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: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper aimed at Grassland-Cropland change detection, it seems the major objective of the paper is to select the optimal feature for the change detection, but the description of the feature importance estimation method is not clear. In addition, the change-detection accuracy is low in my point of view as the UA of change is lower than 10%.The authors should show their novelty and applicability clearly.
Although I am not a native English, I found the language still need some improvement, such as line 240 and 255.
Experimental design: When training the classifier (SVM), only 200 change and 200 no change samples were used, does these training samples enough for training the classifier?
Validity of the findings: As the UA of grass-crop change is low, and the change was significantly overestimated, I donot think the result support the findings well.
Additional comments: 1.	Line 21, what does the sentence mean? It is not clear
2.	Line 65~68, what is the meaning of ‘false one’, I think this sentence should be improved.
3.	Line 107~110, I think it is the objective of this study. But it should be like “to find the optimal variable for grass-cropland detection”.
4.	Line 174, can you show the correlation matrix?
5.	Line 173~179, how do you prove the 41 features are redundant, it is not clear reported.
6.	Line 181, can you introduce GLM in detail ?
7.	Line 193, you used SVM as classifier, but you should introduce the classifier more clearly. In addition, you should introduce how did you tune the parameter for the classifiers.
8.	Why no-change were underestimated, and grass-crop change are signiicantly over estimated?
9.	Line 245~246, what’s the sentence mean?
10.	It seems the UA of ‘change land’ is too low.
11.	Figure 5, you just showed four result maps, you should show the 2013 and 2016 image here.


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

Input:
Title: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: The study tested many spectral and spatial variables for change detection. The overall quality of the manuscript is good. There is still room for improvement on how to deal with unbalanced dataset. It is important to use unbiased estimators for accuracy assessment and calculate standard deviation and 95% confidence interval. Blue lines and confidence intervals in Figure 4 do not make sense. Instead, the confidence interval should look like a boxplot with the unbiased estimation in the middle of it, for each selection of variable number. Please re-do accuracy assessment and provide at least one confusion matrix in the response for checking.

Please present at least one resulted map for the whole study area so people can compare it with the reference to see if the distribution of changed areas is similar. This is important for a very unbalanced classification problem.

For the simplest model with only three variables, it should be very helpful to make a 3D scatter plot to demonstrate the separability between the change and non-change classes.

Line 91: texture metrics should be spatial variables.
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: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Title: I suggest to remove the first part of the title (before the colon) which is redundant.

There is some confusion in terminology such as Land Use VS Land Cover (e.g., line 42, grassland and cropland are land cover types)  indicating that the authors may not have a complete mastery of the concepts they use. Another example is the reference to Blaschke, 2010 to support the claim that ‘a per-pixel classification … is … a more suitable approach for the high resolution data than the object classification’ (line 288) while you can read in the paper of Blaschke (actually in favor of object-based approach): ‘high resolution: pixels are significantly smaller than object, regionalisation of pixels into groups of pixels and finally objects is needed.’ (see legend Fig. 1).

Figure 2:
Define ‘second moment’. NDVI and second-moment plots are unreadable because of poor contrast and the overlapping polygons. I suggest adding a plot with LPIS polygons at the left side of the figure and removing overlapping polygons to improve readability. You may also want to add a color legend on NDVI and second-moment plots.

English:
I am not a native speaker. Therefore, I understand that is not necessarily easy to write in perfect English. However, I think that the English language of this paper can be largely improved to ensure a smooth reading of the text. Furthermore, in several places in the text, the quality of the language prevents the reader from correctly understanding what the authors mean. I suggest that you request a native English speaking colleagues to review the manuscript.
Examples:
Line 48: ‘helps with the accumulation of greenhouse gases’
Line 75: ‘many papers reviewing’ -> ‘many papers have reviewed’  (try to avoid putting the verb at the end of the sentence).
Line 94: ‘variables of’, end of the sentence is missing.

Structure:
Some tables with results are presented in ‘Materials and Methods’ section in place of ‘Results’ section. Examples: Table A, Table 3.
Line 296: I suggest moving this paragraph to the introduction.
Experimental design: Major issues:
Line 159: This section is essential for the understanding of the paper but poorly described. Please detail the variables used, Table 1 is not sufficient. As an example, what does ‘simple ratio’ means? It could be the ratio of any band… Furthermore, the authors should explain the rationale behind the choice of these variables/indices. Some of them are not obvious regarding cropland and grassland discrimination (e.g. Normalized Difference Built Up Index) or Landsat images (e.g. WorldView Improved Vegetative Index). As it is, it looks like the authors have tested a lot of random indices without prior consideration on the relevance for their objective. This section has to be seriously improved before publication.

Minor issues:
Line 107: Please explain the rationale behind the assumptions described.
Line 124: According to USDA (https://ipad.fas.usda.gov/rssiws/al/crop_calendar/europe.aspx), in August, half of the main crops in the Czeh Republic are still not harvested. Please discuss.
Line 144: FYI, surface reflectance products exist for Landsat 8 and are available at https://lta.cr.usgs.gov/L8Level2SR
Line 155: Please discuss this limitation.
Line 166: Not clear. I guess you mean ‘for each field’.
Line 183: Not clear how you have selected the number of variables.
Line 193: Please provide the parameters of the SVM (kernel…).
Line 195: Why did you use the central pixel of the LPIS unit? I do not understand. Here, you have two options either working by object (pre- or post-classification) or by pixel and then compare all classified pixels with the reference (similarly, the discussion line 292 should be revised).
Line 198: Figure 3 should appear earlier (at the beginning of the Method section).

Discussion: discussion about the added value of temporal series and higher resolution such as Sentinel 2 data (and Radar data) would be interesting.
Validity of the findings: Major issue:
Results and Discussion about the confusion matrix miss the most important point. Change user’s accuracy is, for the best model, around 7% which is not mentioned/discussed in the text. Looking at the confusion matrix of the ‘best model’, 4735 units are classified as change while only 458 are real change + out of these 458 only 350 are actually correctly classified as change and included in the 4735 units. For change detection (the purpose of the study as stated in the title), the proposed method performs poorly (which is not surprising using only one date of data for each year). And you can see it clearly in Figure 4 by comparing the ‘best model’ with the validation data. This point is very important and completely misunderstood in the discussion line 259.
Additional comments: To assess the validity of the findings the method should be more explicit and better described. It seems that the authors have used some kind of feature selection based on logit regression  (using the 658 change + 33191 no-change units ? Object-based (zonal stats)?) and then a SVM classification (using 200 change + 200 no-change units, pixel-based) but it is not easy to understand.
Second, the authors have to put their results into the perspective of change detection as explained above. The author may refer to the work of Olofsson et al. (2014) for the best practices for assessing the accuracy of land change.

Reference:
Olofsson, Pontus, et al. "Good practices for estimating area and assessing accuracy of land change." Remote Sensing of Environment 148 (2014): 42-57.


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: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: It seems that the paper has improved significantly.
Experimental design: The experimental design of the revised version is fine.
Validity of the findings: The results support the findings of the paper.
Additional comments: The authors used images in August in 2013 and 2016 for change detection and also claimed that "appropriate time of the acquisition of satellite images is important for grassland to cropland change detection". But, in other study regions, the growing season of grass and crops may vary, the selected time phases for grassland-to-cropland change detection will be different from this paper. I suggest the authors address this issue.


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: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Issues have been addressed and I have no further 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: SELECTING APPROPRIATE VARIABLES FOR DETECTING GRASSLAND TO CROPLAND CHANGES USING HIGH RESOLUTION SATELLITE DATA
Review round: 2
Reviewer: 3

Output:
Basic reporting: While efforts have been made to improve the paper compared to the first version, it is still poorly written and sometimes hard to understand. Several statements are made without being supported by any scientific evidence or reference.

In particular, the authors failed to address one of the major issue raised in my original review regarding the section "2.4. Selection and calculation of the variables" (line 179). The only explanation found in the text to support the choice of the considered variables is that "these [...] variables represent, in our opinion, all potentially used spectral and spatial indicators for change detection in the ENVI software by a common user" (line 182). This a poor justification to support what is supposed to be the main focus of the paper (see the title: "Selecting appropriate variables..."). Furthermore, the authors overstate several times what has been done by claiming to "comprehensively address the effect of spectral and textural variable" (line 114) and to "provide a complex analysis of the utilization of remote sensing variables" (line 353). Both statements are false. The theoretical background that led to the choice of the feature selection method (AIC comparison of logistic regression) is also omitted.

Regarding the results, if I understood well (not sufficiently clear in the paper), it seems that using the Landsat bands alone give the best results for the SVM classification ("to achieve the highest accuracy, it is necessary to use original Landsat 8 bands" (line 358 + Table 4, Figure 5)). If correct, this has major implications for this study (so far as to question its purpose) which are completely omitted by the authors.

