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: GENES FOR DEGRADATION AND UTILIZATION OF URONIC ACID-CONTAINING POLYSACCHARIDES OF A MARINE BACTERIUM CATENOVULUM SP. CCB-QB4
Review round: 4
Reviewer: 2

Output:
Basic reporting: No further comments
Experimental design: No further comments
Validity of the findings: No further comments
Additional comments: No further 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: AGE CONSTRAINT FOR THE MORENO HILL FORMATION (ZUNI BASIN) BY CA-TIMS AND LA-ICP-MS DETRITAL ZIRCON GEOCHRONOLOGY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This manuscript reports important new data on detrital zircon geochronology of Cretaceous terrestrial units in the North American Western Interior Basin.  The authors report high-quality data, with excellent figure quality, and as a whole, this should be published in PeerJ.
Experimental design: The methodology through which new data were obtained , reported, and discussed are excellent throughout.
Validity of the findings: High quality data analysis and presentation throughout.

I did find a few minor faults to bring up:

Line 54: McElwain & Sageman (2010) are not included in the references.
Lines 65-66: Delete the sentence referring to Albright & Titus (2016). Meaning is unclear.
Lines 68 to72: This goes to the heart of this contribution. Two other relevant papers on this approach are Ross et al. (2017) in Science China Earth Sciences 60:44-57, and Joeckel et al. (2019) in Geol. Soc. London Spec. Pub. 498:75-100.
Line 105:Adamkin is misspelled.
Pages 20 and 22: The meaning of citations of Cilliers et al. 2018 unpublished data are unclear. Should this be included?
Lines 936 to 953: The citations of Hook et al. are out of alphabetic order.
Additional comments: I commend the authors for an excellent contribution to the problem of improving the chronostratigraphic understanding of Cretaceous terrestrial strata in the North American Western Interior Basin.  The writing shows an impressive command of the historical development of the relevant scientific literature--almost to the point of distraction with lists of parenthetic citations that extend for several lines of text. This is thorough documentation, but disrupts the narrative--something to think about.


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: AGE CONSTRAINT FOR THE MORENO HILL FORMATION (ZUNI BASIN) BY CA-TIMS AND LA-ICP-MS DETRITAL ZIRCON GEOCHRONOLOGY
Review round: 1
Reviewer: 2

Output:
Basic reporting: See attached pdf
Experimental design: See attached pdf
Validity of the findings: See attached pdf
Additional comments: See attached pdf


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 EXTENT OF CARBAPENEMASE-ENCODING GENES IN PUBLIC GENOME SEQUENCES
Review round: 1
Reviewer: 1

Output:
Basic reporting: This is a nice and clearly written manuscript by Janse et al who have searched for carpapenemase–encoding genes in the public database PATRIC.  Overall, the structure and language of the manuscript are good and I only have some minor comments.

1.	In line 149, you write “…Acinetobacteria (3074 genomes: 4783 genes), here it is unclear if you mean that all genomes belonging to the genus Acinetobacteria contained a total of 4783 genes or if you have discovered 4783 CEGs in these genomes. This is repeated a couple of times throughout the manuscript (line 165, 166 and 170 for instance). The authors need to be consistent with the nomenclature, if you mean CEGs, please write so, and if you mean genes, please clarify.

2.	Line 90-92: I had trouble understanding the purpose of this sentence (and approach). Firstly, it becomes unclear when using the word “host” in this context, since when searching for ARGs a host is mostly considered the bacteria carrying the ARG. Secondly, why is the taxonomic lineage of the isolation source needed when you have the keywords to classify the sources? I can’t find any part in the manuscript where this information is used. These lines need to be clarified.

3.	As there often are many duplicates in genomic databases and, especially, the genomes of some species make up big parts of these databases, I miss a number stating the taxonomic diversity of the analyzed data. Since this most definitely will reflect on the number of species you will find CEGs in, it is important to know how many species that were present in the data to begin with.

4.	In the abstract you write “...antibiotic resistance gene occurrence in (pathogenic) bacteria”. However, you have searched all genomes in the PATRIC database, and it is my understanding that this database not only includes pathogenic bacteria. I therefore recommend to either clarify this sentence or remove the word pathogenic.

5.	Line 118. It is stated that “from the collection of 2 million antibiotic resistance genes in over 170 000 bacterial genomes…”. Here I wonder what the collection is?  Is it that it was 2 million genes annotated as ARGs by PATRICs automatic annotation or is it based on the number you got after you had extracted those annotated as ARGs by PATRIC and then analyzed them using RGI?

6.	Line 86-88: The information about how the suffixes “sp.” and “subsp.”  were removed is somewhat trivial information that rather complicated the reading than giving important information about how the study was conducted. I suggest removing this sentence.

7.	Line 88: The sentence about the free text field seems misplaced. Please elaborate or remove it.

8.	Lines 143-147: Please spell out numbers lower than 11 (i.e. write “one” instead of 1).

9.	The abbreviation CEG is introduced in every section except in the introduction where you use both carbapenemase-encoding genes and CEGs. Please go through the article so that it is consistent with the introduction and use of CEG. Also, sometimes you spell it “carbapenemase-encoding” and sometimes you omit the hyphen.

10.	In table 2 you list three footnotes but I can only see two referenced in the table (* and ***).
Experimental design: 1.	The authors have identified CEGs in the genomic data by first extracting all protein sequences annotated as ARGs in the PATRIC database, then these potential ARGS were analyzed using Resistance Gene Identifier. Those that passed the strict-cutoff by RGI were then subjected to a blast search against a manually collected set of carbapenemases-encoding genes from the beta-lactamase database. The analysis is thoroughly done and I have confidence in the validity of the reported numbers. However, I have some questions regarding the identification of CEGs.

a)	Why did you not search either all genomes or protein sequences in PATRIC for CEGs? I.e. what was the purpose of going through the two intermediate steps of extracting only ARGs as annotated by PATRIC and then RGI? Since you in the end used a TBLASTN against the, from the beta-lactamase database, manually collected set of CEGs with 100% identity and coverage threshold, you can be very certain you don’t get any false positive. Instead, you risk missing true positives by going through the first two steps (PATRIC and RGI).

b)	Since you are using a very strict cut-off, you will only find exactly what you are searching for (i.e. those exact genes you have included in your set of reference CEGs), and there’s a big risk of failing to detect many CEGs that have not yet been included in the database. This needs to be acknowledged. I would furthermore like to know what date these CEGs were collected from the beta-lactamase database as the number of validated CEGs constantly is increasing, and as mentioned, you will most likely only find the CEGs that are included in your set.

c)	It would be interesting to know the class distribution of the CEGs included in your manually collected set (i.e. how many class A, B, C and D), and the class distribution of the ones identified in this study.

2.	In line 148 it is stated that most CEGs were found in the genera of Acinetobacter. Although the absolute number of detected CEGs is the highest in Acinetobacter, it doesn’t necessarily mean that they are the most likely carrier of CEGs but could instead be a reflection of what’s in the database. It would be interesting if the authors could put these numbers in perceptive by comparing them to the number of Acinetobacter, Klebsiella etc. present in the database. I interpret the “3074 genomes: 4783 genes” as that the authors detected 4783 CEGs in 3074 Acinetobacter genomes, but then there’s no information on how many Acinteobacter genomes there originally were in the database.
Validity of the findings: 1.	The authors state on line 221 that “the database approach identified human isolates as the largest reservoir of CEGs”. However, the number of identified CEGs in different environments will largely reflect the composition of the database. As the authors mention in line 222-223, since the database most likely consist of many genomes isolated from humans, it is not strange that you also find many CEGs there. Furthermore, one must also consider that there is a bias in the sequencing where pathogenic bacteria potentially carrying CEGs could be more abundant in the database. However, the study would benefit from actually checking if the proportion of CEGs identified in bacteria from human isolates was significantly higher than the proportion of CEGs identified in bacteria from other sources (this can be tested with for instance Fisher’s exact test). Otherwise, I suggest a rephrasing of the sentence on line 221.

2.	In line 216 it is stated that a large proportion of CEGs have an enhanced capacity for horizontal gene transfer. This statement is based on that 58% of the CEGs detected by the authors were predicted to be plasmid located. However, one has to be careful about drawing conclusions about the real proportion of CEGs that have the potential for horizontal gene transfer. Firstly, the authors have only searched for a limited set of CEGs, using a strict cutoff, where many of these are already confirmed mobile CEGs. Therefore it also likely that many of the, in this study identified, CEGs will be mobile. Secondly, the database is likely biased towards human isolates (See above comment) and there we will probably also find many of the validated mobile CEGs. The authors should therefore rewrite this paragraph.

