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

Input:
Title: THE ROLE AND PROTEOMIC ANALYSIS OF ETHYLENE IN HYDROGEN GAS-INDUCED ADVENTITIOUS ROOTING DEVELOPMENT IN CUCUMBER (CUCUMIS SATIVUS L.) EXPLANTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The English writing has improved a lot, but some mistakes and format problems still to be noticed.
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Authors have carefully revised the manuscript. However, there still have some minor comments.
1. Please double-check the error bar in Fig. 8D. Figure 8 is not of high quality, so it is recommended to redraw.
2. The statement of statistical analysis in Fig. 8 is incorrect, becuase there is no asterisk in the figure. What do the different letters mean? ANOVA analysis was used? How many parallel tests in each independent experiments? It should be included in all figure legends and Materials and Methods.
3. In the legend of Fig. 7, The number of involved proteins and the information of percentage in a term are shown in left and right y-axis respectively?
4. In Fig. 5, C should be in the suitable position.
5. Please double-check the numbers in Line 267-275. What is the mean of 36.935.4%? 4.34.2% 2.22.1% There are three 1312.5?
6. Please notice to the "et al." in Line 54, 73, 77, 79......
7. Please double-check the references. For example, in Line 499, "J Bacteriol, 174" ? in Line 401, "J Exp Bot, 59" ? Line 417, "78" do not be in italics. Line 429, "photosystem II manganese-stabilizing"
8. In Line 325, "H2" should be in the right format.
9. In Line 330, Rubisco, SBPase, TDH, CAPX, PDI and OEE1, total 6 proteins?


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

Input:
Title: THE ROLE AND PROTEOMIC ANALYSIS OF ETHYLENE IN HYDROGEN GAS-INDUCED ADVENTITIOUS ROOTING DEVELOPMENT IN CUCUMBER (CUCUMIS SATIVUS L.) EXPLANTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.	Line 30: “during”
2.	Line 62: “stress”
3.	Line 236: “Although”
4.	Line 268: “All”
5.	Line 269 – Line 274: Firstly, the percentage of all groups were wrong. For example, “Seventeen (17) photosynthesis-related proteins constituted the largest percentage (36.935.4%)”. 17/48 = 35.417%. And so on. Secondly, existing some formatting mistake, such as 36.935.4%, 1312.5%, 6.56.3%, 4.34.2% and so on. Thirdly, Line 274: there were 7 unknown proteins instead of 4. In addition, “six 6 energy metabolism-related proteins” should delete “six”. Please carefully check this section.
6.	Fig 8: the percentage of unknown protein is 14.6% instead of 8.3%.


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: UNDERSTANDING THE SPREAD OF DE NOVO AND TRANSMITTED MACROLIDE-RESISTANCE IN MYCOPLASMA GENITALIUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper is well written in clear English with appropriate referencing.

In the introduction, I suggest that you add a little bit more information about Mycoplasma genitalium infection, including epidemiology and natural history.

Line 157: remove "model"

Line 158 : (Figure 3, left panels), Fig 2?

Figure 3:  Horizontal lines look like black color.
Experimental design: Your compartment model is simple and easy to understand, although this model is oversimplified.

I disagree with the equation 5. φA+T is not the growth rate of the resistant strain because the rate itself includes the prevalence of the resistant strain.

In the equation 6, what is the meaning of "find ki resistant samples in Ni tested individuals"?
Validity of the findings: Table 1: I suggest you to change the column name " number of specimens tested" . because it was a  bit confusing. (e.g. number of mycoplasma genitalium positive)

Please explain a little bit more about fig 2 right panels
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: UNDERSTANDING THE SPREAD OF DE NOVO AND TRANSMITTED MACROLIDE-RESISTANCE IN MYCOPLASMA GENITALIUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: 1. Your first sentence in introduction(lines 32-34) needs more detail. For example, '~ a short history of macrolide usage as treatment of the organism'.
2. The latter part of same paragraph (lines 37-40) need more explanation. It seems NAATs are being used  but also not being used at same time to detect M. geitalium.
Experimental design: 1. Please clarify which criteria did you refer to while you include or exclude the data.(lines 71-76)
Validity of the findings: 1. In the graph of France in the right part Figure 2, the line is barely within the shade. Please provide more data from France which support your results.
Additional comments: Your results are very impressive. Your model is simple and well explaining.
More comprehensive introduction and methods would make this article greater.


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: UNDERSTANDING THE SPREAD OF DE NOVO AND TRANSMITTED MACROLIDE-RESISTANCE IN MYCOPLASMA GENITALIUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Well written
Experimental design: Good
Validity of the findings: Good
Additional comments: After reviewing the updated version of the manuscript, I think this manuscript is ready for publication.


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

Input:
Title: DIFFUSION TUBES: A METHOD FOR THE MASS CULTURE OF CTENOPHORES AND OTHER PELAGIC MARINE INVERTEBRATES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript “Diffusion tubes: a method for the mass culture of ctenophores and other pelagic marine invertebrates” by Patry and collaborators test a method to raise delicate species of pelagic marine invertebrates. I consider the present attempt a good advance in the mass laboratory cultivation of juvenile ctenophores that would facilitate the research with different ctenophore species.

That said, I am adding below few comments and advices.

The authors say that “medium flow outperformed the low flow treatment” (line 215). Can the authors explain in which way? (e.g. length of cydippids, number of cydippids after six weeks). Furthermore, the legend for Figure 6 says that “the high and medium flow treatments ended with significantly greater mean lengths than the low flow treatment” at week 6, while in the text (line196) the authors say that “the medium treatment had the smallest mean lengths”. From the data that the authors have supplied I could see that the mean lengths, after 35 days, were 333 mm and 589 mm for medium and low flow treatments, respectively. Can the authors fix that contradiction?

I encourage the authors to provide more evidence (e.g. growth rate, mortality, fecundity) on the culture of non-ctenophore species (e. g. Nanomia and Oikopleura) with their method. Otherwise, I strongly recommend evaluating if “and other pelagic marine invertebrates” can remain in the title (line3) and abstract (line20) of the article.

The authors supply the number of cydippids that their method can yield as a proof of their method success (line189). Baker and Reeve 1974 (doi.org/10.1007/BF00389086) also provide the fecundity of the animals to test their method to culture ctenophores. Could the authors add some data on animal's fecundity?

The authors include the protocol to culture the ctenophores in the conclusions (lines 250 – 267). I think (IMHO) that a separated box with the protocol will help the reading. Also, a scheme of the culture indicating the initial number of parents, the days that the cydippids spend in the diffusion tube before to be transferred to the Kreisel tank, etc. would help the readers.

Figures and Supplemental material

Figure 4. The legend for Figure 4 should say that the mean of the two measurements taken on opposing sides of the inner tube is a dot, not a line. Besides, the authors should clarify if the line is showing the tendency of the measurements or only is joining the dots.

Figure 6. The title of the Figure 6 is “Pairwise comparison of means for low, medium, and high flow treatments at week 6” and the figure is a box and whisker diagram. As normally box plots depict groups of numerical data through their quartiles and mean is not represented in them, I would like that the authors clarify what is the box and whisker diagram representation, and if the mean is not represented in the diagram, could the authors add it? Otherwise, an easy to confuse reader like myself could get wrong.
Additionally, Could the authors clarify if week 6 is refering 35 days data? If not, Could the authors add the measurements for the week 6 in the supplementary file “Raw data for total length measurements of Hormiphora”?

Supplemental file “Raw water parameters data for T, pH, O2”. In the sheet “High Flow Profile Data” authors say that the turnover rate is 5.5 l/m in the text (line85) is said that is 4.5 Lpm. Can the authors check it?

Other comments.

Line 29. Clarify if citation “Presnell and Browne, 2019” should be “Presnell et al. 2019”.

Line 146. Change “PST” by “PM” or “noon”.

Line 184. Can the authors give the standard error for the probe?

Line 189-190. Change “medium flow (1.1 Lpm)” by “medium flow (2.2 Lpm)” and “low flow (2.2 Lpm)” by “low flow (1.1 Lpm)”.

Line 225. Change “using” by “the use of”
Experimental design: The methods of spawning and culturing section needs more detail. I suggest that the authors improve the description between lines 118 and 147 to include the initial number of cydippids to be raised in each diffusion tube (as animal density can affect the food clearance, and consequently growth rate), how many replicates did they perform for each treatment, how long takes to cydippids to produce eggs (Is there a possibility that juveniles have produced eggs in the diffusion tube during these 6 weeks?). Besides, an estimation of the number of cells of live algae (line 121) (or at least, the optical density of the algae cultures), at the moment of feeding, will help other laboratories to replicate present system. Finally, I also suggest the authors include a short description of how they cultured the other non-ctenophore species (e.g. Nanomia and Oikopleura) at the end of the section (Do you also transfer non-ctenophores animals to Kreisel tanks?). It will be very interesting to see the survival rates of Oikopleura in this system.

I agree with the authors that the initial number of parents might impact in the yield of the culture. The authors say that they obtained higher yields in previous/subsequent trials with more initial parents than the experiment presented in this article (line 222). For that reason, I think that would be worth it to present that data in this article. In the case that these data have been published, they should cite it.