In the conclusions, the authors claim that "the results confirm the principal hypotheses that [...] the appropriate time of the acquisition satellite images is important for grassland to cropland change detection". While it is, indeed, a significant limitation of this work, I was not able to found where it was discussed in the paper.

Minor issue:
line 212: What are the implications of using > 50% of plots w/ change and ~3% of plots w/o change for training data?
line 269: What are “green” crops?
Figure 3: convolution matrix should be confusion matrix
Experimental design: -
Validity of the findings: -
Additional comments: No additional comments


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

Input:
Title: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is clear in general, although some basic English errors were found:
- ...the next “none” (line 484) should be the next one.
- ...Genre (line 296) should be gender.
- TAM should be spelled out in full upon its first use (line 270)

In terms of literature references, herewith some issues and suggestions:
1. The reasons of increasing dementia phenomena should be mentioned, at least with a reference (line 35). Is it related to the advancement of detection technology?
2. “Organization” is not the last name of “World Health Organization” (line 39).
3. Relevant types of patient functional status (line 42) should be spelled out, e.g. physical, psychological, social and role.
4. The notion of “state-of-the-art” should be removed (line 18, 42, ), since the level of novelty in machine learning classification techniques (line 18) and diagnostic procedure (line 42) is relative and maybe subjective. In fact, the references used (Lezak, 2004; Howieson & Lezak, 2010) are more than five years old.
5. The notion of “novel” should be discarded (line 60) since novelty in video game will normally run out in a year.
6. The claim of being “fully” accurate is subject to criticism because the alpha set for statistical test was not absolute zero.
7. Limitations encountered during the preliminary study were not discussed between line 62 and 84. The discussion of the preliminary study was about methodological review, as opposed to specific limitations.
8. The notion  of “recently” should be discarded (line 87) because the references used were over five years old.
9. Do “senior adults” (line 91, 96 & 453), “seniors” (line 168), “senior people” (line 180) share the same meaning as “older adults”? If so, it would avoid confusion to use either one of these phrases, else define each of them accordingly in Sect. 2.1.1 Sample description.
10. Is the Spanish version of CVLT (line 139) same as the Spanish version of CVT (line 137)?

As for the presentation of figs, the screens on tablet shown in C and D of Fig 2 are hardly visible for understanding the game features of semantic and procedural memory.
Experimental design: The psychometric criterion predictive validity should be the key focus of this study. However, the paper included results of a TAM questionnaire survey that collect participants' perceptions. These are indeed two non-complementary studies--one measured the predictive validity of Episodix, while another compared perceptions of three types of senior adults--with AD, with MCI, and HC. The former examined Episodix as a tool, while the latter used Episodix to examine perceptions of senior adults.

Only one research question (Is the Episodix game a valid tool─ with a statistical significance for older adults─ to discriminate between MCI, AD and healthy individuals?) was found in the paper (line 79-81), while the issues raised in the article were also dealing with usability of Episodix, which deserves a standalone research question.

In terms of participant recruitment, the usual onset of AD is 65 years old (Mendez, 2012), but the “older adults” involved in this study were over 55 years old (line 105-106), who were 10 years younger than the usual onset.

A definition of “older adults” as target users should be included with due justification in relation to the age of usual onset (line 110).

Technophobia was used as an exclusion criterion in the study without description on how the phobia was measured (line 111). Without the use of rigorous measuring tool (e.g. Hogen, 2009), excluding potential participants could be unethical.

A mixture of “subjects” (used 20 times) and “participants” (used 34 times) was found in this paper. This implies mixed methods research design but without any elaboration.

The count of “state-of-the-art” machine learning classification techniques (line 18) and the count of relevant metrics (line 19) should be stated.

.
Validity of the findings: The “best” classification result (line 22 ) should be limited to one as opposed to two (Extra Trees Classifier & Gradient boosting algorithm).

The types of limitations detected during the preliminary study should be mentioned in the Abstract (line 24).

The type of “additional research” (line 26) or “more research” (line 31 & 74) should be spelled out.
Additional comments: A study of psychometric criterion predictive validity of Episodix should not include a general usability study of Episodix. A comprehensive usability study of a serious games should cover at least the following five constructs: likeability, efficiency, control, learnability and helpfulness. Although TAM model is acceptable for examining the general use of serious games, rigorous usability instruments should be used to examine specific use of serious games.


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: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 1
Reviewer: 2

Output:
Basic reporting: Very interesting paper from the technological point of view and with therapeutic relevance
Experimental design: Appropriate methodology
Validity of the findings: Appropriate
Additional comments: Using games as an iinterview that mau also improve treatment for Alzheimer's disease


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

Input:
Title: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 1
Reviewer: 3

Output:
Basic reporting: The paper reports on an empirical study around the validity of Episodix, a serious game, as a potential tool for discriminating among seniors who are health or suffer from Alzheimers or Dementia.

A major issue with the paper is that it never makes it quite clear what the difference is between the previous PeerJ paper and this one; as it is the game itself is not described in this paper (I assume it is described in the last paper) and this makes this paper a bit poorer.
Experimental design: 1) Did each session with a senior happen in one single day? How long did the session last?

2) "Missing data was addressed by mean substitution by cognitive group (i.e., AD, MCI or HC)." What does this mean? What data was missing? Why?

3) What are the threats to validity?
Validity of the findings: The Results section repeats the numbers in the Tables - there is no need for that.

The Discussion section also starts by repeating some text from previous sections (on the sampling for example) and it does not go deep enough in the interpretation of the results and their implications.
How do the authors propose that the games be used?
What do the health professionals think?
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: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 1
Reviewer: 4

Output:
Basic reporting: The basic reporting components adhered to the PeerJ standard.
Experimental design: The main contribution of the article is on the psychometric and usability study of Episodix.

The psychometric study involved prediction using machine learning using Episodix labeled data. This was aimed to measure psychometric prediction validity. The author, however, did not expand on what does psychometric prediction validity really means.

Machine learning (ML) algorithms are predictive tools and they are black-box. They work based on the concept of supervised learning where data inputs were already labeled into classes to be predicted e.g MCI, HC, and AD. The algorithms then classify and return the performance metrics (termed as classification validity by author) such as accuracy, recall etc.

The concern was on the relatedness of psychometric prediction validity and classification validity. The author should explain how are they related or do they actually mean the same.

From the results reporting, it seems like the author used binary classification which distinctly separates between MCI, HC, and AD. Can overlapped cognitive condition happen? If yes, have author thought about using multi-class classification to do psychometric prediction validity?

The author later performed Feature Selection using chi-square and ANOVA-F which was a good step. Rather than simply concluding on the 10 most informative features, the author should re-run ML with those features and measure the accuracy; and further discuss how by reducing the number of features affect psychometric prediction validity? It could either increase or decrease the accuracy.

On the usability studies, TAM is an acceptable model to be used in the context since users are mostly registered patients. However, there are other models that could be mentioned and maybe more suitable that the author could review a bit. This is just an optional suggestion.

Looking at the questionnaire questions, I could acknowledge only one question as ATU. The question "I liked this game very much" doesn't fit as ATU. Has the author referred to any sample questionnaire using TAM before coming up with these questions?

The TAM questions were limited. A few more questions on ATU especially will give you a better indicator of the user willingness to use the game.
Validity of the findings: In the discussion section,  the author mentioned that "participating senior adults felt comfortable during the evaluation sessions embedded in their daily routines", a strong conclusion without reference to any of the experiment instruments. I thought at first, this was concluded based on the TAM, specifically ATU's, questions but there were no questions on this.

The findings on ML are valid if they are based on binary classification.
However, it is still not clear what was the author intention on the psychometric prediction validity.

The author mentioned that EPISODIX is able to predict the MCI etc. But this really depends on the training set. ML in Episodix has to be trained first with sufficient data before prediction could be done. And as more users are added, the training set should be updated. Have the author thought about this? Yes, based on the EPISODIX data gathered and labeled, the prediction can be made quite accurately. How would EPISODIX work as a diagnostic tool when it relies on training data? Just some thought that came across my mind on this.

Overall, the findings are there but the storyline gets a bit distorted at the ML usage. There was no clear aim on what the psychometric prediction validity.

The conclusion has also not revisited the main contributions.
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: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 2
Reviewer: 1

Output:
Basic reporting: All issues and suggestion highlighted in the previous review were addressed when revising the paper.
Experimental design: All issues related to the experimental design were addressed when revising the paper.
Validity of the findings: All issues related to the validity of the findings were addressed when revising the paper.
Additional comments: All issues and suggestion highlighted in the previous review were addressed when revising 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: EPISODIX: A SERIOUS GAME TO DETECT COGNITIVE IMPAIRMENT IN SENIOR ADULTS. A PSYCHOMETRIC STUDY
Review round: 2
Reviewer: 2

Output:
Basic reporting: Revised accordingly. The revised version is now recommended for publication.
Experimental design: Revised accordingly. The revised version is now recommended for publication.
Validity of the findings: Revised accordingly. The revised version is now recommended for publication.
Additional comments: Thank you for thorough explanation in the rebuttal.