3.	Line 225. The last sentence here seems a bit out of context. Do the non-human reservoirs have more genomes that contain more than one type of CEG compared to the human reservoir? And if so, what would that implicate? This result is not presented in the main text and Table 2 as the authors refer to here does not contain any information about the reservoirs.
Additional comments: No additional comments


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

Input:
Title: THE EXTENT OF CARBAPENEMASE-ENCODING GENES IN PUBLIC GENOME SEQUENCES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript failed in reporting clear and unambiguous data. Indeed, there is lots of confusion throughout since the authors seem not to possess a sufficient knowledge in carbapenemases. A significant confusion comes from the lack of differentiation between the intrinsic genes (or naturally-occurring in certain species), and the acquired genes. Without addressing clearly that differentiation, and allaying data accordingly, the whole manuscript is meaningless.
Some examples; OXA-66-like enzymes are ALWAYS present in Acinetobacter baumannii. Thus not surprising to see an association with OXA-23 when that latter is acquired !
Experimental design: Unclear whether only whole genome sequences deposited have been retained, or the analysis actually included also incomplete genomes ?
What was the cut-off value retained in term of sequence identity to retain some sequences and not others when variants (close or more distant) are identified ?
Validity of the findings: The validity of data cannot be assessed, since this analysis is a pure in-silico analysis. Therefore, whether a given gene was indeed identified in a given bacterial species remains always doubtful. To the experience of the reviewer, it is very common to have genomic data deposited with inaccurate bacterial species identification. This makes the whole analysis doubtful very often.
Additional comments: Poor manuscript resulting from a poor analysis of in-silico data, without corresponding basic knowledge in the field.


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

Input:
Title: THE EXTENT OF CARBAPENEMASE-ENCODING GENES IN PUBLIC GENOME SEQUENCES
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: The paper describes the distribution of carbapenemases by host species and by environment and collates valuable data. The authors also predict whether genes are chromosomal or plasmid borne. However I couldn't really see the analysis or the results reported regarding chromosomal contexts of the important clinical genes that are plasmid borne and responsible for resistance in key pathogens. Could more information be extracted on likely origins of these genes such as NDM1. Which species carry these genes on their chromosomes, and are they associated with MGEs on the chromosome. Can the authors identify likely original hosts of genes?

In addition if a lower cutoff was used the authors might identify divergent gens that have not already been identified and this might also give information on  progenitor genes and original hosts of these genes. Perhaps this could be done just for some of the NDM genes for example?
Validity of the findings: More could be made of the differentiation between plasmid and chromosomal location, particularly if chromosomal location without MGEs such as integrons, transposons, IS elements can be determiend.
Additional comments: This is an interesting paper but I was disappointed that  analysis and discussion of the origins of these genes was not developed further given the amount of information available. Some further analysis focusing on chromosomal contexts (ie. potential origins) and also of divergent genes (85-90% similarity?) could reveal extremely important insights into the origins, evolution and transfer of clinically important carbapenemases. This would dramatically increase the significance of this study.


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

Input:
Title: THE EXTENT OF CARBAPENEMASE-ENCODING GENES IN PUBLIC GENOME SEQUENCES
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: No additional comments


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

Input:
Title: THE EXTENT OF CARBAPENEMASE-ENCODING GENES IN PUBLIC GENOME SEQUENCES
Review round: 2
Reviewer: 2

Output:
Basic reporting: The revised version has been improved with respect to the original version, but is still of very limited interest according to the referee's perspective.
Experimental design: Seems appropriate, even we must highlight here the strong bias created by the selection of complete genomes. Indeed, when it is said for instance that Klebsiella and Acinetobacter are the genera in which the highest number of carbapenemase genes were identified, this is basically resulting from a higher number of sequenced genomes I guess. Indeed, these are the genera for which we do encounter multidrug resistant isolates most often, and researchers are consequently focusing on those. Thus the distribution issue as presented in a dedicated paragraph is of limited interest.
Validity of the findings: Data are likely valid, and the analysis properly performed.
Additional comments: Still the same feeling I had with the former version. Of extremely limited interest.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT THE MORTALITY RISK IN ELDERLY PATIENTS WITH ARF
Review round: 1
Reviewer: 1

Output:
Basic reporting: Dear authors, I enjoyed reading your manuscript. I believe you report on a relevant topic and think the proposed nomogram is potential useful in clinical settings. However, I believe some points should be further clarified.

The report is well structured.

The professional use of the English language should be checked by a native English speaker. An example can be found in line 44.

Figure 3: The Figures of the calibration curves are blurred (in my review version, so please check)
Experimental design: Line 55 Research question:
Please clarify the precise survival (30d) you assess with your study.
Validity of the findings: Line 68:
Please add name the name of the institution responsible for waiving the informed consent.

Line 69 following:
Please report the methods you used for the recruitment of participants in the validation set within the appropriate method section. Also please report the responsible ethical committee.

Line 69 following:
Please state how the predictors and the outcome were assessed within the validation data set.
Additional comments: Line 83:
Please add the reference for the WHO statement

Line 95:
Please add the reference to the “rms” package.

Line 97:
Please use the full name "concordance index" for the first time.
Please provide a guide how you interpreted the C-index.

Line 97:
Please add the guide, which was used to assess the calibration curves.

Line 98:
Please provide an interpretation guide and the package or statistical software application you used for the decision curve analysis.
It would be helpful if you could reference a method paper, which supports or recommends both statistical procedures (C-index and DCA) for the evaluation of prediction models.

Results section:
Line 108 or elsewhere:
Please report the amount of missing data for the predictors and the outcome.

Line 108 or elsewhere:
Please indicate if any participants were excluded from the analysis?

Line 110-117 or Table 2
Please report your regression models in more detail. There exist several guidelines to report regression analyses. For example, Lang T, Altman D. Statistical Analyses and Methods in the Published Literature: the SAMPL Guidelines.
Link:
https://www.equator-network.org/reporting-guidelines/sampl/

Line 119 and Figure 2:
The nomogram is the key of this paper. I believe it should be reported in more detail. How are the points awarded to the different predictors? Is a summary score build? Or is each item scored individually? Please expand your reporting to allow the replication of the Nomogram in clinical and research settings.

Line 122:
I suggest adding the reference to both nomograms within brackets (such as additional information 1 and 2).
Similar to the comment above (comment line 119). Could you please provide a short description how the nomograms can be used? For example, it looks like a SOFA score of 22 points equals 10 Total points. But what is the difference between “Total Points” and “Points”?

Line 125-127
As mentioned above (comment line 97): please provide more information on how you categorised the calibration curves into “good” or “better”.

Line 133:
If you refer to significantly, this sounds like you performed a statistical test to compare the calibration curves?

Line 137-140:
Your statement regarding the clinical utility is relatively vague. Could you clarify what aspects you consider under the term “clinical utility”.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT THE MORTALITY RISK IN ELDERLY PATIENTS WITH ARF
Review round: 1
Reviewer: 2

Output:
Basic reporting: The tables and figures are well presented. However, the p-values should be listed in Table1.
Experimental design: 1.	It is suggested that the authors give a name of their final model instead of calling it nomogram, otherwise it can be easily confused with the plot.
2.	In the statistical analysis section, the authors wrote ‘The Student t-test, Mann-Whitney U test, chi-squared test, or Fisher’s exact test was performed where appropriate. ’ It is suggested to describe this in a neat way.
3.	The criteria of candidate variables entering into multivariate model is not clear in the statistical analysis section. It wasn't clear on the meaning of ‘showed statistics relationship’.
4.	Was there any variable selection method implemented? In Table2, it seems that there are 5 out of 11 variables are insignificant. A variable selection, either a stepwise or purposeful variable selection process, should be implemented in order to get the final model with the least amount of variables.
5.	It wasn’t clear if the authors did any transformation to the laboratory values in the model, or checked the linearity between variables.
6.    What are the missing percentage of the candidate variables?
Validity of the findings: 1.	In the section of ‘Prognostic nomogram for 30-day mortality’, the authors calculated the C statistics for different models, however, the 95% CIs of the APS-III model and the proposed nomogram model have some overlaps, the authors should be cautious about  stating that ‘our nomogram performed a better predictive power than APS-III score model’. A test to compare the C statistics could be added to strengthen this conclusion. Same for the validation cohort.
2.	In figure 2, it is hard to tell from the plot that ‘the nomogram was better than the calibration curves for SOFA and APS-III’. The authors should report the Hosmer-Lemeshow GOD chi-square statistics and its p-value to strengthen the conclusion. Same for the validation cohort.
3.	It would be interesting to see the subgroup results in different risk factor groups or different time periods.
Additional comments: This is an interesting study to identify the common prognostic risk factors in predicting short term mortality in elderly patients with ARF. The authors constructed the logistic regression models to predict the risk of 30-day mortality and compared the final model with two established risk scores. Although the problem and the clinical importance of this work is well stated, some minor points need to be addressed and further discussed to make it suitable for publication.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT THE MORTALITY RISK IN ELDERLY PATIENTS WITH ARF
Review round: 2
Reviewer: 1

Output:
Basic reporting: ok
Experimental design: ok
Validity of the findings: ok
Additional comments: Dear authors and editors,

Thank you for giving me the opportunity to review the resubmitted manuscript. The authors have responded to my comments and I propose to accept the paper.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT THE MORTALITY RISK IN ELDERLY PATIENTS WITH ARF
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: Minor suggestions:
1. In the statistical section, 'The frequency (proportion) for categorical variables. ', this should be revised as 'Categorical variables were presented as frequency(proportion).'
2. The name of test used for comparing C-indices should be included. The corresponding reference should also be inserted.
3. Table2 should only include the final subset of variables for multivariable results, not all the candidate variables.
Additional comments: Thanks for the feedback. The manuscript looks good to me.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF A NOMOGRAM TO PREDICT THE MORTALITY RISK IN ELDERLY PATIENTS WITH ARF
Review round: 3
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Thank you for addressing my questions. The manuscript looks good to me and no further questions from my end.


