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: HIV-1 PROMOTER IS GRADUALLY SILENCED WHEN INTEGRATED INTO BACH2 IN JURKAT T-CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In their manuscript entitled “HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells, Inderbitzin and colleagues report the development of initial characterization of a system to investigate the impact of integration site choice and HIV gene expression (latency). I found the introduction to be clearly written and to explain the knowledge gap being addressed. The Figures were easy to follow and the Materials and Methods section is detailed enough, in my opinion, to allow independent replication of the experiments. Overall the English is high quality and accessible for international readers.
Experimental design: The authors employ dual reporter viruses that have previously been published from their group. These constructs were integrated into the genome of Jurkat T cells using CRISPR/Cas9 technology via the addition of arms for homologous recombination into the known BACH2 gene that has been shown by several independent groups to be a “hot spot” for HIV integration.

The work certainly suits the broad scope of the journal and will be of use to others working in the field. One question for the authors, in the section “Availability of materials” the authors have marked not applicable. Given the use of new viral constructs and the creation of useful cell lines in the work, why is this the case?
Validity of the findings: The experiments are in my view well controlled and the results are compelling. The authors show that HIV expression is gradually reduced in the BACH2 locus over time in an orientation-independent fashion, without significantly disturbing the expression of BACH2 protein levels. These results contrast with the integration into AAVS1 “safe haven” genomic site where HIV remains active over time. It should be fascinating to use the system in the future to further explore other genomic sites.

Although I found the authors discussion of the results to be reasonable, I had two minor points that should be addressed for clarity to readers. Potential limitations of the system include the fact that an immortalized cancer cell line was employed. Given the fact that genomic preferences are only found in primary cells, certain elements of the system may not recapitulate the selection seen in vivo. A second limitation that should be mentioned is the fact that other viral genes such as vpr are not in these constructs. Since cell cycle impacts HIV gene expression and latency, and vpr manipulates the cell cycle, the absence of these genes could have importance on the future findings.
Additional comments: Overall, I commend the authors on a significant amount of careful work and a useful contribution to the field.


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

Input:
Title: HIV-1 PROMOTER IS GRADUALLY SILENCED WHEN INTEGRATED INTO BACH2 IN JURKAT T-CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Inderbitzin et al. presents a study of HIV-1 integration into introns of the BACH2 gene in Jurkat cells. The author’s approach is using CRISPR insertion of a dual HIV-1 reporter, one gene reporting HIV-1 LTR activity, and another constitutively active. Authors insert this vector into two introns of BACH2 in sense and antisense orientation (relative to the gene) as well as an alternate locus in Jurkat cells. Authors then iteratively sort integrated cells to obtain monogenic clones. Authors observe a limited impact on the gene, BACH2, in terms of RNA and protein expression. Authors also observe a gradual decline in the expression of the HIV specific reporter. While a very interesting study, the approach is limiting to the general applicability of the results and interpretation must be made with caution. The authors should address that it is possible that the finding is because of insulator insertion rather than HIV insertion into BACH2 (if not experimentally justified).
Experimental design: Overall the authors spent respectful effort in inserting this cassette into the BACH2 site with several controls.
Validity of the findings: 1.	Investigators have used a reporter with genomic insulating elements flanking promoters. While desirable for generating a dual reporter, authors have not demonstrated that these do not impact the ability of the HIV-1 LTR from impacting the integrated gene. The cHS4 element is specifically investigated in viral vector gene therapy because of its ability to block chromatin modifications and enhancer promoter interactions (see Emery et al. PNAS 2000). Enhancer blocking activity was later discovered to be due to a CTCF binding site (Bell et al. Cell 1999), which has been implicated gene expression changes in other human retroviruses (Satou et al. PNAS 2016, Melamed et al. eLife 2018). Given these known impacts, it is unclear that authors have demonstrated the impact of the HIV-1 LTR on the inserted gene. To make this claim, authors would need to generate clones without these insulator elements. Further, the vector used in this study contains elements that would likely interfere with read through HIV-1 driven transcription, such as the additional reporter promoter and the polyadenylation site. Or, alternatively, can the authors address these limitations of having significant transcriptional interference by the insulator cHS4, that the results of may be caused by insulator insertion?
2.	Authors did not functionally validate the impact of HIV-1 integration into BACH2. While changes may not be appreciable at the qPCR and western blot level, integration into BACH2 may provide additional competitive advantages or clonal expansion. Authors should conduct competition experiments to determine if this is the case. Authors could perform these experiments either directly (by mixing parental cells with integrant cells and watching the ratio of parental to integrant cells overtime) or indirectly (by starting several wells of clones with the same number of cells and plotting expansion over time. Or, alternatively, can the authors address these caveats in their results?
3.	Authors could strengthen their claim that this system is physiologically relevant by showing their system recapitulates production of known chimeric RNAs such as those reported in Cesana et al. Nature Communications 2017 and Liu et a., Science Translational Medicien 2020. Or, alternatively, can the authors address this caveat, that without knowing whether this HIV inserts is still able to produce aberrant splicing to BACH2, it is unclear that whether the phenotype observed is because of HIV integration into BACH2 or the insulator insertion into BACH2?
4.	Investigators are unclear when they state “BACH2 mRNA nor BACH2 protein expression measured 120 days post sorting”. In figure 1D authors show these experiments occurring 25-30 days post second sort, which is 50 days post initial transfection which only adds to 75-80 days. One of the numbers included must be incorrect, and should be rectified for consistency and clarity. Can the authors revise?
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: CLASSIFICATION OF OVARIAN CANCER ASSOCIATED WITH BRCA1 MUTATIONS, IMMUNE CHECKPOINTS, AND TUMOR MICROENVIRONMENT BASED ON IMMUNOGENOMIC PROFILING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The author provides background that the therapeutic effect in ovarian cancer is not satisfactory. References is also sufficient. However, there are some gramma errors.
In the PDF file:
1. Methods part on page 4. ‘we classified ovarian cancer…’, ‘w’ in ‘we’ needs to be capitalized.
2. Results part on page 4. ‘Interestingly, though tumor mutation burden (TMB) was no significant
difference among three subtypes, but all patients with BRCA1 mutations were detected in Immunity_H
subtype.’ This sentence doesn’t read well, it is wrong in gramma. Should be ‘was not significantly different’. Also, usually ‘though’ and ‘but’ is not used together.
3. Line 45 United Stated->United States
4. Line 48 ‘Although we have made…’, it’s better to say ‘Although researchers have made…’ if the cited reference is not the author’s work.
5. Line 67-68, ‘In the phase II…. show that…’, this sentence has error in gramma.
6. Line 77-78, ‘ssGSEA was used…, then classified…’, this sentence has error in gramma.
7. Line 81-82, ‘Our study findings …immunotherapy’, this sentence has error in gramma.
8. Line 213 ‘the same mothed’ should be ‘the same method’.

This article also has some other gramma errors in Result section starting from line 136, the authors need to carefully double check before re-submission.
Experimental design: The experiment design was good. The research question is well defined, the authors wanted to check the immune landscape of patients to improve treatment efficacy. The research could also fill the gap in the treatment.
The methods are also described in details.
Validity of the findings: The authors provided the validation of results with another dataset and shows consistency. The results were clearly explained. Figures and legends are also clear. But it would be great if the authors can provide more description of the figures. For example, figure 2 and 5.
In the result for BRCA1 mutation and BRCA2 mutation, the subgroup number for BRCA2 mutation is 11 out of ~430 total patients. And then 11 patients distribute in 3 immunity groups. Even the statistical test result shows that the difference is not significant, 11 is a small group number. It would be great if the author could find larger datasets to confirm this.
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: CLASSIFICATION OF OVARIAN CANCER ASSOCIATED WITH BRCA1 MUTATIONS, IMMUNE CHECKPOINTS, AND TUMOR MICROENVIRONMENT BASED ON IMMUNOGENOMIC PROFILING
Review round: 1
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: 1. Please define the following terms in the text: immune score, tumor purity, stromal score, estimate score. How are they derived in the ESTIMATE package? What do they mean? What is the importance of the numbers?
2. Line 45: Missing a 'the' before the United States.
3. Line 45: Due to a lack of symptoms...
4.  Line 62: Give the full names of CTLA-4, PD-1, and PD-L1.
5. Line 62: ...PD-L1 receptors have...
6. Line 63: ...malignant melanoma, lung cancer, and bladder cancer...
7. Line 65: However, the response rate to ICIs for patients with ovarian cancers remains unsatisfying, in which the objective response rate (ORR) was less than 15%.
8. Line 77: It should be cancer and not the cancer.
9. Line 139-141: Please restructure this sentence.
10. Line 142: ...we got the immune score, stromal score, ESTIMATE scores, and tumor...
11. Line 145: These results indicated that immune and stromal cellnumbers...
12. Line 154: ...CTLA4, TIM-3, and LAG-3...
13. Line 163: ...important role...
14. Line 166: ...3 subtypes ovarian cancer were...
15. Line 169: ...have shown...
16. Line 194-196: How are the parameters mentioned associated with a better prognosis? This statement is not clear.
17. LIne 210: what is the function of chlorophyll metabolism in human cells? How can a human cell line have an enrichment of the chlorophyll-metabolism pathway?
18. Line 214: ...a similar...
19. Line 246: ...immune checkpoint plays a crucial role...
20. Line 251: Restructure the sentence.
21. Line 267: What is tumor immune? Tumor immune cells?
22. Line 269: Sentence not clear.
23. Please use either tumour or tumor. There are both spellings present in this manuscript. Please check with the journal recommendations for British vs. American spellings.
24. Figure 1. Define red-->blue gradient. How is the tree constructed? Figure legends should be descriptive enough to sufficiently understand the figure.
25. Figures 2 and 3. Please provide a descriptive figure legend. Also, the texts in the graphs are too small to read properly.
26. Figure 4. Once again, a proper figure legend is missing. The authors should explain the terms of activated and resting dendritic cells. What type of plot is this? How about error bars? How was the statistical significance calculated?
27. Figures 5 and 6. Texts are too small.
28. Figure 6: The term enrichment score should be defined mathematically. Does this plot only convey a list of pathways? Anything more?