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: EARLY HOLOCENE MORPHOLOGICAL VARIATION IN HUNTER-GATHERER HANDS AND FEET
Review round: 1
Reviewer: 1

Output:
Basic reporting: Data Check:  PeerJ requires that the raw data is available.  The current version of the supplementrary tables contains summary statistics and a variety of details for other statistical analyses but not the raw measurements and indices.  Please inclue a data file containing them.

Everything else looks good here.
Experimental design: Please see my comments to the authors.  The key points relevant to experimental design are:

The most important criticism I can offer is that the measurements taken and indices calculated should be clearly described (and probably illustrated to make them as clear as possible) within the text.  It was unclear what, exactly they were.

Second, please describe (perhaps following line 305 in the current text) which variables were used to characterize “shock absorbing” bones and which were used for “weight bearing” bones and provide a brief justification.
Validity of the findings: Please see my comments to the authors
Additional comments: This manuscript presents an interesting and innovative analysis of the dimensions and proportions of hand and foot bones of male and female hunter-gatherers from the early through mid-Holocene site of Windover in Florida. The authors present a very nice summary of the site’s cultural context and relevant data from studies of modern hunter gatherers on variation in patterns of sexual division of labor.  The authors report a larger than expected range of variation in females and higher averages for some indices in females than males, contrary to expectations.  Little sign of lateralization is apparent in the Windover hands or feet.  They interpret these findings as evidence of a heavier activity load in females.  Overall, this manuscript is a useful and stimulating contribution.  A few details should be clarified before it is accepted.

Data Check:  PeerJ requires that the raw data is available.  The current version of the supplementrary tables contains summary statistics and a variety of details for other statistical analyses but not the raw measurements and indices.  Please inclue a data file containing them.

The most important criticism I can offer is that the measurements taken and indices calculated should be clearly described (and probably illustrated to make them as clear as possible) within the text.  It was unclear what, exactly they were.

Second, please describe (perhaps following line 305 in the current text) which variables were used to characterize “shock absorbing” bones and which were used for “weight bearing” bones and provide a brief justification.

Around lines 374-378 and line 408, more caution on the causes of DJD is warranted.  It has a complex etiology, sometimes traceable to differences in activity, but sometimes not.  Please see Jurmain et al. (2012 – see below) for an overview.

In the paragraph under the header “Carpal Variation” (line 413 and following), please be more clear if absolute size or the values for an index are being discussed.  The text usually implies absolute sizes are under discussion and this may be misleading.

More minor critiques:

Around the current lines 298-302, please note what software package(s) were used to analyze the data.

Line 435 – word choice: perhaps “intensive foraging” rather than “intense foraging”?

Reference cited:

Jurmain R, Alves Cardoso F, Henderson C, Villotte S. 2012. Bioarchaeology’s holy grail: the reconstruction of activity, in A.L. Grauer (ed.) A Companion to Paleopathology: 531-552. Crichester, West Sussex: Wiley-Blackwell


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: EARLY HOLOCENE MORPHOLOGICAL VARIATION IN HUNTER-GATHERER HANDS AND FEET
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing could be more clear and unambiguous. For example, it is not always clear if all carpals or tarsals are being used or only the most diagnostic elements. The literature cited is relevant, however several of the references are not particularly specific to the archaeological context under discussion, or are a little dated. The figures are difficult to interpret and need more detail on what data is being presented.
Experimental design: No information is provided regarding ethical protocols, therefore it is not clear if ethical standards were followed. The methods could be more clear to allow for replication. Additionally, the authors could better clarify why this is the best possible method for this particular investigation.
Validity of the findings: Some of the statistical conclusions are difficult to interpret and could be presented with greater clarity. Speculation is evident in the conclusions but could be more clearly identified as such. As this method is not widely used, and the context is not ideal for inferring past lifeways and how they affect morphology, the authors could be more cautious with their interpretations.
Additional comments: In line 28, the bracket following tarsal should be closed.

In line 29, what is the nature of the differences that suggest Windover occupants engaged in heavy impact mobility.

In several cases, such as line 45, citations or comments that belong within one set of brackets are shared between multiple sequential bracketed texts.

Ethnographic research is referenced several times in this study broadly, are there any ethnographic or historic records specifically pertinent to this group from this region? If there are, please provide further details, and if not, that could be clearly stated.

The term listed on lines 47-48 is conventionally ‘musculoskeletal markers’ or ‘musculoskeletal stress markers.’

Throughout the text and starting on line 71, the author refers to the tarsals collectively and does not always clarify which tarsals, in particular, are under discussion. As they outline that different tarsals have particular functional contributions, why refer to them collectively and not consistently identify the tarsals under investigation or discussion?

In line 87, the foot may be differentially shaped by daily logistical mobility?

The authors reference Hiatt (1978) in line 100, are there more recent studies that look into these proportions? Additionally, is there any research specific to the region under investigation in this present study?

In lines 132 and 149, are the authors referring to all tarsals and carpals?

In lines 134, the authors note that forensic studies and some bioarchaeological research have investigated size variation in carpals and tarsals? Are there particular studies that have looked at variation between groups known to have different activity patterns? Is there any clinical research on this topic? If so, it should be mentioned specifically. If not, and this study is the first to undertake this approach, this should be explicitly addressed. Additionally, it should be acknowledged that it is being tested on a group without known activities, which may present some interpretive issues.

In line 140, the authors note that these bones are heavily implicated in daily activities. Are there other studies demonstrating that this is the case among prehistoric groups? Is there direct evidence suggesting they may be more diagnostic of activity than long bones, which are frequently used in investigations of past activity patterns.

In line 144, there are more recent reviews and critiques of long bone variation than Pearson (2000). The authors could include additional references on this topic if they are aiming to demonstrate that carpals and tarsals are better proxies than other conventionally used bones.

In line 147, carpals and tarsals may be more frequently recovered in poorly preserved skeletons. The authors could note that such research could be more broadly applied after further testing in groups with either known activities or strong archaeological or ethnographic evidence of likely daily actions.

In line 171, a definition of what a mortuary pond is and what it conventionally contains would be helpful. This is not clearly stated in the methods section. Also, if the materials are complete, why not cross-reference across the skeleton using different methods?

In line 210, only male graves contain atlatl?

On lines 231-232, what are the different methods used by Smith and Wentz to look at DJD? Additionally, were there no limitations to these studies? Were the sample sizes large? Were only adults analysed? Also, the bones analysed are not outlined, except when results are mentioned. Were whole skeletons analysed? Or only axial elements and long bones?

In line 282, was skeletal sex assessment conducted as part of this study? If so, what methods were used? If not, are sex assessments from previously published reports? If measurements were taken on both sides to allow for directional asymmetry measurements, was one side biased in the comparisons between units? Also, why was Grubb’s statistic used in particular?

In line 289, in addition to sexual dimorphism and trait size variation, could additional factors like physiological stresses or diet be impacting size variation in this group?

Throughout the text, for example on line 325, the authors could reiterate the bones in question when referring to shock-absorbing bones.

In line 339, what do the authors mean by potentially significant differences? What are the criteria used in this paper for significance testing? If a particular criteria (i.e. a=0.05) was used to determine significant differences, please indicate, and be consistent when describing when results are significant or not.

In line 400, is it possible that the greater range of variation in females may relate to the larger sample size? Also, future avenues of research might involve looking at these variables in known populations or further investigate reaction norms in carpals and tarsals in response to activity?

In line 417, increased DJD in what elements?

In line 451, variation may relate to female tasks, but it is worth reiterating that this is may only be the case once factors related to size are taken into account.

In line 462, is the wrist particularly implicated in spear throwing? Studies that have looked at living subjects engaging in spear throwing seem to predominantly implicate the shoulder. Why are carpals a better proxy for such activity than, for example, the shoulder girdle? Also, handedness has not been previously observed in the carpals, a) why was it expected in this study, and b) is it possible that insufficient force is felt at the carpals in particular to reflect handedness? This could be explored in more detail.

In line 468, it would be helpful if the aims of the study were restated before being summarized.

In line 473, if females experienced worse health, would this not potentially indicate some degree of inequality between males and females?

In line 502, what ethical approval process was used to undertake this study? Are there any institutions to be acknowledged?

In the figures in the tables, listing the carpals and tarsals involved in the calculations would be extremely helpful to the reader. For figure 1, are the labels of left and right correct? Also, is the Y-axis the mean of log-transformed ratio data? What is the measure or what are the units involved with this data? These graphs are not informative without more details on what the numbers used actually represent. In Figure 2, why is there a line connecting males and females? This indicates some direct or transformative relationship. Also, if the axes represent a similar measure, the axes should be numerically aligned. In Figure 3, the icons used to identify the different carpals used (also, if these are the only carpals used this should be clarified earlier in the study) are extremely similar and difficult to distinguish. Other images like a circle or a square would be easier to identify.