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: ARSENIC TRANSFER ALONG THE SOIL-SCLEROTIUM-STROMA CHAIN IN CHINESE CORDYCEPS AND THE RELATED HEALTH RISK ASSESSMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors may improve the clarity and discussion of the paper and send this paper to an English-speaking editor.
Experimental design: Authors did not perform statistical analysis
Validity of the findings: 'no comment'
Additional comments: This paper presents information regarding arsenic speciation in tissues of Chinese cordyceps, habit soils, and its risk assessment. Authors found that this fungus was not an arsenic accumulator and the results of risk assessment at acceptable levels.
The topic of the manuscript is interesting. However, in its current state, it is not ready. The authors may improve the clarity and discussion of the paper and send this paper to an English-speaking editor.  It is missing statistical analysis and quality control.

Introduction
Line 99: use (Huan et al., 2011) instead of (Huan and Carew, 2011). Line 103: explain and give examples of As hydrocarbons. Line 105: move “in Chinese cordyceps” (Line 107) after "(4.00 to 5.25 mg/kg)". Line 108: include a coma after -MS. Line: 116 use “finally,” instead of "ultimately". Line 117: use “determine” instead of "clarify". Line 120-124: this section may improve.

Materials and methods
Line 128: use “endemic areas” instead of "native habits". Line 130: begin the sentence without "And".  Line 130: indicate the soil sample number. Line 132: include “samples” after "cordyceps". Lines 132-138: authors may improve this paragraph. Line 145 it is missing the procedure digestion.
Line 151: eliminate “it was”, and use "at" instead of “to”.  Lines 152 – 158: this paragraph may be improved, please check the use of prepositions. Line 156: add the ascorbic acid formula. Line 158: add the equivalence 8000 rpm in (g), and use "filtered" instead of "passed". Line 162: authors may improve this sentence. Lines 171-173: define AsB, MMAV, and DMAV. Line 82: Authors may include information regarding the standards used for quality control, % recovery, coefficient of variation, limit of detection. The samples were analyzed by duplicate? Line 187: use “were” instead of "are". Line 199: Is this the correct citation?.

Results
Lines 224-225: Authors may explain this sentence further. Line 227: "National, 2014" is a correct citation. Line 231: use "(Wei et al., 1991)" instead of "(Wei and Zheng, 1991)". Lines 262-276: Authors may improve these sentences. Line 281: Authors may indicate if HQ and CR were calculated using total or extracted arsenic.

Discussion
Line 302: include proportion. Line 314: eliminate “reduced”. Line 325: was a statistical determination conducted? Line 329 and 333: include “total” before "As". Line 335: include “lower” before "that found" and change "that" for "than those found in..." Lines 336-339: Authors may improve this sentence, also figure 3A is missing the black dashed line. Line 341: figures 2B and 3C are missing a red dashed line. Lines 342-345: authors may improve this sentence. Line 347: include “total” before "As in". Lines 347-371: authors may improve this section.

References
Line 418: Should this reference (CFDA, 2016) be quoted in this manner? Reference (2016b) is also missing. Line 421: Should this reference (CFDA, 2016a) be quoted in this manner?. Line 453-457: authors may check the correct way of citing these references.

Tables
Table 1: Include the number of samples for SC, ST, and A, B, C. Tables 2 and 3 are missing HQ and CR results.

Figures
Fig. 2- it is possible that As was not properly separated?
Figure 3A is missing the black dashed line.
Figures 3B and 3C are missing red dashed line.


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: ARSENIC TRANSFER ALONG THE SOIL-SCLEROTIUM-STROMA CHAIN IN CHINESE CORDYCEPS AND THE RELATED HEALTH RISK ASSESSMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: Review of the manuscript: PeerJ #51522
Title: Arsenic transfer chain in soil-sclerotium-stroma of Chinese cordyceps and its health risk assessment for human exposure via soil
The authors measured the As concentrations in the soil and in the various part of the endoparasitoid fungus Chinenesis cordyceps to assess the toxicological and carcinogenic risk associated to the consumption of this fungus that is wide spread in the Chinese medicine. They also determine the different species of as in the different part of the fungus using   HPLC-ICP-MS. They found that the fungus dilutes and detoxifies the As from sol to sclerotium to stroma. One drawback is the absence of statistics analysis of the data in the tables and figures, the authors should perform the ANOVA with a post hoch test on those numbers to strengthen their results.
The study is very interesting and well explained. Only few questions:
1)from where did the standards of arsenic for the analyses come from?
2) cloud the author clarify the equations 1,2, 3 explaining the meaning of all the parts of the equations?
3) Do the authors have an explanation theory about why the ratio of iAs/oAs and AsV/AsIII increase from sclrerotium to stroma whilst in the stroma there is less of all the As species in comparison to the stroma?
The figures and tables are of good quality and readable. The bibliography is in keeping with the subject of the manuscript.
Review of the manuscript: PeerJ #51522
Title: Arsenic transfer chain in soil-sclerotium-stroma of Chinese cordyceps and its health risk assessment for human exposure via soil
The authors measured the As concentrations in the soil and in the various part of the endoparasitoid fungus Chinenesis cordyceps to assess the toxicological and carcinogenic risk associated to the consumption of this fungus that is wide spread in the Chinese medicine. They also determine the different species of as in the different part of the fungus using   HPLC-ICP-MS. They found that the fungus dilutes and detoxifies the As from sol to sclerotium to stroma. One drawback is the absence of statistics analysis of the data in the tables and figures, the authors should perform the ANOVA with a post hoch test on those numbers to strengthen their results.
The study is very interesting and well explained. Only few questions:
1)from where did the standards of arsenic for the analyses come from?
2) cloud the author clarify the equations 1,2, 3 explaining the meaning of all the parts of the equations?
3) Do the authors have an explanation theory about why the ratio of iAs/oAs and AsV/AsIII increase from sclrerotium to stroma whilst in the stroma there is less of all the As species in comparison to the stroma?
The figures and tables are of good quality and readable. The bibliography is in keeping with the subject of the manuscript.
Experimental design: No comment
Validity of the findings: 1)from where did the standards of arsenic for the analyses come from?
2) cloud the author clarify the equations 1,2, 3 explaining the meaning of all the parts of the equations?
3) Do the authors have an explanation theory about why the ratio of iAs/oAs and AsV/AsIII increase from sclrerotium to stroma whilst in the stroma there is less of all the As species in comparison to the stroma?
The figures and tables are of good quality and readable. The bibliography is in keeping with the subject of the manuscript.
Additional comments: The authors measured the As concentrations in the soil and in the various part of the endoparasitoid fungus Chinenesis cordyceps to assess the toxicological and carcinogenic risk associated to the consumption of this fungus that is wide spread in the Chinese medicine. They also determine the different species of as in the different part of the fungus using   HPLC-ICP-MS. They found that the fungus dilutes and detoxifies the As from sol to sclerotium to stroma. One drawback is the absence of statistics analysis of the data in the tables and figures, the authors should perform the ANOVA with a post hoch test on those numbers to strengthen their results.
The study is very interesting and well explained. Only few questions:
1)from where did the standards of arsenic for the analyses come from?
2) cloud the author clarify the equations 1,2, 3 explaining the meaning of all the parts of the equations?
3) Do the authors have an explanation theory about why the ratio of iAs/oAs and AsV/AsIII increase from sclrerotium to stroma whilst in the stroma there is less of all the As species in comparison to the stroma?
The figures and tables are of good quality and readable. The bibliography is in keeping with the subject of the manuscript.


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

Input:
Title: ARSENIC TRANSFER ALONG THE SOIL-SCLEROTIUM-STROMA CHAIN IN CHINESE CORDYCEPS AND THE RELATED HEALTH RISK ASSESSMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The new document is better, and the authors have included the recommendations. However, the quality of English grammar should be improved. Some examples where the language could be improved include lines 37-38, 40-41, 42-44, 88, 90-92, 120, 137-138, 148-149, 155, 162-164, 204-205, and 245.
Experimental design: 'no comment'
Validity of the findings: 'no comment'
Additional comments: The new document is better, and the authors have included the recommendations. However, the quality of English grammar should be improved. Some examples where the language could be improved include lines 37-38, 40-41, 42-44, 88, 90-92, 120, 137-138, 148-149, 155, 162-164, 204-205, and 245.


