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: MAGNITUDE OF NON-ADHERENCE TO ANTIRETROVIRAL THERAPY AND ASSOCIATED FACTORS AMONG ADULT PEOPLE LIVING WITH HIV/AIDS IN BENISHANGUL-GUMUZ REGIONAL STATE, ETHIOPIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Comments
Abstract
1. In the last sentence of the introduction section of your abstract (from line 24-26) you showed your objective that is good. But my comment is regarding your title. Here you want to show the prevalence of non-adherence to ART. So better to modify  your title” Magnitude of non-adherence to ART treatment and associated factors among people living with HIV in Benishangul-Gumuz Regional State,2 northwest Ethiopia”
2. Methods section
Line 27: Do you mean all sampled population participated in the study? Your response rate was 98.9 % which means from your sampled population around 389 participants were participated. So try to see it clarity
3. Conclusion: First conclude about the outcome variable then you can recommend for your outcome or independent variables. You try to mention the recommendation but better to put recommendation as a title with conclusion.
Methods
1.	Line 119-120: you exclude patients with cognitive problem,i.e. good but my concern is How do you know this population to exclude? Were you use tool to screen or what?

Results
1.	Line 230:……. >95% (what is 95%? It is not clear,is that to mean confidence? And correct though out your data)
2.	Line 242-243: ……..other socioeconomic factor strongly found impeding ART adherence (AOR: 2.1, CI: 6.1–56, p <0.001) .The confidence interval is too large. This might be because of small number of patients who have food insecure and that is why your confidence interval is high. The other thing is you measured food insecurity by HFIAS as continuous variable. Your model was logistic regression. How you manage/enter to SPSS both categorical and continues variable simultaneously and how you used AOR for continuous variable to explain risk? As we can see here majority of participants were near to zero i.e. below mean. So better to clear with your analysis and description.If you have reference/possibility better to dichotomize HFIAS rather than taking as continuous variable.
3.	found impeding ART adherence (AOR: 2.1, CI: 6.1–56, p <0.001) and malnutrition was among factors deterring PLWHA to attain the required >95% ART adherence level (AOR: 0.9, CI:0.82–0.98, p = 0.018).This AOR shows being malnourished is protective? this is contrary to a logic even difficult for recommendation. So better to have a look the analysis.
4.	Line 282-283 and 321-323: recommendation is better to be at the end with conclusion
5.	Table 2: 1-In your table add frequencies for each variable since we can not be sure whether your COR is correct or not
     2- show the AOR some variables like gender,Religion......I know this variables were not eligible for multivariate but better to show its AOR  with  its CI or if you were taking  bivariable, no need of listing this variables here

6.Regarding ethical consideration there is no information about the study from the regional health biro or any concerned body from Ethiopia. Add informations who allow the study from the region health biro. To whom you summit the ethical clearance after you got from College of Human Sciences at the University of South Africa.


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: MAGNITUDE OF NON-ADHERENCE TO ANTIRETROVIRAL THERAPY AND ASSOCIATED FACTORS AMONG ADULT PEOPLE LIVING WITH HIV/AIDS IN BENISHANGUL-GUMUZ REGIONAL STATE, ETHIOPIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: No
Experimental design: No
Validity of the findings: No
Additional comments: Comments to the authors
Abstract
Methods section
1.	Line 27: Do you mean all sampled population participated in the study? Your response rate was 98.9 % which means from your sampled population around 390 participants were gave a response. Still it lacks some clarity. “We studied 394 peoples living…..” For me all 394 participants gave a response. So better to mention with only with 98.9% response rate rather than mentioning the frequency (394) to avoid the confusion. Then avoid “s “from people…….”
Conclusion:
2.	How do you conclude about magnitude of non-adherence here? What factors influence non adherence? But most of the statements seems recommendation rather than conclusion. So either add recommendation with conclusion as a title or modify your conclusion. Even some contents seems out of your objective.
Methods
1.	Line 125-126: you exclude patients with cognitive problem, i.e. well but my concern is how do you know this population to exclude? Were you use tool to screen or what?
Were you exclude known mental health problem or were you screening during interview?
If you were screening during interview your data collectors were not appropriate to screen all mental health problems. But if already patients were known and diagnosed before that is possible. Otherwise you needed to use screening tool.
2.	Line 279-280: ……..other socioeconomic factor strongly found impeding ART adherence (AOR: 2.48, 95% CI: 11.3, 3.2; p <0.0001). You tried to manage large confidence interval but sill reversed. So have a look at again.
3.	Table 2: 1-In your table add frequencies for each variable since we cannot be sure whether your COR is correct or not. Yes off course, I see the frequencies but I want to say the crosstab frequencies. For example gender (female (non-adherence/yes---?, no--?) ….male non adherence( yes-----?,No…..?) and your table needs modification. It is not necessary to put p-value of COR.
4.	2- show the AOR some variables like gender, religion......I know these variables were not eligible for multivariate but better to show its AOR with its CI. I know this variables are not associated at multivariate but it fulfil the minimum criteria. So you need to show further this variables is not associated at multivariate with its AOR and CI.
5.	 “Permission to conduct the study was obtained from the Benishangul Gumuz Regional State Health Bureau. The respective health facilities gave permission prior to data collection” In what way you get permission from Health Bureau written or verbal? Because you said twice permission i.e  health facility and Health Bureau. So state what type of permission you got.


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: MAGNITUDE OF NON-ADHERENCE TO ANTIRETROVIRAL THERAPY AND ASSOCIATED FACTORS AMONG ADULT PEOPLE LIVING WITH HIV/AIDS IN BENISHANGUL-GUMUZ REGIONAL STATE, ETHIOPIA
Review round: 3
Reviewer: 1

Output:
Basic reporting: This manuscript titled "Adherence to antiretroviral therapy among people living with HIV in Benishangul-Gumuz Regional State, Ethiopia" is very well drafted and written. However, there is no novelty in this paper, but maybe it will help in planning future antiviral therapy program in Gumuz Regional State, Ethiopia
Experimental design: No comment
Validity of the findings: No comment
Additional comments: This manuscript titled "Magnitude of non-adherence to antiretroviral
therapy and associated factors among adult people
living with HIV/AIDS in Benishangul-Gumuz Regional
State, Ethiopia" is a resubmission of the previous article "Adherence to antiretroviral therapy among people living with HIV in Benishangul-Gumuz Regional State, Ethiopia"  has been very well-drafted and written. The authors have corrected most of the issues raised by other reviewers. This paper will help in planning future antiviral therapy program in Gumuz Regional State, Ethiopia. I would recommend this paper 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: MAGNITUDE OF NON-ADHERENCE TO ANTIRETROVIRAL THERAPY AND ASSOCIATED FACTORS AMONG ADULT PEOPLE LIVING WITH HIV/AIDS IN BENISHANGUL-GUMUZ REGIONAL STATE, ETHIOPIA
Review round: 3
Reviewer: 2

Output:
Basic reporting: no
Experimental design: no
Validity of the findings: no
Additional comments: I am satisfied with your respones


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: MECHANICAL NOCICEPTIVE ASSESSMENT OF THE EQUINE HOOF FOLLOWING DISTAL INTERPHALANGEAL JOINT INTRA-ARTICULAR ANESTHESIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Overall, one of the best written papers that I have read in quite a while.  Interesting application of pressure algometry to address an important clinical issue.
Experimental design: Line 45 – I am not sure that ‘dorsal sole’ is a correct anatomical description.  To me, all aspects of the sole are on the palmar surface.  Need to recheck to confirm appropriate terminology.

Line 75 – Need to clearly state if the limbs were loaded or unloaded for all measures.

Line 78 - How was 6 kg determined to be an appropriate level to prevent tissue injury?

Figure 1 – I am quite surprised that the majority of your MNT values are < 1 kg, hence do not provide any room for discrimination of values.  It seems that the probe tip used was too severe in this model.  If all values are < 1 kg then there is no real possibility of identifying any treatment effects (local anesthetic) given the intra-horse and inter-horse variability in MNT values.

Did you do pilot work to define the probe tip used in this model or was it borrowed from another research group?  All of this needs to be added to the discussion as a possible factor for no significant differences.

I would also recommend doing power calculations to provide some guidance to future researchers using this model as to the sample size required to show significant results.

The legend also needs to let the reader know that each marker represents a single horse.

Figure 3 – Need to explain why the baseline coronary band MNT values are so low (<1 kg/cm2), compared to values reported in Haussler, Behre 2008 and others.

Need to add some discussion of the strengths and weaknesses of the algometry technique.  It seems that you do not question the MNT values at all and attribute all changes to effects of the local anesthetic, which may or may not be true.

