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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 1
Reviewer: 4

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Line 147 Could you the authors consider adding the following sentences?

With the successful passaging of the cultures, the cells are considering a cell line, following the convention of the Society of In Vitro Biology (Schaeffer, 1990).  The cell line has been designated Ptskf. (Ptskf is just a suggestion).

Lines 302 Could you the authors consider adding the following sentences?
The cell line can be considered the first constituent of the peccary invitrome and a resource for future studies in many disciplines (Barioch, 2018; Bols et al., 2017).

Lines 353-354 Could the authors recast the sentence as follows?  However, fibroblasts detach in response to trypsin more rapidly than epithelial cells and adhere more quickly (Bai et al., 2012).

Could the authors please consider adding the following references?
Barioch A (2018). The Cellosaurus: a cell knowledge resource.  J Biomol Tech 29: 25-38.

Bols NC, Pham PH, Dayeh VR & LEJ Lee (2017). Invitromatics, invitrome, and invitroomics:  Introduction of three new terms for in vitro biology and illustration of their use with the cell lines from rainbow trout. In Vitro Cellular & Developmental Biology-Animal 53: 383-405.

Schaeffer WI (1990) Terminology associated with cell, tissue and organ culture, molecular biology and molecular genetics. In Vitro Cell Dev Biol 26: 97-101.


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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed my comments and suggestions.
Experimental design: The methods have been improved accordingly.
Validity of the findings: Results are now supported by the images.
Additional comments: The authors have addressed my comments and suggestions. The manuscript has been improved accordingly.


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: ISOLATION, CHARACTERIZATION, AND CRYOPRESERVATION OF COLLARED PECCARY SKIN-DERIVED FIBROBLAST CELL LINES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have adequately responded to the proposed corrections and suggestions, have resolved the errata and have successfully discussed some problematic aspects of the article.
Experimental design: ok
Validity of the findings: ok
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: IDENTIFICATION OF DEGS AND TRANSCRIPTION FACTORS INVOLVED IN H. PYLORI-ASSOCIATED INFLAMMATION AND THEIR RELEVANCE WITH GASTRIC CANCER
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1. This manuscript needs extensive English language editing. The current version is full of ambiguous and inaccurate expressions that seriously impede understanding.

2. Figure 4 and 5 are extremely difficult to read and understand. There are many good examples of making a good/legible figure of gene regulation networks. The authors are encouraged to look them up online and see how to adjust figure parameters such as font size, node shape/size, edge line width etc. to improve their network representations.

3.  Non-atrophic gastritis (NAG) and atrophic gastritis (AG) are two important concepts and are referred to frequently in the paper. However, the authors did not give enough background information regarding them in the introduction, e.g. what are they? What are the relationships between the two? How they are related to H. pylori infection? In addition, I think the claim that H. pylori infection relates to NAG and AG (Line 62) is not supported by the cited reference (i.e. Hatakeyama 2017). This reference mainly focuses on a H. pylori protein not the forms of gastritis caused by its infection.

4. The authors need to reformat their references in accordance with the style of PeerJ. In the current version, all authors of the cited papers are listed (and with no separation between names), e.g. in Line 101-102, this makes the paper difficult to read. Plus, the first reference (Line 303) is of a different form than the others.

5. In Line 71 the authors need to cite the original publication where the Correa model was proposed.
Experimental design: 1. The authors used two sets of microarray data from the public database GEO. However, the original label of these datasets are different from what the authors described in the paper (Line 93-97). For the data set with the GEO accession number GSE27411 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27411), indeed there are three balanced groups, namely non-infected, NAG, and AG, but only three patients were chosen for each group and biopsies were collected from two different places from each selected patient. This means the six tissues in each group are not entirely independent. Assuming independency of the tissues could lead to wrong hypothesis testing results. The authors need to disclose how exactly they analyzed this set of data and what error correlation structure they used to model the random errors.
For the data set with accession number GSE60662 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60662), the actual label of the samples are: four replicates of the control, four replicates of mild gastritis, four replicates of severe gastritis, and four replicates of intestinal metaplasia. The authors seem to equate mild and severe gastritis with NAG and intestinal metaplasia with AG. Is it justified? If so, please provide justification and cite supporting evidence in literature.

2. In line 130, the authors describe how they identify differentially expressed genes from the TCGA database by using the same criteria as they used for the two GEO datasets, i.e. FDR<0.05 and |logFC|>1. The problem is that the calculated FDR is not dataset independent, meaning that the larger the dataset, the more pairwise comparisons and statistical hypothesis tests need to be performed, the more correction for the false discovery due to performing multiple tests, and thus the harsher the criteria becomes. That is to say, many DEGs identified in the two GEO datasets would not be identified to be differentially expressed in the TCGA dataset simply because TCGA (data acquired by RNAseq) contains expression information of a lot more genes than the two microarray datasets and not because they are no longer differentially expressed between GC and non-GC samples. A better way to assess DEGs in NAG and AG samples is to directly perform hypothesis test of these selected genes to see if they are differentially expressed in TCGA dataset without the need to resort to FDR.

3. I am not sure how the survival analysis shown in Figure 6 were performed. The authors claims that there are 18 GC specimen with H. pylori infection found in TCGA, however, it is obvious from Figure 6 that the number of data points used to generate the survival curves are far more than mere eighteen. Where are those extra points come from? Are they from GC patient without H. pylori infection? If so, how relevant the lessons learned from them are to H. pylori infection?
Validity of the findings: 1. There are 234 genes identified to be DEGs from dataset GSE27411(Line 141) and yet more than 10 fold more identified from dataset GSE60662 (2425, Line 143). Can the authors provide explanations for this discrepancy, in particular does it mean one study is superior to the other and is it still valid to simple take intersection of DEGs between the two studies?

2. Line 151 says 292 DEGs showing lower gene expression were obtained by intersecting 1376 (Line 149) and 143 (Line 147) DEGs. How can it be possible?
Additional comments: The authors purported that the goal is to understand critical gene regulation in different phases of GC development caused by H. pylori infection. To achieve the goal, the authors made several pairwise comparisons, i.e. NAG vs normal, AG vs normal, NAG vs GC, AG vs GC. I wonder what are the justifications for some of the comparisons. If the development of GC follows (according to the Correa model mentioned in Line 71): H. pylori infection to NAG, then to AG, and then to GC, should it be more informative to compare NAG vs AG than normal vs AG?


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

Input:
Title: IDENTIFICATION OF DEGS AND TRANSCRIPTION FACTORS INVOLVED IN H. PYLORI-ASSOCIATED INFLAMMATION AND THEIR RELEVANCE WITH GASTRIC CANCER
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors identified key genes that play an important role in the molecular mechanism of H. pylori-associated inflammation to cancer. Overall, the manuscript is very poorly written and lacks clarity with no complete detail description of the methods. The data presented seem to be difficult to interpret for the readers.
Experimental design: In the material and methods section, the authors do not provide a detailed description of the methods. Please provide all the details for complete understanding.
Validity of the findings: No comments
Additional comments: 1. The research problem is not presented clearly. The background, purpose, and conclusion of the study are missing from the abstract section. Overall, the abstract section is written poorly.
2. Results and Discussion section in the manuscript are poorly-written; very hard to interpret the text. Re-write this section.
3. I found a research article (Hu et al Helicobacter. 2018;23:e12530) which also highlighted the important genes in Helicobacter pylori-associated gastric cancer based on The Cancer Genome Atlas database and RNA sequencing data. Please cite this paper.
Also, comment on the difference in the identified genes in your study compared to their study.
4. References are not properly cited. Authors have missed important published research articles supporting their findings. For ex., there is a published article that highlights the role of high expression of FoxP3 in tumor cells predicts better survival in GC. Please cite this research article: Ma, G., Miao, Q., Liu, Y. et al. Br J Cancer 110, 1552–1560 (2014).


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

Input:
Title: IDENTIFICATION OF DEGS AND TRANSCRIPTION FACTORS INVOLVED IN H. PYLORI-ASSOCIATED INFLAMMATION AND THEIR RELEVANCE WITH GASTRIC CANCER
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. The manuscript is much improved but there are still places with obvious grammatic errors and vague expressions that may impede and mislead readers. Here are a few of them. Please proofread the manuscript more carefully.
Line 37, “association of JUN were positively” should be “association of JUN was positive”
Line 52, “investigating … is of great importance for (understanding) the occurrence and progression of GC”
Line 257, “It implied us that the prognosis of patients …” should be “It indicates that the prognosis of patients …”

2. Figure 4 & 5 look much better. But it is hard to read the name of those genes that are placed on top and at the bottom of the big circle. If these names are staggered or placed to an angle, they would be offset from each other and not overlapping with their neighbors.
Experimental design: NA
Validity of the findings: NA
Additional comments: NA


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

Input:
Title: IDENTIFICATION OF DEGS AND TRANSCRIPTION FACTORS INVOLVED IN H. PYLORI-ASSOCIATED INFLAMMATION AND THEIR RELEVANCE WITH GASTRIC CANCER
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The revised manuscript is now very clear with the proper usage of the English language. The authors have now addressed all the critical issues raised by both reviewers.
However, I noticed some minor grammatical errors which are highlighted in the attached pdf file which needs to be fixed in the final manuscript file.


