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

Input:
Title: A MOLECULAR PHYLOGENY OF GEOTROCHUS AND TROCHOMORPHA SPECIES (GASTROPODA: TROCHOMORPHIDAE) IN SABAH, MALAYSIA REVEALS CONVERGENT EVOLUTION OF SHELL MORPHOLOGY DRIVEN BY ENVIRONMENTAL INFLUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: English is good, but should be finetuned.
Literature should be a bit extended, see in my general remarks.
Experimental design: see my comments in General comments for the author.
Validity of the findings: see my comments in General comments for the author.
Conclusions: Yes, conclusions are fine, such as a genus is not monophyletic, or a species is nested in another species. However, I, as a taxonomist, don't like to stop here, and strongly suggest to transfer your phylogenetic data into taxonomic changes.

Discussion could be extended a bit with what I suggested.
Additional comments: This is a nice study, definitely worth publishing, however, there are certain problems I can immediately see and I report below.

(1) Molecular phylogeny: Before you analyse the entire matrix, you should produce separate mitochondrial and nuclear trees, and check whether their topology matches. I may have been overlooked, but I could not find any traces of this. Mitochondrial and nuclear trees should be made separately available, even if as supplementary material.

(2) While you present a useful and important phylogeny, I was disappointed to see that unfortunately this is again a study with good phylogenetic information, but insufficient morphological/taxonomic conclusions.

First, the type species of Geotrochus van Hasselt, 1823 is Helix conus L. Pfeiffer, 1841 from Java. The type species of Trochomorpha Albers, 1850 is Helix trochiformis L. Pfeiffer, 1842 (complicated situation, H.B. Baker, 1941 established Trochomorpha typus [from the Society Islands] as a new species, and subjective synonym of Helix trochiformis Pfeiffer, 1842).

You cannot talk about genera without examining the type species. Genera are based on type species. Both type species are probably available in museums, at least you should examine and publish photographs, and arrive to a conclusion based on morphology without molecular phylogeny. I already know what answer I will receive (this is beyond the scope of the present paper, etc.), but please try not to stop at stages like "this and this genus is not monophyletic". With these kind of publications you just increase the phylogeny-taxonomy gap (Franz 2005).

It is OK that you did not include those species in your phylogentic analysis, but I am sure nobody will be able to perform a phylogenetic study with those two type species in the next 50 years or so. So you would need some explanation on the morphology and arrive to a conclusion. I would strongly suggest to synonymize these two genera, or state that you suggest using Geotrochus because its types species occurs in the same geographic area, or whatever, so at least until more information becomes available, make some order and get rid of one of the generic names that causes troubles.

Also, the type locality of the type species of Trochomorpha is not on the map. I understand that that record might not have contained in the database you used, but that means your database is wrong, and you are strongly suggested to make a map with reliable localities.

Second, T. thelecoryphe and T. haptoderma are almost certainly synonyms. I don't see why you don't synonymize them in this paper. Both species were described recently by you!

(3) "The physical shell is deemed to be the by-product of adaptation to their environmental attributes": Well, if you want to support this view, you can find citations for sure. However, there are papers proving that shell morphology is often a result of random genetic drift, and the traits are not to be automatically explained as adaptation to the environment (e.g. Fehér et al. 2018).

(4) Role of rough upper (dorsal) surface: In Plectopylidae, most living species have reticulated sculpture on the dorsal shell side, which is often covered with soil and this may be of value in providing camouflage (Páll-Gergely et al. 2015). This paper should be cited. Are trochomorphid specimens clean or dirty in the wild?

(5) I am not good at statistical analysis, but Fig. 4 about precipitation does not seem to be convincing. The mean for S1 and S2 is roughly 2500 mm, whereas that of S3 and S4 is 2300 m with huge interval. This 200 mm differences is nothing, and can easily be sampling error (in other words you cannot have precise enough data on precipitation for these kind of studies).

References

Fehér, Z., Jaksch, K., Szekeres, M., Haring, E., Bamberger, S., Páll-Gergely, B., Sattmann, H. & Sólymos, P. (2018): Range-constrained co-occurrence simulation reveals little niche partitioning among rock-dwelling Montenegrina land snails (Gastropoda: Clausiliidae). — Journal of Biogeography 45: 1444–1457.
Franz, N.M (2005): On the lack of good scientific reasons for the growing phylogeny⁄classification gap. Cladistics 21: 495–500.
Páll-Gergely B, Hunyadi A, Ablett J, Lương HV, Naggs F, Asami T (2015) Systematics of the family Plectopylidae in Vietnam with additional information on Chinese taxa (Gastropoda, Pulmonata, Stylommatophora). ZooKeys 473: 1–118.


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

Input:
Title: A MOLECULAR PHYLOGENY OF GEOTROCHUS AND TROCHOMORPHA SPECIES (GASTROPODA: TROCHOMORPHIDAE) IN SABAH, MALAYSIA REVEALS CONVERGENT EVOLUTION OF SHELL MORPHOLOGY DRIVEN BY ENVIRONMENTAL INFLUENCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Main concerns:

The fact that the geographical scope of the study is restricted to one

single state of Malaysia (northern tip of Borneo Island) and not the whole

range of the genera in question makes the final conclusions much weaker.

Furthermore, conclusions are even weaker if not all Sabah species is

involved in the study.

my largest concern about this study that authors claim to 'demonstrate' a

relationship between shell traits and environmental factors. This should

be consulted with someone who is more versed in statistics. But I  suspect

that it is not OK that the number of elements per species are different.

If we are curious about intra-specific issues, several specimens of ONE

species should be compared. If we are curious about the genus-group level,

we should include ONE specimen per each species (something suggested by

Fig 7). Maybe one can argue that it makes sense to use one specimen per

POPULATION, so species of larger area are better represented. But using

ALL available specimens when species are unequally represented in the

sample - that seems problematic to me. But, again I think a statistics

expert could better tell what is the problem with it.

some other points:

measured dimensions. Only shell and aperture width values are measured, but based on the pictures these species can be distinguished by many other shell features, e.g. the convexity of the cone, shape of shell, the strength of the keel, etc. these traits are all ignored by the stat analysis.

Discussion

Lines 189-190: Geotrochus and Trochomorpha are not reciprocal
monophyly --> not reciprocally monophyletic

Lines 190-191: "This result is contrary to the current taxonomy of the two

genera that based on the shell characters, especially the shell upper

surface sculpture"   This sentence should be the most important conclusion

of this study. i.e. morphology-based current system is not in consensus

with the phylogenetic tree. I agree. However, see my note about the phylo

signal and table 5. Sculpture is a trait where Blomberg K indicates

phylogenetic signal!!!  So how can you interpret this???

Line 205: I do not clearly understand what the authors wanted to mean

here...

Lines 211-213 If I understand well, here the authors call a species "new"

if it was described in 2015. They are obviously not  "old" species, but in

the literature a taxon is called "new" if it is described in the same

publication.

Conclusions:

I am not convinced that "...The coarser shell character
290 may be an adaption of the land snails to highland habitat with a more

humid condition in the area"  I still believe that excluding the role of

pure chance needs stronger statistical evaluation.

"...the current taxonomy of the two genera
294 need further revision and the future attempt should consider more

samples that cover the entire
295 distribution of the two genera"

I strongly lack any kind of taxonomical/nomenclatural conclusion at the

end. I feel that the statistical support for the relationship between

environmental factors and shell traits is weak, so this remains the only

possible conclusion. If a tree is non-monophyletic it can primarily due to

(i) bad taxonomy (ii) problems with the chosen markers in tree

reconstruction (see the review of Funk and Omland  2003). If I am right,

authors vote here for the first case. If so, they should make a

conclusion. Otherwise, why did they make this study? A conclusion like

"...need further investigation" I feel too little for a renowned journal

like PeerJ.

Lines 151-153: I think a species can have a sister species and a family

can have a sister family...   a species can hardly be a sister of a whole

family unless it is a monospecific family

Lines 183-185
other K's are closer to 1 than 0, although p's are poor. But this might be

due to different other factors, like the number of studied specimens not

only the lack of signal.    And also some lambdas are pretty high (though

p's are poor).
Another point, several experts argue that these metrics for phylosignal are

quite outdated, see e.g. https://www.carlboettiger.info/2013/10/11/is-it-

time-to-retire-pagels-lambda.html

Table 5.  Contrary to the authors' claim, the sculpture's K value seems

significant. Some other lambdas and Ks are also quite high (although p-

values are non-significant). But being unable to demonstrate the presence

of phylogenetic signal does not necessarily equal to the absence of the

phylogen signal. Might also be due to low N, or generally due to the fact

that many authors believe that Pagel's test is really outdated.