What is the reliable lowest range of values that the algometer used in this study can reliably detect?  Is it sensitive enough to repeatably differentiate between 0.5 versus 0.7 kg/cm2, as an example?
Validity of the findings: No comment
Additional comments: No comment


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

Input:
Title: MECHANICAL NOCICEPTIVE ASSESSMENT OF THE EQUINE HOOF FOLLOWING DISTAL INTERPHALANGEAL JOINT INTRA-ARTICULAR ANESTHESIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: General comments to the authors:

This is an interesting manuscript which will merit publication. The information provided in this manuscript may be useful for veterinarians that deal with equine lameness and use lidocaine to perform these diagnostic blocks. Therefore, the authors must include in the discussion that the results of the study are valid only if LIDOCAINE is being used to block the distal interphalangeal joint. Most of the literature cited and used to contrast the results of the current study have used a long lasting local anesthetic solution (mepivacaine) which could have been a different effect under the described study set up/design. Please keep that in mind as you cite those papers.
Specific comments to the authors:

Abstract or summary: please remove “However, signs of analgesia in the dorsal lamellar tissue, beginning 15 minutes after administration, suggest that anesthetic diﬀusion may decrease the speciﬁcity of DIPJ anesthesia in some horses” as this was not demonstrated in the current study.

Line 3: Please clarify what you mean by “associated with errors”.

Line 4-8: Please reword to be more specific: there is no proven diffusion of local anesthetic solution from the distal interphalangeal joint into the periarticular tissues (if so please provide a reference). My understanding is that because the palmar [plantar]outpouching of the DIPJ is in close juxtaposition to the digital neurovascular bundles, this block can desensitize the digital neurovascular bundle and indirectly desensitize the extra-articular structures (i.e. the sole, navicular bone, etc).

Line 10: please define IA.

Line 17: please define MNT.

Line 32-43: please consider including images or illustrations of the hoof defects created for pressure algometry. These images will make readers understand your set up.

Lines 190-191: Your results do not support this statement so please reword. You stated in the first line of the discussion “This study demonstrates that IA injection of the DIPJ with 5 mL of lidocaine does not result in significant increases in MNTs within the dorsal lamellae, sole, coronary band, or heel bulb”.

Discussion: The authors must include in the discussion that the results of the study are valid only if LIDOCAINE is being used for intra-articular anesthesia of the distal interphalangeal joint. Most of the literature cited and used to contrast the results of the current study have used a long lasting local anesthetic solution (mepivacaine) which could have been a different effect under the described study set up/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: MECHANICAL NOCICEPTIVE ASSESSMENT OF THE EQUINE HOOF FOLLOWING DISTAL INTERPHALANGEAL JOINT INTRA-ARTICULAR ANESTHESIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have addressed all prior reviewer comments appropriately.
Experimental design: No comment
Validity of the findings: No comment
Additional comments: Thank you for making the suggested edits and clarifications.


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: MECHANICAL NOCICEPTIVE ASSESSMENT OF THE EQUINE HOOF FOLLOWING DISTAL INTERPHALANGEAL JOINT INTRA-ARTICULAR ANESTHESIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment.
Experimental design: No comment.
Validity of the findings: No comment.
Additional comments: Dear authors, I am satisfied with the changed made to the revised manuscript, however as I suggested before including images or illustrations of the hoof defects created for pressure algometry would be useful to the final manuscript. I consider this point important to illustrate the manuscript and provide clarity.


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: ANTIBACTERIAL ACTIVITY AND MECHANISM OF SANGUINARINE AGAINST PROVIDENCIA RETTGERI IN VITRO
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript describes antibacterial activity and mechanism of sanguinarine against Providencia rettgeri. The manuscript presents potential valuable information on novel antibiotics, but it suffers from some lack of consideration of some aspects. Major revision is needed.
Major comments:
1. The results revealed that the MIC of SAG against P. rettgeri was 7.8 μg/mL. Accurately, i do not think this is a strong antibacterial effect. Do authors have done experiment of SAG acting on other bacteria? Or any references?
2. This manuscript must be edited for proper grammar and overall style. For examples, the sentence of line 58-59 is too elusive to read. The subsequent sentences (line60-62) also presents grammar problems.
Experimental design: The authors only mentioned Sanguinaria was purchased. Was SAG extracted from Sanguinaria? The key process was missed in 'Materials and Methods'.
Validity of the findings: Line 73, authors mentioned 'this study aimed to investigate the antimicrobial and antibiofilm activities of Sanguinaria against P.rettgeri'. Is SAG equal to Sanguinaria?
Additional comments: 1. Line 64-65,  SAG is extracted from the rhizome of Sanguinaria canadensis. In fact, SAG can be extracted from Chelidonium majus, Macleaya cordata, et al. Please find more references and revise this paragraph.
2. Line 222-225, which specific statistical method was used in MS?
3. Line 30-31, Whether decrease in intracellular ATP could definitely cause damage of the cell membrane?
4. The authors did several experiments of SAG on P. rettgeri. However, each experiment seemed independent and unconnected. Authors need to integrate all experimental results and explain how SAG inhibite P. rettgeri in 'Discussion'.
5. Figure 2 need to recreate, especially 2A and B.


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: ANTIBACTERIAL ACTIVITY AND MECHANISM OF SANGUINARINE AGAINST PROVIDENCIA RETTGERI IN VITRO
Review round: 1
Reviewer: 2

Output:
Basic reporting: The overall expression of this article is clear, but the words or sentences in many places are not professional and fluent. Please seek a native English translator or professional translation service to help modify the language of this article.
Given that this article is about using sanguinarine as a potential food preservative, by studying its antibacterial activity and antibiofilm activity, the safety of SAG is not mentioned in the introduction, which limits the significance of the research.
Some references are incorrectly marked in the article, please carefully find and correct them.

Table 1 is missing the MIC experimental  data of SAG, please add.
Experimental design: The experimental design of the article is very comprehensive. It not only studies the antibacterial activity and mechanism of SAG, but also the antibiofilm  activity of SAG, which provides extensive data for its application as a food preservative. However, the description of the MIC experiment is not accurate enough. Choi et al used the disc diffusion method to measure the MIC and you used the microbroth dilution method. This is the wrong reference, and why is there no reference for the standard MIC measurement method (Clinical and Laboratory Standards Institute. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard (9th Edition). Wayne: Clinical and Laboratory Standards Institute)?
Validity of the findings: None
Additional comments: 1. line 30, "The gradient diffusion method" is an inaccurate expression,It should be microbroth dilution method according to MIC test.
2. lines 53,182, 370, 379, Incorrect reference notes.
3. line 78, Please provide the city and country of the reagent manufacturer used in this article.
4. lines 79-81, After ten-fold dilution, the SAG concentration was 1 mg / ml and the dmso was 100% v / v. It is not difficult to infer that the highest concentration in the MIC experiment was 500 ug / ml, and the dmso concentration was 50%. In fact, only when the dmso concentration is lower than 2%, the bactericidal effect will not be affected. Have you considered the effect of DMSO on the antibacterial effect?
5. line 94, The bacterial concentration is 10^2 CFU/ml and the reference is 5x10^6 cfu / ml. Why is it diluted to this concentration? The dilution is likely to affect the result of the MIC measurement.
6.line 96, replaced "visible" by "viable".
7. line 102, "1:8, 1:4, 1:2", 1/8，1/4，1/2 are more appropriately.
8. line 109, 163, Reference citation malformed.
9. line 167, repalced "alcohol" by "ethanol".
10. line 233, delete "Table1".
11. lines 233-234, "The results of the antibacterial activities  ... against P. rettgeri." is useless description.
12. lines 234-235, "SAG was effective with an MIC  of 31.25, 15.6, and 7.8 μg/mL against P. rettgeri." could be described as "SAG exhibited excellent antibacterial activity against  P. rettgeri 1-8, with the MIC values of 31.2,..., respectively." for better.
13. line 242, What is "this" mean?
14. line 274, delete "exhibited".
15.lines 312-313, " eDNA and proteins red" As both eDNA and proteins were stained to red, how to separate them?
16. line 337, antifungal??
17. lines 342-345, "A similar study found... peppermint essential oil." What is the purpose of these citation? These reagents are not related to SAG. These are just quotations of others' results without further discussion!
18. lines 350-353, " A similar study by... to plant extracts." These are just quotations of others' results without further discussion!
19. line 361, Previous studies? not this paper?
20. line 377, observed ??
21. line 215, What is LUT and P.R cells？Do not be metioned above.
22. Figure 7,  (A–C; magnification, 10,000×) and CLSM (D–F; 3D). Wrong label, A-D for FESEM; E-H for CLSM.