The authors say (line 65) that they have observed several physical parameters including; feeding rates, biofouling, and analyzed fluid flow. However, the results supplied do not include feeding rates or biofouling measurements (that I can see). Have these analyses been done already? If so, make that clearer in the text (results section).
Validity of the findings: The authors should show that they have repeated this experiment more than once (is not a long experiment, 35 days). Specially for the medium flow treatment where they have unexpected results. If they did the experiment more than once, could they make it clear?
Additional comments: In my point of view the reported method seems to be adequate to growth juvenile ctenophores in the lab and could help to spread the use of that phylum as a model organism. However, the conclusions presented in the article are more based in the author’s experience cultivating ctenophores for 4 years (line 246) than in the experiments presented (E.g. the experiment was done with 3 initial parents but the given conclusions are to use 1 to 10 parents).


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: DIFFUSION TUBES: A METHOD FOR THE MASS CULTURE OF CTENOPHORES AND OTHER PELAGIC MARINE INVERTEBRATES
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript describes the construction of a double cylinder rearing tank, a protocol for raising ctenophores in these tanks, and best practices for mass culture of ctenophores using this new design. Research on ctenophores has been historically limited due to the difficulty of maintaining the animals in the laboratory, but with the availability of genomes and techniques for studying ctenophore biology in the lab, there is a clear need and demand for a solution to culturing these delicate animals. The authors are are world's experts in rearing ctenophores and the insight from this paper will have a substantial impact on researchers and aquarists alike.

* The paper is mostly well organized and well written, but there are some issues regarding the instructions and figures for building the tank. The authors should make building one of these as easy as possible. An IKEA-like step-by-step instruction set should be provided. This should include an illustration of the final product, tools needed, list of parts, and step-by-step assembly procedures. Here is a nice example: https://dokit.io/wp-content/uploads/2019/05/instructions_IKEA_billy.jpg
Figure 1 is an attempt at this, but was very confusing to me as I was trying to build one. It was not step-by-step. For example, the instructions on glueing the mesh (bottom of the inner cylinder was fitted with 55 μm nylon screening) was not detailed enough. In addition to instructions for gluing etc. it should be included how long to wait, importance of soaking materials overnight before usage, etc.

* A table with each part, vendor, part number, approximate cost would be extremely helpful.  It would be helpful if parts were lettered and referred to in the diagram and in the "Materials" part of the paper (Table 1A). It would also be helpful to mention where potential replacements could be made and what would be the limits (e.g. 40-75 micron mesh?) either in this table or a supplement.
NOTE: A vendor and part number for the 2 main acrylic tubes should be provided.

* I recommend separating the "Materials" section from an "Assembly" section. The assembly section could walk through the step-by-step figure.

* With so many parts from so many vendors, it is inevitable that part numbers will change and perhaps some parts are no longer carried by particular vendors. To ensure that the manuscript is able to be useful for a long time, the authors could maintain a markdown document on a github repository where the links to particular parts could be kept up to date (and alternatives (perhaps cheaper) from users can be posted).  Since GitHub is a software repository, a record of changes is kept and this would enable the historical nature of the paper to be conserved, while allowing the authors to keep an up-to-date set of links, etc.

* building one of these devices costs around $500 USD. It would be useful to mention this up front to save people time who can not afford to make one. Something like: "At the time of the writing of this manuscript, using the specified parts, the cost of building one of these devices is $XXX.YY."

* The model # for the rigid plastic mesh (Model# N1020 80 PentairAES, Inc.) is for a 48" by 50' spool of fencing $202.56.  A user needs about .0001% of this and there are far cheaper options available (e.g. https://www.homedepot.com/p/Everbilt-3-4-in-x-3-ft-x-25-ft-Green-PVC-Poultry-Fence-889241EB/205960879 would probably work). Alternatively, a whole could be drilled near the top of the inner cylinder and a rod going through the tube could suspend the inner tube from the rim of the outertube making this mesh unnecessary. In addition the Model #N1670 PentairAES mesh is also for a huge amount and is $96.23 USD. It would be nice if there a smaller quantity/cheaper option. At the very least, it should be mentioned that these will work, but come in large quantities and replacements could be potentially found in local hardware stores?

* Regarding flow rates, it would help to include a table with optimal (or approximate) flow rates per species.

* Line 133 - it would help to have a section heading (e.g. post-diffusion tube rearing) to make it clear that the supplies in this section are not for the diffusion tube. A table 2 with these supplies and a figure showing how they are used would make it more clear.

* Line 140 - it is not clear as written whether Model #24244SC refers to 4mm or 6mm pieces

* Line 191 - These data would be better in a table.

* Figure 5. - Ideally this graph would communicate the difference between the treatments. Something like the boxplot described here: http://t-redactyl.io/blog/2016/04/creating-plots-in-r-using-ggplot2-part-10-boxplots.html
would be more informative.

Line 210: "that is inexpensive" - It would be helpful to contrast the actual price of a kreisel vs. the diffusion tubes.
For example https://aquaticnow.com/schuran/kreisels/breeding-kreisel.html

Line 213: "Using a higher turnover rate..."
-- Should this be "Using the highest turnover rate" since the lower rate was superior to the medium rate?

Line 214: "It is not clear why the medium flow outperformed the low flow treatment"
-- I'm confused. I thought the order was highest flow was best, followed by lowest, then medium?

Line 225: "It is also possible that lower yields may reflect using fewer parents (n = 3) than previous/subsequent trials (n = 10)."
-- Perhaps due to inbreeding depression due to increased levels of self-fertilization?

* A table with recommended inner and outer tube diameters and thickness (with models) for each species would be helpful.
* Likewise, making an updatable csv file of this table in a repo like GitHub that could be updated by the authors as improvements to suggested sizes/flows etc. would be a wonderful addition.

Line 257: "gently remove them from the tube using ladles or dishes"
-- I assume "them" refers to adults, but that is not clear as written.
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: DIFFUSION TUBES: A METHOD FOR THE MASS CULTURE OF CTENOPHORES AND OTHER PELAGIC MARINE INVERTEBRATES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Patry and collaborators have addressed the previous reviewers' comments rightly, improving the clarity and comprehension of their manuscript. However, new typo errata, that authors should correct, have risen during that process:
- The authors say (line 246) that p-value for the Wilcoxon signed-rank test between low and high flows is 0.00046, while in figure 6 is 0.0046.
- Legend figure 7. Change “srages” by “stages”
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no cmment


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: DIFFUSION TUBES: A METHOD FOR THE MASS CULTURE OF CTENOPHORES AND OTHER PELAGIC MARINE INVERTEBRATES
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: I had 2 very minor points.

1.	On line 118 “single diffusion tube setup is ~$465; on line 305 “diffusion tube setup costs ~$300”; Assuming $300 should be $456, should line the phrase on line 306 be changed from “can cost twice that of a diffusion tube setup” be changed to “usually cost more than $600”?
2.	Line 315: “Should flows need to be minimized in a future lab setting, perhaps replication will provide better resolution.”
>>> This sentence should be removed. It is redundant with the next sentence and is superfluous as more replicates always provide higher resolution.


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: LIVING (STAINED) FORAMINIFERA IN THE LESSER SYRTIS (TUNISIA): INFLUENCE OF POLLUTION AND SUBSTRATUM
Review round: 1
Reviewer: 1

Output:
Basic reporting: No Comment
Experimental design: No Comment.
Validity of the findings: No comments
Additional comments: The Manuscript ID 41510-v0, entitled “Living (stained) foraminifera in the Lesser Syrtis (Tunisia):
inﬂuence of pollution and substratum” of the authors Akram El Kateb, Valentina Beccari, Silvia Spezzaferri, and Giovanni Coletti, is an interesting work. It aims to evaluate the negative impact of phosphate treatment industries along the Gulf of Gabes (Lesser Syrtis, Tunisia) on the marine environment, based on living foraminifera.
The work is based on the analysis of a significant number of stations located in well selected areas depending on proximity to source area of pollutants. The authors apply an excellent methodology for acquisition of data of living benthic foraminifera and present the location of the study sites and the foraminifera database. The work contains new aspects.
The text is presented in a manner that scientists in other disciplines will understand. It is presented and arranged clearly and concisely.
In the methodology, the authors should mention how they obtained authorization to sample in the studied area and which authorities gave permission to sample in Tunisia.
Many species of foraminifera have been photographed and the photos are very good allowing to clearly identify the photographed specimens. The identification of foraminifera is presented as supplementary material which is therefore of great interest.
Figure 3 is not readable. It should be subdivided and the information contained therein readable. The authors apply statistical analyzes and identify groups of stations characterized by differentiated composition of living foraminifera assemblages.
The work does not present physicochemical data measured in water and sediment nor sedimentological, such as particle size, TOC, metal concentrations. These data are important for explaining foraminifer-based station groups. If possible, this information should be added. The lack of this information puts the work at the level of a foraminifera checklist, which by itself is important.
The authors based the interpretation of station groups on the basis of living foraminifera species ecology citing old works based on total association (living+dead) (eg Vilela et al.). That methodology is contested by the FOBIMO group. The works based on total association cited and used to explain the ecology of the species must be changed by others analyzing living assemblages.
Works with living foraminifera in the Bizert Lagoon should be cited e.g.:
Martins, M.V.A et al. 2016. Organic matter quantity and quality, metals availability and foraminifera assemblages as environmental proxy applied to the Bizerte Lagoon (Tunisia). Marine Pollution Bulletin 105, 161–179, https://doi.org/10.1016/j.marpolbul.2016.02.032.
Martins, M.V.A. et al., 2015. Environmental quality assessment of Bizerte Lagoon (Tunisia) using living foraminifera assemblages and a multiproxy approach. PLoS ONE. http://dx.doi.10.1371/journal.pone.0137250

I think that the provided comments should be considered.