The authors used ssGSEA projections to enrich a number of genes associated with ovarian cancer from the TCGA database. They divided the ovarian cancers into 3 classes based on their respective ssGSEA scores and analyzed these classes at finer details. In my view, methodology, experimentation, and results of this study are valid. What needs work is the writing and language. Also, the authors wrote this manuscript like a list of genes/pathways that are enriched in the different sub-types and definitely missed the opportunity to functionally analyze the data. If the sub-types are formed based on immune scores, then isn't it expected that they would have differential enrichment of immune genes? The title of the paper emphasized on the BRCA1 mutations, but that mutant pool is only >3% of the ovarian cancer dataset. The authors need to explain these issues better in the discussion.


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

Input:
Title: CLASSIFICATION OF OVARIAN CANCER ASSOCIATED WITH BRCA1 MUTATIONS, IMMUNE CHECKPOINTS, AND TUMOR MICROENVIRONMENT BASED ON IMMUNOGENOMIC PROFILING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed the comments.

New comments:
In results part, 'Analysis of the TMB and BRCA mutations among the 3 subtypes of ovarian cancer'. The authors mentioned Fig. 3A, FIg 3B and 3C. However, in Fig. 3, there is no A, B, C parts. I would suggest the authors to double check the figures.
Experimental design: The authors addressed the comments. The paper is fine for this part.
Validity of the findings: The comments are addressed. The paper is fnie for this part.
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: CLASSIFICATION OF OVARIAN CANCER ASSOCIATED WITH BRCA1 MUTATIONS, IMMUNE CHECKPOINTS, AND TUMOR MICROENVIRONMENT BASED ON IMMUNOGENOMIC PROFILING
Review round: 2
Reviewer: 2

Output:
Basic reporting: .
Experimental design: .
Validity of the findings: .
Additional comments: I am happy with the revisions and the rebuttals. I would still suggest the authors revise their manuscripts for typos and grammatical errors or take editorial help. I am happy to recommend this revised manuscript for publication.


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

Input:
Title: THE PANDEMIC PARADOX: DOMESTIC VIOLENCE AND HAPPINESS OF WOMEN
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript was well-written and no comment on this part.
Experimental design: Research design, data analyses, and data report were very clear.
Validity of the findings: No significant issue in this part.
Additional comments: Thank you for the opportunity to review such an interesting study. This manuscript is very well-written, data analyses were very solid, and results of this study will contribute to the literature. I enjoyed reading this paper.
1. Line 56, some edits are needed.
2. Authors mentioned, “we observe from the data that very 295 insignificant number of women reported such violence (including physical, emotional and verbal) before lockdown.” I wonder why authors did not control for this variable, and how this could affect their safety during COVID. The authors should try.
3. The authors should present all the variables included in regression models in the descriptive table.
4. Table 3: Income adequacy. The base category should be “strongly agree” instead of “agree.”
5. How many women had zero violence during COVID-19? It would be helpful to note the number of zero cases in Table 3 for the zero model.
6. Examining safety would be interesting enough for this paper, but the authors further examined factors associated with happiness. Since authors further examined factors associated with happiness, a stronger link between safety and happiness should be made, such as why subjective happiness was linked to exposure to violence. I can see some connections here, but it should be elaborated better using some empirical evidence or theoretical perspectives regarding the relationships between these two variables.
7. Also, authors should compare significant results from violence as a DV and subjective happiness as a DV.
8. Implications for preventing domestic violence for women should be further discussed.


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 PANDEMIC PARADOX: DOMESTIC VIOLENCE AND HAPPINESS OF WOMEN
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors clearly make a case for their work, using clear, technically correct writing, anchored to topical literature references. I will defer to a copyeditor for grammatical and syntax corrections. Several statements need completion, rephrasing, or grammatical edits for clarity. Citation formats vary widely. The authors should confer with Peer J on a specified citation format.

The article does not include enough citation - particularly in the background and discussion sections. Several COVID + DV papers are now in publication and can serve to reinforce literature references. If anything, citations here are scant, while not a central issue, additional citations will help contextualize this study by way of comparing/contrasting it with current reports on spikes in domestic violence across in the world - what is now described as a 'shadow pandemic'.
Experimental design: The authors should elucidate on their human subjects protection protocol, COVID has made it precarious to do DV research with women in lockdown. The authors should state what steps were taken to guarantee safe and confidential data gathering (though online) during this time.

It is not clear if one survey instrument captured all outcomes (demographic, economic, socioeconomic, and psychological aspects of married women). On the other hand, it is clear they used a survey by Chinn (2014) to measure overall happiness. The authors should explain what surveys were used, and also define psychometric (reliability and validity) measures for all distinct outcomes instruments used.

The authors mentioned surveys were completed online ("we fielded the survey in the first three weeks of June 2020"), how were participants recruited? What online tools were used?  Were participants compensated? This information is pertinent to the literature and should be included in the methods section.

The authors should clarify what this method is - "iterative BFGS method." Other acronyms should be clarified in their first use.

It is clear there is a substantial emphasis on data analysis methods used, while important for a methodological paper, this hyperfocus on quantitative methods makes it laborious to read portions of this paper. It does not serve the reader to know that "There exist several discrete distributions to model count data such as Poisson, Negative Binomial, zero-inflated Poisson (ZIP) and zero-inflated Negative Binomial (ZINB) distribution." Among other things, all the statistical models are distracting. Consider keeping only information relevant to this paper.
Validity of the findings: The authors state: "Although, the generalizability of our research remains intact for those who cannot use technology or perhaps more domesticated women but even if there is a limit to generalizability we are more interested here to identify the issue of violence and happiness for the particular groups of women who are able to use the technology to fill up the survey. " There are various reasons why women with digital access will be different from women without this access. Thus, the generalizability of findings is in question, given known gender parities with technology access. The authors should consider this a limitation of their data and contextualize their findings as such.

Contradictory finding - the authors should clarify "Unemployed women and women who are not working have higher odds of zero violence as compared to the working woman. These women also had higher odds of facing violence which means that these unemployed and not working women are a vulnerable group"

Results - Findings on anxiety are scattered about the document - the same can be said for empowerment and some other variables under review. The authors should put findings in the same place - under the same header or paragraph to improve readability. For example, it will help if the authors collate the same outcomes under similar topic headers to explain variables. For example, explain demographic, economic, socioeconomic, and psychological variables under Partner Violence dynamics and again under Overall Happiness metrics.
Additional comments: There are several notable strengths of this manuscript. The authors use a zero-inflated negative binomial model to compare the impact of family settings, type of relationships, and age on positive counts of violence during the lockdown; as well as across several crucial metrics as they concern current domestic violence in Pakistan. They also establish determinants of overall happiness - an important and overlooked issue - related to COVID and domestic violence contexts based on demographic, economic, socioeconomic, and psychological attributes of married women in Pakistan. If the aforementioned critiques and commendations are carefully considered, this paper will make an important contribution to the literature at this time.


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 PANDEMIC PARADOX: DOMESTIC VIOLENCE AND HAPPINESS OF WOMEN
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this review, the authors examined the psychological well-being of women and the factors associated with their well-being in Pakistan during the pandemic. The authors provide a strong literature background. This article is well structure. The authors well addressed the research questions in the manuscript. There are several grammar errors in the manuscript. The authors may need to edit it.