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: ANTIBACTERIAL ACTIVITY AND MECHANISM OF SANGUINARINE AGAINST PROVIDENCIA RETTGERI IN VITRO
Review round: 2
Reviewer: 1

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


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

Input:
Title: ANTIBACTERIAL ACTIVITY AND MECHANISM OF SANGUINARINE AGAINST PROVIDENCIA RETTGERI IN VITRO
Review round: 2
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Although this manuscript has been revised thorough, the following issues should be further clarified before publication.

1.	Materials & Methods  Minimum inhibitory concentration (MIC) test, the authors used the agar dilution method to determine the MIC by mixing the bacterial samples and SAG into 96-well plate rather than 9-cm Petri dishes according to the reference. As we know, 96-well plate is normally used in the broth microdilution and 9-cm Petri dishes is used in the agar dilution. Why do you choose the 96-well plate in this study?
2.	In the previous manuscript, the bacterial concentration used in MIC test is 10^2 CFU/ml. You've changed it to 106 CFU/mL in the revised manuscript (line 105), but without any description of repetitive experiment for the influence of bacterial concentration on the MIC values. So the results of the MIC test are strongly doubtful.
3.	Line 432, “Inhibits” should be “inhibits”, and “Candida albicans and Staphylococcus aureus” should be italicized.
4.	Caption of Figure 1, replace “1:8, 1:4” with 1/8, 1/4.


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: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 1
Reviewer: 1

Output:
Basic reporting: In table 3 the p-value for the association between hypermobility at various site and sex should be reported
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: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is clearly written in professional English. It is appreciated that the raw data was provided and that the structure conforms to the standards of the journal.
The Introduction is clear overall. There are a few suggestions to improve the clarity of the text.
-In line 48, the term “flexibility” should be removed in this sentence and just state “normal range”. As the authors correctly state in the next sentence, joint hypermobility is related to ligamentous laxity, but the term flexibility implies mobility of musculotendinous tissue.
-In line 74, the authors also should mention other cutoff scores for GJH that are applied in populations of children. These are mentioned in the Discussion, but it would be useful to provide here in the Introduction to prepare the reader for the Methods where cutoff points of 4 and 6 are used.
-In line 78, a reference is needed to support that GJH in children is related to developmental delay in children.
-In line 83, the authors should indicate why the Majimaah region is of particular interest for this study.
Experimental design: The investigators aim to examine the prevalence of GJH and associated factors in school-aged children in the Majmaah region. This study appears to be within the scope of this journal. Although prevalence of GJH and associated factors have been studied in other populations of children around the world, this study is unique in that it is the first to examine this condition in the Saudi Arabia /Gulf region. This paper would add important information to this body of literature.

Overall, the research question is clear, but the analytic approach appears to deviate somewhat from the study objective. Specifically, it is not explained why the data were analyzed by sex (male/female) for some analyses but not shown for the whole sample; the research question and statistical analysis do not describe a plan for stratifying by sex. Another concern about the analyses by sex is that numbers in some cells end up being quite small (<5). This makes the results imprecise, and approaches using exact tests are needed. Also, variables that are continuous, like age and BMI, could be kept as continuous in models, particularly since the age categories of 8-11 and 11-14 may not be clinically meaningful for GJH and since the number of participants in the higher BMI categories is low, which leads to very small numbers for analyses.

The methods for data collection are presented with enough detail that this study could be replicated. To improve the rigor, the reliability of the two physical therapist examiners should be presented (line 122). Details are needed about how many schools were approached and agreed to participate and how many students were invited. These data would help with determining the success rate with enrollment and determining whether the results from the study sample are representative of the broader population.
Validity of the findings: The underlying data were provided and appear to be robust.

The authors should comment in the Discussion on why they think the prevalence of GJH was different in their region as compared to some other locations.

The conclusion that the prevalence of GJH in the present study was lower than other studies worldwide (line 240) differs somewhat from the literature presented in the Discussion. Line 182 suggests that the present study had similar results to Denmark, Egypt, and the United Kingdom, but was lower than Iceland, Italy, and India. The conclusion should reflect that the present study did agree with results from some populations.
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: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 1
Reviewer: 3

Output:
Basic reporting: The manuscript is well written and in the according format. However the relavance of the study as well as the basic rationality is not well enough described. Reporting prevalances in a region from which no prevalance data has been described is in my opinion not a really strong argument. In addition why authors try to predict the occurence of GJH is unclear. GJH does not equal pathology. The reasons for this study should be detailed more clearly
Experimental design: The design and statistical analyses are correct considering the research objectives (which are not yet logical in the current text). Still i find some methodological issues considering the choice of cut-off points for GJH. In 2017 Juul-Kristensen published the international consensus for classifying GJH and in the current manuscript older and outdated cutt-offs are utilized. This is a major concern for the design of the study, next to the previously mentioned relevance
Validity of the findings: As mentioned previously
Additional comments: The study is well written and logically constructed in terms of analysis. But i fail to see the utilty of prediciting the occurence of GJH. In my opinion this is a variation of normality and not the same as pathologgy

THe study of Jacky Clinch is often mentioned however the findings of this study was not that BMI is a predictor for GJH. But children with GJH and obesity are more vulnarable to pain.


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: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: Author reported in the table 3 p-value unadjusted for multiple comparisons.
Author must checked the data in the table because the totals are incorrect


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

Input:
Title: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 2
Reviewer: 2

Output:
Basic reporting: No concerns. The authors have been receptive to prior comments and have added appropriate references and clarified their work.
Experimental design: No concerns. This study appears to be within the scope of this journal, and the research question and analytic approach are clearly presented.
Validity of the findings: The Discussion and Conclusions are reasonable and well-supported based on study results and the literature.
Page 13, line 235: There is an error where the term females is used twice in the sentence, "Using cut-off ≥6, the prevalence of GJH was associated with BMI in females and not in females."
Additional comments: The authors' responsiveness to prior suggestions from the reviewers is greatly appreciated. The revised paper is much clearer.


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: GENERALIZED JOINT HYPERMOBILITY AMONG SCHOOL-AGED CHILDREN IN MAJMAAH REGION, SAUDI ARABIA
Review round: 2
Reviewer: 3

Output:
Basic reporting: When considering the revions small points have been adressed.

Still the most important issues still remain

I still fail to see the relevance of the study as the manuscript still is not consise enough
Experimental design: as mentioned previously
Validity of the findings: as mentioned previously

In my first comment
Additional comments: i still fail to see the relevance of the 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: VALIDITY AND RELIABILITY OF INERTIAL SENSORS FOR ELBOW AND WRIST RANGE OF MOTION ASSESSMENT
Review round: 1
Reviewer: 1

Output:
Basic reporting: This study seems like a technique report, rather than a scientific paper because the study needs is very weak.
Insufficient introduction and background descriptions.
Poorer article structure in Discussions.
Experimental design: The research question and rationale did not well defined.
“…new sample of 29 asymptomatic adults participated in the study…” this is an inappropriate description. Insufficient demographic data was showed in this study.
Validity of the findings: The rationale and impacts of this study do not state clearly.
The authors concluded that the “…the advantage of capturing the joint ROM digitally, making them a powerful tool to register the progress of treatment or implement physical therapies based on biofeedback (e.g. rehabilitation videogames)…” for people with disabilities. It is not appropriate because this study did not provide this information and techniques in this manuscript.
Additional comments: This study seems like a technique report, rather than a scientific paper because the study needs is very weak.

The authors did not point out the key point that why they have to focus on the elbow and wrist chronic conditions and their relationship with the pain and poorer muscle function in this (the study needs and rationale do not strength enough).

This study claims the inertial sensors are low-cost and accurate digital measurements, but it is not really because several inertial sensors with 3-D high accuracy and sensitivities are pretty expensive from 100-1500 USD.

Poorer scientific writing in Abstract. The authors should objective showed the compared data in results sentences, rather than state “…These values were within the normal range when compared to literature studies…” Furthermore, this problem also found in conclusions sentences “…Particularly noteworthy is their high inter-rater reliability, often questioned in measurement tools…”. The authors should reveal scientific conclusions based on their findings.

The authors concluded that the “…the advantage of capturing the joint ROM digitally, making them a powerful tool to register the progress of treatment or implement physical therapies based on biofeedback (e.g. rehabilitation videogames)…” for people with disabilities. It is not appropriate because this study did not provide this information and techniques in this manuscript.

In the introduction, this study did not show the study rationale very clearly and too roughly.

The scientific paper should focus on the needs in clinical, many clinical and reach tools can be used to evaluate the ROM, such as motion analysis system, Fastrak system…. The authors should compare their advantages and disadvantages in this study, rather than only state the “Technological advances…”

The authors reviewed many studies and revealed large descriptions about the technical information for Inertial Measurement Units (IMUs), robotic platforms and mobile smartphones. I suggest the authors should focus on study needs.