Figure 7 caption: "Figure 6 Visualization...." this was probably

overlooked. But more important points here: what about S4 type?? that is

not illustrated. in contrast with Fig 4, where there is S4.
Why G. paraguensis is represented by two entities when others only by one?

does it mean that N=10 was the number of elements in the statistical test

(and not 155)?
Experimental design: see above
Validity of the findings: see above
Additional comments: No additional comments


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

Input:
Title: A MOLECULAR PHYLOGENY OF GEOTROCHUS AND TROCHOMORPHA SPECIES (GASTROPODA: TROCHOMORPHIDAE) IN SABAH, MALAYSIA REVEALS CONVERGENT EVOLUTION OF SHELL MORPHOLOGY DRIVEN BY ENVIRONMENTAL INFLUENCES
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is brief and concise in style, the language used, although grammatically not always perfect, is sufficiently clear and understandable. Language editing could help getting rid of some linguistic imperfections, but I don't accept this as my responsibility.
Experimental design: The problem is clearly described, relevant literature is considered, the methods and statistical tests are suitable to address the problem. The have been executed properly.
All relevant data is made available.
Validity of the findings: The conclusions of the paper are reasonable and supported by the evidence presented.
Additional comments: Nice work. With a little bit of linguistic polishing, I am sure that this has the potential to become a good example study demonstrating that sole reliance of shell features can be problematic in land snail systematics.

I suggest minor revision to give the authors the opportunity to work on the linguistic aspects of presenting their 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: A MOLECULAR PHYLOGENY OF GEOTROCHUS AND TROCHOMORPHA SPECIES (GASTROPODA: TROCHOMORPHIDAE) IN SABAH, MALAYSIA REVEALS CONVERGENT EVOLUTION OF SHELL MORPHOLOGY DRIVEN BY ENVIRONMENTAL INFLUENCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have read the explanations of the authors, and I accept them. I still "like" if studies like this have stronger phylogenetic/taxonomic base, but I understand that in the present case the scientific question is not taxonomic.
Experimental design: I am satisfied with the explanations of the authors.
Validity of the findings: I am satisfied with the responses of the authors.
Additional comments: No additional comments


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

Input:
Title: A MOLECULAR PHYLOGENY OF GEOTROCHUS AND TROCHOMORPHA SPECIES (GASTROPODA: TROCHOMORPHIDAE) IN SABAH, MALAYSIA REVEALS CONVERGENT EVOLUTION OF SHELL MORPHOLOGY DRIVEN BY ENVIRONMENTAL INFLUENCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: a short note on literature references. Some new papers were added to the revised text. I have found that Pall-Gergely et al 2015 is, for example, mentioned in the text but not listed among references. Please double-check the whole text. Also, keep an eye on the alphabetic order of References
Experimental design: no comment
Validity of the findings: see comments below
Additional comments: I still have some of the concerns written in my first review, However, it has to be admitted that authors improved the ms significantly and gave (or in some cases made a significant effort to give) answers to all the points. So this improved version seems  worthy of publication


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

Input:
Title: EFFECTS OF MIXING TWO LEGUME SPECIES AT SEEDLING STAGE UNDER DIFFERENT ENVIRONMENTAL CONDITIONS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript contain new and significant information to justify publication. It is a study with overall adequate presentation of experimental results,
It is a complementary experiment study useful in agriculture and in research academic. The concept of the study is well investigated and approved by different trials in lab, pot and field.
Sufficient field backgroung/context provided.
Some additions are needed: this paper has potential and is interesting in productivity for adaptation to climate change, however in the current form needs to be reviewed.
Concerning title: I suggested to the authors to adjust the title. I propose (potential use of association (mixture) legumes species as a sustainable technique at seedling stage under the different environmental condition for the intercropping system)
The abstract clearly and accurately describes the content of the article.
Experimental design: The experimental and/or theoretical methods described comprehensively.
Methods described with sufficient information.
The statistical procedure that you used is the most correct for what you are analyzing.
Validity of the findings: The statistical procedure that you used is the most correct for what you are analyzing.
Conceptual of manuscript could be more original if author pay more attention particularly in the conclusions.
Because the study is agronomic investigation give highlights at farmer level the agronomic potential of association pasture legume introductions on droughty soils.
Additional comments: I invite the authors to submit a major revision of the manuscript before a final decision is reached


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

Input:
Title: EFFECTS OF MIXING TWO LEGUME SPECIES AT SEEDLING STAGE UNDER DIFFERENT ENVIRONMENTAL CONDITIONS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The title of the article is misleading, only two legumes were used. Proofreading is required. In the introduction I am missing current articles on this topic: DOI: 10.1111 / grs.12257; 10.1002 / agj2.20045; 10.1111 / jac.12337; 10.2134 / agronj2017.12.0753; 10.1139 / cjps-2017-0183 etc.
Experimental design: Methods, resp. in particular, the design of the experiment is completely unclear. Proper and clearly repeating field experiments would suffice. According to the results and discussion, the others are completely irrelevant to the topic and hypotheses of the thesis.
Why was 3 times in 2016 and 4 times in 2017. Cleary explain pot (samples), 9 variants? 8 section and 3 blocks - how area?. You used just only two legumes and their mixture... Missing table and design of experiment.

In my opinion, laboratory and pot experiments are not suitable for confirming or refuting hypotheses. They cannot simulate abiotic and biotic stresses in all directions.

Statistical models, resp. evaluation is based only on coefficients? Have they been successfully verified somewhere before? Published?
Validity of the findings: The validity of the results is OK. But in general they are very unconvincing given the hypotheses. But even that is the result.

I consider this chapter (Potential ecological mechanisms: How mixing species may affect germination) a literary review. Most results are not significant or have a large variance. Causality cannot be inferred from them.
Additional comments: The article should be clarified incl. methods. Comment only on clearly conclusive results and draw clearly verifiable conclusions from them.


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

Input:
Title: EFFECTS OF MIXING TWO LEGUME SPECIES AT SEEDLING STAGE UNDER DIFFERENT ENVIRONMENTAL CONDITIONS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I thank editors for the confidence given to me to review the manuscript on “Effects of mixing legume species on germination” (Manuscript ID: #50696)
I think authors for patience and time are given for improve the manuscript. The manuscript is revised carefully.
Comments and suggestions of reviewers were taken into account and have included all the reviewers’ comments responded to them individually in a point-by-point response, in preparing the revised version of the manuscript.
Changes made in the manuscript and responses of authors are satisfactory and that the newly revised manuscript is acceptable for publication.
The two versions of the revised manuscript, with track changes and with all track changes accepted are revised and updated. The required additions have been added to the manuscript.
The title was adjusted as suggested and improved. Comments and remarks through the manuscripts taken into account in the revised manuscript.
The abstract clearly and the experimental and/or theoretical methods described comprehensively and improved in the revised version and supplemented file for explanations added.
Sentences needed to be deleted: Line 50- Line 52: "Cover crops are frequently sown during March-April in temperate climates and October-November in subtropical areas according to the fallow-period duration determined by the cash-crop succession"
Field FU9: please define composition to be clear to the reader, is it ready substrate, what is composition? Precise for each texture sandy clay loam or sandy loam or….
References are well checked only there is a minor revision in yellow color comments in added manuscript version revised. Please check the version uploaded for a minor revision. All points are already made in the revised manuscript.
A graphical abstract prepared demonstrates the high probability of increasing the germination percentage in the case of mixing the two legume species that can be added to supplementary data for publication.
I advise as perspectives to identify and analyze the crop mixtures grown in farmland (with the different farmers) with a view to creating resources to help farmers to adopt and adapt this practice to their own context.
The manuscript contains new and significant information (even research needed to be improved in the field in farmland in the future) to justify publication. The study with an overall adequate presentation of experimental results discussion; it is a complementary experiment study useful in agriculture and in research academic. I suggest for publication after taking into account minor revision.
Experimental design: The study with an overall adequate presentation of experimental results discussion; it is a complementary experiment study useful in agriculture and in research academic.
Into revised manuscript author improved this part with one figure
Validity of the findings: Authors improved all parts of the manuscript as suggested by reviewers
the benefit to the reader is clearly stated.
Conclusions are well stated and improved
Additional comments: I advise as perspectives to identify and analyze the crop mixtures grown in farmland (with the different farmers) with a view to creating resources to help farmers to adopt and adapt this practice to their own context.


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

Input:
Title: EFFECTS OF MIXING TWO LEGUME SPECIES AT SEEDLING STAGE UNDER DIFFERENT ENVIRONMENTAL CONDITIONS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors improved the manuscript very conscientiously and carefully.
Experimental design: After the revision, the methods are clearly explained incl. scheme of experiment in the S
Validity of the findings: Now is clear and better explained.
Additional comments: Thank you for revised version. It is clear that a thorough revision of the manuscript has helped significantly.