The demographic table presents detailed information on the sample recruited in this study. It may be clearer to combine the minimum and maximum into “range” in the table 1. In addition, it is not clear about the variables: “father’s years of schooling” and “mother’s years of schooling”. Authors need to give more definition in the manuscript.
Experimental design: no comment
Validity of the findings: The findings were important for clinical practitioners to better serve local communities. Also, it is crucial for policymakers to make relevant police that women stay safe and happy at home during this challenging time.

The authors may need to more discuss how the findings are implemented in the field.
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 PANDEMIC PARADOX: DOMESTIC VIOLENCE AND HAPPINESS OF WOMEN
Review round: 2
Reviewer: 1

Output:
Basic reporting: Reporting has been improved significantly.
Experimental design: Authors have made efforts to improve the readability of research design.
Validity of the findings: Authors have made efforts to clarify their findings.
Additional comments: The authors addressed most of my comments, but I have a few more minor comments.
1. Variables that measured using Likert scales should be presented in the descriptive table as well.
2. Authors combined results and discussion sections in this revision; however, authors should not do this. The authors should have a separate results section and could combine discussion and conclusions.
3. For the abstract, authors only have backgrounds, methods, and results three sections; however, your main text has four sections (intro, methods, results, discussion and conclusion), you should make changes accordingly. I encourage you to have a discussion subheading in the abstract.
4. Since your other research question was about women’s happiness, your title should reflect that you have examined violence against women and women’s happiness during COVID-19.
5. Authors said, “and for mothers is graduation.” Could you please clarify this?


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

Input:
Title: EXPOSURE TO LOW CONCENTRATIONS OF PESTICIDE STIMULATES ECOLOGICAL FUNCTIONING IN THE DUNG BEETLE ONTHOPHAGUS NUCHICORNIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 42 Remove W.
72-75 Could benefit from a little more detail and more recent references e.g. pasture fouling, greenhouse gas reduction, and pest and parasite control. Although I cannot find studies which specifically focus on O. nuchicornis, similar benefits will be provided by other Onthophagus species for which studies have been completed.
96 Agreed
98-102 For predictions b and c please add reasoning behind these predictions.
170 paper is not in sections, remove.
170 I think this is clearer “The same beetles were used for the second experiment, which began immediately after the first experiment. Dung was defrosted forty-eight hours prior and subsequently homogenized for 10 min, as described above.”
182-183 Remove sentence as described above.
260 missing full stop.
339 beetle*s*
Experimental design: 116 was this horse dung collected from the same facilities? Please specify.
124 how was the dung homogenised?
136 where was the builders sand from, could this have contained any pollutants? Possible cause of high mortality in the control. Worth using children’s play sand in subsequent experiments.
I’m assuming there was no mortality during the first experiment? State this here.
178 – was the paint mixed cleaned before each homogenisation to remove residue ivermectin? How was it cleaned? Was the control group homogenised first?
Validity of the findings: For all analyses, why are beetles divided into ‘small’ and ‘large’? Please give reasoning for not using size (pronotal width) as a continuous trait.
268-269 would be useful to state how ivermectin concentration affected survival in text.
274 Surprising result! Add the effect at the two highest concentrations of ivermectin.
363 What kind of treatment was this? I think it is important to mention that higher concentration of IVM will be present in dung if the treatment is applied topically eg Sommer et al. 1992. Here they found concentrations of 9 mg kg-1 dry weight 24 hours after treatment. This translates to around 2 mg kg-1 wet weight if dung is 20% dry matter.
Sommer, C., Steffansen, B., Nielsen, B., Grønvold, J., Jensen, V.K., Jespersen, B.J., Springborg, J. and Nansen, P. (1992). Ivermectin excreted in cattle dung after subcutaneous injection or pour-on treatment: Concentrations and impact on dung fauna. Bulletin of Entomological Research, 82, 257-264.
365-377 Good to mention here high mortality in control group.
365-377 Although long-term studies looking at several generations of dung beetles have not been completed (to my knowledge), field studies have which compare organic and conventional farms. For example:
Sands B and Wall R (2018) Sustained parasiticide use in cattle farming affects functionally important dung beetle assemblages. Agriculture Ecosystems and Environment 265, 226–235.
I think the possible long-term impact of ivermectin on dung beetles should be mentioned with more emphasis.
Additional comments: Overall an enjoyable read with interesting results. Clear aims and results.


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

Input:
Title: EXPOSURE TO LOW CONCENTRATIONS OF PESTICIDE STIMULATES ECOLOGICAL FUNCTIONING IN THE DUNG BEETLE ONTHOPHAGUS NUCHICORNIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 162 remove space between for and seven

Line 176 replace or with and

Line 303 space before "The greater..."

Line 401 please correct "suppressed"

Please check reference list, e.g. Wohde et al (2016) is cited in line 364 but not listed in reference chapter.
Experimental design: Add moisture content of sheep dung at least or in best case add concentrations of ivermectin also per dry weight dung. This might increase comparability with other studies.
Validity of the findings: No comments
Additional comments: No 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: EXPOSURE TO LOW CONCENTRATIONS OF PESTICIDE STIMULATES ECOLOGICAL FUNCTIONING IN THE DUNG BEETLE ONTHOPHAGUS NUCHICORNIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a well written manuscript and I can find very little to criticize. The experimental approach is nice although, as the authors acknowledge, there are some areas in which it could have been made larger and more comparisons could have been included. The authors do a very nice job of bring in past literature and showing how it could have been used in a similar set of comparisons. This manuscript is also makes use of the data in an ecological manner which is a rather novel approach to using data which is more often used as toxicological information.

I have a number of minor queries that should be addressed.

The following is from the PeerJ guide and should be followed throughout. “For three or fewer authors, list all author names (e.g. Smith, Jones & Johnson, 2004). For four or more, abbreviate with ‘first author’ et al. (e.g. Smith et al., 2005).”

Does dung beetle burial of dung ‘impair development’ of horn fly larvae? I find that this statement is rather odd. I suspect the authors are a bit to cautious; why not call a spade a spade.

Use of the phrase ‘due to’ is not correct. This phrase is used in banking to refer to funds that are ‘due to’ an account and is not indented to be used in any other context.


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: EXPOSURE TO LOW CONCENTRATIONS OF PESTICIDE STIMULATES ECOLOGICAL FUNCTIONING IN THE DUNG BEETLE ONTHOPHAGUS NUCHICORNIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: N/A
Experimental design: N/A
Validity of the findings: N/A
Additional comments: Thank you to the authors for addressing each of my comments. I am now satisfied that the paper is of publishable quality.


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: DETERMINATION OF THE REFERENCE GENES FOR QRT-PCR NORMALIZATION AND EXPRESSION LEVELS OF MAT GENES UNDER VARIOUS CONDITIONS IN ULOCLADIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. The English in this manuscript needs to be greatly improved for overall readability and comprehension.
• Some examples where the language could be improved include (but are not limited to) sentences on lines 41-43, 205, 209, 217, 267-269, 284-286, and 315.

2. The introduction could use some improvement.
• In many places, more recent literature can be cited. Some examples are:
i. Lines 85: Most recent literature cited was from 2006
ii. Lines 89 & 90: Most recent literature cited was from 1998 & 2004
iii. Line 90: No reference provided for “… and fungi.”.
iv. Line 329: No reference provide for “… functional in Ulocladium.”

• The introduction needs to better explain the sexual strategy used by these species. For example, it is not immediately clear that the Ulocladium species being studied exhibit primary homothallism and harbour both MAT1-1-1 and MAT1-2-1 in a single isolate.

• The reference list needs to be checked and corrected in many places. Some examples are:
i. Line 388: “Insights” should be capitalized
ii. Line 414: “The” should be capitalized
iii. Lines 455 – 456: “Diaporthe”, “Phomopsis” and “in vitro” should be italicized, “Their” should be capitalized
iv. Line 493: “mat” and “Neurospora” need to be italicized
v. Line 503: “Aspergillus” needs to be italicized

3. The structure does confirm to PeerJ standards as well as discipline norms.

4. The figure are relevant and illustrate the results. However, the titles and descriptions of the figures are insufficient.
• Figure 1: “Environmental concentrations”  “Environmental conditions”.
• Figure 1: In each graph, the title is “Average expression stability value of remaining control genes”. In this context, what does “remaining” mean, as all 7 genes are included in each condition? Furthermore, these titles can be removed as the overall figure has a relevant title.
• Figure 1: The y-axis labels should include “M” in brackets.

• Figure 2: Each graph has its own title (Determination of the optimal number of control genes for normalization) which can be removed as the overall figure has a relevant title.
• Figure 2: The y-axes need labels and the x-axis labels are not descriptive.

• Figure 3: The y-axis needs a unit of measurement (ΔΔCt)
• Figure 3: Correct “Expression levels of the MAT1-1-1 and MAT1-2-1 genes in Pleospora tarda”.
• Figure 3: What is the relevance of “CK” as the abbreviation for P. tarda?
• Figure 3: Although error bars are shown, statistically significant differences should also be indicated on the graphs- ie: if there is a significant difference in MAT1-1-1 expression between -20'C and 0'C, this should be indicated using either asterisks (*) or symbols (a, b).