If Windover is a mortuary pond, and as described in the conclusion, does not have an associated archaeological site, are the skeletons recovered from the pond best described as occupants? Additionally, what greater contextual information from other sites in this region have informed hypotheses and lifestyle inferences? These details could be clarified in greater detail.


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: EARLY HOLOCENE MORPHOLOGICAL VARIATION IN HUNTER-GATHERER HANDS AND FEET
Review round: 1
Reviewer: 3

Output:
Basic reporting: The main goal of the presented articles to differentiating the mobility and subsistence patterns between males and females in the prehistoric hunter-gatherers from North America. In general, because of abundance of local resources studied population was limited to explore large geographic area. The grave goods demonstrate tool choice based on the sex. Both women and man participated in the domestic economy. DJD studies of Windover population clearly demonstrate separation of labor or different activity patterns between males and females. Difference between left and right DJD frequency also was demonstrated in the previous studies. This study greatly benefits if authors could directly compare DJD studies results.
I cannot judge English unfortunately
I recommend remove or shorten introduction before “Aim” and move up in introduction material section and bland into Windover population mortality, mobility, Subsistence economies… with evidence of another hunter-gatherer population. It allows authors to demonstrate Windover population difference from other hunter-gatherer groups and to define unique aspect why this population will be important to study labor and activity difference between male and female. Authors are providing the research questions, but they do not provide the predictions on the base of subsistence strategy of this population and why these predictions are made and what are their arguments.
Tables and figures are fine. Authors only share ratio and not raw data.
Authors choose carpal and tarsal bone for following reason (1) because both tarsals and carpals heavily implicated to the activities; (2) they are less influenced by genetic factors (3) carpals and tarsal are well preserved in archeological samples. All these arguments can be good for any upper and lower limb bone as well. It is not clearly justified why carpals and tarsals are not influenced by genetic factors, also in this particular population it not necessary to study carpal and tarsal in order do not lose the information about mobility patters when they have the most complete skeletons of hunter-gatherers.
Even DJD demonstrate site specific difference between male and females, it does not exclude that both males and females are caring heavy weight and are mobile in the defined geological area. Present work greatly benefits if authors can indicate how they can distinguish on the tarsal bones L versus W mobility related difference if any. How tarsal measurements are correlated to AP versus ML diameters of long bone mid-shaft?
Experimental design: Authors would like to see on bone length and breadth ratio how it is differentiating labor difference as well as heavy load carrying and high mobility or running. They are using log transform length-to-width ratio for each tarsal and carpal bones. When ratio is used most of time it is difficult to distinguish which of two variable affects to the value of ratio. I strongly recommend using bi-variate plot for L versus W measurements to explore real difference between male and female as well as right and left. I also do not convince that variable that are chosen are correct to differentiate load bearing sites. I recommend using each articular surface diameter to calculate surface area for each bone and scale of same bone product L and W or use body weight and compare them between sex and left and right. This will show if compression forces acting on the carpal bone during different manipulation of the hand.
For tarsal, it is unclear how authors can demonstrate shock-absorption using L/W measurement of bone. Probably subchondral porosity or microdamage will be best indicator is one would like to distinguish between shock-absorption difference in males and females. Please describe how one can distinguish on the one bone (not complex structure) weight bearing versus shock-absorption? And, please provide literature supporting this.
Validity of the findings: Previous DJD study of same population demonstrate sex difference of affected site of skeleton on the hand bones. This strongly demonstrate division of labor as well as difference of heavy load caring difference between different sex. I expected if measurements for this study are chosen correctly the result needed to be paired previous results of DJD of same population.
https://academic.oup.com/rheumatology/article/44/4/521/1774828
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266544/

After plotting Log transformed mean ration and SD for each bone (See file attached). Means are almost identical. SD in female is larger when male, but patters for each bone is similar. Presented result do not support any activity or mobility difference, simply because they are sampling general bone size. Here it will be interesting to see body weight distribution of this population in the male and female pool.
Additional comments: line 28 (4 carpal and 6 tarsal. Change (4 carpal and 6 tarsal).
Line 31 “shock-absorbing bones” I do not understand what does it means and how one can depict shock-absorbing on bone using size of bones? If they can describe cartilage degeneration and subchondral porosity or microdamage, but presented data do not support this. In addition, the maximum absorbed energy of subchondral bone is approximately 4 times lower when cartilage (https://www.sciencedirect.com/science/article/pii/S1751616113001628?via%3Dihub#f0020)
Line 45 (Frayer & Wolpoff 1985) (Frayer 1980). Change to (Frayer & Wolpoff 1985; Frayer 1980).
Line 46 ascribed to each of the sexes change to ascribed status to each of the sexes
Line 208 after (Hiatt 1978) put space and eliminate one dash line
And many others


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: EARLY HOLOCENE MORPHOLOGICAL VARIATION IN HUNTER-GATHERER HANDS AND FEET
Review round: 2
Reviewer: 1

Output:
Basic reporting: The paper meets PeerJ's criteria for Basic Reporting: the language used is clear and unambiguous; sufficient references and background are provided; the article has a clear, professional structure, and the raw data is provided; the relevant results are provided for the hypotheses.
Experimental design: All of this is OK.
Validity of the findings: All of this looks OK.
Additional comments: This review concerns the revised version of "Early Holocene morphological variation in hunter-gatherer hands and feet" (#26634) by K. C. Hoover and J. C. Berbesque.  The paper presents an interesting analysis of whether lateralization in hand and foot bones reflects work loads at the early Holocene site of Windover in Florida.  The authors have done a good job of addressing the criticisms I had of the first version of the paper.  I urge the Editor of PeerJ to ACCEPT the revised version or to ACCEPT it after MINOR REVISIONS.

The final revision I can suggest concerns the text on lines 524-526 of the revised manuscript in which the authors summarize the effects of barefoot walking/running during development on the shape of the foot and its arches (longer & narrower feet with lower arches as a result of barefoot running).  Please be aware that Daniel Lieberman has argued (with supporting data) precisely the opposite - that barefoot walking & running leads to wider feet (although perhaps much of this is ligament rather than bone) and higher arches.  (Lieberman, D. 2013. The Story of the Human Body: Evolution, Health, and Disease. New York: Vintage.).  As a result, please double check these facts (or simply delete them since they are somewhat tangential to the data in hand).


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

Input:
Title: MOLECULAR IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPES H5N1 AND H9N2 IN BIRDS FROM FARMS AND LIVE BIRD MARKETS AND IN RESPIRATORY PATIENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: English of the manuscript should be improved throughly.
Experimental design: The research is not met a high technical standard. Beside the cleavage sites, more molecular information of these viruses, such as genome sequences of eight segments, and their phylogenetic analysis should be provided to reveal the relationship among the strains.
Validity of the findings: The conclusions of the manuscript were not supported very solidly by the results.
Additional comments: The manuscript reported the molecular identification of AIVs in poultry from farms, LBMs and patients in Egypt. In general, little new knowledge has been gotten through the manuscript. Maybe it is appropriate to publish it in a local journal rather than in an international journal.

Special comments:
1.English of the manuscript should be improved throughly.
PCR -> RT-PCR
Line 44: identity -> identities
Line 57: types -> subtypes
Line 65: fresh -> freshly
Line 67-68;
LIne 74-75;
Line 80: Although -> Despite
Line 92: since 2003 till 2017
........
........
........

2.The positive rates of H5N1 and H9N2 in poultry from farms were 13.6% and 3.4%, respectively. Is there any relationship between positive viral results and the clinical and postmortem examination results of the birds? In other words, are pathologies of the birds which were showed in Fig. 1 caused by AIV infection?

3.Line 216-217: The birds from farms were admitted to clinic due to their diseases, and the birds in LBMs should be healthy. So, it might be meaningless to compare their positive rates of AIVs.

4.Line 220-227 and Fig 3: There were two kind cleavage sites in nine H5N1 strains. The virus origins (farms or LBMs) should be showed.

5.Phylogenetic analysis: Beside the cleavage sites, more molecular information of these viruses, such as genome sequences of eight segments, and their analysis should be provided.

6.Human patients: The viral genetic information of the H5N1 strains from patients should be obtained to establish the linkage between the virus strains from human and poultry.


