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

Input:
Title: A LONGITUDINAL STUDY OF THE IMPACT OF COVID-19 RESTRICTIONS ON STUDENTS’ HEALTH BEHAVIOR, MENTAL HEALTH AND EMOTIONAL WELL-BEING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is clear and unambiguous. The background provided allows the reader to understand the overall situation during COVID in Florida and at Florida Gulf Coast University. It is well referenced throughout to support what is written. The structure of the article and the tables/data are clear. The results are detailed and address the hypothesis.
Experimental design: The research question is well defined, relevant and timely. The study provided needed information about the behaviors, health and wellbeing of college students during COVID-19 using a comparative study with pre-COVID 19 data. The authors intent to continue to chart students' journeys longitudinally will provide further essential detail on this topic. Publishing this article will allow others to replicate the study in other contexts. This is essential as each state in the US and countries globally have been impacted in different ways.
Validity of the findings: All underlying data has been provided and the finding/conclusions are well stated and link to the original research question around the behaviors, health and wellbeing of college students pre-and during COVID-19. They are clearly outlined and easily accessible for the reader to understand.
Additional comments: I think this is an excellent, relevant and timely article. The topic of the study is essential. It will be interesting to see the development in the study authors continue to further investigate the topic. On a personal note, I think it would be interesting to see a comparison of what has been done here to a state that had stricter lockdown measures/restrictions, such as California.


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

Input:
Title: A LONGITUDINAL STUDY OF THE IMPACT OF COVID-19 RESTRICTIONS ON STUDENTS’ HEALTH BEHAVIOR, MENTAL HEALTH AND EMOTIONAL WELL-BEING
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript provides unique information on how health outcomes and behaviors of young adults have changed from pre-pandemic to during the pandemic. In particular, the study data incorporates experiences of young adults that were collected over a one-year period of the pandemic whereas most prior studies have focused only on the initial few weeks or months of navigating precautions to slow COVID-19 transmission in the U.S. The methodology of the study reported in this manuscript has a number of limitations with regard to generalizability, but nonetheless publication of the results will make an important contribution to the literature. The authors should also be commended for the thorough review of literature related to their study results and the manner in which it is summarized to put their results into context. I have some suggestions for strengthening the manuscript with regard to presentation of the results and the clarity of some points made in the introduction and discussion. The authors do a strong job of discussing limitations of their data.
Lines 59-64. The text is a run-on sentence and needs editing to improve clarity.
Line 141. Need to spell out the acronym the first time it is used.
Lines 429-431. The reference comparison makes the sentence a little confusing.
Table 1. Please add titles for each column.
Table 2. Please add data to show effect size.
Experimental design: Lines 388-390. The overall low response rate should also be mentioned here.
Validity of the findings: Lines 210-213. The difference should be described more clearly in this sentence and the directionality of the difference should be indicated. It is not clear why the authors refer to a proportion. Similar issues with language occur throughout the results.
Line 262-264. This is another sentence that lacks directionality with regard to the reporting of differences. The entire results section should be reviewed to edit for clarity.
Lines 266-268. This line reads very well and is easy to understand – an example of how it would be nice to similarly reword other text in the results.
Lines 316-319. It would be helpful to start to the discussion with a brief restatement of the research questions and a summary of how the results are novel. This sentence does not describe the directionality of changes.
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: A LONGITUDINAL STUDY OF THE IMPACT OF COVID-19 RESTRICTIONS ON STUDENTS’ HEALTH BEHAVIOR, MENTAL HEALTH AND EMOTIONAL WELL-BEING
Review round: 1
Reviewer: 3

Output:
Basic reporting: This reviewer has a few comments on Basic Reporting of the manuscript:

1) Currently within the Methods – subtitled COVID-19 related restrictions in Florida – Please clarify the years for each Phase of the state orders. Additionally, this information (related to the Florida orders) would be better fit in the Introduction section of the manuscript to offer context to the restrictions experienced by the target population.
Experimental design: This reviewer noted a number of comments and questions related to the methodological and design of the present study. Most importantly, the Methods section should be reorganized to follow standards similar to other observational studies of mental health. This reviewer suggests including relevant subheadings such as “Study Design”, “Participants”, “Measurement” to improve the clarity of the manuscript. For example, the Measurement subheading should include detailed information about the outcomes selected and reported in the Results section without having to jump to a table/Appendix.

1. Methods line 129 – would clarify that it is a bi-annual survey.

2. Response rate of 6-10% (line 153): This is a fairly low response rate – how do responders are different from non-responders?

3. Methods line 141 – Please add information what the acronym FGCU means.
     Note: it is clarified later (line 160), but it should be added to the first time the acronym is introduced.

4. Lines 162 – 165: This demographic breakdown is interesting, but does not need to be discussed in this section. This reviewer would be more interested in findings related to how the analytic sample compares to the overall breakdown of the school (particularly with respect to responders and non-responders).

5. This reviewer suggests adding more information about how the survey was modified, and to what extent.

6. Were the samples the same cohort who were measured twice? Or were they two unique samples? It would be important to clarify this for the reader. Additionally, it would be important to note, if these were two unique samples, how they differed.
6a. If these are two unique groups, and the respondents were not matched across surveys, this should be revised to indicate that this is a repeated cross-sectional study that was measured at different timepoints and compared.
6b. If these surveys were matched to participants, the analysis needs to take into account the violation of independence and will need to be revised to account for this within-subject correlation.

7. How are the two groups defined? Are all surveys conducted prior to the COVID-19 pandemic included in Group 1?
Validity of the findings: The authors of this manuscript should temper the language of the implications and conclusions of these findings given that the study is not longitudinal, but rather (as presented) is a repeated cross-sectional study (i.e., the same survey measured with different samples). This is a major limitation of the current analysis and should be addressed thoroughly. This authors highlight the longitudinal nature of the study as a strength, but the analysis does not match this proposed design.

Results:

1. Line 204: Since the authors are comparing to the general student population, it would be useful to know the percentage of males and females in the sample (in addition to the reported statistical significance).

2. What is the definition of physical activity in the context of this study? Could be a result of less access, e.g., Pre-COVID cohort had access to gym but also could report increased physical activity because of in-person learning.

3. Please report results (percentages, means/SD, p-values) in-text, rather than referring to the Tables.

Discussion

1. Lines 334 – 338: Do the authors have additional data about this rationale? If so, this reviewer suggests adding this information to the Results. If this data is not available, this reviewer suggests tempering the proposed reasoning as it is outside of the scope of the current study.

2. Line 339 - 340: This reviewer is uncertain how the authors drew this conclusion based on the information presented.

3. Line 362: Given the cross-sectional nature of the data, the authors are unable to make a conclusion about the impact on food consumption and should revise the language of the manuscript to align with the scope of this work.
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: A LONGITUDINAL STUDY OF THE IMPACT OF COVID-19 RESTRICTIONS ON STUDENTS’ HEALTH BEHAVIOR, MENTAL HEALTH AND EMOTIONAL WELL-BEING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The text of the results section remains somewhat confusing. The authors were modestly responsive to requests with regards to improving ambiguous language, but the directionality of findings is not consistently indicated and the language regarding percentages is awkward in multiple sentences. It is very difficult to interpret sentences such as "The proportion of respondents reporting average hours of sleep over the past seven days differed between the pre-COVID group and the COVID group (Table 3)." The results text describing vaping, alcohol, and marijuana use is an example of strongly written and unambiguous language but the authors did not completely eliminate problematic language within other sections of the results. The authors provide some estimate of the magnitude of changes in behaviors with regard to substance use (i.e., getting at effect size) and for some other health outcomes but this is not done consistently throughout the results.
Experimental design: The details of COVID-related orders in Florida and at the FGCU is somewhat excessive and the key aspects of these orders is missing as context in framing the research questions within the introduction.

The authors were not responsive to requests to clarify language regarding the study design. The discussion text needs extensive editing to improve the interpretation of results based on a repeated cross-sectional survey design vs longitudinal design.
Validity of the findings: The conclusions are difficult to understand because they reference the severity of outcomes reported by other studies and no direct comparisons can be validly made to the effect sizes reported within the current study.
Additional comments: No additional comments


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

Input:
Title: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors have used clear and professional English throughout the manuscript.
Experimental design: The experimental design is standard.
Validity of the findings: The study is comprehensive and provides all underlying data.
Additional comments: Dear Authors,
Congratulations for this interesting study. The manuscript by Jia et al. reported the genomic profiling and expression analysis of the 20 DGK gene family members from the wheat genome. The manuscript is well written. The results and conclusions of the papers are interesting and convincing. However, I found some concerns that need to be addressed before considering this manuscript for publication.