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

Input:
Title: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 1
Reviewer: 1

Output:
Basic reporting: I find the writing to be unclear in various key sections (including methods). I elaborate on this in my comments to the author.

The referenced literature does not adequately address the large body of work in which a predation risk framework is used to predict and interpret behavioral responses to human disturbance.

The article has an adequate structure and raw data are shared.

The manuscript is self-contained and an appropriate unit of publication.
Experimental design: The manuscript represents  original research within the aims and scope of the journal.

Research questions are well defined and relevant. The text identifies the knowledge gap being addressed.

While the investigation is generally rigorous, I do raise issues with some of the methodology used (see comments to author).

Not all aspects of methods were described adequately. (see comments to author)
Validity of the findings: While the study is potentially important for evidence-based marine conservation, I do raise questions about the validity of some of the findings (see comments to author).

Data are provided and their collection appears to be sound (with some clarifications required). I do question, however, the validity of some of the analyses (see comments to author)

Conclusions vary in the extent to which I agree with them in terms of support from the results (see comments to author)

Speculation is not unreasonable.
Additional comments: This manuscript reflects a lot of intricate data collection occurring both in the field and while processing stereo camera images, and I commend the authors for it. The study’s objectives can be summarized as 1) testing whether the minimum approach distance (MAD) between divers and individual fish is higher where fish are exploited by divers with spearfishing gear than in protected areas, and 2) whether the explanatory power of species distribution models increases if MAD is used as a predictor. The first objective is corroborative of earlier studies, while the second is more novel.

While the manuscript has the potential to contribute to evidence-based marine conservation, its current form is insufficient to do so for the following reasons. First, the manuscript is written in a vacuum of the predation risk framework used to predict and interpret the behavior of fish or other animals in response to disturbance by humans (e.g. (Frid & Dill 2002; Samia et al. 2019; Spitz et al. 2019). The application of this framework may help interpret the extent to which species distribution models reflect actual abundances vs bias in survey counts, and potentially lead to correction factors (see, for instance, Frid et al (2019)). Additionally, a predation risk framework could guide discussion on the extent to which spatial variation in MAD may reflect the costs of antipredator behaviour on body condition and reproduction (Heithaus et al. 2008; e.g. Spitz et al. 2019), and how these factors (in conjunction with fishery removals) may influence the results of species distribution models.

I am also concerned about aspects of the analyses which were explained poorly or which are potentially inadequate. Additionally, the writing is unclear or insufficient on various conceptual and methodological aspects essential to the study. Below I elaborate on these points.

Introduction.
In general, the context of predation risk and its applications to the study’s objectives should be added (see my earlier comments).

L38-40 state that “Fishing pressure is a primary driver, not only of fish distributions (Jennings and Polunin 1996, Friedlander and DeMartini 2002), but also of fish behavior (Kulbicki 1998).” As implied by the referenced literature, this statement applies to tropical reefs where spearfishing is the dominant exploitation method. In most other parts of the world, however, most fishing occurs via deployment from vessels of other gear types (nets, hooks). Thus, all aspects of the paper (including title and abstract) should be re-written more transparently about the more limited scope of the paper (i.e., explicit to areas where spearfishing by divers is the dominant exploitation method).

L52-64: while the rebreather vs non-rebreather example is interesting the inherent mechanism on MAD differs from that of risk from spear fishers, and this needs to be contextualized.

L57-58: The definition of ‘minimum approach distance’ (MAD) is essential, yet here it is introduced merely as “the distance between the diver and the fish at its closest point”, which is ambiguous and potentially problematic. The definition should specify that MAD is the distance from the observer at which the individual fish engage on avoidance behavior (i.e. flee). This definition makes it explicit when fish chose to incur the costs of antipredator behavior. If the authors expect fleeing to not occur in a subset of observations, then they should specify so, which would lead to additional analyses of fleeing vs no fleeing as a binomial response variable and including only fleeing observation in analyses of MAD as a continuous variable. This is important, as individuals that are approach to a given distance and do not flee are choosing a very different set of trade-offs than individuals approached to the same distance but that stay in place.

L73-79: This paragraph should be written to reduce redundancies.

Field Methods
L82-91: It would be useful to have more detail on the level of protection/permitted activities in protected vs unprotected sites, and whether enforcement/monitoring resources differed between protected areas. The discussion brings up potential differences in fisher compliance; if there was an a priori reason to suspect these differences, it would be good to specify that here.

L94-91: While the length and width of transect are clearly specified, the height is not. Fig 2 shows a cuboid for the transect design, implying that a height and volume are inherent to transects; details/clarification on this should be provided. (Lines 107 refer only to standardized area surveyed, not volume).

L110-113: Here and in Fig 2 there is ambiguity on how the 10-m-length limit of observations related to the 25-m-length of belt transects. Are you dividing the 25-m-length into 2.5 segments? How are you then combining the segments into measures that represent the transects?

L114: define MHI

L134-136: Again, this is an incomplete definition of MAD. See my earlier comments.

Analysis methods
L144-145, If “site” refers to one of two locations (Pūpūkea vs. Hanauma), each including a no-take reserve and an adjacent control area where fishing is permitted, then specify so. Also specify that management refers to protected vs fished (otherwise it is unclear that each “site” includes 2 “management” treatments.)

L148-152 states that “Linear mixed models (LMMs) were used to compare patterns of MAD between sites and management types and assess relationships with fish body length, angle of approach, and water depth. These variables were included as fixed factors in the models, while transect (location) and species were included as random factors. LMMs were developed with the combined data from both sites and for each site separately.”
--First, if what you mean is that site and management type were the main predictors of interest, but other predictors also had to be considered then clarify.
--If the site and management type are the main predictors of interest, why was the above approach taken when it would have been more parsimonious and direct to use a single LMM including management type (protected vs fished) and site (Pūpūkea vs. Hanauma) as fixed effects? Also, form the results tables I see that an interaction between site and management type was included, but this is not clarified in the text.
--It is unclear why transect is used as a random effect. Is this to account for spatial autocorrelation between fish responses recorded at the transect level? Given that transects are nested within sites, this would affect the model’s ability to test for differences between sites (i.e. random effects are somewhat confounded with the fixed effects). An alternative would be to use lat-long matern function that uses the distance between dive sites to account for spatial autocorrelation .

L152-154: It is generally unadvisable to transform the response variable without changing the model structure. (i.e. using glmmPQL instead might be warranted: https://ase.tufts.edu/gsc/gradresources/guidetomixedmodelsinr/mixed%20model%20guide.html

L154-158: It is unclear an ANOVA framework was used to test the relative weight of evidence for various predictors when there are more modern approaches available (i.e. AICc model selection), and why 2 separate sets of GLMMs had to be used. My view is that much more succinct and parsimonious approach would be if a single LMM is used with site, management type, the interaction between the latter two variables, fish body length, angle of approach, and water depth as fixed effects and species as random effects, and then eliminating unnecessary fixed effects via AIC model selection procedures. After fitting the GLM, spatial patterns in the residuals can be examined; if they are significant, then investigate the lat-long matern function.

L165-165: Briefly explain why fourth transformation was required.

L167-168: briefly explain the model simplification process.

L178. Readers are referred to Stamoulis et al. 2018 and to Table 1 for an explanation of habitat variables, but more detail should be given. An appendix with brief definitions of each habitat variable would be helpful.

L185-193. A subset of species distribution models included both management type and MAD. Yet, following the main premise of the paper, MAD and management type are expected to be correlated. The text should clarify how the model selection procedures address this issue.

L215-223. Given my earlier comments on the analysis of MAD as a response variable, I am not confident that these results provide the best statistical inferences from the data.

Discussion
L242-261. Broader context should be given on how MAD response to divers may differ in exploited areas where non-spearfishing methods are used (see my earlier comments).

L264-268. It is good to see reference to Clark 1994 to bring a predation risk context in which larger fish have more reproductive assets to protect and therefore are expected to take less risks. An alternative explanation, which is not mutually exclusive with the former, is that larger fish are targeted for exploitation, and therefore larger fish are more willing to incur fleeing costs than smaller fish.

L269-271: the effects of approach angle are interpreted as “… likely a result of the methodology as opposed to a behavioral response.” While this may be partly true (MAD cannot be smaller than the nearest distance between the fish and the trajectory of the diver), it is also important to state that the predation risk framework predicts a greater MAD when approaches are more direct because a direct approach may convey detection and intent to capture (e.g. Frid and Dill 2002).

