B capsid protein.
4. The plates were readied at 450 nm; please correct the title of the Y-axis in figure 5 and its legend.
5. Please add the host of SAT 2-specific antisera inline 213.
6. In line 117, change Luria broth (LB) to  Luria-Bertani broth medium
7. please improve the quality of figure 5.


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: EXPRESSION OF 1B CAPSID PROTEIN OF FOOT-AND-MOUTH DISEASE VIRUS (FMDV) USING BACULOVIRUS EXPRESSION SYSTEM AND ITS VALIDATION IN DETECTING SAT 2- SPECIFIC ANTISERA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Good design
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled
Additional comments: In this manuscript, the authors describe the expression of 1B capsid protein of FMDV using baculovirus expression system and validation of its antigenicity against SAT2 antibody. Toward this end, the authors constructed plasmid, performed transfection, infection, WB and ELISA assays. The results demonstrate the expression and specific reaction of the 1B capsid protein to SAT2 antibody, suggesting the expressed 1B capsid protein could be used for detection of FMDV due to infection or vaccination.
Overall, the manuscript is well written and the design of this study is straightforward. The results are encouraging and would be interesting for readers who work in FMD field.
Specific comments & questions:
1)	After transfection, the author examined the presence of viral DNA in infected Sf9 cells by PCR. However, did they verify that by sequencing of the PCR product? If they did, describe it briefly.
2)	How many passages did the authors conduct after transfection to propagate the recombinant baculovirus in Sf9 cells. Usually, a couple of passages would be need for a purpose of generating sufficient recombinant viruses.
3)	Did the authors purify the 1B capsid protein expressed? It looked like the author purified the 1B protein, but no descriptions on purification in the manuscript. The purification procedures are suggested to be included.
4)	In the M&M, the authors stated 100ul/well of the obtained r1B protein was used for iELISA, however, in the Result section, the authors stated a serial dilution of media ranging from 1:1 to 1:32 was used. What exact dilution was used? The volume of antigen does not make sense, an optimal quantity (weight, e.g. µg), rather thanvolume of the r1B protein, finally used for iELISA should be stated.
5)	In the iELISA results shown in Fig 5, how many times or did the author repeat the expt? There was no error bar in the graph.
6)	In the Discussion, more descriptions on the antigenicity of the expressed r1B are suggested to be expanded. In addition, further work, in particularly the potential application of the r1B protein in clinical detection should be described.


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: EXPRESSION OF 1B CAPSID PROTEIN OF FOOT-AND-MOUTH DISEASE VIRUS (FMDV) USING BACULOVIRUS EXPRESSION SYSTEM AND ITS VALIDATION IN DETECTING SAT 2- SPECIFIC ANTISERA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: this manuscript is interesting and valuable, suitable for publication. the methodology is clear.
however, as a minor comment, I would prefer if the authors provide the sequence analysis showing the similarity of selected sequence of peptides in 1B capsid in SAT2 to the other FMDV subtypes. but the data provided is sufficient 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: ADDITIONS TO THE PHYLOGENY OF COLUBRINE SNAKES IN SOUTHWESTERN ASIA, WITH DESCRIPTION OF A NEW GENUS AND SPECIES (SERPENTES: COLUBRIDAE: COLUBRINAE)
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper’s English is well-written and professional. The authors show subject-level expertise in Iranian Herpetology and provide sufficient literature references. All contents of the paper are relevant to their original research questions. However, some of the figures and references section need re-formatting.
Experimental design: The paper fits the aim and scope of the journal well, given PeerJ’s history of herpetological submissions dealing with systematics. I have no objections to their research questions and analyses used. Their methods all seem repeatable. Some of the descriptive information is placed in the results portion of the paper (i.e. how ventral scales were counted) and should be moved to the methods section.
Validity of the findings: It is clear that the authors have a new taxon. The materials and paperwork they provided appear authentic.
Additional comments: The article "Additions to the phylogeny of colubrine snakes in Southwestern Asia, with description of a new genus and species (Serpentes: Colubridae: Colubrinae)" provides an important contribution to the systematics of Western Palearctic Colubrid Snakes and should be accepted after the authors complete minor revisions. The authors of this paper have clear evidence for the description of a new genus/species of snake and for the transfer of Hierophis andreanus to the genus Dolichophis. However, there are a few key issues that the authors should address in the manuscript before it is slated for publication.

I offer the following broad suggestions:

1. The generic diagnosis and comparison of Persiophis fahimii Gen. et sp. nov. is completely based on osteological data. The authors should also include external characters in these sections as well to aid in field identification (i.e. pholidosis + color pattern). This would significantly improve the generic diagnosis.

2. Methods on how morphological characters were counted for the description of Persiophis fahimii Gen. et sp. nov. should be moved to the Materials and Methods section of the paper. Some important information is also missing in the materials and methods (i.e. How was the tissue collected from these specimens? How are the tissues preserved?).

3. The English name Andreas’ Racer is used throughout the paper. This should be changed in most instances to its scientific name so it reads as Coluber (s. l.) andreanus and then later Dolichophis andreanus comb. nov. The current generic position of the species is in Hierophis so the authors should also justify in the introduction why they use Coluber instead.

4. Some of the figures for ancestral state reconstructions (Figs. 7–9) should be edited and redone in higher resolution so they look more professional.

There are other minor issues/suggestions that should be addressed by the authors as well, which I have noted in my comments on the attached PDF. I also cleaned up some of the references in their bibliography so they are consistent in formatting.

– Justin L. Lee


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: ADDITIONS TO THE PHYLOGENY OF COLUBRINE SNAKES IN SOUTHWESTERN ASIA, WITH DESCRIPTION OF A NEW GENUS AND SPECIES (SERPENTES: COLUBRIDAE: COLUBRINAE)
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Missing literature pointed out in my comments

2. Additional tables required, some raw data (measurements & counts) not provided (Figures 7-9 tabulate raw data in supplementary material), photographs of the preserved specimen and its condition. Photographs, measurement and counts for Andreas’ Racer (add in supplementary material). Provide a GenBank table for the sequences used in this study at least for the "Old world racers"

Check general comments section and suggestions in my review for more details.
Experimental design: Comparative morphology sections (external morphology and osteology) needs improvement. It is incomplete now. Therefore not a rigorous investigation.

