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

Input:
Title: THE DETAILED DISTRIBUTION OF T CELL SUBPOPULATIONS IN IMMUNE-STABLE RENAL ALLOGRAFT RECIPIENTS: A SINGLE CENTER STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written with only occasional typos and a few less clear sentences. For example, in the METHOD section, the authors stated "all participants received written informed consent" when it should be " all participants provided or gave written informed consent".

References are appropriate and background are sufficient.

The manuscript is professionally structured and the data presentation is very clear.  The authors listed tables and figures as subtitles throughout the manuscript which is rather unconventional.  They should be referenced in the text without the captions.  Analyzed flow cytometry data were provided as an excel workbook, although the true raw data should the the fcs files themselves, which may not be what PeerJ is looking for.

The manuscript is self-contained, with a hypothesis, an approach to test the hypothesis, results, and a conclusion.
Experimental design: The research is within the scope of PeerJ.

The objective of the study is to identify, among peripheral blood T cells, markers of immunosuppressed and yet stable immune state versus normal.  It is unclear why this is important.

The experimental design has a significant flaw of not controlling for a potential confounder. It is known that renal failure and dialysis change patient's immune profile (Betjes, Michiel GH. "Immune cell dysfunction and inflammation in end-stage renal disease." Nature Reviews Nephrology 9.5 (2013): 255.).  This work compares immune profile of patients with previous renal failure with normal subjects. Although renal function in the enrolled subjects have been corrected with the transplant, it is unclear if transplant also revert immune dysregulation.  Thus, differences the authors find may be due to previous renal disease or immunosuppression.  The authors did not consider the impact of previous renal disease and attributed differences only to immunosuppression.  One way to correct this is to enroll patients with renal failure before transplant not on immunosuppression to see if the observation is unique to stable immunosuppressed patients.

The technical aspect of this work is strong.  There are reasonable number of patients in each group (44 to 52).  The flow panels are rationally designed.  The flow cytometry profiles look very robust.  Statistical analyses are appropriate.  The study has minimal risk to patients and it is conducted after obtaining informed consent.

Method is mostly sufficient.  Some clarification needed:
1. How many subjects for each category (Y1 and Y5 after transplant) are screened to identify the 51 and 52 subject who meet the eligibility criteria?  This may help to understand how selective and representative the study enrollees are.
2. Are patients taking immunosuppressive drugs other than tacrolimus?  Typically renal transplant patients are on three or least two immunosuppressive drugs that include a CNI, MMF with or without steroid.
3.  Were the patients on dialysis before transplant?  This is relevant because dialysis can impact immune profile.
Validity of the findings: As stated above, I think this study lacks a critical control group to make the conclusion that the authors are making.

Otherwise, the data and statistical methods are sound.  Presentation of the data is objective, clear, and focused on the research question.
Additional comments: The informed consent is in English.  Is this a translation or all patients in the study are able to read and understand English?


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: MOLECULAR EVIDENCE FOR SEX REVERSAL IN WILD POPULATIONS OF GREEN FROGS (RANA CLAMITANS)
Review round: 1
Reviewer: 1

Output:
Basic reporting: No commets. Comments are in the general report.
Experimental design: No commets. Comments are in the general report.
Validity of the findings: No commets. Comments are in the general report.
Additional comments: This is an interesting work on the study of the relations between phenotypic and genotypic sexes of an amphibian species in the wild.
However, I do not clearly see why they think that sex reversal might be a relatively natural and frequent process in this species. If so, why the direction is not the same across the different ponds?

Major points:

Figure one: It is clear that in some ponds: Laur02, Forest2, Forest6 and Laur01 the authors only found XX-Males and in other ponds, only XY females. I did not find in the text a clear or at least some explanation for this behavior.  This is also true for the ponds of both sex reverse directions were found.

Figure two: Same considerations for the presence of intersex gonads.

Sex determination markers: Is it not possible that the specific sex markers the authors used are not 100% reliable? Thanking into consideration that some genes have been proposed to be involved in the sex determining system in amphibian such as DM-W. I understand there is a diversity of sex determining mechanisms, but it would be nice if the authors include a discussion on this respect.

Minor points:

Introduction. Page 3. Lines 67-68. The citation Yoshimoto et al. 2008 is in the reference section but not in the text. It should be here. I would also consider that the authors should have to add here another citation from the same group: Yoshimoto et al. 2010. Development 137, 2519-2526. 2010.

Methods. Page 8. Line 179. Please change Yoshomoto by Yoshimoto.

References. Page 31. Line 701. Please change “200” by “2008”.


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: MOLECULAR EVIDENCE FOR SEX REVERSAL IN WILD POPULATIONS OF GREEN FROGS (RANA CLAMITANS)
Review round: 1
Reviewer: 2

Output:
Basic reporting: Meets requirements. See General Comments below.
Experimental design: Meets requirements. See General Comments below.
Validity of the findings: Meets requirements. See General Comments below.
Additional comments: This is important work which starts to investigate both the ecological and environmental context of sex reversal in amphibians. Overall I thought this was a well-constructed piece of work. The authors clearly state their hypotheses, and explain how their work fills an important gap in the knowledge. I really appreciated that is work is empirically testing some long-held assumptions in the field – that sex reversal is an anthropogenic effect rather than a naturally occurring phenomenon. I also appreciated that the authors spent time clearly disambiguating two often conflated phenotypes: intersex from true sex reversal.

I have no major corrections, just a question. In frogs, is intersex a terminal or transitional phenotype? Perhaps the authors can further clarify that intersex testes are not evidence of sequential hermaphroditism? (This was mentioned but the paper could clarify this a little more). If intersex is a terminal phenotype, can the authors comment on whether these intersex individuals are likely to be fertile and capable of successfully reproducing as one sex or another?

Minor typographical corrections:

Line 93: “anthropogenic environments and environments contexts with lower degrees of human activities”. - should be “environmental contexts”.
Line 215 : “These criterial ultimately” – should be “criteria”.


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: MOLECULAR EVIDENCE FOR SEX REVERSAL IN WILD POPULATIONS OF GREEN FROGS (RANA CLAMITANS)
Review round: 1
Reviewer: 3

Output:
Basic reporting: In this study, Lambert et al assess the prevalence of sex reversal and intersex in adult green frogs (Rana clamitans) across 16 wild populations sampled along a forest-to-suburban land use gradient. By comparing phenotypic sexes inferred by histological analysis and genotypic sexes inferred by five sex-linked SNP loci, they found evidence for sex reversal in 12 of 16 populations, including all the four populations inhabiting ponds in undeveloped forests. By examining the presence of oocytes in phenotypic male testis, they found that 10 of 16 populations had intersex phenotypic males, and XX-males have higher probability being intersex than XY-males. They also applied Bayesian analyses to examine the associate between land use and the occurrence of sex reversal or intersex, and they found no influence of suburban land use on sex reversal or intersex frequencies. In general, the manuscript is well organized, and the methods are sound. Given that there are few field-based studies assessing sex reversal in amphibians in contrasting ecological conditions (natural vs anthropogenic), the results from this study are important for this field, providing early evidences for understanding the extent to which sex reversal is related to natural or anthropogenic causes in amphibians.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Throughout the manuscript, the authors frequently use the terms “sexually-discordant phenotypic male”, “sexually-concordant phenotypic males”, “sexually-discordant genotypic female” et al to represent sexual several or normal frogs, which sometimes are quite fuzzy, particularly the authors sometimes use different terms to represent the same meaning, such as sexually-discordant phenotypic male vs sexually-discordant genotypic female. I would suggest the authors add symbols like XX-male, XX-female, XY-males or XY-female in parentheses after each term, e.g. “sexually-discordant phenotypic male (XX-male)”, so that readers can readily understand the meaning of these terms without imaging the genotype and phenotype in their brains.

In the abstract, the authors concluded that “our results also suggest that intersex phenotypic males and sex reversal occur independently of each other”. But according to Line 313-319 and Figure 3, XX-males show twice the probability of being intersex compared to XY-males. Doesn’t it mean that intersex is associated with female-to-male sex reversal?

Figure 2: Given that the sample sizes vary greatly among the 16 populations (ranging from 6 to 92), I suggest the authors present the total number of frogs for each pond in this figure, so that readers can judge the reliability of the frequency for each population. In addition, the symbols of the ponds are meaningless for reads, thus I also suggest the authors show the percent of suburban land use for each pond, so that readers can readily find out the ponds entirely surrounded by undeveloped forests or those heavily surrounded by anthropogenic lands.

Figure 3: the bar plot shows that XX-males have much higher probability being intersex than XY-males. However, the statistical significance of this difference was not assessed. Given that the sample size for XX-male (only 11 in total) is quite small, this observed difference may be insignificant and resulted from random effect.

L301-304: It is unclear if the authors want to say suburban land use was slightly associated with frequencies of sexually-discordant genotypic females here. Please improve the description and indicate your finding more explicitly to readers.