L293-295 states that “… management type was not selected as a final predictor for any models meaning it was a comparatively poor predictor of targeted species biomass,” but the interpretation should be more nuanced. It follows from the main premise of the paper that MAD and management type are correlated with each other, and caution is required in interpreting the effects of these variables; it is simplistic to interpret management type (i.e. spatial protection) as a poor predictor of biomass.

L 311-315. Interpretation of the results of species distribution models can be enhanced by using the predation risk framework. As specified in my earlier comments, that framework can guide discussion on the extent to which lower biomass in exploited areas may reflect fishery removals or the combined effects of such removals and the costs of antipredator behaviour, and may also help generate bias correction factors that account for fish behaviors that can be related to characteristics of individual and or species that affect investment in antipredator behavior.

References
Frid, A. & Dill, L. (2002). Human-caused disturbance stimuli as a form of predation risk. Ecol. Soc., 6.
Frid, A., McGreer, M. & Frid, T. (2019). Chasing the light: Positive bias in camera-based surveys of groundfish examined as risk-foraging trade-offs. Biol. Conserv., 231, 133–138.
Heithaus, M.R., Frid, A., Wirsing, A.J. & Worm, B. (2008). Predicting ecological consequences of marine top predator declines. Trends Ecol. Evol., 23.
Samia, D.S.M., Bessa, E., Blumstein, D.T., Nunes, J.A.C.C., Azzurro, E., Morroni, L., et al. (2019). A meta-analysis of fish behavioural reaction to underwater human presence. Fish Fish., 0.
Spitz, D.B., Rowland, M.M., Clark, D.A., Wisdom, M.J., Smith, J.B., Brown, C.L., et al. (2019). Behavioral changes and nutritional consequences to elk (Cervus canadensis) avoiding perceived risk from human hunters. Ecosphere, 10, e02864.


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: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Nicely written.

Introduction is quite clear, but would profit from a more detailed explanation of how SDMs are constructed and used. What predictors are included? How are they combined? What are the outputs? How are they used?

L 63 Need to emphasize that the estimates of biomass differed with survey method, not actual biomass: “…biomass *estimates* of some targeted reef fishes were significantly lower on SCUBA compared to CCR…”
Experimental design: L 82 Study sites: It is important to state clearly whether activities involving divers/snorkelers in the water (such as spearfishing and feeding) are permitted or not in both reserves.

We also need to know the extent to which any prohibitions are enforced within reserves.

Methods: Survey locations are densely distributed within reserves (especially Hanauma Bay), leading to some concerns about pseudoreplication. Please address this possibility, because it could severely restrict the variability in biomass in reserve areas.

Assigning a MAD value of 10m to fishes that were not seen assumes that the fish were present, creating a datum out of no information. Since fishing lowers fish density, isn’t it preferable to simply not record a MAD for fishes that were not seen? To me, this is a major problem, and it also has the possibility of creating an odd variance structure, with a high number of values at the 10m truncation point. How often did this happen?
Validity of the findings: Results:
L 205 Please explain what “targeted by fishers” means. Fishing can include many methods (nets, hook and line) which should not affect fish wariness of divers.

L 209 What is an “open area”? Does it contrast with a closed area?

Discussion:
L 250 – 254 If enforcement at Pūpūkea has been documented as lax, it is not clear why it was included in the binary classification of reserve or no reserve. A better approach that directly addresses the hypothesis would be to estimate the actual fishing pressure on the four areas and use this as a predictor. This would eliminate the need for this speculation.

L 265 “Optimal fitness theory predicts that as reproductive value increases, risk-taking should decrease (Clark 1994).” This is unclear at several levels. First, it is not clear what “optimal fitness theory” is – fitness should be maximized when fitness components are optimized. Second, reproductive value (expected total future reproduction discounted by the probability of being alive) usually is maximum at maturity and declines thereafter, and does not necessarily increase with size. Larger fish are not necessarily more wary because they have higher fecundity. They can detect threats from further away, and they are targeted more heavily than smaller fish.

L 281 “targeted fishes in these marine reserves are likely habituated to non-aggressive human interactions, leading to reduced MAD” This implies that human factors other than fishing affect MAD, yet the introduction and general premise of this paper considers only fishing. Certainly, habituation to humans (or even attraction) has been considered before in the literature, and should be considered here rather than simply mentioned as speculation.

L 293 “In contrast, management type was not selected as a final predictor for any models meaning it was a comparatively poor predictor of targeted species biomass.” True, but this paper points out that management effectiveness differed dramatically between reserve sites, so that a dichotomous “management type” designation is misleading.

L 312 “from the direct effects of fishing pressure, for which MAD provides a proxy.” Again, it is not clear  that MAD is a proxy for fishing only.

L 355 – 358 Since this is a multi-authored paper, it is not clear why the Acknowledgements shifts to the use of first person.
Additional comments: This is a useful demonstration of the potential of using a wariness measure (MAD) to improve the predictive performance of Species Distribution Models. However, I have several reservations about methods and assumptions, outlined above, which may improve the paper.


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: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Overall I found this manuscript to be well-written, however, there is some overuse of acronyms throughout. In particular, I would like to see no acronyms in the figures, table captions, and the abstract. These are areas that are communicated to the public, and acronyms are the quickest path to losing your readers' interest. I would suggest that the authors attempt to remove all acronyms throughout their manuscript as well, as we are not worried about word counts these days.

While the authors are correct in their coverage of the literature of diver effects on species distribution models, there is a wealth of research on the effect of diving and spearfishing on the behaviours of fish. Rather than focus their topic on just the specific case of research on diver relationships with species distribution, I would like to see a broader examination of the literature that discussed the impacts of diving and spearfishing on behaviour. Since this is a journal with a broad focus, I believe that would be more appropriate.

In particular, I think there needs to be some discussion of the relationship between size of fish/diver and approach distance. There's good evidence to suggest larger fishes are less affected by divers/spearfishers but small fishes can still display significant behavioural changes to divers, despite not being targeted by spearfishers (ie Bradley, D., Papastamatiou, Y.P. and Caselle, J.E., 2017. No persistent behavioural effects of SCUBA diving on reef sharks. Marine Ecology Progress Series, 567, pp.173-184. vs Benevides, L.J., Cardozo-Ferreira, G.C., Ferreira, C.E.L., Pereira, P.H.C., Pinto, T.K. and Sampaio, C.L.S., 2019. Fear-induced behavioural modifications in damselfishes can be diver-triggered. Journal of Experimental Marine Biology and Ecology, 514, pp.34-40.) and that smaller divers may have less of an impact, as implied by ROV surveys (ie Raoult, V., Tosetto, L., Harvey, C., Nelson, T.M., Reed, J., Parikh, A., Chan, A.J., Smith, T.M. and Williamson, J.E., 2020. Remotely operated vehicles as alternatives to snorkellers for video-based marine research. Journal of Experimental Marine Biology and Ecology, 522, p.151253.), and I think this will require a bit more discussion.

For clarity, I would also suggest the authors change all references of 'fishing pressure' to 'sprearfishing pressure'. The two are fairly distinct and generally one does not associate fishing pressure with impacts on fish behaviour towards divers (I come from a commercial fisheries research background).

I really like the effort the authors went to present their figures so clearly, well done.
Experimental design: Overall, the experimental design and the methods are well explained. One significant aspect I would like to see discussed more is the size ranges of the fishes at each site, which is a confounding factor that, while size has been included in models, it's not clear whether an interaction term between site and size was included.

If spearfishing is having an impact on populations, presumably it is selecting for larger individuals first. Since there is a clear relationship here between size and approach distance, size selection could have affected the patterns more than changes in behaviour. The authors need to present information (ideally simple stats tests and a figure) demonstrating that there is or is not a relationship between mean size and site, and see if including interaction terms between size and site in their model has a significant effect. Otherwise, the claim that fish behaviour is different rather than biomass and size could be misleading.

Since camera can have an effect on accuracy of measurements, I would like to see the model type as well as the lens (focal length included), resolution and framerate used to capture video.
Validity of the findings: In general, the authors are thorough in presenting their results. As indicated above, I would like to see the potential confounding effects of size discussed more thoroughly so that the authors are certain that they are not providing misleading claims regarding behaviour.
Additional comments: The authors present a study documenting fish approach distance to divers in reefs that are protected from spearfishing and reefs that are exposed. The presentation of the figures and results are professional and thorough. In general, my concerns are minor, but some possible confounding factors with fish size between sites need to be examined and tested in their models to ensure their claim that behaviour is changed rather than fish size is valid.