• Figure 4: All of the same issues as with Figure 3
• Figure 4: The x-axis label is incorrectly spelt: “Clture”  “Culture”

• Figure 5: All of the same issues as with Figure 3
• Figure 5: Correct “Expression levels of MAT1-1-1 and MAT1-2-1 genes under different photoperiods”.

• Figure 6: All of the same issues as with Figure 3
• Figure 6: Correct “Expression levels of MAT1-1-1 and MAT1-2-1 genes under different CO2 concentrations”.

5. Raw data was supplied
Experimental design: 1. The research presented in this manuscript is original and primary research that represents an impressive addition to the work conducted on the MAT genes and sexual cycle of Ulocladium species in Geng et al 2014 and Wang et al 2017.

2. The research question(s), while relevant and meaningful, need to be better defined. I suggest rewriting the final paragraph of the introduction to better describe what the study aimed to do. It was unclear whether the main aim was to identify usable reference genes or to determine the expression of the MAT genes under different environmental conditions. It is certainly clear that this study aims to address a particular knowledge gap and this was more clearly explained.

3. The research was performed to a high technical standard.

4. Overall, the methods are described with sufficient detail. However, the methods could improve upon in the following ways:
• Line 116 & 256: Two different Ulocladium species are used- however, it is not possible to determine which species is represented in the results. In the discussion, it is further stated that “different fungal species” showed different gene expression levels but this data is not presented.
• Line 117: Explain the relevance of using Pleospora tarda as a sexual representative.
• Under what conditions was P. tarda grown to get the MAT gene expression values?
• Lines 121 – 122: The final sentence of the first paragraph of the materials and methods seems misplaced as RNA is extracted after each of the environmental conditions.
• Line 125: Petri dishes (9cm)- is this the plate diameter?
• Please explain why the treatment experiments were conducted for different time periods: temperature (7 days- Line 127), medium and photoperiod (14 days- Lines 136 & 142), and CO2 (10 days- Line 146).
• What does “proper temperature” (Lines 135, 141 & 148), “optimum media” (Lines 140 & 146) and “proper photoperiod” (Line 148) mean in this context?
• Line 152: What exact material from P. tarda was used to extract RNA? The asci? Or the ascomata? (see Major Flaw comments below)
• Lines 158 – 159: Please add more information regarding what PCRs were done to confirm that no gDNA contamination was present.
• Lines 172 – 174: Move the sentence “Melting curve … reactions” to the results section
• Line 176: Correct the equation to read 2-ΔΔCt and not “2- ΔΔCt”.
• What statistical methods did you use to compare the expression values across experimental treatments? Ie: how did you come to the conclusion that, for example, MAT genes are expressed at a statistically significant higher level on HAY media than on PCA media?
• Line 182: Add a sentence better describing the expression stability measure (M)- what is considered a low/high value?
Validity of the findings: 1. All the underlying data was provided.

2. The majority of the concluding points, including the importance of temperature, media type, photoperiod and CO2 concentrations to the sexuality of Ulocladium species, should be identified as speculative. Additional, the authors should include a statement on what further studies can be done to supporting these speculative conclusions.

3. Many of the conclusions are supporting the results.
• See below the “Major flaw” section in the general comments for author
Additional comments: The manuscript entitled “Low expression levels of MAT genes may be one of the major causes of asexuality in Ulocladium” is a primary research article that makes two important contributions to the field:
1) The manuscript provides important and novel knowledge regarding the use of various housekeeping genes as reference genes for RT-qPCR in Ulocladium species.
2) Additionally, the manuscript sought to identify important environmental conditions that may be conducive to sexual development in Ulocladium species.

Below, I have detailed some suggestions and changes I think are necessary to improve the quality of the manuscript.

Major Flaw:
• One of the major flaws of this manuscript is that the authors have strongly linked high expression levels of the MAT genes to the potential for sexual reproduction. Unless they have reference to support this theory, I suggest that, in fact, MAT gene expression is fairly low in vegetative mycelia and increases upon the onset of sexual development. Thus, I do not think that their claim that “low level expression of the MAT genes is a major cause of asexuality” stands true.

• One of the “supporting results” for this low level expression is the comparison of MAT gene expression in the two Ulocladium species to the expression of the MAT genes in P. tarda. However, from what I can gather from the methods section, RNA extracted from P. tarda was extracted from sexual tissue- which would explain the high levels of MAT gene expression. Thus, this comparison is not biologically relevant.

• Similarly, the authors go as far to use MAT gene expression as a proxy for sexual reproduction. On a number of occasions (Lines 229, 244, 288, 296, 310, 319,), the authors say that conditions that showed the highest MAT gene expression are conducive to sexual reproduction. This is not true, because sexual reproduction and the production of sexual structures was not achieved during the course of this research.

• The authors thus need to tone down the relationship between MAT gene expression levels and the potential for sexual reproduction. Instead, the manuscript should focus on the important contribution of potential HKG for RT-qPCR analysis as well as the different environmental conditions that are not conducive to sexual development.

Minor Flaws:
1. In many cases, the authors use the terms anamorph and teleomorph incorrectly. These terms refer to particular morphs of an isolate- not sexualized tissue.
• Line 152: “teleomorphs of P. tarda” – do they just mean sexual tissue? Ascomata?
• Line 244: “teleomorphs are not sensitive” – teleomorphs were not seen?
• Line 288: “production of teleomorphs” – teleomorphs were not seen?

2. Similarly, the authors mention that sexual structures have been found in asexual species. This clearly means that these species are no asexual. Where this has been stated, adding phrases like “thought to be asexual” would better describe these species (Lines 56, 64, 271 and 272).

3. The authors state at various times (Lines 239 & 310) that the results indicate that light is necessary for sexual development- however, the results actually indicate that a light/dark cycle is important. MAT gene expression is highest when a light/dark cycle is used, not when only light is provided.

4. Line 154 and elsewhere: Please ensure that there is more consistency in your use of “real time”, “reverse transcriptase”, and the abbreviations “RT”, “RT-qPCR” and “qRT-PCR”. Once you have mentioned the technology and its abbreviation once (Line 81), use the abbreviation for all other mentions.

5. Lines 53 & 278: Remove the use of “etc” and replace instead with something like “and others”

6. Lines 326 – 328: The authors state that they “performed extension mating testing by confronting cultures of the same and different strains”. This is never referenced in the methods. Instead, the only methods include plating out a single isolate. This should either be expanded upon or removed.

7. Minor grammatical/spelling errors
• Line 59: Producted  produced
• Line 71: “… and MAT1-2, with”
• Line 73: “… domain, respectively”
• Line 74: lots of  many
• Line 79: “… MAT locus was functional”
• Line 96 – 97: “They are involved in basic”
• Line 100, 201 & 262: “… control gene that is expressed”
• Line 102: “… reference genes under”
• Line 119: “Utrecht, The Netherlands.”
• Line 198: “The seven candidate…”
• Line 292 – 293: “Hypomyces solani var. curcurbitae”
• Line 249: Remove “evolutionary”
• Lines 270 – 271: “… recognized as strictly”
• Line 315: “… production of asexual spores”


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: DETERMINATION OF THE REFERENCE GENES FOR QRT-PCR NORMALIZATION AND EXPRESSION LEVELS OF MAT GENES UNDER VARIOUS CONDITIONS IN ULOCLADIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript may be of interest but this cannot be verified since legends are missing and the figures are by no-way self-explanatory.

The introduction contains a long paragraph on housekeeping genes that seems to directly belong in a textbook, while 76 references occupy nearly one third of the entire document.

The authors do not comply with the one-fungus-one-name paradigm effective as of 1 Jan 2013.
Experimental design: The MS is rather methodological and the results are not very convincing. Both mat1-1-1 and mat 1-2-1 show similar expression patterns under all conditions. Moreover, the structure of the idiomorphs (homothallic vs. heterothallic structure) is missing, which makes things difficult to follow.
Validity of the findings: The authors show qualitative data on housekeeping genes that seem robust.

The expression profiles of both mating type genes does not seem to vary significantly.
Additional comments: The authors are encouraged to have their manuscript reviewed by native English speaking researchers before submission in the future.

Some remarks in the accompanying file might be helpful in improving the document.


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

Input:
Title: DETERMINATION OF THE REFERENCE GENES FOR QRT-PCR NORMALIZATION AND EXPRESSION LEVELS OF MAT GENES UNDER VARIOUS CONDITIONS IN ULOCLADIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Lines 43 – 44: The sentence “Of all known… 76 genera” does not make sense. Please rewrite.

Line 56: The sentence should state “MAT1-1-1 and MAT1-2-1 encode proteins…” – It currently states “MAT1-1 and MAT1-2” which are the idiomorphs, not the genes.