In many of the correlation analyses, it would be easier to follow if the authors could provide the plots.

I would suggest the authors summarize the results in the subtitles. And the discussion is very lengthy making it hard to follow the purpose for some of the discussion. For instance, the authors basically did not provide any new clue on the mechanisms for sexual reversal. The discussion is quite redundant and verbose. Also some of the contents in the discussion are unnecessary background that not related with this work. I would suggest the authors to shorten the discussion section and more focus on their current results.

I cannot find the figure legends for all the supplemental figures.


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: MOLECULAR EVIDENCE FOR SEX REVERSAL IN WILD POPULATIONS OF GREEN FROGS (RANA CLAMITANS)
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This is a nice piece of work. Congratulations.


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: TAMILOKUS MABINIA, A NEW, ANATOMICALLY DIVERGENT GENUS AND SPECIES OF WOOD-BORING BIVALVE FROM THE PHILIPPINES
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: In this study the authors combine anatomical, morphological and molecular data to provide compelling evidence of the existence of a new genus and species in the Teredinidae. The ms is in general clearly written. Below are some minor comments and suggestions on some specific points of your ms. I hope you find them helpful.
Line 82- There are no accession numbers in Table 1.
Line 169-170 “under accession numbers reported in Table 1”. Again, there are no accession numbers in Table 1. A column for them should be already there although they might be supplied at a later stage.
Line 214- “Kidney bean shaped stomach” is there a semicolon missing before stomach?
Line 224- After “faeces” shouldn’t it be a semicolon instead of a comma?
Line 224- Does Tamilokus have more than one caecum? Check if “ceca” is correct here.
Line 228- misspelling for “caecum”. There is also a space too much before the semicolon.
Line 300- Should be “(…) rRNA gene sequences”. As it is, it implies that RNA was sequenced, which I think it is not the case.
Line 238- Different spelling for caecum than in line 228. Should be uniform throughout the text.
347-348-  Instead of “intestines”  should be "intestine" as in line 373.
Line 368-372 is repetition of line 300-304. It should be rephrased in the Discussion to avoid repetition.
Line 401- Take “and” before “intestine”.
Line 401- Again check the use of  “ceca”.
Line 407-411- In my opinion this new information should be in the Introduction and not in the conclusions. In the conclusions the authors shouldn’t bring in anything new.
Line 413- Treneman et al. 2018 do not describe a new species. This reference can be added to line 414 where it refers that additional works suggest new species.

Almost all references I looked at have issues with the format. This section needs attention.

Figure 2- commas after abbreviations not uniform. In addition, “ca” and “gi” are missing from the list.
Figure 5- After “A” there is a comma and after “B” a dash. It needs to be uniform.
Table 1- There is no reference to accession numbers.


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: TAMILOKUS MABINIA, A NEW, ANATOMICALLY DIVERGENT GENUS AND SPECIES OF WOOD-BORING BIVALVE FROM THE PHILIPPINES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This is a well developed and clearly structured manuscript.
Experimental design: The range of morphological and molecular techniques is appropriate and the use of CT techniques particularly interesting in this context.
Validity of the findings: The conclusions are well stated and supported.

I noticed a few technical issues that I suggest addressing:

The most important one is the need for better documentation of the voucher and type material. Lines 105-108 state that voucher specimens are at ANSP and the Ocean Genome Legacy Center. The type material is described (lines 269-272) as having “PMS” numbers, with the holotype to be deposited in Manila and paratypes “deposited at ANSP, MCZ and OGL.” The type locality is stated to have coordinates “13.758, 120.925” (line 274). This refers, minimally, to the collecting site of the holotype, which is explained (line 269) to be specimen number “PMS-3916Y.” Table 1, meanwhile lists eight specimens (7 in Manila, one at OGL -- none at ANSP or MCZ) and, most importantly, indicates that the holotype specimen PMS-3916Y is not from the given type locality coordinates. Please provide a comprehensive listing that links the “PMS” numbers (and any other potential vouchers) referred to in this manuscript to the acronyms and museum collection numbers of the various repositories. Also, please identify the type specimens in the figure captions.

Collecting sites and Figure 1 (map): I cannot discern any individual collecting sites in the map. Table 1 gives a range of different, but adjacent, coordinates for the specimens (was this a transect?). Can you plot them on the map?

I suggest including some additional explanation with the new genus and species names:  I assume these are of “masculine gender” and “used as a noun in apposition,” respectively?

Anatomical measurements: On how many specimens where the statements based?  Please add “n=” where appropriate.

Table 3: The author/year information of genus-group taxa should not be in brackets or parentheses.

The paper is very well written – I only encountered one part that I could not quite interpret (“unique calcareous pallets which cannot be characterized within any existing genera” – lines 29-30). Is this just meant to emphasize that they are unique within the family, or is there something particularly difficult in the character assessment for the other genera?
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: TAMILOKUS MABINIA, A NEW, ANATOMICALLY DIVERGENT GENUS AND SPECIES OF WOOD-BORING BIVALVE FROM THE PHILIPPINES
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript purports to describe a new taxon of teredinid from the Philippines, complete with soft parts and CT Scans.  Although it shows the internal anatomy and provides genetic contrast with other named members of the family, additional work is essential.  The text needs to be revised to eliminate the grammatical errors (e.g. line 58) and mis-statements such as that the shell is covered by ridges (line 61).  Phrases such as “the anterior excavation face” (line 64) should be simplified.  The references are not cited either alphabetically or chronologically in the text, in the few instances I checked, the year cited in the text are not the same as those provided in the references.  Papers that are less than appropriate are cited, for instance Distel et al. 2011 is not a great reference for the economic impact of the teredinids. The format of the references differs within the section.  PMS, apparently the specimen depository, is not defined.  Not all the abbreviations shown on the figures are defined in the captions. The map figure doesn’t identify the island as being in the Philippines, it is cropped too far.  Is the scale bar in each figure the same for each of the images in that figure?  “Variation” is used where I suspect they mean “difference”.  Line 103 “were utilized for anatomical characterization” could you please say this more simply?

Line 105 you say where voucher specimens deposited, but not where the type specimens are deposited.  You say you used partial specimens for anatomical studies & DNA making the identification of those partial specimens based on the pallets.  So you never actually looked at what you cite as key characters in the type specimens?

The Diagnosis isn’t a diagnosis.  A diagnosis should succinctly state the characters that can be used to identify a specimen of the taxon without reference to anything else.  Usually it is written in telegraphic style.  I don’t know what a “Definition” section is in reference to the taxonomic literature. There should be a “Remarks” section in which one contrasts the new taxon to those overtly similar or with which the new taxon could be confused.  Where is the new species actually named?  Line 197 “mid-point of distal shell valves” is unclear. As is “ending hook-shaped posterior in the stomach” Line 204

Teredora & Uperotus are suggested to be open ocean animals – does this impact your understand and the comparison?

Those issues must be fixed before the manuscript can be considered for acceptance, but two additional issues must be raised.  The authors here make the case that this taxon is strongly different from those heretofore named, but it appears that it differs in characters that have not been cited in taxonomic treatments, e.g. the crystalline style length, the soft parts associated with the valve.  This creates a problem, as who knows what the character states are among taxa with older taxonomic names characters?
In terms of the style length, the authors present one set of measurements for each of 9 taxa, but report the source only once in the methods.  Turner 1966 whether she fully documented the style is unknown.  It would be better to measure multiple individuals and calculate allometric relationships.  Ratios are extremely suspect and should be avoided.  In re. Turner 1966, Fig. 5C labels the Mantle Collar on T. malleolus although the present manuscript says that taxon does not have a mantle collar.

The authors appear to weigh the shape of the cecum more strongly than any other character (line 323 which needs a citation!) apparently because this character state is unusual or at least not often reported. Table 3 reports diagnostic features of taxa sharing this character state.  The abstract however begins with mention of the Cephalic hood.  Pallet shape has been the character of choice in the family.  How does the new taxon compare to taxa which share overtly similar pallets?  Remember please pallet morphology can change over time…  My point here is that the previous names were erected on a set of characters; you are devising new characters that justify erecting a taxon without addressing the traditional taxa.  No one would say that it isn’t high time something was added to the character matrix, but rejecting or worse yet, ignoring, the older characters without considering them is inappropriate.  List comparative material examined and their character states.
The phylogeny shown in the figure indicates that this taxon is different from all others in the matrix, but given paraphyletic genera in the tree, this information is less than compelling.  It makes me wonder whether creating another monotypic genus is going to contribute to resolving the group’s phylogeny.  There is somewhere in a Turner paper a depiction of coloration and complex cilia in siphonal openings.

Table 1 is hardly worth having, this information can be put into the text, or indicated on a modified Fig. 1.

The paragraph from Lines 350-362 attempts to argue polarity of character states from the phylogeny in Fig. 6 rather than from outgroup comparison. This is inappropriate.  The paragraph from lines 364-375 hardly contributes anything to the manuscript. Why is it here?