1.	I am wondering about the purpose of this study. Most of the previous studies with different plants reported the similar results. The authors could have discussed why this study is important and how these findings added significantly to the already established facts about the DGK gene family.
2.	The introduction part is well written but I think the authors should include more details about the DGK family isoforms, classes and structural insights. The authors should add – how the stresses involve important signaling molecules related to diacylglycerol metabolism.
3.	Methods have been described with sufficient details but I think in the “Plant transformation” section clarity is missing. Authors should add more details to it.
4.	The authors have primarily used the BLAST program to identify the TaDGK genes. How did the authors make sure that they are not missing any TaDGK genes which are distantly matching to Arabidopsis and rice gene queries?
5.	What criteria the authors have followed to shortlist the four genes for expression analysis? Why were no genes from cluster III considered?
6.	In the phylogenetic analysis the DGK genes from plants like Malus prunifolia, Brassica rapa, Cicer arietinum, Coffea canephora, Solanum tuberosum, Sorghum bicolor etc were missing. Any explanation for these exclusions.
7.	The Figure 4 (multiple alignments) should be placed prior to the figures related to phylogenetic analysis (figure 2/3) in the text.
8.	Kindly move figure 2 or figure 6 to the supplementary figure section. They are basically depicting the similar results. And it is hold true in between figure 1 and table 1.
9.	The genes used for RNA-seq analysis (figure 8A) must be used for tissue specific expression analysis (figure 8B).
10.	The qRT-PCR results of TaDGK genes under salt stress and drought conditions (figure 9) showed that gene expressions level after 24 h were lower than 12 h. What is the possible explanation? Why is gene expression after 12 h is maximum for all the genes studied, why is it dropping when the authors increase the treatment time? Please discuss this down-regulation in the discussion section. I could not find the expression level after 48 h of treatment in the figure but it is mentioned in the figure legend.
11.	Please provide the NCBI accession number of all the 20 TaDGK genes.
12.	Why were only two genes from the same cluster used for subcellular localization study? At least one gene from each cluster should be used.
13.	The “Result and Discussion” section should be improved. The authors should discuss the findings instead of only reporting the outcome of the experiments.
14.	For the salinity and drought treatments, 200 mM NaCl and 20% PEG6000 was used respectively. How were these concentrations fixed at these levels? Did the authors perform concentration gradient experiments? The citations should be mentioned if the authors followed any previous reports.
15.	Please discuss about the wheat BT009326 gene. Is it related to any of the 20 genes discussed here?

Minor Comments:
1.	Line 34- Change the parentheses style for phosphatidylinositol 4,5-bisphosphate. It should be [PI (4, 5) P2].
2.	Line 54 and 55 need a citation.
3.	Line 71- Please provide the web address of BLASTN.
4.	Line 88- Please provide the web address of Evolview tool.
5.	Line 117- Please number the tables according to their mention in the text e.g. here S5 should be S1.
6.	Line 119- Please italicize “Nicotiana benthamiana”.
7.	Line121- Please correct the enzyme writing style e.g. no space between “Spe” and “I”.
8.	Line 127- Please italicize “Agrobacterium tumefaciens”.
9.	 Figure legend of figure 3- The number instead of “he number”


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: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: This manuscript described by Jia et al. performed genomic profiling and expression analysis of the diacylglycerol kinase (DGK) gene family in wheat. They identified 20 TaDGKs and found that several TaDGKs were strongly induced by salt and drought stresses. Because many DGK genes have been already identified in several plants including Arabidopsis thaliana, rice, tobacco and soybean, the findings in this manuscript are substantially novel. However, their findings will be added to the database of DGK in plants and provide some insights into the putative functions of plant DGKs.

There are several concerns that should be addressed by the authors (generally lack of explanation).
(1) Line 177–185: Are exon/intron boundaries of catalytic domains conserved among all TaDGKs?
(2) Line 192: What are the motifs? Are the motifs related to domains of DGK proteins (e.g., catalytic and C1 domains)?
(3) Line 209–224: There are some contradictions between microarray-based expression pattens and those obtained by real-time PCR (e.g., TaDGK3A: roots (microarray) vs. stems (real-time PCR). The authors should discuss.
(4) Line 229: 10 min is not correct. This would be 6 min (0.1 h in Fig. 9).
(5) Line 231 and 236: The authors mentioned the results (increases) after 12 h (Fig. 9A and B). However, the values were decreased after 24 h. Why? The authors should discuss.
(6) Line 239–240: The authors compared expression pattens of TaDGKs under abiotic stresses with only TaDREB2. In addition, the authors should compare with DGKs in other plants.
(7) Fig. 3: What is the pink domain?
(8) Fig. 10: What is DIC? Where are the nucleus and cytoplasm? The authors should indicate them with arrows.


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: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 1
Reviewer: 3

Output:
Basic reporting: - The article provides sufficient literature, but adding suggestions regarding the application of the findings would be helpful. It is important to identify the significance of these findings/work.

- Figure 1: Chr7D and TaDGK7D are missing in the figure
- Figure 2: What do the values mentioned in the tree branches signify?
- Figure 3 legend: Line 2 - "T" is missing in the first word
- Figure 5: Labels for each colored box (blue, light green, dark green, black) can be listed
- Figure 6A and 6B are the same as supplementary fig 1 (fig S1) and therefore redundant.
- Figure 8B - the purple color box is denoted for "Ears" but in the figure legend its mentioned as "spikes"
- Figure 9: Since the qPCR values are compared to 0hr data (it's understood by default that the relative expression level at 0hr is 1), authors can remove the 0hr time point data from the graph.
-Figure 9: Figure legend mentions the 48hr timepoint but the data is absent from the bar graph
- Figure 10: scale bar is missing, labels should be outside the images
- Figure 10: figure legend mentions the brightfield image but the images are labeled as DIC. The author needs to correct this.
Experimental design: Methods section
2.4 RNA seq data: (line 97-99) Authors need to cite appropriate references and the study IDs (if any) for the three datasets used.

2.6 RNA extraction: Line 112-113. The first line mentions that total RNA was extracted, but in the second line gDNA is mentioned. The authors need to correct this method section.
Also, details on methods used to calculate the relative expression levels need to be described (2^-ΔΔCT method?)

2.7 plant transformation: Authors need to cite the reference, as the protocol for transformation is not described in detail.
Validity of the findings: Authors need to provide details in the method section regarding the statistical test/s applied for the qPCR data.
Additional comments: Authors identified 20 diacylglycerol kinase genes, an important enzyme in the Phosphatidylinositol cycle in the hexaploid wheat. The authors performed a detailed bioinformatic and expression analysis of the genes and showed that they were significantly induced under environmental stress of salinity and drought. This is an interesting work that is well performed with all the raw data organized in the supplementary files.

Major comments:
1. Identification, chromosomal location, and gene:
- Line 77: some DGK genes lacking catalytic domain are ignored - authors should list the number and name of genes ignored. Can authors also comment on why the following genes were not included in the analysis
TraesCS7A02G297200 - Chr7A
TraesCS7B02G198600 - Chr7B
TraesCS7D02G291600 - Chr7D
TraesCS6B02G428500 - Chr6B

- Did authors name the genes (TaDGK1-6A/B/D, TaDGK7B/D), or were they already named? It looks like the naming system is based on chromosome number. It is confusing since TaDGK4A/B/D and TaDGK5A/B/D are all on chr 5 and not on Chr 4. If the authors have named the genes, the naming should be corrected (i.e for eg. TaDGK5A1 and TaDGK5A2)

 2. Subcellular localization
- The authors mention that TaDGK2A and 3A are localized in the cytoplasm. But looking at the images, it seems the protein is present in the cell periphery (cell membrane). The authors should explain this.
- TaDGK2A in addition to its localization in the cytoplasm, is also localized in the nucleus. It would be good to confirm the nucleus localization with DAPI and its colocalization with TaDGK2A-GFP.

Minor comments:
Line 13: Full form of PI
Line 22: "express" word can be deleted
Line 36: "These derivatives and catalytic enzymes play....." - Authors should mention which catalytic enzymes?
Line 107: Timepoint 1/2 or 0.5hr is missing. Timepoints for salinity and drought stress needs to be corrected throughout the paper and supplementary files. At some places it is mentioned as 0.1hr,0.5hr, etc., and at others, as 10min, 30 mins. Just make sure 10 min is not the same as 0.1hr.
Line 135-136: Is it 14 chromosomes or 17 chromosomes
Line 169 and figure 4A: The authors mention ATP binding site with a GXGXXG consensus sequence (red box of fig 4A). But the red box in the figure does not match the GXGXXG consensus. This needs to be corrected.
Line 217-240: Why were only genes TaDGK2/3/4/5 selected for qPCR and not TaDGK1/3/7?
Line 254: what do authors mean by "its close relatives"?
Line 254-261:  As stated earlier, it is important to add 1-2 lines on the application or use of this data and/or future work.


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

Input:
Title: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have used clear and professional English throughout the "revised manuscript". Authors have provided proper literature references.
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The authors have satisfactorily addressed all of the concerns raised by me. The "revised manuscript" is now in acceptable form. Congratulations to the authors for this nice Job.


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

Input:
Title: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have modified the manuscript accordingly to the suggestions proposed, and this has improved in clarity. I have no more issues to address.