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

Input:
Title: MOLECULAR IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPES H5N1 AND H9N2 IN BIRDS FROM FARMS AND LIVE BIRD MARKETS AND IN RESPIRATORY PATIENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The text is clear and well structured. English could be improved (i.e. line 74, line 90).
In Line 96 authors should cite the H9N2 subtype cases detected in Morocco and Burkina Faso adding the appropriate references.
Experimental design: The manuscript is not original but it is interesting because it describes the co-circulation of H5N1 and H9N2 subtype viruses in poultry in Egypt, in addition it reports the data collected from patients of a hospital.
Methods described are clear and detailed.
The phylogenetic analysis would have been more accurate if the authors had considered a longer fragment (amplicons of 311 bp are quite short) but it can be considered acceptable because the characterization of the viruses is not the first aim of the work.
Validity of the findings: The manuscript describes the situation in Egypt in 2015-2016, it refers to two-year-old data so it does not have a big impact but it can be useful for the comparison with more recent sequence data and for the understanding of the evolution of the avian influenza viruses in Egypt and in the African continent in general.
Results are discussed properly.
The authors describe accurately the findings in relation to the different species cosidered and the different environments analyzed (farm, LBM and hospital).
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: MOLECULAR IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPES H5N1 AND H9N2 IN BIRDS FROM FARMS AND LIVE BIRD MARKETS AND IN RESPIRATORY PATIENTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript was clearly reported, with sufficient field background and professional article structure. In addition, the manuscript is self-contained with relevant results.
Experimental design: The study aimed to investigate the dynamic evolution of H5N1 and H9N2 avian influenza viruses in Egypt. The experiments were well designed in this study. The study was performed with high technical & ethical standard, and the methods were described with sufficient detail.
Validity of the findings: The authors in this study determined the epidemic situation of H5N1 and H9N2 influenza viruses. Although the evolution of H5N1 and H9N2 influenza viruses has been reported by different researchers, this manuscript demonstrated that the circulation of H5N1 and H9N2 viruses are still undergoing in Egypt. Therefore, this study suggests that intensive surveillance of avian influenza viruses should still be strengthened in Egypt. The data is convincing, and the conclusion is well stated.
Additional comments: 1. line 83, "genotypes", should it be "clades"?
2. lines 82-87, the authors mentioned that two clades of H5N1 viruses were present in Egypt. However, they mentioned three clades, 2.2.1.1, 2.2.1/C and 2.2.1.2. What is the relationship among these three clades?
3. line 153, change "primers" into "PCR amplification".
4. line 367, change "finding" into "findings".


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

Input:
Title: MOLECULAR IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPES H5N1 AND H9N2 IN BIRDS FROM FARMS AND LIVE BIRD MARKETS AND IN RESPIRATORY PATIENTS
Review round: 1
Reviewer: 4

Output:
Basic reporting: In this study by Tolba et al., the authors have characterized the avian influenza viruses which are circulating in the commercial farms and the live bird markets during 2015-2016. The samples were taken from ducks, pigeon and quail that were admitted to the Clinic of Avian and Rabbit Medicine Department. The authors did a systematic screening of the samples by Real-time RT-PCR for high pathogenic avian influenza (HPAI) H5N1 and low pathogenic avian influenza (LPAI) H9N2 virus targets. The positive samples were subjected to virus isolation and molecular identification. They have identified the viruses as H5N1 and H9N2 on the basis of HA sequencing and phylogenetic analysis.

The strength of the study is the animal human interface aspect. The authors made an attempt to identify the virus subtypes from the respiratory patients. They have shown that 3.4% of patients were positive for H5N1 virus, but none for H9N2 virus.

In general, this is an important study considering that HPAI H5N1 and LPAI H9N2 viruses are endemic in Egypt. Importantly, HPAI H5N1 virus poses a threat to both animal husbandry and human health and with the circulation of the LPAI H9N2 virus there is a risk of generating recombinant viruses. The study is well planned and the manuscript is well written. However, it is not clear whether the authors made any attempt to isolate viruses from the human samples that are positive for H5N1 virus by Real-time RT-PCR. It is important to sequence and characterize these viruses and determine the phylogenetic relation with the viruses isolated from the birds. In addition, there are few points which need to be addressed and the manuscript needs to be revised on those lines.
Experimental design: Specific comments:
1)	Abstract: Line 26- I suggest inclusion of ‘High-pathogenic avian influenza (HPAI)’ for H5N1 subtype and ‘Low-pathogenic avian influenza (LPAI)’ for H9N2 subtype.
2)	Abstract: Line 45-‘Out of 89% respiratory patients, 3.4% were positive for H5N1’. I wonder what percentages of patients were positive for H9N2. This is missing in the abstract. Only after reading through the whole manuscript we know that there are none. A line will be informative.
3)	Abstract: Lines 48-51- ‘We recommend the ….vaccine studies’. It is well known that application of proper hygienic measures helps in control of the AI virus. I feel that the authors are not adding any new change in the system. Thus ‘we recommend’ can be removed and the statement rephrased as ‘The application of proper hygienic measures in farms and LBMs to control the exposure of birds and humans to the source of infection along with continuous monitoring of the circulating viruses will provide information on understanding the evolution of the virus and selection of viruses for vaccine studies’.
4)	It is not clear if the authors came across any reassortants of H5N1 and H9N2 viruses in the bird samples in Egypt. This may be added in the discussion.
5)	Introduction: Lines 56-57; the author mentions about 16 HA and 9NA subtypes and I am surprised about the mention. Two new HA and NA subtypes have been isolated from bats recently and that has been added to the influenza HA and NA subtypes. (Reference# Proc Natl Acad Sci U S A. 2012 Mar 13;109(11):4269-74. doi: 10.1073/pnas.1116200109. Epub 2012 Feb 27; PLoS Pathog. 2013;9(10):e1003657. doi: 10.1371/journal.ppat.1003657. Epub 2013 Oct 10; Virology. 2015 May;479-480:234-46. doi: 10.1016/j.virol.2015.03.009. Epub 2015 Mar 24.) I suggest rephrasing the sentence with the updated information and references.
6)	Materials and Methods: It is important to know whether the birds (pigeon, quails and ducks) were housed in the same cage or kept independently. This should be mentioned in the respective section as well as in the discussion.
7)	Materials and Methods: Though the authors have quoted references for the primers, it will be useful for the readers to have a list of the primers used as a Supplementary table.
8)	Materials and Methods: Line 156; Does conventional reaction mean conventional RT-PCR? Correct the text. Which kit was used for the conventional RT-PCR?
9)	Results: Line 200- What was the HA titer of the viruses isolated and the HA unit for the 13 pigeons, 14 ducks and 1 quail samples. This could be presented as a Supplementary Table. It is also not clear whether the authors attempted to isolate virus from the human samples which they got positive for H5N1 target?
10)	Results: Line 215- Table-1 only depicts the % positivity however what was the Ct value is not clear. In my opinion showing the profile of Real-time RT-PCR or mentioning the Ct values of the positive samples would be informative.
11)	Results: Lines 230-238; I wonder whether the authors included the strain mentioned in the text in the phylogenetic analysis since the reviewer could not locate the strains (Potsdam/1402; Egypt/1435; Egypt/2; Egypt/Sharkia-22 and Egypt/1171SG) in the tree Fig .3.
12)	Results: Line 240- In this section, as mentioned above, it is not clear whether the human samples were subjected to virus isolation (same point raised above in #9). What was the HA unit? What was the Ct value of the human samples that were positive for H5N1 virus? How can the authors be sure that it was not due to contamination from the handling of the H5N1 positive bird samples? It is important to show the Ct value and the Real-time RT-PCR profile of the human samples.
13)	Results: Considering the important relevance of animal human interface of avian influenza viruses, why were the human isolates not included in the Phylogenetic tree for analysis? It is important to sequence and characterize these viruses and determine the phylogenetic relation with the viruses isolated from the birds.
14)	Discussion: Well written, however too lengthy, can be reduced by a page and some points such as about the reassortants need to be discussed. The authors need to add if they came across any reassortants of H5N1 and H9N2 viruses in the bird samples in Egypt.
15)	Discussion: Lines 308-311; ‘The present study was the only in Egypt…. H5N1 subtype’. It is too much of an extrapolation to comment from the present study. I suggest that the statement be rephrased. Regarding the next line about co-infection I wonder how the authors have confirmed the co-infection of H5N1 and H9N2 viruses. Have they isolated both the viruses from the same bird samples? Or was the appearance of H5N1 a mere speculation which could be due to contamination during RNA isolation or Real-time RT-PCR? The co-infection must be established. The Real-time RT-PCR profile of all the targets H5, N1, H9 and N2 may be shown together and secondly, this can be clarified by isolating both the viruses from the same sample and then determine the HA unit of the viruses.
16)	Figure 3: The vaccinal strains can be color coded for easy identification in the tree. Many strains mentioned in the text are missing from the tree. No human samples have been included in the analysis.