The authors need to look at this manuscript carefully.  Double check all the statements, correct the references or find ones that are more appropriate.  Rethink the approach – and the format of the manuscript.  Perhaps read a few species descriptions in peer-reviewed journals to better understand how to go about preparing one.   If exclusive reliance on literature sources is absolutely necessary, then make sure that you look at those references carefully and thoroughly.  Describing species is not easy, and when you describe a species from the Philippines please be aware that most readers will not be native English-speakers, requiring that you be as clear as possible.
Experimental design: none.
Validity of the findings: This probably is a new species.  As to new genus, that level is arbitrary.  Is it so well described that I could identify a specimen from the wild?  no.
Additional comments: Pay attention to the details to make the manuscript as good as it can be.


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: TAMILOKUS MABINIA, A NEW, ANATOMICALLY DIVERGENT GENUS AND SPECIES OF WOOD-BORING BIVALVE FROM THE PHILIPPINES
Review round: 2
Reviewer: 1

Output:
Basic reporting: text could benefit from cutting out redundancies.
Experimental design: No comment
Validity of the findings: it's apparently new.  Is it a contribution to the field and the best it can be is the question.
Additional comments: This manuscript still needs revision.  The point of the description is to allow easy differentiation of this taxon from others.  The characters that have been used for sp. identification in this family relate to the pallets.  The description of the genus does not mention the pallets.  It therefore defines the group on characters that are unknown in other taxa.  This unnecessarily complicates its utility for future workers.
	The authors should imagine themselves as a non-English speaking marine biologist who has found a teredinid.  Painstakingly, the biologist translates every word of the description and compares it to the unknown.  They may have prepared the pallets to allow comparison to previously named taxa, but, faced with this description are stuck with having to prepare the guts, without a micro-CT Scanner available… without knowing the anatomy.  What characters can they look at quickly & simply to put this in this taxon?  That information is the diagnosis.  See this work Lyrodus mersinensis sp. nov. (Bivalvia: Teredinidae) another cryptic species in the Lyrodus pedicellatus (Quatrefages, 1849) complex.  A full treatment of multiple characters goes into the description.  A discussion of how this taxon compares to other taxa that are most similar to the new one goes in the Remarks. In addition when comparing the new taxon to others, one should list the material examined, that is what specimens did you use for the direct comparison?
	As a generality in the description proper, because we are conchologists, the discussion of hard parts typically goes first, then any discussion of soft parts.
	Authorities should be cited with a comma.
If your statements about other genera are all based on Turner’s 1966 monograph – which treated lots more than taxonomy - cite that work.  If they are based on your examination of specimens, cite the specimens examined.
	The authors need to follow journal instructions about citing the Zoobank information:  Include the name.
Does MCZ have a “Turner Collection”?
	The description of the genus and species are about 110 lines including double hard returns, and the discussion is another 100 lines.  It should be shorter, as it begins by saying the new taxon is distinct from all others.  Well, yes – you feel this otherwise you would not have attempted the description. In addition, there is no need to repeat characters in the discussion, it is not a re-hash of the description.  Just emphasize what to you are the interesting features that might be of interest to someone else. Don’t repeat the self-evident.
Why not list the type specimens with their museum numbers in association with the description?  One assumes even if they are catalogued at ANSP those numbers will always reflect their present status that is post transfer to their native country.
In the text there is consistent mention that this taxon differs from previously named taxa.  This is risky as many of those names are now synonymized and their soft parts are unknown.  You can say that this is distinct from all currently accepted taxa, but there are more synonymized taxa (many of which do not have soft parts known) than there are accepted ones.
Lines 299-306 go from defining the cephalic collar, to saying its use is unknown, to proposing a use for it.  This could easily be shortened.
Line 326 needs a reference.
A manuscript with an abstract at this length does not also need conclusions.
You report the apparent association of large labial palps with filter-feeding, but regardless of their ability to do so, they also function one would think to move wood shavings into the gut.  Small or large, they need to aid ingestion of whatever.
How was pallet in Fig. 4 prepared?  Dry? Alcohol stored?
Report doi for references
Your expressed expectation that a reviewer provide you with a reference is demeaning.
The bottom line is that this is likely a new taxon.  Is this description as clear and concise as it can be?  Is it a contribution to the field?  Just because you have micro-CT data that no one else has had, doesn’t mean that it helps with all those previous species descriptions that lack such data.


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: CO-EXPRESSION OF HIF-1Α, MDR1 AND LAPTM4B IN PERIPHERAL BLOOD OF SOLID TUMORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Rehman Z. et al. presented a work “Co-expression of HIF-1α, MDR1 and LAPTM4B in Peripheral Blood of Solid Tumors”. The results are well explained and the methods are really clear and reproducible. The general structure of the manuscript and the format make it readable also from experts of other medicine fields.
Experimental design: The topic fits with the scope of the journal, by focusing on biomarkers of tumor hypoxia in chemo-resistance in various cancer types, thus addressing a general question in cancer therapy/medicine, and, moreover, by using an innovative method for biomarkers on liquid biopsy, that is generally applicable. Generally, the experimental design is very simple, but well statistically conducted to be meaningful.
Validity of the findings: Authors demonstrated a significant association between HIF-1α, MDR1 and LAPTM4B and their correlation with negative prognostic factors. Results are obtained by comparison to healthy controls and are validated by statistical analysis thus reinforcing the findings.
Additional comments: Minor comments:
-	The hypothesis and the gap of knowledge are not sufficiently clear in the abstract. Authors can add in line 23-26 a sentence about the advantage to find a biomarker detectable on liquid biopsy/blood sample in cancer patients (thus overcoming the issue of tissue re-biopsy) and then state their hypothesis that hypoxia biomarkers can be detected also on blood samples and introduce the correlations’ experiments.
-	In introduction section, lines 46-48 should be re-paraphrased. The main concept is that targeted therapies are helping improving cancer patients’ outcome but chemotherapy still represent a very important part of anti-cancer therapy for solid tumors, including the ones analyzed in the work.
-	In lines 67-69 clarify that they are results on tissue samples.
-	In line 70 consider to specify “as below clarified” since this data is better explained in the subsequent text paragraph.
-	In lines 88-90 authors should state clearly the hypothesis of the study based on the gap of knowledge in terms of data on blood samples for the hypoxia related biomarkers expression in solid cancers.
-	In line 107, authors state that chemotherapy treated samples are “five cycles” of chemotherapy. Please clarify if it means patients underwent blood sampling after 5 cycles of chemotherapy in any cancer types independently from the chemotherapy scheme and which is the rationale for the decision of this time point (median time of clinical or radiological re-staging?).
-	In line 151, there is a lacking of one+ in first parenthesis.
-	In lines 192-194 please describe in details any associations found in co-expression of the three genes.
-	Lines 227-229 re-phrase to ‘HIF-1α expression from TILs (Tumor Infiltrating Lymphocytes), TAMs (Tumor Associated Macrophages) has been studied for its potential role in cancer progression and metastasis (Wigerup et al. 2016).”
-	Lines 227-229 re-phrase to “Finally, LAPTM4B is known to  mediate chemotherapy resistance by enhancing efflux transporters (MDR1) expression”(Futter et al. 2001).
-	In the tables, please consider to use “pre-treated” instead of “treatment experienced”, as this is a more diffused expression. The same expression could be used in the text.
-	Add in discussion a reference on issue of re-biopsy of patients after chemotherapy (performace status? necrosis of tissue?) and the advantage of blood testing (overcome the heterogeneity inter-metastasis).
-	Table 2, 3, 4, 5 should be reformatted to fit the journal printout, they are not completely visible in the merged PDF uploaded.
-	Consider to use a gray color scale/re-shaping for the tables 6-9 to make more impactful the expression of any single gene and their correlations.
-	Generally, some typos errors are present and should be addressed by careful correction.