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: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 2
Reviewer: 3

Output:
Basic reporting: Line 126: Please change ‘ware’ to ‘were’.
Line 138: Change ‘cdna’ to ‘cDNA’.
Line 170: BLAST cannot be used as a verb. Please modify the sentence that uses ‘blasted’.
Line 176: Change ‘was lost’ to ‘were lost’. Change ‘befor’ to ‘before’.
Line 193: Remove repeated phrase ‘as in other studied plants’.
Line 237: Expand IBS, either here or at first usage.
Figure 1, Please replace ‘Clustel’ with ‘Cluster’ in the color key at the bottom of the figure.
Lines 183-184: Move this sentence to the end of the paragraph. Otherwise it seems that the following 2-3 sentences are about BT009326 and TaDGK2B.
Line 634, Figure S2 legend: I believe Fig. S2B is only wheat DGKs. The legend says wheat and rice.
Fig. S3: (A) and (B) labels are missing.
Fig.6: Main figure should stand alone without having the need to refer to supplementary material. The meaning of each capital letter should be provided here in Fig. 6 itself in a small table.
Line 266: Table S7 is cited here while it does not show the data explained here.
Experimental design: Line 144: 2ΔCt should be 2^-ΔCt and 2-ΔΔCt should be 2^-ΔΔCt
Validity of the findings: Lines 259-261: What could be the reason for ABA-mediated repression of TaDGK2,3, and 6 when there is no ABA-sensitive motif (A) found in the upstream region of these genes?
Additional comments: The manuscript should be edited again for any grammar/spelling mistakes. I have suggested a few changes, but there could be more.


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: GENOMIC PROFILING AND EXPRESSION ANALYSIS OF THE DIACYLGLYCEROL KINASE GENE FAMILY IN HETEROLOGOUS HEXAPLOID WHEAT
Review round: 4
Reviewer: 1

Output:
Basic reporting: NA
Experimental design: NA
Validity of the findings: NA
Additional comments: I carefully read the revised version of this manuscript. In the revised version the authors have carefully introduced the changes suggested by the section editor. I found this manuscript can be now published.


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

Input:
Title: COMPARISON OF AFRICAN SAVANNA ELEPHANT (LOXODONTA AFRICANA) FATTY ACID PROFILES IN WHOLE BLOOD, WHOLE BLOOD DRIED ON BLOOD SPOT CARDS, SERUM, AND PLASMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper reports on a method comparison for fatty acid measurement in African elephants. The language is clear and professional, the structure is fine, the raw data is supplied. The results are relevant. In my view, one citation of the 2003 elephant paper of ours is not adequate (as we do not compare with our own "free-range" data in there, and as its results are included in the 2007 review that is also cited), whereas another FA comparison paper of ours (from 2008 on black rhino) would be.
Literature supporting the interpretation that two fatty acids are especially susceptible to elution or inclusion of cells in the sample should be given, or at least a logic why these and not others. Without such a logic / literature, I recommend not to give these hypothetical explanations.
I think there are only minor revisions necessary for the language and flow of narrative, incl. a deletion in the discussion to avoid unnecessary content duplication, and a change in the footnote format for the results table.
Most importantly, the authors must mention that they reported on fatty acid levels in these animals (and from these samples?) already in their 2020 Zoo Biol paper. This is not a problem to the present study, as the method comparison was not part of that 2020 manuscript. However, NOT mentioning this in the Introduction equals concealment; I urge the authors to mention this upfront, incl. a statement of to which results of the 2020 paper (i.e., to which season) the current samples correspond, or, if the present study used samples from the same animals that were not used in the 2020 manuscript, then state that. As a note on the side, this is a habit I am seeing more and more in papers I review and I am not sure whether this is a new writing style - not mentioning that one has done very similar work, or work on the same samples or the same experiment, in the Introduction.
sincerely  marcus clauss
Experimental design: In my view, the study is well within the aims and scope. The question is well defined and meaningful, and the technical standards appear up-to-date to me. I think that the description of statistics requires some modification, but not a data re-analysis.   mc
Validity of the findings: The findings are valid. The underlying data are provided and correspond, in random checks, to the data in the results table. Conclusions are well stated.  mc
Additional comments: I made comments in the provided word documents. If will try to attach them. If that is not possible, I will combine them into one pdf and attache that. sincerely   m clauss


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

Input:
Title: COMPARISON OF AFRICAN SAVANNA ELEPHANT (LOXODONTA AFRICANA) FATTY ACID PROFILES IN WHOLE BLOOD, WHOLE BLOOD DRIED ON BLOOD SPOT CARDS, SERUM, AND PLASMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The researchers explored the usefulness of a dry bloodspot method to determine the serum fatty acid profile of (African savanna) elephant serum. This was compared with other whole blood sampling methods. The application of DBS lies in post sampling storage and handling prior to analysis.
The document was well written and no linguistic errors were detected.
Experimental design: The experimental design is acceptable. Methods used were state of the art. The work was carried out with care. The data was presented in an accepted and interpretable manner.
Validity of the findings: The data was presented in an accepted and interpretable manner. Specific care was taken not to over-interpret the statistical data.
Additional comments: Little information is available on the biochemistry of elephants. New information is presented that contributes to elephant biology.
The document may be published as is.


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: SPATIAL AND TEMPORAL DISTRIBUTION PATTERNS OF TICK-BORNE DISEASES (TICK-BORNE ENCEPHALITIS AND LYME BORRELIOSIS) IN GERMANY
Review round: 1
Reviewer: 1

Output:
Basic reporting: Cunze et al. gathered spatial and temporal patterns of reported cases of
human TBE and LB and some associated factors, in order to find some association between these factors and curve of human TBD cases. Figures, the language, editing are good.
The authors overtook a heavy burden as lot of researhers tried to explain epidemiological data of TBDiseases by some associated factors, but they could not. This is also the case with this paper. Although the manuscript is valuable and worth publishing, but the aimed goal is unaccessable, as TBDs are under preassure of very complex effects of lot of known and still unknown factors.
Experimental design: Other factors (more game animals, migratory birds) could have been included in the study, see later. Otherwise the plan, figures,  are Ok.
Validity of the findings: The authors give interesting details of the LB, TBEV epidemiological processes of the last decade in Germany, which are valuable for researchers, but real findings i.e. association of some of the studied factors to LB TBEV epidemic data I think is missing. As such association have never been done by others so far.
Additional comments: Introduction.
Reading the introduction I could not decide whether this paper was planned as an informative review article for the public, or for collegues from the TBD scientific field. The introduction is full of data which are known and obvious for researchers. When we are citing we must cite those authors which described first the cited data. e.g. bimodal pattern of tick activity was described decades earlier than Estrada-Pena et al. (otherwise one of our ongoing study of ticks of hedgehogs detected 5-6 small peaks for all tick stages, not 1 and not 2). The thing is not so simple as bimodal or unimodal. I would suggest the authors review papers of Mandl et al., 2008, Süss 2003 Vaccine 21, 2011, Gray 1991. (and several others).

Reference Mehlhorn H. 2012. For me, who does not speak German such citations are not understandable, unaccessable, and as such, does not exists. Cite papers avaiable online with minimum an English abstract. Especially when the cited things were described earlier by others, cite the previous author(s).

row 84  Lucius et al., article is missing from the reference chapter

row. 112. I am a little bit doubtful, whether an adult female could spent minimum 2-3 days on a human being to infect him/her with LB spirochaete, whitout being noticed. Human LB infections must be made mostly by nymphs, whose bloodmeal is much faster, and could be remained unobserved throughout their biting. The same for TBE with larvae.
115-116. Larvae can transmit TBEV, even in the first minutes of biting. Could have been mentioned here.
120-121- „plus human beings nearby” is also necessary
Figure 3. It is not easy to evaluate epidemiological processes with lack of 2/3, or half of the necessary data.
254-256- The two illnesses are parallelly move. Should be mentioned underlined, and think of it.
260-261- why the two brackets are necessary here?
267- small mammal host species are also almost inactives.
303- …. is repeatedly suspected, „but still not proven” .
312--313 With both disease subclinical infection exist, especially with TBEV. The reported data is only the number of infections with symptoms. + problem of standardized reporting, different laboratory (detection methods) background make most of our epidemiological data not reliable. We could draw other conclusions if we knew the real number of infected humans.
323-324.- Red voles seems the most important small mammal vector especially for TBEV. Please see papers below, and there are some others, among them articles from German authors.
331 Oh dear, not dear but probably deer
334-335 it is not true. see previously
335- co-feeding. On the base of a badly written, unnatural, laboratory, forced paper of Labuda et al, 1993 Vet. Med entomol.  (the worst paper I have ever read) there is no proof for co-feeding process in nature in case of TBEV or LB transmission (and probably it is also true for other TB agents). This fairy tale successfully were smuggled into hundreds of brains, and researchers (who never did anything with co-feeding just read about it) cite those who cited others. Finally this co-feeding idea came into fashion, and accepted without any proof. No independent author studied the phenomenon in nature, so the whole theory is still a theory. Personally after capturing hundreds of small mammals in nature, and sampled them for ticks, no co-feeding ticks were found, or seen. Some authors (S Randolph ) even writes, that biting viremic hosts is negligable in nature comparing to the role of the miraculous co-feeding (her enduring paradigm). We should forget this co-feeding, till somebody, independent authors really prove it. Please mention this co-feeding if You have any proof that this theory exists at all.
340-342- competent not competent. Large mammals like red deers are able to move 60 km a day. By transporting engorging females deers are able to create new foci, at areas where noone expects ticks, or tick-borne pathogens. Role of large mamals in TBdiseases is not only feeding females but to spread them.