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: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The revised manuscript is much improved but some issues remain (detailed below).
Experimental design: The revised manuscript has clarified a number of issues about the data collection and analysis. However, as detailed below, I am not convinced that both the GLMM and GLM are needed to analyze drivers of MAD.
Validity of the findings: Except questioning the need for the GLM, I find the findings valid and relevant to conservation.
Additional comments: The authors have done a thorough job addressing reviewer comments and the manuscript is much improved. However, now that other matters have been cleared, I can see that the manuscript is hampered by the conflicting results on the effect of management and angle of approach on MAD, which depend on whether a GLMM or GLM is used (Tables 3 vs 4). The manuscript would be clearer and more succinct by describing the drivers of MAD with the GLMM only (Table 3). The GLMM uses “individual measurements of MAD” while for the GLM “MAD was averaged at the transect level.” According to Table 3, in the GLMM the drivers of MAD are site, fish length, depth, and angle of approach. According to Table 4, in the GLM fish length, depth and management are the drivers (angle drops from the model while management stays). This raises two questions. The first is, which model is a better representation of reality? My view is that the GLMM—which accounts (via random effects) for interspecific variability in MAD and for variable numbers of fish observed per transect—is the better representation. The second question: Why use the GLMM and the GLM if one is more appropriate than the other? In lines 207-208 we are told that MAD must be averaged by transect to be used in the SDM, but that does not resolve the above issues. First, the authors should explain why SDM require that transect-averaged value. If it is because the spatial units used for the SDM are the 25m X 5m dive transects, then this should be specified. Second, it still does not explain why two models with the same predictors were chosen to analyse MAD. If the GLMM is the most appropriate model, then why not use only that one and then simply use the transect-averaged values of MAD for the SDM? Unless the authors have a clear answer to the final question, I strongly recommend dropping the GLM and revising the text accordingly.

Other comments are:
L151-153 states that “Fishes located greater than 10 m in front or 2.5 m to the left or right of the stereo-DOV system as it was moved along the transect were excluded based on minimum visibility encountered and transect dimensions.” It is unclear how “minimum” visibility fits here. If visibility was >10 m during better days but only 10 m during poorer days, and therefore—for consistency—you excluded fish recorded during better days >10 m from the cameras, then clarify the text accordingly.

L237-241. As mentioned in my introductory comments, the spatial units used for the SDMs need to be specified. If they the 25 X 5 m dive transects, then say so.

L275-284 and L300-302. If my advice on using only the GLMM to describe drivers of MAD is followed, then these sections could be rewritten with greater clarity and precision.

L309-“fish drive” requires explanation.

L336: I suggest qualifying the statement as “may have more experience”

L352- the prediction from that literature is for greater flight initiation distance (not MAD). While FID and MAD are related, they are not the same thing and the authors should qualify that sentence accordingly.
L373-5 states that “management status explains variation only between management strata.”
 This is very unclear. What is management strata?

Fig 3. It would be better to use the same scales for y axes


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: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Excellent responses to nearly all concerns
Experimental design: No further comment
Validity of the findings: No further comment
Additional comments: In general, the authors have worked very hard to improve this paper. Most of my observations and comments have been adequately addressed. I do have one recommendation to make the paper more palatable to evolutionary ecologists:
L 334 “Optimal fitness theory predicts that as reproductive value increases (Birkeland and Dayton 2005), risk-taking should decrease (Clark 1994).”
As I mentioned before, as far as I can tell there is no such thing as “optimal fitness theory”. Certainly, Clark does not use this phrase, and it does not turn up in any searches I conducted.
As for reproductive value, it can increase with fish size if fecundity increases exponentially with size (as in indeterminant growth) and there is no truncation in survival. This occurs in Clark’s models, and forms the basis for the prediction about risk-taking.
The Birkeland and Dayton paper may be a good reference for dramatic increases in fecundity with size, but it may also contain some misinformation. In the rebuttal the present authors mention “that larger fish produce larvae with higher survivor potential”, and refer to Birkeland and Dayton. This idea remains highly controversial (see, for example, DJ Marshall et al. 2010, The relationship between maternal phenotype and offspring quality: do older mothers really produce the best offspring? Ecology 91:2862-2873). Rather than opening this can of worms, might I suggest a rewording?
“Reproductive value often increases with size in fishes (Birkeland and Dayton 2005), and theory predicts that risk-taking should decrease at higher levels of reproductive value (Clark 1994).”


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: INCORPORATING REEF FISH AVOIDANCE BEHAVIOR IMPROVES ACCURACY OF SPECIES DISTRIBUTION MODELS
Review round: 3
Reviewer: 1

Output:
Basic reporting: To my satisfaction
Experimental design: To my satisfaction
Validity of the findings: To my satisfaction
Additional comments: I am satisfied with the revisions. Thank you for the hard work!


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

Input:
Title: ADDRESSING INITIALISATION UNCERTAINTY FOR END-TO-END ECOSYSTEM MODELS: APPLICATION TO THE CHATHAM RISE ATLANTIS MODEL
Review round: 1
Reviewer: 1

Output:
Basic reporting: A more rigorous approach to defining and testing for chaos and stability is needed. Please see attached document for specific advice.
Experimental design: Generally well designed and carried out, some specifics early in the methods and intro would go a long ways to improving the MS
Validity of the findings: As presented, findings are reasonable and sound.
Additional comments: I appreciate the amount of work that went into this effort, my comments are generally related to clarity. I also suggest a judicious amount  be moved to supplementary material.


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: ADDRESSING INITIALISATION UNCERTAINTY FOR END-TO-END ECOSYSTEM MODELS: APPLICATION TO THE CHATHAM RISE ATLANTIS MODEL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript uses clear and unambiguous language throughout most of the paper. Adequate background is provided. Figures and table are adequate.  Largely this is a well-written manuscript; however, the structure could be improved and some references were missing.
My main concern is about the organization of the manuscript. The title led me to believe that the primary focus of the manuscript would be about approaches to exploring sensitivity to initial conditions of an ecosystem model, i.e., approaches applied to a specific model that could be applied to other similar models. The Abstract followed this logic – general information on sensitivity to initial conditions and the problems that might cause for ecosystem models, information on the specific model being used, and then recommendations on using perturbation analysis for Atlantis ecosystem models. The Discussion also followed this order – general information, specific information, and general recommendations. However, the Introduction dives right into specifics of the Chatham Rise Ecosystem, followed by general information on model stability and sensitivity to initial conditions, then reverts back to specific information on the Chatham Rise Atlantis model, and finally fairly specific recommendations about the Chatham Rise Ecosystem.
The Introduction left me wondering whether this paper is supposed to be about a broadly applicable demonstration of approaches for exploring model stability or just specific approach that can only be used for this particular Chatham Rise model. If the former is the authors’ intention, I recommend restructuring the Introduction and including a bit more general background on Atlantis models.
I also noted several missing reference citations (they just showed up as “?” in the pdf I had access to). These reference concerns and other comments are included in an annotated pdf that I submitted.
Experimental design: The work presented in the manuscript is within the Aims and Scope of the journal.  The research questions are fairly clear, but see concerns noted in the Basic Reporting section of the review. The methods are rigorous and relatively clear, some additional information is needed and noted in an annotated pdf of the manuscript.
Validity of the findings: The authors have interpreted their results in a reasonable manner.
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: ADDRESSING INITIALISATION UNCERTAINTY FOR END-TO-END ECOSYSTEM MODELS: APPLICATION TO THE CHATHAM RISE ATLANTIS MODEL
Review round: 2
Reviewer: 1

Output:
Basic reporting: Much better, but still some changes to be made to improve readability and flow of the paper, see attached suggestions.
Experimental design: Sound and better explained in this version
Validity of the findings: Unchanged from previous version, sound.
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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED METHYLATED GENES IN MELANOMA VERSUS NEVI USING BIOINFORMATICS METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see general comments for the author
Experimental design: Please see general comments for the author
Validity of the findings: Please see general comments for the author
Additional comments: The authors present a bioinformatics study investigating the differentially expressed genes and differentially methylated genes in four datasets re-analysed from the NCBI GEO database. The authors used the NCBI GEO built in comparison tool (GEO2R), to identify significantly differentially expressed genes and significantly differentially methylated genes between benign nevi and malignant melanoma. The authors go on to define a “hypermethylation-low expression” group and a “hypomethylation-high expression” group which they subsequently subjected to gene ontology analysis, STRING analysis and hub gene analysis. Finally, the authors identified nine key hub proteins which they checked against TCGA and GTEx data using another online tool and found them to have a significant effect on survival.
There are several areas within this study that require major attention and/or clarification.

1.	There is an entire dataset missing from the supplementary files. GSE3189, GSE86355 and GSE120878 are all present but the fourth dataset appears to be GSM46517 where it should be GSE46517 (GSE indicated a data series whereas GSM indicates just one sample). Either the wrong data set has been uploaded or the wrong dataset has been analysed from the start.