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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript requires English language editing. The introduction is ambiguous not providing a direct focus of the aim of the study. The results and raw results are presented. However, there are issues with the figures, figure 5 and 6 do not have units. Usually, in RT PCR there is a ratio with GADPH or actin. The discussion is poor and requires rewriting
Experimental design: The experimental design lacks important details. The Jurkat cells were obtained from? What were the conditions of cell growth in culture?, ie, how do we know that the cells were not overgrown? There is 5 % necrosis along with 3.6 % late apoptosis, in total around 9 % of the cells were IP positive in the control, it is too high. Why not perform the assays with a range of concentrations from nanoM to microM? The assays with bcl are not performed correctly, probably cytochrome c release would have been a better option. The cell cycle experiments look ok in principle, but the results suggest that the cells were overgrown. The RT PCR data lacks details of the number of cells used.
Validity of the findings: There seems to be an effect of hirsutine, but the results are not being presented in the adequate form. The cell culture should be synchronized in order to ascertain the effect on apoptosis which is moderate, and some assays like RT PCR or bcl are not very convincing.
Additional comments: Please restructure the paper. Perform experiments with cells with less than 2 % IP in the controls, perform curves with the compound from nanoM to microM, either assess caspase activity or cytochrome c release from the mitochondria. Finally, the introduction and the discussion should be rewritten with specific aims and direct conclusions. English language editing 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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English is clear.

	The references are sufficient.

	The Figure 2 is necessary changed because the asterisks are not in the correct position

	It is important to precise the innocuity because in this manuscript there are not experiments to confirm the not toxicity.
Experimental design: The aims are clear
The research question is clear and the methods to answer correct.
Methods was described with sufficient detail to be replicate.
Validity of the findings: The results obtained are important to considerer the hirsutine as probably leukemia treatment.
I suggest check the statistical analysis on the first to bars of the figure 5 bax/bcl-2.
To improve the abstract, I suggest including the concentrations studied.
Conclusions are well stated supported by the results.
Additional comments: The manuscript need minor reviisions in order to improve the article.
I think it is important to pay attention to figure 2.


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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The hypothesis modeled by the authors is well clear; the methods are proper and well defined; the data are complete; the manuscript adheres to the relevant standards for reporting and data deposition
Experimental design: Appropriate research techniques and data analysis were employed
Validity of the findings: All underlying data have been provided; they are robust, statistically sound, & controlled.
Conclusions are well stated, linked to original research question & limited to supporting results.
Additional comments: Jie Meng et al investigated the antitumour activity of hirsutine isolated which is isolated from Uncaria rhynchophylla and its mechanism of action. The scope of the manuscript is clearly defined, and the title is adequately interpreted. The abstract presented the aims, objectives, methods, results as well as conclusion adequately. Introduction, there is strong evidence of engagement with relevant and current literature, and all the references listed were cited in the text/body of the thesis. Appropriate research techniques and data analysis were employed, and the manuscript is highly structured and argued. However few questions need to be addressed.

What is the source of hirsutine? What is its purity?
What is the role of free radicals in hirsutine-induced apoptosis?
What would be the effect in normal cells at the tested concentration of cancer cells?
The apoptosis induction is mitochondrial dependent or independent?
In general, activation..i.p..cleavage of caspases are more important than the gene expression. Authors may repeat the experiment with western blotting using cleaved caspases.
It would be better to include antibody catalog number in the methodology
Gene accession number may be included in the methodology.
Discussion part needs to be strengthened


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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript has improved partially. There are some changes which were suggested and not performed. The English language revision is still required. As an example in the new sentence in the abstract of the manuscript states, Western blotting were utilized to discover the expression, which is not scientifically correct. There are may other grammatical mistakes in the corrected manuscript.
The experimental design was modified, but still, there are gaps in how part of the cell cycle experiments were performed.
Finally, the discussion was modified but it is still lacking a proper structure. The effects of hirsutin are after 48 hrs of culture at micro Molar concentrations suggesting that the compound is unable to directly affect cell viability. Thus, cell control mechanisms should be discussed.
Experimental design: The authors made most of the required changes, but still, a description of cell cycle analysis is not detail as requested
Validity of the findings: Most of the crucial issues remain unsolved. Certainly, the authors have data pointing out the effect of the compound, but the data still not well presented. First, the most important element is to ascertain the effect of the compound intrinsic of the extrinsic apoptotic pathways which were done poorly. The effect is observed at 48 hr at mico M concentrations. Figure 3 there is a conflict between the flow cytometry assessment, performed poorly, and the figure which the percentages do not match. Figure 4, there is a clear decrease in the DNA density; however, the interpretation in the figure below is not correct. Figure 5 was improved showing the importance of cytochrome c, but the results on caspase 3 and 9 are very similar. In figure 6, based on the previous data, it is impossible that the values of caspase 3 and 6 are the same upon treatment. Thus, the authors claim a decrease in chromatin structure based on mitochondrial induced apoptosis but show that the extrinsic apoptotic induction is also present without even analyzing CD95. The results do not support this conclusion.
Additional comments: The paper is still lacking the conditions to be published. It still requires mejor 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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 3
Reviewer: 1

Output:
Basic reporting: The article was improved significantly. The use of the English language has been improved as well as other important points
Experimental design: Experimental design now is more understandable and is according to the journal guidelines.
Validity of the findings: The findings are better presented and sicussed.
Additional comments: The manuscript is highly improved with all the detailed responses.


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: INHIBITORY EFFECT AND MECHANISM OF ACTION (MOA) OF HIRSUTINE ON THE PROLIFERATION OF T-CELL LEUKEMIA JURKAT CLONE E6-1 CELLS
Review round: 3
Reviewer: 2

Output:
Basic reporting: The English is clear and the references enough, The figures is necessary to improve the figure 6 in order to precise if it is RT_PCR or qPCR.
The results are important and could be useful in the leukemia treatment.
Experimental design: The aims of the manuscript is clear and the experimental design is ok, the question is clear in order to know if hisutine has effect on the leukemia cells studied and the results important to think in the use of this molecule in the treatment.
Validity of the findings: The results are important and novelty for leukemia, the data are robust but it is important to precise the type of PCR used, the conclusions are well stated because they used as control a normal cells.
Additional comments: I will like to comment about the basic report: the manuscript has been write in clear English and have sufficient references. The experimental design is good and the results importan to think in the possibility to add hirsutine on the leukemia treatment.
In order to improve the manuscript I suggest introduce a reference about the hirsutine  safety because it has been used in others sickens.
A main point to attend is to precede in abstract, methods, results , discussion and figure if the PCR used is qPCR, because now is confuse.
Warm regards
Guadalupe


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: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Taxonomic, functional and expression analysis of viral communities associated with marine sponges
Nguyen and collaborators did an interesting work, where they studied the viral communities associated to the microbial cells of six marine sponges’ species from Australia. They used published data of metagenomes and metatranscriptomes of unfractionated viral pools (microbial cells plus associated viruses) of sponges and the surrounding seawater, and used bioinformatics tools to obtain viral contigs from these dataset.
A strong point of this article is that they analyzed the viruses associated to the microbial cells (attached to prokaryotic cells, virions present inside cells, prophages) of the sponge holobiont, and not the free viruses in the sponge. With this approach, they identified AMGs related to specific interactions between viruses and the microbial cells in the sponges. A weakness is that a small number of viral contigs (N: 451) could be recovered with confidence from the metagenomes, then limiting an overall extrapolation of the article´s findings.
Anyways, the article addresses the knowledge gap of sponges’ associated viromes using metagenomic and transcriptomic approaches, to analyze the taxonomic and functional profile of these communities. The methodology employed here, to retrieve “active” viruses associated with microbial cells was interesting and contributes with meaningful insights to the literature of sponge holobionts.   Authors could have explored more in the discussion what functions are enriched in sponge viromes compared to seawater viromes, if there were any features that were significantly enriched in one of those. Maybe employing the same statistics used to analyze taxonomic structure.

The article is well written and the methods are adequate to the answer the research questions. Although, no raw data was supplied or indicated in the manuscript, and the figures need a better resolution.

Some recommendations to improve:
l. 52 It would be appropriate to add an explanation of how AMGs work here, in the introduction.
l. 76 I recommend to add a flowchart summarizing the methods (as supplementary material). As the present work uses data from published articles, with different methodologies, a flowchart can help the reader to understand how these data were generated, which data was used, and how these data were analyzed in this present article.
l. 79 and 81 When these sponges were collected (year and season)? Please, add this information.
In lines 109-114, you classify contigs into categories and choose only contigs that met categories 1 and 2 for further analysis. Maybe you could’ve kept contigs from category 3 and showed their taxonomic classification and functional properties as well, specifying that these contigs are “possible” viral predictions, since your stringent criteria only kept a few contigs for further analysis.

l. 114 Are the identified viral contigs deposited in the VirSorter database or in the NCBI? Please, add the link or NCBI id to these data.
In lines 157-165 you argue that high variability among replicates and different hosts was observed, and no significant differences between viral assemblages of sponges and seawater was found.  You could’ve explored more this observation. Maybe this high variability is due to HMA/LMA dichotomy. LMA pumping rates may be higher than HMA sponges (Weisz, 2007), therefore their viromes may be somewhat similar to seawater. You could at least indicate if the sponge species in your work have been previously classified as HMA or LMA, since this is a relevant feature of sponge microbiomes and have important ecological implications.

l. 239 Another explanation can be ecological (temporal and spatial) differences between sponges, as they were sampled from different sites. I recommend the authors to explore this possibility.
l. 246 I recommend to add a consideration that the findings here (viral roles in the sponge microbiome) are valid and relevant for the studied sponges, but the extrapolation to other sponges and environments should be made with prudence, because the small number of recovered viral contigs.
l. 273 Another explanation can be that these bacteriophages are not specific to these bacterial genera, and may be infecting others similar bacteria in the sponge.
l. 355 The conclusion is well written and readily summarizes the main findings of the work. It would be interesting if the authors could add a graphical figure (model) summarizing how the viruses can affect the sponge and its microbiome, through the AMGs.
l. 365 The figures lack resolution, and the texts in the figures are difficult to read, especially in the Figure 1. I recommend enhancing the resolution of the figures.