General comments:
What I missed.
migratory birds. What about the role of migrating birds in TBD foci in Germany. It has been shown that migratory birds carry not only ticks, but TBD infected ticks. REF Hasle, Wanderström. Recently TBEV strains (with almost identical genome sequences) were isolated in Bayern (XY), Finland (XY) and in Hungary (Egyed et al., 2018). All the three isolation sites were waterfowl habitats, and probably bound by migrating birds, as Hungary is the main resting place of Central-East-European migrating birds crossing the Bosporus. Are there places in Germany where large body sized migrating birds (storks, cranes, Anseriformes) accumulate, rest? How these places are related to the human TBD cases?

females/males. What about the sex/gender of human patients in Germany? In a similar brother-paper from Hungary (which should have been read and cited) 70% male dominance was found among TBEV patients, but a slight female majority 54% among LB patients. Is this similar in Germany, or not? For both, what could be the reason?
Zöldi, V., Juhász, A., Nagy, Cs., Papp, Z., Egyed. L. 2013. Tick-borne encephalitis and Lyme disease in Hungary: the epidemiological situation between 1998 and 2008. Vector borne and zoonotic diseases. 13(4):256-265. doi:10.1089/vbz.2011.0905

why wild bores? Selecting only one (2) species could be misleading. Wild ruminants especially roe deers probably are better hosts for ticks than wild pigs. Roe deer live in a relatively small area (in contrary to boars) lies a lot on the ground regularly, daily, in the grass or in remote bushy areas. In my country we have 1.2 boars /km2, but 4 roe deers/km2. From your country Król et al., 2020 Parsitol res) roe deer was also found as good target for tick infestation,  Haut et al, 2020 Microroganisms) found correlation from German red foxes and human TBEV cases. An Italian work also found corelation between roe deer abundance and human TBEV cases but these case swere not corelated with any climate factors tested (Rizzoli et al., 2009, Plos One). Swedish authors found positive correlation between TBE human cases and population densities of roe and red deers and hares, but not with fallow deer, elks, red fox, lynx and wild boar. So densities of certain game animals are correlate with human TBD cases, others not. Similarly, more (or ruminant) wild species should have been studied instead of wild boars. It would have been interesting to compare German and Swedish data.
Jaenson, GTT., Petersson, H.E., Jaenson, G.E.D., Kindberg, J., Pettersson, H-O.J., Hjertqvist, M., Jolyon M Medlock, M.J., Bengtsson, H., 2018. The importance of wildlife in the ecology and epidemiology of the TBE virus in Sweden: incidence of human TBE correlates with abundance of deer and hares. Parasit Vectors 11(1):477. doi: 10.1186/s13071-018-3057-4.

What about vaccination (TBEV) of German people? What percentage of them are vaccinated and in which provinces (states)? Vaccination of the population against TBEV is a decisive tool to decrease TBEV human cases, and to change completely the epidemiological situation. In two European countries (quite close to Yours) vaccination almost annihilated the TBEV epidemics. In my country the 3 hospitalized TBEV patients/100000 inhabitants after 1995-96 was sharply dropped to 0.2-0.6 due to a large-scale vaccination champaign.

Figure 3. It is very difficult to draw reliable conclusions, when half, 2/3rd of the neccesary data are missing. If all provinces of Germany would release their LB data some time in the future this work should have been repetead probably with better results.

Climate change 126-128- in our published papers (I not cite them) we never found correlation between abundance, activity of ticks, and any weather conditions (min. max. mean daily temperatures, rain humidity etc.). Jaenson et al + Rizzoli, and lot of others (above) also could not. Someone can assume lot of things.
Like with co-feeding authors should be more careful with this climate chance-tick abundance moving Northward idea. No black and white proof for that is exists. During an EU project (2005-10) dozens of TBE researchers from Europe believed it, and tried to prove it. Finally the project leader must admitted, that altered human behavioural habits, leisure-time activities (mushroom picking) is much probable reason for changed human TBEV epidemiological data, the increased human cases than climate change (Sarah Randolph 2010, You cited). Even Your own data and sentences, rows 232-233, 236-238 (cold does not has effect on human TBD cases) rows- 296-298 298-301 (warm also not) admit, and what is assumed, does not work. If climate change (warming) would increase human TBEV cases or tick abundance, moving northward etc. in the Nothern half of Germany TBEV would not be almost unknown. (Your Figure 2).
Even the references You cited (Semenza and Suk, Medlock) are full of assumptions. There are no proven effect of climate change onto the objects of this manuscript (TBEV, LB, ticks ). There is no need to wash together things which are not belong together (mosquitoes and ticks, West Nile/chikungunya and TBEV/LB).
The authors should indicate both in Abstract and in the Discussion chapter, that correlation between human TBEV, LB human cases and any of the tested climate factors were not established.

VOLES

Zöldi,V., Papp, T., Reiczigel, J., Egyed L. 2015. Bank voles and adult rodents show high seropositivity rates in a natural TBEV focus in Hungary. Infectious diseases. Mar 2015, Vol. 47, No. 3: 178–181.

Michelitsch A, Tews BA, Klaus C, Bestehorn-Willmann M, Dobler G, Beer M, Wernike K. In Vivo Characterization of Tick-Borne Encephalitis Virus in Bank Voles (Myodes glareolus). Viruses. 2019 Nov 15;11(11):1069. doi: 10.3390/v11111069.

Grzybek M, Tołkacz K, Alsarraf M, Dwużnik D, Szczepaniak K, Tomczuk K, Biernat B, Behnke JM, Bajer A. Seroprevalence of Tick-Borne Encephalitis Virus in Three Species of Voles (Microtus spp.) in Poland. J Wildl Dis. 2020 Apr;56(2):492-494. doi: 10.7589/2019-02-048. Epub 2019 Dec 27.

Tonteri E, Kipar A, Voutilainen L, Vene S, Vaheri A, Vapalahti O, Lundkvist Å. The three subtypes of tick-borne encephalitis virus induce encephalitis in a natural host, the bank vole (Myodes glareolus). PLoS One. 2013 Dec 13;8(12):e81214. doi: 10.1371/journal.pone.0081214. eCollection 2013


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: SPATIAL AND TEMPORAL DISTRIBUTION PATTERNS OF TICK-BORNE DISEASES (TICK-BORNE ENCEPHALITIS AND LYME BORRELIOSIS) IN GERMANY
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript describe a well conducted and informative analysis of the spatial and temporal variation of two diseases, Tick-Borne Encephalitis (TBE) and Lyme Borreliosis (LB), in Germany and some possible underling drivers. The manuscript is well written and clear, and figures are nice. The authors provide the necessary background for the study, so that, even non-specialists in the system can understand it.

The only important issue, but easy to solve, is that some statements that are assumptions of the study are provided in the introduction without proper references. See this and other minor suggestions bellow.
Experimental design: The study is well designed, the research question is well defined and relevant.
As far as I can tell, the data is appropriate for the question addressed, the analysis is well conducted, resulting in meaningful results.
Validity of the findings: My main criticism to the manuscript is that the discussion is a bit disconnected from the results presented.

The study did not find a relationship between climate and other factors and the temporal variation of the diseases cases. But most of the discussion is written as if the pattern was detected. Because of this disconnection, the reader might feel that the absence of pattern is an unwelcome finding. And that is unfair to the good study described in the manuscript.

The authors should, of course, present other perspectives, acknowledge the limitations of their study and try to explain why the results did not match the expectations. But the discussion must be mostly focused on what they found, not on what they expected to find.
Additional comments: Specific suggestions:

Line 35 – As several mild winters did not resulted in increased case numbers and cold winters did not resulted in decreased case number, linking the low case numbers of 2012 with the previous cold winters is a too unsupported speculation.

Line 42 – The conclusion of the study should be that, given the analyzed evidence, weather conditions did not influence the diseases’ prevalence.

Line 52 – Probably a typo here.

Line 112 – I suggest reformulating the sentence to separate what is known, from what is the possible consequence, for instance “Being more numerous than adult ticks, nymphal ticks are responsible for approximately 80% of tick bites in many areas (Heyman et al., 2010) and, thus, we may expect them to cause most of LB cases”.

Line 114 -115 - These are too important information to be provided without references.

Line 122-128 – These are the main assumptions that justify the study. Authors should provide references for them.

Line 130 – The prevalence of the pathogens depends on the prevalence of the pathogens (?)

Line 132 – It is assumed by whom? If this is a common approach, the authors should provide references. Otherwise, the author should state “Here, we assume …” and justify why their assumption is reasonable.