2.	The Venn diagrams presented in figure 1 could not be reproduced with the data provided (disregarding the incorrect dataset provided outlined in 1st point) (appendix 1, incorrect dataset shaded in red). The entire datasets provided in the supplementary files were uploaded to the online tool the authors describe in their methods to recreate the Venn diagrams. Although no cut-off filter was used (as they outline in their methods), less overlap was achieved (highlighted with yellow circles), where equal or more overlap would be expected.

3.	The STRING figures 4 and 5 appear different than would be expected from STRING or cytoscape. Figures 4 and 5 do not match the theme of either cytoscape or STRING and therefore the authors should specify how these networks were visually created. If they were produced using additional software, the authors should note this.

4.	The authors report on non-significant findings without specifying their significance value. Lines 141:145 and 193:194; although the authors don’t specifically state significance, they should explicitly indicate that three of the five pathways they mention are not significant (table 2: pvalues=0.056, 0.085, 0.079)

Other areas of consideration include:
1.	Line 86, authors state “corresponding DNA methylation microarrays are…”. This is misleading as it implies the methylation data has come from the same samples as the gene expression data, which I believe is not true.

2.	No clarification of the parameters used for STRING analysis. The authors should clarify what parameters the string analysis was set to i.e. interaction score threshold and indeed, interaction sources used.

3.	Line 159; “nine hub genes were screened out”. How were they screened out?

4.	Table 3 appears in the manuscript after table 4.

5.	Consider removing module 2 from figure 4(c). very weak module and the authors do not discuss.

6.	Figure 1: the authors should make the labels in the same place for each Venn diagram to aid readability.

7.	Figure 2 & 3: invert the figure. The lower p-values (i.e. higher log10 p-values) should be at the top of their respective groups, not the bottom. Furthermore, text size appears to be different between ontology groups, this should be rectified.

8.	Figure 4: would aid in understandability if authors would highlight modules within the network. Furthermore, a scale is needed to indicated what the colours of each node mean (edges per node?).

In conclusion, I believe the most interesting aspect of this study is the identification of hub genes and their affect on survival in TCGA and GTEx data. Prior analyses, although some required to obtain the hub genes, are rather unremarkable and mundane. Furthermore, other studies have tried similar approaches, with no mention or comparison (PMID: 29302192, GEO:GSE31879).


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

Input:
Title: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED METHYLATED GENES IN MELANOMA VERSUS NEVI USING BIOINFORMATICS METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The paper was well written with very few typos. The authors provided sufficient background knowledge and references for the context of the paper. The structure of the article is clear and the figures reflect the results generated from the experiments of the paper. All raw data was provided or was referenced to. Most of the results were relevant to the hypothesis of the paper although the methodology needs to be improved.
Experimental design: Uncovering the differences between nevi and primary melanoma is an important question in the melanoma field. As the transition from nevi to melanoma is not a very well understood process, this question is important and could have implications for the risk factors for developing melanoma. The authors attempted to integrate DNA methylation and gene expression data from published GEO datasets to address the differential methylation and gene expression between melanocytic nevi and primary melanoma to better understand the etiology and pathogenesis of primary melanoma.

The methods used in the paper were mostly correct. One of the main weaknesses in the methodology is not having paired samples for DNA methylation and gene expression. To circumvent this problem the authors analyzed 4 independent datasets separately to obtain the differentially expressed genes or methylated regions and then overlapped the gene sets.

For the target gene cut-offs, I would recommend using logFC as a cutoff instead of the moderated t-statistic, it is more biological meaningful. I would also suggest the authors perform further analysis of the differential methylation data. Using the moderated t-statistic or logFC as a cut-off is fine as a first step, but it doesn't reflect of the biological methylation status of the group since it is just a ratio and the absolute values of methylation aren't considered. Generally in DNA methylation, B values of less than 0.3 are considered hypo-methylation and B values of more than 0.7 are considered hypermethylation. For instance, if the FC between the groups is 3, it could signify a B value difference of 0.1 vs 0.3 or 0.3 vs 0.9. In the first scenario, I wouldn't consider it as a biological difference in the methylation status since 0.1 and 0.3 would both be considered as hypomethylation, whereas 0.3 vs 0.9 would give an actual biological difference in the methylation state. Due to this change, the authors will have to repeat the downstream analysis as the genes from the differential methylation analysis will change. It is also unclear how the authors took into account of the multiple probes from the methylation array that match to a single gene and if they considered CpG structures such as CpG Islands and Shores.

The authors use of cBioportal for validation is puzzling to me. As far as I know, the paired methylation data and gene expression data from cBioportal for melanoma are based from the TCGA which are tumor only samples and do not contain nevi. I don't think this is the correct dataset for validation as the authors want to look for correlation in nevi and primary. For survival analysis, the tool GEPIA uses TCGA data for survival analysis. Most of the melanoma samples in the TCGA are from metastatic patients, I would recommend the authors just use the primary samples for survival analysis, as it would be more relevant to their research question.
Validity of the findings: Since the methodology for differential methylation needs to be corrected and as well as the gene selection for differential gene expression using logFC, the current findings aren't interpretable.
Additional comments: I would recommend consulting a bioinformatician experienced in DNA methylation analysis to help analyze and interpret the differential methylation data. There are other specialized tools available for differential methylation analysis of the Illumina 450k platform. Although limma is a valid tool for log2 transformed B values


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

Input:
Title: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED METHYLATED GENES IN MELANOMA VERSUS NEVI USING BIOINFORMATICS METHODS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: I question the biological significance of the study if there were very few genes that passed the logFC cutoff. Although t-statistic can be used as a filter, as the authors suggested it has a tendency to choose genes with low expression. This would suggest that the differentially expressed genes between melanoma and nevi are not highly expressed which makes me doubt if these genes have a functional role in the disease.

Regarding the methylation analysis. There are several pipelines that analyze Illumina 450k data, here is are 2 review that summarizes a few of them.
https://dx.doi.org/10.1186%2Fs12859-018-2096-3
https://doi.org/10.1016/j.ymeth.2014.08.011

One of the issues still standing is the differential methylation analysis. Typically, CpG islands are more reliable than a single CpG probe and these CpG islands should be in the promoter regions of the gene of interest. I've attached a reference regarding this type of analysis. https://www.nature.com/articles/ng.865

Illumina provides a reference database for their probes in context to the gene. In the GSE86355 dataset this in under the column UCSC_RefGene_Group, GSE120878 is missing this column. I inspected the 9 hub genes in GSE86355 and CDC6, CENPF, GPI, VARS have significant down regulated probes located in the gene body or 3' UTR. See attached pdf.

I would suggest that the authors redo the methylation analysis by looking at differentially methylated regions by filtering for CpG probes in the 5'UTR and the TSS200, 1000 and 1500 regions. If the authors still prefer to analyze their data by probe, I would recommend to have at least more than 1 significant probe per gene as a very lax filter for hypo or hypermethylation.

As for the analysis of just the primary cases in the TCGA dataset. The authors can use this workflow to extract just the primary cases for SKCM from the GDC and analyze the survival differences. https://f1000research.com/articles/5-1542
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: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED METHYLATED GENES IN MELANOMA VERSUS NEVI USING BIOINFORMATICS METHODS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The background provided is sufficient to clarify the basis of the paper and the references are relevant and plentiful. Some clarification needed at points within the paper that lack good grammar (see comments for authors). Very few typos. The figures are relevant to the paper but the quality and resolution of figures 2 and 6 are very poor (I can’t read figure 6 plot titles to identify the genes).
Experimental design: The biggest issue with this paper is the inability to recreate the results from the data provided. The supplementary files for methylation and gene expression data offer different gene identifiers and so Venn diagrams obviously cannot be reproduced. It also warrants an explanation as to how the authors did this analysis themselves (with different identifiers). The authors should check the supplementary files (specifically the methylation data) and upload the exact data that was used to create the Venn diagram as until this point, it is unclear how the authors achieved the initial results, which subsequently puts the rest of the paper into question. I suggest the authors use gene symbols as the identifiers as most readers would prefer this format.

Nonetheless, as long as this clarification is addressed, the authors present a bioinformatics study which investigates the gene expression/methylation differences between naevi and primary melanoma ultimately identifying hypomethylated-high expression and hypermethylated-low expression genes of importance which they then go on to highlight  that several have an effect on survival in melanoma patients. The analysis is straight forward and takes a sensible approach to answer the question at hand.
Validity of the findings: The authors describe their results with good detail and in a concise manner. The authors discuss their results and relate them to findings in the current literature to an acceptable degree. There are several issues with the proposed figures (outlined in comments for authors).
Additional comments: The area requiring attention from the authors are as follows:

Major:
The authors must provide the data used to create the venn diagrams so that the reader can reliably reproduce the results. To do this the identifiers must be consistent as at the moment, the expression uses gene symbol and the methylation data uses RefGene name.