Check general comments section and suggestions in my review for more details.
Validity of the findings: The new genus is described based on one female specimen because it is rare which is understood. Therefore, it is the responsibility of the authors to do a thorough comparison with all the genus in the lineage and all the closely related sister species.

Some claims ("smaller head elements may be accompanied by a lower number of covering scales including labial and temporal scales")  in the discussion section are not statistically sound. Therefore, I suggest these to be excluded or do a thorough comparisons to back these claims.

Check general comments section and suggestions in my review for more details.
Additional comments: This manuscript require a lot of improvements. I have made my comments and suggestion in the manuscript. These are some of the most important components to be dealt with in this manuscript.

Molecular: 1. Mitochondrial genes tend to have faster rate of molecular evolution and therefore higher genetic difference. I think it is important to add at least one nuclear gene in this study,  2. I see you have done only a ML analysis and the bootstrap support is "80" for the new genus and its closest sister, while this support is "89" for Scaphiophis and Muhtarophis but you have less confidence on the phylogenetic placement of the latter pair than the former. Therefore, it is better you run a bayesian analysis for the “old world racer” dataset to have more than one line of evidence to support this relationship. 3. Recent studies shows that Argyrogena is also within the old world racer group add this taxa to your phylogeny

Morphology: 1) Provide detailed morphological data table for the new taxa and figures of the preserved holotype 2) Provide a comparative osteology table for all old world racer genera, apart from this compare osteology of the new genus with all the sister species in the genus (Rhynchocalamus, Wallophis, Wallaceophis and Lytorhynchus). 3) Provide a comparative external morphology table for all old world racer genera.

Ancestral state estimation is incomplete, needs further research: 1) Add information for all the old world racers and then do a ancestral state estimation if not tabulate the scalation data. 2) If your idea is to show that dwarfism has evolved multiple times in this group do so by showing them in the phylogeny of all the old world racers.


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: ADDITIONS TO THE PHYLOGENY OF COLUBRINE SNAKES IN SOUTHWESTERN ASIA, WITH DESCRIPTION OF A NEW GENUS AND SPECIES (SERPENTES: COLUBRIDAE: COLUBRINAE)
Review round: 2
Reviewer: 1

Output:
Basic reporting: Writing and grammar is improved from previous draft, references section has been cleaned up and is more consistent.
Experimental design: See first draft for comments, experimental design is sufficient for publication
Validity of the findings: Discovery of new genus/species is certainly valid, other taxonomic changes seem supported by phylogenetic data.
Additional comments: The authors have made improvements on this new manuscript and have addressed the suggestions of my first review adequately. There are some small changes I wish they fix, but I would not go so far to suggest Minor Revisions. My comments are attached as a PDF file.


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

Input:
Title: ADDITIONS TO THE PHYLOGENY OF COLUBRINE SNAKES IN SOUTHWESTERN ASIA, WITH DESCRIPTION OF A NEW GENUS AND SPECIES (SERPENTES: COLUBRIDAE: COLUBRINAE)
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: Some of the analysis I suggested to exclude in the previous revisions are retained although the data is incomplete. I suggested to add 'one' nuclear gene to their dataset, which is not included.
Validity of the findings: The new genus and species as such is valid and the authors have added additional comparative morphological data which I had requested in the previous review.
Additional comments: I appreciate the authors for addressing most of my minor comments and some of my major comments. However, I still have the two major issues in this MS which is not addressed.

1. Additional genetic data was not added. At this day in age when the costs of DNA sequencing are cheap and the authors have cited lack of funds to add “one” additional nuclear gene sequence is unacceptable. I would not be this particular if it was only a species description. The authors are also trying to describe a whole new genera here hence this data is a must

2. I asked the authors to remove sections on the examples of parallel evolution because this is carried out based on an incomplete dataset therefore misleading. I still think this section will be misleading without a complete dataset. I suggest them to remove this.


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 TRANSCRIPTOMICS CHARACTERIZED THE DISTINCT BIOSYNTHETIC ABILITIES OF TERPENOID AND PAEONIFLORIN BIOSYNTHESIS IN HERBACEOUS PEONY STRAINS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Most of the papers' english is understandable, but phrase and paragraph structure needs to be improved  as the information is often not clearly stated. In the material an methods section, the latter are not extensively well described, so the experiments performed are not clear for us to understand as it stands.

Ambigous number of genes are given throughout the article (In the abstract and first part of results: 36,264- further 36,203 (supposedly these are the annotated ones? it is not clear). In the supplemental Table 3 there are 36,267 unigenes.) . Please stick to one number.

Some important references are missing, for instance another transcriptome study in Paeony (Luo, J etal. 2017. Transcriptomic Analysis Reveals Transcription Factors Related to Leaf Anthocyanin Biosynthesis in Paeonia. Molecules, doi:10.3390/molecules22122186).

The structure is overall right, supplemental data are given extensively. Nevertheless, some of the main Figures might be moved to the supplementals as the relevance for the paper is not really clear.

Furthermore, the authors miss phrasing the hypothesis clearly, which would be something like :” we expected the root to contain the paeoniflorin biosynthesis genes highly expressed compared to shoots as observed in Yuan et al. 2013…”.

In the abstract they mention "preferentially expressed" genes, but they should talk about "differentially expressed" instead. The numbers are quite high, but I think this depends on the statistical threshhold used to classify the genes, as I described later.
Experimental design: The research is an extensive analysis with RNAseq data, which complements a previous study, Yuan et al. (2013) often cited by the authors, as this study identifies 19 of the terpene backbone biosynthesis genes from ESTs (probably 454 sequencing).
In the current study, the authors find 14 more genes than Yuan et al., 13 from the same pathway, shown in Figure 4, and another involved in the conversion from "GPP to alpha-terpineol" as they write in the paper but don't show in the Figure 4. I suggest showing this step in a Figure, as PeerJ is not a phytochemistry journal and might be read by non-experts as well.
It might be interesting to use the same nomenclature used for the enzymes as the one used by Yuan et al. (2013), also in the heatmap Fig. 6, as to render them comparable and to be able to distinguish the newly identified genes. It might also be nice to highlight somehow the novel genes in Figure 4. And to go into more detail of the novel genes in the paper and their relationship with the way of sampling- why were these not identified in the previous study even if the tissue sampling was more extensive, is it because other strains were analyzed?
The comparison of the transcriptomes among strains is also nos extensively discussed, event if their analysis shows (PCA) that the strains have significant differences, even if the tissues do account for most of the differences analyzed.