Introduction - The introduction is very well written and is accessible for non-specialist in LB and TBE diseases, and even, for non-parasitologists. All the important information is provided. An easily solvable but important weakness is that essential assumptions for the study are provided without references. Also, I strongly suggest the authors to include a last paragraph on the introduction explicitly describing the aim of the study and briefly explaining the study design. This paragraph will help readers to interpret the material and methods and benefit the flow of reading. Such a section is present in most of PeerJ recently published papers.

Line 138 – An improved version of this paragraph could be the last introduction paragraph.

Line 206 – What do the authors mean by that? Are there different regulations on reporting between federal states on the North and on the South of Germany? Do they suspect that these regulations are the causes for more reported cases in the south? Otherwise, I strongly disagree with this sentence. The spatial pattern (South vs North) is absolutely clear.
Maybe what the authors mean is that it is difficult to assess the spatial pattern in a smaller geographical scale (e.g., among administrative districts within each Bundesland). Anyway, this sentence is unnecessarily undermining their findings.

Line 213 – Here, for LB, the sentence in line 206 make sense.

Line 215 – Incomplete sentence

Figure 5 – What happened to rabbits after 2017?

Line 235 – To be honest, I think there is a huge chance that this seemingly correlation is just coincidental. This is a very weak and too circumstantial evidence. The evidence presented by the manuscript is strongly against, not in favor, of climatic factors driving the temporal variation of the two diseases in the analyzed years.

The authors should not highlight this observation and it definitely should not be in the abstract. The focus here must be on the result that no influence of climate factor was found on the number of cases.
In the current form, the reader might feel as if not finding this expected correlation between climate and disease occurrence is a flaw of the study. It is not. If the data and the analyzes are appropriate (and as far as I can tell, they are) this is a meaningful and important result. Finding the absence of a given pattern is a result as important for the advance of science as detecting the pattern.

Not requiring positive result is one of the most beneficial features of Peer J. I suggest the author to take full advantage of that, and advocate for the validity of the results they present.

Line 252 – The “together with mosquitoes” came out of the blue.
I recommend the authors to use the first paragraph of discussion to interpret their main findings and their implications.

Line 274 – Ambiguous sentence. Not sure whether the author mean that the LB numbers in winter is high compared to the TBE numbers in winter or to the LB numbers in summer.

Line 334 to 340 – I did not understand the explanation. Based on the explanation, when the rodent population density is low, co-feeding should be high, thus, infecting more ticks. So, why not enough ticks are infected?

Discussion and conclusion – I suggest the authors to close the discussion with conclusions that arrive or, at least, are coherent with their findings. In fact, I feel that in many points the discussion is a bit disconnected from the study results. The author should, of course, discuss other perspectives and results on the literature, and provide possible explanations for why their results did not match their expectations, but the focus of the discussion must be their findings. This problem is most evident in the final paragraph, as the authors conclude the manuscript by extensively discussing patterns that they have not found.


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: SPATIAL AND TEMPORAL DISTRIBUTION PATTERNS OF TICK-BORNE DISEASES (TICK-BORNE ENCEPHALITIS AND LYME BORRELIOSIS) IN GERMANY
Review round: 2
Reviewer: 1

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: Since the authors replied my comments and questions and introduced some minor requested, suggested changes, I suggest this paper for publicantion in PeerJ.


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: SPATIAL AND TEMPORAL DISTRIBUTION PATTERNS OF TICK-BORNE DISEASES (TICK-BORNE ENCEPHALITIS AND LYME BORRELIOSIS) IN GERMANY
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript describes a well conducted and informative analysis of the spatial and temporal variation of two diseases, Tick-Borne Encephalitis (TBE) and Lyme Borreliosis (LB), in Germany and some possible underling drivers. The manuscript is well written and clear, and figures are nice. The authors provide the necessary background for the study, so that, even non-specialists in the system can understand it.
Experimental design: The study is well designed, the research question is well defined and relevant.
As far as I can tell, the data is appropriate for the question addressed, the analysis is well conducted, resulting in meaningful results.
Validity of the findings: The issues I pointed in my previous revision were solved.
The authors present sounding analyses and results, and draw appropriate conclusion from them. The findings are valid, relevant and deserves to be published.
Additional comments: Line 114 - “rises” or “increases”, but not both

Line 139 -  This sentence seems weird. Probably some mistake here.
The introduction is well written and accessible for a broad scientific audience. All the important information is provided. The last paragraph included worked well and the other problems I pointed in my last revision were solved.

Line 150 -  “diseases”

Figure 3 -  In the maps, it is very hard to distinguish the absence of data from the absence of cases. I suggest the authors to use another color (for instance, white) to represent the absence of data.

Line 229 -  Why some federal states that has data for LB infections in some years, such as Bavaria, were indicated as “no data” in figure 4b? The authors used some criterium based on the number of years available? This must be clearer.

Line 260 -  These results are of interest, but it is hard to take any lesson from absolute values. Results would be much more informative if the authors provided a comparison against the gender and age structure of the population.
Are males more infected by TBE than females just because there are more males than females on the population? Or the male individuals have an increased chance of being infected?

Line 307 -  Repeated words: “in years in years”.

Discussion -  The discussion is well written and accurate, however, in my view, it remains too long and a bit unfocused. The authors dedicate several large paragraphs to discuss possible drivers of the diseases that were not detected by their study.
I believe that reducing and focusing would benefit the discussion. However, as the discussion is accurate, this criticism should be seem as a suggestion to improve the paper readability, not as a barrier for publishing it.


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: IDENTIFICATION OF HIGH PROTEIN KINASE CK2Α IN HPV(+) OROPHARYNGEAL SQUAMOUS CELL CARCINOMA AND CORRELATION WITH CLINICAL OUTCOMES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Clear writing throughout.
Appropriate references.
Excellent presentation of the data with respect to figures and tables.
Clear hypothesis; clear results; clear relationship between hypothesis and results.
Experimental design: Straightforward retrospective design.
Clear methodology.
Clear IHC and ISH methods.
Clear statistical design and methods.
Well defined research question and hypothesis regarding the interaction between HPV and CK2a.
Original research question and findings within the Aims and Scope of the journal.
Validity of the findings: Novel description of the interaction between HPV status and CK2a as a secondary function of Ki-67. Good data overall, will need validation in subsequent cohorts by an excellent start and contribution to the literature. Robust, statistically sound analysis. Clear and reasonably phrased conclusions.
Additional comments: Trembley et al. discuss the relationship between CK2alpha and oropharyngeal cancer (OPC) outcomes in a Veteran population with a focus on interactions between CK2a and HPV oncogenic drivers in this complex disease. This is a novel study that is well done overall. Good overall cohort size; combined with TGCA dataset. The large number of HPV+ tumors is a good complement to the existing limited datasets. Good correlation of p16 IHC and E6/E7 ISH.
Some clarification is needed with respect to treatment. Presumably all patients received curative intent treatment? How many of the patients completed treatment compliant with NCCN guidelines? This is important since this will impact survival and may skew the correlations with any individual biomarker.
Overall the analysis is clear, well done and compelling. Obviously this will require additional validation in additional HPV+ cohorts but overall this is an important preliminary dataset that will be of value to the field. The images are clear, the tables are clear and well organized. The text is well organized and of appropriate length.


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: IDENTIFICATION OF HIGH PROTEIN KINASE CK2Α IN HPV(+) OROPHARYNGEAL SQUAMOUS CELL CARCINOMA AND CORRELATION WITH CLINICAL OUTCOMES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well written and easy to follow. I have a couple of comments which I hope would add additional clarity to the article.

Reviewer Comments:
==================

Page 12 Line 157/158.
==================
(R = .92, p < 0.0001)

From preceding lines in the paper P16 and E7/E6 RNA are either positive or negative. What hypothesis test does the R and p value refer to? Is this a correlation coefficient (and if so which one)?

Page 12 Section 2.5
================
“Z-score cut-off was set at >1.5 higher expression of 165 CSNK2A1 compared to unaltered levels of CSNK2A1.”

Question: What exactly does the Z-score refer to? It a hypothesis test for gene expression?  Some additional details would help clarify this.
Experimental design: This was a retrospective cohort study including all newly diagnosed primary OPSCC from January 2005 to December 2015.
Exclusions included patients who had other simultaneous active malignancy at the time of diagnosis or lacked follow up.
A limited number of variables were collected:
1.	Demographics: age at diagnosis, gender, smoking history, alcohol consumption history.
2.	Tumor related variables: primary subsite, tumor stage, and treatment modalities
3.	Outcome related variables.
Reviewer Comments:
==================
Why would a patient lack follow-up – for example did they move to another treatment centre? Can you provide any characterisation of why they weren’t followed-up?

Was there a limited number of variables available on the patients? I’m thinking here of co-morbidities (renal or liver for example), co-medications etc. If these were available why were they not included in the analysis in some form to give a broader idea of the patients?
Can you please state explicitly how censoring was handled – I’m assuming that patients were censored at the end of the study, is this correct?