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: CO-EXPRESSION OF HIF-1Α, MDR1 AND LAPTM4B IN PERIPHERAL BLOOD OF SOLID TUMORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript “Co-expression of HIF-1α, MDR1 and LAPTM4B in Peripheral Blood of Solid Tumors” by Rehman Z et al shows an elevated expression of HIF-1α, MDR1 and LAPTM4B in blood of patients with different types of solid tumors and a correlation of such expression with tumor stage, metastasis and chemotherapy treatment. With the present study, the authors want give force to the concept that the high level of the selected proteins in the blood of cancer patients could be a predictor of disease progression and treatment response.
The present study addresses a clinical research topic within scope of the journal and the proposed research question is well defined but there are some comments:
-To better support the conclusion of the study, the authors should indicate the levels of HIF-1α, MDR1 and LAPTM4B in the tumor biopsy of patients thus comparing the results with the expression of the same proteins in the peripheral blood. Furthermore, it is not clear what is the blood fraction isolated to detect the expression of the selected genes. Can the presence of circulating tumor cells give a contribution to detection of HIF-1α, MDR1 and LAPTM4B in the blood of the selected patients?
Experimental design: -Methods should be improved especially in the section referred to the collection and analysis of blood samples. It is not clear what blood fractions were isolated.
-The figures and tables should be described with more deatils both in the legends and in the text.
-Table 4 lacks the age correlation
-The tables indicating the linear correlation between several parameters should be accompanied by a graphic representation thus improving the data understanding and points distribution.
- In the figure 1C and 1D there is a difference between raw data and the value reported in the histograms. In excel raw data file, the average relative expression for MDR1 in the controls is > 1 whereas in the figure it is reported to be < 1. Could the authors comment this discrepancy and eventually do the appropriate correction in the figures ? Furthermore, in the figure 1E and 1F the relative expression of LAPTM4B in the controls should be the same, why do the authors reported a different values?
Validity of the findings: The conclusions of the study are supported by results but some additional experiments should give an additional validity to the findings.
Additional comments: No additional comments


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

Input:
Title: CO-EXPRESSION OF HIF-1Α, MDR1 AND LAPTM4B IN PERIPHERAL BLOOD OF SOLID TUMORS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript by Rehman and colleagues highlights that in peripheral blood of breast, ovarian, prostate and colon cancer patients the expression levels of HIF-1α, MDR1 and LAPTM4B are higher than those of healthy counterparts. The authors also show the correlation between HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy experienced group in peripheral blood of the solid tumor patient’s samples.
The presented paper is clearly written but some main issues have to be clarified in order to validate the proposed thesis in a more accurate and in-depth way.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Major points:

1.	In tables 1-5 are reported the correlations of HIF-1α, MDR1 and LAPTM4B expression with clinico-pathological features of patients samples and the analysis reveals differences in the expression levels of these genes. Can the authors classify the patients for HIF-1α, MDR1 and LAPTM4B levels and specific mutation in oncogenes (e.g. p53, kras, tgfb signalling, etc)?
2.	The authors should mention the survival data of patients with solid tumors in correlation with HIF-1 α, MDR1 and LAPTM4B expression levels.
3.	As further validation of the overexpression of these genes in tumor development, the authors should perform immunohistochemical analysis on tumor specimens.
4.	It is known that not only the ubiquitary HIF-1α is involved in the mediation of the cellular response to hypoxia but also the HIF-2α is a key regulator of this process. Moreover, the HIF-2α gene is commonly analyzed by qPCR, while the variation in the expression of HIF-1α is usually analyzed by western blotting. Have the authors tried to analyze the correlation between HIF-2α and MDR1 and LAPTM4B expression levels?
5.	The high expression levels of the HIF-1α, MDR1 and LAPTM4B where found by analysing the whole peripheral blood. Is there a specific expression of such genes in particular cells subpopulation? Are these mRNA presents in circulating microvescicles/exosomes?


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: CO-EXPRESSION OF HIF-1Α, MDR1 AND LAPTM4B IN PERIPHERAL BLOOD OF SOLID TUMORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The authors well addressed the all raised questions


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: CO-EXPRESSION OF HIF-1Α, MDR1 AND LAPTM4B IN PERIPHERAL BLOOD OF SOLID TUMORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.
Experimental design: The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.
Validity of the findings: The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.
Additional comments: The authors have satisfactorily responded to all my questions and made the necessary changes to the manuscript.


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

Input:
Title: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The English language should be improved. For example: “the exact same splits”  line19, 54-55, 109; “of for” 52; line 61 “these exact methods”; difficult  to understand lines 52-55.

In the Introduction the authors describe the variety of applications of random forest and the problem which arises while nominal  predictors are in data. The description is too detailed and its large part is then repeated in Methods section. There is no literature review concerning the methods (heuristics) proposed to solve the problem with nominal predictors  - the list (without any description) of relevant references is given in Methods section (lines 118-119).
Experimental design: The paper aims at investigation whether a new stage in the random forest induction procedure, concerning the ordering of nominal attributes, can make the algorithm faster with the prediction performance not worse than the standard approach.

The following points need to be addressed:
1.The description of the method of Coppersmith et al. (1999) should be more detailed. Points 1-3 (lines 122-129) are very general. Since the described method is essential in the paper, it would be worth to describe the way of calculating the probability and covariate matrix more precisely.

2.Line137: is “.. we identify the largest quartile…” – is it true? Maybe it should be written “the smallest”.
In case of survival data: What happens if one of the categories will have only censored cases?

Simulation studies:
3. Could you please describe more precisely the way survival data are simulated. I mean the sentence: “ For survival outcomes, the sum of …. a standard normally distributed variable was used as survival time with random censoring” (lines 184-186).

4. The censoring rate was set to 30%. It would be interesting, to investigate how the performance of the applied algorithm depends on the percentage of censored observations.

5. For survival data, the integrated Brier score was used as a performance measure.  From the simulation description I can conclude that test datasets also contain censored observations. In experiments based on simulation data we do not need to use censored test data, since the true survival time can be calculated for all the observations. So even the model is trained using censored data, the performance measure should be calculated  for uncensored test data. This does not apply to real survival data, in which the true survival is unknown.
Please, recalculate the performance measure.
Validity of the findings: 1.In Table 3 and 4  beside the median runtime, also the min and max or upper and lower quartile should be presented.  Please bold the best results.

2.Real datasets were narrowed to classification and regression problems. Since the conclusions concern also survival data, the real survival datasets should also be taken into account.

4.Statistical analysis of the results was not performed. The statement from the Discussion section “The new method for multiclass classification and survival improved the prediction accuracy considerably” was not proved for survival data.
Additional comments: The idea of ordering nominal attributes seems to be interesting and can cause the random forest algorithm to be faster, but the results presented in the paper should be extended with new datasets, especially survival ones. This type of data, due to the presence of censored observations, should be analyzed more carefully (see the points above).


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: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This paper discussed the splitting of categorical predictors problem in random forest.  This paper has shown that ordering of categories before splitting  is subtle but vital in random forest modeling, particularly in multiclass classification and survival analysis.

Generally, this paper is well written and easy to follow.  The research is sound and the simulation and real data results are clearly presented.
Experimental design: Minor comments:
In "Simulation studies"
    Line 198:  "For each dataset, we simulated 50 training observations and 50 validation observations." The sample size for simulated training data might be somewhat small. It might be suitable for regression and binary classification. However, it might  not be appropriate  for multiclass classification and survival analysis. I suggest to set sampsize of training data to at least 100.
Validity of the findings: Minor comments:

Tables in Tables 3 and 4,  and Box plots in Figures 2 and 3 are useful, but might not be the most appropriate method for multiple comparison across different methods. I would suggest  to use multiple comparison statistical tests to check whether there are significant differences among these methods.
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: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: 'Rigorous investigation performed to a high technical & ethical standard.'

In the simulation scenarios, the sample size for the training and validation data sets was small, especially for multiclass outcomes (50 observations in each sets for a total of 100 observations – see Table1 and line 198). Why this single choice of a small sample size to evaluate the performance of the proposed method? How this choice could explain some of the results?

There were also very few scenarios with multiclass and survival outcomes (only two each). There were also two real data sets with a multiclass outcome and none with a survival outcome. The authors should consider doing more simulations with different scenarios to evaluate the performance of their proposed methods.

Why the ‘ignore’ method is not always the method with the lowest runtime and ‘Partition’ with the largest runtime in all cases considered in the manuscript, as expected?

In practice, we expect the ‘Partition’ method to be the method with the best performance. However, according to the results of the simulations (Figure 2) and of the real data sets (Figure 3), it is not always the case. Why? A more in depth analysis of the results should be done to explain these results.
Validity of the findings: 'Conclusion are well stated, linked to original research question & limited to supporting results.'

Lines 70-71: ‘Compared to the standard approach, the performance can be improved while simultaneously reducing computational time.’ There is a need to better explain how the performance can be improved compared to the standard approach. As mentioned above, in general it should not be the case; the chosen simulation scenarios might be too specific.

Lines 282-283: the sentence ‘The new methods for multiclass classification and survival improved the prediction accuracy considerably.’ overstates the results presented in the manuscript.
Additional comments: Other minor comments:

Line 64:  ‘between a category and the outcome’ should be ‘between a categorical variable and the outcome’?

Line 122: what is the meaning of ‘N’ for the k x c contingency table here?


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: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors addressed all the questions.  Only one problem is partially solved - statistical analysis of results. The authors have informed in the Real data analysis section about the statistical tests that were used, but without the value of significance level. It is important information. In the description of results, I haven’t noticed any sentence that contains the information whether the differences are statistically significant or not.
Experimental design: No comments.
Validity of the findings: Comments in the Basic reporting section.
Additional comments: No comments.