Lines 69 – 71: Change the sentence to read “… is the absence of environmental stimuli, including medium…”

Line 78: Incubating  Incubation

Line 101: Genera  genus

Line 112 – 113: CBS is now called The Westerdijk Institute

Line 147 – 149: Please indicate which PCRs (ie: what primers were used) were performed to determine whether genomic DNA was present. This was not adequately dealt with in the first review. Simply stating that no bands appeared does not confirm the absence of gDNA if the readers don’t know what primers were used.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: No additional comments


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

Input:
Title: EFFECTS OF WHOLE-BODY VIBRATIONS ON NEUROMUSCULAR FATIGUE: A STUDY WITH SETS OF DIFFERENT DURATIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well written overall.
I only have few comments.

Background in the abstract - increasing evidence ...induce a decline of neuromuscular parameters. I could be improved with an acute impairment in neuromuscular function

Introduction , line 34. Not sure about the use of reflectors here. Check english

Lines 48-51. The authors should also mention the differences in protocols and subjects with De Ruiter's findings (in their experiment they had untrained students) but in Maffiuletti's work there was external load added. I suggest the authors clarify this aspect and/or only cite literature with similar protocols and subjects to make their case
Experimental design: The cross-over design is appropriate, the experiment is well described and written so it can be replicated.

The statistical analysis as well, however I am not sure why the authors decided to add the magnitude based inferences approach. I think this makes the findings confusing. I suggest the authors to report the results only in the context of the two-way factorial ANOVA reported and discuss the effect sizes (eta squared) to interpret the magnitude of change rather than adding the magnitude of effects.
Validity of the findings: It is clear that there is no difference between trials and trial x time interaction and therefore the discussion should be focused on that aspect.

What is the biological meaning of a likely small to possibly small fatiguing effect on single twitch?

The comparisons with the results on central fatigue with De Ruiter's work should be discussed possibly due to the difference in the subjects used (the subjects in this study were recreationally trained athletes vs students untrained in De Ruiter's work).

Underlying mechanisms. Improve the first sentence. Probably the authors mean that there seems to be no beneficial effects of using WBV on neuromuscular function due to the lack of difference observed?

Lines 336-339 are a bit confusing, this sentence needs improving it is not clear what the authors are trying to say.

Line 340. Poor English. Are the authors attributing the observed acute decrease in force production in both treatments to an impairment in Ca2+ handling?
line 342- Following the intervention.

The following sentences are a bit too long and have few improvements needed. Also this part should be discussed in the context of the findings. The way it is currently written it is assuming that the intervention (WBV) caused an impairment in neuromuscular function, while the statistics analysis reveals no difference with the sham intervention. So the authors should clarify the last part better and make clear that there was no difference and therefore, the observed reduction over time is possibly caused by the same mechanisms of the sham intervention?

Limitations
While the authors explain well the limitations of the study. I am not sure if the work/rest ratios is an actual limitation. The study was designed for this scope and therefore it was a design choice? I am surprised as well that there was no larger evidence of fatigue with this protocol (180s work - 120s rest).

Conclusions.
As indicated above, results should be discussed in light of the statistical findings and not with the MBI. I have the impression that at all costs there is the willingness to identify a positive aspect when the data are clearly showing the opposite. I am confident the authors can address this properly as negative findings are as important as positive ones.

Also, I am am a bit puzzled of how the findings can be used by practitioners. Since there is no difference between treatments on the acute measures of neuromuscular function, what is the advantage of using vibration with the protocols and equipment used? This should go in the conclusion

My first impression is that probably the sample size was too small despite the a-priori power calculation (Jordan's study used 24 subjects) and they only found a difference in voluntary muscle activation. On what parameter was the power analysis conducted for the Jordan 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: EFFECTS OF WHOLE-BODY VIBRATIONS ON NEUROMUSCULAR FATIGUE: A STUDY WITH SETS OF DIFFERENT DURATIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study is very clear, well written.
Experimental design: No comments.
Validity of the findings: Studies show that more than 10 minutes of WBVE can cause fatigue; why do you use 9 minutes ?
Additional comments: Congratulations. This study is very important to our comunity. We need to know about WBVE and fatigue.


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

Input:
Title: EFFECTS OF WHOLE-BODY VIBRATIONS ON NEUROMUSCULAR FATIGUE: A STUDY WITH SETS OF DIFFERENT DURATIONS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The reviewed paper adheres to all PeerJ policies and within the journal scope. The manuscript is written in the proper English style and conform to professional standards of courtesy and expression. The introduction is sufficient with relevant referenced literature. The structure of the article is typical for an original research article.
Experimental design: The authors define the clear research question, connected with the identified gap of knowledge, presented at the broad background in the introduction. Methods and protocols are described very well, also with very detailed statistical analysis. The measurements performed with high technical and ethical standards.

Minor comments:
Line: 104: „different visits with at least seven days rest in-between.” – please check it with Figure 1A, there is „separated by 6 days rest in between”.
Validity of the findings: The data have been provided, but some of them weren't compatible (I described this at the end in detail) and the figures should be revised. The results are enough to verify hypotheses and make conclusions. The discussion section is prepared nicely. Conclusions are well stated, linked to the research question. The authors have noted some limitations of the study.

Minor comments:
Line 270: „…statistically insignificant for TWPT”. – did you mean „…for TWPT”? {last two letters in subsricpt}

Line 325 and 326 – there is no explanation of ‘LFF’. Is it low-frequency fatigue? Add abbreviation in line 319 - should be enough.

Description of Figure 4: you put one black triangle with one white circle together as p<0,05.

I have one question for the research protocol: only right legs were measured, so are they all were right –legged persons? Did you check it? If yes, describe how; if not, please put some opinion about the lower limb lateralization and its influence on your results.

The other minor comment on data presentation at figures is about showing native units at graphical parts. It suggests that you present raw data or maybe raw differences for readers who didn’t read carefully the description of the figure. As you noted – the graphics show ES as d Cohen value, so that value hasn’t any unit. Please verify this for Figures 3 and 5. Please consider is it necessary to show the same both in numerical and graphical parts? What is ‘MBI’ abbreviation?

The major comment is about data compatibility. I compared the relative changes from baseline between Figure 2 and Table 1 for MVC and %VA. The graphical representations didn’t match numerical values. For example, MVC(%) at Figure 1A-30s set and connected black triangles for WBV. I clearly see that the relative change is bigger at tf15 then tf, and the biggest at tf30 but data from Table 1 are -12.59% at tf, -12.03 at tf15, and -12.41 at tf30; that’s why I’m confused. Look also for %VA where for every set (30, 60, and 180s) I found differences. For example (Fig. 1B, 180s set) relative difference at tf for WBV is a positive value (around 2-3% based on the graph) but Table 1 showed -1.20 value.
I didn't check all values at Figures 2 and 4 with connection to Tables 1 and 2 – now it’s your job to do it precisely.
Additional comments: Summarise my review – the paper is very good but necessary is a major revision on data compatibility between graphical and numerical representation. Other comments are minor.


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

Input:
Title: EFFECTS OF WHOLE-BODY VIBRATIONS ON NEUROMUSCULAR FATIGUE: A STUDY WITH SETS OF DIFFERENT DURATIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The reviewed paper adheres to all PeerJ policies and within the journal scope. The manuscript is written in the proper English style and conform to professional standards of courtesy and expression. The introduction is sufficient with relevant referenced literature. The structure of the article is typical for an original research article.
Experimental design: The authors define the clear research question, connected with the identified gap of knowledge, presented at the broad background in the introduction. Methods and protocols are described very well, also with very detailed statistical analysis. The measurements performed with high technical and ethical standards.
Validity of the findings: The data have been provided. The results are enough to verify hypotheses and make conclusions. The discussion section is prepared nicely. Conclusions are well stated, linked to the research question. The authors have noted some limitations of the study.
Additional comments: Summarise my review – the paper is very good. All previous major and minor recommendations (especially revision on data compatibility between graphical and numerical representation) were made.