Page 12 Line 171
==============
What was the structure of the logistic regression equation and could you put in a reference to where it is reported please?

Page 13 Line 184/185
==================
“OS time was defined as from treatment initiation to death. PFS time was defined as from treatment initiation to disease progression.”
Did any patients die before treatment commenced and if so, how where they handled in the analysis? Was there an untreated group?
Validity of the findings: I feel that the paper has been caught between a possible study of the mechanics of the disease / interaction with the HPV, and an observational study of the disease in a particular cohort. And this is the paper’s weakness. I will make some initial comments first and then address where I think the paper has issues that need to be fully addressed.

Some initial comments:
====================
Can you please specify that the age in Table 1 is the median age and add the IQR or range.

Page 14 Line 182
==============
“In analysis of CK2α scores with the Ki-67 index in tumors, there was a positive correlation of higher CK2α score with higher percentage of Ki-67 positivity (P < 0.0003; Table 2).”

In Table 2 this is claimed as a Pearson chi-square test?

Page 14 Line 222 and following
=========================
“The average time frame for event occurrence was 3.2 years for progression free survival (PFS) and 3.6 years for overall survival (OS). PFS rates and OS rates among the CK2α scores were very similar, and OS was highest for patients with tumors having a CK2α score of 3 (P = 0.004, Table 2).”

What does average time mean in this context? Do these times take into account censoring? Is this the median as calculated from the KM analysis, or just an average of all survival times?

Why is a Pearson test used here when survival times generally should be analysed through survival analysis (KM or Cox regression)? I do not think the Pearson’s chi-square test is the best choice here. I would remove this section from Table 2.

Page 15 Line 231
==============

Please state exactly the multi-variate model fitted and associated HRs – could have this as a table. Did you check he proportional hazards assumption for the Cox models?

=====================================================

More detailed comments:
====================
The most interesting claimed result in the paper is:
“High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort.”

but we also know that

“the current increase in OPSCC incidence is considerably linked to HPV presence”

So HPV is related to an increase in OPSCC cancer but the associated increased CK2α improves overall survival, while for HPV(-) OPSCC an increased CK2α is associated with poorer overall survival.

Confounding is an issue all observational studies have to address, especially so when the results are unexpected. For any observational study the question is: Could bias or confounding explain the results?

This needs to be properly addressed in the paper to strengthen the claim. For example:

1.	All the participants are male. So the results actually only apply to OPSCC cancer in men and not more generally. But more than this, gender is considered typically an important variable in these types of studies and the fact that it could not be included has weakened the results.

2.	All participants come from one hospital, a potential section bias issue.

3.	For the cohort none of the treatments appear to have an effect on overall survival (Table 3) – is this similar to reported clinical trial results for OPSCC? Are the HRs similar to the trials? If not then it is likely that the cohort is biased.

4.	Were HPV(+) patients healthier than HPV(-) patients? Are there lifstyle differences? Could this or similar be the reason for the increase in overall survival. Was this investigated? Are there systematic differences between HPV(+) and HPV(-) patients in the cohort?

5.	Treatment choice for the patients was likely not random – could this have an effect?

A convincing or at least plausible biological explanation is needed as an alternative. Have similar results been reported in the past?
Additional comments: I enjoyed reading the article and found it to be well written and an interesting study. My comments detailed above are mainly in the area of the role of possible bias and confounding in the study. The paper does not make any causal claims but I think that a robust analysis of the possibility of confounding needs to be included.


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: IDENTIFICATION OF HIGH PROTEIN KINASE CK2Α IN HPV(+) OROPHARYNGEAL SQUAMOUS CELL CARCINOMA AND CORRELATION WITH CLINICAL OUTCOMES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Given that 94% of patients were current or former smokers, is it really possible to "control" for smoking history in this patient population statistically?  This shortcoming should be noted and the authors should suggest testing the external validity of their finding on a patient population with a more heterogeneous patient population in terms of smoking history.
Additional comments: The authors should explain their use of Ki-67 in the Introduction and provide their justification for inclusion of this marker in this study.

Lines 105-109 at the end of the Introduction read as results/discussion points.

How many patients were excluded due to simultaneous malignancy?

Were patients that had distant mets at presentation not excluded?  Were patients that did not receive curative intent treatment (i.e. palliative treatment) also included in the analysis?

Line 160: consider adding "including those patients with discordant p16 results."

Line 185: add "or death." to the end of the sentence

Line 252: I believe the second HPV (+) was meant to be "HPV (-)"

Line 302: I'm not sure how effectively you can control for smoking statistically when 94% of patients have a smoking history.

Overall, this is a very organized and presented work with meaningful value to the head and neck oncology literature.  I feel that with the above revisions, this article will make an excellent additional contribution to the current body of literature.  I thank the authors for their submission.


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: IDENTIFICATION OF HIGH PROTEIN KINASE CK2Α IN HPV(+) OROPHARYNGEAL SQUAMOUS CELL CARCINOMA AND CORRELATION WITH CLINICAL OUTCOMES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Changes or explanations as per the first review were made by the authors.
Experimental design: Changes or explanations as per the first review were made by the authors.
Validity of the findings: Limitations (possible bias and confounding) were discussed by the authors. I think this places the paper correctly in the literature.
Additional comments: The authors have addressed the issues I raised in as far as this is possible and have adequately discussed the issues with regard to possible bias and confounding.

One thing to note: In the pdf of the revised manuscript Table 1 does not look like it has been updated from the previous version.(The table is not included in the tracked word version).


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

Input:
Title: TRANSCRIPTOMIC ANALYSIS OF MELON/SQUASH GRAFT JUNCTION REVEALS MOLECULAR MECHANISMS POTENTIALLY UNDERLYING THE GRAFT UNION DEVELOPMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript entitled “Transcriptomic analysis of melon/squash graft junction reveals molecular mechanisms underlying the healing development ”, by Xu et al. was reviewed.
The authors are interesting in the gene regulatory mechanism and involvements of graft formation of melon/squash, they performed transcriptome analysis to describe the morphological/transcriptional changes during graft formation in melon/squash.
In addition, the authors also analyzed endogenous levels of phytohormone and lignin of melon/squash graft, discussed about the involvement of auxin, GA, ABA, cytokinin and lignin on graft union .
The authors are discussing about relationship between morphological/physiological changes and gene regulatory network that occur during graft formation, this work contains new and important information for graft formation of plants.

Specific comments;
They used “IL, CA, and VB” as abbreviation for grafting stage, but these wording are not appropriate and difficult to understand. I propose to use “2DAG, 6DAG, 9DAG” or “Early, Mid, Late” or something similar in manuscript.

Table1) I propose to move this table to Supporting Information, because it is not informative.

Fig.1) These photographs should be revised. The orientation of each photos is different, and more explanation for results and abbreviation for symbols should be added in photo and figure legend. The authors also should indicate which is a scion or rootstock. In addition, the reviewer is wondering how authors determine callus formation and new vascular bundles. They described about it in text (line 176-191), but I cannot see such morphological changes from these images (B, C). The photograph should be replaced with better images and explain in more detail.

Fig.3,4,5) The text is too small, and the resolution is too low to read. These figures should be revised, higher resolution, split, or simpler, because it is difficult to see the data.

Fig. 5, 9) These figures need more information. It will look better if a gene name or deduced function were included in each graph.

I propose to cite these related-papers about plant grafting “Notaguchi et al., 2020; Science” and “Matsuoka et al., 2018; Plant Cell Physiol.”in the introduction or discussion, for the reader's better understanding.
Experimental design: Fig. 6) One section of the paper concerns about the relationship between phytohormone and graft formation. The authors showed that endogenous levels of phytohormone, but there are some concerns about experimental design. The reviewer is wondering why authors determine endogenous phytohormone levels using ELIZA, instead of LC-MS or LC-MS. Furthermore, they analyzed levels of auxin, GA, ABA, cytokinin. Although GA3 is one of major bioactive GA, but generally, not detected in wildtype of higher plant. The reviewer is also wondering why such amount of GA3 was detected in this work. In addition, zeatin riboside is not bioactive form in higher plants. Please explain why authors used this method and analyze these hormones, and I strongly recommended that authors should confirm experimental condition, data analysis, and perform quantitative analysis of endogenous bioactive GA and cytokinins. Moreover, there is no control experiments. Analysis of phytohormone using non-grafted plants should be done.

They mentioned hormone content during the graft process and discussed about the relationship with gene expression and morphological changes. There were statistically significant changes, but I am concerned about whether these are biologically meaningful changes, except for ABA. Explanation needs more detail.
Validity of the findings: While the results are intriguing and may potentially lead to greater understanding of molecular mechanisms underlying melon/squash graft formation, no direct connection between gene function and graft union formation has been shown and there are some concerns about the interpretations of the results and illustration /description of figures.
Additional comments: The text is clearly described, but the illustration, description and legends of each figures were not clear, so an effort should be made by authors to clarity 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: TRANSCRIPTOMIC ANALYSIS OF MELON/SQUASH GRAFT JUNCTION REVEALS MOLECULAR MECHANISMS POTENTIALLY UNDERLYING THE GRAFT UNION DEVELOPMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: see my comments in general comments.
Validity of the findings: no comment
Additional comments: The study is nice and adds to the existing information. My minor comments are:

Line #111: What are the control conditions in sample collection?