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

Input:
Title: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The paper has been greatly improved after major revision. New data and comparison results make the work solid. I suggest its 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: SPLITTING ON CATEGORICAL PREDICTORS IN RANDOM FORESTS
Review round: 2
Reviewer: 3

Output:
Basic reporting: The revised manuscript meets the standards.
Experimental design: The revised manuscript meets the standards.
Validity of the findings: The revised manuscript meets the standards.
Additional comments: The revised manuscript is much improved.


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: INTRAUTERINE GROWTH AND THE MATURATION PROCESS OF ADRENAL FUNCTION
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general fine -

a number of small issues around the raw data:

In the raw data there is an unrealistic number of digits in some measurements that go far beyond the actual accuracy of the measurement.
In the raw data the units of the measurements are not given.
In the raw data, the column steroid use is present twice

and two small textual issues:
l.69 What is meant with ‘the remote period’?
l.177: ‘recent studies’ refers to two papers that are >20 years old
Experimental design: Descriptive, cross sections, shortcomings are discussed properly.
Validity of the findings: no comment
Additional comments: This manuscript describes the salivary cortisol levels in newborn infants, as a function of postnatal age (< 5d; > 14 d), and a number of aspects during pregnancy and first days of life, focussing on complications. The main findings are a decrease in cortisol levels over time, and quite strong interactions between age and maternal high blood pressure/ intrauterine growth retardation / C-section. In these three conditions cortisol is higher shortly after birth, and much lower after the age of two weeks.
The authors suggest that these changes indicate reprogramming of the hypothalamus-pituitary-adrenal axis, and that this may be relevant for disease vulnerability in later life.

The manuscript represents a further analysis of earlier reported studies. It is purely descriptive but seems well-controled (this reviewer is not a methodological specialist in this area). And: the research question is in fact a very interesting one, and bears relevance beyond the acute setting of the obstetrics ward. There is a substantial research community focusing on HPA axis changes in relation to adult stress (PTSD) and on ‘early life stress’ in relation to behavioural changes.  The initially higher cortisol levels, developing towards lower levels may well bear relevance to those fields.

I have only very minor comments on the manuscript as is.

It struck me that glucocorticoid therapy (outside the exclusion period of  one week earlier) has no significant impact by itself. Can the authors discuss this?
It would also be of interest to know what the effects of GCs would be at shorter notice (apparently a week suffices for the HPA axis to normalize, but how fast does this happen?). But probably these children were excluded beforehand, and no measurements are available.

l.69 What is meant with ‘the remote period’?

l.177: ‘recent studies’ refers to two papers that are >20 years old


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: INTRAUTERINE GROWTH AND THE MATURATION PROCESS OF ADRENAL FUNCTION
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article failed to meet in standards in providing enough evidence, rationale and background for both the study rationale and interpreration of the results.

Overall the article flow was hard to follow
Experimental design: The research question needed to be reworded for clarity (i.e. maternal stress, see comments below) and it wasn't clear how it filled a gap nor was substantively different from their previously published work

Methods and Statistical Analysis needed more detail
Validity of the findings: Impact and novelty of data weren't properly discussed and test statistics were missing for all presented data
Additional comments: Title
1)	Doesn’t seem IV is antenatal stress, but rather prenatal clinical conditions- see comment 15 as well
2)	Association is also likely a more accurate term than Influence.
Abstract
3)	In Intro, permanently make be too strong of a word as the HPA system is plastic, perhaps long-term consequences or alters may be more accurate terms
4)	In Aims, Would not use the word dependence, but rather association
5)	In Methods, would be useful to know when the infant cort levels were collected since results were analyzed overtime and that is a strength of this study
6)	The relationship in the Results is not clear
7)	The conclusion is concise and clear
Introduction
8)	Should be a space between the last word preceding a citation
9)	Lines 47 and 48 should be clear if referring to maternal or fetal cortisol/glucocorticoid levels
10)	There should be a brief discussion of the consequences of IUGR and preterm labor to strengthen the impact of the current study
11)	More rationale is need at the beginning of the second paragraph to understand why looking at the temporal changes in adrenal function are needed (lines 61-63).
12)	Add the word exposure in line 63 before pathological and in line 67 before maternal
13)	Line 64, it is unclear what authors mean by sporadic observations- perhaps cross-sectional is a better term?
14)	Not clear what remote period refers to in line 69.
15)	More citations are needed to demonstrate that “several studies have shown associations between perinatal stress and attenuated adrenal function” on lines 68-69. Only one study is cited and more evidence is needed. Also, It is unclear if maternal stress is the correct word to use here (and throughout the manuscript), as the authors are not referring to psychological stress, but rather than physiological stress or maternal health conditions. This should be made clear.
16)	The last point on lines 70-74 is not clear. The conclusion of a rapid postnatal decline in cortisol levels does not seem to be supported by the first half of the sentence. Instead, it would be more helpful for the authors to speculate on why this juxtaposition may occur in prenatal vs. postnatal cortisol in offspring with evidence from the literature.
17)	Again, the aim that the changes in postnatal cort will change in response to antenatal stressors, does not seem to be the correct term to orient readers the tested research questions.
18)	Overall a stronger case needs to be made to look at the interactions with postnatal age and maternal clinical outcomes on infant (see comment 11)
Material and Methods
19)	Line 86, the cortisol levels are referred to as independent variables when in fact they are the outcome, thus dependent variables. Also diurnal rhythms aren’t addressed here.
20)	It is unclear what is meant by lines 87-91 and how they related to the current study. It is odd to include an exploratory analysis in a Methods section. How did this work inform the current study?
21)	Under study population, if the recruitment, sample collection (amount) and cort assay have been reported previously then that needs to be clearly cited.  And sample size for this cohort needs to be included here.
22)	The times, means ± sd of the ages of saliva collection for the infants should be included in the Methods
23)	Was only one saliva sample collected? This seems not in concert with the above statement from the exploratory analysis that suggests a fetal-type diurnal pattern (again unclear what this means and is especially curious because much other work suggests infants don’t develop a diurnal cort pattern right away)
24)	Why was postnatal age grouped and not used as a continuous variable?
25)	The description of how clinical variables were operationalized needs to be clearer. It seems each clinical variable was tested along with postnatal age on infant cortisol as a multiple comparison correction was used? Is this correct? What stats were used? Was a Two Way ANOVA- clinical dichotomous outcome x postnatal age category -on cortisol levels or change in cortisol levels performed? The Statistical analysis section needs much clarification.
Results
26)	Table 1 does not include a full demographic table. Were any demographics considered as covariates like maternal age and/or BMI? Also, it isn’t clear if the numbers refer to percentages or whole numbers and where the comparison numbers are located?
27)	The large postnatal range seems to be an issue, especially if postnatal cortisol decreases quickly as suggested and samples were collected within a week apart.
28)	The main effects should not be mentioned or reported here as they were previously. These should be included in the introduction and/or discussion where a stronger case needs to be made for this study.
29)	Both the tables and text are missing test statistics for the statistical test run (ie, F-value, df, etc).
30)	The description of the interactions were unclear, again with the use of the word dependence.
31)	Table 2 should report the non-transformed means for cortisol for comparison with other studies, especially since Figure 1 (or Table 3?) has the ln transformed means and comparisons and currently is just a replica of Figure 2 in graph form- thus repeating data.
Discussion
32)	Begin the Discussion with a summary of results from the current study, not previous work. Though authors can go on to describe how these fit together. The current summer is still confusing as mentioned in the Abstract presentation of the results above.
33)	The importance of IUGR is included here, but would also be useful in the Introduction
34)	The second paragraph in the Discussion is nicely written, though overall the Discussion is hard to follow. For example, facts presented in line 173 and 175/176 seem unrelated and their inclusion isn’t clear nor is the relevance to the current study clear.
35)	The third paragraph does not include any references nor refer how this work compares with other studies


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: INTRAUTERINE GROWTH AND THE MATURATION PROCESS OF ADRENAL FUNCTION
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comments - I found it clear.
Experimental design: I am not a specialist on the type of analysis performed here.

Given the weight that is given in the literature to early life regulation of the HPA axis this is very interesting data in a carefully considered naturalistic setup.
Validity of the findings: The results are discussed in a careful and conservative manner.