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 USE OF ARTIFICIAL SUBSTRATE UNITS TO IMPROVE INVENTORIES OF CRYPTIC CRUSTACEAN SPECIES ON CARIBBEAN CORAL REEFS
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nicely written paper with only a few minor English issues. It is well constructed, covers the appropriate literature, etc.
Experimental design: I have some problems with the design of the study.
The research question is an interesting one and worthy of investigation.
Here are some specific comments.
l. 95. Nice that all organisms were removed by sun drying, but rubble in the sea is not sun-dried, usually, so the collectors won’t start to replicate the rubble zone until there is colonization by microbes and micro-algae, etc. Perhaps having left them in place for two months would have allowed that to happen, but I think that could explain some of the results.
l. 96. I have no problem with the collectors per se, although finding a way to standardize the collector might have been better. But I do wonder about placing the cinder block on the collector in the plastic crate. That will influence access to the rubble in some way, I would expect. Also, the collectors were placed adjacent to the back reef zone, but not in the back reef zone, or for that matter, on the rubble, a part of which was collected for comparative purposes (Fig. 1D).
The biggest problem with this study is that there were too few collectors to accommodate the variability of colonizers. Only two replicates each of two times. Would have been much better to deploy minimum of three collectors, but probably more, up to six, each of two times, maybe three times. I recognize that the person power to handle all those crustaceans might not be present or affordable, but I think one could also narrow the scope of the groups dealt with, or look for the 10-20 most commonly occurring species. It is nice, but not necessary to identify everything that is in the collector as many species are rare, as the authors have seen.
The second problem with the collectors is that they were placed near the back reef zone and not on or near the rubble they were intended to replicate. That most likely cut off a lot of potential colonizers. None of the peracarids have water borne larvae, and some, like the cumaceans, tanaids, etc., tend to be sedentary unless conditions get bad enough to make them move.
The data in Table 1 also suggests that the different crustacean groups respond to the relative newness of the rubble in the collectors in different ways. Cumaceans and taniads like a nice layer of microalgae and fine sediment particles on which to feed. On the other hand, the decapods are more likely looking for space in which to live away from predators.
l. 155-158. There should be some discussion of the taxa that were more abundant in the collectors vs not. Why are they there? I note that tanaids and gnathiids and a few other crustaceans were more abundant in the collectors.
l. 156. No confidence intervals visible in the Fig. 2
l. 193 ff. That is probably due to the small amount of rubble material, thus limiting the habitat space. More rubble collectors would undoubtedly collected more of the species known from the area.
l. 198-199. I don’t agree with this sentence at all. Peracarids are not typically found in early successional stages of habitats. Some species are, but the vast majority have very specific habitat requirements. In studies of recovery of bottom habitats after trawling, for example, it has been shown that cumaceans are the last group to arrive, long after the worms. Same with a number of the amphipod taxa.
l. 202-205. Yes, but it is also likely that rubble gets well “glued” to the bottom as it were during periods of relative calm due to microbial and microalgal growth. Those periods can last for months or, in the absence of strong winter storms or hurricanes, maybe even years. During those periods one would expect the rubble community to reach some state of stability with a different cryptofauna than what might be expected show up in rubble baked in the sun and placed in bags.
l. 212-226. Exactly the point.
l. 230. Chondrochelia dubia is probably a weedy species. Early and fast colonizer of new habitat space.
Validity of the findings: In the end, I am not sure that these collectors do what I think the authors want them to do. That is, provide an inventory of species from the rubble without destructive sampling. Perhaps a better designed, and aged collector would do a better job. For example, the collector could be dipped in fresh water to remove all the macrofauna, then placed in bags with 0.5 mm mesh, or smaller, and then allowed to incubate in the water for a month or two to allow the turf to develop. Then they could be placed on the bottom to see what they collect. If the objective is to get a good inventory of the cryptofauna without destructive sampling, then the cryptic habitat provided needs to be a good approximation of the real thing.
Additional comments: This is a very promising preliminary study in my view.  I think you have the potential to develop a cryptofauna method, similar to the ARMS for larger organisms (that also have problems in my opinion) that can be made by everyone and used all over the world. The advantage is standardization that allows comparison.
I would urge you to continue to develop your method.


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 USE OF ARTIFICIAL SUBSTRATE UNITS TO IMPROVE INVENTORIES OF CRYPTIC CRUSTACEAN SPECIES ON CARIBBEAN CORAL REEFS
Review round: 1
Reviewer: 2

Output:
Basic reporting: While the majority of this manuscript is well written, there are several sentences that could use reworking. Similarly, the manuscript is well-cited and the authors possess an good understanding of the literature, though there are some gaps that should be corrected, particularly with reference to prior studies that use artificial reef substrates to assess decapod diversity.
Experimental design: I am extremely impressed with the amount of work that has gone into identification of the decapod specimens collected in this study. I have some concerns with replication and the clarity of the goals, specifically the definition of what a "good" collector is. It's not clear to me if the aim is to collect the most species or minimize variance, or create a natural habitat with real-world assemblages of cryptic organisms.
Validity of the findings: Please see specific comments below with respect to replication and recommended additional analysis, as well as clarifications.
Additional comments: This paper examines communities of cryptic decapod crustaceans associated with rubble and artificial collectors in order to assess the biodiversity of this community, as well as the efficacy of the aforementioned methodologies. This manuscript focuses on an extremely understudied community, especially in the Caribbean where they have been sampled less than in the Pacific. As such, this paper represents an important contribution and great effort was taken to identify and process so many individuals from so many species. I have several concerns pertaining to sampling effort and design, as well as the results and corresponding conclusions. I hope and trust that they can be sufficiently addressed before publication.

Line 1. Suggest: “Are artificial substrates effective tools for estimating crustacean cryptofauna diversity on coral reefs?”
Line 16. They comprise a group of organisms largely overlooked in biodiversity estimates because they are hard to collect and identify.
Line 18. That may not be sustainable in light of the widespread degradation of coral reef habitat.
Line 20: examines
Line 23: the Simpson’s … and the Shannon-Wiener
Line 24: Separate species or taxa? If not all identified to species, could say at least 178 different species, belonging to five orders of Crustacea were collected.
Line 26. Again, “taxa richness” should be substituted with “richness” or “species richness”
Line 28: reword
Line 31: “and sampling of animals associated with coral rubble”
Line 38 “Cryptofauna are”
Line 38: The part of this sentence on fishes and reef-building organisms is not needed an arbitrary. There are numerous other taxa on reefs and therefore highlighting just these two groups is ineffective.
Line 40: largest is not specific, you mean most biodiverse? Most speciose? Most abundant?
Line 41: replace “either in” with “including within gaps”
Line 47-50: This sentence needs to be cleaned up, perhaps broken into two
Line 53: Technically, I would say that Enochs et al. 2011 was more sustainable than Enochs and Manzello 2012 or Enochs 2012 (Marine Biology, not cited) in that it just used rubble in bags, that were then returned. The other studies involved sampling of framework and are therefore more destructive, though similar to this study, rubble was a large component of that...
Line 58: I think it is critical that you incorporate the work of Plaisance and others that have used NOAA and Smithsonian artificial reef monitoring structures (ARMS) to monitor cryptofauna biodiversity (primarily decapods like this study). These structures are probably the most widely-used, ubiquitous structures around the world, and are definitely worth discussing in this paper.
Line 62: Inappropriate use of the word factual.
Line 63-64: This makes it seem like the references are about artificial collectors that use rubble, rather than just the rubble itself.
Line 62-68: It strikes me that Enochs et al. 2011 employs artificial bags of rubble, directly as discussed here, rather than references to the rubble. There are also other studies that have used rubble bags as well, both in the Pacific and to a lesser extent in the Caribbean. I would suggest highlighting them as you are introducing the topic here.
Line 95: Are there permitting issues with this type of collection, drying, and redeployment? Numerous organisms naturally grow on this substrate and aerial exposure can be somewhat destructive (and smell bad).
Line 96: Are there shading concerns from the block?
Line 98: From this I am interpreting that the effective sample size is two for each of these timepoints and eight in total? Please clarify here and in the text. This is a little lower than I am comfortable with given the high variability observed within this community (especially over time) and may have ramifications for statistical analysis.
Line 101: Can you report the average depth of the rubble collected? Not water depth, but the actual depth of the layer of rubble on the sea floor. 50cm high rubble bag collectors may provide more shelter per unit area, but they also may have more restricted water exchange, and less epibenthic flora/fauna for organisms to feed on. In essence, if you sample three kilograms of rubble, how spread out of an area are you talking about vs. the 40 cm diameter of the collector?
Line 101: Can you speak to the structural similarity, perhaps size frequency distribution or volume of the rubble used in bags vs. collected? Were they the same general sizes? I think this is important for exploring the similarity of the approaches, as well as differences in the habitat/sheltering potential. Also, a detailed description of the rubble is necessary for general understanding of the habitat sampled. What species of coral and sizes? Was it highly eroded or recently dead and intact? Was it covered with algae or sponges, or bare and uncolonized?
Line 108: Was the rubble reused in the next deployment of the bags? If so, was there mortality of the sessile organisms or did that community mature over the subsequent sampling events throughout the year.
Line 119: Is there a citation for Hill numbers? I am not familiar with them.
Line 123: Why extreme transformation?
Line 126: This statement isn’t clear to me? All time points were pooled or they were pooled only within time points?
Line 127: P values equal to or less than
Line 137: great!
Line 141: wording
Line 143: Wow, well done.
Line 144: The taxonomic composition of the samples taken using the two methodologies
Line 152: , as well as animals belonging to Mollusca, Polychaeta, and Echinodermata
Line 155 and Line 163: Are you confident in the statistical power and sample sizes used to assess this or do you think the lack of significance is reflective of replication
Line 157. nMDS or other multivariate ordination would be an effective method to assess variation in community composition, showing overlap and separation between the two methodologies.
Lines 160: This underscores my question of habitat depth and the similarities in substrate porosity/ shelter potential
Line 185: reword
Line 189: I don’t agree with this. Just because they are not common in the samples does not mean that they were transient or opportunistic. Maybe they are rare, or their preferred food source wasn’t on the rubble. Maybe sampling effort wasn’t high enough to collect patchily distributed communities. There are lots of potential reasons this could be the case
Line 208: et al.
Line 209. Great paragraph and discussion
Line 218, not just algae. Other sessile organisms may be important food sources as well.
Line 224: Do you have data on this?
Line 228: I think just tanaids is preferable as their classification as shrimps is colloquial and factually incorrect.
Line 233: Yes abundant species are recruiting to them, but also rare. I also think the last part of the sentence is not necessarily correct. There could still be an effect in their colonization as their abundances, sizes, etc. could be different.
Line 237: reword
Line 241: How do you know that great replication or longer deployment time would have yielded the same results? Do you have enough data to state that the substrate/collector is the difference, not the amount of material or time of community development/succession?
Line 241: I think you need to be more clear on what the actual goal is. Are you trying to collect the maximum number of decapod species possible or are you trying to determine if the 2-month collector deployment reflects the same species composition, diversity, and abundances? These are different goals and the conclusions as to the effectiveness of each are different. It also strikes me that part of the “effectiveness” of the collectors is their community variance and sensitivity to environmental differences/change. Are successive collections similar enough that they can be used to detect the influence of environmental gradients? That would be very valuable or “effective” and wouldn’t necessarily result in the highest number of species collected. I think community similarity should be investigated further, and I think ordination would be helpful.
Line 266: substitute access for sample
Line 254: What about flow, porosity, and benthic cover as investigated in Enochs et al. 2011?