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: LIVING (STAINED) FORAMINIFERA IN THE LESSER SYRTIS (TUNISIA): INFLUENCE OF POLLUTION AND SUBSTRATUM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Language needs to be polished in several parts of the paper. In several parts of the discussion, paragraphs need to be rearranged in a way that the environmental conditions are described first and then the micropaleontological data are presented in order to support the various environmental conditions.
Several references to be added would improve the documentation.
Fig. 3 needs to be enlarged.
For further comments, see the attached pdf.
Experimental design: The originality of the research is well justified and research question is well defined.
Geochemical data need to be included and a correlation between main geochemical components and species deistribution will better support the foraminiferal environmental preferences. For further comments, see the attached pdf.
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: LIVING (STAINED) FORAMINIFERA IN THE LESSER SYRTIS (TUNISIA): INFLUENCE OF POLLUTION AND SUBSTRATUM
Review round: 1
Reviewer: 3

Output:
Basic reporting: - Professional English language was used
- References are updated and relevant
- The structure of the manuscript is adequate; figures are relevant (plots of Figure 3 are hard to be read); Table 1 needs to be improved with water depth of sampling stations raw data are shared
- This study is not self-contained because only the use of abiotic data publushed elsewhere (El-Kateb et al. 2018) would support conclusions
Experimental design: - The research is original and falls in the aim of the journal
- The research question is not clear. The Authors declaired the aim to "to trace the pollution produced by the phosphate industries in the Gulf of Gabes, to document assemblage  composition variations in relation to the pollution sources" but they did not supply quantitative data on sediment and water parameters.
- The investigation on Benthic Foraminifera was rigorously conducted, according to recent international protocol
- Details on statistical analysis are missing. In particular, methods about the preparation of dataset and statistical analysis should be supplied.
Validity of the findings: 1.	This study consists in a very detailed and accurate documentation of living Benthic Foraminifera (BF) in the marine coastal area of Lesser Syrtis. A valuable taxonomic work was done reporting synonymies; also the huge work of illustrating species in photographic plates is appreciable. The utility if this work is considerable because it supplies a baseline for the classification of foraminiferal species in the area.
2.	The study was rigorously conducted as regards sampling methods and quantitative analysis of BF, according to the FOBIMO protocol.
However, taking into account that the aim of the study is “to use the response of living BF to trace pollution ….. to document assemblage composition variations in relation to pollution sources” I consider this study affected by many weaknesses described below.
1.	Although water and sediment abiotic parameters were mentioned in the interpretation of clusters obtained on the basis of foraminiferal results, no quantitative data, were reported; these data are available for the same sampling stations and published by El-Kateb et al. (2018b). In my opinion, this approach is not suitable to reliably demonstrate the actual role of abiotic factors on BF, in order to meet the aim of the study. Moreover, the response of BF to contamination should be demonstrated by a comparison of foraminiferal data with sediment/water data by means of suitable statistical techniques. Differently, the nMDS used by the Authors to enforce the environmental interpretation seems applied exclusively on foraminiferal data and the interpretation was given on the basis of the ecological features of foraminiferal taxa.
2.	Also the attempt to supply environmental interpretation of clusters based on the autoecology of dominant taxa in each clusters was sometimes weak, because not supported by literature concerning the same species found in this study, but only dealing with the same genus (see below for more precise indications).
3.	Quantitative data of species abundance (absolute or relative) were completely missing in the text of the manuscript (available exclusively from supplementary material).
4.	Results on foraminiferal density and diversity, which are of basic importance while using BF for environmental assessment, were not reported at all.
5.	The ecological interpretation of stressed environment for cluster A seems weak, because it was based on the supposed pollution tolerant character of Ammonia beccarii, while this species is very common in unstressed shallow-water Mediterranean environment (see below for references). Also reference to abiotic environmental parameters which may be the origin of such stressed conditions (phosphorous contamination, salinity changes) was not supported by quantitative and, on the whole, it is not clear which is the main environmental driver for this cluster.
On the whole, this study, as it is now set up, is not able to scientifically demonstrate what it was stated in the conclusions (the distribution of some species is related to different pollution degree), nor to achieve the purpose declared in the introduction (to document assemblage composition variations in relation to the pollution sources).
Additional comments: I have two main suggestions for re-addressing the work, because it could potentially be an excellent application of BF as bioindicators for the rigorous methodological approach adopted, but it needs considerable improvement finalized to support the given conclusions.
1.	The interpretation of data should be carried out by integrating the biotic (in this study) and abiotic data (published in El-kateb et al. 2018b) by means of a suitable statistical approach.
2.	I would also recommend the Authors to apply the Foram-AMBI index, according to Jorissen et al. (2018), and to compare the obtained values with the concentration of contaminants, in order to enhance the value of BF as tools for the environmental assessment.

Some minor observations are listed below.

Abstract
Lines 16-17. Because the audience of the journal is not limited to foraminifera specialists, I think that more preliminary information on BF as applied as environmental indicators should be supplied.
Line 21. I would specify “sediment samples”.
Lines 26-29. Which is the criterion for considering these species as good bioindicators? Please, specify.
Line 27. Eggerelloides scabrous is reported in the taxonomic list as Eggerelloides scabrus. However, according to the Worms database, the accepted name is Eggerelloides scaber.

Introduction
Line 49. Please, substitute “radioactivity” with “radioactive isotopes”.
Line 59. Zaouali et al. 1993 is not reported in reference list.
Lines 61-62. This sentence is not clear to me.
Lines 89-93. I would specify that the FOBIMO protocol was aimed at the standardization of methods for the application of BF in biomonitoring activities for the assessment of the ecological status, according to the Marine Strategy Framework Directive (MSFD). The cited papers may not be considered as an assessment of the contamination impact on marine environments, but studies on the response of living BF to specific environmental factors; in particular, Polovodova et al. (2015)  did not consider living BF because it studied samples from sediment cores.
Lines 95-95. What does it mean “to trace pollution”? Please, clarify.
Although several earlier works examined sediment contamination in the study area, no quantitative concentration data for specific contaminants were reported from literature. More detailed reference to these works, including their location with respect to the present one would be needed.

MATERIALS AND METHODS
Because seasonality largely influences the living assemblage, the sampling period should be specified (and should be the same for all samples).
Lines 108-112. The range of water depth of transect stations should be supplied.
Details on the preparation of dataset (were all species included or rare species were not considered? Which was the principle for defining a species as rare?) and methods of statistical analysis should be given here.

DISCUSSION
Lines 152-153. I would not base the interpretation of stressed environment on the dominance of Ammonia beccarii, because assemblages dominated by this species are common in Mediterranean shallow-water unpolluted environment (see for example Walton and Sloan 1990 Journal Foraminiferal Research 20, 128-156; Murray 1991 Ecology and Paleoecology of Benthic Foraminifera. Longman Scientific & Technical). I would also suggest to critically consider literature reporting information on the tolerant character of this species because, especially in past times, several Ammonia species (mostly A. tepida and A. parkinsoniana) were classified as beccarii.
Lines 155-156. Concentration ranges of phosphorous should be supplied.
Lines 157-159. Because, in general, different species of the same genus have different ecological requirements, the ecological interpretation given by the Authors should be based on information regarding the species found in their samples, particularly Quinqueloculina laevigata, not “some quinqueloculinids”.
Lines 160-161. Salinity values should be supplied.
Lines 167-168. Please, supply quantitative data. Foraminiferal density is influenced by several environmental parameters such as sediment texture, nutrients, oxygen. How can the authors attribute the low foraminiferal density to contamination when these parameters were not considered?
Line 174. Please, write “Sen Gupta” instead of “Gupta”.
Lines 184-186. Which heavy metals showed high concentrations? Please, supply quantitative data. El-Kateb et al. (2018b) did not analyze heavy metals, but only mineralogical sediment composition.
Lines 191-192. Are the Authors speaking of statistical correlation? Please, specify.
Line 202. Please, supply data on mud percentage.
Lines 204-206. Organic matter is a potential food resource for all foraminifera species. Which are conditions favoring the abundance of Asterigerinata mamilla?
Line 215-2016. I don’t understand how sediments samples may be considered as hard substrate. El-kateb 2018b reports results of grain size analysis  for sample CST-05. I would not define this cluster as related to hard substratum, but to coarse bottom sediment.
Lines 251-252. Please, add a reference.
Line 256. Because the Authors carried out the Cluster Analysis, a two-way (Q and R-mode) plot would have been sufficient to identify the dominant species in each cluster, instead of a Non-metric MultiDimensional Scaling consisting of 15 plots very hard to be read.
Lines 267-270. How the authors compared abiotic data with the plot based on foraminiferal abundance? this is the crux of all the work, because this comparison must be conducted with a statistical approach and based on a matrix containing all the data. How the Authors obtained the scale of grain size and pollution at the base of the nMDS plot (Fig. 4)?
Line 275. Table 2 reports only geographic coordinates. Water depth should be included.
Lines 268-270. These statements are not supported by the results of this study. The role and importance of abiotic factors on BF may be demonstrated only applying a statistical analysis which considers together biotic and abiotic data. At least the range in each cluster of these parameters, published by El-kateb 2018b, should be supplied in this study.