Line #161: Why did you selected only IAA, ZR, GA3, ABA?

Line # 175: If you did microscopy continuously then where is the remaining data?

Line # 176:  The graft healing formation was divided into three recognizable developmental stages, so what was the basis of division of the developmental stages?


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

Input:
Title: TRANSCRIPTOMIC ANALYSIS OF MELON/SQUASH GRAFT JUNCTION REVEALS MOLECULAR MECHANISMS POTENTIALLY UNDERLYING THE GRAFT UNION DEVELOPMENT
Review round: 1
Reviewer: 3

Output:
Basic reporting: Xu et al present a transcriptomic study of the graft interface of melon/squash grafts at 2, 6 and 9 days after grafting, this study is complemented by some microscope images and hormone analysis. The manuscript is well-written and the results are interesting. However, some references need to be modified and more details are required in the methods section.

In the abstract, the authors need to define what DEGs, IL, CA and VB are.
Experimental design: Methods
More details of the grafting technique should be given, what stage were the grafts performed? What were the growth conditions? What was the grafting success? Etc.
More details are required in the methods section such as how was the graft interface sampled? For example, 2 mm above and below the graft interface? Where the same samples used for the RNAseq, lignin and hormone analysis?
For the RNAseq analysis, it is interesting and novel that the transcripts were assigned separately to the scion and rootstock, but more details of the methods need to be given, such as, how were transcripts assigned to the melon scion or squash rootstock? What reference genomes were used? It seems that more reads mapped to the stock than the scion, is this due to the sample? Did it contain more rootstock than scion tissue? What percentage of transcripts was ambiguous in their assignment? What did the authors do with the transcripts that could not be assigned to either the scion or rootstock genotype?
The different gene expression patterns in the scion and rootstock should be discussed in relation to the study of Melnyk et al 2018.
If the RNAseq, lignin and hormone analysis samples are the same, it could be worth doing a WGCNA analysis of the data to find transcripts strongly correlated to hormone/lignin concentration.
Validity of the findings: The validity of the findings is generally OK, but a few changes are required.
Title: change to “Transcriptomic analysis of melon/squash graft junction reveals molecular mechanisms POTENTIALLY underlying the GRAFT UNION development”
Lines 282-285, lines 323-324, lines 388-389, lines 413-415: confirmatory statements should be made more modest with works like suggests/may/could etc.
Additional comments: The different gene expression patterns in the scion and rootstock should be discussed in relation to the study of Melnyk et al 2018.

Figure 1: The images should be better annotated, the graft interface, scion and rootstock should be labelled, etc. It might be good to have better images of the beginning of callus cell formation and vascular bundles and some idea of where in the graft interface the images were taken. This would also help the reader see what you want them to see in the images.
Figure 8: Error bars missing
The details of the data repository (NCBI) should be given in the methods section.
What is CTK signaling? (line 282)
What is the reference for this statement “In addition, a blockage in auxin transport due to vascular damage lead to decreased expression of GH3 genes during the early stage, suggesting a response for wounding during graft healing.” (Line 348-350)
Reid and Ross 2011 is a commentary on a paper, it is preferable to cite original research.
Line 399: should be Cookson et al 2014


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

Input:
Title: TRANSCRIPTOMIC ANALYSIS OF MELON/SQUASH GRAFT JUNCTION REVEALS MOLECULAR MECHANISMS POTENTIALLY UNDERLYING THE GRAFT UNION DEVELOPMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I was still confused with the words of "“IL, CA, and VB” as abbreviation for grafting stage, in this figure and the text.
The reviewer understood the “IL, CA, and VB” as a key events in the process of grafting healing, but these wording are not appropriate for abbreviation of grafting stage this way.
The authors use the abbreviation of “IL, CA, and VB” in two means, name of structure and grafting stage. It would be clearer to give each abbreviation used with its explanation.
Again, I propose to use “2DAG, 6DAG, 9DAG” or “Early, Mid, Late” or, at least, “IL stage, CA stage, and VB stage” for grafting stage or something similar in manuscript.

The authors described the reason of using ELIZA, instead of LC-MS or LC-MS, to determine endogenous phytohormone levels, but did not explain why such amount of GA3 was detected in this work. The authors may have misunderstood GA3 for another GA or may be an artifact of experimental design.
As pointed out previously, GA3 is one of major bioactive GA, but generally, not endogenous GA, and not detected in wildtype of higher plant, including melon/squash Solanaceae plants, these results are not justified. If GA3 was detected, the authors should give some references and/or convincing evidence.
Validity of the findings: no comment
Additional comments: The revised manuscript entitled "Transcriptomic Analysis of Melon/Squash Graft Junction Reveals Molecular Mechanisms Potentially Underlying the Graft Union Development, by Xu et al. " was reviewed. I feel that this manuscript has been improved satisfactorily since some sentences and figures were added and improved, but there are still some points 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: TRANSCRIPTOMIC ANALYSIS OF MELON/SQUASH GRAFT JUNCTION REVEALS MOLECULAR MECHANISMS POTENTIALLY UNDERLYING THE GRAFT UNION DEVELOPMENT
Review round: 2
Reviewer: 2

Output:
Basic reporting: The basic reporting of this manuscript is correct.
Experimental design: The experimental design is OK, but care should be taken in the interpretation of the data: as only the graft interface is compared over time, the authors cannot separate changes due to development and those due to graft union formation. Ideally scion and rootstock tissues should have been studied at the same time to identify graft union formation specific genes.
Validity of the findings: The findings seem to be OK, comments have been added to the word document of the manuscript.
Additional comments: I have some comments about the figures:
Figure 1: This figure could be improved, the different cell types are not particularly clear.
Figure 3: This figure is not very readable, the data may be better presented in a Table and maybe focus only on the over-represented categories and not the under-represented categories (like reproduction, etc).
Figure 4: the text is too small and unreadable. It is very surprising to find differences in circadian rhythm, were the plants all harvested at the same point in the photoperiod? Or is it due to changes in carbon availability? If they were not harvested as the same point in the diurnal cycle this should be added to the methods section
Figure 5: the text is too small and should be made simpler.


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

Input:
Title: THE INFLUENCE OF RHIZOSPHERE SOIL FUNGAL DIVERSITY AND COMPLEX COMMUNITY STRUCTURE ON WHEAT ROOT ROT DISEASE
Review round: 1
Reviewer: 1

Output:
Basic reporting: General comments

This is an interesting manuscript in which the authors report a correlation between soil fungal community richness and symptomatic or non-symptomatic plants at the heading and filling stages of hexaploid wheat. However, there are inconsistencies between the importance of soil fungal diversity as mentioned in the Introduction and the analyses reported in Results and Discussion. It would be interesting to see the diversity if the OTUs were parsed by year as well as by host developmental stage. The Discussion is redundant with Results in some places (e.g., L352-359). The authors should consider discussing what the results mean, and what might be the next steps to explore. Also, the manuscript lacks certain details that would improve the utility and significance of the data to the broad readership of PeerJ, as described in Basic Reporting and Experimental Design.

Basic Reporting

The authors mention the importance of soil microbial diversity and soil structure to sustainable agriculture in the Introduction but their annotation to the genus level does not distinguish between beneficial and wheat-pathogenic fungal species within the genus. In this reviewer’s opinion, soil health can indirectly be conditioned by crop and crop rotation, root exudate quality and environmental factors.

Is there any information on the types of root rot observed at the Xiangyang Original Farm in previous years. For instance, what information is available for the causal agents in the diseased plots.

The authors should state whether alpha diversity was analyzed using R, and describe how the soil physical and chemical properties were quantified.

References for data processing steps will be useful to readers who have not used Trimmomatic or FLASH.

Do the statements in the Discussion L292-297 refer to references cited immediately before? If not, please provide new references.

The English grammar and spelling are excellent. However, certain words are unclear, such as exhaustive (L57); ears on L 136 (do you mean heads); endemic (L248); monotonous (L297).

L72 – Include Rhizoctonia oryzae or omit R. solani and substitute Rhizoctonia spp.
L90 – Nutrients are also drivers for rhizosphere soil community structure.
L199-200 - High community richness can also be a factor in disease suppression.
L273 – Explain briefly what soil density represents—water saturation, or clay/sand composition?
Fig. 1 – Describe to the readers what the plants are showing. Any stunting, chlorosis?
Figures 2 and 3 appear to be switched as presented.
Experimental design: The author should clarify how the 100 samples were selected for the 5 sequencing reps. Were any samples pooled before extraction or the extracts pooled for sequencing.

The authors could consider identify the OTUs to species using UNITE and Blastn, especially to look for known soilborne pathogen species of the cereals.
Validity of the findings: The findings have moderate validity regarding the correlation between host floral/head development and soil community members.