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 USE OF ARTIFICIAL SUBSTRATE UNITS TO IMPROVE INVENTORIES OF CRYPTIC CRUSTACEAN SPECIES ON CARIBBEAN CORAL REEFS
Review round: 2
Reviewer: 1

Output:
Basic reporting: This second version is much more modest in its goals. The paper is well written with only a few sentences that need checking for English. There were no page or line numbers but an example is "Cumacea was...".
Literature sufficient, article structure very professional, paper easy to read.
Figure legends are missing from revised draft.
Experimental design: The authors have reduced their goals due to the very limited sampling.
The design obviously cannot be changed at this time, so how to make the best of the data they have. I think they have done what can be done but need to add a few more thoughts to the Discussion.
Validity of the findings: The MDS tells us something the authors could point out. They do say that the ASU and the CG provide mostly different fauna lists. They admit that could be due to their limited sampling. But they should also talk about sampling issues for the cryptofauna in general. Why would the two sampling methods produce such differences in faunal composition. I disagree with the authors that there is much overlap of the two faunal lists, but that's not as important as understanding the differences. There is some discussion of that but that discussion avoids a question of how many samples would it take to get all the fauna.

Figure 3 suggests that it would not take many in order to account for most of the species. The rarefaction curve, however, is an estimate of species accumulation. The MDS was run on species abundance data, not presence-absence data, and it shows that the ASUs don't help to estimate community structure of the cryptofauna, i.e., there is very little overlap of the two data sets. That is a difference that I think should be pointed out. Might also be worth running the MDS on presence data. I suspect there would be a greater overlap. The raw counts for the samples are not provided in the supplemental data so that can't be checked. In any case, it does seem like it would take a lot of ASUs to provide estimates of the community structure as seen in the coral gravel samples.
Additional comments: This is much better and more modest. I think the idea of using ASUs to sample this habitat is an interesting idea, but you need to strengthen your case that it will be better than sampling the coral gravel itself, which is the conservation goal (I am very interested in conservation but I am not so sure that much is lost by sampling the coral gravel... you could take 10 samples of 250 cc and not make much of an impact on that habitat, although that might not be legal, as you point out). To replace that sampling, many more ASUs will need to be used.

All that said, I am pleased that someone is paying attention to the cryptofauna. Please keep working on this topic.


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 USE OF ARTIFICIAL SUBSTRATE UNITS TO IMPROVE INVENTORIES OF CRYPTIC CRUSTACEAN SPECIES ON CARIBBEAN CORAL REEFS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I think this manuscript is greatly improved, minor comments are provided directly below.
Experimental design: The shift in direction and design of the current study are entirely appropriate and I commend the authors for making the difficult decision to do so, as well as the work involved with revising the paper. I think it has been a worthwhile endeavor.
Validity of the findings: See comments below. Findings are sound.
Additional comments: Suggest: The use of artificial substrate units to the improve species inventories of …
-or- improve inventories of cryptic crustacean species on …
Line 23: can cause disturbance that is unsustainable in light of widespread
Line 25: coral rubble
Line 27: the composition
Line 31: rubble not gravel, here and throughout.
Line 34: with sampling of the surrounding…
Line 44: comma after citation, before “with”
Line 44: Suggest checking out the following reference: Small AM, Adey WH, Spoon D (1998) Are current estimates of coral reef biodiversity too low? The view through the window of a microcosm. Atoll Res Bull 458:1-20.
Line 55: write out numbers less than 10… here and throughout.
Line 58: crustaceans
Line 61: Not entirely fair to single these authors out as the only destructive samplers. Numerous people have sampled rubble (including Monroy-Velazques et al. 2017) and live corals as they did.
Line 72: Wrong paper. Enochs et al. 2011 used artificial rubble bags.
Line 78: Suggest checking out: Valles H, Kramer DL, Hunte W (2006) A standard unit for monitoring recruitment of fishes to coral reef rubble. J Exp Mar Biol Ecol 336:171-183.
Line 79: Suggest different word than “dominant.” Not clear what you mean. I’m guessing most abundant substrate, though the references cited indicate “play an important role in harboring diverse cryptofaunal communities.
Line 116: Length? Width? Circumference?
Line 136: Need to give brief description here so the reader can figure out what you did without reading the other paper. Okay to provide less details and point the reader to the other paper but still need a synopsis.
Line 146. Suggest new sentence
Line 149: I would suggest sqrt transformation to decrease the potential for extremely abundant species to strongly skew your assemblage comparison. This is pretty common practice. It may also help to deal with the very high stress in your nmds plot.
Line 188: italicize.
Lines 194: and 205: Odd spacing.
Line 235: Looking for space is conversational. Suggest “recruiting to ASUs for shelter from predators” …though previous studies have found abundant cryptic predator populations. I also think that it is entirely possible that herbivorous, detritivorous, and carnivorous species were actively foraging within the ASU’s. Trophic consideration of the decapods considered may be helpful in making this case.
Line 251: reference?
Line 278: Not sure if I follow. What if the most abundant species are dependent on a food source that takes time to recruit to the ASU’s?


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: IDENTIFYING MMP14 AND COL12A1 AS A POTENTIAL COMBINATION OF PROGNOSTIC BIOMARKERS IN PANCREATIC DUCTAL ADENOCARCINOMA USING INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript meets basic reporting criteria. The current literature in the field is nicely described with relevant citations. The research question is clearly outlined.
Experimental design: Authors use previously published data and perform a comprehensive bioinformatics analysis to study the differential expression in genes under PC vs normal tissue. All the methods are well described with enough details for the data reproducibility and transparency.
Validity of the findings: The findings are interesting and may help understand the biology of PC in more details, but certainly lack biochemical validation by authors.
Additional comments: Manuscript by Ding and colleagues, presents a bioinformatics study using the previously published microarray datasets on Pancreatic ductal adenocarcinoma (PDAC). Authors identify several differentially expressed genes (DEGs) followed by attempts to understand their biological functions and relevance. The authors further show that the expression of two DEGs viz. COL12A1 and MMP14 was correlated with a poor prognosis in PDAC and poor overall survival; presenting these two DEGs as potential biomarkers for PDAC.
1) The manuscript presents some interesting findings, but it certainly lacks biochemical validation of the differential expression of the genes identified. It mostly represents a consolidation of the previously published dataset and previously published literature (data mining). The manuscript would gain much if authors validate these findings by some experimental evidence like RT-qPCR, immunoblot analyses. Authors may also take a few candidate genes and show any mechanistic data that these are somehow regulated/regulating in PC development. For example – lines 196-207 and lines 208-223 describe speculative mechanistic implications of the findings. Can authors test some of these ideas?
2) Authors should explain the rationale of using the GEO dataset viz. GSE28735, GSE62165, and  GSE91035. Why did they select these datasets? Also, it would be very relevant to discuss what were the main findings from the papers that generated these datasets.
3) Figure 1: Are these overlaps statistically significant?
4) Figure 7: Can authors quantify the signals?
5) What are the Y-axes in Figure 6?