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: DIFFERENCES IN THE ENDOPHYTIC FUNGAL COMMUNITY AND EFFECTIVE INGREDIENTS IN ROOT OF THREE GLYCYRRHIZA SPECIES IN XINJIANG, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article meet the standards of Peer J. However, revision is needed for improvements
Experimental design: Statistical analysis needs authors attention
Data shown in Figure 1 must be analyzed through one way ANOVA and authors need to mention which posthoc test was applied
Correlation study is misleading. Zero means no correlation
Validity of the findings: Findings are valid
Additional comments: The MS reports the effect of root depth and plant species on fungal diversity and root flavonoids, glycyrrhizic acid and liquiritin. It was found that liquiritin content is affected by plant species but not by root depth. However, fungal diversity varied with root depth and plant species.
There are several issues in the study which need author’s attention
1.	The MS is written in poor English and need substantial improvement
2.	How can total flavonoids be quantified by HPLC as mentioned in abstract?
3.	liquiritin contents were not affected by root depth, correlation showed that differences in fungal diversity were due to liquititin. Interestingly, root depth had effect on endophytes diversity. How can authors justify this?
4.	In tabe 1, Figure 5 and 6 abbreviations are not explained
5.	In Figure 1, DATA must be analyzed by ne way ANOVA? It is not clear which posthoc test is applied?
6.	L53-55: How does genetic background of other plants affect bioactive compounds of a medicinal plant?
7.	Methods section contains some unnecessary details such as L116-121. Delete such details
8.	In method section mention Detector, mobile phase and Elution rate of HPLC
9.	L214-216: what is meant by W, D and G? explain on first appearance
10.	L220 and elsewhere: 0 means no correlation at all?
11.	Discussion needs improvement. Include logical reasoning i.e. why fungal diversity varies with root depth? What is the possible reason of ascomycetes abundance ect.
12. conclusion is a mere repetition of results.


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

Input:
Title: DIFFERENCES IN THE ENDOPHYTIC FUNGAL COMMUNITY AND EFFECTIVE INGREDIENTS IN ROOT OF THREE GLYCYRRHIZA SPECIES IN XINJIANG, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1.1. Figures are relevant, but show various styles, they seem to come from various software (Fig. 1. and 3a show a different style vs the others). Please address. Additionally, taxonomic names should be italic in captions and statistical tests should also be included.

1.2. English needs minor corrections, but otherwise the paper is clearly written. Please correct this non-exhaustive list of errors:
L44: new sentence, ". Its dried roots"
L46: "Glycyrrhizic acid, the richest content of triterpene saponins [5]," - perhaps you meant "Glycyrrhizic acid, the chief triterpene saponin"
L65: there is no need to write hormone names with capital letters
L75: typo "officinale"
L82: "on routinely used media"
L382: can regulate

1.3. I do not understand why G. uralensis is abbreviated "W", G. inflata as "D" when G. glabra is "G".

1.4. Introduction is good. I'd add that a few relevant literature remained uncited: 10.1016/j.cpb.2020.100154, 10.21276/ap.2016.5.2.19. As no metagenomics is done in these, novelty is not compromised.

1.5. Structure conforms to PeerJ standards, but the applied reference format is not author - date format.

1.6. Raw data were shared.
Experimental design: 2.1. The applied methods confirm accepted methodologies, with the exception of no p-value threshold adjustment for doing a high number of statistical tests. This is a major issue. Such a high amount of correlation tests requires the adjustment of p-value thresholds to set the hypothesis (or better, study-) level error chance to 0.05. See 10.1007/s11306-006-0037-z. This adjustment will tag many of your current results as false-positives. To cope with the issue, the amount of statistical tests can be reduced by subjecting the dataset to statistical tests after reduction of dimensionality.

2.2. I'd speak of correlations above an absolute value of 0.6 - 0.7. With n = 3 you simply do not have the statistical power to test these relationships successfully. The low values in Table S7 and a fault in experimental design makes the claims in L300-316 unsupported in my opinion. A more prudent discussion of the results is warranted.

2.3. Minor notes:
L177: Please state what was the amount of sequences of plant origin in your raw dataset.
L196: Why is such an old R version used? Did you accidently write the version of a used R package?

2.4. An original primary research within Scope of the journal. No ethical issues. Methods are descrbied in sufficient detail. Research question is well defined and relevant. There is not much information available on the microbiome of this important medicinal plant. I think the investigation of the depth gradient is especially interesting.
Validity of the findings: 3.1. The biggest problem with the experimental design is that each soil type was represented by a single species only that limits the possible conclusions that can be drawn from the findings. The authors are fortunately aware of this limitation, as written in L387-391. But this, unfortunately this prevents drawing conclusions about the effects of the soil type and the plant species, as the interaction term cannot be estimated from these data. The situation is worsened by the low statistical power (n=3). Perhaps this is why we see extreme within-sample variability, as presented in the db-RDA plot in Figure 7. This also questions the usefulness of Table 1. I understand that the answers can be answered only by introducing all test species to all sampling sites, which is not realistic. Therefore, I suggest conversion of the claims regaring the effects of soil parameters and species to speculative sentences, which is accepted by the journal. The undoubtedly well-supported data are limited to the fungal species list in each Glycyrrhiza, and the depth-gradient values. Please revise the study accordingly.
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: DIFFERENCES IN THE ENDOPHYTIC FUNGAL COMMUNITY AND EFFECTIVE INGREDIENTS IN ROOT OF THREE GLYCYRRHIZA SPECIES IN XINJIANG, CHINA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The article is substantially improved and is suitable for publication now
Experimental design: Good
Validity of the findings: Results are valid
Additional comments: Please give attention to my previous comment no. 3.
In abstract you mentioned that root depth had no effect on liquititin contents, in reply to comment you pointed that root depth had significant influence on liquititin.


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: DIFFERENCES IN THE ENDOPHYTIC FUNGAL COMMUNITY AND EFFECTIVE INGREDIENTS IN ROOT OF THREE GLYCYRRHIZA SPECIES IN XINJIANG, CHINA
Review round: 2
Reviewer: 2

Output:
Basic reporting: 1.1. Fig.1. caption shows letters for the post-hoc test but the figure has '*' in the caption. Please address.

1.2. Abbreviating G. uralensis as "W", G. inflata as "D" and G. glabra as "G" is (still) disturbing. In my previous point, I did not argue against using abbreviations, I just meant why not use Gu, Gi, Gg, or something similar? One always has to check again which letter is which species.
Experimental design: 2.1. The 2.1. point of my previous comments was not adequately addressed. In particular, I still see no p-value threshold adjustment for doing a high number of statistical tests - Fig. 5. is the same as before, the authors apparently misunderstood my comment, but this is an important issue. Such a high amount of correlation tests requires the adjustment of p-value thresholds to set the hypothesis (or better, study-) level error chance to 0.05. Again, see 10.1007/s11306-006-0037-z.

In particular, in Fig. 5., and in section L296-313, p < 9.52381e-05 would be required to pass a significance test if Bonferroni correction was truly applied (0.05 / n, where n is the number of statistical tests). Logically, this adjustment will tag many of your current positive results as false-positives. Doing hundreds of tests logically results in some p<0.05 results.

To cope with the issue, the amount of statistical tests can be reduced by subjecting the dataset to statistical tests after reduction of dimensionality (e.g. grouping multicorrelating groups together). Anyway, if such high amounts of statistical tests are done, you should not claim high significane for phenomena with p ~ 0.01. Also see 3.1.

The most sound solution would be to accept that you did not have the experimental setup and/or sample size to study soil parameter - microbial composition correlations that lead to statistically significant results - you still have one species per site. This would result in removing results in L296-313, but this would not reduce the value of your work as you perhaps think.

2.2. Response to previous 2.2.
Statistical significance of a correlation coefficient means you have enough data and good signal-to-noise ratio (hence, statistical power) to be able to say the correlation is truly there, it's "real". This does not make a correlation "strong" or "weak". Therefore, phrases such as "highly significant but negative correlation" (L300) are perhaps signs of misunderstanding. Don't get me wrong, I am not expecting 0.99 R2 values from a biological experiment. But you should ONLY discuss correlations that passed the statistical tests, the rest should be considered noise. I doubt you have enough data to support the claims in L296-313. Please give four-digit R2 values throughout the results section instead of writing "r > 0".
Validity of the findings: 3.1. The Discussion section seems to be cautious enough in its current form.
Additional comments: None.


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

Input:
Title: COLORECTAL CANCER SCREENING BEHAVIORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In general well written, some typo - in specific comments
The literature review is sufficient
I would skip most of the details of table 5
Experimental design: Well designed
Needs clarifications about the sample size calculation, and correction for multiple comparisons
Validity of the findings: The findings are valid
Additional comments: To the Editor
PeerJ
Re: Health beliefs of patients at a primary health care center in relation to attitudes to colorectal cancer screening (#48107)
General
Colorectal cancer screening is a very important measure to improve morbidity and mortality rates in the general population. We should look for any possible way to overcome obstacles and to increase compliance rates.
Abstract
Introduction is too long and should be shortened substantially, I would leave only:
CRC screening program compliance in Turkey t is around 20-30%. Factors that may affect preventive health behaviors, mainly individuals’ values, beliefs and attitudes should be explored. To explain insufficient participation of some individuals at screening programs, a “Health Belief Model” (HBM) scale was developed in 1950. In this study we aimed to investigate attitudes toward prevention from CRC using the HBM scale.
Methods
Need more details about the research tool, scales, subscales etc. You cannot use in the result section terms that were not defined in the methods section. – Please revise
Results
It is not clear why you choose to study young patients because screening is recommended 50-75 years old and not earlier – please clarify the rationale and add to the methods section more details about the participants age and gender and refusal / compliance rates of the questionnaire filling
"Significant differences" is not enough – You should give the numbers in each group and the p Value
Conclusion
"Regardless of their area of expertise or the health system level worked those who provide healthcare should practice the idea of CRC screening for their patients especially among young people. They should talk about screening program at every meeting." – should be omitted as it is not a conclusion of the current study.