I have to refer to other reviewers for judgement of the statistical analysis
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: PHYSICAL ACTIVITY DURING PREGNANCY AND ITS INFLUENCE ON DELIVERY TIME: A RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 1

Output:
Basic reporting: Please see the uploaded review document
Experimental design: Please see the uploaded review document
Validity of the findings: Please see the uploaded review document
Additional comments: Please see the uploaded review document


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

Input:
Title: PHYSICAL ACTIVITY DURING PREGNANCY AND ITS INFLUENCE ON DELIVERY TIME: A RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 2

Output:
Basic reporting: The objective of the paper is to evaluate the effect of water exercise on labour duration.
The topic is relevant and the role of “movement” in pregnancy and birth not negligible.
Experimental design: The study is well described in the method section and the characteristics of exercise well standardized, however the section regarding results shows major limits and it is lacking regarding “obstetric” points.
Power analysis is not reported.
In particular because the variability of the duration of labour is enormous, influenced by an innumerable number of factors, and the diagnosis of the beginning of labour is not easy, the definition of the beginning of labor and the cm of dilation at admission to hospital and at the beginning of partograph compilation should be reported.
Otherwise the conclusion could be completely (and anyway interesting) different: a woman performing water exercise spends less time in labour in Hospital respect to a woman not performing such exercise
Validity of the findings: First question: all the deliveries were at the same hospital? In hospital?
I cannot understand the total number of CS or operative vaginal delivery, how many elective and how many in labor? How many induced? How many admitted for labor? Reason for induction?
The overall percentage of CS of the Hospital/s? Underline eventual difference that could be due to different setting
Which is the meaning of : “birth in progress” , and At the beginning of labor, the rate of spontaneous birth was higher in EG than in CG (70.8% versus 60.9%, respectively)
Data on neonatal outcome should be reported, neonatal weight is another important potential confounder present in the dataset, include in the results.

The sentences reported below are redundant and the definition of duration in days obvious: report only the duration of gestation in days
The duration of gestation in EG and CG was examined to determine whether physical
exercise during pregnancy produced any alteration in this respect. To perform this comparison, the duration of gestation (in weeks) recorded in the partograph was converted to days of gestation. The resulting distribution did not differ significantly (p=0.996) between EG (281 days [277 - 286.50]) and CG (281 days [275.25 - 286.75]) (see Figure 2).

Conclusions regarding neonatal outcome should be supported by results, otherwise removed

Statistical analysis  should be supervised
English should be reviewed
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: PHYSICAL ACTIVITY DURING PREGNANCY AND ITS INFLUENCE ON DELIVERY TIME: A RANDOMIZED CLINICAL TRIAL
Review round: 1
Reviewer: 3

Output:
Basic reporting: The data are presented in a confused way. In particular, there is no correspondence between the numbers shown in the abstract and those reported in the results.
Experimental design: The experimental design is correct and appropriate but the evaluation criteria of the results have to be defined more.
Validity of the findings: The conclusions is welcome, but they  should be certain.
Additional comments: The study of physical exercise during pregnancy is an important topic which has not been explored enough. Therefore, this paper could make a contribution. However, the results could be imprecise because the Authors did not define the first stage of labour accurately. The first stage of labour is divided into the latent phase and the active phase. The length of the latent phase varies physiologically from pregnant woman to pregnant woman and is difficult to define.  I suggest the Authors review the results taking into considerations the active phase of the first stage of labour and identifying an objective point in the active phase, such as 4 cm or 6 cm cervical dilatation. More precise data are required to demonstrate that physical exercise significantly reduces the length of labour, in order to be sure that these results are not due to physiological variations.


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: PHYSICAL ACTIVITY DURING PREGNANCY AND ITS INFLUENCE ON DELIVERY TIME: A RANDOMIZED CLINICAL TRIAL
Review round: 2
Reviewer: 1

Output:
Basic reporting: The referee feels that the authors have appropriately addressed the questions raised during the reviewing process in the new version of their manuscript and in the rebuttal letter.
Experimental design: see above
Validity of the findings: see above
Additional comments: see above


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: PHYSICAL ACTIVITY DURING PREGNANCY AND ITS INFLUENCE ON DELIVERY TIME: A RANDOMIZED CLINICAL TRIAL
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The study of physical exercise during pregnancy is an important topic, which has not been explored enough.
In this new rewrite, the authors have more accurately specified the different stages of labor. Now the results are consistent and deserving to be published.
In Figure 1 to translate the sentence  “Complicaciones durante la gestación” into English


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: SNAKE VENOM NAD GLYCOHYDROLASES: PRIMARY STRUCTURES, GENOMIC LOCATION, AND GENE STRUCTURE
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well organized and well written. The authors familiar with the progress in the field and provide sufficient background.
Experimental design: In Figure2:
The authors compare the structure of soluble venom NADase with human homolog CD38 using predicted 3D structure of two proteins. Actually, the structure of human CD38 extracellular domain has been solved in 2005 already (PDB ID: 1YH3). Why authors using the predicted structure to do analysis? It is more reasonable to analysis the structure based on a solved structure than predicted one.
Validity of the findings: The title of Figure2 is “The predicted 3D structure of a soluble venom NADase is more compact than its membrane-bound human homolog.”
The title of Figure4 is “Predicted 3D structures of short (left) and long (right) NADases from Protobothrops mucrosquamatus venom.”
Why the panel A of figure2 and figure4 looks exactly same?
It is unreliable that the human CD38 has exactly same structure with Protobothrops mucrosquamatus venom.
Additional comments: In this manuscript, the authors searched 10 elapid and crotalid species’ venom gland transcriptomes and reported all snake venom gland transcriptomes contain sequences of NAD glycohydrolases. The authors found the key residues requisite for NADase activity are conserved in snake venom. In addition, structural analysis revealed that a disulfide bond that related with cyclase activity is present in all snake venom NADases for which they have sequences. This manifest the Aird’s group earlier snake envenomation hypothesis: “purine nucleosides comprise core elements of the envenomation strategies of most advanced venomous snakes”. The results may provide new clues for the community to study the NADase activity and envenomation strategies of venomous snakes

The authors describe the detailed sequence difference on line 130 and 131. Mention the residue number is meaningless unless coordinate it with the protein structure. It is better if the authors could label these residues on the structure and the discuss the potential influence of these residues on the function.

The authors mentioned: “Snake venom NADases also all have Glu146 (Glu151 in
191 Figure 1), which is essential for NADase activity and is present in all CD38 molecules.” Why Glu146 is important for NADase activity? It is helpful if author could label the residue on the structure. Similarly, where is the 31 amino acids (delete in short form) locate, what's the possible function of this part?


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

Input:
Title: SNAKE VENOM NAD GLYCOHYDROLASES: PRIMARY STRUCTURES, GENOMIC LOCATION, AND GENE STRUCTURE
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment, see "General Comments".
Experimental design: No comment, see "General Comments".
Validity of the findings: No comment, see "General Comments".
Additional comments: 1. Although it’s appreciated that snake venom enzymes are soluble instead of membrane-bound, it is still not obvious to tell from the Kyte-Doolittle hydrophobicity surface plots shown in Fig. 2B that the snake protein is more hydrophilic than the human homolog. It would be more helpful if the authors can provide an overall hydrophobicity score (e.g., GRAVY value) of each protein and see if the snake protein (at least in the N-terminal region) is more hydrophilic compared to the human homolog.
2. In Figure 1, it would be better to explain color coding scheme. Line 159, “venom NADase could be membrane-bound in exosomes”, does this statement have literature support? Line 131, “G/D254” should be “G/D253”; also what about I/T217 and N/S219? Line 154, “in” should be “is”.
3. It would be helpful if the authors can annotate the predicted disulfide bonds on the Micrurus NADase model shown in Fig. 2A.
4. To support their claim in Line 166 “venom enzymes apparently have a slightly more compact shape than human CD38”, it would be better if the authors can provide some numbers characterizing the overall shape of the protein, for example, distances between conserved amino acid residues, and annotate on their models.
5. It would be more informative if the authors can point out in the introduction challenges obtaining structures for snake venom NADases.


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: SNAKE VENOM NAD GLYCOHYDROLASES: PRIMARY STRUCTURES, GENOMIC LOCATION, AND GENE STRUCTURE
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I have gone over the revised manuscript and the comments, and I am happy with this version. I recommend 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: SNAKE VENOM NAD GLYCOHYDROLASES: PRIMARY STRUCTURES, GENOMIC LOCATION, AND GENE STRUCTURE
Review round: 2
Reviewer: 2

Output:
Basic reporting: See General Comments.
Experimental design: See General Comments.
Validity of the findings: See General Comments.
Additional comments: In the revised manuscript, the authors nicely addressed the previous reviewers' comments. GRAVY scores were nicely incorporated into the manuscript and supported the authors' claim. Their efforts to modify the wording and figure legends are also appreciated. Overall, this revised version is more informative and provide more evidence to back up 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: A SURVEY ON ZOO MORTALITY OVER A 12-YEAR PERIOD IN ITALY
Review round: 1
Reviewer: 1