Minor comments:
1)	Line 74: delete ‘is’
2)	Line 78: change losses to ‘loss’
3)	Line 80: delete ‘the’
4)	Line 100: add ‘virus’ after H9N2
5)	Line 119: delete ‘lonza’
6)	Line 125: delete ‘s’ from ‘samples’
7)	Line 134: Add ‘the’ before ‘QIAamp…
8)	Line 135: Change ‘manufacturer’ to ‘manufacturer’s’
9)	Line 137: Add ‘Real-time RT’ before PCR…
10)	Line 138: Add the company name for the Quantitect
11)	Line 146: Change ‘real-time PCR’ to ‘Real-time RT-PCR’. The reviewer suggests that this should be changed at all places in the text.
12)	Line 156: Delete ‘was’
13)	Line 178: change ‘routs’ to ‘route’
14)	Line 211: Add ‘the’ before farm origin
15)	Line 212; Add ‘subtype’ after H9N2
16)	Line 216: Add ‘subtype’ after H5N1
17)	Line 217: Change ‘in’ to ‘birds from’
18)	Line 220: Change ‘belonged’ to ‘belonging’
19)	Line 230: Change ‘96.1-94.1%’ as ‘94.1-96.1%’
20)	Line 269: Add ‘viruses’ after H9N2
21)	Line 284: Change ‘plays’ to ‘play’ and delete ‘a’
22)	Line 290: Add ‘virus’ after H5N1
23)	Line 295: Add ‘virus’ after H5N1
24)	Line 301: Add ‘virus’ after H5N1
25)	Lines 303, 321, 323: Add ‘viruses’ after H9N2
26)	Line 314: Add ‘virus’ after H5N1
27)	Line 390: Change ‘continues’ to ‘continuous’
28)	The title in Table 1, please add ‘H5N1’ and ‘H9N2’subtypes
29)	In Table 2 line 2, add ‘virus’ after H9N2 and ‘Real-time’ before RT-PCR
Validity of the findings: Virus isolation from the human samples.
Confirmation of coinfection of H5N1 and H9Nr viruses in bird samples.
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: MOLECULAR IDENTIFICATION OF AVIAN INFLUENZA VIRUS SUBTYPES H5N1 AND H9N2 IN BIRDS FROM FARMS AND LIVE BIRD MARKETS AND IN RESPIRATORY PATIENTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: Validity of coinfection in birds due to H5N1 and H9N2 viruses not clear.
Additional comments: In this revised manuscript by Tolba et al., the authors have either addressed to or answered the questions raised by the reviewer. There is only one query this reviewer is not clear about.
1)	The authors mentioned in response to query R4.16 that both H5N1 and H9N2 viruses were isolated from the same bird samples. Each PCR was run separately. The H5N1 viruses were isolated, while, H9N2 were not successfully isolated due to low virus quantity (High Ct values), but to confirm that the H9 subtype was present, conventional PCR was carried out to ensure that the product was amplified.

The reviewer has a point that if due to low virus quantity H9N2 virus could not be isolated, how conventional PCR was successful. The reviewer would like to see the amplification results of both H5N1 and H9N2 viruses from the same bird samples.


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

Input:
Title: CHANGES IN METABOLIC PROFILING OF SUGARCANE LEAVES INDUCED BY ENDOPHYTIC DIAZOTROPHIC BACTERIA AND HUMIC ACIDS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: This manuscript presents a study of the metabolic profiling of sugarcane leaves induced by endophytic diazotrophic bacteria and humic acids composting. Although the topic studied in the manuscript is interesting, the paper needs to improve in some aspects.
General Comment:
1. The experimental design of this interesting. However, the manuscript is not concise. Specifically, the Introduction section should be shortened and provide a better justification of why an investigation on metabolic profiling of sugarcane leaves is relevant and necessary. On the other hand, in my opinion, the Discussion section contains much repeated or redundant information.
2. The authors provided a schematic chart (like Fig 3), which is good. However, most discussions are not (sufficiently) conclusive. The authors do have a solid understanding of mechanisms but they need find a better way to present their thoughts.
Specific Comments:
1.	The abstract better to be refined. Please introduce the aim and innovation of this study and bring to the abstract the better as possible of facts/data/description of each research objectives that were accomplished. Avoid being too general.
2.	As the research defects described in this article are not prominent, leading to the innovation is not clear.
3.	Please give a summary after each chapter.
4.	Further refine the important conclusions of this paper.
5.	Please provide details of three colors of cycle in Fig 1. The big or small cycle whether have different meaning in this figure.


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: CHANGES IN METABOLIC PROFILING OF SUGARCANE LEAVES INDUCED BY ENDOPHYTIC DIAZOTROPHIC BACTERIA AND HUMIC ACIDS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The writing is clearly structured, but the text contains a number of sentences with error (e.g. missing words) and terms usually not found in a manuscript. An example would be the sentence 'However, the farm use of beneficial bacteria in non-leguminous is wispy compared with the rumbling success of soybean-rhizobia bioinoculants'. I get the meaning, but the clarity could be improved.

Data (integrals rather than raw data) is shared, but more information would improve it (see point 3).

The representation of the data needs quite a bit of improving in my opinion.

Tables: The changes in the table are expressed as % of control. Log fold change is a better value, as 400% looks more important than 25%, while there are indeed the same magnitude of change.

Figures: Figure 1 is confusing (what does the size of the circles mean, the caption 'Schematic representation of the effect of the treatments on the levels of amino acids in
respect to control' is not informative). Some of the labels are in Spanish. During glycolysis, GA3P is not converted to DHAP in a linear fashin, both can be interconverted by TPI, but it is the GA3P, which is converted to 3-PG (not 6-PG, as stated in the figure).

Similarly, Figure 3 is puzzling. What do the numbers on the legend mean? Why do some compounds have one square, others three and malate has four?
Experimental design: The method section (L163) states that the four treatments were carried out in four replicates. However, I can only see three replicates shown in the raw data. Do these three replicates correspond to three out of the four biological replicates?
Validity of the findings: I have my doubts about the GC-MS assignments, particularly for the carbohydrates. Carbohydrate assignment of GC-MS spectra is known to be tricky and I was particularly surprised to see that sucrose was missing from the list of annotated metabolites.

It would be better if raw data were reported with retention times and Validation and Quantification ions, which would make validating the assignments easier. Also, a representative chromatogramm, zoomed in on the most significant changes would be beneficial.

I am also suprised that the authors report glutamine and glutamate, as both are at least partially converted to pyroglutamate during derivaitisation.
Additional comments: Minor comments:
L116 - 1H NMR, not H1 NMR
L149 - nM, not ɳm


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: CHANGES IN METABOLIC PROFILING OF SUGARCANE LEAVES INDUCED BY ENDOPHYTIC DIAZOTROPHIC BACTERIA AND HUMIC ACIDS
Review round: 2
Reviewer: 1

Output:
Basic reporting: I think the reporting of the raw data still needs improving. While the authors have provided the raw data, no information about retention times for the reported compounds was given, neither about the quantitation and validation ions that are a feature of Fiehn library matching.

Despite the point about figure 1 and 3 being confusing and the authors' statement that those were modified, the figure legends were not modified according to the track changes document.
Experimental design: My point has been addressed.
Validity of the findings: My comments about the partial conversion of glutamine and glutamate to pyroglutamate (or oxo-proline as it also called) is not a knock on the Fiehn library, which a fantastic resource.

It is, however, an acknowledged fact that partial conversion happens in both GC-MS and LC-MS for these compounds (see, for example https://pubs.acs.org/doi/abs/10.1021/ac501451v for LC-MS and https://www.ncbi.nlm.nih.gov/pubmed/17052933 for GC-MS) and should therefore being acknowledged in the discussion

Another point concerns the sugars found in the experiment. While I can follow the major changes, some of the identified compounds seems to have no biological connection to sugar cane, e.g. salicin is found in willow bark and melezitose is a product or aphids and other insects. Are these confirmed components of sugar cane as well?
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: CHANGES IN METABOLIC PROFILING OF SUGARCANE LEAVES INDUCED BY ENDOPHYTIC DIAZOTROPHIC BACTERIA AND HUMIC ACIDS
Review round: 3
Reviewer: 1

Output:
Basic reporting: There is still no RT in the raw data, but otherwise it is all reported. The RT would have been helpful to ascertain if a peak had been assigned multiple compound identities.
Experimental design: fine
Validity of the findings: fine, with caveats outlined above
Additional comments: No additional comments


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

Input:
Title: TRANSLATIONAL EFFECTS OF ROBOT-MEDIATED THERAPY IN SUBACUTE STROKE PATIENTS: AN EXPERIMENTAL EVALUATION OF UPPER LIMB MOTOR RECOVERY
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1.	The article uses clear, unambiguous and technically correct text. It conforms to professional standards of courtesy and expression.
2.	The article includes sufficient introduction and background to demonstrate how the work fits into the broader field of knowledge. I suggest the authors to enrich what they state in lines 51-55 with more references.
3.	The structure of the article conforms to an acceptable format of ‘standard sections’.
4.	Figures are relevant to the content of the article, of sufficient resolution, but I suggest the authors to increase the description of them. Moreover, a figure showing the markers location would help the reader. Otherwise, the authors could show markers locations by figure 2, but comprehensive description is needed.
5.	All appropriate raw data has been made available in accordance with Data Sharing policy.
Experimental design: 1.	The research question is not well defined
2.	Methods describe no sufficient information to be reproducible by another investigator.
•	What kind of pre-processing did the authors use?
•	How onset and offset of movement were defined?
3.	Could the authors explain me how HPR parameter provide information about the compensation strategy?
4.	Why the authors did not consider an accuracy index?
Validity of the findings: 1.	The study could be considered a meaningful replication. The parameters are widely accepted but the authors did not consider some parameters such as accuracy (please, refer to Balasubramanian et al (2012)). There is no novelty in the analysis proposed. I would spent some words about the motion smoothness measure. Normalized jerk is, probably the most common measure of smoothness. However, recent studies have suggested other type of parameters such as SPARC (see Balasubramanian et al. (2015)).