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: IDENTIFYING MMP14 AND COL12A1 AS A POTENTIAL COMBINATION OF PROGNOSTIC BIOMARKERS IN PANCREATIC DUCTAL ADENOCARCINOMA USING INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Ding et.al. identified 20 differentially expressed genes in Pancreatic ductal adenocarcinoma patients, wherein, MMP14 and COL12A1 is associated with poor overall survival, and these might be a combination of prognostic biomarkers in PDAC. In addition, FBN1 and COL8A1 are probably novel biomarkers of PDAC. Overall, the authors listed a lot of work in the text, I would suggest the author to purify their result and clearly show the most interesting ones.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: IDENTIFYING MMP14 AND COL12A1 AS A POTENTIAL COMBINATION OF PROGNOSTIC BIOMARKERS IN PANCREATIC DUCTAL ADENOCARCINOMA USING INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors conducted extensive bioinformatics analysis to identify key genes and cellular pathways that play a role in development and progression of pancreatic ductal adenocarcinoma. The authors analyzed 3 different microarray datasets to identify differentially expressed genes. They further used STRING to establish protein-protein interaction networks and performed GO analysis and KEGG pathway enrichment analysis to identify the most significantly enriched biological processes, molecular functions, cellular components, and cellular pathways in PDAC. They validated their findings using the TCGA and GTex databases as well as the expressed proteins using the HPA website. They also investigated the impact of the expression levels of their top 20 hub genes on the overall survival. Considering the notorious lethality of PDAC, it is important to perform bioinformatic studies such as the work in this manuscript to contribute to the current efforts to identify prognostic biomarkers. This work is within the scope of PeerJ journal. The manuscript is written in professional English and the structure conforms to PeerJ standard. Most figures are of publication quality and appropriately annotated. Most of the raw data relevant to the manuscript is available. A few points are noted below which must be addressed prior to acceptance.
1.	Figure 2C: Please add a color-key explaining what the different shades indicate in terms of the scores for each hub gene.
2.	Figure 6: The individual images are of good quality, however, the ones in the PDF are not clear. The images in the PDF need to be improved in terms of their readability. Also, please describe what the colors (red and gray) indicate.
3.	Section 3.4: Please provide overall survival data for other hub genes as well.
Experimental design: The authors have defined the objective of the study well. The bioinformatic analyses are well structured and seem to be performed accurately following standard procedures in the field. However, the authors must clarify the following points to improve the overall clarity and transparency of the manuscript.
1.	Section 2.1: The authors have used three data sets- GSE28735, GSE62165 and GSE91035 while there are several other datasets related to pancreatic cancer available in the GEO database. The authors must provide the rationale for choosing these particular datasets.
2.	Section 2.2: Please explain the method used to obtain the adjusted p-values
3.	Section 2.3: Please provide the version number of the cytoscape software used
Validity of the findings: Overall, the results are detailed and presented in a clear manner for the most part. Appropriate statistical analyses have been performed and reported in accordance with standards in the field. The discussion of results is thorough, and the conclusion answers the original research question. There are some points outlined below which must be rectified/ clarified prior to acceptance of the manuscript.
1.	Line 149: “Enrichment of KEGG….” This sentence is not required here and fits better in the next section where the authors have already explained this in lines 161-163.
2.	Section 3.3: Is there a set cut-off based on which certain functions or pathways are designated as “enriched”? Based on figure 3 & 4, it appears that to call a certain function or pathway “enriched”, the p value needs to be less than 0.0001. The authors should clearly state what the criteria is. If it is the case that anything with p<0.0001 is considered “enriched”, then why is ‘endodermal cell differentiation’ not considered in the hub genes?
3.	Section 3.4: Provide overall survival data for other hub genes as well. Line 168 should say “…demonstrated that the high expression …”
4.	Section 3.5: CXCL12 is missing from this list of genes. Is there any specific reason for this or is this an accidental omission? If it was accidentally omitted, please, add a panel for this gene in figure 6 and comment on the expression trend.
5.	Section 3.6: Certain panels in figure 7 do not support the conclusions drawn. For FBN1, both normal and cancer panels look identical. I believe, the same image might have been pasted in both cases. The separate images supplied as supplemental files with the manuscript looks correct. Please correct the ‘normal’ panel for FBN1. Based on the images, CDH1 looks to be overexpressed in cancer tissue whereas the results state that this protein was not expressed in PDAC or normal tissues. Please clarify this ambiguity. Similarly, ALB appears to be upregulated in cancer tissue compared to the normal tissue. Please clarify this ambiguity.
6.	For tables 1 & 2: Add another column listing the different genes involved in each BP, CC and MF terms as well as pathways. This makes it easier to tie it back to the discussion section.
7.	Line 205-207: The basis for this statement is not clear. The authors should elaborate how they concluded that interaction of MMP14 with Ca2+ can serve as a biomarker. Please provide overall survival data supporting this claim.
8.	Line 246: please cite the full reference.
9.	Line 291: “FBN1 and COL8A1 might be biomarkers of PDAC”. The basis of this statement is not clear. How do the authors explain this conclusion given that in section 3.6 they mention that the protein product of COL8A1 was not expressed in cancer or normal tissue? Please provide a more detailed justification for this statement.
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: IDENTIFYING MMP14 AND COL12A1 AS A POTENTIAL COMBINATION OF PROGNOSTIC BIOMARKERS IN PANCREATIC DUCTAL ADENOCARCINOMA USING INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript meets the basic criteria of reporting.
Experimental design: The manuscript employs sound experimental design.
Validity of the findings: The findings are valid and supported by the experimental results.
Additional comments: In this revised version, the authors have addressed the concerns raised by the reviewers. I recommend this article for publication 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: IDENTIFYING MMP14 AND COL12A1 AS A POTENTIAL COMBINATION OF PROGNOSTIC BIOMARKERS IN PANCREATIC DUCTAL ADENOCARCINOMA USING INTEGRATED BIOINFORMATICS ANALYSIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript and the rebuttal letter address the previously raised concerns appropriately.
Experimental design: The revised manuscript and the rebuttal letter address the previously raised concerns appropriately.
Validity of the findings: The revised manuscript and the rebuttal letter address the previously raised concerns appropriately.
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 META-ANALYSIS CONTRASTING ACTIVE VERSUS PASSIVE RESTORATION PRACTICES IN DRYLAND AGRICULTURAL ECOSYSTEMS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This is a global meta-analysis and synthesis of research on restoration practices in agricultural dryland ecosystems. It looked at the outcomes and effectiveness of active vs passive restoration practices. The meta-analysis resulted in 42 peer-reviewed articles containing 1,427 independent observations (or data entries) from which the restoration practice, goal, and response for each practice was assembled. Passive restoration focused on soil, vegetation , and grazing exclusion, while active restoration focused on soil, vegetation and water supplementation.

The paper is clearly written and the results are important and compelling.  I just have just one suggestion for improvement:

A bit more explanation and contextualization of the results would help. For example, it would be good to add a few specific examples from the actual studies to highlight the restoration effects that are summarized in the meta-analysis results.  Give a case study or two that shows how and why soil restoration was done and how it produced the desired effects.


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 META-ANALYSIS CONTRASTING ACTIVE VERSUS PASSIVE RESTORATION PRACTICES IN DRYLAND AGRICULTURAL ECOSYSTEMS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The work is to a high standard, but perhaps missing some small details to add some depth to the analysis.

Results section: A table listing the countries (to complement the map) and No. of cases per country and categories of practice per country would be useful.

A paragraph explicitly acknowledging the limitations of the study and the methodology would be welcome at some point in the text.

L 303 – 305 (Word doc) This needs to be stated much sooner, if biodiversity is not considered and instead only abundance. As you state the importance of drylands ecologically in the introduction, and it is not mentioned in the methods that biodiversity was not considered.

L 51 – 52 (Word doc) It is important here to contextualise between intensive agricultural activities and extensive/traditional agricultural activities in dry lands as causes of degredation

L88 – 91 (Word doc) While agricultural intensification is responsible for degradation, traditional livestock systems can be used to increase biodiversity and the exclusion of livestock causes a decrease in biodiversity and an increase in abundance of a few species. Please try to contextualize the difference between traditional and intensive agricultural land use.

L 91 – 93 (Word doc)  Please mention here if this land is to be retired because it is unproductive or if there are other factors socio-economic factors such as an aging population and lack of succession options. Due to the previous sentence it reads like this land in California is going to be retired exclusively because they are no longer productive.

L136 (Word doc) These categories represent your goals well, but I think differentiating those systems that could be considered intensive vs traditional or extensive would add depth to the analysis. Equally a list of the crops or species uses in the systems prior to restoration activities could  help contextualize your study.