Output:
Basic reporting: •	Although some sections of this paper are very well written, I feel that the authors would benefit from having a native English speaker re-read and edit the text. There are a number of sentences that do not flow well and this detracts from the content of the paper. In particular, a number of sentences throughout the abstract and results. Particular sentences in other sections include: lines 54, 98, 114,142 and 256.
•	There does appear to be some bias in the introduction towards the positive aspects of zoos (specifically in lines 30, 41, 48 and 57), and although this is understandable, there needs to be more referencing and a more balanced view including the negative points of captivity to depict a more realistic view.
Experimental design: Although the idea behind this paper is a relatively good one, I feel that there is not really a specific question asked and answered.
•	More detail is needed lines 166 and 167 as to which microscopical examinations were performed and their methodology.
•	Clarification is needed of the term “complications” in line 170 and again in the discussion – line 284.
Validity of the findings: Within the results section, although there is a lot of information presented here it is not easy to determine the most relevant points. If the number of tables was reduced and collated into one table per zoo it would make it much easier to read through.
•	I found the text in the results section rather confusing in some places e.g. line 183 – although significance is reported what is the importance of the significance? If not clarified here this needs to be further addressed in the discussion.
•	The text in the results also describes results for different groups in different zoos (rather than the same groups in each zoo) which becomes confusing – it might be easier to just have one simple table showing all the groups and their causes of death from each zoo.  Also, it might be easier to report where statistical significance was present rather than the areas where there was none as this again becomes confusing.
•	It would also help to have a much greater number of references for the paragraph starting on line 256 as it contains many wide ranging disease statements and a large number of the conditions mentioned are not due solely to diet as seems to be implied in the text.
•	Although it is interesting to link climate change to increase in infectious respiratory diseases in the paragraph beginning on line 267, the multifactorial nature of these conditions does not appear to be discussed which can appear somewhat misleading.
Additional comments: •	The word “table” either needs to be standardized throughout the text to start with a t or T.
•	Line 101 – this statement also needs to stress that this work can only be carried out with correct licensing.
•	Line 105 – this needs to be tempered (as mentioned previously) by saying that not all information from captive animals can be applied to their wild counterparts.
•	Tables 3, 4 and 5 seem to be the most useful of the group.  Information needs to be collated into a smaller number of tables as it is currently complicated to cross reference between them. The Tables showing details of the post mortems are also very interesting as there are very large varieties of species in each group so it is interesting to know which species are actually represented.


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 SURVEY ON ZOO MORTALITY OVER A 12-YEAR PERIOD IN ITALY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Very many spelling and syntax mistakes. I suggest that you have a native English speaking colleague review your manuscript.

2. References: spelling mistakes and incorrect citation.
- line 78: WRI/MCN/UNEP/UNESCO should be WRI/IUCN/UNEP/FAO/UNESCO
- line 311: States's should be State's
- line 312: Practice should be Practise
- line 316: Johnstoni should be johnstoni / Report should be Reports
- line 328: Phatology should be Phatology  / Fourteen Year should be Fourteen-year
- line 329: insert Wild Animal Park between Diego and Zoological
- line 341-343: this is an incorrect reference for line 290
- line 345: Smithsosian Institute should be Smithsonian Institution
- line 360: Startegy should be Strategy
- line 363: WRI/UNC/UNEP/FAQ/UNESCO should be WRI/IUCN/UNEP/FAO/UNESCO

3. Raw data and tables: there are many different numbers in the raw data and the tables.
Some examples:
- numbers of traumas in tables 6+12, 8+13, 10+14 are different
- numbers of zoo 1 and 2 are different in raw data and table 6 and table 8

4. Title is about a 12-year period, but line 240 says 11-year period.

5. Line 175: p<0.5 ??

6. Line 182: 96 should be 14

7. Line 201, 203, 218: monogastrics should be monogastric herbivores
Experimental design: I believe that it is more valid to consider the animal inventory in relation to the number of necropsies.
This inventory takes also into account the number of new animals arriving in the zoo, the number of births, the number of animals sent to other zoos, and this all influences the number of dead animals.
Validity of the findings: 1. The numbers used in the results and discussion will change when the correct numbers are used in the tables.

2. Line 242-244: please add the type of zoo of the private collection (which type of the 6 described types on lines 62-63 ?).

3. Line 254-255: this is incorrect because in zoo 1 96% were monogastric herbivores and 70% were ruminants.

4. Line 256-263: this reference of Fox et al. 1923 is incorrect and outdated.
Additional comments: Dear colleagues,

You put a lot of work into this survey, which could be a worthwhile contribution to zoological medicine.
Please, consider all my suggestions carefully in rewriting your manuscript.


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

Input:
Title: A SURVEY ON ZOO MORTALITY OVER A 12-YEAR PERIOD IN ITALY
Review round: 2
Reviewer: 1

Output:
Basic reporting: •	Although some sections of this paper have been very much improved, I feel that there are still a number of areas that would benefit from further review by a native English speaker. In particular, a number of sentences throughout the abstract and results would benefit from reassessment, e.g. line 21 and the phrase “where to study”, line 25 “pledge the health”, line 31 “died for” rather than died from which is used frequently throughout the paper- particularly in the results section. Also, lines 103, 104 and 157 which are somewhat confusing in their present form. Although I have mentioned specific sentences, the entire manuscript would benefit from a further full review to ensure all points can be clearly understood.
•	It is a shame that no further data could be gathered for analysis from the three institutions, but I understand the reasons why this was not possible and the explanation in the paper is a useful addition.
•	I would remove the “…” from lines 108 and 185 as these lists only require a few examples and should not be left open ended in this way.
•	In a number of places there is a lack of spaces between words – this is likely just due to formatting but should be checked fully.
Experimental design: No comment
Validity of the findings: The data presented in Table 1 is also repeated in Table 2 which presents the information clearly and concisely, so I would remove Table 1 completely.
It would be helpful if some of the other tables could be combined to give a broader and more comprehensive picture rather than splitting out the data as they currently do – ie combine Tables 3 and 4 so it is possible to see what the most common causes of death were in each category for Zoo 1. The same for Tables 5 and 6 for Zoo 2 and then Tables 7 and 8 for Zoo 3. Combination of Figures 1 and 2 would also show more clearly the causes of death for each type of herbivore at each zoo.
Combining the tables in this way could also help to simplify the results section and make it easier for the reader to understand and compare between the zoos.

•	Although respiratory disease has been described as multifactorial, the strong bias towards climate change as a cause is still somewhat questionable as vegetation management, species choice and issues such as stress affecting the immune system and therefore increasing the chance of respiratory disease are not mentioned at all. Particularly in Line 287 where the line “Risk assessments should focus on looking for combinations of factors that may be directly influenced by climate changes, or that may be indirectly affected through changes in human activity, such as transport and movement of animals, intensity of livestock farming and habitat change” as risk assessments should cover all aspects of particular diseases rather than focus on particular ones.
Additional comments: The authors have put a lot of work into improving the content of this paper and I feel that with a few more changes, it could be a useful addition to the literature.


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 SURVEY ON ZOO MORTALITY OVER A 12-YEAR PERIOD IN ITALY
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1. Still many spelling, synthax and typing errors.
E.g. : to die of/from infection and not to die for infection, areas of expertise and not application fields, abnormal behaviour such as stereotypies and not abnormal behaviour such sterotypes, how diseases may be spread and not how diseases may be diffused, C. perfringens (in italics) and not Cl. Perfringens, E. coli (in italics) and not E.Coli, Dystocia and not Distocya, Trichostrongylus (in italics) and not Thricostrongylus, aspiration pneumonia and not Ab ingestis Pneumonia, enteritis and not entheritis, hypothermia and not ipothermia, Taenia saginata (in italics) and not TaeniaSaginata, omit rows 2007-2012 in Table 8, ...

2. Numerical errors.
Incorrect numbers in Table 1 and Table 2: total must be 282 and not 279: missing in column/row "Other" of 1 case in zoo 1 and 2 cases in zoo 3.
Experimental design: I do understand that the authors are not allowed to report the data concerning the number of animals in the zoos (inventory).
I do not agree with line 158 "There are potential criticisms in this paper". I have fundamental and existing criticism. The authors must not use statistics and percentages, because it is incorrect to compare zoos, categories and years using the bare numbers of the observations (necropsies). The authors can interpret each number only for each individual zoo, category and year. Ergo, the authors can only rewrite this manuscript as an observational report without comparison between zoos, categories and years.
Validity of the findings: I do not agree with the statistics and percentages and the interpretation of the authors (see my comment under experimental design).
Additional comments: I still feel that your observations can be a worthwhile contribution to zoo veterinary medicine.
But please, carefully rewrite your manuscript.


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

Input:
Title: TURTLES OF THE GENERA GEOEMYDA AND PANGSHURA (TESTUDINES: GEOEMYDIDAE) LACK DIFFERENTIATED SEX CHROMOSOMES: THE END OF A 40-YEAR ERROR CASCADE FOR PANGSHURA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is clearly written and is to the point. It addresses an important hypothesis in that it corrects a long standing misinterpretation of the sex determining mode in a clade of turtles. The coverage of the literature is adequate to establish the problem addressed and its significance. The paper is succinct and addresses a well defined question.
Experimental design: The authors reinterpret the karyotype of earlier workers and present an alternative interpretation that does not require proposing ZZ/ZW chromosomes in a hard-shelled turtle, particularly where a sister taxon has XX/XY chromosomes or GSD.