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

Input:
Title: LIVING (STAINED) FORAMINIFERA IN THE LESSER SYRTIS (TUNISIA): INFLUENCE OF POLLUTION AND SUBSTRATUM
Review round: 2
Reviewer: 1

Output:
Basic reporting: Clear and unambiguous, professional English was used throughout.
Literature references, sufficient field background/context were provided.
Professional article structure, figures (I’m still convinced that small writing of Figure 4 result unreadable), tables were supplied.
Raw data were shared.
I consider this second version of the manuscript self-contained with relevant results to hypotheses.
In my opinion, the study changed completely perspective with respect to the first version due to the introduction of abiotic water and sediment data. t is now coherent with the declared aim and, in the present version, the conclusions are adequately supported by the results. Also, the more careful discussion of the ecological significance of  species improved the quality of the manuscript.

LINE 387. Field et al. (1982) was not reported in the reference list.
LINE 737. “Jorissen” instead of “Jorisse”.
CONCLUSION. I would say something concerning the important role of TOC on foraminiferal distribution
Experimental design: ok
Validity of the findings: The statistical analysis aimed at highlighting relationships between foraminiferal species and environmental parameters, carried out in the revised version of the manuscript, added scientific value to the study.
Additional comments: In my opinion, the study changed completely perspective with respect to the first version, due to the introduction of abiotic water and sediment data. The statistical analysis aimed at highlighting relationships between foraminiferal species and environmental parameters added scientific value to the study. It is now coherent with the declared aim and, in the present version, the conclusions are adequately supported by the results. Also, the more careful discussion of the ecological significance of  species improved the quality of the manuscript. I appreciate very much the photographic plates.


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: DEEPBINDPOC: A DEEP LEARNING METHOD TO RANK LIGAND BINDING POCKETS USING MOLECULAR VECTOR REPRESENTATION
Review round: 1
Reviewer: 1

Output:
Basic reporting: The publication requires significant revisions in grammar and overall use of the English language. Almost every sentence from the 'Introduction' section on, presents an issue with number concord or is missing a determinant. Some examples are:
Line 85: "Our method is not mutually exclusive with those traditional method that include spatial information."
The line after, line 86, reads:
"Since the way we used molecular vector have ignore the spatial information..."
In some cases, the grammar errors make it hard to understand the actual meaning of the text.
Experimental design: The construction of the datasets needs some clarifications. Besides that, it is nowhere explained in the text why some pockets from Fpocket are included in the negative set. The fact that there is not a ligand present in that pocket, does not mean that the pocket is druggable.
Overall, the methods section needs some further explanations on how the datasets were created and the role of Fpocket on each of the steps.

It is also somewhat confusing, that Fpocket is used to generate the datasets which are then used for the training, but then the performance of DeepBindPoc is precisely compared against Fpocket.
Validity of the findings: The idea of the work is the implementation of a machine learning protocol to detect the pocket in a protein for a given ligand. The input set contains over 16000 proteins from the PDBbind database. It is, however, unclear how many were rejected due to RDkit parsing issues and how many proteins ended up on each dataset. The construction of the datasets needs further explanations, it is unclear for the reader to see where the datasets from Strategy 1 and 2 ended up in Tables 1-3. The comparison against other programs such as Fpocket, also needs further information. Table 3 lists some pdbs and compares the ability to find the correct pocket in the first 5,3 and first guesses. It is however nowhere stated why these PDBs where selected.

Overall, the publication needs major revisions and I do not favour its publication at this stage.
Additional comments: The implementation of an algorithm (DeepBindPoc) that detects binding pockets for a given ligand in a protein has tremendous applications in the early drug discovery pipeline. The fact that the program has been made available as a web server is also a very nice feature. However, the paper as it is in its current state needs major revisions and should be rewritten. The fact that the neural network is trained on a set of pockets obtained with Fpocket, but then later Fpocket's performance is compared against DeepBindPoc does not sound like a rigorous method or should be better explained.
The method section needs a better explanation of how the datasets were created, and why they were created this way. This process should be better reflected in Figure 1, and the Strategies used for the creation should either be better explained or integrated later in the Results tables.
Lastly, the use of the English language needs a major revision. Almost all sentences present a grammar error.


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: DEEPBINDPOC: A DEEP LEARNING METHOD TO RANK LIGAND BINDING POCKETS USING MOLECULAR VECTOR REPRESENTATION
Review round: 1
Reviewer: 2

Output:
Basic reporting: In this paper, authors have proposed a new deep learning method, DeepBindPoc for identifying and ranking ligand-binding pockets in proteins. They built DeepBindPoc by utilizing information about the binding pocket and corresponding ligand. The authors have extensively tested and validated the model with standard procedures on multiple datasets, including a dataset with G-protein Coupled receptors. The article is well written and relevant references were provided. Hypotheses were backed by data. Overall, I consider this work very important in the field of structure-based drug design. I recommend this article for publication in PeerJ.

0.+
Experimental design: No comment
Validity of the findings: No commnent
Additional comments: 1. The performance of DeepBindPoc on membrane proteins was not that good. What could be the possible reason? How do you want to address it in the near future?

2. line 87, page 2: "have ignore....". it should be "have ignored".

3. there are article mistakes.. for eg. line 88-90: "To sum up, this method has strong complementary advantage over existing traditional methods, because we incorporated ligand information and effective molecular vector representation in our model". It should be "......has a strong....... "


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: DEEPBINDPOC: A DEEP LEARNING METHOD TO RANK LIGAND BINDING POCKETS USING MOLECULAR VECTOR REPRESENTATION
Review round: 1
Reviewer: 3

Output:
Basic reporting: The authors introduce a deep-learning-based tool DeepBindPoc for the identification of binding sites. The model is built using both information about the binding site and associated ligand using the mol2vec tool. The model was trained on structures in PDBIND 2018 and tested and validated on PDB structures which were deposited in 2019.

  I reviewed basically the same text about a half a year ago for the Bioinformatics Journal. Although the authors have addressed some of the major issues remain.

  	* The paper needs to be proofread by a native speaker. For example:
  	   * Our method is not mutual exclusive with those traditional method that includes spatial information. ---> Our method is not mutually exclusive with those traditional methods that include spatial information.
  	   * Since the way we used molecular vector have ignore the spatial information, ... ---> Does not make sense at all
  	* As the point of the method is to do rescoring of existing predictions with the ligand information present, I believe it should be also compared with rescoring using protein-ligand scoring functions available in existing structure-based drug design tools. There even exist deep-learning-based approaches such as https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479431/ (the virtual screening scenario).
Experimental design: * Common issue with the evaluation of ML methods is information leakage. I strongly believe this is exactly the case here. It is very probable, that the train and test sets contain virtually the same structures because no effort what so ever was put into filtering out homologs. The least one should do is to use sequence identity to filter out similar sequences. And even then, since we are not interested here in the whole structures, but only in active sites it is quite possible that there can be almost identical binding sites in both test and train sets since the structure similarity, we are interested in is only local. So to really be sure there is no information leakage, one should use some local structure similarity measure.

* The AUC and other measures are computed based on whether a true binding site was found among the top 3 predicted pockets (that is three selected from Fpocket results). However, one should also show the baseline, that is what are the numbers for Fpocket only to see what numbers DeepBindPoc actually tries to improve. I.e. the authors should pick top 3 pockets from Fpocket and show the numbers for that.

* At one point authors compare to P2Rank. When looking into P2Rank paper, one can see that the authors have chosen Top-N+k evaluation strategy. That is if the protein has N pockets and you choose k=0 you look at top N pockets. Since here the authors search for binding sites of a specific ligand, the N is always 1 and k = 2. To paint the full picture, the authors should also provide at least the Top1 and Top2 or TopN in general, where N is the number of predicted pockets given by Fpocket.

* I don't really see the point of testing on the 6 cases where FPocket failed to generate the pocket since in real-world scenario, DeepBindPoc wouldn't have anything to rescore.