Typing errors:
l. 261 “Phycodnaviruses” the capital letter is not needed here.
l. 298 “et al. 2002) .” remove the extra space before the dot.
l. 325 “(see above}” put this sentence between parentheses.
Experimental design: OK
Validity of the findings: OK
Additional comments: OK


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: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 1
Reviewer: 2

Output:
Basic reporting: Generally clearly reported and well-written. Small comments below.

Line 119: Awkward “and, when present, being single-copy in ≥95% of all genomes”

Line 247: Change “is indicative” to “indicate”

Lines 260-261: Awkward “The taxonomic identification made here…being closely related to”

Lines 265-268: break into two sentences

Line 273: Fragment “sponge, however this requires further investigation”

Line 356: Add comma before “as well as”

Line 358: Clarify “this”

Fig. 1 legent: Add “(B)” before describing Fig. 1B.

Fig. 1A: Text for taxonomic annotations might be too small; legend is missing for top dendrogram and heat map shading.

Fig. 2A & B: Consider adding legend to heat map shading. Are the rows ordered by fold difference between viral and whole assemblages? If not I would consider reordering by the degree of functional enrichment in viral assemblages.

Fig. 2C: On the heat map legend, typo “fouth root”

Fig. 3A & B: Legend is missing for the top dendrogram and heat map shading.
Experimental design: Research fits well within the scope of the journal. Metagenomic and metatranscriptomic workflows for viral genome curation, transcript mapping, and annotation look reasonable.
Validity of the findings: Lines 203-208, 286-291, 316-326, 339-349, and possibly others: major sections of the discussion interpret putative AMGs observed in viral sequences. How reliable are KEGG pathways in determining gene functions? Previous work suggests false positives in inferring gene function from viral metagenomic annotations, particularly for AMGs involved in antibiotic resistance. See doi:10.1038/ismej.2016.90. It’s unclear to me if the domain-specific annotations discussed in this referenced paper differ from KEGG pathways used for functional annotations in this manuscript. If KEGG annotations were more reliable, I would add this justification here or in the methods. Otherwise, I would reword this section towards a more cautious interpretation.

Lines 243-245: Some clarification needed. It’s unclear to me here why cell-associated viruses would be more variable through kill-the-winner dynamics, if these dynamics assume that cell-associated viruses are concurrently producing free-virions. My initial interpretation of the text is - do you mean that free viruses that are “stably embedded” are simply filtered by the sponge, are not infecting any cellular hosts inside the sponge, and are therefore stable through inactivity? If so, this idea seems inconsistent with lines 246-248.

Lines 271-273: “One potential explanation for this observation is that the ‘active’ bacterial viruses and their bacterial hosts may be derived from mammalian/terrestrial contaminants that are accidentally filtered into the sponge” – it’s unclear to me how this explanation is relevant to finding active viral transcription in a sample without detectable host sequences. Regardless of origin, how can a virus be transcriptionally active without its host?

Lines 289-291: “Analysis of the viral transcripts from the (meta-) transcriptomic data further revealed the expression of genes associated with antibiotic biosynthesis, photosynthesis and thiamine metabolism (Figure 3)” – since virus-encoded AMGs are derived from their host, how do we know that these transcripts are viral? Are there protein alignment trees showing that viral AMGs partition into their own “viral” clade that are distinct from their hosts? Please clarify.

Lines 303-305: “A high relative abundance of aegerolysin genes was detected in viruses found in the Scopalina sp. metagenomes (average abundance of 7.8 copies per prokaryotic genome; Figure 2C). Proteins of the aegerolysin family are widely distributed in fungi and bacteria…” – similar to the comment above, how do we know that these 7.8 copies are viral? Are they co-located on contigs with viral marker proteins? Broad in silico identification programs, such as VIRSorter, could pick up other mobile genetic elements that have distinct genome structures from that of prokaryotes, generating false positives in viral contig ID. Additional stringency measures, such as only counting AMGs if they are co-located on a contig with a known viral marker protein, would support these quantitative statements about viral AMG copy numbers per genome.
Additional comments: This manuscript exploring cell-associated viruses in sponge microbiomes is generally clear, well-written, and well-justified. A couple of sections in the discussion requires clarification - more details provided in the “validity of findings” section.


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: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: I believe the interpretation of the findings are sound - however, see my detailed comments on several minor points.
Additional comments: PeerJ review:
In this manuscript the authors have performed phylogenetic and functional characterization of sponge associated viruses through analysis of metagenomics and metatranscriptomic datasets. The manuscript is well-written with sufficient details on methods and the results. I don’t have any major suggestions – only a few minor details that the authors can take care of.

Minor comments:
Line 93: rRNA should be RNA
Line 97: I understand that the authors tried to designate different terms for the two fractions, but simply using ‘transcriptomic’ might be a little confusing – since metatranscriptomic is a general term for environmental RNA-seq. For example, what does ‘metatranscriptomic’ mean at line 187? Is it only the rRNA depleted fraction?
Perhaps they could assign ‘polyA metatranscripotmic’ and ‘non-polyA metatranscriptomic’ for these two fractions?
Line 177: Please rephrase this statement to clarify that the virus is a ‘close relative to O. lucimarinus virus 7’, not exactly the same virus.
Line 182 – 183: Any indication that the exact same viral contigs found in metagenome were expressed? It might be interesting to match the similarity of the metagenomics and metatranscriptomic viral contigs to investigate this.
Line 258-259: I would suggest that the authors specifically mention that no NCLDV infecting diatoms is known yet. However, I understand that the possibility cannot be completely ruled out.
General comment regarding the “Functional contribution of the viromes to the sponge holobiont” section:
While I appreciate the detailed discussion on the functional implications of the viral genes – an important point is, many of these genes might have a completely different function relevant to the viral fitness and in the context of host-virus interactions. The discussion if fine as it is, but the authors might want to add a short paragraph discussing the caveats of interpreting the functions of AMGs in the viruses – most of these have not been confirmed in lab yet, and these genes might perform a different function in the virus than the known functions.


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: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors have implemented all suggested modifications.
Experimental design: corrected accordingly.
Validity of the findings: corrected accordingly.
Additional comments: congratulations on the relevant study.


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

Input:
Title: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 2
Reviewer: 2

Output:
Basic reporting: Original comment and response in double quotations, and my new comment below.

““
Comment:
Line 119: Awkward “and, when present, being single-copy in ≥95% of all genomes”

Response:
We have rephrased this as follows:”… and being found as single-copies in ≥95% of all genomes.”
””

Change being to were?
Experimental design: ok
Validity of the findings: The following two responses did not fully address my initial discussion points. Original comments and responses are in double quotations, and my new comments are below.

““
Comment:
Lines 289-291: “Analysis of the viral transcripts from the (meta-) transcriptomic data further revealed the expression of genes associated with antibiotic biosynthesis, photosynthesis and thiamine metabolism (Figure 3)” – since virus-encoded AMGs are derived from their host, how do we know that these transcripts are viral? Are there protein alignment trees showing that viral AMGs partition into their own “viral” clade that are distinct from their hosts? Please clarify.

Response:
All viral transcripts presented here belonged to category 1 (“most confident” viral predictions) or 2 (“likely” viral predictions) by meeting Virsorter’s criteria 1 (presence of viral hallmark genes) and/or 2 (enrichment in virus-like genes) (see Methods). The identified expressed AMGs were therefore part of a polycistronic viral transcript.
””

VIRSorter would identify longer viral contigs, not individual short transcripts. It is unclear to me how one can determine that transcripts (short mRNA reads) themselves are viral unless one is certain that the gene (in this case AMG) that the transcript is mapping to is distinct from other non-viral sequences. My concern is that, considering that AMGs are derived from viral hosts, it is possible that host and viral AMGs are similar enough that host transcripts (short reads) are mapping to the viral AMG, generating false positives. Going back to my original question, have you looked at gene alignments indicating that viral AMGs are distinct from that of their hosts (at least below the mapping identity threshold)? If not, I am not convinced that one can conclude that these AMG transcripts are viral.