The authors then use contemporary techniques of CGH and FISH in an attempt to demonstrate sex specific chromosomal differences in 2 males and 2 females of their target species, and fail to identify any differences. They also examine the distribution across metaphase spreads of two common repeats -- GATA and telomeric repeats. I found this a bit odd, as novel repeats on de novo chromosomes could be based on any motif, and not necessarily one of the two motifs chosen. A more exhaustive examination of potential microsatellite repeats  would have been more appropriate.

Overall, though, I found the approach to be appropriate and the results convincing.
Validity of the findings: This paper reports a negative result in the context of previous work that had yielded a positive result. Making such a case is always difficult, but in this case the reinterpretation of earlier work and the application of more contemporary approaches to detecting sex linked chromosomal markers is convincing. The authors do not overspeculate.
Additional comments: This is an important paper because it reverses what is clearly a misinterpretation of the chromosomes of P. smitthii. It will be good to see this work published.


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

Input:
Title: TURTLES OF THE GENERA GEOEMYDA AND PANGSHURA (TESTUDINES: GEOEMYDIDAE) LACK DIFFERENTIATED SEX CHROMOSOMES: THE END OF A 40-YEAR ERROR CASCADE FOR PANGSHURA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Please see General comments for the author below
Experimental design: Please see General comments for the author below
Validity of the findings: Please see General comments for the author below
Additional comments: I am pleased to receive this manuscript to review again. I was positive the first time I reviewed it, and the authors corrected most of the issues raised in the previous review. There are still some pending issues or new issues that need addressing and which I detail below. Thus my review contains some of text from my previous review updated to bring up the issues that still need resolving.

The present manuscript describes the results from a molecular/classic cytogenetics study of three turtle species from the family Geoemydidae in search of sex chromosomes. Some of the data are first time reports for some of this taxa. The data appear robust and indicate that Geoemyda japonica, Geoemyda spengleri, and Pangshura smithii all lack a sex chromosome system detectable at the resolution of the CGH methods used in this study. Importantly, the new data from Pangshura smithii refute a previous report of ZZ/ZW in this species from 1975 that used only Giemsa staining and what appears to be a erroneous assignment of homologous chromosomes. Thus, the results of this study constitute an important contribution not only to cytogenetics but also to evolutionary biology as they alter our understanding of the evolution of sex determination and sex chromosomes in turtles, and I am enthusiastic in seeing this work published. However, the manuscript suffers from some deficiencies, mainly in presentation and grammar, that need addressing before publication. I detail my concerns below.

Table 1. The provenance of each of the animals examined must be included in the text or table. Were these animals from the wild (and if so from which location), or captive bred (and if so, from a zoo, a pet store, a private collection)?

Ln 77: Please clarify why the authors are certain that Chaowen studied G. spengleri? They do explain nicely why Nakamura studied G. japonica, but the same clarification would be useful for Chaowen’s study.

Ln 112-117: The methods here need more detail. The blood culture and chromosome preparation protocols needs description so that the study can be replicated by the scientific community. The authors should include a brief description even if they cite Pokorna et al 2010 as the source of the protocol as this should be a standalone manuscript. Same for CGH in Ln 144. Also, the other version of this paper that I reviewed stated that the C banding was done with modifications to Sumner’s C banding but those were not described? This version simply omitted the mention of the modification, when the authors should instead include the modifications that were done. This again is crucial for the reproducibility of the study by others.

Ln 150: Replace ‘our study material’ with ‘the specimens examined here’.

Figure 3. Panels a and c should be kept as they are. However, all the reassignments of chromosomes to pairs need to be clearly shown in panel b (not just mention this in the text where the description is harder to follow), but only the ZW chromosomes are traced in panel b). For instance, the authors moved the W from the ZW pair to pair 6, and so you should show where the chromosome from pair6 that is no longer there move to (which seemed to have been assigned to pair 9). Furthermore, a new pair 6 is now assigned in panel b compared to panel a.  And so on and so forth. Perhaps panel b would be better as a two column (or two row) figure containing the original and the new arrangement of chromosomes, where the correspondence of chromosomes to pairs is denoted by lines connecting them. Attached is a crude (and incomplete example of such a figure, showing the correspondence for only some of the chromosomes).

Ln 202-204. Odd wording. I suggest replacing “identified as a chromosome from the pairs 16 to 26’ with ‘better reassigned as a homolog of one of the pairs 16 to 26’, or something to that effect.

ln 204-206. Likewise, please reword as: ‘Additionally, the metacentric chromosome identified by Sharma et al. (1975) as the W chromosome should be reassigned as a homolog of pair 7, 8 or 9’.

ln 207. Again, I suggest rewording here. Perhaps ‘After simple rearrangement of the original
karyogram, no obviously heteromorphic pair of chromosomes is detectable (Figure
3b), consistent with our own karyotyping of new specimens (Figure 3c).

Ln 216. Replace ‘and’ with ‘or’.

Ln 218. Add ‘the’ before ‘incorrect’.


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

Input:
Title: THE IDENTIFICATION OF GENE SIGNATURE AND CRITICAL PATHWAY ASSOCIATED WITH CHILDHOOD-ONSET TYPE 2 DIABETES
Review round: 1
Reviewer: 1

Output:
Basic reporting: 'no comment'
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: I was confused a few moments.
1. The manuscript is devoted to the study of the identification of gene signature and critical pathway associated with children-onset type 2 diabetes. But analysis based in the data of  24 healthy  and 12 young patients with T2D.
2. It remains unclear how old healthy people are? If they are adults, how likely is the next? Differences in gene expression are caused by physiological age-related features. For example, enrichment up-regulated genes in cytokine metabolic process, cytokine biosynthetic process, interleukin-1 beta production, positive regulation of cytokine biosynthetic process may be caused by large activation of the immune response to a large number of new antigens in childhood. In adults, the immune response is less active.
3. Under the action of insulin, glucose is utilized by cells from the blood. Therefore, the phrase «In a diabetic patient, blood glucose levels are high as the body either fails to produce insulin or does not utilize insulin effectively.» seems to be not entirely successful. Maybe replace it with another? «In a diabetic patient, blood glucose levels are high as the body either fails to produce insulin or cells are not sensitive enough to insulin.»
4. «Based on pathogenesis, diabetes can be classified into three types: gestational diabetes, type 1 diabetes (T1D), and type 2 diabetes (T2D).» It seems that at least it is necessary to mention the monogenic forms of diabetes.
5. It seems that in the discussion it is necessary to explain that visfatin is a product of the NAMPT gene.
My opinion as medical doctor is related only to clinical aspects of this work.
However expert statistician or bioinformatician sould estimate the research.


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

Input:
Title: THE IDENTIFICATION OF GENE SIGNATURE AND CRITICAL PATHWAY ASSOCIATED WITH CHILDHOOD-ONSET TYPE 2 DIABETES
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Please clarify: The dataset GSE9006 reports only 43 newly diagnosed T1D subjects but the manuscript reports it as 81 subjects

2. Please change "Islet B cells" to beta cells of islets in line 46

3. T1D group is misspelled as TID in line 76

4. Please clarify: There are two platforms (GPL 96 and GPL 97) reported in the original dataset but the manuscripts mention only one (GPL 97)
Experimental design: No comment
Validity of the findings: 1. In table 3, information is provided for only 10 genes (PROK2 is not listed)

2. Result sections should be more descriptive

3. Please adopt or list the demographics of at least healthy and young T2D subjects as the supplemental table 3 and 4 does not have information about age, BMI and Hb1Ac levels

4. Bottom label in figure 1, that is mentioned as gene symbol is not seen in the figure

5. In figure 1, please include the bar that indicates the range of expression level based on color codes

6. Please provide legends for supplementary tables

7. Figure legends are also missing in the word file submitted and it is only available in the pdf file
Additional comments: The manuscript by Keren Jia et.al., is well written and it provides a good rationale for the study. However, the results section can be described in detail with some more clarification on how they are derived. Incorporating some speculations about significance of the findings in the discussion section will add more value to the manuscript. Hence, listed corrections should be considered/made before publication.


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

Input:
Title: THE IDENTIFICATION OF GENE SIGNATURE AND CRITICAL PATHWAY ASSOCIATED WITH CHILDHOOD-ONSET TYPE 2 DIABETES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: I agree with the corrections made with the exception of two points.
It is necessary to correct the error in the lines 34-35: In a diabetic patient, blood glucose levels are high as the body either fails to produce insulin or cells are not Islet B cells (sensitive enough to insulin).
It is necessary to replace “healthy individuals” in to “healthy children” in the line 96.


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

Input:
Title: THE IDENTIFICATION OF GENE SIGNATURE AND CRITICAL PATHWAY ASSOCIATED WITH CHILDHOOD-ONSET TYPE 2 DIABETES
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1. Please change "Islet B cells" to beta cells in line 37 and always refer it as beta cells

2. Yellow text in line 36 and 37 is inaccurate and must be changed
Experimental design: No cooment
Validity of the findings: No comment
Additional comments: No additional comments