* Related to the previous point - I tried to pick one of the pockets (6mt8) which FPocket failed to predict and run the prediction with [2] and the correct pocket was found as the first one. This might show that choosing Fpocket as the underlying main pocket detection tool might have been not the best choice and the authors should really see how DeepBindPoc would improve on state of the art approaches.
Validity of the findings: See the previous section.
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: DEEPBINDPOC: A DEEP LEARNING METHOD TO RANK LIGAND BINDING POCKETS USING MOLECULAR VECTOR REPRESENTATION
Review round: 2
Reviewer: 1

Output:
Basic reporting: My comments have been addressed.
Experimental design: My comments have been addressed.
Validity of the findings: My comments have been addressed.
Additional comments: My comments have been addressed.


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: GENETIC EDITING OF THE VIRULENCE GENE OF ESCHERICHIA COLI USING THE CRISPR SYSTEM
Review round: 1
Reviewer: 1

Output:
Basic reporting: good
Experimental design: good
Validity of the findings: good
Additional comments: Authors have used a two-plasmid system of CRISPR for the Genetic editing of the virulence gene of Escherichia coli and can be used for deleting, replacing, or mutating the ETEC associated virulence genes on plasmids and chromosome. This is a good mechanistic study with clear objectives. However I suggest some minor comments
1.	Despite the fact that authors have mentioned ‘the editing efficiency is closely related to the homologous arm length, and the efficiency is the highest when the length of the homology arm is approximately 300 bp’. This high efficiency means to edit/replace coding sequence? Have you checked any non-coding DNA too?
2.	Another important change should be made in the statement of the objectives. Actually, there is no such statement of the objectives of the study; in case there is one, it failed to catch my attention as a reader. In your revision, include, in a sentence or two, a clear statement of the objectives of the study. Because this technology is used in various articles for different deleting, or replacing purposes of virulence genes. What is the impact of your study?
3.	You have examined the ETEC associated virulence genes editing. Or have you checked any clinical virulent strain? And did you check any lethality of the test strains? If so please include the lethality test results, as VAGs are not considered virulent based on molecular data only.


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: GENETIC EDITING OF THE VIRULENCE GENE OF ESCHERICHIA COLI USING THE CRISPR SYSTEM
Review round: 1
Reviewer: 2

Output:
Basic reporting: Although the study is interesting the structure and the grammar are ambiguous. It should be edited from professional English service.
The structure of the manuscript should be improved.
Please rearrange your results.
Experimental design: The objectives are not clear.
Experimental design should be improved.
Methods are not sufficiently described.
Validity of the findings: Impact and novelty not assessed.
Conclusions are not stating the research question & limited to supporting results. It should be improved.
Additional comments: 1. The authors mentioned that a two plasmid system (pCas/pTargetF) that contains Cas9 and λ-Red recombination elements constructed by Jiang. Who is Jiang? I think it should not be mentioned as the name in the abstract. You can write it in the method section.
2. The introduction is inconsistent. The paragraphs line 72 to 79 are unnecessary. Should be removed.
3. The authors mentioned that the E. coli O142 and E. coli DN1502 were isolated from the bovine rectum. How they isolated from the rectum directly? It is not logical. Please make it clear.
4. Please re-write the lines 124-128.
5. The authors mentioned experiment numbers throughout the manuscript. I could not understand about these experiments. Which experiments have been numbered in this manuscript?
6. There repetition of plasmid curing methods in the results. Please remove this paragraph from the results .
7. The results are few but the discussion section is too long and non-logical. The discussion must be re-written and it should be in a logical flow that compares your results.
8. The English language are poor. It should be edited by a professional or native English speaker.


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: GENETIC EDITING OF THE VIRULENCE GENE OF ESCHERICHIA COLI USING THE CRISPR SYSTEM
Review round: 1
Reviewer: 3

Output:
Basic reporting: Figure 2 requires detailed annotation；The electrophoretic bands in Fig. 3 and Fig. 4 need to be marked clearly with maker size；The annotation of the figure should be below the figure
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The study seems to be quite interesting and is quite valuable to report on CRISPR/Cas9 coupled λ-Red recombination technology, which was firstly invented by Liu et al. As far, model strains such as E. coli K12 or E. coli BL21 have been employed when using CRISPR/Cas for genetic manipulation in E. coli, but nobody has performed genetic manipulation in virulence genes of wild-type Escherichia coli. In this study, the authors focus on the application and efficiency of the CRISPR/Cas system in enterotoxigenic E. coli (ETEC) virulence genes. The improved gene editing technology provides great convenience for the study of virulence genes of wild-type Escherichia coli. The manuscript is suitable for publication in “PeerJ” but in order to improve the quality of the text, following “minor changes” are suggested:
1. in line 44-45, How to understand this sentence “Gene recombination efficiency ranged from 0 to 77.78% when the length of the homology arm was from 50 to 300 bp” Is the efficiency of gene editing high or low？
2. in line 96-98, It is mentioned that “our laboratory selected the two-plasmid system constructed by Jiang to perform genetic editing in E.coli”, both are gene editing technology in these two article, what is the main innovation of this article.


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: DIVERSITY OF RHIZOSPHERE AND ENDOPHYTIC FUNGI IN ATRACTYLODES MACROCEPHALA DURING CONTINUOUS CROPPING
Review round: 1
Reviewer: 1

Output:
Basic reporting: The authors used culture-independent Illumina MiSeq to evaluate the endophytic and rhizospheric fungi upon continuous cropping of A. macrocephala. The objectives of the paper are clearly stated in the introduction. The introduction is well structured and referenced as is the discussion. The authors shared the raw data and I suggest indicating in the manuscript the deposit of the data in some bank.
Experimental design: The material and methods are unclear about the experimental design. The authors need to clearly indicate how many biological replicas and how many technical replicas have been sequenced. Apparently, the authors pooled samples. Statistical analyzes are committed to this approach. Other comments are in the general comments.
Validity of the findings: I indicated my comments on data validation in the item “General comments for the author”
Additional comments: Line 47: term “microflora” is not appropriate. I suggest replacing the term microbiota.

Line 53: Authors could insert recent references to the first sentence.

M&M:
The caption of fig 1 is confusing. Consider the abbreviations used in the material and methods: 1-year fallow soil (S_F1), 2-year fallow soil (S_F2), 1-year healthy rhizospheric soil (RS_H1), 2-year healthy rhizospheric soil (RS_H2), 2-year root-rot diseased rhizospheric soil (RS_D), blank control soil with no A. macrocephala planted (S_CK)

Improve the text so that it is clear: five zones? How many plots? Replicates?
How deep was the soil sampled? Composite sampling (how many subsamples?)?
How long were samples processed after storage?

Line 116: Were the samples sequenced separately or pooled for sequencing? Make it clear how many biological replicas and how many technical replicas were used.
Line 117: The authors used which parameters to filter the reads: size, phred value, etc:
Were chimeras excluded? Have mitochondrial sequences been deleted? Communities have different amounts of reads. Did the authors rarefy and normalize data before analysis? Rarefaction is essential for comparing libraries with different amounts of reads.
How many reads have not been identified?
Did the authors adjust the significance value in multiple comparisons, for example using false discovery rate (FDR)?

Results:
The material and methods need to be clear in the sample design. The variation indicated in fig2d came from how many sample units? Did these sample units come from sample pools?
Fig2d: Which parameter to select a dominant genera?
Line 157: Indicate the statistical test together with the value of p.

Line 160: “The fungal diversity index was the highest in blank control soil, and the fungal diversity of the fallow soil samples were also higher compared with samples from continuous cropping”: How to state in larger or smaller values without statistical analysis?

Line 166: What criteria for choosing 25 most abundant fungi?

Discussion:
The discussion needs to clearly indicate whether or not there was a limitation in the sample design.


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: DIVERSITY OF RHIZOSPHERE AND ENDOPHYTIC FUNGI IN ATRACTYLODES MACROCEPHALA DURING CONTINUOUS CROPPING
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall English language needs to be improved for clarification.

More references for background introduction are needed.

Experimental methods and data presentations need to provide more details and support for the conclusions.
Experimental design: The experimental design is OK. However, the method needs to provide more details, for instance:

1. The soil sample collection, how to collect rhizosphere vs. endosphere samples; and how the Furasrium involved in the related study, any related experiments and the plants showed any related symptom?

2. How the sequencing library was set up and how the related bar codes were used?

3. All the soil content analysis did not show the standard deviation. A more detailed statistical analysis needs to be provided for clarification.
Validity of the findings: The methods, data presentation, and statistical analysis need to be improved and clarified to draw more accurate conclusions.
Additional comments: No additional comments


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

Input:
Title: DIVERSITY OF RHIZOSPHERE AND ENDOPHYTIC FUNGI IN ATRACTYLODES MACROCEPHALA DURING CONTINUOUS CROPPING
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors used culture-independent Illumina MiSeq to evaluate the endophytic and rhizospheric fungi upon continuous cropping of A. macrocephala. The objectives of the paper are clearly stated in the introduction. The introduction is well structured and referenced as is the discussion. The authors shared the raw data and I suggest indicating in the manuscript the deposit of the data in a gene-bank.
Experimental design: -	If I understand correctly, there were 54 samples totally. However, Figure 2-3 and Table 1 indicates there were 18 samples, I am very confused about this point，although I read you explanation in the responds letter.