The investigation appears rigorous, but the lack of detail in the Materials and Methods section gives the impression of superficiality in the analysis. A couple of details need to be included in the statistical analysis, as to why a cutoff of p=0.1 is used, further they mention Fold change, and it's rather log10FC. They have biological replicates, which is seen in the supplemental tables and the PC, but they don't mention it clearly in the text.

Could they please show how the gels/expression profiles of the GAPDH reference genes? In Yuan et al. (2013) they used PIActin, why did they not use the same one?

Please indicate the estimated efficiency from the standard curves for each primer (supplemental table 1). This is needed to be able to use the delta-deltaCT quantification Method mentioned previously.
The authors should please describe if two- or three- step cycles (95C denaturation, 60C annealing, etc) for the qPCR.
Validity of the findings: The underlying data are all provided, but they appear not to have been submitted to the public databases yet, NCBI- This needs to be done before re-submitting and the accession numbers given instead of, or additionally to, attaching the data.

There are novel data, as new genes in the paeoniflorin biosynthesis pathway were identified, and multiple transcription factors. It's important to annotate TFs, but the authors miss to show which of these are correlated (co-expressed) with terpene- paeoniflorin biosynthesis, to round-up their hypothesis.

Reference to the other potential interesting hypothesis to test should be made at least in the end of the discussion (strain comparisons, annotation o transcription factors related to paeoniflorin biosynthesis, etc.)

In the conclusions the authors should go into the hypothesis "comparing differentially expressed genes among tissues" which they don't go back to.
Additional comments: Dear authors,

The presented work is of general interest to the phytochemistry public, and the novel genes identified are important completion of the picture from previous work on paeoniflorin biosynthesis.
I hope the comments previously made will help you to improve the paper.
I attach the corrected pdf, for individual changes to be considered as well.

best regards.


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 TRANSCRIPTOMICS CHARACTERIZED THE DISTINCT BIOSYNTHETIC ABILITIES OF TERPENOID AND PAEONIFLORIN BIOSYNTHESIS IN HERBACEOUS PEONY STRAINS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is very hard to decipher. The Introduction fails to explain where the biosynthesis takes place and why then root and shoot tissues are selected for this study and not others. Furthermore, there is no short and clear statement about what to expect in every strain/tissue with respect to the production of the substances and/or their biosynthesis. Supplementary figure 1 should be included and explained in the main text, since it helps understanding the whole experimental set up.

The bioinformatic analysis is not thoroughly described, so reproducing the results would not be possible.

The whole description of the differential analysis (From line 214 on) is not understandable. Suggestion: “For each strain, we compared shoots VS roots and extracted lists of root/shoot specific upregulated  genes. We then found 322/886 genes that were upregulated in all three strains, regarding them as tissue specific.“ In line 217 correct please the number 332 to 322 or viceversa.

In line 141 : differentially expressed genes are defined to have FC >2, pval < 0.05 and FDR<0.1 On line 216: FDR < 0.05: Which is correct?

Figure 2A please fix the labels for XT roots since they are all marked as rep2 and explain why one XT Root replicate is grouped outside the rest of  root and shoots replicates from XT and PB.
Experimental design: The experimental design involves two different tissues from 3 strains producing different amounts of the substances of interest and 3 biological replicates per condition. This allows differential expression analysis between conditions to determine candidate genes involved in biosynthesis. A discussion of the reasons why roots and shoots are the best tissues for this purpose is missing.
Validity of the findings: The bioinformatic analysis is not thoroughly described, so reproducing the results would not be possible. Furthermore, it is almost impossible to validate the results with the lacking description of the analysis and results:

1. The big amount of de novo transcripts call my attention: did the authors post-process the transcriptome after Trinity to remove redundancies (e.g. CD-Hit) and artifacts (lowly expressed transcripts in all samples).

2.  In order to be able to validate the assembled transcriptome, some important measures and their comparison to those in published transcriptomes should be shown and discussed. E.g. transcriptome statistics (N50, etc), transcript length distribution  (Fig 3A),  BUSCO completeness scores, etc
2.1 How were the reads aligned to the transcriptome (name of aligner and parameters), how were the reads counted to make a counts matrix for DESeq2, did the authors account for multiple alignments? How was the mapping percentage of raw reads to de-novo transcriptome?

3. The whole description of the differential analysis (From line 214 on) is not understandable. Suggestion: “For each strain, we compared shoots VS roots and extracted lists of root/shoot specific upregulated  genes. We then found 322/886 genes that were upregulated in all three strains, regarding them as tissue specific.“ In line 217 correct the number 332 to 322 or viceversa, it doesn't match Figure 4.

4. In line 141 : differentially expressed genes are defined to have FC >2, pval < 0.05 and FDR<0.1 On line 216: FDR <0.05: Which is correct?

5. The gene lists discussed from line 214 on are not clearly explained. Not all of the found genes should be used in the next section. If no biosynthesis is expected on GS, I would remove from the candidate genes all those up-regulated in GS roots. If this does not make sense, please discuss.
5.1 The authors also show lists of genes up-regulated in shoots. From supp. figure 1, there is almost no substance in shoots of any strain. Since it is not clearly explained which genes were used from line 222 on, I assume you used all the up-regulated genes and that would be counter-intuitive. Please, explain which genes you used for enrichment analysis in the identification of genes in biosynthesis pathway (from line 222 on) , e.g. 'We used  total of 2597 (XT-root) + 677 (XT and PB-root ) + 3737 (PB-root) for identifying candidate genes in the biosynthesis of ...'