Introduction
Line 10 – usually it is either FOBT or Colonoscopy and not both, please clarify
Line 34 – "In this study we aimed to evaluate the health beliefs of people before the age of screening in relation to attitudes to colorectal cancer screening" – This should be clarified also in the abstract
Methods
Well and clear written
Line 40 – "The sample size was calculated as to be 200 and we enrolled 215 volunteer responsive participants" – please clarify for what outcome you calculated the sample size and also clarify if it was calculated to find gender or age differences in this outcome / outcomes
Results
Line 70 – "Average age of participants were.." – should be "was"
Line 84 – "“It is not pleasant to have regular controls for early diagnosis of colon cancer" – I would use tests instead of controls, here and along the text
Lines 92-114 – You did a lot of subgroup analyses – I believe that some of the statistically significant differences you found are only a result of multiple comparisons. You have one of two options – to reanalyze the finding with correction for multiple comparisons or to shorten substantially this part of the results – I would recommend option 1.
Table 2 – The content is not coherent – I would remove from the table the family history of CRC and move it to table 1 as one of the background data on participants.
Table 3 – Add the maximum points for each statement.
Discussion
Line 172 and forward, in accordance to my remark above (lines 92-114) I would recommend keeping in the discussion only the differences that are related to age.
Conclusion
The same sub analyses interpretation as above should be limited in the conclusion 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: COLORECTAL CANCER SCREENING BEHAVIORS
Review round: 1
Reviewer: 2

Output:
Basic reporting: I suggest revision on title to improve readability.

Needs language editing. There are grammatical mistakes such as: “Of the participants’ 62.8%”.

L3 Comma is needed when you place three digits “862 000”

L4: Incostintent citation style for (Turkey Cancer Statistics, 2015). This is also not present in the reference list. The authors are encouraged to follow the instructions from PeerJ for styling.

L34: Please change “evaluate” to assess.

L75 space needed between “58.1%agreed”

Since the authors use HBM scale in the study, I do not see a point to give item by item results as presented in the table 3 and the section starting line 78. This overly complicates the reading of the manuscript, and makes it long.
Experimental design: I am confused with the participant selection. In the beginning of the methods section, the authors explain the participants as anyone who was admitted to Primary Care Clinics but on L64 they say “students.” Please clarify this. The authors also refer to the participants as patients. Please try to use consistent naming throughout the study.

For the data collection part, you mentioned 14 CRC and sociodemographics but the raw data has 15 different independent variables, as follow: “age, sex, marital status, education, business statement, occupation, height, weight, security, alcohol, exercise, nutrition, colon ca history, fec. occult blood, colonoscopi” Please provide your data collection form as a supplement to evaluate this.
Validity of the findings: There are multiple missing data in the raw data. The SPSS output and raw data do not match.

I did not see the original cronbach alpha of the scale as well as the cronbach alpha of the scale used in the current study. Authors should provide reliability results for the scale.

As data is not intact, I was not able to reproduce the analysis. I encourage the authors to give more details on the analysis, explain and justify how you handled the missing data.

What is the original source of the scale? You only mentioned that this scale is a Turkish version. You mentioned the HBM scale developed on L19, but I am confused if it is the original scale. Was it adapted from this scale developed by Hochbaum, Kegeles, Leventhal and Rosenstock? Or, is this an original scale constructed in Turkish? Please explain in the methods section.

How many negative items were there in the scale? How did you transform them and get a total score for the subscales and overall scale. More details are needed for the scale.

In the results section, you presented the statistical results but you did not write the statistics. When you compare the groups, it is not enough to present p value only, you should provide the test statistics such as t-test, anova along with their effect size such as Cohen’s d.
Additional comments: Dear Authors,

Thank you for your submission to PeerJ. I have read your manuscript with interest. I am a PhD scientist in the field of medical education. I know less about the topic that you covered in the study. I accepted to review this article because of the methods that you applied. I was not able to assess the content area and its importance to your field. Please consider my reviews as a method and statistical expertise.

I wanted to repeat the analysis to confirm the results but there were missing scores in the raw data, and you did not describe the analysis thoroughly. Therefore, I could not evaluate the results.


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

Input:
Title: COLORECTAL CANCER SCREENING BEHAVIORS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well reported
Experimental design: Well designed
Validity of the findings: Seems valid, the authors may try to handle better some confounders
Additional comments: To the Editor
PeerJ

Re: Colorectal cancer screening behaviors by Selda Tekiner Corresp et al.

Dear Editor, I would like to thank you for the opportunity to evaluate the revised manuscript.
Here are my detailed comments:
Abstract
Results section:
Please the total number of participants, and if you have data on the participation compliance rate
In general and in the abstract – Age can be presented with only one digit after the point (ie. 35.4 instead of  35.42)
Conclusion section:
Please “affected by” to “associate with” as in such a descriptive study we cannot establish cause and result effect and can only point association between two phenomena.
Introduction – no comments
Materials and methods
Line 94: “Based on this, we know that our patient population consists of those who are not at the screening age for colon cancer, which was the age group we wanted to study.”
Please clarify – if this is not the group you wanted to study why you choose this group. I would rephrase it in this way: “Based on this, we know that our patient population consists of those who are not at the screening age for colon cancer, but these are the years when we can intervene and change the health beliefs.”
Results
Table 1 - the legend – “Table 1: Some characteristics of participants” please change to: “Table 1: Sociodemographic characteristics of participants”
Table 2 legend – referral (page 27) is a mistake referring to the author's details, and the mistake continues to all tables headings.
Please start the results section with the number of participants, and add information (if available) on compliance rates.
Line 183:” There was a difference between work status and barrier scores (p<0.01). The barrier scores of the participants who were students were found to be significantly higher than those of working and retired participants (p=0.001, p=0.013).” – It can be a confounder – as age have a strong relation to the work status, students are usually much younger. Please discuss it
Discussion:
Line 198:” This study was conducted in a reference hospital in the capital city of Turkey, for the purpose of evaluating the health beliefs of people in relation to CRC before the age of screening for CRC.” – You have to clarify as 15.3% of your study population was older than 50 years, the age screening is recommended.
Line 286 – the conclusion: “As a conclusion, we can say that it is better to give specific messages and specific barriers according to personal characteristics instead of general messages about conducting screening tests beginning from youth.” – I would say “it may be better” instead of “it is better” as to prove this we need an intervention study and not a descriptive study like the current one.


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: COLORECTAL CANCER SCREENING BEHAVIORS
Review round: 2
Reviewer: 2

Output:
Basic reporting: L154 please revise Kruskal-Wallis test with right spelling.
please remove white spaces in the paper (e.g, L185 ( p )
Please use lower case p value L181
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


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

Input:
Title: EXPOSURE-RELATED, GLOBAL ALTERATIONS IN INNATE AND ADAPTIVE IMMUNITY; A CONSIDERATION FOR RE-USE OF NON-HUMAN PRIMATES IN RESEARCH
Review round: 1
Reviewer: 1

Output:
Basic reporting: See comments below

The article is well written and the introduction was clear.
Experimental design: See comments below

I highlight point 4 - I had difficulty in understanding how the data in figure 7 was generated. My best interpretation is that the authors correlated cell counts or cell percentages in the peripheral blood of three groups of animals.

It gives the impression that there is some functional or (epi)genetic link that generates the correlations. If this is not so that the data should be presented in a less misleading manner.
Validity of the findings: See comments below.

Fundemenatly the premise is interesting and that animals should be assessed prior to starting a new experiment. Experienced animals may be more useful sample as this reflects animal populations in the real world.
Additional comments: Dear Editor,

Thank you for asking me to review the manuscript “Exposure related global alterations in innate and adaptive immunity; a consideration for re-use of non-human primates in research” by Francois Bates et al.

The authors compare four groups of rhesus macaques, naïve animals, animals that were vaccinated with a live attenuated dengue virus, those that were vaccinated with a malaria antigen and those that were vaccinated with a non-replicating human adenovirus containing the same malaria antigens.
The authors tracked changes to serum cytokines, CD14+ monocytes, HLA-DR+ NKT cells, CD20+ B cells, total and HLA-DR+ Th and Tc cells over a six month period. They found residual changes to all of these measurements that lasted for up to six months post challenge.

The authors highlight an important point in non-human primate research and suggest that animals exposed to multiple experiments will diverge from naïve animals that may confound future experiments. Conversely, they may make the animals more representative of a human population that is constantly being immunologically challenged.