-	Is it possible used abbreviation for the samples like this?
1-year fallow soil (FS_1),
2-year fallow soil (FS_2),
1-year healthy rhizospheric soil (RS_H1),
2-year healthy rhizospheric soil (RS_H2),
2-year root-rot diseased rhizospheric soil (RS_D)
1-year-old healthy root/stem/leaf/tuber (R/S/L/T_H1),
2-year-old healthy root/stem/leaf/tuber (R/S/L/T_H2),
2-year-old root-rot diseased root/stem/leaf/tuber (R/S/L/T _D)

-	Bioinformatics analysis is not part of statistical analysis, usually. (see details in the file attached)

-	More details of the statistical analysis could be added. (see details in the file attached)

-	the endophytic fungi determined by plants much more than soil. So, what is the point to analysis correlation between endophytic fungi and soil?
Validity of the findings: It is very import to described how statistical analysis conducted and show the results clearly. Because in this article it is not difficult to evaluate the validity of the findings without statistical analysis.
Additional comments: I indicated the comments in the file attached.


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: ELMO2 ASSOCIATION WITH GΑI2 REGULATES PANCREATIC CANCER CELL CHEMOTAXIS AND METASTASIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The paper is well written overall. The references used are sufficient.
The figures are prepared professionally and the raw data is present as well, however it is not always clear how this relates to the experiments. For example the F actin content looks like the raw data is given, however the figure shows relative F actin content without any indication how this was calculated. Without labelling which data point belongs to which condition, it is impossible to see whether the derived curves are reflecting the raw data. More information about the raw data and the actual manipulation and calculation of the data is needed
For the adhesion data for the PANC cells, there is an error in the calculation for si at 0ng in the raw data, it is not a huge difference, however that does not fill me with confidence that all calculations are correct
Experimental design: Overall the experimental design is good, however in particular the chemotaxis assay is lacking details in description of methodology. There are no details about what type of plates are used, whether there are transwell or others. Also, there is no description of how the adherent cells were counted, adherent cells do not migrate through a membrane, they will stick to the membrane, but the methodology described gives no details about the procedure.
Validity of the findings: The data looks robust, the migration data is very tight, which is a bit surprising since all experiments were done as an n=3 and woundhealing assays in particular show some variety of response, but the raw data provided supports this.
I don't like seeing the input bands in Figure 3 used twice, if all experiments are done on the same membrane, it is valid, however I'd appreciate if the input is only shown once.
Additional comments: It would be helpful to have the raw data annotated better, so it can be seen when each experiment was done and also how relative values were calculated.


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: ELMO2 ASSOCIATION WITH GΑI2 REGULATES PANCREATIC CANCER CELL CHEMOTAXIS AND METASTASIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: In lines 254-256, please list all the reference articles about ELMO3 study.
Experimental design: In order to give the reader a direct impression, the author is suggested to present a picture of immunofluorescence about F-actin Polymerization. Please refer the paper of PMID: 30345300
Validity of the findings: (1) The authors should explain why PANC-1 and AsPC-1 were chosen in this study. In Fig 1(A), it seems ELMO2 revealed a weak protein expression in PANC-1 cells. In addition, both two cell lines presented low invasiveness in normal condition in Fig 2(A). I was wondering if other suitable cell lines can be used such as Capan-1 cell. Or the author should optimize the experimental conditions.
(2) I suggest that the authors make a statistical analysis of whether silencing ELMO2 affects cell chemotaxis, migration, invasion and F-actin polymerization without CXCL treatment, at least make a mark on the Fig. 1 and Fig. 2.
Additional comments: Invasion and migration are important reasons contributing to the death of pancreatic cancer patients. In this manuscript, the authors thought ELMO2 could promote pancreatic cancer cell chemotaxis, migration, invasion and F-actin polymerization. Furthermore, they also found chemokine CXCL12 triggered Gαi2-dependent membrane translocation of ELMO2. Generally, this study is critical in exploring the role of ELMO2 in pancreatic cancer. However, these issues should be concerned by the authors.


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: ELMO2 ASSOCIATION WITH GΑI2 REGULATES PANCREATIC CANCER CELL CHEMOTAXIS AND METASTASIS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Introduction:
Line19: “knockdown” instead of “knockout”

The role of the ELMO protein (ELMO1, ELMO2 and ELMO3) family is described (line 40 - 55). It is however, not clear why the authors specifically focus on the role of ELMO2 in pancreatic cancer (as stated in line 55), and not on the role of ELMO1 or ELMO3.
For instance, is there any evidence/suggestions from the literature that links ELMO2 with pancreatic cancer? What about redundancy between the function roles of the three ELMO isoforms? I suggest the authors elaborate a bit more on this topic and clearly state why they choose to study ELMO2 in the specific context of pancreatic cancer.

The authors state that ELMO family members have been implicated in a variety of malignant cancers including glioma, breast cancer etc (line 52-53). I think it would be of interest to mention for the different types of cancer if these data were obtained from in vitro studies or in vivo studies.
Experimental design: Description of the Materials and Methods:

Transient transfection:
1. Line 105: western blotting is mentioned. However, the protocol for western blotting  (including a reference to the antibodies used), is not included in the manuscript. Please include.
2. Line 106 - 109: ELMO2 siRNA sequences are shown. It is not clear which of the three siRNAs is actually used throughout the experiments. Did the authors use only one siRNA to knockdown ELMO or did they use a combination of all three siRNA at the same time? Also, how specific are these siRNAs for ELMO2 (Is there any change of also targeting ELMO1 and ELMO3 with these siRNAs?)

Exogenous co-IP
Line 118: Please describe how cell the lysates were prepared.
Line 124: Please better specify the used antibodies (e.g. add catalog number)

Immunofluorescence:
What primary and secondary antibodies were used in the study.

Chemotaxis assay:
Line 148: The number of migrated cells in the chemotaxis assay seems rather low (Figure 1). Please mention how many cells were added to the upper compartment?  Also, include a reference for the type of transwell plate(s) that is used in this experiment.

Cell Invasion assay:
Also here, it is not mentioned how many cells were used and when they were added to the plates. Also include a reference for the type of plates that were used.

Adhesion assay:
Line168: How many cells in 100µL?

Statistical analysis:
Line 184: Add the number of replicates per experiment.
Validity of the findings: In Figure 1 and Figure 2 it is stated that all data are represented as the mean of three independent experiments. How many replicates were performed per experiment? Also, the raw data of these experiments are made available as supplemental files. However, it seems that the data of only one (out of three) experiments is given for each type of experiment. Can you clarify this, and if needed, add the missing data?

In Figure1B and 1C: Does the “siELMO2” condition refer to cells in which siELMO2_1,  siELMO2_2 or siELMO2_3 siRNAi was used? Or a combination of all the siRNA sequences?

It appears in Figure 1B that the siELMO2 condition by itself (thus, without CXCL12 stimulation, 0 ng) already gives a decrease in the number of migrated cells. Is this decrease statistically significant? If yes, can you speculate about a possible explanation for this?

Line 208-209: “CXCL12 generated a transient F-actin accumulation in PANC-1 and AsPC-1 cells, in line with previous findings.” Please, add a reference to the literature for this statement.

In Figure3A and 3B: Please specify what “Input” and “IP” means in the figure legend. My interpretation is that when PANC-1 cells are transfected with GV362-ELMO2, a clear (and expected) increase in expression of ELMO2 (70kDa) is observed in the input sample  compared to non-transfected (normal) cells. It seems, however, that also the expression of GNAI2 has increased in the GV362-ELMO2 condition compared to normal, which is rather unexpected. Is this interpretation correct? How can this be explained?
Similarly, when overexpressing GNAI2 it seems that also the expression level of ELMO2 increases?

Figure 4A: Adding the DAPI stain to all panels could help in giving a better visualization of the data.

In Figure 4A and Figure 4D: Where do the arrows point at? Pleas describe in the figure legend.
Additional comments: This manuscript is well-written and data are clearly presented and structured. It is, however, not immediately clear what the novelty and importance of the work is, given that the role of ELMO proteins in GPCR-mediated chemotaxis has been described previously. The novelty and importance of your work should be more clearly stated in the introduction part.


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

Input:
Title: ELMO2 ASSOCIATION WITH GΑI2 REGULATES PANCREATIC CANCER CELL CHEMOTAXIS AND METASTASIS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Although some defects cannot be made up due to the outbreak of new coronavirus pneumonia in China, this manuscript is generally acceptable.


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: ELMO2 ASSOCIATION WITH GΑI2 REGULATES PANCREATIC CANCER CELL CHEMOTAXIS AND METASTASIS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The authors have included the clarification and additional information in the Introduction and Materials&Methods section of the manuscript as requested. Figure legends were also updated as requested. Please note that the legend of Figure 3 was updated in the text part of the manuscript, but not yet in the separate Figure 3 file.
Experimental design: The Materials&Methods section has been updated and includes now all relevant information with sufficient detail. Missing information (e.g. western blotting procedure and references to used antibodies) is added.
Validity of the findings: Importantly, the confusion concerning the number of replicates per experiment and the number of independent experiments is solved. Missing raw data are made available and are better annotated.