““
Comment:
Lines 303-305: “A high relative abundance of aegerolysin genes was detected in viruses found in the Scopalina sp. metagenomes (average abundance of 7.8 copies per prokaryotic genome; Figure 2C). Proteins of the aegerolysin family are widely distributed in fungi and bacteria…” – similar to the comment above, how do we know that these 7.8 copies are viral? Are they co-located on contigs with viral marker proteins? Broad in silico identification programs, such as VIRSorter, could pick up other mobile genetic elements that have distinct genome structures from that of prokaryotes, generating false positives in viral contig ID. Additional stringency measures, such as only counting AMGs if they are co-located on a contig with a known viral marker protein, would support these quantitative statements about viral AMG copy numbers per genome.

Response:
Please see our response to the previous comments. Yes, AMGs would have been on the same transcript as known viral marker genes.
””

A single transcript (transcriptomic short read) cannot contain a full-length AMG and known viral marker gene. Similar to the comment above, I’m not sure that one can make quantitative assessments about viral gene abundance ratio (copies per genome) without information that the gene is specifically viral, and not recruiting host reads.
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: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 2
Reviewer: 3

Output:
Basic reporting: The authors have taken care of all the concerns that I raised. I believe the manuscript is suitable for publication.

Thank you.
Experimental design: I originally did not have any concern regarding the experimental design.
Validity of the findings: The authors have incorporated the improvements that I suggested in the results and conclusions.
Additional comments: No additional comments


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

Input:
Title: TAXONOMIC, FUNCTIONAL AND EXPRESSION ANALYSIS OF VIRAL COMMUNITIES ASSOCIATED WITH MARINE SPONGES
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Thank you for clarifying the misunderstanding.
Additional comments: I support this paper for publication. Best of luck!


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: INCREASED PRECIPITATION ENHANCES SOIL RESPIRATION IN A SEMI-ARID GRASSLAND ON THE LOESS PLATEAU, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study investigated the impact of precipitation changes on soil respiration in a semi-arid area of the Loess Plateau, China. The authors provided some background information related to this study. But the manuscript was poorly written. There are too many awkward sentences that significantly hinder the readers’ understanding. Some examples are showed below. The authors need to seek help from a native English speaker or a commercial English editing company.
Experimental design: The research is original and fit the aims and scope of the journal. The authors provided some rational in the Introduction. Materials and methods were provided in details in general. But the number of replication (or block) of three was a little bit too low, particularly considering that there were only three precipitation treatments. It was not clear if the transparent polyethylene plates were installed (in reverse V) in the control and R150 plots to avoid the influence of light.  For data analysis, the description of correlation analysis (Lines 175-185) was confusing. Ggplot2 seems like a plotting R script. Why was this one used for correlation analysis? I don’t understand why only one variable was selected? I think multiple regression should be used here.
Validity of the findings: The authors provided the results, and results are generally sound. But some conclusion were not fully supported by the results. For example, the authors claim that precipitation influenced aboveground biomass, however, above-ground (shoot and litter) and total biomass were not significantly influenced by precipitation (Fig. 3).
Additional comments: I have a few major concerns with this manuscript. One was the experimental design. The experiment did not use block control, and the plots were not randomly assigned to the plots (Fig. 1). I also feel that the replication number was too small. The second one was data analysis. The authors mentioned ANOVA in the data analysis. But the results were not presented. In the regression analysis, relationship between soil respiration and temperature is usually not linear. Nonlinear regression should be used for soil temperature. The 3rd one was English and presentation. The manuscript needs to be significantly improved. I recommend Reject or Major revision.
Specific comments:
L29: was precipitation change a part of climate change?
L29: have an impact on, or impact (delete on)
L30: In dry ecosystems?
L32: delete (i.e., … grassland)?
L36: across precipitation treatments
L36: soil moisture was influenced by precipitation treatment
L38: there was (Please use past tense to describe current results. In the manuscript, both past tense and present sense are used).
L41-43: replace are with were, is with was
L44-46: run-on sentence. Please revise or separate it into two sentences.
L68-76: The logic flow is not good here. Please move the L72-74 to where soil respiration was introduced above.
L117-119: awkward sentence. Please revise.
L117: No block control was used in this study. The design only considered replication (3 plots for each treatment), but did not apply block control, and did not randomly assigned treatments to plots.
146: GOOD?
L165-166: not clear here.
L169: repeated measure?
L174-180: I’m confused by the data analysis here. It seems that ggplots is R script for plotting. Why used this for correlation (regression) analysis?
L178: why only one variable was selected? I think multiple regression should be applied.
Results: I would suggest to present soil temperature, moisture and soil respiration before Biomass of shoot, litter and root.
Where were results of ANOVA? Treatment effects, depth, date, interactions?
L251: use past tense for the results
L256-257: “So other factors were also analyzed using a linear model”: why?
L259 variables
L259-261: Revise this sentence.
L267-269: Why? So was there a significant treatment effect or not on above-ground biomass?
L280: was
L283: hole? Hold?
L286: The journal of .. has studied?
L333-341: This is background info. Too long here.
L349: what’s Akaike analysis?
L369-371: This is not new.
L372: if not significant, how can the authors say that precipitation can reduce soil nutrient?


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: INCREASED PRECIPITATION ENHANCES SOIL RESPIRATION IN A SEMI-ARID GRASSLAND ON THE LOESS PLATEAU, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The English language should be improved to ensure that an international audience can clearly understand the text.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Abstract:
The results don't support the conclusion that more precipitation can produce more root biomass. because the highest root biomass was found in R100.

Introduction:
The authors made a review of the recent studies of precipitation on CO2 flux and the need to take a study on grassland in semi-arid areas. however, it failed to descript the knowledge gap and put forward scientific questions or the objectives of this study.
line 97: the font of “—” should be fixed to “-”, and the same comment for line 99, 104, 105, etc.

Material and methods:
line 174: the varIdent function here was used to allow each timepoint rather than precipitation to have a different variance.

Results:
line 196: how did the 23% calculate?
line 198: how does the author define topsoil? 0-9.9 cm or 0-20cm
line 199: what is the meaning of "Soil pH was greater at depth in all three precipitations"
line 204: the text is not consistent with Fig 3A, As showed in the figure there is no significant difference between the three treatments but the authors said "significantly "lower by 45% in R50.
line 208-209：The same comment as line 204
line 211: the Fig.4 showed the Root biomass at different soil depths of three precipitations rather the "volume of roots"
line 215:The same comment as line 204
line 220: be consistent with the Table2, "0-10cm" or "0-9.9cm"
line 256: where is Fig.6?
line 261：no models showed in Table 6 had an AICC<2.

Discussion:
line 269-275: I don't think the authors give a robust explanation of why the R100 had the highest root biomass.
line 280-291: The interpretation may not seem to be robust, As it showed in Table 3 that precipitation showed a significant effect on soil temperature and moisture, however, it is not consistent with the authors’ statement here.
line 290-300: Another reason might that the water has a higher specific heat volume, thus the treatments with more soil water are more likely to have a low soil temperature.
line 328-332: However, more root and above ground biomass may lead to more water was absorbed and evapotranspiration by plants.
line 525: the first letter of the journal name should be capitalized.

Figures and tables:
Fig.1: only the R50 treatments were designed with shelters. how to avoid the shelters' effect on the solar radiation interception, it may differ between R50 and the other two treatments.
Fig.4: Please show the sample size
I don't think it is appropriate to do one-way ANOVA. you have two factors: soil depth and precipitation, so a two-way ANOVA is recommended.
Table 4: Why the sample size was 33？The soil CO2 flux was measured from April 2019 to October 2019 as mentioned in line 151, thus 11 measurements were taken during the experimental period.


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: INCREASED PRECIPITATION ENHANCES SOIL RESPIRATION IN A SEMI-ARID GRASSLAND ON THE LOESS PLATEAU, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: English and writing have been significantly improved.
Experimental design: Experimental design was adequate.
Validity of the findings: Results are clearly presented mostly.
Additional comments: Comments
The authors made efforts and addressed most of my concerns. There are still a few minor issues with the revision. Please see the specific comments below. I recommend Minor revision.

Specific comments:
(Line numbers in Word file with tracking)
L37: add "of ambient precipitation" after 150%.
L91: delete "the soil CO2 flux"?
L117: We set three blocks with three 6 x 6 m plots in each block, to make ...
L118: change interfere to intercept
L127: after each snowfall
L168: The company of IBM SPSS is IBM, not SPSS now
L173-188: I feel this part is too long. Please shorten this paragraph.
Two references are not found
Fig. 1: The three plots slope can be considered as one block. This study had three blocks (replications).
Table 2. Since only soil depth influenced most variables here, the main effect of soil depth could be presented. There is no need to present the interactive effect of precipitation and soil depth, as the interaction was not significant.
Table 4. Add significant test results to means.