Minor:
Line 50: The sentence “While most melanomas are primary tumours, approximately 33% develop from nevi” should be altered. I understand what the authors mean but all cancers arise from a primary tumour (even if that primary tumour arises from a naevus). Consider changing this to “approximately 33% of all primary melanomas arise from naevi”

Line 105: As per my previous recommendation, the authors have detailed the cutoff and evidence used in the STRING analysis. Whilst this is good, text mining, neighbourhood, gene fusion and co-occurrence is not ideal to use as evidence. I would suggest the authors try the analysis without these features and see how much it affects the results. If it makes little difference the authors should either state this or consider re-analysis. If the differences are major, the authors can leave the results as they are (the data is still reproducible as the authors have specified the parameters).

Line 138: The sentence beginning “But there are little significant pathways…” is grammatically incorrect and requires a re-write.

Line 148: The sentence beginning “Primary melanoma samples from TCGA was…” is grammatically incorrect and requires a re-write.

Line 192: I appreciate the authors removed the second module which had no significant enrichment but on line 192 where they state there are two significant modules, then only go on to talk about module one may confuse the reader and make them confused as to where the second is. I advise a clarification somewhere; i.e. “ two significant modules were identified within the hypomethylation-high expression genes, of which, one module exhibited enrichment for several gene ontology terms”.

Line 200: It is unclear what the authors mean in the sentence beginning; “in this study, we screened out genes….”.

Line 207: typo; glioms – glioma/gliomas

Figure 1: I advised in the previous review that the authors make the labels on the venn diagrams consistent between A and B as to aid in cross-reading between them. This has not been done. For clarification, the left most circle on both A and B plots should read the same GEO data set (i.e. GSE86355), so should the next circle, and the next, etc...

Figure 2 & 3: The authors have put figure 3 in decreasing -log10 pvalue, as per my initial recommendations but have not done this for figure 2, please could the authors do this. The axis titles have been cropped from both but need to be present for the viewer to understand the units. Furthermore, the image is of very low resolution and the text size is different between ontologies, this should be corrected and a higher quality image obtained.

Figure 4 & 5: The authors must provide a colour scale. The nodes of the network are colours but it is unclear what they are coloured by and so a scale or legend of some sorts is needed.

Figure 6: Almost unreadable. The authors must acquire higher quality images as it is almost impossible to read the graph titles and thus which graphs represent which genes. All text within this figure is extremely hard to read.


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

Input:
Title: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED METHYLATED GENES IN MELANOMA VERSUS NEVI USING BIOINFORMATICS METHODS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The study is reported in a clear and understandable manner.
Experimental design: The authors justified their use of the t-statistic for the gene cutoff and performed survival analysis on only the primary cases in the TCGA melanoma dataset.
Validity of the findings: This analysis will be useful for future studies for identifying risk factors in genes associated with nevi and primary melanoma.
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 LINKAGES OF PLANT, LITTER AND SOIL C:N:P STOICHIOMETRY AND NUTRIENT STOCK IN DIFFERENT SECONDARY MIXED FOREST TYPES IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This article reports C, N, and P concentrations and stoichiometry of tree, shrub, herb, litter, and soil pools in three secondary forest types in the Qinling Mountains of central China.

The results presented were not particularly novel, so I felt that some of the background/context was over-explained.

The authors pointed out in the introduction that these types of studies are often done on only one stock or in only one forest type, and effort should be made to make connections between these, but I think the authors could have done a better job of tying it all together themselves.

The hypotheses given were so vague; it made this seem like an exploratory study.

The results could be better articulated.

The figures and tables were good.
Experimental design: I don't think that specific, valuable hypotheses were given with a solid reason for this study.

I did have some questions as I read the methods, but they seemed generally well thought-out.

The authors may want to provide a citation or more detail about lab methods to facilitate reproducibility.
Validity of the findings: I think this article might benefit from embracing the fact that these results are not presenting a lot of new information. By couching these results in similar results from around the world, in different species, in different ecosystems, this study could advance us towards finding global patterns.

The conclusions given were just a recap of the discussion. I think this section could use some work.
Additional comments: Please see attached document for additional general and specific 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 LINKAGES OF PLANT, LITTER AND SOIL C:N:P STOICHIOMETRY AND NUTRIENT STOCK IN DIFFERENT SECONDARY MIXED FOREST TYPES IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript cited from references in relevant field. I found several format mistakes in the references: genus and species names should always be italicized, the authors did well in the Material & Method section, as well as in the supplementary material, but the authors should double check the format in the References section.

The raw data is presented in a very neat way, please format the ‘percentage’ in Tables 2,3,4. The first letter of ‘Net’ were hidden in Tables 3 and 4.
Explain what are ‘DBH’, ‘BM’, ‘BCM’, ‘CM’ abbreviation for in caption for table.

Some terminologies should be checked, for example, ‘storage characteristics’ in the Background section; ‘plant layers’ in line 30; ‘stoichiometric characteristics’ in lines 89-90, 'undersoil' in line 270.

Some grammatical errors and inconsistency issues could be corrected, for example, “stoichiometric ratios’ and ‘storage’ in line 22.
Experimental design: The objectives are clear and the experimental design fulfil the requirements in order to achieve them. However, the two hypotheses should be revised. Additionally, the third objective in this manuscript is the implication of the result, rather than the objective.

The introduction section is not attractive enough, the novelty should be highlighted, for example, this study maybe the first study to compare stoichiometry ratios and concentrations of C, N and P in different component in three forest types. I use 'maybe' because I don't have expertise in forest research.

My suggestion for the data analysis is to do liner regression or Pearson’s correlation to see C, N and P in plant organs are stoichiometric correlated with C, N and P in soil or not. This would improve the novelty of this manuscript, and to me, it’s more related to Ecological Stoichiometry Theory.

Please add references in the Method section.
Validity of the findings: In the results section, present with quantitative data. Also, summarize the results section, and please highlight just the significant difference, for example, in line 234, ‘no notable difference’ means significant or non-significant?
In the Discussion section, avoid repeating results, for example, in lines 332-334, lines 358-360.
I found a couples of long sentences in this manuscript, it's not easy to understand, it’s better to shorten into simpler ones, for example, in lines 325-328, lines 447-451.
Additional comments: The article entitled “C:N:P stoichiometry and nutrient storage at the ecosystem level in different secondary mixed forest types in the Qinling Mountains, China” is an interesting study about the stoichiometry ratios and stocks of carbon and nutrient in different component at the ecosystem level, and the study were conducted at three secondary mixed forest. However, to my view, the data presented was insufficiently treated and the discussion and could be improved. Still some comments present in the attached PDF file. For these reasons, I can only refer this work for publication after major revision.


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 LINKAGES OF PLANT, LITTER AND SOIL C:N:P STOICHIOMETRY AND NUTRIENT STOCK IN DIFFERENT SECONDARY MIXED FOREST TYPES IN THE QINLING MOUNTAINS, CHINA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: The references of physical and chemical index determination methods should be supplemented
Validity of the findings: no comment
Additional comments: Based on the Qinling Mountains, this paper studies  C, N, and P concentrations and  nutrient storage in trees, understory plants, litter, and soil layers in three mixed forest secondary forest , which can provide important theoretical basis for the rational management of secondary forest.The experimental design of this paper is reasonable, the data is detailed and reliable, the analysis is rigorous and the discussion is clear, but there are still some problems.
1.The three research objectives proposed in the introduction are simple, and the scientific significance of the article needs to be further condensed and summarized.
2.Relevant references should be supplemented for the temperature, precipitation and other parts in the study site description.
3. Is the collected litter sample decomposed?
4.Why the soil samples were not collected according to the occurrence layer of forest soil?
5.The meaning of the letters in the charts in the supplementary materials shall be explained, such as Fig. S1 and table S2


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 LINKAGES OF PLANT, LITTER AND SOIL C:N:P STOICHIOMETRY AND NUTRIENT STOCK IN DIFFERENT SECONDARY MIXED FOREST TYPES IN THE QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors have incorporate changes to reflect most of the suggestions provided by the reviewers. It is accepted to be published 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: THE LINKAGES OF PLANT, LITTER AND SOIL C:N:P STOICHIOMETRY AND NUTRIENT STOCK IN DIFFERENT SECONDARY MIXED FOREST TYPES IN THE QINLING MOUNTAINS, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The author modified some problems in the manuscript and improved the quality of the manuscript. The revised manuscript meets the requirements and criteria of the Journal.


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: REAL-TIME REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF SARS-COV-2
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a well-written submission. I have a few minor recommendations to improve the language in the article.

Line 72 - 73 - "the limit of detection (LODs) were" should be either singular or plural.

Line 73 - "previously published method" should be either singular or plural.

Line 77 - please define "LAMP-LF" before using an acronym.