However, it seems that the raw data for the invasion assay may not be entirely correct. When looking at the mean number of invaded cells with 100ng/mL CXCL12 under normal conditions, this number seems to higher than represented on Figure 2A of the manuscript. Please clarify.

The authors commented on Question 3 (the reason why siELMO2 by itself already gave a decrease in the number of migrated cells) and Question 5 (the interplay between the expression levels of ELMO2 and GNAI2) in their rebuttal letter, though they did not include these comments in the manuscript itself. Although I don’t consider this as a major issue, it could have enriched the discussion section.

As the authors suggest themselves, few small additional experiments (e.g. testing the specificity of siRNA towards ELMO2 by also including ELMO1 and ELMO3 samples) may have further strengthened the data, but I understand that the outbreak of Corona virus may have hampered this.
Additional comments: The manuscript has certainly improved in clarity and readability and now contains sufficient experimental 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: PREVALENCE OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE AT HIGH ALTITUDE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Basic reporting adequate, including components of the manuscript, professional structure, short manuscript.
Experimental design: Design is adequate, question clearly defined, impact of altitude in COPD prevalence is relevant from the scientific point of view and also from a practical point of view as there is a substantial proportion of individuals living at high altitude especially in some countries.
Limitation of the analysis derives from a limited number of original studies analyzing the question, although adding to a large number of individuals.

Methods are well described although not enough detail on obtaining adjusted OR in each study, and what variables were adjusted,  what is important for the summary adjusted OR
Validity of the findings: The summary analysis is correct (except previous observation on adjusted OR´s with unclear variables adjusted for)
Additional comments: Interesting review on the impact of altitude on prevalence of COPD, analyzing crude and adjusted OR. Study well described and including all important items.
Crude OR suggests altitude is a risk factor, but adjusted OR did not, as there was confusing factors better explaining it. Not clear what variables were adjusted for in the individual studies (should be clarified)
A recent review analyzing almost the same original manuscripts. Horner A, Soriano JB, Puhan MA, et al. Altitude and COPD prevalence: analysis of the PREPOCOL-PLATINO-BOLD-EPI-SCAN study. Respiratory research 2017; 18, worked on individual data.

In several parts of the manuscript the authors talk about incidence but all studies analyzed are cross-sectional and the manuscripts describe prevalences.

Several limitations, as ethnic composition changes with altitude, often with native (amerindian in America) ethnicity increases with altitude, also socioeconomic status, use of biomass as a fuel, and others,  that may change susceptibility.


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: PREVALENCE OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE AT HIGH ALTITUDE: A SYSTEMATIC REVIEW AND META-ANALYSIS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study “Prevalence of chronic obstructive pulmonary disease at high altitude: a systematic review and meta-analysis ” is a well-written article that identifies an important gap. However, to make this paper publishable, the author needs to respond to the following points.

Abstract.
Include the objective of the study.
If the objective was to analyze previous studies to conduct a meta-analysis and summarize the prevalence of COPD at high-altitudes and explore whether altitude is an independent risk factor affecting the prevalence of COPD; Why is the conclusion in terms of incidence?
Experimental design: Methods. The study "Prevalence of chronic obstructive pulmonary disease at high altitude: a systematic review and meta-analysis" is original and there are new data. Methods were described in detail, but there are some points that must be addressed:

1. The bibliographic search in four databases (PubMed/Medline, Cochrane Library, WoS, and Embase) is very limited. They should include other databases (SCOPUS, OVID, ScieLO, and LiLacs).
2. What was the definition of high altitude?
3. Was high altitude defined as >1500 m above sea level?
Validity of the findings: Results:
1. The analyses are robust, and statistically sound.
2. If the authors included cross-sectional (prevalence) studies. Why were determined the results and the conclusion in terms of incidence?
3. I suggest include Mexico in Table 1 study of Ana Maria Menezes/Country “Americas”
4. For a better understanding of the readers, in the Forest plot (Figures 2, 3 and 4) include the prevalence rates (values) of COPD.
5. For in study of Andre´s Caballero (Colombia), how they have synthesized the prevalence rates for COPD in Medellin and Bogotá, (height 1538 and 2640 m, respectively).

Discussion:
1. Revise. Line 175. “...incidence of COPD at high altitude…” in results is as prevalence.
2. The conclusions should be appropriately stated in terms of prevalence (not incidence)
Additional comments: The manuscript is clearly written. If there are comments as I have noted above, which should be improved upon before Acceptance.


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

Input:
Title: LOOK OUT: AN EXPLORATORY STUDY ASSESSING HOW GAZE (EYE ANGLE AND HEAD ANGLE) AND GAIT SPEED ARE INFLUENCED BY SURFACE COMPLEXITY
Review round: 1
Reviewer: 1

Output:
Basic reporting: This paper explores the relationship between gaze angle, gait speed and the complexity of surfaces by simultaneously measuring the orientation of the eyes with an eye tracker and the orientation of the head with an IMU.

The paper is well written and the design of the study is adequate for the aim of the study. In addition, I agree with the authors that when studying gaze in natural conditions head movements should be considered. Practically, this is not trivial and it can be challenging to measure head and eye movements simultaneously. Furthermore, it is good to see that the IMU was compared to a MOCAP system, this demonstrates that the authors are well aware of potential issues in their set-up.

However, I was slightly disappointed after reading the results section. I feel that the authors oversimplify their dataset by only analyzing the average head angle and average eye angle during the different surface conditions. This is a pity considering the rich dataset the authors have at their disposal. In addition, due to the limited analyses, I feel that the conclusions drawn in this paper are not yet supported by the data.
Experimental design: The design of the study is adequate, but I do have serious concerns about the analyses. Please find specific comments below.
Validity of the findings: Due to the limited analyses, I feel that the conclusions drawn in this paper are not yet supported by the data. Please find specific comments below.
Additional comments: 1.	The main reason for this study appears to be that previously most studies only analyzed the eye movements of the participants and neglected the head movements. I certainly agree that this can be problematic, especially in natural conditions and in relation to surface complexity. However, in this study, the eye movements and head movements are completely separated. And although the authors mention in the introduction that: “[while] gaze is often used by researchers to refer only to eye movements, here we define gaze as the combined position of the eye and the head”, this appear to be completely overlooked in the results of this study. The only reference to gaze in the results section is in the first paragraph, in which is referred to Figure 2 and Figure 3. However, in both figures, the gaze is not shown, only the head pitch and elevation angles of the eye movements are plotted. It is difficult to interpret both figures without this information.

For example, the plot in Figure 3 of the relative frequency distribution for the “indoor uneven” surface could be the result of participants keeping their gaze steady and effectively cancelling the head movements with their eye movements (i.e. head about 20 deg down from the average position, while counter rotating about 20 deg up with the eyes to keep gaze stable using the vestibular ocular reflex, thus a negative correlation between eye and head), or by vertically scanning the environment (i.e. a large eye movement of the head accompanied by a large eye movement in the same direction, thus a positive correlation between eye and head), of by uncorrelated movement of the eye and head.

In addition, the results of the ANOVA reported in the first paragraph of the results (lines 200-203) do suggest that the gaze was calculated, but further inspection reveals that the main effect of the ANOVA and the subsequent reported values of “gaze” are only the mean of the average head and eye angles. And as I just pointed out that does not necessarily reflect the behavior of the participant. The same appears to be the case for the contribution to gaze from head pitch in lines 203-204.

In sum, instead of inferring gaze based on average head and eye angles,
the authors should first calculate the gaze orientation based on the individual raw (or filtered) eye and head traces, before averaging the angles for different surface. Which is in fact exactly what Tomassi et al, 2016 did (the authors do suggest that they followed their approach in line 67, but they actually do not).

2.	Furthermore, I feel that only presenting the averaged angles is an oversimplification of the behavior of the participants. I assume that they did not keep a fixed eye and head orientation during the trials. It would be beneficial to analyze the data further, for instance looking into the variance of eye and head movements during trials. I can imagine that they explored the complicated surfaces more, probably reflected in more saccades and/or larger variance in gaze orientation (and probably also in head/eye angles). In addition, I would like to see some data traces of eye, head and gaze angles during trials on the different surfaces (representative trials would be a good start, trying to visualize the data of all participants even better).
In addition, it might be interesting to have a look at gaze projection on the ground, i.e. how far ahead they directed their gaze, especially in combination with the speed of gait.

3.	In the abstract it is stated that: “Eye and head pitch angles did not correlate across any surface, thus can be considered independent measures.” However, only the average head and gaze angles of the participants were correlated. I assume that the participants actually did coordinate their eye and head movements during the trials, at least to some extent, to keep a stable image on the retina. You made the data of the head and eye movements independent by first averaging them, so it is not a surprise that your analysis does not show a dependence. The same goes for the statements in the abstract that: “[it] provides the novel finding that head movements provide important independent contributions to gaze location.” You cannot conclude this based on your current analyses.