2.	Data are robust, statistically sound, & controlled. However, I believe that showing the arm in the results, without having considered it in the statistical design, is confusing.

a.	Figure 5. What are the error bar?
b.	Table 3. Please explain what symbols mean.
Additional comments: MAJOR
- The submission do not clearly define the research question. It is not clear, if the authors contributes to increase the knowledge gap of robot mediated therapy efficacy or if they propose the kinematic evaluation instead of/in support of clinical scales. Which are the hypothesis of this study?
- Introduction:  I suggest clearly declaring which is the primary outcome of the study and the secondary outcomes. If authors’ intention was to proof the efficacy of RMT, then the study lacks in the design of the experiment because of the absence of a control group undergoing only a conventional rehabilitation therapy. While if the intention was to propose the kinematic evaluation of motor performance, then I would spend more words about the advantages and disadvantages of clinical scales assessing motor performance and describing what is the state of the art about quantitative assessment of motor performance.
- Methods: Are movements comparable between subjects? Did the authors normalize with respect to arm length, time or something else?
- Because of the confusion, defining the original research question, Discussion section is difficult to read.
a.	First, I suggest to briefly describe the scientific questions declared in the introduction.
b.	Line 227. How can the authors assert that the affected arm is comparable with the unaffected arm? Maybe it would be sufficient to point out that this is just a qualitative impression and that obviously a further quantitative analysis is needed.
c.	Line 285. The authors did not measure accuracy.
d.	Lines 287-291. This paper would acquire more value if the authors had used a system more ambulatory affordable. I agree with the authors that clinical scales could lack in objectivity but I believe that the alternative should be low cost, easy-to-use, reliable, etc..

- Please find here some useful references:
o	Reinkensmeyer, D. J. "How to retrain movement after neurologic injury: a computational rationale for incorporating robot (or therapist) assistance." In Engineering in Medicine and Biology Society, 2003. Proceedings of the 25th Annual International Conference of the IEEE, vol. 2, pp. 1479-1482. IEEE, 2003.
o	Squeri, Valentina, Angelo Basteris, and Vittorio Sanguineti. "Adaptive regulation of assistance ‘as needed’ in robot-assisted motor skill learning and neuro-rehabilitation." In Rehabilitation Robotics (ICORR), 2011 IEEE International Conference on, pp. 1-6. IEEE, 2011.
o	Kahn, Leonard E., Peter S. Lum, W. Zev Rymer, and David J. Reinkensmeyer. "Robot-assisted movement training for the stroke-impaired arm: Does it matter what the robot does?." Journal of rehabilitation research and development 43, no. 5 (2006): 619.
o	Colombo, R., I. Sterpi, A. Mazzone, C. Delconte, and F. Pisano. "Robot-aided neurorehabilitation in sub-acute and chronic stroke: Does spontaneous recovery have a limited impact on outcome?." NeuroRehabilitation 33, no. 4 (2013): 621-629.
o	 Balasubramanian, Sivakumar, Roberto Colombo, Irma Sterpi, Vittorio Sanguineti, and Etienne Burdet. "Robotic assessment of upper limb motor function after stroke." American journal of physical medicine & rehabilitation 91, no. 11 (2012): S255-S269.
o	Balasubramanian, Sivakumar, Alejandro Melendez-Calderon, Agnes Roby-Brami, and Etienne Burdet. "On the analysis of movement smoothness." Journal of neuroengineering and rehabilitation 12, no. 1 (2015): 112.


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: TRANSLATIONAL EFFECTS OF ROBOT-MEDIATED THERAPY IN SUBACUTE STROKE PATIENTS: AN EXPERIMENTAL EVALUATION OF UPPER LIMB MOTOR RECOVERY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1) The article is well-written and use technically correct text
2) Please cite also:

Wisneski KJ, Johnson MJ. Quantifying kinematics of purposeful movementsto real, imagined, or absent functional objects: Implications for modelling trajectories for robot-assisted ADL tasks. J Neuroeng Rehabil. 2007;4:7.
Panarese A. et al. Model-based_variables_for_the_kinematic_assessment_of_upper-extremity_impairments_in_post-stroke_patients. Journal of NeuroEngineering and Rehabilitation  (2016) 13:81 DOI 10.1186/s12984-016-0187-9

which is relevant prior literature to be referenced.
3) The figure captions must be rewritten as they not provide enough information. Statistical results must be included in Figure 5.
4) The article is self-contained
Experimental design: The research is within aims and scope of the journal
The research question of this work is not well defined. The study seeks to find evidence that RMT for upper arm rehabilitation after stroke is beneficial to some extent. However, this was assessed in previous studies, with similar design. The authors must reformulate the abstract and introduction with a clear hypothesis within.
The  reseach was conducted rigorously and to a high technical standard.
Methods are described with sufficient detail and information to replicate.
Validity of the findings: Without a clear hypothesis stated it is difficult to say whether the presented results are a 'pointless' repetition of well-known, widely accepted results.

The authors find significant improvement - on average - in the scores of clinical scales, which is to be expected because all patients are in their subacute phase. How can the authors disentangle from such result the effects of RMT?

The idea of a standard procedure based on kinematic analysis of a reaching task to compare the results of different protocols is not new (see Arch Phys Med Rehabil. 2014 Feb;95(2):338-44. doi: 10.1016/j.apmr.2013.10.006. Epub 2013 Oct 23.
Impact of time on quality of motor control of the paretic upper limb after stroke.
van Kordelaar, van Wegen, Kwakkel. Can the authors explain the added value of their research?

Data provided is robust. Conclusions are well stated.
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: TRANSLATIONAL EFFECTS OF ROBOT-MEDIATED THERAPY IN SUBACUTE STROKE PATIENTS: AN EXPERIMENTAL EVALUATION OF UPPER LIMB MOTOR RECOVERY
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: GDF11 INDUCES DIFFERENTIATION AND APOPTOSIS AND INHIBITS MIGRATION OF C17.2 NEURAL STEM CELLS VIA MODULATING MAPK SIGNALING PATHWAY
Review round: 1
Reviewer: 1

Output:
Basic reporting: The overall quality of the text needs improvement, especially concerning English writing and syntax. E.g line172, 411, 414, 418…
The structure of the manuscript is fine and follow the journal requirements.

The raw data for the ERK WB (Figure 4d) is missing.
Experimental design: The experiments fit and follow a logical thought based on the initial hypothesis. It fits within the debate on the potential role of GDF11.
The experimental section is clear enough to allow researchers to reproduce the experiments.
Validity of the findings: The results support a new possible application for GDF11 as therapeutic target which is a new input in the field. It brings some new arguments in the current debate on the role of GDF11 as anti-aging agent.
The data are robust and conclusion drawn according to the findings.
Additional comments: The paper shows the involvement of the GDF11 in neural cell differentiation and apoptosis, the experiments and results support the initial hypothesis raised by the authors. The link with the MAPK pathways have been well highlighted and more generally, this work opens new  questions about contradictory role of this protein on anti-aging.