Line 137 - Rather than "a cheap," I recommend "inexpensive."

Line 140 - Rather than "poor countries where resources are limited," I recommend "resource-limited countries."
Experimental design: Can you add information regarding how you obtained the nasopharyngeal swabs from patients? Did you use RNA extracted previously for RT-PCR and freeze the sample prior to testing, or did you obtain your own samples from COVID-tested patients? Etc.
Validity of the findings: Can you talk a little more about why you used the SARS-CoV2 N1 fragment as your choice for LAMP primer design?  Typically researchers design LAMP primers to very highly conserved regions because LAMP primary cannot function if even a single SNP is present. Viruses mutate rapidly, how can you be sure that this region is highly conserved enough that future mutations will result in the primer set becoming non-functional?  What evidence do you have that this region is highly conserved?

Can you expand the written component in your results section to include the names of the negative control viruses and the number of samples you tested so that the reader does not need to read through the full results section?
Additional comments: Thank you very much for contributing to the COVID-19 literature during this worldwide pandemic.  In your report, please expand briefly on the down sides of the LAMP procedure compared to the gold standard for detecting microorganisms (RT-PCR).  Specifically, it is important to note that the primers are HIGHLY specific and that any mutation can throw off the entire reaction.  You also recommend the use of LAMP in under-developed countries, but can you discuss the limitations that some of these countries may have in completing this full protocol?  How could those limitations be overcome?  Finally, there is much recent evidence that SARS-CoV-2 RNA could be found in other biologic samples such as sputum, urine, throat swabs, and pulmonary lavage samples--a future option to study would be whether the LAMP reaction can take place in these types of samples.


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

Input:
Title: REAL-TIME REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF SARS-COV-2
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.	There are no figures. Please show the color change for the LAMP reaction (and preferably compare it to running on a gel) and some quantification of standards and samples from the RT-PCR reaction.

2.	Please specify when the HNB was added to the RT-LAMP reactions for the experiments shown in the paper. The paper would be improved by showing that HNB added before amplification does not affect the reaction.

3.	Please describe how RNA extraction was performed.

4.	Please describe in detail how the in vitro transcribed RNA SARS-CoV-2 standards for RT-LAMP were made: primer sequences, conditions, etc.

Minor revisions:
Line 41: Specify U.S. CDC.
Line 53: Missing period
Line 72: limit should be limits
Line 97-98: “Addition of HNB to the LAMP reaction solution did not affect the time taken for amplification (data not shown).” Please move to the Discussion if not showing data.
Experimental design: Please see point 1. More description of the methods is required to replicate.
Validity of the findings: See point 1 - underlying data are not included.
Additional comments: This is an important contribution – isothermal protocols for COVID-19 diagnosis are critical. The high sensitivity and specificity of this RT-LAMP protocol are promising, and the use of HNB for assessment could be an important contribution. However, much more underlying data need to be shown and methods described so that this protocol can be fully assessed and replicated.


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: REAL-TIME REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION FOR RAPID DETECTION OF SARS-COV-2
Review round: 2
Reviewer: 1

Output:
Basic reporting: This revised article is clear and the points of clarification raised in the reviews were well-addressed. The results as described are so promising, and the development of a specific and sensitive RT-LAMP assay for SARS-CoV-2 that does not require opening the tube is really fantastic. However, sufficient raw data are not included. The following claims lack sufficient raw data:
1.	The tubes of positive and negative samples shown in Figure 1 have a clear color difference, which makes this contribution very exciting. However, we need to see the tubes for all the assays reported in Supplementary Table 2 if those results are to be included throughout the paper (e.g., as part of the sensitivity and specificity calculation described in the abstract).
2.	Similarly, all of the RT-PCR traces from Supp. Table 2 need to be shown. If possible, the quantitation from the trace should be shown as a bar graph or other readout in Fig.2 and the raw traces put in as a supplementary figure.
3.	Still, technically, no raw data is shown for the claim that HNB does not inhibit the RT-LAMP reaction. However, now that the authors have clarified that all RT-LAMP reactions presented had HNB added at the beginning, this claim can just be cited as “data not shown.”
4.	The raw data for the other coronaviruses tested is not shown.
5.	The experiments used to determine the LOD for qRT-PCR and RT-LAMP are not shown.
Missing information from the methods: what primers and kits are used for qRT-PCR?
Missing information from the figures: please label the RT-LAMP primer locations in the clustal-w readout in Supp Fig 1.
Experimental design: The only point of clarification not yet addressed (I apologize that it wasn’t explicitly included in my earlier review) is how the samples were chosen for inclusion in the RT-LAMP experiments. For example, were positive samples with especially high titers by RT-PCR chosen? From looking at the current Figure 2, this doesn’t appear to be the case, which is fantastic, but a brief description of how the samples were chosen would put any concerns to rest.
Validity of the findings: See point 1. Much raw data is still missing.
Additional comments: This is an important contribution and PeerJ does not consider impact. If you no longer have any raw data I asked for, please consider how the statements of the paper can be changed so that they are supported by the data and the work can still be published.


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

Input:
Title: GINSENOSIDE PANAXATRIOL REVERSES TNBC PACLITAXEL RESISTANCE BY INHIBITING THE IRAK1/NF-ΚB AND ERK PATHWAYS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Authors study the role of a ginseng related molecule in sensitisazion of paclitaxel resistant cells to drug treatment.
The topic is important, the english is acceptable in the text but legends are poorly understandable, background can be made clearer, in particular stressing why the ginseng derived molecule is making breast cancer cells more sensitive.
The results are relevant to the hypothesis, however.
Some items to be addressed:
1) Ginsenoside PT has to be briefly described also in the abstract
2) Ginsenoside family and structure has to be summarized in the introduction. For instance I found in another paper this description: "The ginsenosides are the major active pharmacological components of ginseng. Ginsenosides, also known as steroid-like saponins, are unique to ginseng species. There are more than 100 ginsenosides etc.
Abundant literature is available on several ginsenosides and it sould be expanded.
3) The production of the resistant cell line has to be described, not only cited. Only one line is employed.
Raw data are shared but the definition of "Full-lenght uncropped blots" is erroneous, since what presented are portions of western blots and not the entire one. Please provide.
Experimental design: The research is experimental and within the scope of the journal.
Figures are not easilyy readable. Since Paclitaxel is abbreviated as (PTX) I suggest that Ginsenoside PT is abbreviated with a different acronym than PT, because it makes the figures very confusing (GPT? GiPT?). The abbreviations are never used in the text, I suggest that after the first mentioning of ginsenoside PT the abbreviation is used for faster reading.
In Fib 1 C at day 8 a rather catastrophic fall of cell viability occurs with GPT. The explanation given in the results, is not satisfactory. How is that the parental cells are even more sensitive to GPT than to PTX? those compounds are supposed to have a limited toxicity. I suggest to repeat the experiment or to come up with a molecular explanation.
Methods, besides the preparation of the resistant cell lines, are sufficient.
There is no control sensitizing drug, this is a major limit, so a second molecule, already known for sensitization of PTX resistant clones should be tested at least in some experiments
Validity of the findings: The findings are valid, but they would appear more novel when the Ginsenoside PT is compared to other well studied ones.
Data are sound, with the exception of the viability assay.
Discussion should include a part on the use of plant derived preventive drugs for sensitisation to chemotherapy as general concepts as well as other pathways addressed by ginsenosides should be mentioned (for instance PMID: 32067911.
PubMed PMID: 30772396. PubMed PMID: 32020217. PMID: 29035827 etc)
Additional comments: The paper is approaching an interesting topic. In the future I think in vivo studies would be necessary.
More resistant cell lines should be also employed
At least a comparison with a different agent is required.


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: GINSENOSIDE PANAXATRIOL REVERSES TNBC PACLITAXEL RESISTANCE BY INHIBITING THE IRAK1/NF-ΚB AND ERK PATHWAYS
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors analyzed to the effects of ginsenoside PT as a molecule to revert paclitaxel resistance in an aggressive breast cancer cell line model. The manuscript is potentially interesting and the results valuable for readers in the field. However, some issues need to be adjusted before final acceptance.
My major concern is based on the results in Figure 1, particularly in Panel 2B and 2C. MB231-PR cells have been created to be resistant to paclitaxel. But from Figure 1C, it is evident that MB231-PT cells (parental) seem not to be sensitive to paclitaxel at all. How can we accept the validity of these observations and the following ones if parental cells are not sensitive to paclitaxel??? This needs to be clearly addressed by the authors.
The second major comment refers to Figure 2: to demonstrate the effect of combination treatment on IRAK1 and NF-kB pathways, qPCR has been performed. However, the effects rely on the fact that PTX increases these two signaling pathways (as seen in qPCR experiments), even if at protein level p-IRAK1 and p-p65 did not increase in response to PTX (both panels 2A-2B). The authors need to convince the reviewers these two main mediators are differentially regulated in response to PTX; for instance, given p65 and generally NF-kB proteins shuttle between cytosol and nucleus, maybe the level of phosphorylation does not change, but the localization does it to stimulate the expression of downstream targets. Also, other pathways can converge on these regulations (i.e., STAT3). Please clarify this.
Third: the authors have used only MDA-MB-231 as TNBC cellular model. To better generalize their results, at least one set of the experiments should also be confirmed in another model for TNBC, such as for example MDA-MB-468, BT-459, or Hs578T cells among others.