4.	Data from 9 of the 20 participants was excluded. Although I know that these kind of set-up can be technically challenging and sometimes eye trackers have troubles tracking certain eyes etc, excluding almost half of the participants is too much to justify it only based on the vague reason of technical errors. Please elaborate on the reasons why the measurements failed for these participants. In addition, I noticed that there is actually data on all surfaces for these participants in the dataset, was all data useless, or did you exclude participants if the data quality was insufficient on one or more surface (and if so, based on what kind of criteria).

5.	I tried to have a look at the raw data. However, without additional information, I could not explore the data myself. Please provide preprocessed data, e.g. head pitch and eye orientation over time with time stamps that allow to synchronize the data.

6.	In the introduction it is stated that “For example, Matthis and Fajen [1] & Marigold and Patla [9] only considered eye movement during walking over complex surfaces.”. And although most studies did not specifically analyze the kinematics of the head and eyes simultaneously, several studies actually did measure the 3d gaze orientation, thus incorporating simultaneous measurements of head movements and eye movements. See for instance reference [3]. Mathis et al., 2018, in which they looked at the 2D projected position of gaze on the ground. In addition, there is more literature about eye-head coordination in other tasks. Please discuss these studies in the introduction.

7.	In Figure 3, the distribution of head and eye angles are plotted for the different surfaces. I noted that, although the relative frequency is low, that there are measured eye angles of >30 degrees. Considering the normal range of eye movements (see e.g. Lee et al. 2019, EYE, Differences in eye movement range based on age and gaze direction), I wonder whether these might be artefacts rather than actual eye movements.

Furthermore, please change the label on the y-axis. You do not plot gaze, but only the orientation of the eye and head.

8.	Supplementals: Please provide more detail about your set-up. What kind of IMU’s did you use and what kind of MOCAP system? And please provide additional information about the marker placement, i.e. number of markers and placement of markers, and how you calculated head pitch and speed of gait from that.


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: LOOK OUT: AN EXPLORATORY STUDY ASSESSING HOW GAZE (EYE ANGLE AND HEAD ANGLE) AND GAIT SPEED ARE INFLUENCED BY SURFACE COMPLEXITY
Review round: 1
Reviewer: 2

Output:
Basic reporting: It is clear and well written with sufficient background and a good structure.

I can confirm that raw data is shared on fig share.
Experimental design: This is original research which addresses a meaningful question. It is performed to a rigorous standard.

Methods-wise, I think more information could be given about how the gaze angles are calculated as at present it would be hard to replicate this. The eye tracker presumably gives eye-in-head data and/or the point-of-regard in the scene camera. I'm not sure whether this is what is analysed or if the authors have head position or just work out the offset relative to gravity.
It is also not clear how the "percentage contribution of eye or head" to gaze is calculated.
It is also not clear how the eyetracker was calibrated.
Validity of the findings: Everything seems robust here.

I didn't really understand why eye and head were correlated in the way that they were. It seems like we would expect an anti correlation (because if the head points down the eyes would have to point up maintain fixation on the same point.
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: LOOK OUT: AN EXPLORATORY STUDY ASSESSING HOW GAZE (EYE ANGLE AND HEAD ANGLE) AND GAIT SPEED ARE INFLUENCED BY SURFACE COMPLEXITY
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have comprehensively responded to my comments and have sufficiently addressed all my major concerns. I am particularly pleased to see the authors have added the gaze data to the manuscript, that they have changed their conclusions accordingly and that they have added the scripts and additional data to facilitate exploration of their data by others.

There are a few minor issues that needs attention, but they should be quite easy to fix.

General:
-	Please be consequent in terminology about gaze/head/eye direction. Now different words are used which do not always have the same meaning in this context: orientation, position (which is not the same as orientation in this case), angle , pitch, and direction.
-	I appreciate that the authors added the data on the gaze projection on the ground with the speed of gait in the supplementary materials. I think that it would be a valuable addition to the main manuscript and would therefore encourage the authors to move the table from the supplemental materials to the manuscript.

Title:
-	I prefer “speed of gait” instead of “gait speed”. Please change throughout the manuscript. I suspect that you made this choice because of the word count of the abstract, but I think that this can be fixed easily by removing one sentence in the significance section of the abstract.

Abstract:
-	Lines 29-31: wording is a bit awkward. It is now insufficiently stated that previously only the eye movements were measured, but that head movements contribute to gaze as well and that therefore it is important to measure both simultaneously. In your introduction you already mention that: “it is important to independently assess head as well as eye movements to understand how surface complexity influences gaze, and to see how this relates to changes in gait.” Please rephrase the background section of the abstract.
-	Research question: I think that the wording in sentence I mentioned above is stronger. Please consider rephrasing the research question
-	Results: I would change the order, by first describing the results of surface complexity on gaze and then indicate that head movements play an important part in the changes in gaze.
-	Significance: Considering the limited word count I would suggest removing the last sentence.

Introduction:
-	Lines 66-67: “Lastly” to “head movements.” Your definition of gaze is not clear enough. Gaze is the combination, or result’ of head and eye movements. Gaze is effectively the orientation of the eye in a world reference frame, in this case calculated based on eye-in-head data of the eye tracker and head-in-space of the IMU.

Methods
-	I appreciate the addition of the reason why you had to exclude the data of 9 participants.
-	Lines 147-148: “Vertical pupil” to “they walked”. This is rather vague. I assume that you used the eye-in-head data in degrees. And you need to use the gaze data to calculate how far ahead on the ground participants were looking, i.e. combination of eye and head data. For me, vertical pupil movement refers to the uncalibrated data of the movement of the pupil in pixels in the video data.
-	Repeated measures ANOVA only on eye and head data? Why not on the gaze data as well. That will provide the answer to your main research question, i.e. to understand how surface complexity influences gaze. Edit: Based on the results it appears that you actually performed the ANOVA on the gaze data. In that case, add it to the methods.

Results:
-	Line 210. This heading should be rephrased for clarity. For example something like this: Analyses of the orientation of the eye, head and gaze
-	The positive correlations between eye and head angles for the indoor flat and outdoor paved surface are a bit surprising. In my opinion, it indicates a coordinated scanning pattern (i.e. head lowered accompanied by lowering of eyes). I think it would be good to explain this in the result section and maybe elaborate a bit more on this in the discussion section.
-	Lines 240-249: Why not calculate the correlation between gaze and speed of gait? Again, that is in line with your main goal.

Discussion:
-	Lines 252-253: Here the aim is clearly stated, but it differs slightly from what is stated in the abstract and introduction.
-	Lines 263-265: I think this is a relevant addition to your results and therefore it belongs in the results section, not in the supplementary materials.
-	Lines 274-275: I think that the statement that “eye and head pitch angle measures were not
predictive of one another” is a bit too strong considering the analyses you did. I suspect that in reality, periods of positive correlations (e.g. making a gaze saccade to the next point of interest) are alternated with periods of negative correlations (e.g. keeping gaze stable at point of interest with the VOR to counteract the movement of the head due to walking). You only looked at the mean correlation. And therefore, perfect alternation of positive and negative correlation periods, and thus perfect coordination of eyes and head, would in your analysis result in an average correlation of zero.
Experimental design: No additional comments.
Validity of the findings: No additional comments.
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: LOOK OUT: AN EXPLORATORY STUDY ASSESSING HOW GAZE (EYE ANGLE AND HEAD ANGLE) AND GAIT SPEED ARE INFLUENCED BY SURFACE COMPLEXITY
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: The authors have addressed the minor analysis/method points I raised and the manuscript is now acceptable.


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: PREVALENCE AND RISK FACTORS OF HELICOBACTER PYLORI INFECTION AMONG CHILDREN IN KUICHONG SUBDISTRICT OF SHENZHEN CITY, CHINA
Review round: 1
Reviewer: 1

Output:
Basic reporting: I review of the English applied in the manuscript should be revised. Nothing very problematic but there are some sentences not well explicit. Very good manuscript with interesting results. However, I suggest demonstrating the association and non-association with other risk factors, if questioned and analyzed.
The discussion should be more developed and supported in more scientific articles.
Experimental design: Good methodology applied. The questionnaire should be more explicit and the questions should be better described in the Materials and Methods section.
Validity of the findings: Nothing to add.
Additional comments: Congratulations for the present manuscript. The authors have found very interesting results on the risk factors and prevalence of H. pylori among children which is a very important public health issue. I suggest to verify the adding of other risk factors associated with the presence and non-presence of H. pylori and explain the non-association between some risk factors analyzed.


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: PREVALENCE AND RISK FACTORS OF HELICOBACTER PYLORI INFECTION AMONG CHILDREN IN KUICHONG SUBDISTRICT OF SHENZHEN CITY, CHINA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: There is no novelty of this research as the study aims to explore different well known socioeconomic and demographic risk factors for Helicobacter pylori infection and its results only replicate the current knowledge on the subject.
The only part of the study that can be considered as novel is the estimation of the prevalence of Helicobacter pylori infection in a subdistrict of one city in China, therefore the article should be published in a medical journal on a national scale.
Validity of the findings: The validity of the findings for the international audience is questionable .
Additional comments: No additional comments