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: INTESTINAL FATTY ACID-BINDING PROTEIN, A BIOMARKER OF INTESTINAL BARRIER DYSFUNCTION, INCREASES WITH THE PROGRESSION OF TYPE 2 DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: Although the manuscript has been proof red an edited by Medjaden bioscience limited, there are still several sections of the paper that require careful editing for English language.  For example in the abstract the statement "however this association disappeared in followed multiple linear regression analysis" needs to be corrected and in the discussion line 293 the word "wildly" needs to be replaced by "widely"
The introduction needs to discuss the sensitivity of I-FABP as a marker of intestinal per me ability or barrier dysfunction rather than as a marker of intestinal epithelial cell damage.  We does of the article will wish to know the relative sensitivity of the blood test compared to other biomarkers of permeability such as saccharide absorption tests

The article structure, figures, tables and raw data are appropriate
The conclusion regarding the progression of diabetes is not in place it to the association with duration of hyperglycemia
Experimental design: This research study is within the Aims and scope of the Journal
The research question it is well defined and relevant, however the title needs to specify that the study was conducted exclusively in type 2 diabetes and that type 1 diabetes as well as patient below the age of 18 years were excluded
The investigation is performed in at technically adequate and ethical manner
The methods are sufficiently detailed
Validity of the findings: The discussion includes appropriate for text regarding the limitations of the study specifically the concomitant pathological conditions that may have been impacting the results in the cohort studied as in patients, and therefore the associated comorbidity may have impacted the result in serum I-FABP.  A 2nd major limitation that is acknowledged is the presence of confounders, such as the use of anti diabetes drugs as well as the exclusion of severe nephropathy, which may impact the conclusions of the study.
The underlying data have been provided in the supplementary files specifically the EXCEL spread sheet with all the data available
The conclusions are somewhat suspect that since the only positive finding that remains significant after multi very it analysis is the duration of hyperglycemia, and the conclusion statement of the abstract which suggests that duration of hyperglycemia implies progression of diabetes is not documented in the cohort.  For example it is not probe in that duration of type 2 diabetes in this cohort actually caused a more diabetic tropathy or vascular complications.
Additional comments: 1.  The title needs to specify that the study was conducted exclusively in type 2 diabetes
2. The conclusions are somewhat suspect that since the only positive finding that remains significant after multi very it analysis is the duration of hyperglycemia, and the conclusion statement of the abstract which suggests that duration of hyperglycemia implies progression of diabetes is not documented in the cohort.  For example it is not proven that duration of type 2 diabetes in this cohort actually caused a more diabetic tropathy or vascular complications, and in addition, the study shows no association of serum I-FABP with insulin levels which would be an appropriate marker of progression of diabetes..
3. The discussion includes appropriate text regarding the limitations of the study specifically the concomitant pathological conditions that may have been impacting the results in the cohort studied as in patients, and therefore the associated comorbidity may have impacted the result in serum I-FABP.  A second major limitation that is acknowledged is the presence of confounders, such as the use of anti diabetes drugs as well as the exclusion of severe nephropathy, which may impact the conclusions of the study.
4. In addition the discussion needs to place in perspective the relative contribution of hyperglycemia to the abnormal levels of I-FABP;  thus, if the correlation coefficients with duration of hyperglycemia are approximately 0.33, the variance in serum I-FABP attributable to duration of hyperglycemia is estimated at only approximately 10%, which reduces the importance of hyperglycemia as a risk factor
5. The introduction needs to discuss the sensitivity of I-FABP as a marker of intestinal per me ability or barrier dysfunction rather than as a marker of intestinal epithelial cell damage.  We does of the article will wish to know the relative sensitivity of the blood test compared to other biomarkers of permeability such as saccharide absorption tests
6. Although the manuscript has been proof red an edited by Medjaden bioscience limited, there are still several sections of the paper that require careful editing for English language.  For example in the abstract the statement "however this association disappeared in followed multiple linear regression analysis" needs to be corrected and in the discussion line 293 the word "wildly" needs to be replaced by "widely"
7. In line 104, use of the word "had" to describe the age of the patients is incorrect, and S1H abbreviation does not appear to be explained anywhere


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: INTESTINAL FATTY ACID-BINDING PROTEIN, A BIOMARKER OF INTESTINAL BARRIER DYSFUNCTION, INCREASES WITH THE PROGRESSION OF TYPE 2 DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: While the article had a good structure and flow, I would suggest that the manuscript should be proof read again to ensure clarity of expression, correct tenses etc. I have detailed a few examples of what I mean by this:

Line 49-50: “Serum I-FABP is positively associated with the duration of hyperglycemia, which suggested that the IB dysfunction got worse with the progression of diabetes.”
Reviewer comment:
=================
Mixture of tenses here – possibly better as:
“Serum I-FABP is positively associated with the duration of hyperglycemia, which suggests that the IB dysfunction gets worse with the progression of diabetes.”

Lines 63 – 64: “since hyperglycemia has existed long before other diseases happen in diabetic patients,”
Reviewer comment:
=================
This may be better as “since hyperglycemia exists long before other diseases occur in diabetic patients,”

Lines 68 – 69: “Nowadays intestinal barrier (IB) dysfunction induced by hyperglycemia was considered to be”
Reviewer comment:
=================
Here the tenses do not appear to be correct: was/is:
Instead I would suggest:
“Nowadays intestinal barrier (IB) dysfunction induced by hyperglycemia is considered to be”

Lines 69 – 70 “was considered to be the underlying mechanism of systemic infection and inflammatory in diabetes.”
Reviewer comment:
=================
Ii looks to me like there is a word missing after “inflammatory”?  Possibly “response”?

Reviewer comment:
=================
Lines 100 – 101: “who had been tested hyperglycemia”
Possibly missing word: “for”
“who had been tested for hyperglycemia”

While it is possible to understand what is being said, clarity would be increased for the reader with some further changes like these examples.
Experimental design: This is an observational study on both inpatients and outpatients with a relatively straightforward statistical analysis. At the centre of the discussion is the relationship between IB dysfunction (as expressed through I-FABP) and duration of hyperglycemia (duration).

The main conclusion is:
Line 49-50: Serum I-FABP is positively associated with the duration of hyperglycemia, which suggested that the IB dysfunction got worse with the progression of diabetes.

There are a couple of points to be made here about the relationship.

One line 74: “Thaiss’ study, hyperglycemia was confirmed to be the independent risk factor of IB dysfunction”

On line 68 – 70 we have: “Nowadays intestinal barrier (IB) dysfunction induced by hyperglycemia was considered be the underlying mechanism of systemic infection and inflammatory in diabetes.”

This seems to indicate that some that there is some sort of causal relationship between hyperglycemia and IB.

The study in this paper states (lines 280 – 281):
“The association between hyperglycemia and IB dysfunction was clarified in our study, but the causal relationship between them is yet to be discussed.”
(Reviewer comment:  As it is duration of hyperglycemia that is being investigated, I would expect that this should be ““The association between the duration of hyperglycemia and IB dysfunction…”

So no causal assumptions for the analysis. Just an observational investigation.

Reviewer comment:
==================
In the multiple linear regressions (lines 138, 152) I-FABP is always the response or independent variable and duration is the explanatory variable, which could be considered to at least indicate that the causal direction is as discussed lines 74, 68-70. In particular, in Table 1.3 both duration and age are explanatory variables, with age always affecting I-FABP.
As the causal relationship is not discussed in this paper, when the model is introduced it should be stated more clearly that this is just way of determining possible associations between I-FABP and the other variables – with, for example, some further discussion of how age may effect both I-FABP, duration.

Study participants (97 - 107)
=======================
Were all patients who agreed to the study and met the criteria included in the analysis or did any drop out (as this is a cross-sectional study drop may not be an issue). While the inclusion and exclusion criteria are clearly given I think a small statement about possible bias in the study population would be helpful.

Statistical Analysis (132 - 140)
========================
Lines 133 – 134 “Non-parametric tests were performed to determine the normality of continuous variables, whereas the non-normal distribution variables were expressed as median (interquartile range).”

Reviewer comment:
================
This sentence contains two things: assessing normality and summary statistics for non-normal variables. I would suggest this be changed into 2 sentences, for example: Continuous variables were assessed for normality using the <test-name> test. Non-normal variables are…

Results (Line 142 – 153)

Table 1.1
Reviewer comment:
================
The Male/Female p-value reported is from a Fisher exact test not chi-square test.

Lines 150 – 152 and (Table 1.2)
Reviewer comments:
=================
Some of these correlations are weak, for example FPG is   -0.19. I think a comment on the strengths of the relationships is need.

Lines 152 – 153: “Further, multivariate linear regression analysis showed that serum I-FABP was positively associated with age and duration of hyperglycemia in all patients (Table 1.3).”

Reviewer comments:
=================
I-FABP is right-skewed and looks to be non-normal and therefore model assumptions are violated. Any estimates and inferences are likely to be biased.

I suggest you try a transformation of the data to reduce skewness e.g. log transform, and re-run the analysis.

A general question: what model was fit here? No intercept is reported in the Table.
Were all variable including intercept fitted or just duration and age?  This is what I would expect. If so then they should also be reported in the Table.
Please state clearly the model.