Poorer scientific writing in the last paragraphs in the Introduction

“…new sample of 29 asymptomatic adults participated in the study…” this is an inappropriate description. Insufficient demographic data was showed in this study.

Poorer organization for the “Statistical analysis” in Methods

The explains for study findings did not enough and without appropriate comparing with previous studies in Discussions,

The conclusions should be reorganized.

English editing would be needed.


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: VALIDITY AND RELIABILITY OF INERTIAL SENSORS FOR ELBOW AND WRIST RANGE OF MOTION ASSESSMENT
Review round: 1
Reviewer: 2

Output:
Basic reporting: I think that this is a well done study describing the reliability of an IMU method in comparison to a traditional goniometer method. However I think the structure of the paper would be improved by making sure that the correct details and specifics, of interest to the reader, are stated in the research paper. Some information could actually be removed (see some examples in my comments below) to give more room for discussion and interpretations on your results.
Experimental design: I have no comments on the experimental design other then a question on why three repetitions were used? Can you motivate why this number was selected? In many reliability studies, especially when using ICC statistics, a larger number of repetitions are recommended.
Validity of the findings: On row 317-318 you state that “The lowest values for r2 were found in pronation (0.47), during the first session and in particular for examiner B, although examiner A obtained higher results.” Does this mean that elbow pronation-supination ROM more variable/less reliable than elbow flexion-extension ROM for example? Given how the different motion directions are measured, it is not surprising if flexion-extension turns out to be more reliable and easier to measure. I think this should be reflected on and discussed in comparison to other studies analyzing elbow ROM.

In row 338-340 you state that the Bland-Altmann plots show good agreement between the methods. The number on the y-axis are however too small to judge whether this is true or not. Please revise the figures 7-8 so that the numbers can be read.

Row 379 – The references given here are about ten years old. More recent studies have been done examining upper body limbs using inertial sensors.  A few examples: “Monitoring of upper-limb movements through inertial sensors – Preliminary results” (2019),  "Motion capture sensing techniques used in human upper limb motion: a review" (2019)."A new way of assessing arm function in activity using kinematic Exposure Variation Analysis and portable inertial sensors--A validity study (2016).
Additional comments: -	The abstract do not state which movement directions you evaluate (elbow flexion-extension, supination-pronation, and so on). Please add this information.

-	The introduction is very general, starting with describing all musculoskeletal disorders (row 58-64), and all different ways that IMUs can be used for (row 97-123).  Please instead focus only on musculoskeletal disorders that impair the upper limb and specifically elbow and wrist movement which is the scope of your study (e.g. row 66-76). Also, the section about IMUs (row 97-123) should be shortened to focus on IMUs and upper limb. Perhaps a short section in the discussion about how this method can be generalized to other disorders in other parts of the body can be added if you think this is necessary.

-	Row 143-150 should be removed (all research studies are structured in the way you describe). Instead the aim of the study (3ow 136-141) should be the final word of the introduction.

-	Please also clarify and evaluate the aim further: We aimed to validate… by assessing criterion-related validity, intra-rater reliability and inter-rater reliability.

-	Row 183-185: I don’t think it is necessary to state that each person is given a code number; this is common practice in order to fulfill demands about anonymity etc.

-	Row 188- “Measures were taken by a universal goniometer” – please extend this description and add the name of the goniometer here instead (row 199-200).

-	Row 250: I think you should add a short desricption about how “ROM end-feel” is defined, since not all readers are physiotherapists (e.g. that end-feel means that the joint has full ROM and the range is stopped by the anatomy). Also, add that ROM stands for Range of Motion the first time you use this abbreviation.

-	Row 279 – Please do not specify which options you selected in SPSS, this may change over time due to software updates and is unnecessary information. Also, some information about the statistical choices you have made is more appropriate for a text book than a research paper even if the information is correct, e.g. row 283 “The use of SEM in reliability studies is justified because when a test administered to 284 one individual is repeated a number of times the observed value will vary between repetitions 285 due to the error in the measuring process. These responses are related to the mean. It can be proven that the true value of the measure falls within the observed value ± SEM 68% of the 287 times (McKenna, Cunningham & Straker, 2004).”  Perhaps this is enough information to the reader of this paper: “The SEM and the MDC at the 90% confidence level were calculated for both measuring instruments. SEM can be estimated from the ICC ….”

-	Row 334-336 – is something missing in this sentence? “as it was expected”? What was expected?

I did not find any raw data, i.e. an excel file with ROM measurements or similar? Have you considered to add such a file?


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: VALIDITY AND RELIABILITY OF INERTIAL SENSORS FOR ELBOW AND WRIST RANGE OF MOTION ASSESSMENT
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have reorganized added more exhaustive literature review in the introduction and discussion sections, well structure paragraphs, and sufficient context was provided to show the objectives and relevance in this study.
Experimental design: Research questions well defined, relevant, and meaningful.
Method sections described with sufficient detail.
Validity of the findings: Conclusions are well stated and strongly linked to the original research question.
Additional comments: I suggest this article is suitable and ready to publish.


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: EVALUATION OF COMPUTATIONAL METHODS FOR HUMAN MICROBIOME ANALYSIS USING SIMULATED DATA
Review round: 1
Reviewer: 1

Output:
Basic reporting: Studies comparing metagenomic algorithms are useful to both experts in the field, and researchers who are just considering applying metagenomics. Therefore, to help both groups, I'd suggest the following changes:
-	Discussing beefily the difference/similarities between binning and profiling approaches to metagenomic analysis.
-	Line 152: DIAMOND can align a large number of reads to the large databases, thanks to the spaced-seed. Buchfink, B., Xie, C., & Huson, D. H. (2014). Fast and sensitive protein alignment using DIAMOND. Nature Methods, 12(November), 59–60. https://doi.org/10.1038/nmeth.3176.
-	You mention the diversity of the dataset as an important factor impacting performance of the analysis pipelines. However, it would be good to discuss the concept of diversity mentioning the relative abundance of the species.
-	I’m not too familiar with the Oral microbiome. It would be nice if you could add a couple of sentences describing the general structure of the oral microbiome, how many phyla? How much inter-personal diversity etc.
-	The authors explained well that the profiling algorithms can only be as good as robust and comprehensive as is the underlying reference database
-	The authors describe in detail the differences of the selected pipelines, but I believe a graphical representation would better serve as an overview and comparison of the analysis pipelines.
-	In conclusions: you could attempt to give a simple recipe to the metagenomics newbies, which tool to use regarding the data, available time, computational power and diversity.
-	In your opinion, could there be value in using some combination of tools?
Experimental design: -	Why did you pick 75-100 bp as the read length? This way you are leaving out the popular sequencing technology such as Illumina MiSeq with paired-ends.
-	You also have not mentioned the long reads technologies like PacBio or Oxford NanoPore. Although their application to metagenomics is still in the beginning of the development, I think you should better justify the read length.
-	Kraken is being rapidly developed, I think using the latest versions of the software (Kraken2) would be much more useful.
-	Using perfect data constitutes a good baseline to pick the best suited tool for the problem. But this way you do not compare how robust the tools are regarding the error rate, which is an important factor.
Validity of the findings: - The authors thoroughly analyze the results and pose well justified conclusions.
- Graphical representation of the simulated profiles (e.g. a stack chart) and read length would be quite informative.
-Using the newest Kraken, adding longer reads, adding non-perfect reads would much improve this publication.
Additional comments: a.	Lines 201 – 208 I’d add the references here.
b.	Figure 2: It is hard to distinguish the shapes and colors.
c.	Figure 3: if the x-axis represents the different pipelines, what does it mean if the data-point is located between two ticks on x-axis?
d.	Figure 4: I suggest to flip this plot, namely to show how does the performance of pipeline change with the diversity.


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: EVALUATION OF COMPUTATIONAL METHODS FOR HUMAN MICROBIOME ANALYSIS USING SIMULATED DATA
Review round: 1
Reviewer: 2

Output:
Basic reporting: Clear, unambiguous professional language used throughout.

For the most part, clear language is used. However, there were a few noted instances of unclear language:

Line 84 – ‘of one sort or another.’
Line 397 – ‘nearly the entire gamut’
	Line 402 – ‘break this trend’
	Line 425 – ‘vary very little’
	Line 694 – ‘buck that trend’
	Line 660 – ‘occupies a space in between’

The authors may benefit from reading through the manuscript and clarifying language for an easier read.

Intro and background to show context.

The introduction does a great job showing the progression of taxonomic classifier strategies over time, effectively showcasing the differences among classifiers and their strengths/ weaknesses. Also, presenting the manuscript as a strategy for future comparisons as more classifiers are released shows the sustained impact this manuscript can have.