A few points should be considered:
(1)	There are inconsistencies in the investigation of post DENV and post malaria/ adenov vaccine effects in the monocyte (figure 1) and T cell subsets. In the former CD14+ cells were compared with DR+CD14+ cells. In figure 5 CD4+ T cell data was not shown in the DENV group.
(2)	It is unclear what total populations of cells the percentage cell frequency is determined throughout the paper. For example, I assume that the % frequency of total T cells is derived from a different cell total population that that used to calculate the % CD3+ CD8+ T cells.
(3)	The lymphocyte analysis is relatively superficial. It would be useful to see naïve and memory B cell subsets. It would be useful to see CD25+CD4+ T cell subsets as this would have relevance in auto immune models.
(4)	I am confused by the data generated in figure 7. I do not understand how correlations can be generated on the basis of cell counts and how this could be interpreted to correlate with immune cell function.


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

Input:
Title: EXPOSURE-RELATED, GLOBAL ALTERATIONS IN INNATE AND ADAPTIVE IMMUNITY; A CONSIDERATION FOR RE-USE OF NON-HUMAN PRIMATES IN RESEARCH
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this article the authors make a case that NHPs that are re-used for multiple experiments should be carefully evaluated before assigning to experimental groups in order to avoid biassing and to enhance reproducibility. In order to evaluate the impact of prior immune stimulation on the resting immune response the authors used blood drawn from three groups of rhesus macaques at defined time points after immunization and challenge with dengue virus or after immunization or boosting with two different malaria vaccine regimens. Blood samples were taken as far as 6 months after their last exposure to antigen and compared against age and sex-matched naïve controls. The authors noted transient as well as long-lasting changes in immune cell frequency, cell numbers and cytokine production in the blood. The overall changes in biology varied depending on the type of antigenic stimuli each group was exposed to. Hence, the authors conclude that antigenic stimulation of the immune system leaves unique patterns of immunological imprints in NHPs that could affect the consistency of subsequent data when the NHPs are re-used in separate experiments. The authors suggest that their method of evaluating cytokines and immune cell phenotypes in fresh blood samples derived from NHPs at various times after the conclusion of their study will help aid in determining an appropriate time when these NHPs can be re-used in new experiments.

This is a well written manuscript that provides a clear objective, results and discussion of relevant findings and interpretation of the findings. The authors do a sufficient job of using the literature to support their study design and rationale, and their conclusions. The results were adequately summarized at the end of each results section, making it easy for the reader to understand the significance of the findings.
Experimental design: The authors provide the necessary information regarding the use of the NHPs in this study. Specific animal protocol numbers for the studies that the NHPs were apart of were provided in the methods section. A statement indicating that all procedures and protocols were approved by the institutes IACUC was provided in the methods. Also, an animal ethics statement was provided in the methods section. The authors provided information about the sex, age and number of rhesus macaques used in each group in the study in the methods section. Lastly, the research question was defined and addressed adequately by the authors in the manuscript.
Validity of the findings: While the authors emphasize that epigenetic changes in innate immune cell populations such as monocytes could impact future immunological readouts in secondary experiments due to the phenomenon of trained immunity, they do not investigate this idea in this study. However, they do a sufficient job of using the literature to support this idea in the introduction and discussion sections of the manuscript. They also allude to the need of future studies to determine how different antigenic stimuli can induce tolerance or trained immunity.

One point that the authors should briefly address in their discussion is in regard to the limitations of their study. This was one study conducted using rhesus macaques from their facility at Walter Reed. Although the use of three separate groups exposed to three different antigenic stimuli increased the rigor of the study, this is just one study. Do the authors anticipate that that other NHP species will response similarly as the rhesus macaques? Only one of the three groups was challenged with the agent that the NHPs were vaccinated against. Would similar results as seen with group one be predicted after live challenge with other pathogens? Would the authors anticipate similar findings using blood derived from NHPs used in studies conducted at  different animal facilities?
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: DEVELOPMENT AND VALIDATION OF BIOIMPEDANCE PREDICTION EQUATIONS FOR FAT-FREE MASS IN UNILATERAL MALE AMPUTEES
Review round: 1
Reviewer: 1

Output:
Basic reporting: A generally well-written manuscript with acceptable English although with flaws as noted below. Cited references are mostly recent, although not always the most recent,  and provide adequate background to the study. Raw data have been provided. The manuscript is appropriately structured.
Experimental design: The experimental question is clearly stated and background provided. The experimental design is acceptable and execution allowed required raw data to be obtained. Unfortunately, the manuscript lacks necessary methodological detail to allow full replication and the data analysis could be improved.See below for details
Validity of the findings: The authors have not described adequately, exactly how the FFM has been calculated. The  multiple regressions appear to have been undertaken simply to maximize the correlation and minimize error without thought as to the appropriateness of inclusion of variables. For example, it is hard to see why different predictor variables are required for the left versus the right arm.The conclusions are not justifiable. The authors appear to equate a strong correlation with predictive performance. The clinical value of the data and the clinical impact of errors are not discussed. These points are amplified in the section below.
Additional comments: Line 78 onward. BIA is a generic term used to describe the overall methodology. Within this there are single frequency BIA (SFBIA), multiple frequency (MFBIA) and spectrometric approaches (bioelectrical impedance spectroscopy, BIS). Each of these can be used for whole-body or segmental measurements and are not always exclusive, for example BIS can provide SFBIA data but not vice versa. Methods of transformation of measured impedance/resistance to body composition does not always use empirically-defined prediction equations. Hence this explanation of BIA is simplistic. The authors should rewrite this section to reflect the above comments and provide suitable citations to recent reviews. They may wish to highlight the approach to be used in the present study. It is not clear from the text exactly what type of device the S10 is.
Line 84. This is vague. What "independent variables"? I assume that you are referring to , weight, sex etc. in addition to BIA parameters but this is not clear.
Lines 89/90. Again this vague and potentially misleading. A simple BIA prediction of body composition based on wrist-ankle measurement on the non-amputated side could predict perfectly well (within typical BIA-related errors) since stature is used not individual segment lengths. It would, however, be in error if the ERE included weight as a variable since presumably the ERE would have been generated on a population of non-amputees, i.e. including amputated limb weight. The point being made is that the text is simplistic.
Line 94. While ERE is defined sERE is not.
Line 102. Why were the 10 females dropped from the analysis? Please justify, line 222 states simply the sample was not large enough, on what basis? Were they all unilateral amputees, were any bilateral? On what side were amputations, dominant or non-dominant?. Humans are bilaterally asymmetric, having more FFM on their dominant side, particularly in the arms. For upper-limb amputees, work-induced hypertrophy of the remaining limb may be a confounder. Was this considered?
Line 111. The description of DXA is limited. For example, it is well recognized that inter-device, inter-software differences exist. provide more details. Were these single measurements? Was a soft-tissue calibration performed (not simply for BMC/BMD)?
Line 118. This again lacks detail. For example, is the S10 device a stand-on, hand-held or electrode type device? Line 125, suggests the latter. What exactly was the measurement protocol? Were the participants lying down, if so for how long before measurements? Were measurements in replicate or single measurements? Since it is unlikely that manufacturer's instructions covered amputees where were electrodes located in these cases? The authors state that the device can perform measurements at adjustable frequencies in the range 1-1000 kHz. While it is implied, but not explicitly stated was 50 kHz the frequency of measurement chosen? How was the system calibrated and what QC procedures were in place? More detail is required.
Segmental impedance measurements. The use of resistance/impedance quotients is supportable by theory and is standard practice. My question here is why consider Z, R, Xc and phase? The aim was to produce EREs for FFM. Impedance is based on measuring the opposition to current flow through the body, i.e. body water and, assuming either implicitly or explicitly, an hydration fraction for FFM. The appropriate measure that relates to water volume (FFM) is R. Xc  and phase relate to cell membranes and Z is a composite or R and Xc (Z^2 = R^2 + Xc^2). So all three parameters are intimately related. Hence there is a high potential for multi-collinearity. Similarly, because of this relationship it is generally considered inappropriate simply to include multiple combinations of these parameters in regression equations (Table 2). It also appears from Table 2 that absolute values for R (or Xc or Z) as well as the length-related quotients are included. Why?  The authors explain this (line 247 onward) by simply saying that since the early studies additional variables have been included to improve accuracy. This is not good scientific reasoning. Others may have included additional variables wrongly. There should be valid reasons for their inclusion. Basic impedance theory, provided in many reviews, demonstrates that the quotient should be used not simply the raw R. This section needs to be re-written to align with the theoretical basis of BIA.
Since it has not been made clear where electrodes were actually placed, it is equally not clear how these align with limb lengths. For example, if electrodes were placed along the limb e.g. at the wrist and acromion then this length should be used. If the method of equipotentials was adopted then the proximal virtual electrode position is unknown and hence a surrogate length measurement should be used. Line 143, implies that electrodes were located at the length measurement locations and these were then also used for DXA ROI but it is not made clear.
Line 154. Again more details are required. What was the order of measurements, DXA then BIA or vice-versa? Was it consistent? If not, the time interval for the subject (assumed to be supine) lying down prior to BIA would be variable, and hence the potential for variable fluid shifts. More details needed.
Line 162. Statistical analysis is not well described. Multiple regression was used. Was normality assessed? What criteria for multi-collineraity was used - VIF threshold? Were potential outliers evaluated and if present what strategy was used to account for this. Step-wise can be forward or backward, what criteria were used to remove variables from the regression, indeed what variables were included and assessed? Fig 1 is a regression/correlation of predicted versus measured. What type of regression was used? Where both variables are subject to error then a procedure such as Deming or Passing-Bablok regression should be used. Correlation should be assessed using Lin's concordance correlation not Pearson. Some details are provided but in Results (line 191 onward). This should be in statistical analysis. The B&A plots are not a representative validation. All data were used to generate the ERE and then again used in the B&A plot. This is circular and will not provide a true measure of validity. Validation should either be via a split group design, K-fold or LOOV-type procedure. For example, an ERE in say 50 randomly-selected participants could be generated and the its predictive power tested in the remaining 25. There is ample published literature on these methods, including specifically for validating impedance predictors.
Results.
Table 2 and text. It is not made clear how the amputations are distributed between the different regression equations. For example, for upper limb amputees were they all the same side? Is a the distribution a contributor to why quite different equations (variables and coefficients) have been generated for the right and left arms? What is the reason that might explain height and age being significant predictor variables for the left arm but not the right? This would not seem logically plausible for non-amputees and would appear unlikely for this group also unless amputation was much more prevalent on one side than the other. Explanation is required.
Figs. Again it is not explicitly explained how these data are obtained. Is it a result of a whole-body wrist-ankle prediction or is it the sum of the segmentally-predicted values. Fig 2. On what criteria is a bias of 4.5 kg considered poor? What is clinically acceptable (minimal clinically important difference, MCID)?
Discussion
Line 212. The authors appear here to be confusing correlation with predictive performance. A high correlation does not necessarily indicate a good predictor in terms of accuracy. The authors have not actually assessed this for the segments only whole body. Bias was 4.5 kg for a mean FFM of around 50-52 kg. This is a bias of around 10% is this acceptable? More importantly there is systematic bias, well demonstrated in Fig 1. Why? This needs to be discussed. The LOA are -10.26 to 1.064 or around +/-6.0 kg, again around +/- 11%. Note that this is based on a circular B&A (see above), a true cross-validation would likely produce worse results. Is this clinically acceptable? This is important since in a clinical setting accuracy in an individual is of paramount importance.  Again this requires discussion and a reasonable correlation (line 310) should not be used as justification for adequacy of the method.
Line 228. 50 kHz is not a standard, it is a commonly used frequency largely for historic reasons and from the widespread use of SFBIA devices. A strong argument can be made that if a single frequency is to be used it should be a the characteristic frequency or a frequency higher than 50 kHz where current penetration of the cell membrane is greater.
Lines 234 onward. This is misleading. The authors imply that measurement systems are 4 or 8 point. This is incorrect. ALL measurements are tetrapolar, two current drive electrodes and two proximal voltage sense electrodes. The so-called 8-point systems use the same measurement approach but simply swap in turn pairs of electrodes to measure the different body segments. A typical "4-point" system can achieve exactly the same by manually connecting electrodes to measure the segments according to the equipotential principle. There are many publications attesting to this. The use of the "8-point" system as implemented in the In-Body device is not  justification for the claim of highly accurate sERE (line 246).
Line 293. I do not see the basis for the claim of "perfect positive correlation of y=x". This requires justification.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF BIOIMPEDANCE PREDICTION EQUATIONS FOR FAT-FREE MASS IN UNILATERAL MALE AMPUTEES
Review round: 1
Reviewer: 2