This model really doesn’t really tell much more than Table 1.2 where duration and age were the two largest positive correlations with I-FABP.

Why might it be the case than some of the variable with correlations to I-FABP are not significant in the model?

The headings for the Table need to be explained. Is B the estimate of the parameter from the model? (This looks to be taken directly from the SPSS output).

Serum I-FABP in outpatient subgroup
===============================
Reviewer comments:
=================
Lines 169 – 171 / Table 3.2 “However, the duration of hyperglycemia was the only metric that statistically associated with serum I-FABP in the followed multiple linear regression 171 analysis (P<.001) (Table 3.2).”

Same comments apply as for the previous regression.

Serum I-FABP in inpatient subgroup
================================
It is not clear how Table 3.3 is formed from the supplementary material.
This needs to be explained further please.
Validity of the findings: I have concerns here about the overall conclusion statement.

Taken together the data overall does show an association or correlation between I-FAPB and hyperglycemia duration.

However, the two subgroups show different behaviours.

For outpatients all the durations are 0 (or rounded to 0). There is no variation in the data, the correlation can’t be calculated and no (statistical) relationship is established between the two variables.

The case is different for the inpatients where durations have a median of 10 years.
My concern here is that the relationship between I-FAPB and hyperglycemia duration reported for the overall population is driven by the sub-group sizes. One could imagine a situation where, if a larger number of outpatients were recruited, it would eventually mask the signal in the pooled data.

Looking at the outpatient duration, even large values of I-FABP (max 5221.39) have 0 durations, whereas for inpatient the durations tend to be non-zero, with positive association. (Is the rounding of duration for outpatients hiding systematic changes in duration?)

The conclusion needs to be adjusted for the different behaviours in the sub-groups, with discussion of why this is so. Given the 0 durations for the outpatients for high levels of I-FAPB, what is driving the relationship for the inpatients data?
Additional comments: I enjoyed reading the article and found it to be an interesting study. My comments detailed above are mainly in the area of the statistical analysis, where some clarification is needed, with some additional comments on the grammar, but more importantly in the area of the different behaviours of the subgroups and how these should be reported in the findings.


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: INTESTINAL FATTY ACID-BINDING PROTEIN, A BIOMARKER OF INTESTINAL BARRIER DYSFUNCTION, INCREASES WITH THE PROGRESSION OF TYPE 2 DIABETES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Authors reported that serum I-FABP was significantly associated with duration of hyperglycemia. The manuscript is well written and easy to read.  Additionally, the results are interesting but some issues should be solved. I think the data are sound and this manuscript can be published in "PeerJ" after major revision.  In the section of introduction, The sentence " Generally, the nature......... to evaluate IB dysfunction in our study" in the line 87-89, please check the citations of references 8 and 22. Are these two references belong to authors' studies ? . Please correct study to studies.
Experimental design: Please add the country name of ELISA kit in the line 129.
Validity of the findings: Please describe "subtitles "more detail.
Additional comments: 1. Please cite the references in the section of discussion in the lines 220-230.
2. Please discuss the role of I-FABP in oxidative stress, inflammatory response, and infection.


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: INTESTINAL FATTY ACID-BINDING PROTEIN, A BIOMARKER OF INTESTINAL BARRIER DYSFUNCTION, INCREASES WITH THE PROGRESSION OF TYPE 2 DIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have had the manuscript proof read.
Experimental design: Statistical Analysis (Lines 150 – 161) needs some additional work:

Line 152: Continuous variables were assessed for normality using the nonparametric test.

There is no single non-parametric test, there are non-parametric tests for all sort of hypotheses. The query here was with regard to which test was actually used – a name or a reference is all that is required.

Line 153: Variables with a non-normal distribution were expressed as the median (interquartile range).

Better would be:
Variables with a non-normal distribution are summarised by the median and interquartile range.

Line 153 – 155:
Then, the difference of variables with a non-normal distribution between two groups was assessed by the Mann-Whitney test, the categorical data were assessed by the chi-squared test, and the relationship between two continuous variables was assessed by the Spearman correlation coefficient.

The “Then” at the start of the sentence is not needed. Non-normal distribution also applies to categorical variables. Better to re-state with something like:

Differences between two groups of continuous variables with non-normal distributions were assessed by the Mann-Whitney test. Differences between categorical variables were assessed by the chi-squared test. The relationship between two continuous variables were assessed by the Spearman correlation coefficient.

Line 157: Logarithmic transformation was performed to generate data with a normal distribution.

Was the transformed data assessed for normality?
Validity of the findings: While I realise that duration is just one aspect of a hyperglycemia episode it is used in the paper's title and to convey the results and conclusions.

The authors state in their rebuttal:

"However, our conclusion has not changed."

The conclusions (line 58 - 62) is:
The serum I-FABP level was positively associated with the duration of
hyperglycemia and glycemic variability but negatively associated with islet beta-cell function in type 2 diabetes patients; moreover, the serum I-FABP level was higher in patients with retinopathy than in those without retinopathy, suggesting that the IB dysfunction got worse with
the progression of diabetes.

Let me explain the issue I have with this statement.

1)	for the inpatients there is a (statistical) relationship between duration and I-FAB,

2)	for the outpatients here is no evidence of a relationship.

As the sub-groups have been pre-defined, this is a planned finding for the experiment.

When the data is combined there is statistical evidence of a relationship, but we know that for the outpatients no such relationship has been established.

This may occur in the combined group for a number of reasons - group sizes, effect sizes etc. So, while there is statistical evidence (i.e. small p-value) this does not change the outpatient result from the outpatient subgroup analysis and therefore, despite the statistical evidence, we cannot say that the relationship exists for all patients. This needs to be reflected in the conclusions and results.

Are there are systematic differences between the groups that are not captured in the analysis of all patients together? It appears that the analysis for all patients is confounded in some manner.

In my opinion, it is not enough that this is discussed at lines 365 – 367 / 321 – 322, it needs to be acknowledged in the Conclusions and Results (and possibly also the title).

For example: You could state that when all patients are combined there is some evidence of a relationship but on further investigation it turns out that there appears to be different behaviours in each subgroup with some further discussion.

Given that there are 3 groupings, inpatients, outpatient and all patients, why are only the results for inpatient and all patients reported, the result for outpatient is of interest too. You need to mention the results for all groupings and any discussion of “all” patients with regard to duration needs to acknowledge this.

I would recommend that the following need to be changed to reflect this difference between inpatient/outpatient/all:

Conclusions (line 58 - 62)

Results (46 - 56) Only inpatient and all patient results are reported - outpatient results are need too.

Discussion (209-210)
Additional comments: I would like to thank the authors for the revisions made in respect to the comments on the first revision of the article. Many of the issues raised in the comments have been resolved.

However, I feel there are still a couple of remaining issues that need to be looked at, detailed in the Validity of findings. These are related to how the results are reported.


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: EPIGENETIC IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE 10 AS A FUNCTIONAL TUMOR SUPPRESSOR AND CLINICAL SIGNIFICANCE FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	English language should be improved and typos are present in the main text; English proofreading is recommended.

2.	Thanks to the authors for uploading the raw data; however, some raw data included as supplementary material are not commented in the main text; see the section "General comments for the author"