However, some information presented in the methods would benefit to clarify the descriptive information about how the classifiers work as added detail in the introduction. Classifier background information could be integrated in the paragraph on line 145. For more information, please see Experiment Design: Methods described with sufficient detail and information to replicate portion of the review.

Also, the background about different methods of creating complete metagenomic reference sets on line 104 could be further synthesized to the most relevant information. This paragraph and subsequent small paragraphs (starting at line 122 and line 128) can be combined and synthesized as well.

Structure conforms to PeerJ standards, discipline norm, or improved for clarity.

The results and discussion section were combined, and it would benefit the manuscript to separate them. Also, a separate funding statement is required outside of the acknowledgements.

Figures are relevant, high quality, well labeled, and described.

Figure 1 is not necessary if a link to the GitHub page is included.

Figure 2 text is too small to read, specifically the axis and legend. The dot size can be increased, and transparency of the dots reduced. For the bottom two panels especially, can the y-axis range be reduced (noting that in the figure caption) so it shows a clearer picture.

Figure 3 – The title is unnecessary. Also, can the authors need to clarify how outliers were determined in the figure caption.

Figure 4 – The text and data points should be a little larger.

Figure 5 – Axes and legend labels should have larger text.

Raw data was supplied.

Raw data were supplied on Github. Data provided are sufficient to replicate results.
Experimental design: Original primary research within the Scope of the journal.

Primary research was within the scope of PeerJ. This manuscript has relevance to biological, environmental, and medical sciences as selecting tools to characterize the microbiome have broad reaching impact. This research article is scientifically sound and identifies an important knowledge gap relevant to many fields.

Research question well defined, relevant, and meaningful. It is stated how the research fills an identified knowledge gap.

Research fits the aforementioned criteria. It fills a research gaps as there is limited research on the topic, however the results are useful to many different fields.

Rigorous investigation performed to a high technical and ethical standard.
The authors did a very thorough job documenting how the taxonomic profilers differ and comparing their performances. They included several different parameters for a vigorous comparison. Technically, the methods are sound and effective. There are no ethical concerns with this study.

Methods described with sufficient detail and information to replicate.

Overall, the methods described are detailed enough for replication. The authors should include code for running each taxonomic classifier on the GitHub page, to make the work a more useful resource for readers.

The methods overall were well justified and rigorous. Microbial/metagenomic attributes were informed by biological conditions, as well as read length and sequence depth by current technologies. Including the equations for RRMSE and AVGRE were useful for reader understanding and replication. The logic presented on excluding certain pipelines for downstream analyses was sound. Adding an unknown taxon and determining the effect on relative abundances has real-world significance, as well as addressing run time, which is often neglected in bioinformatic studies.

However, descriptions of the taxonomic profilers in the materials and methods read like background information. Some of this information might be more effective in the introduction or synthesized into the most important information in material and methods. For example, lines 218-221 and lines 223-230 could be further synthesized down to the most important information. Emphasizing the justification as to why each taxonomic profiler was chosen would make that section more useful for readers.

Line 312 – Because you have one biological scenario to mimic microbial data, i.e. acute infection, are there scenarios that reflect the small group of species show high abundance and half of species account for a majority of reads?

Line 331- why was +/- 50% of the actual abundance chosen as a match?
Validity of the findings: Impact and novelty not assessed. Negative/inconclusive results accepted. Meaningful replication encouraged where rationale and benefit to literature is clearly stated.

The structure of the results/discussion is effective. However, further rational and benefit to literature could be explored in the result/discussion section, as a lot of data were presented that could benefit from further discussion/summarization.

All underlying data have been provided; they are robust, statistically sound, and controlled.

Underlying data meet these criteria. There are a lot of data presented, and it would benefit the reader to summarize information and present main take home points.

There are not a lot of statistical tests are included in the manuscript, but sensitivity and specificity among classifiers with mean and variances test?

Speculation is welcome, but should be identified as such.

Speculation is not a large portion of this manuscript.

Conclusions are well stated, linked to original research question and limited to supporting results.

The conclusions are a little unclear, but could be due to the combined results/discussion format. However, conclusions are linked to the original research question and presented results.

Additional discussion points could improve the overall manuscript:

As the taxonomic profilers were compared under perfect base calling conditions, it might be useful to add to the discussion a comment about how the profilers would be affected by real data with sequencing errors.

A more clear summary of the tradeoffs among the pipelines and under which scientific scenarios would be best for each taxonomic classifier (presented in a table might be most effective) is needed. Also, based on the taxonomic profiles strengths/weakness there should be a better link back to the biological significance presented earlier in the manuscript (e.g., MetaPh1An2 would not be useful for disease states).

While the null state (presence of species in roughly equal number) was tested, the authors should add more discussion about what the results mean for the null case?
Additional comments: This manuscript has a lot of potential and will be an excellent contribution to high throughput sequencing methods; however, the overall manuscript is dense and long, and could be streamlined (especially in the methods and results/discussion) to make a better, tractable read for a more general scientific audience.

Line 210 – splitting up this paragraph when MetaPhlAn is introduced (line 218) will add clarity.

Line 328 – Definitions of sensitivity and specificity could be more clearly explained.

Line 497 – ‘little to be said’ sentence is unnecessary


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: EVALUATION OF COMPUTATIONAL METHODS FOR HUMAN MICROBIOME ANALYSIS USING SIMULATED DATA
Review round: 1
Reviewer: 3

Output:
Basic reporting: Given the increasing number of metagenomic analysis tools, it is very important to have a comprehensive evaluation/comparison of different tools. Thus I believe that this work is important and timely.
Overall, the writing is good but some parts are hard to follow, which can be particularly hard for readers who are not familiar with the methods. Several parts read like a laundry list. Better organizations should be applied to highlight the most important findings.
Experimental design: The authors did experiments using a large number of simulated metagenomic data and thus the results should be helpful for users to choose an appropriate tool. But one concern is that all the data are simulated. Although using simulated data can help quantify the performance more accurately, lacking results on real data is worrisome. Usually, the performance on real data and simulated data can be different, sometimes much worse. And this is not only because of the sequencing errors.

Can the authors compare these tools on one or two real data sets? Just comparing the findings among different tools will provide important information, if lacking ground truth is a concern.

Some tools were designed for slightly different purposes. For example, a couple of them can conduct strain-level analysis while others cannot. So, in this case, should the utilities specific to each tool be considered in the comparison? Or at least provide a table summarizing that information for users?
Validity of the findings: No comment
Additional comments: Some detailed comments are listed here:
1. I think it is not accurate to call Bowtie a heuristic. When conducting exact match, BWT-based tools can return correct matching positions. When errors are allowed, Bowtie may only return some of the matched positions. But overall, its main indexing structure (BWT) is not a heuristic.
2. The authors chose Bowtie but did not give justifications. Some people prefer BWA. They do have different outputs under their default parameters (such as local vs global). The authors need to discuss this because using different read mapping tools can lead to different downstream analysis output.
3. Line 331, any justification for choosing 50%? This sounds like a very permissive threshold.


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: EVALUATION OF COMPUTATIONAL METHODS FOR HUMAN MICROBIOME ANALYSIS USING SIMULATED DATA
Review round: 2
Reviewer: 1

Output:
Basic reporting: It is a second-round review, therefore I have only a couple of general remarks:

I believe the reviewed publication in naming and explaining the challenges in the short-read metagenomics, and is going to benefit scientists both new to metagenomics, thanks to the extensive introduction), and experts seeking the best tool. A continuously updated benchmarking tool is an important innovation. I hope authors consider extending their benchmarking efforts on the one side to 16S rRNA sequencing studies, and on the other side to the long-reads analysis.
Experimental design: The experimental design fits the question - the benchmarking of the pipelines from taxonomic profiling of the whole-genome metagenomic studies. The authors justified their choice of the:
- read length
- error rate (the reads are perfect because they wanted to compare the pipelines regardless of the error rates)
- the taxonomic composition (mock human oral microbiome)
Validity of the findings: Hope the authors commit to maintaining the benchmarking git repository,
Additional comments: Some language issues that I believe could be clarified that I managed to find:
-	Line 86:  I think “in turn” misleads the reader in this context, I would suggest a rewrite.

With taxonomic binning, individual sequence reads are clustered into new or existing operational taxonomic units (OTUs), obtained through sequence similarity and other intrinsic shared features present in reads. In turn, taxonomic binning can contribute to taxonomic profiling. With taxonomic profiling, the focus is on estimating the presence and quantity of taxa in a microbial population as well as the relative abundance of each species present.

-	Line 153:  consisted in reducing a multiple sequence
-	Line 167:  Other taxonomic profilers have therefore been developed around the desire to use entire genome libraries for more accurate taxonomic profiling and better functional inference, Is too long and convoluted.
-	Line 181: .. before classification can take place.