Minor issues:

- There are some typos throughout the manuscript that need to be fixed (i.e., "resistant" instead of "resistance" line 148; missing full stops; few words misused).

- Methods and Results sections are correctly subdivided in paragraphs, but with a useless numbering. This is not needed, and it would have been more coherent to use it for the whole manuscript, not only in 2 out of the different sections. Please remove it.

- The authors abbreviated the "MDA-MB-231" cell line to "MB1231" throughout the manuscript for simplicity. Given this is not the official name of the cell line, at least once (preferentially in the methods section) they need to use the official name.

- According to the methods, it seems the authors did not use Glutamine in their cultures; however, it is an essential supplement I'm pretty sure has been added to the culture medium. Please revise this.

- There are some problems with supplementary data... There are 2 Word documents... S1 and S2... But each of them names Fig. S1 and Fig. S2. This is confusing.
Moreover, in the first paragraph of Results (lines 152-153) the authors refer to apoptosis induction due to the combination treatment indicating Fig. 1E and Fig. S1 panels. However, while Fig. 1E shows sub G1 and other cell cycle fractions, in Fig. S1 there is not any FACS experiment! I assume Fig. S1 should have been confused with Fig. S2 of the Supplemental Data S2. The authors need to make more clarity to guide the readers to explicative Figures better.

- Second paragraph of Results lines 164-166: the results presented in this part deserve a citation (potentially it is an article already cited before or later in the text).

- The closing sentence at the end of the second paragraph of Results (lines 167-169) can be misleading and need to be re-written. Maybe it might be acknowledged that in panel 2B MCL1 reduction is not that evident.

- Figure 1: Panel A; Given it represents cellular viability, it would have been more informative to express everything related to DMSO (set at 100%) and plotted as %. This may also help the reader to visualize IC50.

- From the Legends to Figure 2 it is not indicated that PTX was added alone and the combination treatment is not mentioned as well. Please adjust this.

- Figure 3B: MDA-MB-231 is not the best model to study cellular behavior in 3D. As it is possible to appreciate from the images, spheres are very dishomogenous, not well shaped, and spheres' roundness is limited. It is clear the authors did this additional experiment with this cell line to be consistent with the other results, but they need al least to mention this.

- From the legend of Figure 3B right panel, it is not clear what the luciferase activity refers to; and at what time point it was tested... day 6 or day 12?


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: GINSENOSIDE PANAXATRIOL REVERSES TNBC PACLITAXEL RESISTANCE BY INHIBITING THE IRAK1/NF-ΚB AND ERK PATHWAYS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The manuscript has been revised according the suggestions of the reviewers, some novel additional experiments have been included, and it has gained more clarity and consistency. The Figure Legends have been improved and in the current version the reader can better appreciate the results from them.

However, there are some minor issues that need to be fixed prior final acceptance.

1. There are still some typos, particularly in the parts with new text. Please check this carefully.
2. Although in the rebuttal letter the authors mentioned they agreed with my comments for the seventh minor issue (about lines 167-169 of the original version) and have corrected the sentence, in the current version the sentence is still the same, it is still convoluted and the comment on MCL1 has not been included. The authors are requested to do it accordingly.
3. Although the authors agreed with my eight minor issue (Figure 1A presentation), and set the control condition (DMSO) to 100% as suggested (for viability), the axis name is still reporting Luciferase (RLU). Please modify this.
4. The new version of Figure 3 with the additional data on SUM159 needs to be adjusted. In my opinion, panel 3G and panel 3I can be reduced in size and the panel numbering changed as follows: current 3I can become 3H, and the two quantification plots respectively 3J (MB231 cells) and 3I (SUM159). In this way the numbering will be more linear.
5. In the Supplementary files, there are two pieces of data which seem unrelated to this story, or not requested for this submission. Raw data of a gene expression profile with the compounds used in the manuscript (excel table) and a heatmap summarizing this. What does these data refer to? Why are they not included in the original submission? Are there simply for a mistake and they will be part of a follow-up study? Please clarify at least the editor.


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: ASSOCIATION OF FKBP5 POLYMORPHISMS WITH PATIENT SUSCEPTIBILITY TO CORONARY ARTERY DISEASE COMORBID WITH DEPRESSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study investigates the potential association between 7 SNPs within the FKBP5 gene and depression in a population of 123 patients with coronary artery disease (CAD) and comorbid depression, 147 CAD patients with no depression and 113 healthy controls. A significant association was found for rs9470079- GA genotype conferred decreased risk for CAD. Further associations were reported for two additional SNPs with sex effect on the association (significant for females but not for males).
Experimental design: The strengths of the study are the reasonable sample size and the recruitment of healthy controls, the standard clinical assessment and measures for depression, and the extensive study of a single gene- looking into seven different SNPs.
Validity of the findings: The limitations of the study are the narrow focus on a single gene:
1.    To my knowledge candidate gene approach is not scientifically popular anymore. Instead the GWAS approach is more standard. I understand that the sample size is underpowered for conducting GWAS. However, the authors should refer to results of GWAS studies of depression and possibly also of CAD in the paper.
2.    Are any of the SNPs with any known functional significance on the protein or are they all synonymous change with no effect on the protein? If the latter is true what is the significance of the findings?
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: ASSOCIATION OF FKBP5 POLYMORPHISMS WITH PATIENT SUSCEPTIBILITY TO CORONARY ARTERY DISEASE COMORBID WITH DEPRESSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The authors designed a case-control study, that included seven polymorphisms of FKBP51 gene in susceptibility to comorbid CAD and depression. I suggest modifying tables 3 to 6 as they are saturated with results and it is confusing to understand them. However, the information presented from the associations is correct.
Experimental design: The experimental design is adequate considering the objective of the study.
Validity of the findings: No comment.
Additional comments: It would be interesting to perform the linkage disequilibrium analysis and include possible haplotypes that are associated with the disease.


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: ASSOCIATION OF FKBP5 POLYMORPHISMS WITH PATIENT SUSCEPTIBILITY TO CORONARY ARTERY DISEASE COMORBID WITH DEPRESSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised the manuscript to meet my comments. I have no further comments
Experimental design: .
Validity of the findings: .
Additional comments: .


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

Input:
Title: ASSOCIATION OF FKBP5 POLYMORPHISMS WITH PATIENT SUSCEPTIBILITY TO CORONARY ARTERY DISEASE COMORBID WITH DEPRESSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised manuscript is now more interesting.

I don't have more questions.
Experimental design: The experimental design is adequate with the hypothesis of the paper.
Validity of the findings: The authors are aware of the limitations of the study; however, they are working to improve future manuscripts
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 EFFECT OF JOURNAL GUIDELINES ON THE REPORTING OF ANTIBODY VALIDATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a great study that evaluates the reporting of antibody validation in studies that are published in journals that require validation in their instructions to authors.

As a researcher who previously worked with antibodies during my time on the bench (western blots and flow cytometry), I understand the fickle nature of antibodies as reagents, and the importance in validating antibodies for use in scientific assays.

This paper nicely describes the background and previous studies related to this topic, the literature is well-referenced and cited.

The writing is clear and unambiguous.

The figures are very clear and professionally done, the raw data is shared. Please include a data dictionary to describe all column headers in the supplemental data.
Experimental design: The research question is well defined and very relevant to current issues in scientific reproducibility (ie the reproducibility crisis).

The execution of this study is well done. The methods are sufficiently reported and this study should be reproducible based on the reporting and supplemental data.

I recommend sharing your data in a public repository, in addition to sharing it as supplemental data.
Validity of the findings: The findings seem valid and robust. The conclusions are well supported and the discussion does a nice job of pointing out the limitations of the study.
Additional comments: It is encouraging to see that the percentage of authors who report validation studies for their antibodies has increased over time. To be honest, I would have expected these numbers to be higher, but you give good reasons in the paper for the lack of adherence to the policy.

I hope that more journals adopt these policies and find ways to enforce them. I hope there is a shift in the way research is performed, to recognize the importance in validating antibody reagents prior to performing experiments. Antibodies are valuable reagents for research, but the lack of reliability and inconsistency in their results is really a detriment to science.

This study is a nice contribution to the scientific community and I hope it will push further change, on the part of journals and researchers, to improve the validation of antibodies used in research.