3.    Overall, the results as presented in this manuscript do not fully support the initial hypotheses
Experimental design: Some experiments were not performed respecting the technical standards for the used experimental procedures, please see the comments in the "General comments for the author"
Validity of the findings: Overall, the data provided in this manuscript are not very robust to fully support the conclusions as stated (please see in "General comments for the author")
Additional comments: In the current study, Tang and colleagues have focused their research on investigating the expression and the role of MAPK10 in human HCC. By performing immunohistochemistry analysis, the authors could detect a downregulation of MAPK10 protein in HCC samples when compared to the adjacent non-tumor tissues; more importantly, negative expression of MAPK10 in HCC was associated to high AFP levels, advanced tumor stage and shorter overall survival of HCC patients following liver resection. Further experiments showed that MAPK10 expression is reduced in human HCC by promoter methylation. MAPK10 overexpression in vitro led to inhibition of colony formation and apoptosis induction. The authors concluded that MAPK10 might represent a new therapeutical target in a clinical setting.
Overall, the data as presented in this manuscript do not fully support the conclusions stated by authors; therefore, the manuscript needs to be revised in many aspects and complemented with new experiments in order to be considered for a new revision.
Here the major points, which need to be addressed:
1.	The authors performed semi-quantitative RT-PCR for detecting MAPK10 expression in human HCC cell lines as shown in Fig.3; however, in the image presented in the figure it is possible to see additional upper bands, which might indicate not specific primer binding to any other sequence; could the authors comment on that? It would be helpful to include in the same analysis any type of positive control (human normal hepatocyte?). Additionally, detection of protein levels in the same cell lines is necessary to complement the expression data, considering also the data presented in Fig.4B (see comment below).
2.	In Fig.4A/B positive controls for unmethylated and methylated DNA must be included in the assay in order to determine the quality of the bisulfite conversion and identify artifacts such as mispriming. In addition, the authors should add any comment about the detection of unmethylated MAPK10 in all the HCC samples used in order to better integrate these results  with the IHC results; indeed, are additional mechanisms involved in MAPK10 expression regulation? The same comment is valid for the results presented in Fig.4B; do they authors know or speculate why MAPK10 promoter in the HuH4 is unmethylated but there is no expression of MAPK10?
3.	The authors stated in the main text that cells were treated with 5-Aza and TSA (line 241-242), but in the legend for figure 5 (line 474) the only treatment specified is with TSA and in the image labels is again A+T; which is the treatment performed in those cells? In the case that Hep3B and HepG2 cells were treated with Aza and TSA, no changes were observed at MAPK10 methylation level when compared to the untreated control cells in the MSP results, especially for Hep3B, meaning that promoter methylation is not playing a role in the regulation of MAPK10 expression, how to explain these results?
4.	 In Fig.6A, Flag signal overlaps mostly with DAPI especially in HepG2 cells, which does not support the conclusions drawn by the authors. Any comment for these results? Analysis of MAPK10 protein in fractionated cytoplasmatic and nuclear protein lysates would be helpful for describing the cellular localization of MAPK10.
5.	The technical approach used for performing the colony formation is not right; indeed, it would have been more appropriated to transfect first the cells with MAPK10 and control plasmid and select the cells, then seed the same number of cells after selection for each condition and let them to grow for 10-14 days, leaving out with this approach the unwanted difference in transfection efficiency of different plasmids (empty vector and vector with the gene insert), therefore difference in number of cells which can form colonies.
6.	Apoptosis data presented in Fig. 7A should be confirmed by detection of cleaved PARP and cleaved Caspase 3 proteins.
7.	English language should be improved and typos are present in the main text; English proofreading is recommended.
8.	Excel file for raw data includes some data not displayed or commented in the main text (figure 7G?); can the authors explain these results?

Minor points:
1.	The panel C in Fig.6 shows the relative MAPK10 protein expression; do the data include three biological replicates? The raw data or images for the three replicates should be supplied.


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: EPIGENETIC IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE 10 AS A FUNCTIONAL TUMOR SUPPRESSOR AND CLINICAL SIGNIFICANCE FOR HEPATOCELLULAR CARCINOMA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Please see Line 53. The word “finding” should be “find”.
2. Please describe the known tumor suppressor genes frequently methylated in HCC. Compare those known tumor suppressor genes with MAPK10 or include them in this study as positive controls.
3. Are there any known tumor suppressor genes which belong to JNK family implicated in HCC?
4. In Line 75-77, the provided reference no.18 indicates JNK3 deficiency induced cell apoptosis in the brain, which is opposite to what the authors observed in the current study. Please explain why MAPK10 (JNK3) overexpression induced cell apoptosis in the current study.
Experimental design: 1. Please provide the reference of the scoring method for the IHC staining of HCC liver sections.
2. Please explain why using histone deacetylase inhibitor TSA in this study. Results of DNA methyltransferase inhibitor Aza treated alone can better elucidate the effect of MAPK10 methylation in HCC cell lines.
3. Please provide the reference to support the methylation sites detected in this study (Supplementary Table 1).
Validity of the findings: 1. It’s hard to see the association between MAPK10 expression and methylation in HCC cell lines in Figure 4B. Please add graphs to show the association clearly.
2. Please add colony forming assay, apoptosis assay, and cell viability assay for HCC cell lines after Aza treatment to better address the causal effect of MAPK10 methylation in HCC progression.
3. In Figure 3, please provide a non-cancer hepatic cell sample as a control for MAPK10 expression.
4. Please add an internal control to Figure 4A.
5. In figure 5, the methylated MAPK10 expression seems to be unchanged after A+T treatment in Hep2B cell line. Please explain.
Additional comments: I commend the authors for their extensive data set, compiled over many years of detailed fieldwork. In addition, the manuscript is clearly written in professional, unambiguous language. There are some issues which should be improved upon before Acceptance.


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: EPIGENETIC IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE 10 AS A FUNCTIONAL TUMOR SUPPRESSOR AND CLINICAL SIGNIFICANCE FOR HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 1

Output:
Basic reporting: 1. English language has been improved

2. additional literature references should be provided (see comments)

3. The new results and the new comments are not exhaustive for supporting all their conclusions.
Experimental design: Please see the comments
Validity of the findings: The validity of the findings (in particular in Fig.5) is not fully supported by the data and the comments provided in the revised version of this manuscript by the authors.
Additional comments: 1. Previous comment: 2. In Fig.4A/B positive controls for unmethylated and methylated DNA must be included in the assay in order to determine the quality of the bisulfite conversion and identify artifacts such as mispriming. In addition, the authors should add any comment about the detection of unmethylated MAPK10 in all the HCC samples used in order to better integrate these results with the IHC results; indeed, are additional mechanisms involved in MAPK10 expression regulation? The same comment is valid for the results presented in Fig.4B; do they authors know or speculate why MAPK10 promoter in the HuH4 is unmethylated but there is no expression of MAPK10?
"We included the YccB1, a TSG hypermethylated in the breast cancer, in our study as positive controls. At the same time, we also included the negative control in it. We are sorry that these controls were not shown in the previous versions and we have already corrected the Fig.4A/B in our revision. Among the 18 fresh primary HCC tissues, 15 of them underwent immunohistochemical staining at the same time and we found that 11 of them lost expression of MAPK10 and the corresponding Mapk10 promoter methylation was positive. We can see a band indicating the low expression of MAPK10 in the HuH4 RT-PCR results, but there is no obvious methylation detected in its promoter. We think some additional mechanisms maybe involved in Mapk10 regulation."

New comment: The authors included YccB1 as positive control gene for their analysis; could the authors please provide explanation about this gene and references? NCBI gene database doesn´t retrieve any result about this gene. In addition, the section of M&M should be updated accordingly. Additionally, the correct controls for the MSP are 100% methylated DNA and 100% unmethylated DNA samples (commercially available), which the authors have to include in their experiments.

2. Previous comment: 3. The authors stated in the main text that cells were treated with 5-Aza and TSA (line 241-242), but in the legend for figure 5 (line 474) the only treatment specified is with TSA and in the image labels is again A+T; which is the treatment performed in those cells? In the case that Hep3B and HepG2 cells were treated with Aza and TSA, no changes were observed at MAPK10 methylation level when compared to the untreated control cells in the MSP results, especially for Hep3B, meaning that promoter methylation is not playing a role in the regulation of MAPK10 expression, how to explain these results?
"We are sorry that there is a mistake in the legend (line 474), the cells were actually treated with 5-Aza and TSA in our experiment and we have already corrected that in our revision. In addition, the MSP result showed that there was the decrease in Mapk10 methylation level for HepG2 and the increase in Mapk10 unmethylation lever for Hep3B after treatment with 5-Aza and TSA, the inhibitors of methylation, which indicated that the transcriptional silence of Mapk10 for these two cell lines was caused by promoter methylation."

The answer provided by the authors doesn´t explain the results reported in Fig. 5; cell line treatment with 5-Aza and TSA should lead to decreased methylation and increased unmethylated levels in a similar way in both cell lines by MSP detection and the MSP results should be in line with the PCR results showed in the upper panel of Fig.5. Since the results presented in Fig.5 are not convincing, the authors should provide an additional proof that methylation and histone acetylation are involved in the regulation of MAPK10 expression in HCC (pyrosequencing?)

3. Which antibody was used for detection of PARP?


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: EPIGENETIC IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE 10 AS A FUNCTIONAL TUMOR SUPPRESSOR AND CLINICAL SIGNIFICANCE FOR HEPATOCELLULAR CARCINOMA
Review round: 2
Reviewer: 2

Output:
Basic reporting: Thanks to the authors for the correction of typos.
Experimental design: Thanks to the authors for adding the references.
Validity of the findings: 1. Please explain why mRNA expression of MAPK10 is not negatively correlated with its methylation in each HCC cell line shown in Figure 4B.
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: EPIGENETIC IDENTIFICATION OF MITOGEN-ACTIVATED PROTEIN KINASE 10 AS A FUNCTIONAL TUMOR SUPPRESSOR AND CLINICAL SIGNIFICANCE FOR HEPATOCELLULAR CARCINOMA
Review round: 3
Reviewer: 1

Output:
Basic reporting: Still some English typos or ambigous sentences are present in the "Discussion" section
Experimental design: Please see "General comments"
Validity of the findings: See "general comments" section
Additional comments: Thanks to the authors for replying to comment 1 and 3.
Regarding the comment 2, it is more matter of 5-Aza concentration and time treatment; the authors should add more details about the method used for interpretation of results (thickness of band, M or U) in the corrisponding section in materials and methods.
Additionally, the authors should add a sentence in the discussion that additional mechanisms beside methylation are involved in the regulation of Mapk10 expression.