6. The qPCR are ot shown in the main text but in supp. figure 2. The conclusion in lines 250-251 does not explain the fact , that except for AACT1, the rest genes don't seem to prove the hypothesis (if this is it!) that biosynthesis is occurring in roots and not in shoots and in XT, PB and not in GS. This qPCR results, instead of showing what one would expect, show almost the opposite. Otherwise, state please clearly what is the hypothesis.

7. Figure 6 is also not very useful the  way it is. I would suggest use a color mapping red-green for low-high expression, which is standard and group columns according to the pattern we would like to see. Or plot log2FC, maybe will be clearer.
Additional comments: The authors have sequenced and de-novo assembled a reference transcriptome for the study of the biosynthesis of terpenoid and paeoniflorin in 3 different peony strains. The experimental design involves two different tissues from 3 strains producing different amounts of the afore mentioned substances and 3 biological replicates per condition. This allows differential expression analysis between conditions to determine candidate genes involved in biosynthesis. The work behind this paper has a great potential to serve as reference for further studies in this area.

However, the manuscript is not written in an understandable way, since no clear hypothesis is made, the analysis is not well described and thus not reproducible, the transcriptome is not backed  up with a good validation.

I have big concerns about the validity of the candidate genes, since not even qPCR or expression heatmaps show a reasonable trend. I also doubt about the enrichment tests. If all genes found as differentially expressed (about 23K) were used for enrichment analysis, even by chance one could at a high probability choose 32 Unigenes related to biosynthesis.


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 TRANSCRIPTOMICS CHARACTERIZED THE DISTINCT BIOSYNTHETIC ABILITIES OF TERPENOID AND PAEONIFLORIN BIOSYNTHESIS IN HERBACEOUS PEONY STRAINS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I still had problems deciphering many of the phrases. I indicate most of the problems I had in an additional document.

Same with the references, I indicated one to improve their manuscript. Although missing ones in the previous version were included this time.

The structure is still not professional. I indicate the problems in the additional document.

The hypothesis were stated a bit clearer this 2nd time, but it still lacks a better connection among the strains and tissue analysis, which needs to be stated in the abstract and conclusions.
Experimental design: I think the research is very interesting, therefore it is crucial to make it more accesible to the public with the corrections needed.

Research question was better stated with re-submission, can still be improved.

Technical standards seem to have been followed, with exceptions, in the qPCR. The number of upregulated genes is still high, clarification is needed in the analysis. A higher cut-off might be needed for FDR, 0.01.

Methods need to be described better, e.g. Tissue dissection scheme, primer efficiencies in qPCR.
Validity of the findings: The results maybe novel. The significance, for the hypothesis, of the detected genes is not clear as it stands.

Data were made publicly available this time.

Conclusions need to be restated. Still many unclear points, for instance the number of transcription factors, the numbers of unigenes and differential expressed genes, which are not cited in either the main text or the Figures.
Additional comments: Dear authors,
Your study and data are very interesting and highly relevant for the lay public. Nevertheless, I have a complete review with still many issues to fix, so that the readers can get the crucial information and publication standards are met.

Best regards.

Complete review follows as an attached document.


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 TRANSCRIPTOMICS CHARACTERIZED THE DISTINCT BIOSYNTHETIC ABILITIES OF TERPENOID AND PAEONIFLORIN BIOSYNTHESIS IN HERBACEOUS PEONY STRAINS
Review round: 3
Reviewer: 1

Output:
Basic reporting: It is clear and unambigous. English is professional.

Background and Literature are good.

Ok, more comments later to improve paper readability.

Results are relevant to the hypothesis.
Experimental design: Correct.
Validity of the findings: Valid findings for an explorative study.
Additional comments: Dear authors,

This is a very interesting explorative study on the paeoniflorin biosynthesis in three Paeony strains.
I have a few corrections to point out to improve paper readability and/or quality.

L. 82-84. Please provide the abbreviations for the enzymes in the MVA pathway that will be used later in the text. This will help the reader to identify them quicker later, i.e. acetyl-CoA acetyltransferase (AACT1), hydroxyl methylglutaryl-CoA synthase (HMS), HMG-CoA reductase (HGMR) etc.

Correction in Figure quality, Fig3. Labelling of Subfigure C is not visible. Subfigure B, Correct to SwissProt, which is the correct name of the database (also in the text please!).

Figure 5: IMO: Enzyme name (EC:...) and Unigene Name should go in separate table. Else it will be too small to be read in a publication.

Other corrections:
L. 64 the correct name is of the deaseae is “typhoid fever”
L. 277-279: “These results ...” should be moved to Discussion section, as this is a very important speculation about the results.
L. 284 space missing: Spartina alterniflora

Else, it might be worth in a future study to determine the ploidy level of Paeony, as many transcripts are found in more than one copy, which might be an indication of polyploidy.

Best wishes!


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 TRANSCRIPTOMICS CHARACTERIZED THE DISTINCT BIOSYNTHETIC ABILITIES OF TERPENOID AND PAEONIFLORIN BIOSYNTHESIS IN HERBACEOUS PEONY STRAINS
Review round: 4
Reviewer: 1

Output:
Basic reporting: Everything is ok.
Experimental design: Well explained.
Validity of the findings: All is well.
Additional comments: Dear authors,

After a careful review, I remarked that all previous concerns have been well addressed.
I suggest just a careful proofreading for spelling mistakes, but nothing critical anymore.