Output:
Basic reporting: -	This paper is relevant, nicely written and has a logical structure.
-	Relevant use of references.
-	Use the abbreviation BC for body composition throughout the paper.
-	The abbreviation of phase angle (PhA) should be changed to PA since this is the accepted abbreviation in the bioimpedance literature.
-	Decide how to report figures in the paper, with 2 or 3 decimals. In your paper, I would use 2 decimals for R-values and kg (e.g. -4.598 kg).
-	Line 119 and 226: Write 1000 kHz instead of 1,000 kHz
Experimental design: -	In general, the experimental design is well described.
-	Line 118-128: However, the use of the BIA technique needs more attention when reporting in order to obtain valid measurements. E.g. was measurements made in triplace?, was the device calibrated prior to use?, how was skin prepared before placement of electrodes?, how was the subjects positioned during measurements? etc. The importance of standardised bioimpedance measurements have been proposed by following authors:

https://www.tandfonline.com/doi/abs/10.1080/03091902.2016.1209590?journalCode=ijmt20

https://www.tandfonline.com/doi/full/10.1080/03091902.2017.1333165

https://pubmed.ncbi.nlm.nih.gov/15556267/

-	Line 162-: Was data normally distributed?
-	Line 163: Please add a p for statistical significance, e.g. p ≤ 0.05.
Validity of the findings: -	Relevant to study prediction equation for use in amputees.
-	The number of subjects included seems relevant.
-	The data analysis models (Linear regression and Bland-Altman) are relevant when developing prediction equations.
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: DEVELOPMENT AND VALIDATION OF BIOIMPEDANCE PREDICTION EQUATIONS FOR FAT-FREE MASS IN UNILATERAL MALE AMPUTEES
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have satisfactorily addressed the issues raised in my original review. In places the English is somewhat idiosyncratic but the meaning remains generally clear.
Experimental design: The authors have satisfactorily addressed the issues raised in my original review. Additional methodological detail has been provided.
Validity of the findings: The authors have satisfactorily addressed the issues raised in my original review. The authors have adopted revised/improved statistical analysis.
Additional comments: The authors have satisfactorily addressed the issues raised in my original review.


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

Input:
Title: DEVELOPMENT AND VALIDATION OF BIOIMPEDANCE PREDICTION EQUATIONS FOR FAT-FREE MASS IN UNILATERAL MALE AMPUTEES
Review round: 2
Reviewer: 2

Output:
Basic reporting: -	This paper is relevant, generally nicely written and has a logical structure.
-	Relevant use of references.
Experimental design: -	In general, the experimental design is well described.
-	Line 111-119 “Participants”. Why is there no control group?
-	Line 136-137 “Experimental device (BIA)”: “After checking reproducibility and the precision error of FFM…” If you have checked reproducibility, then only write this since reproducibility is a precision measurement. Precision is the overall term containing repeatability (same person) and reproducibility (different persons).
-	Line 132-152 “Experimental device (BIA)”: Was measurements made in triplicate?
-	Line 168: Change to Heymsfield and remove region of interest and only keep ROI.
-	Line 248: Write FFM in the subheading, since the terms BIA and DXA are used.
-	Line 284: “SFBIA is better for measuring cell membrane properties through Xc”. Better than what? Why? Please elaborate on that statement.
-	Line 287: “Only an SFBIA of 50 kHz was used for the calculation of total body water. Why that? In line 283 it is stated that “MFBIA can more accurately measure intra- and extracellular body water than SFBIA of 50 kHz. From the intra- and extracellular body water the total body water can be calculated. Please explain how this is related to the knowledge and potential of MFBIA and BIS techniques?
-	Line 381-382: “We used only 50 kHz to characterize the cell membrane. We will proceed with the MFBIA study as a future research project”. Now you write that you only used 50 kHz to characterize the cell membrane. The use of “only” indicates that you are not sure whether 50 kHz is “enough” for measuring the property of the cell membrane. Once again, in line 284 you wrote that “SFBIA is better for measuring cell membrane properties through Xc”. Please explain how all these statements are related. Also, what is the MFBIA study?
-	Figure 1: The picture has a bad resolution. Try to find another one.
Validity of the findings: -	Relevant to study prediction equation for use in amputees.
-	The number of subjects included seems relevant.
-	The data analysis models (Linear regression and Bland-Altman) are relevant when developing prediction equations.
Additional comments: -	Due to the numerous abbreviations and parameters used in the paper, which makes it a little hard to read the paper, please add a Table summarizing these.
-	The first time an abbreviation is used, it must be written in full. Be consistent with abbreviations throughout the paper.
-	I find the use of phase angle (PA) a little awkward in the paper. Maybe it should be analysed separately in later paper?


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

Input:
Title: THREE-DIMENSIONAL DATA-TRACKING SIMULATIONS OF SPRINTING USING A DIRECT COLLOCATION OPTIMAL CONTROL APPROACH
Review round: 1
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: The work does not support the conclusion that this model of sprinting is suitable for research on sprint performance. At the very least, the limitations should be discussed. See general comments for details.
Additional comments: This manuscript presents simulations of sprint running, developed using a model of multibody dynamics with contact. The joint torques and contact model parameters were optimized to track kinematic data, measured ground reaction forces, and measured joint torques. It was found that the simulations could closely reproduce the data, with low dynamic inconsistencies. The authors concluded that the model is ready to be used for predictive simulations to study the cause-effect relationships in sprint performance. The paper was technically sound, well written and a pleasure to read.

I agree with the authors that a data-tracking optimization, as presented here, is a necessary and important test to determine whether a model has the capability to replicate a measured performance. Until a model passes this test, applications and predictive simulations should not be considered. However, I feel that this capability was not sufficiently tested, see comments 1 and 2, below.