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

Input:
Title: A VALIDATION OF EMOTIV EPOC FLEX SALINE FOR EEG AND ERP RESEARCH
Review round: 1
Reviewer: 1

Output:
Basic reporting: - The first sentence of the introduction is plain wrong. Note that action potentials cannot be the biophysical origin of the scalp-recorded EEG signal, as they do not sum up to open fields (in contrast to post-synaptic potentials or pyramidal neurons). Please consult well-established references in the field of EEG physics for details (e.g. the works of Paul Nunez).

- Repeatedly, statements in the manuscript imply ignorance of the inverse problem in EEG interpretation (direct inferences sensor to source cannot be made). Note that EEG was not measured „in the right hemisphere“ as stated in the abstract, but „over“ the right hemisphere. Indeed, the measurement of EEG asymmetries is already a highly complicated issue, and only the statement „over“ correctly expresses that signals measured over the right hemisphere may (or may not) originate in the right hemispheric regions of the brain. Again, the authors might benefit from consulting literature for details (e.g.: https://sapienlabs.org/the-inverse-problem-in-eeg/)

- The introduction ignores early work on signal quality of EMOTV systems. Specifically, Debener et al., (2012, Psychophysiology) presented the precursor of the EMOTIV Flex system, by combining the EMOTIV amp with easycap electrodes and reporting high-quality auditory oddball P300 ERPs even in outdoors free walking conditions (replication published by De Vos et al., 2014, Int J Psychophysiology). The same group also compared the EMOTIV amps with a research-grade amplifier in a P300 speller BCI paradigm and found very similar online performance (De Vos et al., 2014, J Neural Eng). Given that the Flex EMOTIV system is based - to a large extent – on the very same hardware as the EMOTIV epoc system, it is necessary to at least discuss those papers in the introduction. In fact, given that the most critical specs of the Flex and the epoc system are identical (14 bit, 128 Hz AD rate, etc), the introduction should also briefly summarize similarities and differences between Flex and epoc hardware.
Experimental design: - While the overall design is convincing, two issues captured my attention. First, can the authors really guarantee that bringing Flex and neuroscan electrodes next to each other did not generate low impedance electrical bridges? Otherwise the limitations resulting from such electrical shortcuts should be discussed (i.e. is cannot be guaranteed that the Flex system would generate the same signal quality when used alone). Please provide at least a close-up picture of the electrode arrangement and discuss this limitation. Another issue is the use of different filter settings. Why was the neuroscan data recorded with a 1 Hz high-pass filter? It would have been much better to use a filter matching the EMOTIV filter settings (at least offline). This issue requires some discussion as well, as a 1Hz filter clearly alters the P300 morphology (i.e. attenuates P3 amplitudes).
- Using the number of remaining trials as a measure of EEG quality is not convincing, in particular when a rather dull amplitude-based rejection criterion was used. Note that the amplitude of EEG signals depends on many factors (for instance, the distance to the reference location, which will be larger for a person with a larger head even if identical 10-20 locations were used) and does not separate signal from artefact very well (e.g. some individuals have eye blinks below 120 uV, others have alpha oscillations above 150 uV). A much better criterion for ERP comparison is the signal-to-noise-ratio (signal amplitude divided by the rms of the pre-stimulus signal, for instance). Please replace your trial-number results with a proper report of SNR values.
Validity of the findings: - While the ERP morphology comparison at single channels is well justified, this analysis missed another key feature of EEG signal quality, namely the topographies. Unfortunately, topographies are only provided for some analyses. Please add topographic illustrations and a statistical comparison of the topographies. Very similar topographies would support your claim that the Flex system can collect research-grade signal quality.

- the acknowledgments state that the paper was supported by the company EMOTIV. The authors should add a statement on potential conflicts of interest.
Additional comments: This is an interesting paper comparing ERPs, SSVEPs and resting-state EEG spectra that were recorded concurrently with a new consumer-grade EEG system and a research-grade system. Overall, the results reveal very similar signals, which led the authors to conclude that the consumer-grade system allows recording research-grade EEG signals. This finding is not really surprising, given previous reports on the issue.

There is much to like about this paper, which is well written and easy to follow. Moreover, the experimental design is straight-forward and  the Bayesian approach used for the statistical analyses makes perfect sense, as it allows interpreting similarities and differences. However, the following issues require consideration:


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

Input:
Title: A VALIDATION OF EMOTIV EPOC FLEX SALINE FOR EEG AND ERP RESEARCH
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript is written well and presents data clearly within the presented tables and figures. The authors have also provided a good literature review of EEG technology and the emergence of low-cost EEG alternatives. I feel the rationale around the choice to validate the Flex Emotiv EEG could be communicated more strongly in the introduction. For example, the importance of why the Flex model has been chosen for validation has not been communicated other than “providing greater sensor coverage” (line 156) compared to three other brands of portable EEG device. If the specifications of the Flex EEG device were stated within the introduction (e.g., sampling rate, number of electrodes, method for reducing and measuring impedances, details of the data acquisition software used, etc) it would help establish clearly the pros and cons of this system relative to a lab-based EEG set-up, and hence, the importance for the present validation study.

Some other points are included below for your reference:

-Line 133 – Could the authors specify which “involved set-up procedures” and “lab-based limitations” are being alluded to regarding lab-based EEG, and describe how Flex may overcomes these problems?

-Line 288 – I appreciate the transparency of reporting the sampling rate of your specific Emotiv Flex device as being 129.05 Hz. The accuracy of an EEG systems’ sampling rate is highly important when suggesting an EEG device to be research-quality, so feel this should be considered in more depth in the body of the manuscript rather than in a footnote. It might be helpful to discuss what variability in sampling rates one might expect from low-cost EEG devices, and what impact (negligible or not) that such variations in hardware may have on research accuracy. Further, it would be highly beneficial to describe your method for determining the precise sampling rate of your Flex unit to allow other researchers to do the same to their own devices.

-It would be fruitful to discuss the trade-off of using low-cost EEG devices compared to traditional, lab-based equipment for research. For example, the added flexibility of the Flex system is off-set by a lowered sampling rate of 128 Hz compared to the much higher sampling possible from research-grade EEG. A more balanced assessment of the strengths and weaknesses of the Flex EEG device should be offered in the discussion to concede some of the current limitations of portable EEG technology for research.

-Line 283. Please provide more comment regarding the Flex’s impedance measurement system. What does a connection of “green” in the Flex acquisition software equate to in kΩs?

-There are inconsistencies regarding specifics of the method used to different analyses and the subsequent data presented in the results. For example, Lines 362 and 363 imply assessing the N170 only at P8, but data for both P7 and P8 electrodes is presented in Figure 5 and discussed later in the manuscript. Please check for consistency between these details explained in the method and subsequently reported output.

-Lines 171, 302, 309 and 315 – The citations/ URL’s provided in text in these lines of text appear to be broken. Please revise.

-Line 509 – remove the second “is” in the sentence “it is the implicit aim of validation studies such as the current one is to determine …”

-Line 276 – recommend “ml” be written as “mL”

-Line 113 – remove period at the end of citation “(…2015 for a review). and represent a ”...
Experimental design: The experimental design used to produce the desired ERP components and EEG power spectra were sensible. I feel this is a valuable research area and the manuscript is working towards addressing a relevant and meaningful research question. I have no major comments as to the methodology used, but have included some suggestions regarding the reporting of the methodology to improve the ability to replicate.

-Lines 318, 328, 331 and 333 – Please provide explicit description of the settings used within the Fieldtrip functions described at these lines. For example, line 318 should describe details of the hanning window used, including the length, step and overlap of the window used to allow precise replication of this FFT process in future.

-Line 231 – Please describe in what location relative to the Flex electrodes the Neuroscan electrodes were placed adjacent to each other on the scalp. For example, the direction and approximate distance of Neuroscan adjacent to Flex electrode would be valuable for replication.

-Line 191 to Line 200 – The number of blocks, number of tones-per-block, and overall MMN task length, should be reported in this section for replication of the MMN paradigm.

-Line 213 – Wording appears to suggest the N170 delivered in a single block of 300 trials, with a self-timed break after 150 trials. Can the authors please confirm/ elaborate upon the administration of the N170 task? For example, what was the task length for the N170 paradigm used?

-Line 225 – Please describe what materials were used in the SSVEP paradigm. For example, was the same monitor placed at 50cm away as in the N170 paradigm used here?

-Line 226 – Duration of the eyes-open RS task should be stated. Were any instructions provided to participants relating to minimising eye-blinks and movements during this task? Furthermore, procedure for the ‘eyes-closed’ RS paradigm was not described at all. Please confirm details of this task, including the duration of the eyes-closed block, and specifying any instructions provided to participants.