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: IMMUNE RESPONSE OF HUMAN CULTURED CELLS TOWARDS MACROCYCLIC FE2PO AND FE2PC BIOACTIVE CYCLOPHANE COMPLEXES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Information available in the literature on the immune response to exposure of cyclophanes is very scarce. Therefore the work presented in this paper, dealing with the immune response of cultured human cells (Human embryonic kidney (HEK) 293T cells) when exposed to Fe(III) complexes of two different cyclophanes short-named by the authors Fe2PO and Fe2PC is a valuable contribution to a little-explored field.
There are several aspects in the paper that should be improved: The English, especially in the abstract and the introduction is rather poor.
I would like to disagree with the use/abuse of the term ‘biomimetic’. Here I find it to be totally inappropriate as common sense identifies as biomimetic molecules that have significant structural similarity to biomolecules (e.g. a peptide made up of non-naturally occurring amino acids). If one adopts the criteria mentioned in this paper to define a molecule as biomimetic, then even sodium chloride is ‘biomimetic’. Hence we have an adjective that does not mean anything because it can be applied to everything. I think the authors do not need to use fashionable (but in this case meaningless) adjectives to ‘sell’ their work.
I think it is unacceptable that one can go through the whole paper without seeing the structural formulas of the investigated compound (although a reference (Salazar-Medina AJ., 2013) showing these is cited.
Experimental design: I noticed that the dosage used for the tests is quite high (120 mM) but there is no indication as to why such dosage was selected. There is no dose/response investigation. I think the authors should explain why, and also mention why they selected (HEK) 293T cells for their tests.
Validity of the findings: There is an articulated discussion on the correlations between certain genes being over or down-regulated and the related biological pathways. The data appear consistent. However, one is left to wonder as to what could be seen if the cyclophanes ‘as such’ and not their Fe(III) complexes were used in the experiments. The authors recognize this weak point in one of their conclusive remarks. Therefore, why was nothing done about it? An explanation should be provided.
Additional comments: These are covered in the above fields


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: IMMUNE RESPONSE OF HUMAN CULTURED CELLS TOWARDS MACROCYCLIC FE2PO AND FE2PC BIOACTIVE CYCLOPHANE COMPLEXES
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: More experimental methods should be employed, such as WB or Elisa.
Validity of the findings: no comment
Additional comments: Major revision before publish
Major comments:
1. The mechanism study about the thermal effect on cells is not accepted. Authors just tested two proteins and speculate the potential mechanism. It is strongly suggested to conduct more cell culture to block the signal pathway and test the downstream gene expression of the stem cells related to osteogenic differentiation.

2. There is no quantitative and statistics analysis for the results. For example, authors claimed that “The gene for the adenosine receptor (ADORA2A) is related to apoptosis, and it was strongly down-regulated by…”. The statistics analysis should be performed and P value should be provided.

3. Author tried to evaluate the immune gene expression changes in human cells exposed to metal particles. However, why is Fe2PO or Fe2PC? Authors should give more information about the reason they used Fe2PO and Fe2PC in the introduction.

4. It is an uncompleted work. It can’t be get these conclusions just depend on the gene expression. Western blot or Elisa should be performed to evaluate the expression of relevant proteins.

Minor comments:
1.	Authors should carefully organize the manuscript. Many inconsistent descriptions were existed. For instance, “eight” should be corrected as 8 In line 174.
2.	There is a writing error in line 48


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: IMMUNE RESPONSE OF HUMAN CULTURED CELLS TOWARDS MACROCYCLIC FE2PO AND FE2PC BIOACTIVE CYCLOPHANE COMPLEXES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The structure of the article conformed to an acceptable format now.
Experimental design: More experimental methods should be employed, such as WB or Elisa.
Validity of the findings: The data on which the conclusions are still absence.
Additional comments: The authors adressed most of the problems, but some critical ones still yet to be done.
1.As I said, it can not be got the conclusions made in this study just based on genes expression. Authors refused to add experiments and deleted the relevant conclusion. However, this kind of correction make the paper lack of central topic and important conclusions.
2."Conclusion" should be re-eidted and exhibited the most important findings in this study. Currently, much unrelevant contents were involved in Conclusion part.


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: EFFECTS OF BIODEGRADABLE PLASTIC FILM MULCHING ON SOIL MICROBIAL COMMUNITIES IN TWO AGROECOSYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Overall, the manuscript is well-written, with appropriate support from previous studies. The comparison of PE with BDM in the same study is critical for increasing understanding of the impact of these management systems on agroecosystems, as the authors state.
•	Line 67: Could the authors provide a source for this statement? What regulatory agencies are involved in these decisions?
•	Line 85-86: “….we compared the impacts on soil microbial communities of BDM and PE mulch in two-year….
•	Line 88: “measurements of a suite of soil….”
•	Line 388: typos?
•	Line 439: “….this suggests that this is in response to the incorporation…”
Experimental design: •	Line 147:  I assume the May collection was with plastic and the September collection was after the plastic was removed or tilled? How long before plastic was applied were the samples collected? How long after plastic was tilled were samples collected? How far apart from each other where the 30 soil cores per plot?
Validity of the findings: •	Line 444: Why are fungi important colonizers and degraders of BDMs? Is there any relation to changes in soil nutrient content? Carbon? The authors discuss carbon inputs are briefly at the beginning of the Discussion section, but it’s not clear how those inputs might be tied (if at all) into a potential impact/difference between BDMs and PE.
•	The ambiguity in the collection times (see Experimental Design) made it a little difficult to follow what comparisons were made between locations. The authors indicate that some of the differences between their findings and that of another BDM study may have been the time of interaction between the soil microbes and mulch. Clarification on what this time period was for this study would also help put the authors results into a better context with this other study.
Additional comments: Again, this is a well-written study. However, in the introduction, greater clarification on the link between soil health and microbial communities may strengthen the overall research question. The authors indicate that a previous study found that the “effect on soil health was minimal” (Lines 88-91). As microbial communities are a component of soil health (though not necessarily “officially” measured as such), it would be good to make a clear distinction on the differences between the previous “soil health” measurements and those being addressed in this study.


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

Input:
Title: EFFECTS OF BIODEGRADABLE PLASTIC FILM MULCHING ON SOIL MICROBIAL COMMUNITIES IN TWO AGROECOSYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article meets the standards and all aspects included in this basic reported are satisfactory.
However, there are other relevant improvements to incorporate. These are included in the attached file.
Experimental design: The research is relevant and meaningful. The technical standards of the research carried out are high. However, the experimental design covers knowledge gaps only partially, see attached file.
Validity of the findings: Research standards are met; results are robust and statistically sound.
Revision of the paper and improvement is required due to the paper experimental design.
Some conclusions are not directly derived from the paper results and are to be refined.
See details in the attached file.
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: EFFECTS OF BIODEGRADABLE PLASTIC FILM MULCHING ON SOIL MICROBIAL COMMUNITIES IN TWO AGROECOSYSTEMS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have done a good job responding to previous comments.