The raw data deposition to is valid. Raw data on the soil properties is provided in a supplementary table.
Additional comments: No additional comments


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

Input:
Title: THE INFLUENCE OF RHIZOSPHERE SOIL FUNGAL DIVERSITY AND COMPLEX COMMUNITY STRUCTURE ON WHEAT ROOT ROT DISEASE
Review round: 1
Reviewer: 2

Output:
Basic reporting: In general, the research has been carried out with scientific rigor, the proposed methods are adequate to achieve the goals.
Experimental design: The methods are adequate, as well as the organization of the manuscript.
There are some suggestions, I will show them shortly:

Line 72: ;Rhizoctonia is spp or what specie?
Line 100: microflora --> microbiota
Line 125: Reference the method of collecting the samples or mention if it is from this study.
Line 136-137: Although it is not totally necessary, you could place an image to make the comparison between the diseased and healthy plant (perhaps in supplementary material).

Fig. 2. If possible, enlarge panels B and C; since it is difficult to visualize them.

Lines 216-224: How would the differences that exist in that short time be explained?

What explanation exists for this relationship that exists between a greater quantity of total nitrogen and a greater quantity of pathogenic fungi?

Line 277: Why is there that relationship?
Line 291: What are some examples of these metabolites?
Line 297: Is there any reference for this statement?
Line 309-310: Reference
Line 377-380: So if microbial diversity increases, would it tend to a balance between species and therefore a decrease in infection?

Line 387-390: Reference.

Line 397-398: Is there an explanation for this? Or is there any reference to it?
Validity of the findings: The proposed conclusions are supported by the results achieved.

Some references are needed, I indicated them previously.
Additional comments: Review the suggestions shown in the previous sections.


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: DEVELOPMENT AND PRIMARY VALIDATION OF THE SCHOOL HEALTH ASSESSMENT TOOL FOR PRIMARY SCHOOLS (SHAT-PS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: a.	Abstract : Adequately written
b.	Introduction: Adequately written
c.	Methodology: Kindly refer to the statement “Exclusion criteria were individuals with less than five years of job experience and those 240 who did not intend to participate in the research.” There is no need to mention it in the exclusion criteria as the authors had already mentioned it in the inclusion criteria as “at least five years of job experience is needed for inclusion.” It implies that those who had less than five years of experience will be excluded.
d.	Why the authors keep five years of work experience for inclusion? Is it an arbitrary selection or on the basis of any evidence?
e.	Any abbreviations used in table and figures need to be mentioned with expanded version as foot notes
Experimental design: Adequate and appropriate
Validity of the findings: Appropriate
Additional comments: None


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: DEVELOPMENT AND PRIMARY VALIDATION OF THE SCHOOL HEALTH ASSESSMENT TOOL FOR PRIMARY SCHOOLS (SHAT-PS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is a well-written article. I found it interesting. The research question is well mentioned based on the background. The references are adequate and updated.
Experimental design: It is novel research with a fundamental impact on the school health system in Iran.
Validity of the findings: The impact of the study is long-term and significant on the community of countries like Iran. Also, the nearby countries can utilize and adapt the same instrument and/or same procedure.
Additional comments: I was wondering to have a small para on the schooling system in Iran in Introduction so that readers can get an idea from the article. Secondly, authors could discuss from the below-mentioned paper on psychometric validation
Arafat SY, Chowdhury HR, Qusar MM, Hafez MA. Cross cultural adaptation & psychometric validation of research instruments: A methodological review. J Behav Health. 2016;5(3):129-36. doi: 10.5455/jbh.20160615121755


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

Input:
Title: A NEW AND VERY SPINY LIZARD (GYMNOPHTHALMIDAE: ECHINOSAURA) FROM THE ANDES IN NORTHWESTERN ECUADOR
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: Evidence presented strongly support conclusion that they have discovered a new species. The authors go far beyond what generally constitutes a species description.
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: A NEW AND VERY SPINY LIZARD (GYMNOPHTHALMIDAE: ECHINOSAURA) FROM THE ANDES IN NORTHWESTERN ECUADOR
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I suggest to to add a discussion about the hemipenial morphology of the new species.


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

Input:
Title: A NEW AND VERY SPINY LIZARD (GYMNOPHTHALMIDAE: ECHINOSAURA) FROM THE ANDES IN NORTHWESTERN ECUADOR
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is clearly written with good English throughout (minor exceptions below).  The literature references are sufficient—there is a small body of literature on these taxa and they have included all that was necessary.  The article structure was good and clear.  They have some excellent useful figures.  There are a lot of figures but I believe they are all useful.  The ones comparing among the species will be incredibly useful to readers.  The skeletal figures are a step above what has ever been done in the past with these taxa.  I think there are too many tables and some of the tables could use some work (more below).  The paper is self-contained with results relevant to their project.
1.	L89-99 and elsewhere in the Methods. Change all of this to active voice to make it easier to read.
2.	L155, 158. Poor grammar: “was ran” is incorrect.
3.	L231. Tables are referred out of order. You refer to Table 3 here but Tables 1 and 2 later.
4.	Table 1. Write out character names. The abbreviations are not defined in this table.
5.	Table 2. I’m not sure this table is necessary.  They are mostly the same and you can describe the variation in the text.  I would omit this table.
6.	Table 3. Put the columns in a logical order. I would put the new species first and the rest in alphabetical order (or some other order that makes sense).
Experimental design: This paper contains original research describing a novel species.  More importantly for most readers, it clarifies differences among Echinosaura species with helpful tables and figures.  It gives excellent skeletal information that is lacking for most gymnophthalmids.  The methods are described in sufficient detail to replicate the morphological and molecular analyses.
Validity of the findings: The data appear to be robust and evidence that the species is novel is abundant.  The Discussion and Conclusions are quite brief, but I do not think that more needs to be said.  I have two comments for the authors to consider that need to be clarified in the text.
1.	L177-180. You give ND4 distances for the taxa closely related to your new species but 16S distances for the distantly related ones.  Why?  Give 16S distances for all of them so that they are comparable in this paper and across other papers.
2.	Figure 4. Did you take these photos immediately after they were preserved?  If some were preserved longer than others then the patterns may have faded (Doan and Adams, 2015 Zootaxa). The live photos of Figure 6 show the variation but you still have to be careful when displaying patterns of preserved specimens.  You should state how long these had been preserved and discuss if there is variation in preservation times.
Additional comments: I look forward to seeing this in print.  I would be happy to discuss any of my comments with the authors.


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: CURRENT FREEZING AND THAWING SCENARIOS EMPLOYED BY NORTH ATLANTIC FISHERIES: THEIR POTENTIAL ROLE IN NEWFOUNDLAND AND LABRADOR’S NORTHERN COD (GADUS MORHUA) FISHERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The contents of the article is clear and unambiguous, but the overall the language phrasing is more of academic nature. Although the article confirms to the scope of the journal, there is space for improvement with more broad and cross disciplinary interest. Introduction adequately introduce the subject in more of an academic pattern. Author can take up an analytical approach to the past and current researches based on the particular subject (eg.. broad analysis on studies under a different freezing methods). Overall the idea and suggestions were appreciable and will help to satisfy the cross disciplinary readers.
Experimental design: Article satisfy the scope of the journal. The review is organized logically with sufficient subsections. The first part of the review focused more on theoretical aspects and need more cross referencing in each subsection.
Validity of the findings: Conclusion identify the unresolved problems and suggestions for improving the current gaps in the value chain with reference to the emerging Newfoundland and Labrador's emerging cod fisheries.
Additional comments: Line 93 “To the best of our knowledge, this paper is the first comprehensive review of this broad scope.” Justify ?….Give focus on North Atlantic fisheries
Line 265 to 282 the word “Freezing” has been used in multiple times
Certain references were too old (eg: line no 405) can be revised
Line 508 –line 514: reframe the sentences to give more focus to the principle rather than specification of the equipment mentioned
Line 537-552 Authors can cite more references in the emerging high pressure thawing technologies
Line 615-616 Reframe the sentences in a professional language
Line 626- It is advisable to cite recent references in the article


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: CURRENT FREEZING AND THAWING SCENARIOS EMPLOYED BY NORTH ATLANTIC FISHERIES: THEIR POTENTIAL ROLE IN NEWFOUNDLAND AND LABRADOR’S NORTHERN COD (GADUS MORHUA) FISHERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: I find the article interesting, and it gives a good overview of the industrial technology status within freezing and thawing.  Also, nice to learn more about Newfoundland and Labrador challenges. Nearly all the figures and all the tables seem adequate.  I only have one comment regarding the manuscript.
•	In line 314 there is a reference to Fig.2 that I don’t understand.  Fig.2 shows an air blast freezer and a spiral freezer, both depend on air as cooling medium(direct) and not indirect.
Experimental design: The introduction present the content and purpose of the review in an structured manner.  The sources are cited correct, and the structure of the paper is logically.
Validity of the findings: no comment
Additional comments: No additional comments