-Please provide citation/s supporting the methods used for calculating the SNR as described in lines 320 to 322.
Validity of the findings: It is unclear why the researchers recorded EEG from 16 scalp electrode sites, but only report comparisons of single electrodes or small groups electrodes for most of their experimental paradigms. A more comprehensive manner to demonstrate Flex being a robust EEG device would demonstrate comparable signal quality to Neuroscan at all the selected International 10:20 electrodes sites. For example, the ICCs achieved during the N170 paradigm were lower at P7 compared to position P8, indicating that different electrodes may have different recording quality relative to the Neuroscan. It may be that the further away an electrode is placed from central scalp locations, or locations where ERP components are reliably detected, that the SNR of the Flex diverges significantly from a research-grade device like Neuroscan. Given the amount of data acquired across 16 electrode sites, I feel the authors have the opportunity to provide more comprehensive analysis of the EEG across all the different International 10:20 electrodes recorded from to conclusively show Flex and Neuroscan do not differ across any of the chosen electrode locations used.
Additional comments: Thank you for the opportunity to review this manuscript. The authors present a thorough, well-devised experiment to validate a low-cost EEG device detecting both ERP and temporal EEG date. This is a valuable research area, and the breadth of data collected across several common EEG paradigms is an intelligent way to compare the Emotiv Flex EEG device’s signal quality against a research-grade Neuroscan EEG device. The manuscript provides a good overview of the relevant literature supporting the five paradigms used but is less clear in establishing the advantages that the Emotiv Flex EEG device offers over traditional EEG and other low-cost EEG devices. This leaves the importance of validating this specific EEG device somewhat in question. For example, the authors could elaborate as to the technical specifications of the Flex EEG system in comparison to the other portable EEG systems (e.g., Neurosky, Muse 2 and EPOC) including the number of recording electrodes, sampling rate, data acquisition hardware and software), and methods of data acquisition (e.g., use of saline compared to electroconductive gel, methods for reducing electrode impedances, method for and reliability of recording electrode impedances) to demonstrate Flex as a credible research-grade EEG system.

The paradigms chosen to illicit a MMN, P300, N170 and the detection of alpha power and SNR ratios across 1 to 30 Hz power-bands were intelligent and demonstrate the Flex EEG system’s capabilities across several measures of EEG. However, I am unclear why such a small subsection of all the recorded EEG data acquired from these paradigms were presented within the results of this paper. It appears that limited/ no analysis was conducted on data collected from many of the electrode sites recorded from (i.e., Fp1, Fp2, F3, F4, FT7, FT8, CP3 and CP4) in preference of comparing signal quality at sites known to robustly show an ERP component of interest (e.g., Fz to assess MMN detection). I feel a more comprehensive demonstration that the Flex EEG system has comparable signal quality to the Neuroscan system at all 16 recording sites across numerous paradigms in order to be genuinely considered a potential research-grade EEG device. Additionally, some aspects of the procedure and specific aspects of the EEG data processing should be stated more explicitly to allow independent replication of findings.

Overall, further methodological explanation and statistical evidence is needed to convincingly meets the stated aim of the manuscript in showing the the Emotiv Flex is equitable to Neuroscan for research-standard EEG.


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

Input:
Title: A VALIDATION OF EMOTIV EPOC FLEX SALINE FOR EEG AND ERP RESEARCH
Review round: 2
Reviewer: 1

Output:
Basic reporting: •	Line 69: the allusion to “different questions using different techniques” is vague. Could examples of such questions be explicitly/ immediately stated, or else a stronger segue to EEG oscillations provided?
•	Lines 73 and 74: it is unclear why the Delta power is described here given you do not explore deep-sleep or detection of the delta band specifically in any analyses.
•	Line 75: “myriad processes”
•	Line 114: Replace comma with period after “(Eimer, 2012),”
•	Line 151: Please describe some of the ways these systems have increased portability and easier set-up than lab-based devices.
•	Line 154: Could you add a comment regarding in what capacity they have attracted the attention of researchers? Are you referring to they attract researchers due to their advantages over lab-based EEG, they are attracting researchers attempting to validate the systems, or both? That is, are low-cost EEG devices being adopted by researchers for experimental studies at present?
•	Line 174/ Line 175: Was this a ‘Muse’ or ‘Muse2’ model as per your earlier listing in Line 152?
•	Line 176: Given your earlier mention of ‘NeuroSky MindWave’ in Line 153, please provide an example (if available) of a validation of this system or else discuss the weighting of the different validations of these systems. That is, since the literature review provided shows that most prior interest has been towards validating Emotiv EPOC systems please comment as to how well validated the different low-cost EEG devices you’ve discussed currently are.
•	Line 196: As stated above, the advantage of the “configurability” of the system is overstated given it has been possible to modify these systems to be configurable. Please comment/ revise.
•	Lines 354 to 364: un-bold text in this section
•	Line 536: “neuroscan” misspelled (missing 'o')
•	Line 556: “Figure 3” on wrong line
•	Line 798/799: un-bolden DOI
Note: I had difficulty aligning some figures with the relevant output being discussed in text. For example, I have missed the distinction between what the top (A, B, C, D) and bottom (E, F, G, H) panels of figure 3 are displaying. For example, it was unclear what the comparison between Panels D and H of figure 3 (as made on line 552) was intended to contrast. Are these deviant signals under the active listening from different 10:20 scalp locations? In addition, panels C and D of figure 5 are not clearly labelled in text (likely an error in the description in line 572). Please double-check all in-text descriptions and figures for clarity and ease of comprehension.
Experimental design: •	As per previous review, the study is conducted to a high empirical standard.

The gap in the literature this validation addresses is more clearly outlined in the introduction, but the advantage of the Flex system as being “configurable” is overstated as other low-cost EEG devices can be modified to be configurable to be used with an EasyCap (see above comment). I also feel the distinction between the Flex model of the Emotiv and prior EPOC+ systems could be discussed further, with the emphasis of the types of analyses possible with a 32-electrode array over the 14-electrode array of other devices being the main advantage of the system. Hence, to show the benefits of this system above others, this unique aspect of the system need to be emphasized and clearly linked to the unique research questions that could be better answered using Flex > EPOC+ versions of Emotiv
Validity of the findings: Line 182: While the rigidity of the sensor placements in base EPOC is true, several of the prior studies you have cited (Debner et al 2012, Debner et al, 2014; Barham et al., 2017) have already validated modified versions of the EPOC in which the ridged sensory bracket was replaced with shielded EEG wires allowing a configurable EEG array. Hence, this identified limitation of low-cost EEG systems is not completely accurate. Please revise.

I feel the authors have demonstrated comparable signal quality between EEG systems across several EEG and ERP protocols, an important limitation of the system that needs to be acknowledged more directly is the colour-coded impedance measurement system. I appreciate the authors personal correspondence finding that a “green” equates to an impedance of “<20 kOhms” (line 341) but the implications of this is not considered beyond this point. To me, the connotations of the conclusion that Flex is “a suitable alternative to research-grade EEG system” means both that is able to acquire accurate EEG and ERP data (demonstrated well), but also that it is possible to publish data acquired with a system as per the typical reporting standards of a field. Given EEG research studies typically aim for (and report) achieving electrodes impedances < 5 (or sometimes < 10) kOhms, I feel the ambiguity both in the actual impedance that was achieved, and the much higher minimum impedance of the Flex cannot be ignored. While not an irreconcilable problem, it is a genuine drawback of the Flex that is not considered at all in the manuscript. I would appreciate the authors comments on this limitation and a slightly more balanced consideration of the trade-off of this low-cost system has over lab-based devices.
Additional comments: This is a well written manuscript with good methodological rigour. I appreciate the inclusion of the comparison Table of the different low-cost EEG systems in the introduction, additional explanation of the methodologies of the different tasks, and additional analyses regarding the SNRs of the systems. Aspects of the basic reporting need to be reviewed (see above suggestions). Regarding the “gap” in the literature, I would encourage the authors to more clearly differentiate the advantages of this Flex model of and Emotiv EEG system from the EPOC+ (standard and modified versions) which have undergone several past validations in the past. Given the better sensor array at the main strength of this system over the 14-channel EPOC+ studies, discussion of the types of studies which can be achieved with 32-electrode arrays that cannot be achieved with 14-electrodes would be a useful. In addition, the “configurability” of the system is overstated given several prior studies which have shown modifications are available to the EPOC+ to allow it to be used with an EasyCap at configurable 10:20 locations. Finally, the Flex does provide appear to have potential as a research-grade EEG system, but some limitations of the system may be overlooked/ downplayed to make the conclusion about the system appear stronger than they potentially are. For example, the subtle, but important limitation of the Flex colour coded “traffic-light” impedance measuring method should be considered as part of the shortcomings of the device and final conclusion about the value of the system.