A minor change could be made to Line 116: "...but generally not explicitly included in assessments of soil health."
Experimental design: The authors have adequately addressed previous comments.
Validity of the findings: The authors have adequately addressed previous comments.
Additional comments: The authors have adequately addressed previous 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: EFFECTS OF BIODEGRADABLE PLASTIC FILM MULCHING ON SOIL MICROBIAL COMMUNITIES IN TWO AGROECOSYSTEMS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript has been substantially improved in basically all aspects required. It is now well written and, especially, better focused on the specific studies carried out. Overgeneralization is now avoided and, where needed, conclusions have been refined.
Experimental design: The experimental design is now clear to the reader. Several little but substantial changes have contributed to make the paper focused on the specific results found.

The research carried out is well defined, relevant and meaningful.

The contribution of the paper to advancing knowledge on the impacts of BDMs on the agricultural soil ecosystem is valued.
Validity of the findings: Regarding the conclusions, limitations arising from the experimental design stay and somehow restrict the impact of the paper findings. However, the authors make a very balanced discussion.

Two aspects are still requiring to be reviewed and modified:
•	L 334-337: “Post hoc testing showed that the richness estimates in TN significantly differed between 2015 and 2016. In WA, Fall 2015 differed in richness from the other time points. In TN, Fall 2016 diversity was significantly higher than other seasons. Diversity estimates were significantly lower in Spring than in the Fall seasons for WA.

The whole paragraph should be revised and limited to really relevant findings. If authors consider they are relevant and worth to be mentioned, they should be supported by the data shown. Table 3 (referred to in line 334) shows significant differences due to location, time and time interaction with location and with treatment, but none of the specific differences mentioned can be inferred. They are also not evidenced in figure 3.

•	L 344-345: “It is important to note, however, that Weedguard completely disintegrated in TN after the field season (Ghimire et al. 2018b)”

The term “disintegration” is unclear. Ghimire et al. 2018b do not determine “disintegration” but “percentage of soil exposure”, and only through direct visual observation. Small mulch fragments could remain imperceptible to the human vision. This is relevant since it has been acknowledged plastic fragments under 1mm size to cause significant effects on the environment.
In addition, Ghimere et al. 2018b do not report complete disintegration of the Weedguard mulch in TN. On the contrary they report percentage of soil exposure after the mulch treatment and crop growing season to be strongly dependent on the year. For Weedguard paper, figure 2 of the paper shows 100% TN soil was exposed (no mulch seen to remain on the soil) in 2015, but in 2016 only ca. 25% of the TN soil was exposed (75% of the soil remained covered by the mulch).
Additional comments: No additional comments


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

Input:
Title: COMPARATIVE TRANSCRIPTOMIC ANALYSES OF POWDERY MILDEW RESISTANT AND SUSCEPTIBLE CULTIVATED CUCUMBER (CUCUMIS SATIVUS L.) VARIETIES TO IDENTIFY THE GENES INVOLVED IN THE RESISTANCE TO SPHAEROTHECA FULIGINEA INFECTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In the submitted manuscript, the authors described the transcriptomic analysis of genes which are differentially expressed in cucumber between the resistant and susceptible cultivated varieties under the control condition and after inoculation with using Sphaerotheca fuliginea which is a plant pathogen that causes powdery mildew on cucurbits (very destructive disease). Introduction and background of the article are well written. Proper breeding technologies for selection of PM-resistant cucumber varieties are an effective way to control the disease but there is still a lack of knowledge about the molecular mechanism underlying the infection.
The authors conducted extensively in silico analyses of the studied genes, the annotation, and assignment to KOG/COG, KEGGO and GO terms. The strong points of the manuscript is a very interesting topic and application of NGS technology. The description of identified mRNA transcripts is very interesting especially with regard to hormone signaling pathway connections. I commend the authors for their extensive data set. The workflow of data processing is proper. In addition, the manuscript is clearly written in a professional, unambiguous language. Figures are rather relevant and high quality. Figure 2 and 4 are not properly labeled and there is a lack of full descriptions in the legend part (see general comments). Raw data are deposited in NCBI SRA database and are available but there inaccuracies of a given method of obtaining the raw reads. Authors should be more precise. The overall structure of the manuscript is proper. References are relevant.
Experimental design: The aim of the study is clearly stated and it was to identify genes and clarify the correlation mechanism of plant response after pathogen infection in resistant and susceptible cucumber.
Proper breeding technologies for selection of PM-resistant cucumber varieties are an effective way to control the disease but there is still a lack of knowledge about the molecular mechanism underlying the infection.
There are several weak points of the article.
It is a good and valued practice to combined RNA – seq data with the experimental validation of gene expression in transcript level by quantitative real-time RT-PCR. In this article, there is a lack of this confirmation. It is a big failure of this work.
The statistical analysis (as I have noted above) which should be improved. The authors do not present borderline data for statistical analyses. They give it only in some cases (see general comments). Statistical analysis presented herein seem to be appropriate but there is no clear description of the used threshold in the analysis of QC of reads, mapping to the reference genome and DEGs analysis. The lack of this description is a problem and should be complied. The description of DEGs using only p-value it could be not enough, Often the statistical approach use also fold change
Authors provide the raw data, and supplementary tables, however, your supplemental files need more descriptive metadata identifiers to be useful to future readers. Although your results are compelling, the data analysis should be improved in the following ways: gene id, functional description, ontology assignment, expression value, p-value, fold change etc…
Validity of the findings: Impact and novelty of the finding relay on the pointing out of genes differentially regulated and not more. The authors emphasize the importance of influencing the results too much. There is a set of data not confirmed experimentally. There is a lack of statistical description and experimental validation (see experimental design). Conclusions are linked to the scientific question.
Additional comments: Line 84 – lack of space: CsERF004expression
Line 134- “The four libraries were used” if they were pooled or they were sequenced separately?
Line136 – “Raw reads were generated in a paired-end format” -what about the length?
Line137 - NCBI Short Read Archive with accession numbers SRP212890 – in the SRA database you show different sequencing technology Illumina Hi seq4000, in the than in presented manuscript
Line:142 – “clean data(clean reads)” – lack of space
Lines 141 – 145 – You should provide more information about thresholds. The statement (line 144): “All the downstream analyses were based on clean data with high quality (Yu et al. 2017).” Is definitely not enough for scientific paper.
Line 148: “Raw sequences were transformed into clean reads after data processing.” – this step should be more descriptive.
Line 149 – you should specify what was your reference, by adding the Accession Number.
Line 150 – “Only reads with a perfect match or one mismatch
were further analyzed and annotated based on the reference genome” – you should give the specification for this task
Line 151 soft were – bad writing
Line152 – Gene function you should write the web source for the programs and databases which were used for this task. Additionally, the scheme of functional analysis is not clear. Did you perform functional annotation for all sets of genes? What about the annotation form the reference genome? Why do you not use it?
Line 156 – lack of space
Line 160 – please provide a standard shortcut for this – FPKM
Line 187 – to what refers the number of 280 mln reads – you should specify
Line 189 – could be mapped or were mapped?
Line 189 – again specify the reference genome
Line 192 – Please specify better how many genes you detected after mapping? And describe the better generation of 21 408 number.
Line 194 – “The largest term” - does it refer to the KOG term?
Line 194-198 - Are genes counted once or are together?
Line 205 – additional mark ‘
Line 215 – DGEs-?
Line223 –Fig 2c - describe the axes (x, y) in the drawing
Line 244 –“a large number” – how many percents?
Line 248 – “1” should be written as “one”, – “2” should be written as “two”  like in other parts of the paragraph.
Line 282 – exactly you write time points but in your study in did not mention time point study
Line286 – tiny variation – could you specify this?
Line 293-294 – “Our data provide comprehensive information
294 on hormone signaling pathway genes under S. fuliginea infection in cucumber” This is a too far-reaching statement. It is now known that the hormone-related gene group is changing, but this is not fully experimentally justified. And it would be worth highlighting in the discussion.
Line 300 – “including 1 AUX1 gene, 1 AUX/IAA gene, 1 GH3 gene” see comments to Line248
Line 306 – nomenclature of gene – the gene name should be written in italic
Line 306 – Is this gene name? Could you specify this?
Line 306 – Not Cas but Csa
Line 314 – see comments to line 306
Line329 – 330 – “In our study, some peroxidase genes (Class I) were only
330 induced in the susceptible cultivated variety, while some other peroxidase genes (Class II)…” – the word “some” is not appropriate to a scientific paper.
Fig 4 – what is a grey box, you should add this to the legend, what is a rich factor – this should be explained.
Table S5 – what is the number of 4219?