1. The model is actuated by joint torques rather than muscles. Muscle-actuated simulation is already state of the art, for example, in the work by Falisse which is cited in the manuscript, and which shares one co-author with this manuscript. Muscle actuation would constrain the joint torques to the dynamic force-generating capabilities of muscles. Without constraints on the torques, almost any movement is possible, making the findings hardly surprising. This also is important for the intended application. If one wanted to study sprint performance, performance is only limited by arbitrary bounds placed on the joint torques, rather than bounds informed by muscle physiology. It is unlikely that such a model would provide insight into the factors that affect sprint performance. This limitation diminishes my enthusiasm for this work, and should, at very least, be properly discussed.

2. In the Introduction, the authors mention correctly that dynamic residuals should not be neglected in sports biomechanics research. However, their model still allows non-zero residuals, although they are minimized during the optimization process. If residuals are allowed, the movement is no longer strictly constrained by the laws of physics. Trajectory optimization with zero residuals is the state of the art, (e.g. the cited papers by Falisse) so there is hardly a good excuse for allowing residuals. This is also important for the envisioned applications. If a predictive simulation allows an external “helping hand” force acting on the pelvis, this becomes a major confounding factor for performance (running speed).  The residuals that were found to be acceptable in this paper (34 Nm RMS in anterior-posterior direction, Table 3) are as large as the air drag force during sprinting, which is known to be a major factor in performance. I suspect that removing the external pelvis actuation would still produce very good results, and I strongly recommend making that change.

3. The description of the model did not mention air drag, so I assume it was not included.  Air drag produces a significant amount of force during sprinting. A simple lumped force, proportional to squared velocity, could be modeled, for instance, based on Quinn, Journal of Sports Science, 2004. The lack of air drag in the model may well be the reason for the large anterior-posterior force residuals (Table 3). This is also important for the intended application: without air drag, performance will be overestimated.  At the very least, this limitation should be properly discussed.

4. Line 201-202, “The generic modified model was linearly scaled to match the anthropometric and inertial characteristics of the athlete by using a measurement-based approach within OpenSim”.  This requires a citation if it was a specific, previously described approach.  If not, a more detailed description is needed.

5. Line 259. The optimization objective has five terms. The first three are data tracking terms, and it is logical to give them the same weight, with exception of the justified lower weight of the torque tracking term. The 4th term penalizes residuals, and it is not automatically clear that its weight (w1) should be the same as the angle and GRF tracking terms. I would prefer not to have this term at all (see comment 2), but if you have it, its weight should not be tied to the tracking term. I suggest defining w1 for angle and GRF tracking, w2 for torque tracking, w3 for residuals, and w4 for accelerations.  Then define w = [0.1 0.01 0.1 0.0001].  This does not affect the results, but would prevent the reader from thinking that residuals need to be weighted the same as tracking data.

6. Line 273. If all variables were normalized to 10% of the range observed in the experiment, a constant factor 0.1 should be added in each denominator in the equation. This seems arbitrary. However, this would simply scale the whole objective function by a factor 100, and not affect the optimization problem in any way.  Consider leaving out this mention of the 10% because it does not matter. If the scale factor of 100 was helpful to make IPOPT perform better, it can simply be mentioned as an overall scale factor, rather than applied to the denominator in each term. But be careful to account properly for the 5 mm normalization in the anterior-posterior translation variable.

7. Line 294-295. The optimization of the contact model parameters is a strong and innovative aspect of this work. The resulting contact model makes sense, and is probably going to improve the validity of predictive simulations. Consider highlighting this a bit more as one of the goals of the work.


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

Input:
Title: THREE-DIMENSIONAL DATA-TRACKING SIMULATIONS OF SPRINTING USING A DIRECT COLLOCATION OPTIMAL CONTROL APPROACH
Review round: 1
Reviewer: 2

Output:
Basic reporting: Suggest avoiding use of “excellent” as a subjective descriptor of the quality of the results (Lines 50, 312, 414, 423, 451) or similarly “great” (Line 443).  Some context should be provided to the reader to gauge this, e.g. are the errors small vs. measurement error, or vs. trial-to-trial variation, or something similar?
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The study used a direct collocation approach to perform data-tracking simulations of sprinting.  It demonstrates a nice alternative to performing inverse dynamics in cases where dynamics residuals are large, among other potential untested use-cases e.g. predictive simulations.

I had five concerns of a technical nature described below that I would like to see addressed in revisions to the manuscript text, and a few other minor suggestions:

(1) Lack of muscles: The planned next step in the research line (as per Lines 57, 113, 131, 147, 443, 558) is predictive simulations of sprinting, which makes the lack of muscles in the present model an important consideration (I'll refrain from citing a bunch of studies on how muscles are important for sprinting).  For a non-specific use-case, it's unlikely that the present model lacking muscles would produce realistic results in predictive simulations (it would be interesting if this was the case, but this would likely also require comparison to results with muscles).  Essentially what is shown is an alternative approach (vs. traditional inverse dynamics) for estimating joint moments from measured kinematics and GRF, with the benefit of lacking residuals (see question #2 below however).  This alone is a fine contribution, I personally would really like to see this become a field-standard approach for doing inverse dynamics, but lacking muscles I have a hard time seeing how the present work demonstrates readiness for predictive simulations.  For that I think it would need to be shown here that there is good tracking of the data well with muscles or something like muscles, realistic muscle excitations etc.  So I suppose my specific suggestion/question here is, should the paper be cast as an “alternative to inverse dynamics” paper, and not a “ready for predictive simulations” paper (assuming the authors are uninterested in a revision in adding muscles, which would be a major effort)?

(2) Residuals confusion: I was confused by the inclusion of “residual forces” on the pelvis in the model.  This may just be a semantics issue and I'm misunderstanding what the authors mean by "residuals", or maybe I’m ill-informed on when “residuals” (F – ma = non-zero) are present in simulations like this, but regardless I though this issue needs to be clarified.  In the typical OpenSim workflow or traditional Bresler & Frankel / Winter-style inverse dynamics of gait, residual forces result from applying measured GRF to the model, which were not generated by the model’s dynamics and are not necessarily consistent with its motion, resulting in violations of the equations of motion (Hatze’s (2002) “fundamental problem”).  In a typical direct collocation problem, these violations are eliminated by enforcing the equations of motion as constraints.  This was done here (Line 286) so it was unclear to me why any additional forces were applied to the pelvis and minimized in the cost function.  It seemed that the “residuals” referred to in the simulations were actually forces applied to the pelvis that help move the model independent of the joint moments (sometimes called "hand of god" forces in modeling), not violations of the equations motion that were computed from an inverse dynamics approach.  More explanation is needed on what these residual forces on the pelvis actually were and how/if they are consistent with the Hatze/OpenSim definition of residuals.  Essentially, I did not understand how a simulation with F = ma as a constraint can have violations of F = ma, which seemed to be the definition of “residuals” in the paper (Line 99).

(3) Optimizing foot-ground contact locations: This approach seemed to deviate from what is generally good modeling practice (assigning model parameters using an independent set of data from the test data).  The optimized element locations and stiffness/damping values should be validated in some way, i.e. were the optimized locations realistic locations of contact, were the stiffness/damping realistic for a human plantar foot/shoe/ground composite?  Aerts & De Clercq (1993) is a common reference for the latter.  The description of the contact model mechanics (Lines 245-253) would benefit from being expanded, e.g. including the mechanics equations and the range of permissible values in the optimization.  The shear contact/friction forces and the associated parameters should be included.  The finding that different locations are determined from optimizing all phases vs. a single phase is important, but at a more basic level I think the motivation/benefit of optimizing the contact element positions vs. the typical approach just assigning the positions ahead of time to reasonable anatomical locations and avoiding the possibility that the optimizer places them in an unrealistic location, needs to be strengthened.  I am not saying "Don't optimize the contact locations", just that I don't see the benefit/need of doing this unless there are too few elements for realistic general tracking, and am wondering if readers will not see it either.

(4) Mesh sensitivity: The choice of the temporal mesh interval density (Line 282) should be justified, e.g. is 80 intervals large enough for “mesh-independent” solutions?  This is important to demonstrate before moving onto predictive work.  If I understood correctly, the solution was evaluated at ~4*80 nodes, which is likely plenty for a partial gait cycle even of a fast movement like sprinting, but some verification that this is so should ideally be done.

(5) Initial guess: I couldn’t find any mention of the source of the initial guess to the optimizations.  This is an important detail in these types of problems.

Minor:
(1) Readers may not understand why it's helpful to have joint torque controls (e.g. why not just compute torques from the state?) and similarly GRF controls.  Consider adding an explanation for why this is helpful.

(2) Some additional discussion on this approach vs. other residual reduction/elimination approaches would be helpful, e.g. pros/cons vs. OpenSim RRA.

(3) On the convergence tolerance (Line 293), some additional context on this value would be helpful for readers who are unfamiliar with these terms and methods, e.g. does this mean the largest constraint violation could be 0.001 N?

References
Aerts (1993): https://pubmed.ncbi.nlm.nih.gov/8301705/
Hatze (2002): https://pubmed.ncbi.nlm.nih.gov/11747889/


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: THREE-DIMENSIONAL DATA-TRACKING SIMULATIONS OF SPRINTING USING A DIRECT COLLOCATION OPTIMAL CONTROL APPROACH
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: The authors have responded exceptionally well to the initial review. I have no further comments on the manuscript.