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: GDF11 INDUCES DIFFERENTIATION AND APOPTOSIS AND INHIBITS MIGRATION OF C17.2 NEURAL STEM CELLS VIA MODULATING MAPK SIGNALING PATHWAY
Review round: 1
Reviewer: 2

Output:
Basic reporting: 	The overall quality of the text is not poor, but there is space for improvement, especially concerning the correct use of the English language. In addition, some vague, not clear wording has been used in some instances (e.g. line 86, 144,152,156-159, 198,230-232,270,316,411,427,489 etc.)-  further proof-reading is suggested.
	The overall structure of the manuscript is generally in accordance with the basic suggested structure, however, some results are presented in the Introduction section. In addition, the background given could be richer.
	Section 2.3 is a bit too dense- it could be broken down into smaller sub-sections.
	The supplementary tables are complete and clear.
	The supplementary figures are generally up to a high-quality standard; however, the Erk1/2 for Fig.4d supplement is missing
Experimental design: 	Overall, the experimental processes used throughout the study are clearly presented in the ‘Materials and Methods’ section and are analytical enough for other researchers to reproduce the work.
	In addition, there is a logical sequence of the experiments, which was followed in order to address the hypothesis and shed light on the controversy over GDF11.
	However, I believe two points should be carefully addressed:
o	The confluence of the cells for the scratch assay may need to be higher, even ~100%
o	In the statistical analyses, it may be a better approach to present cumulative results as mean +/- SEM
Validity of the findings: 	The results of the study are well-presented, organised and seem to be robust, given the statistical analysis presented. This is an in-depth study of the effects of GDF11, which addresses one by one numerous aspects of functional effects and answers a number of different components presented in the hypothesis.
	However, for aesthetic purposes, in the scratch assay results, the use of straight lines to approximately indicate the borders seems a better solution than the curved lines.
	Conclusions have been rationally drawn based on the study’s findings.
Additional comments: Overall, this is a robust and integral piece of work. The group have rationally designed and executed the current study, which has successfully provided some important insight on the role of GDF11 on various aspects of the neural cell life and has, as well, added to the controversy over this specific factor. Therefore, there is indeed some contribution of the study to the field. However, the manuscript itself needs some improvements, especially concerning vocabulary and word structure.


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

Input:
Title: PHYSICAL, MECHANICAL AND ENERGY CHARACTERIZATION OF WOOD PELLETS OBTAINED FROM THREE COMMON TROPICAL SPECIES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The article is well written and informative. literature and references are sufficient, short and specific.Data are seems to be from smaller samples.
Experimental design: Its original primary research and have scope for better utilisation of wood pellets with high calorific values. The correlation between different parameters also well established .
Validity of the findings: Novelty of finding is reconfirming density and IRI of wood of selected tropical species with heating value. the pellet densities are 1.10 to 1.18 g/cm3 in three species but what is basic density of these woods is not mentioned and not a relationship with basic density of wood with their pellet density is unfolded.
Is there any method or formulation to increase the pellet density of inferior or lighter wood biomass/ chips to be used as pellet for better use.
The wood billets or chips from high density or specific gravity has high calorific value/ heating value than why we need to make their pellets, as these can be directly used in improved thermal heaters or wood gasifier. Pelleting has major role where wood not have good calorific value and easily combustible to mix its particle with charcoal or coal and other additives to have briquettes for better use.
I think  pellets and briquette are same terms.
Additional comments: The article is good and concise.


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

Input:
Title: PHYSICAL, MECHANICAL AND ENERGY CHARACTERIZATION OF WOOD PELLETS OBTAINED FROM THREE COMMON TROPICAL SPECIES
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comments
Experimental design: No comments
Validity of the findings: No comments
Additional comments: The manuscript is suitable for publications. The minor corrections are made in the manuscript which may be incorporated before publication. There should be a title in tables and figures which are lacking. The corrected manuscript is sent via mail.


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

Input:
Title: PHYSICAL, MECHANICAL AND ENERGY CHARACTERIZATION OF WOOD PELLETS OBTAINED FROM THREE COMMON TROPICAL SPECIES
Review round: 1
Reviewer: 3

Output:
Basic reporting: Clear and unambiguous, professional English used throughout.
Experimental design: Original primary research within Aims and Scope of the journal.
Validity of the findings: Conclusion are well stated, linked to original research question & limited to supporting results.
Additional comments: The paper is interesting and actual. It is a subject that has direct application.

In my opinion the authors didn’t presented a very well structured abstract and didn’t showed properly the importance that the article may have.

Would be important if the authors could present some examples for the potential uses of this.

This paper must be revised to include a short history about the usage of this type of solutions and the impact that this may have.

The following suggestions are intended to be seen as a help to the authors in order to improve the final version of the paper to be ready for publication.

1.	Introduction can be improved to better explain the importance of the subject.
2.	Authors should review more the references used. It is possible to find more references about this subject or related references and many important are missing.
3.	Tables and figures must be better explained in plain text or modified to be more understandable.
4.	Authors should try not to overkill references using several in the same paragraph without a clear understanding of the interest of each 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: ESTIMATING THE FREQUENCY OF MULTIPLETS IN SINGLE-CELL RNA SEQUENCING FROM CELL-MIXING EXPERIMENTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: - On line 99, “results” should be singular: “result”. Other than that, the writing was easy to read, and I did not notice any other grammar or spelling mistakes.
- Paragraph starting on line 37. Another strong argument for using unequal proportions of cell types when assessing the accuracy of a scRNA-seq technique is that it is more representative of what you’d find inside of an organism. When working with blood or a tissue containing multiple cell types, it’s unlikely that those cell types will exists in equal proportions. Make sure to emphasize this at some point.
- At some point in the Introduction is important to make it clear that other people have intentionally used unequal proportions of cell types in QC analysis of an scRNA-seq technique. For example, see Figure 2F of Rosenberg et al, 2017 (https://www.biorxiv.org/content/biorxiv/early/2017/02/02/105163.full.pdf). However, your work is novel in that it provides a detailed explanation of how to calculate the multiplet frequency in these scenarios.
- Lines 44-47. Give some brief detail about the Poisson distribution for readers who may not be familiar with it.
- A figure with diagrams showing how the scRNA-seq techniques work may be helpful to some readers.
- Instead of calling R code from within Python (which only works for Python 2 and not Python 3, as far as I know) it would be best to just provide two scripts for readers to choose from that each contain the whole analysis pipeline with one script written purely in Python (no R code) and another purely written in R.
Experimental design: - See my previous comment regarding the Rosenberg et al paper.
- How closely do you expect the number of cells per droplet to follow a Poisson distribution? From my understanding of microfluidics devices, there would be some upper limit to how many cells could physically fit into a single droplet. This concern may be negligible statistically, but it should still be addressed.
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: ESTIMATING THE FREQUENCY OF MULTIPLETS IN SINGLE-CELL RNA SEQUENCING FROM CELL-MIXING EXPERIMENTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article is well-structured and clearly written with appropriate references and easy-to-follow code (including an html rendering of a Python Jupyter Notebook) for recapitulating the tables in the paper.
Experimental design: The main purpose of this article was to derive a general equation for evaluating the multiplet frequency in a single-cell RNA-Seq experiment (e.g. from a mixed species experiment). Multiplets represent a major problem in single-cell RNA-Seq (and single cell analysis in general), as they often give profiles that resemble hybrid cell types or states, when they actually originate from a spurious mixture of two or more different cells in a single chamber. The author clearly articulates the problem that the current literature only addresses the case that two species are mixed in equal proportion. He provides an important experimental scenario in which this problem is highly relevant – namely the case that an experimenter includes a small number of cells from a different species as a “spike-in” sample during single-cell RNA-Seq for internal evaluation of the multiplet frequency. He then describes a detailed and straightforward derivation, based on simple Poisson statistics, of an equation that accommodates the more general case that the two species are mixed in arbitrary proportions. The derivation is mathematically sound and provides a straightforward way to evaluate multiplet frequencies from experimentally accessible observables. The methodology used to compute example calculations is easy to replicate, because thoroughly commented code is provided.
Validity of the findings: The findings presented here are valid and useful.  The conclusions are well-supported by a straightforward mathematical derivation – an analytical solution to the proposed problem is provided.
Additional comments: I have a few questions and comments regarding the potential for more general application of the analysis described here:
1)	In many single-cell RNA-Seq experiments, unsupervised clustering reveals very discrete cell types that are readily distinguishable, but from the same species. The author focuses on the case that two cells from two different species are mixed in arbitrary proportion, but could these results be extended to the case that an arbitrary number of cell types is mixed in varying proportions?  As a concrete example, if I am looking at a blood sample, I might expect to readily separate monocytes, B cells, T cells, etc. Could the framework described here be used to calculate an expectation value of, for example, the doublet frequency of each pair of cell types? This may be beyond the scope of the study described here, but may be worth a comment in the manuscript.
2)	Throughout the paper, the author focuses on what I would call “statistical multiplets”.  However, in many single-cell RNA-Seq experiments, this is not the only source of multiplets (and in some cases, not even the dominant source). For example, incomplete cellular dissociation could result in sequencing of multiplets simply because the cells were stuck together in the original tissue and remain stuck together during the profiling experiment.  In my own work, I have noticed an enrichment in apparent multiplets coming from cell types that I know are interacting with each other in the tissue I am profiling. This makes the application described in 1) particularly important.  Having an expectation value for the “statistical multiplet” frequency could provide a framework for evaluating whether or not the observed multiplet frequency for a pair of cell types is higher than one would expect, thereby implying incomplete dissociation. Again, this may be beyond the scope of the study described here, and I leave this to the author's judgement, but I think it would be worth commenting on the distinction between "statistical multiplets" and multiplets arising from other sources.