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

Input:
Title: COMPARATIVE TRANSCRIPTOMIC ANALYSES OF POWDERY MILDEW RESISTANT AND SUSCEPTIBLE CULTIVATED CUCUMBER (CUCUMIS SATIVUS L.) VARIETIES TO IDENTIFY THE GENES INVOLVED IN THE RESISTANCE TO SPHAEROTHECA FULIGINEA INFECTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes a study on identifying candidate differentially expressed genes in cucumber species in response to the infection of Sphaerotheca fuliginea. Authors mainly used a RNA-seq experimental design to answer this question. Main findings of this study could be potentially meaningful, but there are many places authors should do to improve the quality of this manuscript.
1.	The overall English writing could still be improved. There are a few of flaws could be easily caught. For example, L71 “to control” should be “to be controlled”; L109-L110 “one when… one when…” should be restructured; L150, the word of perfect in “perfect match” looks more symbolic, but you should use a more scientific word to describe; L160, you should give FPKM as an abbreviation;
2.	Figure 1, the left border seems to be trimmed a little bit, please modify you figure and update.
3.	Figure 2, Heatmap not heat map; you should label the unit for the scale bar; what is the y scale and y scale unit for the cluster subpanel?
4.	Figure 3, you should also list p-value for each enriched term.
5.	Figure 4, what does it mean the rich factor? What does different size of round mean? What does the red arrow mean? What does the color of round mean?
6.	Figure 5, what does the phylogeny in each heatmap mean? You didn’t explain Class I and Class II in the figure, also I understand you have mentioned that in the main text.
Experimental design: 1.	From authors’ description, I think there were 3 replicates for each treatment per species. However, the expression authors used in the manuscript is confused, please restructure sentences to be concise for better understanding.
2.	The “processing of RNA-seq data” session should be reinforced a lot, such as detailed parameters used, or what detailed filtering parameters employed in the in-house perl scripts.
3.	Which version of reference genome you used? Which should be given detailed information.
4.	For the gene function annotation session, I am totally lost. You listed a number of software for the annotation, but why did you use those software? What are the differences among them? All of these types of information should be given but not jest listed names of software/methods.
Validity of the findings: 1.	Only depending on the p value to define differentially expressed genes is not accurate, you should also consider absolute log2 fold change to avoid instability in low read counts per gene.
2.	For GO analysis, other than looking at which gene associated with which GO term, it is more informative to look at which GO is significantly enriched for different gene populations (such as DEGs), although authors mentioned GO enrichment in the methods session, but I couldn’t find any in the results. I don’t think the current GO analysis result could be used in the main result.
3.	Please also specifying the background gene population in your analysis (GO, if you are going to do GO enrichment; and KEGG); also how did you define the p value in this GO analysis? (one-tail or two-tail?)
4.	The methods of clustering analysis should be solely listed as an additional paragraph in the methods session. Also, you should clarify how did you decide the number of clusters you used.
Additional comments: There are two cultivars were used in this study, except for looking DEGs, it would be very interesting to see or discuss how orthologous genes are regulated differentially between these two species, like the study in the paper “Zhang et al. (2017) Differentially Regulated Orthologs in Sorghum and the Subgenomes of Maize”. Please discuss this point and I think it will be helpful to enhance the significance of the manuscript.


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

Input:
Title: COMPARATIVE TRANSCRIPTOMIC ANALYSES OF POWDERY MILDEW RESISTANT AND SUSCEPTIBLE CULTIVATED CUCUMBER (CUCUMIS SATIVUS L.) VARIETIES TO IDENTIFY THE GENES INVOLVED IN THE RESISTANCE TO SPHAEROTHECA FULIGINEA INFECTION
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this manuscript, Zhang et al. attempted to describe an identification of genes involved in the resistance to Sphaerotheca fuliginea infection using comparative transcriptome approach. However, the authors simply represent their data of bioinformatics with typical analysis, while the manuscript seems to have no scientific hypothesis driven-question. Additionally, it has raised enumerated concerns that need to be thoroughly addressed before consideration further.