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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript by Zhou et al. describes the effects of n-acetylcysteine (NAC) on the oxidative stress- and inflammation-induced hepatotoxicity of polychlorinated biphenyl 52 (PCB 52) in two cell lines.

The manuscript and graphics are clear, the structure of the manuscript is appropriate. For more clear understanding, I recommend to make a scheme of genes and respective proteins and their connections (e.g., Keap1/Nrf2 regulating Hmox1 etc) to illustrate the findings of the second part of the manuscript (Figs 3 and 4).

There is a remarkable overlap with the previous publication by the same authors (Wang et al., 2018, https://doi.org/10.1016/j.toxlet.2018.03.002) in terms of overall approach, cell lines and methods. In the previous publication, authors showed the toxicity of PCB 52 to two cell lines, the ability of PCB 52 to induce reactive oxygen species (ROS) and that 3-Methyladenine suppressed ROS. This part is very similar to the first part of the current manuscript. Authors mentioned their previous paper, mostly in the context of PCB 52-induced hepatotoxicity, but they did not explain clearly, what are the knowledge gaps and what is the main hypothesis of the current manuscript compared to the published paper. Also, it would be useful to discuss their current gene expression and Western blot results in the context of the previous findings.
Experimental design: The research hypothesis is unclear.
Methods are mostly well described, only some details are missing (source and passage number of the cell lines; validation of the antibodies).
Validity of the findings: The interpretations that are based on the results of Figs 3 and 4 are inconclusive. For example, in my view, the statement on the "silencing of keap1/Nrf2 pathway" is too speculative. There is no down-regulation of respective mRNAs (Fig 3) and no convincing proof of silencing. In addition, the data on Hmox1 expression on mRNA vs protein level and the explanation of authors is not convincing and should be supported by further experiments.
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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 1
Reviewer: 2

Output:
Basic reporting: The article structure is in a good shape.
Experimental design: The experimental design is reasonable.
Validity of the findings: The results are conclusive, but more experiments can be done to better understand the mechanisms.
Additional comments: In this manuscript, the authors investigated the effects of NAC to mitigate the hepatotoxicity mediated by PCBs. Pretreatment with NAC was able to ameliorate the oxidative stress and inflammatory responses in hepatocytes triggered by PCBs. Evidenced by the genes and proteins expression profiles, the authors concluded that the hepatotoxicity was induced mainly through the TLR4-mYD88 pathway, but not Nrf2 pathway. The data presentation is clear, but more discussion is needed to discriminate the results. Some detailed comments for the authors to address.

1.	The authors used two hepatocyte lines, one from rat and one from human. The responses in these two cell lines are similar but not the same. What is the rationale to use these two cell lines? The authors should discuss the results.

2.	The names of the genes should be consistent throughout the manuscript.

3.	In this study, NAC was pretreated with the cells. How about co-exposure of PCB52 with NAC? NAC has a short therapeutic time window evidenced by some in vivo studies. It will be interesting to perform experiments in vitro to understand the best therapeutic time for NAC.

4.	The authors found a significant upregulation of TNFα and IL-1β mRNA expression, which indicated the activation of NF-κB. The role of NF-κB activation as down-stream of TLR signaling is implicated in the expression of pro-inflammatory cytokines (TNFα, IL-1β) and assist in the manifestation of inflammation. The authors are encouraged to check the NF-κB activation (phospho-p65) by western blotting.

5.	TLR signaling and Nrf2 pathway may crosstalk with each other through p62 driven autophagy for the regulation of inflammatory responses. The authors could discuss more about the communication between these two pathways.


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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 1
Reviewer: 3

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: In the submitted manuscript, Zhou et al. investigates the mechanism of N-acetylcysteine alleviates PCB52-induced hepatotoxicity. They find that a significant decrease in cell viability and increased ROS levels and malondialdehyde contents in PCB52-treated cells relative to the control, which may be regulated by Keap1/Nrf2 signal pathway and need further study such silencing the Keap1/Nrf2. Overall, the study contributes novel knowledge on the role of NAC in hepatotoxicity caused induced by PCB52. Meanwhile, methods and materials described with sufficient detail and information to replicate. However, there are some points that would need more explanation before publication.

Major comments:
1. Why did the study choose 40 μM of PCB52 for the further experiments? Because it is the IC50 or other reasons.

2. In figure 3, I see some error bar is so high. Is it three independent replicates or three replicates of an experiment? Why did not detect IL-8 in Rat Brl-3A cells and Sqstm1 in human L-02 cells?

3. In figure 4, maybe it’s better to show the protein band size. Because some protein bands are two bands, such as Nrf2 and Keap1.

4. Whether it has time-dependent effect of PCB52 on Brl-3A and L-02 cells, like 24 h, 48 h, 72 h and longer?

5. In the supplementary figure 2, different concentrations of NAC ameliorated PCB52 effects on cell viability in different degree. I wonder why 5 mM and 10 mM of NAC has lower effect than 3 mM under noncytotoxic concentrations?

6. Are there some agonists or antagonists of Keap1/Nrf2 signal pathway for positive or negative control?

7. In the line 132, “And” should be corrected to “and”.


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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 2
Reviewer: 1

Output:
Basic reporting: Authors answered the questions and significantly improved the manuscript. I propose to accept this manuscript.
Experimental design: Correct.
Validity of the findings: Correct.
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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 2
Reviewer: 2

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: The questions and comments have been well 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: N-ACETYLCYSTEINE ALLEVIATES PCB52-INDUCED HEPATOTOXICITY BY REPRESSING OXIDATIVE STRESS AND INFLAMMATORY RESPONSES
Review round: 2
Reviewer: 3

Output:
Basic reporting: no comment.
Experimental design: no comment.
Validity of the findings: no comment.
Additional comments: In the revised submitted manuscript, the author has  a good response to the questions and I don't have other questions. The submission has been greatly improved and is worthy of publication. Finally, I think it is better for author that the change of figures and the revised manuscript was shown in the " Response to the reviwer".


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: COMPARING TRANSCRIPTOME PROFILES OF HUMAN EMBRYO CULTURED IN CLOSED AND STANDARD INCUBATORS
Review round: 1
Reviewer: 1

Output:
Basic reporting: 1) The grammar, spelling mistakes and references in the manuscript need to be checked:

a.	Line 30: We is spelled wo
b.	Line 50: change provide to provides
c.	Line 55: reference missing
d.	Line 59: Replace changing to changes
e.	Line 91: Change u to µ
f.	Line 120: Change zygote to zygotes
g.	Line 131: Please check cDNA production. Do you mean cDNA product?
h.	Line 172: Give full form for TPM abbreviation
i.	Line 193: Change group to groups, add a coma
j.	Line 222: change PH to pH
k.	Line 227: change require to requires
l.	Line 237-239: Please check grammar
m.	Line 244: Check grammar
n.	Line 246: says genes play important roles but no mention of what roles and how it affects the embryos
o.	Line 259: Change significantly to significant
p.	Line 260: Change have to has
q.	Line 270: Inconsistent fonts
r.	Line 274: Change difference to differences, have to has and add an
s.	Line 275: Change need to needs
t.	Line 449: Check grammar
u.	Line 453: Change represent to represents
Experimental design: The main focus of this manuscript was to compare the transcriptomic profiles of 8-cell embryos cultured in standard incubator (SI) to time-lapse imaging incubator (TLI) using RNA-sequencing. Authors found that the global transcriptomic profiles of the 8-cell embryos were similar except for 539 genes that were differentially expressed. Using GO enrichment analysis authors found the different functional pathways that those genes were part of.
Overall, this is an interesting study because it is imperative to confirm that the newer ART/IVF laboratory materials and methods not only have no detrimental effects but also improve the quality of the embryos. RNA-sequencing is a great tool to test the differences between gene expression profiles of these embryos cultured in two different incubators.

1)	Section: Methods, Subsection: Embryo score. The embryos were scored on a scale of 1-3. Which ones were chosen for RNA-sequencing?

2)  Section: Methods, Subsection: Embryo score. For the SI, how often were the embryos taken out for observation? Did that match the usual methodology for embryos cultured in SI?
Validity of the findings: 1)	Although RNA-sequencing generates a lot of useful data, it is incomplete without proper validation. The authors have mentioned this, however, this approach makes the manuscript purely descriptive.

2)	The functions/pathways of the over expressed genes in both groups are generated through bioinformatics analysis. The manuscript should address why they might see the differences in the 2 groups with regards to the cellular pathways and what that might mean for the quality of the embryos. This should be addressed in the discussion.

3)	The paper could be elevated if there is mention of any data that may have looked at the differences between the embryo qualities or even blastocyst rates between the 2 incubators. This can confirm whether functionally, one method might be better than the other. This may also address the differences in gene expression profiles.
Additional comments: No additional comments