Introduction lacks specific information regarding molecular mechanisms of PM-resistant cucumber varieties. How many PM-resistant cucumber varieties obtained so far? What are the obstacles that need to be further investigated? Very scare information of Sphaerotheca fuliginea is described, especially its pathogenesis, epidemiology, life cycle, molecular biology disease, ect.

Additional information has to be included: What are the morphologies and phenotypic characteristics of BK2 and H136? What are their genotypes and resource? Where are they obtained for this study? Ect.

At least 2 biological replications should be performed for each condition of RNA sequencing.

Several time points should be performed to represent the infection. One timepoint is insufficient. Also, infected morphological results have to be included that confirm the gene expression caused by the infections.

How to determine which level will be assigned as differential expressed genes? Log2? How difference will be considered “significant”?

There is no quantitative validation of RNA sequencing. At least one other method has to use for candidate genes obtained RNA sequencing, otherwise, it is meanless.

The use of English in this manuscript is poor and contains typos, grammar errors, etc that would be greatly improved by running by a native speaker.
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: COMPARATIVE TRANSCRIPTOMIC ANALYSES OF POWDERY MILDEW RESISTANT AND SUSCEPTIBLE CULTIVATED CUCUMBER (CUCUMIS SATIVUS L.) VARIETIES TO IDENTIFY THE GENES INVOLVED IN THE RESISTANCE TO SPHAEROTHECA FULIGINEA INFECTION
Review round: 1
Reviewer: 4

Output:
Basic reporting: 1.	Some of the sentences used are too general. For example in the abstract line 42, 47-48. ‘…a few differences..’, ‘the differential expression of prdx genes might contribute..’. The authors should highlight the main findings, at least mention a few genes that may contributed to the resistance of the plant.

2.	In line 68, the author mentioned ‘..massive foundational data..’ what does this mean?

3.	In line 106-111: this paragraph should be in the method section – however it does not tally with the method section where it was stated that ‘..Three independent cDNA libraries for each sample group were constructed..’ . does this mean that the authors pooled the 3 cDNA samples in each of the groups?.  The justification given in the last paragraph is weak. The authors could explain more on the need for identifying potential PM resistance genes and its application.

4.	The way the data is presented may be improved.
Experimental design: The method section needs to be improved.

Line 145: Yu et al is quoted to address which method? Is it the parameters used to trim the data?

Line 148: this has been mentioned in the previous paragraph.
Line 150: what is the reference genome that was referred to? State the ref genome
Line 151: ‘..tophat2 tools soft were…’. Please revise this sentence.

In the differential expression analysis section, the authors did not elaborate on the cutoff point of the fold change of DEG. Does this mean that there’s no cut-off point of FC, why is this so?

Line 175: need to add a citation/reference for KOBAS software.

Statistical analysis: which experiment utilized the ANOVA by Excel? Some of the researchers do not recommend the usage of Excel for ANOVA analyses due to the highly error prone analysis as it requires a unique data format for the built in analysis. Suggest to use other software such as Graphpad prism/spss.

What about the validation of RNAseq expression by qPCR?

How do you compare the DEG between resistant and susceptible cucumber varieties infected with S.fuliginea (ST vs RT)? Do you straight away analyze the ST data to RT data? According to the results, there were 97 DEGs in the control groups (SC vs RC). The method of comparison is not clear in the methodology section.
Validity of the findings: Results were presented thoroughly. However, there are a few queries:
1.	Line 188: Table S1 showed a total of 280.92 million trimmed reads. But in Table S2, the reads that are mapped to the reference genome is a total of 561.8 million reads. Assuming that this is the untrimmed reads, how do you explain this?

2.	Line 192: Please elaborate in the method section on why you chose to annotate the DEG against ~ 7mentioned platforms.

3.	Line 218-223: the details of clustering were not explained in the methodology.

4.	Line 233-224: some of the results were only explained briefly, for example in line 233-224, I suggest to at least state a few of the gene names for ‘flavonoid biosynthesis’.

Discussion could be improved:
1.	For example in line 288-294, the discussion was very brief and too general. Instead of stating the pathways alone, I suggest the authors to mention a few genes that are worth to be highlighted in the study. Please rephrase the sentences to link the previous studies mentioned to your results.

2.	Line 342-346: too brief. How does the PR genes ‘play a role in pathogen resistance?’, please elaborate with mechanism and again mention a few genes from your study.

Conclusion: no need to mention the method in conclusion.  Please summarize the findings without being too general. In the last sentences (352-353), it is good to state the importance of ‘analyzing potential genes related to PM resistance.’
Additional comments: In this study, Zhang et al. have reported the differential gene expression of resistant BK2 and susceptible H136 cultivated cucumbers under S.fuliginea infection. The abstract is only briefly described, especially the methodology and results section. The author needs to mention specifically the main highlight of the study. Furthermore, some of the sections in the body of the manuscript are not explained thoroughly and are confused to read. This manuscript needs to be proofread before submission. Above are the major points that need to be addressed to improve the quality of the study.


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

Input:
Title: COMPARATIVE TRANSCRIPTOMIC ANALYSES OF POWDERY MILDEW RESISTANT AND SUSCEPTIBLE CULTIVATED CUCUMBER (CUCUMIS SATIVUS L.) VARIETIES TO IDENTIFY THE GENES INVOLVED IN THE RESISTANCE TO SPHAEROTHECA FULIGINEA INFECTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: Authors addressed my concerns properly, but there are few things authors need to pay attention in the updated version of manuscript.
1> the expression of log2 fold change is confusing, you could either say absolute log2 fold change or |log2 fold change|
2> the statistical significance of P value reported by DESeq is original p value or adjusted p value?
3> For GO analysis, you should also report GO accession for each term
4> L148, L154, what does the "f" in "average length f 300bp" mean?